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1 INTEGRATED DISEASE MANAGEMENT IN CHICKPEA THROUGH CONVENTIONAL AND BIOTECHNOLOGICAL APPROACHES Sadia Perween, Anand Kumar*, S.P. Singh, Manoj Kumar, Anil Kumar, Satyendra, Ravi Ranjan Kumar1, Mankesh Kumar and Sanjay Kumar Department of Plant Breeding and Genetics, Department of Molecular Biology and Genetic Engineering Bihar Agricultural University, Sabour (Bhagalpur)-813210, Bihar *Corresponding Author:
[email protected]
ABSTRACT Chickpea is grown mostly in South Asia and Sub-Saharan Africa, which accounts for more than 75% of the world chickpea area. The area under chickpea is gradually facing a decline. The major causes being biotic and abiotic stress prevalent in different areas. Diseases causes up to 100% losses in yield. The severity of disease is compounded due to the favorable environmental factors and abiotic stresses. The extent of loss in yield due to wilt and root rot are far more than due to drought and high temperatureIn recent years, genetics of complex biotic stresses like Fusarium wilt, Ascochyta blight have been understood in the chickpea and the genomic regions/QTLs have been identified. Furthermore, several functional genomics approaches such as RNAseq, Massive Analysis of cDNA Ends (MACE) with parental genotypes of mapping populations as well near isogenic lines (NILs) have provided some candidate genes for abiotic and biotic tolerance that are being validated through genetical, genomics and/or TILLING approaches. In 2013, International Chickpea Genome Sequencing Consortium (ICGSC) co-led by ICRISAT, University of CaliforniaDavis (USA) and BGI-Shenzhen (China) assembled the draft genome of kabuli chickpea genotype CDC Frontier, while Next Generation
2 Challenge Programme on Chickpea Genomics (NGCPCG), India assembled genome sequence of desi genotype ICC 4958. After assembling the draft chickpea genomes, efforts to exploit the potential of next generation sequencing (NGS) technology to understand the genome architecture of chickpea were initiated. As part of these initiatives NGS based whole genome re-sequencing (WGRS) of chickpea parental lines were undertaken, which led to identification of 2 million Single Nucleotide Polymorphisms (SNPs) and more than 290K Indels. These SNPs are valuable resource providing enough markers to undertake the genetics research. In addition, NGS based WGRS was used to understand the impact of breeding on genetic diversity and temporal diversity trends in chickpea. For this, WGRS was used to re-sequence more than 100 chickpea varieties released in last five decades and identified 1.2 million SNPs. Global gene banks store the huge germplasm wealth that has the potential to contribute significantly towards the goal of enhancing the rate of genetic gain. Chickpea Genome Sequencing Initiative” to re-sequence 3000 lines from Global Chickpea Composite Collection The genomic resources generated have been successfully deployed for developing superior lines for different traits of interest. Trait mapping and molecular breeding such as marker assisted backcrossing (MABC), markerassisted recurrent selection (MARS) and advanced backcross quantitative trait loci (AB-QTL) analysis, which are routine in breeding programs for major crops, are also being practiced in chickpea. For example, superior lines with enhanced to fusarium wilt and ascochyta blight have been developed. Introgression of the “QTL-hotspot” into several elite varieties in India as well as Kenya and Ethiopia led to development of superior lines with tolerance to multiple diseases and increased yield under rainfed and irrigated conditions irrespective of genetic background. Several molecular, genomics and functional genomics approaches are currently being used for identification, mapping and cloning of biotic stress resistance genes. These resistance genes could be conventionally transferred to susceptible varieties through molecular breeding procedures or modern biotechnological approaches to develop high yielding and biotic stress resistant varieties. Key words: Chickpea (Cicer arietinum L.), IDM, Disease resistance and molecular approaches
INTRODUCTION Chickpea (Cicer arietinum L.), a member of Fabaceae family, is one of the most important rabi pulse crops in India contributing 20% to the total pulse production in the world. Chickpea is the third most important grain legume in the world after dry beans and dry peas. It is grown in Asia, Africa, Australia, Europe, North America and South America. Two main types of chickpea cultivars are grown globally– kabuli and desi, representing two diverse gene pools. The kabuli types are generally grown in the Mediterranean region including Southern Europe, Western Asia and
3 Northern Africa and the desi types are grown mainly in Ethiopia and Indian subcontinent. Desi chickpeas are characterized by angular seed shape, dark seed coat, pink flowers, anthocyanin pigmentation of stem, rough seed surface and either semi-erect or semi-spreading growth habit, whereas kabuli types generally have owl shaped seeds, beige coloured seeds, white flowers, smooth seed surface, lack of anthocyanin pigmentation and semispreading growth habit. Chickpea is a good and cheap source of protein for people in developing countries (especially in South Asia), who are largely vegetarian either by choice or because of economic reasons. Additionally, chickpea is rich in minerals (phosphorus, calcium, magnesium, iron and zinc), fiber, unsaturated fatty acids and β-carotene. Chickpea also improves soil fertility by fixing atmospheric nitrogen, meeting up to 80% of its nitrogen (N) requirement from symbiotic nitrogen fixation. Chickpea returns a significant amount of residual nitrogen to the soil and adds organic matter, improving soil health and fertility. Growing interest in chickpea consumption, coupled with increased preference for vegetable-based protein has led to an increase in global demand for chickpea. Additionally, awareness of both economic (premium prices for large seeded kabuli) and other benefits of chickpea including human health, crop diversification and sustainable agriculture have increased interest among farmers in cultivating chickpea. Conventional breeding approaches have given over 350 improved cultivars, which have contributed to improved productivity, reduced fluctuations in yield, and enhanced adaption of chickpea to new niches (Gaur et al., 2007). It is a self pollinated crop with a basic chromosome number eight and a 738 Mb genome size. Based on seed market type, chickpea is classified into two groups namely desi and kabuli. Grains of desi chickpea are small in size, light to dark brown in color and have a thick seed coat. Grains of kabuli chickpea are bigger in size, have a whitish-cream color and thin seed coat. The desi type is more prominent and accounts up to 80% of global chickpea production. Chickpea is a highly nutritious grain legume crop and is one of the cheapest sources of protein. Chickpea is an important source of protein for millions of people in the developing countries, particularly in South Asia, who are largely vegetarian either by choice or because of economic reasons. In addition to having high protein content (20-22%), chickpea is rich in fibber, minerals (phosphorus, calcium, magnesium, iron and zinc) and β-carotene. Its lipid fraction is high in unsaturated fatty acids. It is an important source of energy, protein, soluble and insoluble fibre. Further, the seed protein contains essential amino acids like lysine, methionine, threonine, valine, isolucine and leucine. On an average,
4 chickpea grains contain 60-65% carbohydrates, 6% fat, and between 12% and 31% protein –higher than any other pulse crop. It is also a good source of vitamins (rich in B vitamins) and minerals like potassium and phosphorus. Chickpea like other legume crops also replenishes soil fertility through biological nitrogen fixation. Chickpea plays a significant role in improving soil fertility by fixing the atmospheric nitrogen. Chickpea meets 80% of its nitrogen (N) requirement from symbiotic nitrogen fixation and can fix up to 140 kg N/ ha from air. It leaves substantial amount of residual nitrogen for subsequent crops and adds plenty of organic matter to maintain and improve soil health and fertility. Because of its deep tap root system, chickpea can withstand drought conditions by extracting water from deeper layers in the soil profile. International Chickpea is grown mostly in South Asia and Sub-Saharan Africa, which accounts for more than 75% of the world chickpea area. Global chickpea production has increased from 7.68 million tonnes (1961) to 13.73 million tonnes (2014) (FAOSTAT, 2016). It is the third largest produced food legume globally, after common bean (Phaseolus vulgaris L.) and field pea (Pisum sativum L.). Amongst the annual seed crops it ranks 14th in terms of area and 16th in production. According to Food and Agriculture Organization (FAO) statistics for 1996–2005, the chickpea area averaged nearly 11 million hectares worldwide (1.2% of total crop area) and production was slightly more than 8 million tonnes (0.34%). However, amongst pulse crops chickpea has consistently maintained a much more significant status, ranking second in area (15.3% of total) and third in production (14.6%). Chickpea is grown in more than 50 countries (89.7% area in Asia, 4.3% in Africa, 2.6% in Oceania, 2.9% in Americas and 0.4% in Europe). India is the largest chickpea producing country accounting for 64% of the global chickpea production. The other major chickpea producing countries include Pakistan, Turkey, Iran, Myanmar, Australia, Ethiopia, Canada, Mexico and Iraq. National East Asia dominates the world’s chickpea production, accounting for 75%; India ranks first in terms of cultivated area and production. However, there is a slight increase in the chickpea cultivation area in India from 9.27 million hectares (1961) to 9.92 million hectares (2014), but production increased significantly from 6.25 million tonnes (1961) to 9.88 million tonnes (2014) due to significant increase in the productivity from 0.67 t/ha (1961) to 0.99 t/ha (2014). India alone produced 65% and the
5 majority (85%) were desi types. The Genus Cicer The Cicer genus belongs to family Leguminoseae, sub-family Papilionaceae and tribe Cicereae. It encompasses 9 annual and 34 perennial wild species. Most of these species are found in West Asia and North Africa covering Turkey in the north to Ethiopia in the south, and Pakistan in the east to Morocco in the west. Of the 9 annual species, chickpea (Cicer arietinum L.) is the only cultivated species. The eight other annual species of chickpea are wild and include: C. reticulatum, C. echinospermum, C. pinnatifidum, C. judaicum, C. bijugum, C. cuneatum, C. chorassanicum and C. yamashitae. Van der Maesen (1987) classified the Cicer species into four sections based on their morphological characteristics, life cycle and geographical distribution. Eight annual species namely C. arietinum, C. reticulatum, C. echinospermum, C. pinnatifidum, C. bijugum, C. judaicum, C. yamashitae and C. cuneatum were placed in Section Monocicer, C. chorassanicum and C. incisum (perennial species) in section Chamaecicer, 23 perennial species in section Polycicer and seven woody perennial species in section Acanthocicer. It has been found that the area under chickpea is gradually facing a decline. The major causes being biotic and abiotic stress prevalent in different areas. Diseases causes up to 100% losses in yield. The severity of disease is compounded due to the favorable environmental factors and abiotic stresses. The extent of loss in yield due to wilt and root rot are far more than due to drought and high temperature. The diseases which are of economic importance are discussed in the given sections: Fungal diseases affecting root/stem base 1. Wilt of Chickpea [Fusarium oxysporum Schlechtend. emend. Snyd.et Hans. f. sp. ciceri (Padwick) Matuo et K. Sato] Distribution: Fusarium wilt disease in now widely spread in most chickpeagrowing areas of Asia, Africa, southern Europe and the Americas. Economic importance: Yearly yield losses are estimated at 10-15% in India and Spain, with losses of 70-100% in years of severe outbreaks of the disease. Epidemiology: Wilt is a seed and soil borne disease. Wilt incidence is generally higher when chickpea is grown in warmer and drier climates (>
6 25°C) and when crop rotations are not practiced. Symptoms: The field symptoms of wilt are dead seedlings or adult plants, usually in patches. The disease can affect the crop at any stage. Seedling stage: The disease can be observed within 3 weeks of sowing. Whole seedlings (3 - 5 weeks after sowing) collapse and lie flat on the ground. These seedlings retain their dull green colour. When uprooted, they usually show uneven shrinking of the stem above and below the collar region (soil level). The shrunken portion may be about 2.5 cm or longer. Affected seedlings do not rot on the stem or root surface. However, when split open vertically from the collar downwards or cut transversely, dark brown to black discoloration of the internal stem tissues is clearly visible. In seedlings of highly susceptible cultivars, e.g., JG 62, which dies within 10 - 15 days after emergence, the black discoloration may not be clearly visible. However, internal browning from root tip upwards is clearly seen. Adult stage. The affected plants show typical wilting, ie, drooping of the petioles, rachis and leaflets. Drooping is visible initially in the upper part of the plant but within a day or two, the entire plant droops The lower leaves are chlorotic, but most of the other leaves droop while still green. Gradually, however, all the leaves turn yellow and then light brown or straw coloured. Dried leaflets of infected plants are not shed at maturity. Affected plants, when uprooted and examined before they are completely dry, show no external rotting, drying, or root discoloration. When the stem is split vertically, internal discoloration can be seen. Around the collar region, above and below, the xylem in the central inner portion (pith and part of the wood) is discoloured dark brown or black. In the initial stage of wilting, the discoloration may not be continuous. Discoloration also extends several centimetres above the collar region into the main stem and branches. If the collar region is cut transversely with a sharp razor blade, black discoloration of both pith and xylem can be seen. Sometimes only a few branches are affected, resulting in partial wilt. In certain cultivars, the lower leaves dry up before the plants wilt. Physiological races of Fusarium oxysporum Fusarium wilt, caused by Fusarium oxysporum f. sp. Ciceris is an economically important disease of chickpea. Seven distinct physiological races of Fusarium oxysporum have been identified so far, of which 1, 2, 3 and 4 are prevalent in India (Haware and Nene, 1982) and the remaining races (0, 5 and 6) are reported from Spain (Jimenez –Diaz et. al., 1989). Race 1 is common in central India, whereas race 2 is common in northern India. Race 3 and 4 appear to be location specific in Punjab and Haryana
7 states of India. The pathogen can survive in soil for more than six years even in the absence of chickpea and causes considerable yield losses. Breeding varieties resistant to fusarium wilt is an eco-friendly, economically viable strategy. Breeding programmes at national and international centres have resulted in the release of several chickpea cultivars resistant to fusarium wilt. But these cultivars do not show resistance across locations (Infantino et al., 1996) due to the prevalence of location specific races of the wilt pathogen. Resistance to the different races of Fusarium wilt fungus in chickpea and pea is controlled by single genes (Muehlbauer and Kaiser, 1994). The genotypes of chickpea differ for their time to develop initial symptoms of wilt that indicates different degrees of resistance. The difference in time taken for the development of initial symptoms appears to be controlled by segregation of a single gene with early wilting partially dominant over late wilting (Upadhyaya et al., 1983). Resistance to fusarium wilt race 0 is governed by one or two independent genes (Tekeoglu et al., 2000; Rubio et al., 2003). Involvement of two independent loci in the inheritance of resistance to race 1 has also been observed (Singh et al., 1987; Upadhyaya et al., 1983). Presence of either of these genes in homozygous recessive form increases time to develop wilt symptoms (late wilting), but both the genes must be in homozygous recessive form to confer resistance. Late wilting chickpea genotypes would be of great value in reducing the yield losses by delaying the wilt development. These late wilting lines may carry useful resistance genes that can confer complete resistance when complemented with other resistance genes. Gumber et al. (1995) reported that resistance to race 2 of fusarium wilt is controlled by two genes, one gene must be present in the homozygous recessive form, and the second gene must have its dominant allele (whether in homozygous or heterozygous condition) for complete resistance. The resistance to race 4 of Fusarium oxysporum is controlled by a single recessive gene (Tullu et al., 1998).Only one resistance gene has been described and mapped for race 5 (Tekeogluet al., 2000; Winter et al., 2000). The genetics of resistance to various other diseases of chickpea has been discussed in detail by Singh and Reddy (1991). Nene and Haware (1980) evaluated 102 accessions representing six annual wild Cicer species for resistance to fusarium wilt. All accessions of C. bijugum and some accessions of C. judaicum, C. reticulatum, C. echinospermum and C. pinnatifidum showed resistance to fusarium wilt, whereas all the accessions of C. yamashitae were highly susceptible. Some accessions of C. bijugum, C. judaicum and C. reticulatum were completely free from wilt damage, and are therefore potential source
8 of genes for Fusarium wilt resistance that could be transferred into the cultivated species. Seventeen lines resistant to race 0 and only one line resistant to race 5 were identified from 1,904 lines of kabuli chickpea tested, showing the paucity of resistance in the cultivated chickpea accessions (Jimnez-Diaz et al., 1991). Race 1 of Fusarium is the most widespread in India, to which 160 resistant lines, including both desi and kabuli, have been identified. Fifty-two accessions representing 11 wild Cicer species were evaluated for resistance to races 0 and 5 but promising source could not be identified in the accessions of the primary gene pool. Resistance to highly virulent race 5 was identified in the accessions of C. bijugum, C. cuneatum and C. judaicum, and resistance to race 0 was observed in accessions of C. bijugum, C. chorassanicum, C.cuneatum, C. judaicum and C. pinnatifidum. Resistance to race 0 was also observed in a perennial species C. canariense. Some accessions of C. bijugum, C. judaicum and C. pinnatifidum with combined resistance to races 0 and 5 of fusarium wilt and ascochyta blight have also been identified. Different accessions of C. bijugum, C. judaicum, C. reticulatum, C. pinnatifidum, C. echinospermum and C. cuneatum were found to be resistant to fusarium wilt in other studies (Singh et al., 1998). Table : Improved varieties of chickpea having wilt resistance Zone
Varieties
North East Plain Zone (NEPZ)
Avrodhi, KPG 59, K 850, Pusa 1003, Haryana chana-1
North West Plain Zone (NWPZ)
KPG 59, Vijay, KWR 108, GNG 469, DCP92-3
Central Zone (CZ)
ICCC 32, JG 315, BGD 72, Gujrat Gram 1, ICCV 10, ICCV 2, SAKI 9218, H 355, BG 244, Phule G 5, Pusa 391
South Zone (SZ)
JG 11, Co3,& Co4, Annegiri 1
In the chickpea growing countries wilt is a one of very serious diseases leading to yield losses from 10-90% (Srivastava et al 1984). It is generally found that early growth stage causes more loss in yield than the late stage. Wilt is accompanied by root and stem rot diseases. This is a vascular, systemic disease which causes xylem browning and blackening and may infect at any growth stage. The susceptible cultivars show symptoms within 25 days after sowing which causes seedlings collapsing. The dull green colour of seedling is retained. In adult plants drooping of petioles, rachis and leaflets occur. In roots external rotting is not visible but on vertically splitting brown discoloration could be seen. Pathogens
9 survive in soil as well as seed as chlamydospores. The pathogen can survive even in the absence of host plant up to six years. At high temperatures the symptoms are more profound and soil environment greatly influences the expression of symptoms. Table-: Chickpea genotypes with moderate to high level of resistance to wilt (19851997) Desi Types
Avrodhi, BG 209,246, 313, 367, BGM 418, 419,432, 436, 439, 443, 444, 451, GF 89-36, GL 86114, GNG 468,469, H 82-2, 8318, 83-23,83-60, 83-84, 84-8, 85-65, 85-124, 86-8, 86-18, 8620, 86-21, 86-39, 86-42, 86-72, 86-73, 86-91, 86-100, 86-142, 86-143, 86-156, 86-158, 86-170, 88-123, 89-50, JG 315, 317, 322, KPG 63,142-1, 143-1, PDG83-39, 84-10, 84-14, 85-8, 8518, PhuleG 87207
Kabuli types
BG287, ICCC 32, ICCC 42
Source: Consolidated Report on Rabi Pulses, Plant Pathology AICRP on Chickpea (19851997)
THE VARIATION IN PATHOGENS The existence of pathogenic races of F. oxysporum f.sp. ciceri is well established with race 1, race 2, race 3 and race 4 reported from the country (Haware and Nene 1982b) and races 0, 5 and 6 from Spain ( Jimenez diaz et al 1989). The race 0 is the least virulent among all the races causing only yellowing symptoms. The race 5 is the most virulent causing severe leaf chlorosis and plant death. In recent times the potential of molecular markers for fingerprinting in studying genetic variability among the four Indian races of F. oxysporum f.sp. ciceriwas accessed using 13 oligonucleotide probes complementary to microsatellites in combination with 11 restriction enzymes (Barve et al 2001). The genomic distribution of microsatellite repeats disclosed race 1 and race 2 as closely related at similarity index of 76.6% as compared to race 2 at similarity index of 67.3%. Race 3 similarity index value of 26.7% makes it very distinct. Chakrabarti et al (2002) on the basis of EcoR1 restriction pattern of the nuclear ribosomal DNA divided Indian races in 3 different groups, race 1 and 4 representing one group, race 2 in a separate group and race 3 in the other. Sivaramakrishnan et al (2002) accessed the genetic variability of 43 isolates of the pathogen using RAPDs and AFLP, and found that race 1 and race 2 are distinct whereas race 3 and race 4 are similar. Future studies may reveal the possibility of existence of more number of races in the pathogen. The identification of the new races could be made possible by the amplification of intergenic spacer (IGS) region and restriction digestion, which could further help in the study of polymorphism in F. oxysporum
10 f.sp. ciceri. SCREENING PROCEDURE Nene et al (1981) have developed and standadarized field, greenhouse and laboratory technician for evaluation of chickpea lines for resistance to Fusarium wilt. There have also been reports for screening under controlled conditions using pot culture method (Haware and Nene, 1980). Steps to follow 1) fungus is derived from single spore 2) it is multiplied on 100g of sand maize meal medium in 250 more flask(incubated at 25 C for 14 days) 3) 100 g inoculums is mixed with 2 kg of autoclaved soil and riverbed sand ( 1:1, v/v) in 15 cm plastic pots 4) susceptible cultivar, JG 62 is grown in two successive batches and allowed to wilt. 5) wilted plant debris is incorporated into pots to increase the inoculum 6) the pots are now ready for screening against Fusarium wilt. In order to screen large number of germplasm , sick plot is developed. STEPS TO DEVELOP A SICK PLOT 1) site selection is done with history of frequent occurrence of wilt. 2) Highly susceptible cultivar example, JG 62 is grown repeatedly at the site for 2-3 years 3) infected plant debris is incorporated into the soil Studies are being done to identify sources of resistance and development of wilt resistant high yielding varieties. It was found out that under field conditions resistance to Fusarium wilt in chickpea lines was monogenic with incomplete dominance (Ayyar and Iyer 1936) or monogenic and recessive (Lopez Garcia 1974, Pathak et al 1975, Haware et al 1983). A systemic study under controlled conditions showed that resistance to race1 of pathogen in CPS 1 and WR315 was conferred by a single recessive allele at the same locus in both genotypes (Kumar and Haware 1982).
11 Further investigations revealed genotypic differences in days taken to wilting which seemed to be controlled by a single recessive gene with early wilting partially dominant to late wilting (Upadhayeet al. 1983a). The other studies revealed the resistance to race 1 being controlled by at least three independent loci, H1, H2 and H3 (Upadhaye et al.1983b, Singh et al. 1987, 1988). Partially recessive alleles in homozygous form at either of the first 2 loci and dominant allele at the third locus delay wilting, but any two of these two alleles together confer complete resistance. This phenomena is still under investigations and further study of the existing races of pathogens in this direction is required. Biochemical Basis of Resistance The biochemical basis of resistance of wilt of chickpea has not been much studied in the past. As a result there is scanty information on this topic. The susceptible cultivars root exudates stimulated mycelial growth while the resistant cultivars inhibited the growth (Satyaprasad and Ramarao 1983). Gurha and Srivastava (2002) reported the effect of wheat and Indian mustard root exudates on the isolates of F. oxysporum f.sp. ciceri. There was reduction in the growth of all isolates. COLLAR ROT (Sclerotium Rolfsii Sacc.) This disease is seen when soil moisture is high and temperature warm at the seedling stage. The incidence of the disease decreases with the age of the plant. The disease development is favoured by undecomposed organic matter on the soil surface. The affected seedlings yellow and die. The seedlings collapse and rotting is observed at the collar region and below. Whitish mycelial strands cover the rotten portion. When chickpea is sown after paddy, the disease incidence increases. Kabuli types are more susceptible to to this disease. Distribution: Collar rot occurs in every region where chickpea is cultivated. Epidemiology: It is a widely prevalent disease and can cause considerable loss to the plant stand when soil moisture is high and temperatures are warm (30°C) at sowing time. The incidence decreases with the age of the crop. The disease is favoured by the presence of undecomposed organic matter on the soil surface and excessive moisture at the time of sowing and at the seedling stage. Disease incidence is higher when chickpea is sown after rice. Symptoms: Drying plants whose foliage turns slightly yellow before death, scattered throughout the field is an indication of collar rot infection. Most
12 often, collar rot is seen at the seedling stage (up to 6 weeks after sowing), particularly if the soil is wet. Affected seedlings turn yellow. Young seedlings may collapse, but older seedlings may dry without collapsing. Leaves do not droop. When uprooted, the seedlings show rotting at the collar region and downwards. The rotten portion is covered with whitish mycelial strands. A white mycelial coating can be seen on the tap root of completely dried seedlings, even several days after death. If affected seedlings are uprooted from moist soil in the earlier stages of infection, rapeseed-like sclerotia (1 mm in diameter), attached to mycelial strands around the collar are seen. The non-affected portion of the root is white inside, as is normal. WET ROOT ROT ( Rhizoctonia solani Kuhn) The disease occurs when there is high soil moisture and warm temperature in the seedling stage. It doesn’t lead to collapsing of seedlings. Above the collar region a dark brown lesson can be seen. It can extend up to lower branches in the older plants. The stem and root area below the lesson show rotting. Distribution: Wet root rot is a minor disease and is reported from several countries. Epidemiology: No-till or reduced-till conditions favour growth of the pathogen and development of the disease. The pathogen can infect at a wide range of soil temperatures, but cool (11-18°C), wet soil conditions are optimum. Symptoms: The field symptoms are almost the same as those of collar rot, i.e., drying plants scattered throughout the field. Like collar rot, this disease is most often seen at the seedling stage (up to 6 weeks after sowing) in soils with relatively high moisture content. However, in irrigated chickpea, the disease may occur at later stages in the crop growth. Affected seedlings gradually turn yellow and petioles and leaflets droop. Seedlings do not usually collapse. A distinct dark brown lesion appears above the collar region on the main stem and can extend to lower branches in older plants. The stem and root below the lesion show rotting, frequently with pinkish mycelial growth. Sclerotia are not usually seen. DRY ROOT ROT [Rhizoctonia bataticola (Taub.) Butler] Distribution: Dry root rot is the most important root rot disease in chickpea particularly in the semi-arid tropics of Ethiopia and in central and southern India. It has also been reported from Australia, Bangladesh, Iran, Kenya,
13 Lebanon, Myanmar, Mexico, Nepal, Pakistan, Spain, Sudan, Turkey and the United States. Epidemiology: The pathogen is a facultative sporophyte and is both seed borne and soil borne. Maximum ambient temperatures above 30°C, minimum above 20°C, and moisture stress (dry conditions) at the reproductive stages favour disease development. Symptoms: The disease generally appears around flowering and podding time in the form of scattered dried plants. The seedlings can also get infected. The susceptibility of the plant to the disease increases with age. Drooping of petioles and leaflets is confined to those at the very top of the plant. Sometimes when the rest of the plant is dry, the topmost leaves are chlorotic. The leaves and stems of affected plants are usually straw coloured, but in some cases the lower leaves and stems are brown. The lower portion of the tap root usually remains in the soil when plants are uprooted. The tap root is dark, shows signs of rotting, and is devoid of most of its lateral and finer roots. Dark, minute sclerotial bodies can be seen on the roots exposed or inside the wood. PHYTOPHTHORA ROOT ROT [Phytophthora medicaginis E. M. Hans & D. P. Maxwell] Distribution: Phytophthora root rot is an important disease in Australia. It is also reported from Argentina, India, Pakistan and Spain. Symptoms: Symptoms of phytophthora root rot in chickpea can develop from seedling emergence to near maturity. The disease is commonly lethal, causing wilting chlorosis and rapid death of plants a week of more after rains. Patches of dead plants are seen in the field. Symptoms on individual plants are yellowing and drying of foliage with basal rot symptom on stem and decay of lateral roots and the lower portion of the tap root. On the upper portion of the tap root, dark brown to black lesions are seen, which in some cases extend to the stem base. The advancing margins of these lesions are often reddish brown. These symptoms can be easily confused with those of wet root rot. The disease incidence is high in low lying areas where water stagnates. DRY ROOT ROT (Rhizoctonia batticola) The disease appears around flowering and podding stages of the plant. The first symptom include yellowing and sudden drying of the plants. Petioles and leaflets of the top of the plant droop. Sometimes leaves on the top are chlorotic and rest of the plant dry. The tap root becomes dark brown
14 and brittle (dry soil) and starts extensive rooting as a result lateral roots are lost. Dark brown or black sclerotia are produced on the infected tissues. The sclerotial bodies can be visualised in the pith cavity as well as the outer surface of taproot aided by a hand lens. When plant is uprooted, often the lowerportion of the taprootis left in the soil. The high temperatures i.e. 30 degree C and above and moisture stress increases the susceptibility of disease development. This disease is manifested as scattered dead plants. Screening Techniques The identification of resistant genotypes through both field and laboratory techniques have been developed at ICRISAT (Nene et al 1981). Paper towel technique is used in laboratory and pot culture technique in green house (Nene et al 1981). Paper Towel Technique 1) 5 day old culture of fungus is macerated and multiplied in 100ml potato dextrose broth at 250C, with 50ml sterilized distilled water. 2) The seeds of chickpea are surface sterilised (2.5% sodium hypochlorite for 5 min) and sown in green house in polythene bags containing sterilized sand. 3) Temperature is maintained at 20-25oC. 4) 5 days old seedlings are removed and washed 5) Roots are dipped in the freshly prepared inoculum for 30 seconds 6) Seedlings are placed in blotting paper keeping their green tops exposed. Blotter ends are are moistened with sterilized water and placed in polythene bags. 7) Seedlings are incubated in Percival incubator at 35o C (12 hours artificial light per day for 8 days ) 8) Blotters are moistened each day 9) The seedlings are scored for root infection 8 days after incubation on a 0-9 scale ( 0- no disease, 9- highly susceptible) BLACK ROOT ROT ( Fusarium solani (Mart.) Appel & Wr.) The disease can occur at any stage of the crop. The plants affected turn yellow and wilt. The roots rot and turn black. The disease is encouraged by excess soil moisture and moderately high temperature.
15 ASCOCHYTA BLIGHT (Ascochyta rabiei) Distribution: Ascochyta blight is a major disease in west Asia, northern Africa, and southern Europe. The disease usually builds up in February and March northern India when the crop canopy is very dense and temperatures are favourable to disease development. In West Asia, southern Europe and northern Africa, such conditions usually prevail in March, April and May. In the winter-sown chickpeas of the Mediterranean region, blight symptoms can be found in November and December when the weather is wet and warm. The disease has been reported from 35 countries across 6 continents and as recently seen in Australia and Canada, it can spread rapidly to new areas of chickpea production. Economic importance: The disease can cause grain yield and quality losses up to 100%. Epidemiology: Ascochyta blight is a seed borne disease. Diseased debris left over in the fields also serves as a source of primary inoculum. Ascospores were also found to play a role in the initiation of disease epidemics. Secondary spread is through pycnidiospores. Chickpea and its wild relatives are the only confirmed hosts of the fungus A. rabiei. Cool, cloudy and wet weather favours the disease development. The disease builds up and spreads fast when night temperatures are around 10°C, day temperatures are around 20°C, and rains are accompanied by cloudy days. Excessive canopy development also favours blight development. Symptoms: It is a foliar fungal disease. It can cause up to 100% yield loss. It affects all the aerial parts of the plant. It can occur at any growth stage. The initial symptoms include occurrence of circular spots near the tip of young shoots and topmost leaves. With the advancement of disease brown spots appear in other aerial parts. On these spots characteristic concentric rings are formed. This concentric ring is due to the development of fruiting bodies of the fungus, the pycnidia. On the leaves and pods the lessons are circular but elongated on stem and branches. The apical twigs, stems and branches show girdling. The plant part above girdle portion break off even before drying. On the pods, the lesions are circular with dark margins. The lessons are formed even on the seed coat. In the field, it appears as patches after 6-8 days of infection, which spread to the entire field under favorable conditions. The disease is usually seen around flowering and podding time as patches of blighted plants in the field. Symptoms are seen on all aboveground parts of the plant. However, the disease can appear at a very early stage of a crop’s growth. When the pathogen is seed borne and conditions at the time of germination are conducive to disease development, the
16 emerging seedlings develop dark brown lesions at the base of the stem. Affected seedlings may collapse and die (damping-off). Pycnidia may be formed on the lesions. Isolated infected seedlings may not be noticed. But at flowering and podding time, when conditions are usually favourable for disease development, the disease spreads from these isolated seedlings, resulting in patches of blighted plants. When the source of inoculum is airborne conidia or ascospores, the disease initially appears in the form of several small water-soaked necrotic spots on the younger leaves of almost all branches. Under conditions favourable for disease development these spots enlarge rapidly and coalesce, blighting the leaves and buds. Pycnidia are observed on the blighted parts. On a susceptible cultivar, the necrosis progresses from the buds downwards, killing the plant. In cases of severe foliar infection, the entire plant dries up suddenly. If conditions are not favourable for disease development (hot dry weather), the plants do not die and the infection remains in the form of discrete lesions on the leaves, petioles, stems, and pods. The symptoms on the leaflets are round spots with brown margins and a gray centre that contains pycnidia, which are often arranged in concentric rings. On the stems and petioles, the lesions are obovate or elongate and bear pycnidia. The size of the lesions varies greatly; some may become 3 - 4 cm long on stems and often girdle the affected portion. The stems and petioles usually break at the point of girdling. If blight occurs at the pre-flowering stage and then conditions for its development become unfavourable (hot dry weather) the crop re grows fast but symptoms can still be seen on the older branches. Fully developed lesions on pods are usually round, up to 0.5 cm in diameter, usually with concentric rings of pycnidia. Several lesions may appear on a single pod and if infection occurs in the early stages of pod development, the pod is blighted and fails to develop any seed. Late infections result in shrivelled and infected seed. The fungus penetrates the pod and infects the developing seed. Symptoms on the seeds appear as a brown discoloration and often develop into deep, round or irregular cankers, sometimes bearing pycnidia visible to the naked eye. Ascochyta blight, caused by Ascochyta rabiei, is the most important foliar disease of chickpea. The disease is particularly severe on winter sown chickpea, favoured by cool and wet weather conditions and attacks the crop at both vegetative and podding stages. The yield losses of chickpea due to blight range from 10 to 100 per cent under severe natural epidemics (Nene and Reddy, 1987; Singh, 1990). Most of the resistances to blight identified so far is under multigenic control (Muehlbauer and Kaiser, 1994). The chickpea lines exhibiting resistance to 3-5 races of Ascochyta rabiei
17 were identified after evaluation of 1,069 germplasm lines (Singh, 1990). Reddy and Singh (1983) observed resistance to ascochyta blight in 0.29 and 0.06% of kabuli and desi accessions, respectively. Total 19,343 Cicer germplasm accessions which includes both kabuli and desi types were screened for resistance to six races of A. rabiei and 14 lines (9 desi and 5 kabuli accessions) of durable resistance at both vegetative and podding stage were identified by ICARDA, Syria (Singh and Reddy, 1993a). More concerted efforts at ICARDA, Syria led to the development of 92 lines resistant to all the six physiological races of A. rabiei, which have registered 33% more seed yield than the original resistant sources. Planting of these highly resistant lines in winter season increases the prospects of achieving higher yields in the Mediterranean region (Singh and Reddy, 1996). The ascochyta blight resistant breeding lines showed no association with late maturity and plant height, as a result 28 resistant lines coupled with early maturity have been developed. These lines are highly suitable for regions with short growing seasons. The pathogen Ascochyta rabiei is inherently unstable and continuously evolves new races in complex interaction with host, which makes resistance ephemeral and limits the effective life of resistant lines under production. The evolution of new races has made the plant breeders to change their strategy from oligogenic resistance to pyramiding of genes to provide durable resistance. It is also equally important to understand the race distribution of A. rabiei in different chickpea growing regions, which will help in selective deployment of varieties to provide effective resistance and to save resistance genes from being defeated by the races of the pathogen. The development of DNA markers for different races of the pathogen help in rapid diagnosis of the pathogens and also to conduct pathogen surveys more frequently and systematically compared to the analysis of pathogens based on differential sets. Resistance to ascochyta blight was observed in the accessions belong to C. pinnatifidum, C. judaicum and C. montbretii (Singh et al., 1981). The wild species C. echinospermum, C. pinnatifidum, C. bijugum and C. judacium possessed high degree of resistance to ascochyta blight (Singh and Reddy 1993b; Singh et al., 1998). VARIABILITY IN PATHOGEN There is existence of variability in A. rabiei in respect of characters like color, rate of growth, pycnidia production pattern, pycnidia and pycnidiospores size. The change in physical factors lead to change in these characters (Aujla 1964, Singh 1990). A. rabiei populations have high level
18 of variability for pathogenicity (Vir and Grewal 1974a, Nene and Reddy 1987). Race 1 race 2, and one biotype of race 2 have been reported by Vir and Grewal (1974a). There are reports for the presence of six races from Syria, twelve from India, and several from Pakistan (Singh 1990). The races reported in India are 1(3904), 2(4080), 3(3844), 4(3492), 5(3522), 6(3968), 7(4064), 8(3560), 9(3744), 10(3904), 11(4088), 12(1744). Udapa et al 1988 found occurrence of three pathotypes: pathotype I (less aggressive), pathotype II (aggressive) and pathotype III (most aggressive). Recently molecular markers have been employed to study the diversity in A. rabiei is isolates of India, USA, Syria and Pakistan. The diversity was found more in the Indian isolates. SCREENING TECHNIQUES The screening techniques developed for resistance breeding program are as follows Pot Culture Screening Technique 1) Test lines are planted in plastic pots (15x10 cm) in glasshouse 2) 20-25 days old plants of test lines and susceptible checks are placed in circular pits (8-10 cm deep) 3)
Water added to pots and pit and inoculation is done with 1x 105 spores per ml
4) The plants inoculated are covered with dasuti cloth chambers which are kept moistened 5) After 48 hours the chambers are removed 6) For nearly 20 days leaf wetness is maintained for maximum disease severity Cut Twig Screening Technique When population is generated from wide hybridization this method is very useful (Sharma and Singh 1995) Moreover this method is very quick with results of screening being used the same season. The steps followed are as follows1) The tender shoots are cut and transferred to test tubes(15 x 100 mm) 2) The twigs are inoculated by spray of spore suspension (1x 105 spores per ml) of fungus and covered with dasuti cloth which is moistened.
19 3) 48 hours after inoculation the chambers are removed and kept wet by water up to 13 days 4) After 6 days symptoms start appearing on susceptible lines and check Detached Leaf Screening Technique 1) It uses detached leaves or leaflets from test lines and susceptible check 2) Leaves are surface sterilized (sodium hypochlorite 5%/ Mercuric chloride 0.1%) and transferred to water agar in petri plates 3) Spore suspension (1x 105 spores per ml) is used to inoculate the leaves 4) Petri plates lid is sealed with paraffin wax and incubated at 20o C (12 hours light and 12 hours dark) 5) Lesions become visible in 4-6 days and observations are recorded after 8 days (Singh et al. 1992) Field Screening Technique 1) 3-5 rows of test lines are planted at 40cm space in replicated trials 2) Rows of highly susceptible varieties ( indicator-cum-infector rows) are planted after every 4 to 8 rows 3) At flowering period i.e. Feb plants are inoculated by spore suspension spray 5x104 spores per ml. 4) High relative humidity should be maintained by perfo spray system for 21 days. 5) In the susceptible checks/ susceptible lines 100% mortality is observed within 15 days of inoculation 6) After 21 days of inoculation observations are recorded (Singh 1987 Reddy et al. 1984) BOTRYTIS GREY MOULD (Botrytis cinerea Pers. Ex Fr.) Gray mold, Botrytis cinerea, is second most important foliar disease of chickpea and is prevalent in sub-mountainous region of northern India, as well as in Bangladesh Chickpea improvement: role of wild species and genetic markers 281 and Nepal. The presence of resistance to gray mold was observed in the accessions of C. pinnatifidum and C. judaicum (Singh
20 et al., 1982).The disease occurs on the aerial plant parts of which flowers and pods aremore susceptible. The affected plant parts show water soaking and softening at the initial stage of infection. Grey to dark brown necrotic lesions are formed in the form of sporophores and mycelium. On the stem, the grey mould is replaced by dark grey and further black sprodochia. When the humidity is low, irregular brown spots appear without any fungal growth. Under humid conditions, all flowers are attacked. The affected crops either do not produce any seed or produce small seeds. It can occur at any growth stage, the most susceptible is the flowering and podding stages. Distribution: Botrytis gray mold (BGM) is a serious disease in parts of Bangladesh, India, Nepal, Pakistan, Australia and Argentina. BGM has also been reported from Canada, Chile, Colombia, Hungary, Mexico, Myanmar, Spain, Turkey, the USA and Vietnam. Economic importance: BGM can cause yield losses up to 100%. Epidemiology: BGM is a seed borne disease. The fungus has a very wide host range. The disease is usually seen at flowering time when the crop canopy is fully developed. Excessive vegetative growth due to too much irrigation or rain, close spacing, and varieties that have a spreading habit favour disease development. Temperatures between 20 and 25°C and excessive humidity around flowering and podding time favour disease development. As temperatures favourable to BGM are slightly higher than those for ascochyta blight, these diseases may occur one after the other with ascochyta blight appearing first. Symptoms: Lack of pod setting is the first indication of the disease. Leaves and stems may not show any symptoms. Under weather conditions highly favourable to the disease, foliage shows clear symptoms and plants often die in patches. The disease is more severe on portions of the plant hidden under the canopy and is obvious if the canopy is parted to expose the symptoms. Shed flowers and leaves covered with the spore mass can be seen on the ground under the plants. When humidity is very high, the symptoms appear on stems, leaves, flowers and pods as gray or dark brown lesions covered with moldy sporophores. Lesions on stem are 10 - 30 mm long and girdle the stem completely. Tender branches break off at the point where the gray mold has caused rotting. Affected leaves and flowers turn into a rotting mass. Lesions on the pod are water-soaked and irregular. On infected plants, the pods contain either small, shrivelled seeds or no seeds at all. Grayish white mycelium may be seen on the infected seeds.
21 VARIABILITY IN PATHOGEN The morphological and cultural characters of B. cinerea reflects it’s variability. The existence of variability can be seen on the basis of various char acters like presence or absence of microsclerotia, microsclerotia, both micro-and microsclerotia and sporulation. There have been report of four race viz., 232, 494, 508, 510 from India (Singh and Bhan 1986). Five pathogens have been screened based on reactions on 5 chickpea varieties (Rewal and Grewal 1989). In recent times variability in B. cinerea has been discovered using molecular techniques. RAPD markers have been used to identify high genetic variability in B. cinerea populations in Korea (Chung et al 1996). In Spain too similar findings have been accounted (Alfonso et al 1996). SCREENING TECHNIQUES Different National Agricultural Research Centers and International Institutes have developed screening techniques to screen chickpea germplasm and breeding materials: Growth Room Screening Technique I 1) Chickpea test lines are planted in pots in glasshouse 2) 15-20 days old plants are transferred to controlled environmental condition growth room 3) Inoculation is done by spray of spore suspension the fungus (50,000 spores per ml) 4) Within 13 days after inoculation results become available Growth Room Screening Technique II 1) In the glass house test lines are planted in polythene bags (15x10 cm)containing sandy loam soil 2) Susceptible check (H 208) and 25 days old plant are transferred to growth chambers (temperature 20o C, relative humidity > 90%, alternate light and dark period) 3) Inoculation with spore suspension (50,000 spores per ml)is done covered with moist polythene covers with iron frame support for 6 days 4) Disease symptoms appear after 24h and 100% mortality is seen in susceptible lines in 6 days (Singh et al. 1985)
22 Cut Twig Screening Technique 1) In this technique 10-15 cm long tender shoots are cut in the evening 2) Lower portion of twig is wrapped with cotton plug and transferred to a test tube (15x100 mm) containing fresh water 3) Tubes are inoculated with twigs of susceptible checks by spray of spore suspension (50,000 spores per ml of 10 days old culture of fungus) 4) Inoculated twigs are covered with polythene chamber which is kept moist and supported by iron frames for 144 hour. Disease symptoms appear after 24 h of inoculation mortality of 100% is seen after 144 hour (Singh et al. 1985). Field Screening Technique For the large scale screening of germplasm and breeding material in segregating generations, field screening technique is used. The following steps are followed for field screening. 1) Test lines are sown in 3-5 m rows with spacing of 40 cm 2) After every 2-4 test rows indicator rows are planted 3) In February, irrigation is done in the morning and spores are sprayed as inoculum (5000 spores per ml of 10 day old B. cinerea.) 4) Sprinkler irrigation is used for maintaining moisture condition in the field 5) After 10-15 days of first inoculation inoculation of crop is repeated 6) At the end of March to first week of April the disease observations are recorded Table:-Wild Cicer species with resistance to various biotic stresses Trait
Source of resistance
Reference (s)
Ascochyta blight
C. echinospermum, C. pinnatifidum, Singh et al., 1981, C. bijugum, C. judaicum and C. montbretii 1998
Fusarium wilt
C. bijugum, C. judaicum, C. pinnatifidum, Nene and Haware, C. reticulatum, C. echinospermum and 1980 and Singh et C. cuneatum al., 1998
Gray mold
C. judaicum, C. pinnatifidum
Singh et al., 1982
Phytophthora root rot
C. echinospermum
Singh et al., 1994
23 Table-: Sources of single and multiple resistance to biotic stresses in chickpea Disease(s)
Lines with resistance
Asochyta blight
ILC 72, 200, 3279, 3856, 5902, 6090, 6188, ICC 1069,MCK 54
Botrytis grey mould
ICC 1084, 1102, 3540, 4065, 6299
Fusarium wilt
ILC 54, 240,256,336, 487, WR 315, ICC 11550, 12467, 14424
Dry root rot
ICC 12237, 12269, H208, C 104
Stunt virus
ICC 12455
Wilt + Dry root rot + Black root rot
ICC 12237,12269
Wilt + Dry root rot + Stunt virus
ICC 12435
Wilt + Dry root rot
ICC 11315, 12241, 1432, 14443
Wilt + Black root rot
ICC 11313, 12236, 12256, 12275
Wilt + Stunt virus
ICC 10136, 10805, 11502, 11551
Wilt + Botrytis grey mould
ICC 11321
Ascochyta blight + Botrytis grey mould
ICC 1069
Source: Ali Masood, Kumar S. and Singh, N.B. 2007. Chickpea research in India. Table: Improved varieties of chickpea with specific traits Specific trait
Released varieties
Tolerance to Fusarium wilt
KWR 108, ICCV 10, H 82-2, CSJ 8963, DCP 92-3, GCP 101, GCP 105, JG 315, GPF 2, Vijay, KGD 1168, JG 74, GNG 663, K 850, Radhey, Pusa 256, Pusa 212, KPG 59, BG 1003, BG 1053, Annegiri 1, Mahamaya 1, Vikas, BGD 72, Gaurav, PBG 1, GNG 469
Tolerance to root rot
Alok, Co-3, Co 4, KWR 108, Pusa 209. Pusa 240, Pusa 244, Pusa 362, Pusa 372, Pusa 391, Pusa 413, Pusa 417, ICCV 5, ICCV 10, Vijay, Vardan, GNG 469, BGD 72, JG 11, L 551
Tolerance to Asochyta blight
GNG 469, Gaurav, PBG 1, GNG 146, C 235, Pusa 261, PBG 5
Tolerance to Botrytis grey mould
Pusa 256, ICCV 2
Tolerance to stunt
Kiran, Pusa 244
Tolerance to root knot nematode
Kiran, Pusa 362, BGD 72
Source: Ali Masood, Kumar S. and Singh, N.B. 2007. Chickpea research in India.
24 Phoma blight [Phoma medicaginis Malbr. & Roum] Distribution: Phoma blight is a minor disease reported from Australia, Bangladesh, India, Syria and USA. Symptoms: It usually affects the crop in the reproductive phase. The field symptom is the appearance of patches of drying plants. The symptoms are somewhat similar to those of ascochyta blight. Irregular, light brown lesions on the leaves, stems, and pods have dark margins. Dark, minute, submerged pycnidia are irregularly scattered in the infected tissue. Seeds from infected pods are discoloured and shrivelled. The conditions favourable to phoma blight are similar to those that favor ascochyta blight. Stemphylium blight [Stemphylium sarciniforme (Cav.) Wilts.] Distribution: Stemphylium blight is a minor disease reported from Bangladesh, India, Iran, Nepal and Syria. Epidemiology: Excessive vegetative growth, high humidity, and cool weather (15 - 20°C) favour disease development. Symptoms: It usually affects the crop from the flowering stage onwards. Defoliation, especially of the lower branches is conspicuous. Lesions on the leaflets consist of roughly ovoid necrotic spots, which may measure up to 6 x 3 mm. The spots are dark brown at the center, with a broad gray border. Minute, dark brown, elongated spots also develop on the stems. Rust [Uromyces ciceris-arietini (Grogn. )Jacz.& Beyer] Distribution: Rust is prevalent in the Mediterranean region, south eastern Europe, southern Asia including India, eastern Africa and Mexico. It is not considered serious as it appears late in the season when the crop is maturing. Epidemiology: Moderate warm weather favours rust development. Symptoms: The severely infected crop looks rusty because the foliage is coated with rust pustules and urediniospores. The rust appears first mainly on the leaves as small, round or oval, cinnamon brown, powdery pustules. These pustules tend to coalesce. Sometimes a ring of small pustules can be seen around larger pustules, which occur on both leaf surfaces but more frequently on the lower one. Occasionally pustules can be seen on stems. Severely infected plants may dry up prematurely. Cool and moist weather favours rust build up; rain does not appear to be essential for the infection to spread.
25 POWDERY MILDEW [lEVEILLULA TAURICA (lEV. ) SALMON] Distribution. Powdery mildew is a minor disease reported from India, Mexico, Morocco, Pakistan, and Sudan. Epidemiology: Cool and dry weather favours powdery mildew development. Symptoms: Like rust, powdery mildew appears late in the season when the crop is nearing maturity, except in highly susceptible genotypes, when it appears earlier. Severe infection of powdery mildew can be easily recognized by the white powdery growth on the foliage, which is a characteristic feature of the powdery mildews. Small patches of white powdery coating initially develop on both surfaces of older leaves. These patches grow and may cover a large area. Affected leaves turn purple and then die. When infection is severe, stems, young leaves and pods are also covered with the powdery coating. SCLEROTINIA STEM ROT [Sclerotinia sclerotiorum (Li b.) de Bary] Distribution: Sclerotinia stem rot is reported from most of the chickpea growing regions of the world. At present, it is a minor disease. Epidemiology: Excessive vegetative growth, high soil moisture, and cool weather (20°C) favour disease development. Symptoms: It can affect the crop at any stage. The pathogen has been observed to cause collar rot of seedlings in North African chickpea-growing regions. Otherwise it usually appears after the crop canopy has covered the ground. The disease is characterized by the appearance of chlorotic or drying branches or whole plants scattered in the field. Such drying plants or branches rot at the collar region or at any point on the branch. The leaves of affected plants/branches turn yellow or droop while remaining green, then dry up and turn straw coloured. A web of white mycelial strands appears at the collar region and above (up to 5 cm) and may cover the base of the branches. Extended grayish lesions with or without mycelial coating can also be seen on the upper parts of the stems. Whitish or brownish irregular-shaped sclerotia can be seen, occasionally mingled with mycelial strands on branches, or inside the stem. VIRAL DISEASES STUNT [BEAN (PEA) LEAF ROLL VIRUS] Distribution: Stunt is the most important viral disease of chickpea prevalent
26 in most of the chickpea-growing countries. Epidemiology: Early sowing (September) and wider spacing favour stunt incidence in India. The aphid vector activity (Aphis craccivora, Myzus persicae) also influences disease incidence. Symptoms: All other viral diseases are minor. Stunting is most conspicuous in early infections. It occurs because of shortened internodes. In later infections, stunting may not be obvious, but plant discoloration and phloem browning are seen. Affected plants can be easily spotted in the field by their yellow, orange, or brown discoloration and stunted growth. The disease is not seed borne. Leaflets are small and yellow, orange, or brown. In some cases, stems turn brown. The tips and margins of leaflets often become chlorotic before turning reddish brown. In general, leaf discoloration is more pronounced in desi types (reddish) than in kabuli types (yellow). The stems and leaves of diseased plants are stiffer and thicker than normal. The most characteristic symptom of stunt is phloem browning. It becomes obvious if the bark is removed at the collar region (by cutting a thin slice length-ways). A transverse cut reveals a brown ring or a split through the collar region and reveals brown streaks of discoloured phloem vessels. The interior wood of the root is white, as is normal, without xylem discoloration. If the plants survive up to the podding stage, pod set is sparse. Many plants dry up prematurely. Sometimes stunt and fusarium wilt infection occur together. In such cases, xylem discoloration, which is typical of fusarium wilt, is also seen. The wilting is the result of combined infection. Mechanical damage to the phloem by chewing insects, which attack the plant at the collar region, can also result in leaf discoloration and stunting similar to stunt. But there will be no phloem browning. PHYLLODY [MYCOPLASMA] Distribution: Phyllody is a minor disease reported from Ethiopia, India and Myanmar. Characteristic symptoms are excessive proliferation of branches with smaller leaflets, giving a bushy appearance to the plant. Diseased plants are scattered in the field and are more easily spotted at flowering and podding time. The flowers are converted into leafy structures. At the time of crop maturity, when the healthy plants are drying, the diseased plants in the field will be conspicuously green. Biotechnological Approaches In recent years, genetics of complex biotic stresses like Fusarium wilt, Ascochyta blight have been understood and the genomic regions/ QTLs have been identified. Furthermore, several functional genomics
27 approaches such as RNA-seq, Massive Analysis of cDNA Ends (MACE) with parental genotypes of mapping populations as well near isogenic lines (NILs) have provided some candidate genes for drought tolerance that are being validated through genetical, genomics and/or TILLING approaches. In 2013, International Chickpea Genome Sequencing Consortium (ICGSC) co-led by ICRISAT, University of California-Davis (USA) and BGIShenzhen (China) assembled the draft genome of kabuli chickpea genotype CDC Frontier, while Next Generation Challenge Programme on Chickpea Genomics (NGCPCG), India assembled genome sequence of desi genotype ICC 4958. After assembling the draft chickpea genomes, efforts to exploit the potential of next generation sequencing (NGS) technology to understand the genome architecture of chickpea were initiated. As part of these initiatives NGS based whole genome re-sequencing (WGRS) of chickpea parental lines were undertaken, which led to identification of 2 million Single Nucleotide Polymorphisms (SNPs) and more than 290K Indels. These SNPs are valuable resource providing enough markers to undertake the genetics research (Thudi et al. 2016a). In addition, NGS based WGRS was used to understand the impact of breeding on genetic diversity and temporal diversity trends in chickpea. For this, WGRS was used to resequence more than 100 chickpea varieties released in last five decades and identified 1.2 million SNPs. These SNPs were used to identify the genomic changes during the history of chickpea breeding suggesting increase in diversity in the primary gene pool as result of recent chickpea breeding programs. In addition to parental lines and varieties, chickpea reference set (comprising 300 accessions) were also re-sequenced using WGRS approach and led to identification of 4.9 million SNPs. These SNPs are being used to undertaking the genome wide association study (GWAS) for identifying the markers associated with traits of interest and understanding the domestication and post domestication divergence in chickpea (unpublished data). Global gene banks store the huge germplasm wealth that has the potential to contribute significantly towards the goal of enhancing the rate of genetic gain. Chickpea Genome Sequencing Initiative” to re-sequence 3000 lines from Global Chickpea Composite Collection The genomic resources generated have been successfully deployed for developing superior lines for different traits of interest. Trait mapping and molecular breeding such as marker assisted backcrossing (MABC), markerassisted recurrent selection (MARS) and advanced backcross quantitative trait loci (AB-QTL) analysis, which are routine in breeding programs for major crops, are also being practiced in chickpea. For example, superior lines with enhanced drought tolerance, fusarium wilt and ascochyta blight have been developed. Introgression of the “QTL-hotspot” into several elite
28 varieties in India as well as Kenya and Ethiopia led to development of superior lines with enhanced tolerance to drought and increased yield under rainfed and irrigated conditions irrespective of genetic background. Further, the available genomic resources also enabled the successful deployment of modern breeding approaches like genomic selection for faster genetic gains. For effective utilization of the available genomic resources in crop improvement cost effective genotyping platforms also play a major role. Towards this direction, for use in foreground and background selection, cost effective SNP genotyping assays like Vera Code assays, KASPar assays were developed. Recently, a precise and cost effective SNP genotyping platform, with 50,590, high quality non-redundant SNPs on Affymetrix Axiom®Cicer SNP array has been developed and is being used for high resolution genetic mapping (unpublished data). This array will also be useful for fingerprinting the released varieties as well as assessing their adoption in addition to genetics and breeding applications. During last 12 years significant progress has been made in terms of developing genomic resources and these resources have been effectively used for attaining faster genetic gains in chickpea. Molecular approaches for disease resistance Understanding the molecular mechanisms of host-pathogen interactions is of primary importance in devising strategies to control diseases. Various molecular techniques canbe effectively used to decipher these interactions by identifying differentially expressed proteins and genes upon pathogen infection and to devise the strategies to control the pathogens. Similary, identification of resistance genes and mapping them with closely linked molecular markers can help in marker assisted breeding to develop biotic stress tolerant varieties. In addition, the complete genomic sequences of a variety of microorganisms and an increasing number of model organisms are being determined, which will help in precisely targeting the pathogen virulance genes or host resistance genes for effective disease control. Analysis of the Arabidopsis genome suggested that~14% of the genes are potentially involved in disease resistance, which could be exploitated to our advantage. Genomics approaches: Molecular markers like ISSR, RAPD and AFLP have proven to be superior in identifying the athogens as compared to the traditional techniques. This in-turn has helped in deploying the right resistant cultivar in order to combat the diseases. Using ITS-RFLP, Phan et al. (2002) distinguished A. rabiei from the other closely related species
29 like A. pinodes, A. lentis and A. fabae. Desai et al. (1992a) used antigens to reveal genetic relationships among four races of Fusarium oxysporum f.sp. ciceri (Foc) causing wilt in chickpea and found that the races 1, 2 and 3 were similar, while race 4 was different. However, further biochemical analysis of the four Foc races revealed variation in total sugar and amino acid content for race 3 as compared to races 1, 2 and 4 (Desai et al., 1992b). Jiménez-Gasco and Jiménez-Diaz (2003) used RAPD and SCAR markers to characterize the races 0, 1B, 1C, 5 and 6; whereas, Barve et al. (2001) used di-, tri- and tetra-nucleotide repeats in combination with different restriction enzymes to distinguish four Foc (1, 2, 3 and 4) races. Recently, various DNA based approaches, viz., gene-specific amplification, internal transcribed spacer region(ITS)-RFLP, inter-simple sequence repeat (ISSR) amplification, AFLP and sequencing of translation elongation factor 1 alpha (EF-1α) have been employed to characterize the four Foc races in our laboratory. Genetic analysis studies have indicated that the resistance to Foc race 0 is governed by two genes (Halila et al., 2009); whereas, resistance to race 1 of the pathogen (Foc1) is governed by up to three genes that segregate independently (h1, h2 and H3). Similarly, three genes have been described for resistance to race 2, two for race 4 and one each for the races 3 and 5 (Sharma and Muehlbauer, 2007). Molecular markers tightly linked to the resistance genes for various Foc races have been identified and most of these resistance genes have been found to cluster on LG2 of the chickpea genome. The resistance gene foc-1 (syn. h1), imparting resistance to Foc1, was initially mapped byMayer et al. (1997). Subsequently, markers linked closely to resistance genes for different Foc races have been identified, including foc-01 (Rubio et al., 2003; Cobos et al., 2005),foc-02 (Halila et al., 2009), foc-1 (Sharma et al., 2004b; Sharma and Muehlbauer, 2005;Gowda et al., 2009), foc-2 (Sharma and Muehlbauer, 2005; Gowda et al., 2009), foc-3(Sharma et al., 2004b; Sharma and Muehlbauer, 2005; Gowda et al., 2009), foc-41(Sharma et al., 2004b; Sharma and Muehlbauer, 2005), foc-42 (Tullu et al., 1999) and foc-5 (Tekeoglu et al., 2000; Winter et al., 2000; Benko-Iseppon et al., 2003; Sharma and Muehlbauer, 2005). However, the genes imparting resistance to the races 1B/C and 6 are yet to be mapped. Rajesh et al. (2004) developed a bacterial artificial chromosome (BAC) library of chickpea to facilitate positional cloning of disease resistance genes and physical mapping of the chickpea genome. Two clones with a combined insert size of 200 kb were isolated after the library was screened with an SSR marker, Ta96, which is tightly linked to a gene for resistance to fusarium wilt caused by Foc3 at a genetic distance of 1 cM. The analysis of the BAC library will facilitate the isolation of foc-3 and allow physical mapping of this genomic region where additional resistance
30 genes against other races of the wilt causing pathogen are also positioned. Cloning and characterization of disease resistance genes is essential not only to study their evolution but also to understand the mechanisms involved in resistance and their exploitation in disease resistance breeding and management. Though several resistance sources have been identified, the chances of breakdown of resistance and the appearance of new pathogenic races remain. Combining resistance to more than one race or pathogen in a commercial cultivar i.e., pyramiding of resistance genes, is expected to provide durable resistance against the disease, as the pathogen has to mutate several avirulence genes to overcome the resistance governed by several major genes (Sharma and Muehlbauer, 2007). Screening of the progeny plants carrying the resistance gene(s) can be facilitated with marker assisted selection (MAS) and chickpea breeders are aiming to exploit MAS for resistance breeding. Efficacy of MAS, however, depends upon closeness of the marker to the gene, projecting the need to saturate the chromosomal region harboring the resistance genes with more markers. To understand the gene networks that underlie plant stress and defense responses, it is necessary to identify and characterize the genes that respond both initially, and as the physiological response to the stress or pathogen develops. Several approaches arecurrently being used to decipher the plant-pathogen interactions at molecular level. PCR based suppression subtractive hybridization was attempted to identify Arabidopsis genes that are differentially expressed in response to bacterial and oomycete pathogens and the signaling molecules salicylic acid (SA) and jasmonic acid (Mahalingam, 2003). Such techniques can help in understanding the stress, pathogen- and hormone-modulated genes underlying stress signaling and responses and may contribute to the characterization of the stress transcriptome through the construction of standardized specialized arrays. Table - List of some QTLs/genes identified for different traits in chickpea Trait
Number & Name of gene/QTL
Marker
Ascochyta blight
AR2, ar1, ar1a,ar1b, ar2a, ar2b,Ar19
SSRs, RAPDs, DAF
QTLAR1, QTLAR2
SCARs, SSRs, RAPDs
13 QTL
SSRs, RAPDs
5 QTL (1-5)
SSRs
3 QTL
SSR
Fusarium wilt
foc-0, foc-1, foc-2,foc-3, foc-4, foc-5
SSRs, STSs, RAPDs,
Botrytis grey mold
3 QTL (1, 2, 3)
SSRs
Rust
1 QTL
SSR
31 Integrated disease Management A single disease management strategy rarely provides complete disease control using a number of Integrated disease management (IDM) techniques which aims to Reduce background inoculums levels through paddock selection, control of volunteers and stubble management. Exclude pathogen through the use of clean seed and farm hygiene Protect the crop by resistant varieties, seed treatments and foliar fungicide. A fully integrated disease management programme should be initiated before sowing and maintained through the growing season to greatly reduce disease impact. CONVENTIONAL APPROACHES: Resistant varieties/ Lines Country
Disease
Resistant cultivars
Seed type
Australia
Phytophthora blight ICC11870, Yorker
Desi
Bangladesh
Root diseases
Sabour 4, Fatehpur 1, Bhaugora
-
Botrytis gray mold
Barichola 5, ICCL 87322
Desi
Bulgaria
Ascochyta blight
Plovdiv 019, Obraztsov chijlik 1 Plovdiv 8
Kabuli
Chile
Root rot
California INIA, Guasos SNA
Kabuli
Cyprus
Ascochyta blight
ILC 3279
Desi
Ethiopia
Wilt
Chefe
Desi
Italy
Ascochyta blight
Ali, Sultano, Califfo,
Desi
India
Ascochyta blight
F8, C 12/34, C 235, G 543, H 75-35, GG 688, GNG 146, Gaurav, BG 261, GG 588, Hima chana-1, Gaurav, Vardan, Samrat, PBG 1 and BG 261
Desikabuli
Ascochyta blight
ICCV 04052, 04509, 04505, 04512, 04513, 04523, 04524, 04525, 04526, 04530, 04537, 05502, 05503, 05511, 05512, 05513, 05514, 05515, 05523, 05530, 05531, 05546, 05551, 05571, 98811, 98816, 98818
Desi
32
Wilt No. 10, S 26, G 24, C 214, BG 244, Pusa 212, Avrodhi, JG 315, JG 14, JG 11, JGK 2, KAK 2, Vijay, Vaibhav, JG 63, Birsa canna-3, WR 315, JG 74, JAKI 9218, Vihar, JG 1265, BG 1053, PDG 4, Gujarat gram 4, Gujarat gram 1, BGM 47, COG 29-1, L551 Root diseases
ICCC 32, GL 769 Botrytis gray mold BG 276, GL 90159, GL 91040, GL 91071 and GL 92162
-
Mexico
Wilt
Surutato 77, Sto. Domingo, Senora, UC 15 and UC 27
Kabuli
Morocco
Ascochyta blight
Pch 46
Kabuli
Nepal
Botrytis gray mold
ICC 14344
-
Pakistan
Ascochyta blight
F8, C 12/34, C 727, C 235, CM 72, C 44
Desi
Ascochyta blight and wilt
AUG 480
Desi
Wilt
Punjab 2000
Desi
Syria
Ascochyta blight
ILC 482, Ghab 2 kabuli Sudan Wilt ICCV 2, UC 15, FLIP 8520C, FLIP 85-29C and FLIP 85-30C
Kabuli and Desi
Spain
Ascochyta blight
Fardan, Zegri, Almena, Alcazaba desi Wilt FLIP 84-43, FLIP 85-20, FLIP 85-29C, ILCs 127, 219, 237, 267, 513 and CA 2954
Desi
Turkey
Ascochyta blight
Guney Sarisi 482, ILC 195
Desi
USA
Root rot
Mission kabuli
USSR
Ascochyta blight
VIR 32, Nut Zimistoni
Intermediate desi
Source: Field Diagnosis of Chickpea Diseases and their Control, Information Bulletin No. 28, (Revised 2012) ICRISAT, Hyderabad
33 Fungicide Disease
Seed treatment*
Wilt
Carbendazim @ 2.5 g/kg
Foliar application#
Belate T®(Benomyl + Thiram) @ 1.5 g/kg Dry Root Rot
Captan Thiram
Wet Root Rot
Captan, thiram, Benlate®
Ascochyta blight
2-h immersion in malachite green 5 mg/l
Wettable sulphur
4-h immersion in formal in
Zineb
12-h immersion in pimaicin 150 mg/L Phenthiuram @ 2 g/kg
Ferbam
Thiram @ 2 g/kg Azoxistrobin @ 1g/kg
Maneb captan
Benomyl @ 2 g/kg
Captafol
Calixin M® (tridemorph + maneb) @ 3 g/kg
Chlorothalonil
Calixin M® (tridemorph + maneb) + thiram (1:1) @ 2-5 g/kg
dithianon (Dosage @ 3 g/l)
Calixin M® (tridemorph + maneb) + Bavistin® (carbendazim) (1:1) Bavistin® (carbendazim) + thiram (1:3) thiabendazole (3 g/kg) Botrytis gray mold
carbendazim + thiram (1:1)
Vinclozolin
Vinclozolin, carbendazim
Carbendazim @ 1g/L + Thiram @ 2g/L
Triadimefon
Carbendazim @ 1g/L
Dithane M 45® (maneb)
Captan
Triadimenol
Chlorothalonil
Thiabendazole
Mancozeb
Iprodione
Thiophonate methyl
34
Thiram
Black Root Rot
Apply at 50 days after sowing or at the first sign of symptoms
Thiram + Benomyl Thiram + Captan
Collar Rot
Rizolex® Vitavax 200®
Pythium root and seed rot
Metalaxyl
Source: Field Diagnosis of Chickpea Diseases and their Control, Information Bulletin No. 28, (Revised 2012) ICRISAT, Hyderabad
CULTURAL PRACTICES: Disease
Cultural Practices
Wilt
Use disease free seed Avoid sowing when temperatures are high (late sowing) Follow 4 years crop rotation
Ascochyta blight
Sow late Remove and destroy dead plants Rotate crops Sow deep (15cm or deeper) Wider row spacing Adopt low seeding Intercrop with wheat and barley
Dry Root Rot
Avoid drought Sow on time so that the crop escapes hot weather.
Botrytis Grey Mold
Use disease free seed Burn infected debris Deep ploughing Adopt late sowing Wider row spacing Intercrop with linseed or wheat
35
Avoid excessive vegetative growth Avoid excess irrigation Use erect compact varieties Alternaria blight
Maintain low plant density Avoid excessive irrigation
Sclerotinia stem rot
Deep ploughing Avoid excessive vegetative growth Use upright growth habit cultivars Wider row spacing
Wet root rot
Avoid high soil moisture at sowing
Black rot
Avoid high soil moisture
Pythium root rot and seed rot
Adopt late planting Treat seeds with Penicillium oxalicum, Pythium oligandnum, Pseudomonas fluorescens and Bacillus subtilis
Collar rot
Avoid high soil moisture at sowing & seedling stage Avoid wider spacing Remove all undecomposed organic matter while preparing seed bed Soil solarisation during summer month
Phytophthora blight
Avoid poorly drainage facility Crop rotation with wheat
Stunt
Adopt close spacing Sow when aphid vector activity is low
*Wherever not specified, the dose is 3 g/kg of seed. #3 g or 3 ml per litre of water Source: Field Diagnosis of Chickpea Diseases and their Control, Information Bulletin No. 28, (Revised 2012) ICRISAT, Hyderabad
RESEARCH STRATEGIES AND FUTURE THRUSTS Chickpea production can be increased significantly by utilizing high yielding varieties against major diseases. There is needed to take up research work on the following aspect.
36 Molecular characterization of genetic diversity in key pathogens including F. oxysporum f. sp. Ciceri, Rhizoctonia bataticola, Ascochyta rabiei and Botrytis cinerea needs immediate attention. Development of efficient plant types suitable for different agroecological zones and cropping system. Development of varieties with multiple resistance and multi-gene resistance to key diseases. Development of resistance genes in spatial and temporal manner through gene pyramiding. Pyramiding of genes for resistance to different races in respect of each pathogen in a desirable background is required for stable resistance. Molecular mapping of economically important genes and their precise transfer through marker aided selection. Introgression of desirable resistance genes from wild species to broaden genetic base of cultivated germplasm. Exploitation of secondary gene pool for resistance against major pathogen. Molecular characterization of pathogenic variability of key pathogen. Identification of race specific and multi-race resistance donors. Mechanism of resistance to major diseases.
Multiple diseases resistance is required for insulating varieties against key pathogen.
Exploitation and integration of available management components in respect of these diseases. Evaluation of germplasm for identification of resistance against disease. An easy and reliable screening technique needs to be developed for large scale screening of germplasm and breeding lines. Basic studies on biochemical basis of resistance, genetics and linkage of resistance genes and their molecular characterization in order to follow marker aided selection for major genes.
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39 Gurha SN, Vishwa Dhar, Naimuddin and Singh RA. (2002). Stable resistance in gram (Cicer arietinum) against Race 2 of Fusariumoxysporum f.sp. ciceri. Indian Journal of Agricultural Sciences 72(2): 88-89. Hafiz A and Ashraf M. (1953). Studies on the inheritance of resistance to Mycosphaerella blight in gram. Phytopathology 43: 580-581. Hafiz A. (1952). Basis of resistance in gram to Mycosphaerella blight.Phytopathology 42: 422-424. Halila I, Cobos MJ, Rubio J, Millan T, Kharrat M, Marrakchi M, Gil J (2009). Taggingand mapping a second resistance gene for Fusarium wilt race 0 in chickpea. European Journal of Plant Pathology 124: 87-92. Haware MP, Narayana Rao J and Pundir. (1992). Evaluation of wild Cicer species for resistance to four chickpea diseases. International Chickpea Newsletter 27: 1618. Haware MP, Nene YL and Rajeshwari R. (1978). Eradication of Fusarium oxysporum f.sp. ciceri transmitted in chickpea seed. Phytopathology 68: 1364-1367. Haware MP. (1998). Diseases of Chickpea. pp 473-506. (in) The Pathology of Food and Pasture Legumes (Eds. D.J. Allen and J.M. Lenne), CAB International ICARDA, Wallingford, UK. Jhorar OP, Mathoda SS, Singh G, Butler DR and Mavi HS. (1997). Relationship between climatic variables and Ascochyta blight of chickpea in Punjab (India). Agricultural & Forest Meteorology 87: 171-177. Jimenez Diaz RM, Trapero Casas A and Cabrera de la Colina J. (1989). Races of Fusarium oxysporum f.sp. ciceri infecting chickpea in southern Spain. pp 515-520. (in) Vascular Wilt Disease of Plants (Eds. E.C. Tjamos and C. Beckman), NATO ASI Series, Volume H28, Springer-Verlag, Berlin, Germany. Jiménez-Gasco MM, Jiménez-Diaz RM (2003). Development of a specific Polymerase chain reaction-based assay for the identification of Fusarium oxysporum f. sp. Ciceris and its pathogenic races 0, 1A, 5 and 6.Phytopathol 93: 200–209. Jiménez-Gasco MM, Pérez-Artés E, Jiménez-Diaz RM (2001).Identification of pathogenic races 0, 1B/C, 5, and 6 of Fusarium oxysporum f. sp. ciceris with random amplified polymorphic DNA (RAPD). Eur J Plant Pathol 107: 237–248. Joshi MM and Singh RS. (1969). A Botrytis gray mold of gram. Indian Phytopathology 22: 125-128. Kaiser WJ. (1991). Host range studies with the Ascochyta blight pathogen in chickpea. International Chickpea Newsletter 25: 25-26. Kumar S. (1998). Inheritance of resistance to Fusarium wilt (race 2) in chickpea. Plant Breeding 117: 139-142. Lopez Garcia H. (1974). Inheritance of the character resistance to wilt (Fusarium sp.) in chickpea (Cicer arietinum ) under field conditions. Agricultura Technica en Mexico 3: 286-289.
40 Luthra JC, Sattar A and Bedi KS. (1938). The control of the blight disease of gram by resistant types. Current Science 7(2): 45-47. Luthra JC, Sattar A and Bedi KS. (1935). Life history of gram blight (Ascochyta rabiei (Pass) Lab. Phyllosticta rabiei (Pass.) Trot.) on gram (Cicer arietinum L.) and its control. Punjab Agriculture Live-Stock, (India) 5: 489-498. Mahalingam R, Gomez-Buitrago AM, Eckardt N, Shah N, Guevara-Garcia A, Day P,Raina R, Fedoroff NV. (2003). Characterization of the stress/defense transcriptome of Arabidopsis genome biology. Genome biology, 4: p.R20.1-R20.14. Mukhopadhyay AN, Shrestha SM, Mukerjee PK. (1992). Biological seed treatment for the control of soil borne plant pathogens. FAO Plant Protection Bulletin 40: 2130. Nene YL and Reddy MV. (1987). Chickpea diseases and their control. pp 233-279. (in) The Chickpea (Eds. M.C. Saxena and K.B. Singh). CAB International/ ICARDA, Wallingford, UK. Nene YL, Haware MP and Reddy MV. (1981). Chickpea diseases, resistance- screening techniques. ICRISAT Information Bulletin 10, ICRISAT, Patancheru, India. Nene YL. (1980). Diseases of chickpea. pp 171-178. (in) Proceedings, International Workshop on Chickpea Improvement (28 February- 2 March 1979), ICRISAT, Patancheru, India. Pandey BK, Singh US and Chaube HS. (1985). Development of Ascochyta rabiei in the leaves of susceptible chickpea cultivar. Indian Phytopathology 38: 779-781. Pathak MM, Singh KP and Lal BB. (1975). Inheritance of resistance to wilt (Fusarium oxysporum f.sp. ciceri ) in gram. Indian Journal of Farm Science 3: 10-11. Rajesh PN, Coyne C, Khalid MK, Dev Sharma, Gupta VS, Muehlbauer F. (2004). Construction of first Hind III Bacterial Artificial Chromosome library and its use in identification of clones associated with disease resistance in chickpea. Theory and Appl. Genet 108(4): 663-669. Rao S and Padmaja M. 2000.Selection of chickpea cell lines resistant to culture filtrate of Fusarium oxysporum f.sp.ciceri. Phytomorphology 50: 41-46. Rewal N and Grewal JS. (1989). Differential response of chickpea to grey mold. Indian Phytopathology 42: 265-268. Rubio J, Moussa E, Kharrat M, Moreno MT, Milla N T, Gil J (2003). Two genes and linked RAPD markers involved in resistance to Fusarium oxysporum f. sp. Ciceris race 0 in chickpea. Plant Breed 122:188–191. Sattar A. (1933). On the occurrence, perpetuation and control of gram (Cicer arietinum L.) blight caused by Ascochyta rabiei (Pass.) Labrousse with special reference to Indian conditions. Annals of Applied Biology 20: 612-632. Satyaprasad K and Ramarao P. (1983). Effect of chickpea exudates on Fusarium oxysporum f.sp. ciceri. Indian Phytopathology 36: 77-81. Sharma KD, Chen W, Muehlbauer FJ (2005). Genetics of chickpea resistance to five races
41 of Fusarium wilt and a concise set of race differentials for Fusarium oxysporum f. sp. ciceris. Plant Dis.89: 385-390. Sharma KD, Muehlbauer FJ (2005). Genetic mapping of Fusarium oxysporum f. sp. ciceris race-specie resistance genes in chickpea (Cicer arietinum L.). In: Abstract of the International food legume research conference—IV, Indian Agricultural Research Institute, New Delhi, India, pp 18–22. Sharma KD, Muehlbauer FJ (2007). Fusarium wilt of chickpea: physiological specialization, genetics of resistance and resistance gene tagging. Euphytica157: 1–14. Singh G and Kaur L. (1990). Chemical control of gray mold of chickpea. Plant Disease Research 5:132-137. Singh G, Pande S, Bakr Abu and Singh SD. (1999). Rating scale for Botrytis gray mold of chickpea. BGM Newsletter 2 (1): 56. Singh G, Sharma YR and Bains TS. (1998). Status of Botrytis Gray mold of chickpea research in Punjab, India. pp 7-14. (in) Recent Advances in Research and Management of Botrytis Gray Mold of Chickpea (Eds. M.P. Haware, D.G. Faris and C.L.L. Gowda), ICRISAT, Patancheru, India. Singh G, Sharma YR and Kaur L. (1992). Detached leaf method of screening for resistance to Ascochyta blight of chickpea in wide hybridization. pp 1-70. (in) Annual Progress Report of Pulse Pathology, Department of Plant Breeding, Punjab Agricultural University, Ludhiana, India. Singh G. (1989). Identification of chickpea lines resistant to Ascochyta blight. Plant Disease Research 4 : 128-132. Singh H, Kumar J, Smithson JB and Haware MP. (1987). Complementation between genes for resistance to race 1 of Fusarium oxysporum f.sp. ciceri in chickpea. Plant Pathology 36: 539-543. Sivaramakrishnan S, Kannan S and Singh SD. (2002). Genetic variability of Fusarium wilt pathogen isolates of chickpea (Cicer arietinum L.) assessed by molecular markers. Mycopathologia 155: 171-178. Srivastava SK, Singh SN and Khare MN. (1984). Assessment of yield losses in some promising gram cultivars due to Fusarium wilt. Indian Journal of Plant Protection 12: 125-126. Tewari SK and Pandey MP. (1985). Allelic relationship between genes for resistance to Ascochyta blight in chickpea. International Chickpea Newsletter 12: 13-15. Udapa SM, Weigand F, Saxena MC and Kahl G. (1998). Genotyping with RAPD and microsatellite marker resolves pathotype diversity in the Ascochyta blight pathogen of chickpea. Theoretical & Applied Genetics 97: 299-307. Upadhaya HD, Smithson JB, Haware MP and Kumar J. (1983b). Resistance to wilt in chickpea. II. Further evidence for two genes for inheritance to Race 1. Euphytica 32: 749-755. Upadhaya HD, Haware MP, Kumar J and Smithson JB.(1983a). Resistance to wilt in
42 chickpea. I. Inheritance of late wilting in response to Race 1. Euphytica 32: 447452. Vir S and Grewal JS. (1975). Inheritance of resistance to Ascochyta blight in chickpea. Euphytica 24: 209-211. Weltzien HC and Kaach HJ. (1984). Epidemiology aspects of chickpea Ascochyta blight. pp. 35-44. (in) Proceedings, Ascochyta blight and Winter Sowing of Chickpea (Eds. M.C. Saxena and K.B. Singh), Martinus Nighoff/Dr. W. Junk Publishers, Hague, The Netherlands.Tekeoglu M, Tullu A, Kaiser WJ, Muehlbauer FJ (2000). Inheritance and linkage of twogenes that confer resistance to Fusarium wilt in chickpea. Crop Sci.40:1247-1251. Winter P, Benko-Iseppon AM, Hu Ttel B, Ratnaparkhe M, Tullu A, Sonnante G, Pfaff T,Tekeoglu M, Santra D, Sant VJ, Rajesh PN, Kahl G, Muehlbauer FJ (2000). Alinkage map of the chickpea (Cicer arietinum L.) genome based on recombinantinbred lines from a C. arietinum XC. reticulatum cross: localization of resistance genefor fusarium wilt races 4 and 5. Theor Appl Genet101:1155– 1163.
43
2 PRODUCTION CONSTRAINTS AND GENETIC IMPROVEMENT OF PULSE CROPS IN BIHAR 1
Anil Kumar, 1Anand Kumar, 3 Kumari Rajani, 1Chandan Kishore, 1 Manoj Kumar, 2Ravi Ranjan Kumar, 2Vinod Kumar and 4 Abhijeet Ghatak 1 2
Dept. of Plant Breeding and Genetics,
Dept. of Molecular Biology and Genetic Engineering, 3
4
Dept. of Seed Science &Technology,
Dept. of Plant Pathology, Bihar Agricultural University, Sabour, Bhagalpur-813210 (Bihar)
INTRODUCTION Pulses are major sources of proteins among the vegetarians in India, and complement the staple cereals in the diets with proteins, essential amino acids, vitamins and minerals. They contain 22-24% protein, which is almost twice the protein in wheat and thrice that of rice. They provide significant nutritional and health benefits, and are known to reduce several noncommunicable diseases such as colon cancer and cardiovascular diseases (Yude et al, 1993; Jukanti et al, 2012. Pulses are the important sources of proteins, vitamins and minerals Table 1) and are popularly known as “Poor man’s meat” and “rich man’s vegetable”, contribute significantly to the nutritional security of the country. Currently, we are in the mid-way of self-sustaining in pulses production as we are world leader in production, consumption and import as well. India import 2-3 million tons (MT) of
44 pulses during 2010-11, causing huge hard foreign earning. We will able to sustain our production and we turned to net importer to net exporter for pulses if everything goes as per plan by the 2050. India is the largest producer and consumer of pulses in the world. Major pulses grown in India include chickpea or Bengal gram (Cicer arietinum), Pigeonpea or Red gram (Cajanus cajan), Lentil (Lens culinaris), Uradbean or Black gram (Vigna mungo), Mungbean or Green gram (Vigna radiata), Lablab bean (Lablab purpureus), Moth bean (Vigna aconitifolia), Horse gram (Dolichos uniflorus), Pea (Pisum sativum var. arvense), Grass pea or Khesari (Lathyrus sativus), Cowpea (Vigna unguiculata), and Broad bean or Faba bean (Vicia faba). More popular among these are Chickpea, Pigeonpea, Mungbean, Uradbean and Lentil. In general, pulses are mostly grown in two seasons: (a) the warmer, rainy season or Kharif (JuneOctober), and (b) the cool, dry season or Rabi (October-April). Chickpea, Lentil, and Dry peas are grown in the Rabi season, while Pigeonpea, Uradbean, Mungbean and Cowpea are grown during summer as well as Kharif season. Among various pulse crops, Chickpea dominates with over 40 percent share of total pulse production followed by Pigeonpea (1820%), Mungbean (11%), Uradbean (10-12%), Lentil (8-9%) and other legumes (20%) (IIPR, Vision 2030). Although India is the world’s largest producer of pulses; it imports a large amount of pulses to meet the growing domestic needs and is the largest importer, producer and consumer of pulses. During 2009-10, India imported 3.5 million tons of pulses from the countries like Australia, Canada, and Myanmar. On the other hand, India is also the largest pulses processor, as pulses exporting countries like Myanmar, Canada and Australia do not have adequate pulses processing facilities. Large shares of pulses import, including desi chickpeas, Pigeonpea, mungbean, Uradbean, and kidney bean come from Myanmar. Importers support Myanmar because it offers varied pulses with qualities similar to those produced in India, low freight rates, and relatively fast delivery. Canada and Australia are major suppliers of dry peas and kabuli chickpeas to the Indian market, each supplying about one-third of India’s pulses imports. Most kabuli chickpeas come from Mexico, Australia, Canada, Turkey and Iran. Nepal and Syria account for the largest shares of Indian lentil imports. It has been estimated that India’s population would reach 1.68 billion by 2030 from the present level of 1.21 billion. Accordingly, the projected pulse requirement for the year 2030 is 32 million tons with an anticipated required growth rate of 4.2% (IIPR, Vision 2030). India has to produce not
45 only enough pulses but also remain competitive to protect the indigenous pulse production. Keeping the ideas in mind, India has to develop and adopt more efficient crop production technologies along with favourable policies to encourage farmers to bring more area under pulses. Table 1: Nutritive value of Pulse Constituents
Magnitudes
Protein
>20-%
Carbohydrate
55 – 60%
Fat
>1.0%
Fibre
3.2%
Phosphorus
300-500 mg/100 g
Iron
7-10mg/100 g
Vitamin C
10-15 mg/100 g
Calcium
69 -75mg/100g
Calorific value
343
Vitamin A
430-489 IU
Pulses Scenario in India India is the largest producer and consumer of pulses in the world accounting for about 29 % of the world area and 19 % of the world’s production. Even more importantly India is also the largest importer and processor of pulses in the world. The country’s pulse production has been on the edge of around 14– 15 MT, coming from a near-stagnated area of 22– 23 M ha, since 1990–91 (Singh et al., 2013a). Major areas under pulses are in the States of Madhya Pradesh (20.3%), Maharashtra (13.8%), Rajasthan (16.4), Uttar Pradesh (9.5%), Karnataka (9.3%), Andhra Pradesh (7.9%), Chhattisgarh (3.8%), Bihar (2.6%) and Tamil Nadu (2.9%). Pulse productivity which was 441 kg/ha in 1950 increased up to 689 kg/ha during 2011, registering 0.56% annual growth rate. Table 2: Compound growth rate achieved in pulses production in India during the period 1950-2011 Particulars
Pulses
Productivity (1950)
441 kg/ha
Productivity (2011)
689 kg/ha
46
Overall compound growth rate (%)
1.011
Compound growth rate (%) Area
0.52
Compound growth rate (%) Production
1.27
Compound Growth rate (%) productivity
0.73
Source: Department of Agriculture and Cooperation, Ministry of Agriculture, Govt. of India
Production of pulses in 2008-09 was 14.66 million tons with an average yield of 655 kg/ha. In the Year 2013-14 India produced record 19.5 MT of total pulses. Share of chickpea (39%), Pigeonpea (21%), mungbean (11%) and Uradbean (10%) to total production has been worked out. Lentil and field pea accounted for 7% and 5% share of total production, respectively. To meet out the demand of the growing population, the country is importing pulses to the tune of 2.5–3.5 MT annually. Strong ascending trend in the import of pulses is a cause of concern, since an increase in demand from India has shown to have cascading effect on international prices, thus draining the precious foreign exchange. Therefore, the domestic requirements would be 26.50 Mt, necessitating stepping up production by 81.50%, i.e. 11.9 Mt additional produce at 1.86% annual growth rates by the 2050. This uphill task has to be accomplished under more severe production constraints, especially abiotic stresses, abrupt climatic changes, emergence of new species/ strains of insect-pests and diseases, and increasing deficiency of secondary and micronutrients in the soil. This requires a two-pronged proactive strategy, i.e. improving per unit productivity and reducing cost of production. The yield levels of pulses have remained low and stagnant, also area and total production. Number of districts harvesting more than 0.8 or 1 t/ ha yield of kharif pulses is very less (Annoymus, 2013). Situation of rabi pulses is better in this regard. The gap between demand and supply has been widening and has
47 necessitated import of pulses of 2.8 million tons in 2007-08. Uttar Pradesh contributes significantly to the pulses production and its share to the national pulses security is 21.8% with 3.196 MT followed by Madhya Pradesh and Rajasthan with 19.5% and 13.6% production share of India. Only eight states contributing 90 % of total pulses production (Table-3). Table 3: State wise contribution in the Indian pulses production State
Production (MT)
Production (%)
Uttar Pradesh
3.196
21.8
Madhya Pradesh
2.859
19.5
Rajasthan
1.994
13.6
Maharashtra
1.407
9.6
Orissa
1.202
8.2
Bihar
1.041
7.1
Karnataka
0.777
5.3
Haryana
0.748
5.1
Other states
1.437
9.8
Total
14.66
100
From the Table 4 it reveals the importance of individual pulse crop in country pulse production. Chickpea is on the top with 26.85 and 38.81 % share with respect to area and production in India, followed by pigeon pea and mungbean, respectively. Lentil occupies only 4.94 % area and corresponding contribution in national pulse production is 6.96%. Table 4: Individual Pulse Contribution in Indian Scenario Name of crops
Area(Mha)
Area (%of Total)
Production (MT)
Production (% of Total)
Chickpea
5.81
26.85
5.69
38.81
Pigeonpea
3.62
16.73
3.07
20.94
Mungbean
2.98
13.77
1.61
10.98
Uradbean
3.18
14.70
1.46
9.96
Lentil
1.07
4.94
1.02
6.96
Other Pulses
6.30
29.11
1.75
11.94
Total
21.64
26.85
14.66
100.00
48 Pulses Profile of Bihar Bihar ranks 9th in terms of production with a contribution of 0.52 million tons to the national pulse pool, population of the state ranks third, the most populous state sharing 2.8 % of geographical area and 8.6 % population of India. Food grains are the major agricultural commodity, produced on about 93 % of cropped area, of which pulses share merely 7.06 %. Adaptability of pulses to diverse climatic and agro ecosystems is exceptional making them victim of human apathy. Pulses are often cultivated on marginal lands under rain-fed conditions on residual soils moistures after harvesting of Kharif crops. Owing to high instability in productivity there has been an incessant decline in area of pulses area, production and productivity during last three and half decades which accounted for about 437.24 thousand hectares, 428.93 thousand tons and 981 kg/ ha respectively in 2014 - 15 against the corresponding figures of 717.2 thousand hectares, 620.7 thousand tons and 865 kg/ ha in 2000-01, registering a compound annual decline of -2.5 % in area and -0.41 % in production but productivity increased by 2.15 Percent annually. It is obvious that there has been a radical fall in pulse area with a corresponding decline in pulse production. There has been a compound annual growth in productivity of pulses during 2000-01 and 2014-15, probably due to adoption of improved seeds and steps taken by the government to accelerate pulse production in recent years. Lentil is only crop which has performed well in Bihar whereas area and production of most of the major pulses have gone down. It is worth noting that there has been a negative correlation between irrigation and acreage of pulses indicating that farmers prefer to allocate irrigated lands to cereals and other crops substituting pulses. Bihar contributes about 3.06 % in production and 2.35 % in area. Important pulses like Lentil, Latyrus, Pigeon pea, Green gram, Chickpea etc are mostly grown in under rainfed conditions on marginal land with poor level of management practices resulting in low production and productivity which was of 897 Kg/ha in 2013-14. Table 5: Area, Production and Yield of total Pulses in Bihar Year
Area (‘000 hectare) Production (‘000 hectare) (‘000 tonnes)
Yield (Kg/ha)
1970 – 71
1644.8
987.4
600
1975 – 76
1531.3
822.4
537
1980 – 81
1367.8
833
609
1985 – 86
1233.1
887.4
720
49
1990 – 91
1175.7
915.8
779
1995 – 96
921.8
560.7
608
2000 – 01
717.2
620.7
865
2005 – 06
596.9
446.8
749
2010 – 11
605.0
556.0
918
2013 – 14
500.0
522.0
1044
Constraints of Pulses Production in Bihar The area under pulses has undergone drastic reduction resulting in low production, although the productivity of major pulses has either increased or remained stagnant but still there is huge gap with potential yield. Pulses are generally grown on marginal lands due to the low productivity-low input nature as rainfed crop, with main focus on high productivity-high input crops like paddy and wheat by most farmers. In a study regarding impact of NFSM on pulse production, marketing it was found that in NFSM and non NFSM district of Bihar about 64% of sample farmers mainly grows pulses for home consumption needs, followed by lack of irrigation and inferior land (14% each) and profitability ranked important only by large farmers (8%). In NFSM and non-NFSM districts pest problem has been mentioned as main limitation (28-30%), other being lower profitability (24%), yield instability (20%) and lack of assured market (16%) were listed as main reasons for shifting away from pulse cultivation (Singh et al., 2016). As a crop of low priority and status they do not get best of management practices of farmers besides being adversely affected by a number of biotic and abiotic stresses, responsible for a large extent of the instability and low yields. The major abiotic, biotic and socio-economic constraints in Pulse production are enumerated below. Abiotic Constraints Drought, water logging, temperature extremes, wind or hail (causing lodging), alkaline and saline soils, acid soils, and deficiencies or toxicities of various mineral nutrients are the common abiotic stresses that limit pulse production in Bihar. Drought: In chickpea, Pigeon pea, green gram, lentil, black gram drought is the main cause of reduction in yield as these are grown in residual receding soil moisture environment or face soil moisture deficit conditions during their reproductive phase and are exposed to terminal drought stress. To overcome such situation better management techniques to conserve soil
50 moisture and maximize crop transpiration over soil evaporation can help to reduce drought effects in pulses. In some situation excess soil moisture in tal and other areas induces excess vegetative growth with more disease incidence as well as lodging in gram and lentil, whereas, Khesari (Lathyrus) has good drought resistance and production even when sown on droughtprone upland soils. Temperature: Terminal drought stress during maturity in chickpea and lentil results in poor pod filling whereas, low temperatures results in frost injury Water logging: Khesari (Lathyrus) and horse gram can establish well in waterlogged soils, even when relay-sown in rice, and can grow until maturity in waterlogged soils. All other pulses do not tolerate water logging condition. Micronutrient deficiencies: Deficiency of micronutrients adversely affects production of lentil, chickpea and other pulses, which become more evident once the biotic stresses are managed. Experiment have shown that deficiency of micronutrients like sulphur and zinc is common in pulse growing regions and application of sulphur and zinc has been found with a cost benefit ratio of 10-21%. Nitrogen fixation: In traditional pulses grown area Rhizobium spp present in soil results in effective nodulation, but when introduced in new areas the host specific Rhizobium also needs to be introduced through inoculation. In Bihar Khesari (Lathyrus) nodulates prolifically meeting its own nitrogen (N) requirements as well as also fix N to the cropping system. Pulses are very sensitive to alkaline, saline, and acidic soil which limits its prospects of cultivation in irrigated areas where there is such increase incidence. Table 6: Important Biotic and Abiotic Stresses Identified in Major Pulse Crops of India Sl.No.
1.
2.
Crop
Chickpea
Seasons
Stress Biotic
Abiotic
Timely sown
FW, root rot, chickpea stunt, BGM, pod-borer
Low temperature
Early sown
FW, root rot, AB, or Terminal drought, chickpea stunt, pod borer salt stress
Late sown
FW, pod borer Terminal drought, cold
Pigeonpea
Kharif-early
FW, PB, pod-borer complex
Water logging
51
3.
Moong
4. 5.
Urad
Medium late
FW, SM, pod-borer complex
Cold, terminal drought, water logging
Pre-rabi
FW,ALB, Pod fly
Cold, terminal drought
Kharif
MYMV, CLS, WB, sucking insect pests
Pre-harvest sprouting, terminal drought
Zaid
MYMV, root and stem Pre-harvest rot, stem agromyza, sprouting, temperature Sucking insect pests
Stress, drought
Rabi
PM, Rust, CLS
Terminal drought
Kharif
MYMV, anthracnose, Terminal drought WB, LCV
Zaid
MYMV, root and stem rot, stem agromyza
Pre-harvest sprouting, temperature Stress, drought
Rabi/rice fallow Spot 6.
Lentil
FW, root rot, rust
Terminal drought Moisture, temperature
FW= Fusarium wilt, PB= Phytophthora blight, SM= Sterility mosaic, ALB= Alternaria leaf blight, MYMV= Mungbean yellow mosaic virus, BGM= Botrytis gray mould, AB= Ascochyta blight. Source: Reddy (2006)
Biotic Constraints: More than 250 insect species are reported to affect pulses, of which nearly one dozen cause heavy crop losses. Pod borer (helicovera armigera) causes the most harm, followed by pod fly, wilt and root rot, important diseases wilt in chickpea, sterility mosaic virus (SMV) in pigeon pea, yellow mosaic virus (YMV) and powdery mildew (PM) are also common and damaging. Another important pest affecting pulses are nematodes, among which root-knot nematodes are important in terms of spread and damage to crop yield, which have been effectively controlled by bio-agents. Recently many successful trials have been conducted to control pod borer through using nuclear polyhedrosis virus (HaNPCV) and Bacillus thruringiensis Berliner (Bt) var. kurstaki, which has been found to be more efficacious in bringing about higher and quick mortality.
52 Table 7: Improved Varieties of Pulses Released from 1991-2005 Crops
Number of Varieties Released
Major Characteristics
1991-97
1998-2005
Pigeonpea
20
13
Tolerance to pod borer, pod fly, wilt, phytopthera blight, pre-rabi, 20-22 q/ ha short duration (95 to100 days), long duration (170-190 days)
Mungbean
21
19
Resistance to YMV, powdery mildew, jassids and whitefly, spring, medium to bold seeded
Uradbean
17
15
Tolerance to YMV, powdery mildew, rabi season, 12q/ha, 70-80 days to maturity
Lentil
9
4
Tolerance to rust and wilt, bold seeded, early maturing, 18-24 q/ha, 110 days to maturity
Pea
5
11
Resistance to powdery mildew, rust and leaf miner, 23q/ha, 95 to 100 days to maturity
Cowpea
11
10
Early maturing varieties, resistance to yellow mosaic virus, 12q/ha, some are for fodder purpose
Chickpea
18
34
Resistant to ascochyta blight, tolerance to wilt and root rot irrigated area, bold seeded, tolerance to pod borer, 25 to 30 q/ha, 75 to 100 days to maturity
Varietal constraints: Lack of high yielding varieties, low harvest index, high susceptibility to diseases and insect pests, flower drops, lack of short duration varieties, intermediate growth habits, poor response to inputs and Instabilities in performances are the few of the varietal constraints needs immediate attention (Singh et al., 2013e and Ramakrishna et al., 2000) Physiological Limitations: There is a general feeling that pulses (C-3 plants) suffer from inherently low yield potential and are a physiologically inefficient group of plants compared to cereals (C-4 plants) such as sorghum and maize. However Aggarwal et al (1997), reviewed the comparative advantages of C-3 and C-4 group of plants and argued that C-3 and C-4 plants seem to compete on fairly even terms in hot dry environments. The fact that C-3 plants usually do better in cool climates suggests that C-3
53 plants are better for rabi season. However, the disturbing future is that the harvest index (HI) in pulses is low compared to cereals. HI is defined as seed yield per unit of recoverable biomass. In pulses it is only 15-20% compared to 45-50% in case of cereals such as wheat and rice. Low HI results from excessive vegetative growth, but can be overcome by early partitioning of dry matter into seeds (Saxena and Johansen 1990) and evolving biotechnology and genomic tools to incorporate good features of C-4 plants into C-3 plants. Pulses in general have a high rate of flower drop. In pigeonpea, over 80% of the flowers produced in a plant are shed; by decreasing flower drop, yield can be increased considerably. This can be done either by breeding lines which retain a large proportion of flowers producing pods or through physiological manipulations, such as spray of hormones which reduce flower drop. Physiological studies at ICRISAT, involving removal of flowers and young pods of pigeonpea, have shown that plants compensate for the loss of flowers and young pods by setting pods from later formed owners, which otherwise would have dropped. This compensatory mechanism provides substantial plasticity of adaptation to intermittent adverse conditions such as moisture stress or insect attack, which are common in warm rainfed areas of south India. Recent increase in yield levels in pigeonpea is due to release of long duration (annual) varieties, which maximise utilisation of assimilates in filling the available sink of a large number of flowers (Rego and Wani 2002). Pests and diseases Although legumes crops are prone to many insect pests and seed borne diseases, a major cause of concern as its incidence, if not controlled, devastates the crop. Fusarium wilt is wide spread in legumes growing regions. In addition, heavy damage to legumes grain is caused by pests during storage. Legumes are in general pest free crop under normal condition if proper crop rotation is follows. However Pod borer, Aphids and Wilt (Fusarium lentis) aremajor insects and disease pests (Singh et al., 2013b and Singh et al., 2013g). Hence the control measures of all three are listed in Table 8. Table 8: Important diseases and insets pests of major pulses Sl.No. Crops
Disease
Insect-pest
1
Chickpea
Fusarium wilt, Ascoochyta blight, Botrytis grey mould, and stunt virus
Pod borer andcut worm
2
Pigeon pea
Sterility mosaic virus,Fusarium wilt, Phytophthora stem blight Alternaria leaf spot and powdery mildew
Pod borer andpod fly leaf
54
3
Urad and mung
Yellow mosaic virus,Cercosora leaf spot, powdery mildew, leaf crinple virus and root rot
White fly, Borer
3
Lentil
Rust, wilt Sclerotinia blight, collor rot
Pod borer
4
Field pea
Powdery mildew, rust,
Pod borer, stem borer, leaf minor
Blue Bull trouble Legumes are vulnerable to attack by Blue Bulls in the IndoGangetic Plains. Because of the widespread menace particularly in Uttar Pradesh, Bihar, Madhya Pradesh, Rajasthan and Chhattisgarh the potential area suitable for taking legumes crops is left uncultivated by the farmers. There is no viable strategy available in the country to effectively the menace. Exploitation of Heterosis Breeding in Pulses: The quantum of yield advances made through breeding in cereals such as maize, sorghum, millets, etc, is much higher than that of pulses. This difference in case of pulses arises primarily due to lack of commercial exploitation of hybrid vigour due to lack of mass pollen transfer mechanism and non-availability of effective male-sterility system. With the exception of pigeonpea (ICPH-8) the hybrid vigour for yield has not been exploited in all other pulse crops. The adoption of these hybrids, however, is limited due to seed production limitations posed by genetic nature of male sterility (Saxena et al 2000) even though ICPH-8 was found to be more promising. It was released for cultivation in the central zone of India in 1981. Evaluation from 100 trials showed ICPH-8 to be superior to controls, UPAS-120 and Manak by 30.5% and 34.2% respectively, in productivity. There is still further scope for enhancing genetic improvement in pulse crops through biotechnology. Mutation breeding has contributed about 10% of the total improved varieties of pulses and is supplementing the conventional breeding programme. The mutant variety, Pant Moong-2, with resistance to YMV disease is very popular in north India (Pawar and Panday 2001). There is good scope for development of ideal plant type especially chickpea and pigeonpea, in line with rice developed by the International Rice Research Institute (IRRI) in collaboration with other research organisations with the leadership of ICAR. Specific efforts in this direction are already in place by both national and international organisations with respect to chickpea. Technological Constraints: A legume is grown under varied agro-climatic conditions (soil
55 types, rainfall and thermal regime) in the country. This calls for region specific production technology including crop varieties with traits relevant to prevailing biotic and abiotic stresses. Even biological fertilizers and pesticides used should be based on strains isolated from regions with similar agro-climatic conditions for them to be effective. Our research and development programme in pulses has yet to appreciate and address this issue adequately. Production technology for a legumes crop has to be soil type/region specific (Singh et al., 2012a) equally applicable for tillage and seeding device/gadgets. Socio-economic Constraints: Pulses production in India is characterised by a very high degree of diversity as indicated both by the number of crops, and their spatial distribution into varied agro-climatic conditions. Most of these crops are region-specific in the sense that a single state or a cluster of few states accounts for the bulk of the area and production of a specific pulse crop. Pulses such as pea, lentil, khesari and even chickpea indicate their regional distribution pattern. This diversity has several implications. In the first place it places serious limits to a single national policy for the promotion of pulses production in the country, and for the promotion of regional crop specific strategies to pulses development programmes. However, in view of the meagre resources available to pulses development as a group, this diversified approach may mean spreading the resources too thinly and in turn making the effort inconsequential. This dilemma may partly explain the absence of any major thrust on research on pulses, which in turn is partly responsible for their stagnation. The structure of pulses production is also characterised by the dominance of two crops, viz, chickpea and pigeonpea, which together account for more than one-half of total pulse area in India. Hence if these two crops suffer from adverse climatic conditions, it significantly reduces the production of pulses. The decline of chickpea in particular and pigeonpea and other crops in major pulse growing states like UP, Punjab, Haryana and Bihar clearly support this possibility. Prospects of Increasing Area and Production Bihar had been a traditional pulse growing state but the focus with time shifted to more remunerative and assured cropping system of cerealcereal. With policy apathy it has reached to low which can be reversed with more focus on pulses. New initiatives can change this trend with following steps:
56 Short-duration Pigeonpea in sequence with wheat: Introduction of short duration pigeon pea in uplands of Bihar can be successfully grown. As this region receives heavy rainfall it is important that sowing should be done in the first fortnight of June with pre-planting irrigation so that by the time monsoon rains start the seedlings are strong enough to combat adverse effects of excess moisture. Development of varieties tolerant to excess soil moisture would help in popularization of short-duration pigeon pea. Spring/summer cultivation of black gram and mung bean: Bihar has good scope of cultivation of kharif spring black gram and mung bean as well as summer mung bean. With nearly 155.1 thousand hectare area under mung bean there is great scope of widening this area. They are very suitable for intercropping with spring-planted sugarcane and sunflower. Under resource constraints, rice-chickpea is found to be more beneficial than rice-wheat and northern Bihar show most potential for this system. In excessive moisture area i.e., in lowland lentil is a more assured crop than chickpea. So the rice-lentil system is also very popular in the lowlands of Bihar. Utilization of rice fallows In Bihar 2.2 million ha rice fallow land in district like Kisanganj, Sahibganj, Gaya, Aurangabad, Katihar and Bhagalpur is there which are most suitable for pulses cultivation. In large area medium and long duration paddy is cultivated and after field vacating due to lack of irrigation facility and delay normally the field remains vacant. Pulses like lentil, mung bean, Uradbean, Lathyrus, peas etc. in rice fallows can increase the productivity as well as sustainability of rice-wheat production system. In low land areas with excessive soil moisture, lentil is more suitable and assured than chickpea so it can be made more popular in the lowlands of Bihar. Groundnut can also be introduced in rice fallows in Char area of Bihar. Area expansion: Besides rice-fallow there is further scope of increasing pulse area by cropping system manipulation, like mung bean and Urad bean as catch crop in summer/spring under cereal-based cropping systems of Bihar, intercropping of short-duration pulses like mung bean, Urad bean, cowpea in sugarcane etc. Mung bean with ratooned Sugarcane during spring/ summer (irrigated), Chickpea or lentil with autumn planted sugarcane and advocating new cropping systems like rice–lentil.
57 Integrated Pest Management: Integrated pest management (IPM) gives wider scope for costeffective control of multiple pests and diseases. It is basically pest management technique which uses one or more management options to reduce pest population below the economic injury level, while ensuring productivity and profitability of the entire farming system. Pulse crop is attacked by more than one disease and pest at a time. However, farmers are hesitant to use knowledge intensive, systems approach IPM as it needs community approach and takes time to yield results. Cultivation of post rainy pigeon pea and common bean: Varieties like Sharad and Pusa 9 are suitable for September planting, are found best for extension of post rainy pigeon pea on uplands of, northern Bihar which receives very heavy rainfall in July causing damage to July planted Pigeon Pea. As the September sown verities are thermo sensitive any delay after mid- Sept. causes heavy loss in yield. Introduction of black gram and mung bean as winter crops: In some part of north-eastern Bihar where the winter temperature are moderate black gram and green gram can be introduced in rice fallow areas utilizing residual moisture and bringing additional area under pulses increasing system productivity too. Another important practice widely applied is that of “growing of short duration legumes” such as moongbean, cluster bean, cowpea and horse gram in widely spaced crops and incorporation of their biomass after harvesting (green manuring), which increases the yield of subsequent crops. Table 9: Productivity of major pulses grown in Bihar Year
Chick pea
Pigeon pea
Lentil
Mung bean
Lathyrus Urad bean
Peas
Kulthi
1965-66
742
855
-
-
-
-
-
-
1970-71
713
896
643
351
581
487
-
-
1975-76
550
705
582
336
612
377
606
352
1980-81
718
971
641
466
619
470
534
387
1985-86
839
1142
772
485
787
456
665
546
1990-91
941
1243
892
556
790
670
747
537
1995-96
651
929
581
557
657
505
554
455
2000-01
1033
1348
981
581
915
634
965
771
58
2005-06
902
1291
705
556
853
770
892
858
2010-11
1182
1515
900
669
998
841
1051
980
2013-14
1147
1667
1147
680
-
912
1041
953
BREEDING METHODS IN PULSE CROPS 1. Conventional Plant Breeding Methods Breeding procedures which are commonly used for genetic improvement of grain legumes include
Introduction
Pure line selection
Backcross method
Pedigree method
Bulk method
Single seed descent method
Mutation breeding
Singh (1991) reviewed the mutation breeding work on pulse crops. Mutation breeding helped in developing improved varieties in various pulse crops such as chickpea (Pusa 408, Pusa 413, Pusa 417 and Kiran), Lentil (Arun), field pea (Hansh), Pigeonpea (Co 5, Co 3, TAT 5 and Trombay), blackgram (Co 4), greengram (Co 4, ML26-1-3, Pant mung 2 and TAP7) and cowpea (V 16, V 3, V 38 and V 240. Standard breeding procedures viz., the pedigree, bulk, backcross and their modifications as applicable to any self-pollinated crop are utilized for chickpea improvement. Lal et al. (1973) emphasized the significance of pedigree method in comparing different procedures. Singh (1987) has suggested the usefulness of different breeding methods as follows (i) pedigree method for resistance breeding (disease, insect, nematode) (ii) modified bulk method for stress situations (drought, cold, heat, iron deficiency). (iii) backcross method for interspecific hybridization. (iv) limited backcross (one or two for desi x kabuli introgression and also for resistance breeding. The major breeding procedures applicable to pigeonpea are as follows (Laxman Singh et al., 1990). Pedigree selection has been useful in the traits such as sterility mosaic disease resistance, seed size, seed colour, growth habit, and number of seeds/pod. For yield improvement bulk hybrid advance by single seed descent appears to be better procedure. Mutation breeding. This has been
59 found successful in creating useful variability in pigeonpea. Pawar et al. (1984) developed a mutant named as T 6 from irradiation of T 21 with gamma rays at Bhabha Atomic Research Centre, Trombay, Mumbai. T 6 has been released for cultivation due to larger seed and higher yield than T 21. Heterosis breeding. There are reports of substantial amount of nonadditive genetic variance and heterosis for seed yield in pigeonpea. The discovery of genetic male sterility (Reddy et al., 1978) and reports on extent of natural crossing being as high as 70 per cent have helped producing pigeonpea hybrids on commercial scale. The genetic male sterile plants are identified by translucent anthers. Use of this technique requires rouging of 50 per cent normal fertile plants from the female rows in hybrid seed production block at flowering. This has been put to commercial use where one fertile pollinator parent is planted after every six male sterile rows (Saxena et al., 1986). The details of hybrid seed production in pigeonpea using genetic male sterility are as follows (Saxena and Ariyanayagam, 1991). Table 10: Recommended varieties of pulses in Bihar Varieties
Source
Maturity (days)
Yield (q/ha)
Remarks
Pigonpea Bahar
RAU, Dholi
230-240
20-25
Resist. to sterility
Pusa9
IARI, Pusa
230-250
22-26
Resist. to sterility
NDA-1
NDAUT, Faizabad 250-260
22-25
Resist. to sterility & Wilt
MAL-13
BHU, Varanasi
20-22
Resist. to sterility & Wilt
230-240 Urad
PU-19
Pantnagar
80-85
8-9
Tolerant to YMV
PU-31
Pantnagar
75-80
10-12
Tolerant to YMV
PU-35
Pantnagar
70-80
08-10
Tolerant to YMV
Moong PM-4 & 5
Pantnagar( 1997)
70-80
12-14
Tolerant to YMV, All Season
Samrat
IIPR (2001)
62-75
10-12
Tolt. to YMV, Mature at time
IPM 2-3
IIPR (2009)
70-80
10
Tolt. to YMV, All Season
HUM-1
BHU,(2001)
75-80
09-11
Tolerant to YMV
Pusa Vishal
IARI,(2000)
70-75
11-12
Tolt. to YMV, Mature at time
60
Lentil HUL-57
BHU,(2005)
112-120
14
Small seeded, rust resistant.
Arun
RAU, Dholi
125-130
20-22
Medium seeded, Tol. to Wilt & Root rot
NDL-1
NDAUT, Faizabad 115-125
15
Resist. to rust & Tol. To Wilt
IPL406
IIPR (2007)
17
Large seeded, rust resistant.
130-140 Chickpea
GCP105
JAU, Junagadh (2000)
130-145
18-20
Medium tall, SE, Tol. To Wilt & Stunt, 17gm/100 seed wt.,
DCP92-3
IIPR, Kanpur (1998)
145-150
19-20
Medium tall, SE, Tol. To Wilt & lodging, 17gm/100 seed wt., Suitable for high fertility & excessive moisture condition
KWR108
Kanpur (1996)
130-145
20-22
Resist. to wilt.
BG372
IARI, (1993)
130-135
15-20
Small seeded (12gm/100 seed wt.), Mod. Resi. To wilt, blight, root rot & tol. To pod borer
PG186
Pantnagar (1997)
125-135
18-20
Small seeded (14gm/100 seed wt.), Mod. Resi. To wilt, root rot & tol. To pod borer
2. MOLECULAR METHOD OF PULSES IMPROVEMENT The incredible advances in biotechnology demonstrably hold great promise for crop improvement. For instance, molecular breeding, the integration of molecular biology techniques in plant breeding, through enhanced efficiencies, has great potentials for changing permanently the science and art of plant breeding. Molecular breeding encompasses both the use of distinguishing molecular profiles to select breeding materials and the applications of recombinant deoxyribonucleic acid (DNA) methods that is genetic transformation, to add value to PGRFA. There are also a number of other emerging molecular biology-based techniques that hold promise for enhancing the efficiency levels of plant breeding activities. We provide some overview of the use of these technologies and techniques in developing novel crop varieties.
61 2.1 Marker Assisted Breeding The exploitation of factors co-segregating with a trait in a simple Mendelian fashion in order to understand its inheritance is an old notion, but these simply inherited morpho-physiological variants are very rare1 (Bergal and Friedberg 1940). They remained of restricted use for practical breeding purposes until the development of biochemical markers in the 1960s (Koebner 2003). However, it was not until the introduction of DNA marker technology in the 1980s, that a large enough number of environmentally insensitive genetic markers could be generated. Restriction fragment length polymorphisms (RFLPs) were the first DNA markers to be suc cessfully used in plants (Helentjaris et al. 1985). However, as these markers are time-consuming, labour-intensive and require large amounts of DNA, their use was gradually supplanted by more user-friendly techniques (Gupta et al. 1999). Indeed, the development of the polymerase chain reaction (PCR, Saiki et al. 1988) hasmade DNA markertechniques quicker and cheaper. Several PCR-based markers such as random amplified polymorphic DNAs (RAPDs), amplified fragment length polymorphisms (AFLPs), simple sequence repeats (SSRs or microsatellites), inter SSRs (ISSRs) and most lately single nucleotide polymorphism (SNP) have been developed and applied to a wide range of crop species including cereals. Under the past decades, the molecular marker technology has rapidly evolved into a valuable tool able to dramatically enhance the efficiency of conventional plant breeding (Peleman and van der Voort 2003). Its various uses, as exemplified in the different chapters of this edition, have given modern crop improvement new potentials unthinkable until recently i.e. association mapping, induced mutagenesis, Tilling etc. Simultaneously as biotechnology produces these efficient tools to assist plant breeders in their enterprise, it also provides them with new possibilities of gene transfer. To breed and/or distinguish genetically modified (GM) individuals, may not differ much from other traits, however, molecular markers is the only technique available capable of differentiating GM transformation-events. The increasing insight provided by the genomics era presents wider possibilities to compare gene structure and function in divergent organisms. Comparative mapping allows the transfer of information among orthologous genes or homologous chromosomes. This is not only useful for better mapping, gene cloning and characterization but also for marker discovery (Sorrells and William, 1997) and the comprehension of the processes’ underlying genetics. Marker aided selection is a tool for breeding, wherein genetic marker(s) tightly linked with the desired trait/gene(s) are utilized for indirect
62 selection for that trait in segregating/non-segregating generations. In its simplest form it can be applied to replace evaluation of a trait that is difficult or expensive to evaluate. When a marker is found that co-segregates with a major gene for an important trait, it may be easier and cheaper to screen for the presence of the marker allele linked to the gene, than to evaluate the trait. From time to time the linkage between the marker and the gene should then be verified. When more complex, polygenic controlled traits are concerned, the breeder is faced with the problem how to combine as many as possible beneficiary alleles for the QTLs that were detected. In this case, the breeding material can be screened for markers that are linked to QTLs. Based on such an analysis specific crosses can be devised for creation of an optimal genotype by combining QTL alleles from different sources. Marker assisted selection, when applied within the current breeding material to enhance a breeding programme, does not solve the problem of limited genetic variability that is often seen in breeding stocks. A different application of marker assisted selection could contribute to a genetic enrichment of breeding material. Marker assisted selection may be used to facilitate a controlled inflow of new genetic material. The wild species often carries desired components that may be missing in cultivated material. Such components can be transferred to elite cultivated material by repeated backcrossing. However, breeders are often reluctant to apply this method because of unpredictable linkage drag. These are caused by other genes, which are unintentionally transferred along with the genes that control the target trait. It may take considerable effort and screening to get rid of the unwanted genes and return the material to an acceptable agronomic value. Markers can be used to pinpoint the genetic factors that are responsible for the desired characteristics in the unadapted material. In a backcross programme, the presence of the desired QTL alleles can be verified continuously by observing linked markers. 2.2 Applications of molecular markers in plant breeding Modern plant breeding is not only based on genotype-building but also on manipulating variation within gene-pools of a cross. DNAfingerprinting of breeding lines using molecular markers, as well as detailed genome analysis of plants, provides in this aspect a very powerful and efficient tool to characterize, monitor and protect germplasms (Lombard et al. 2000). When molecular markers are available, conveniently co segregating with candidate genes, marker-assisted selection (MAS) or marker-aided selection may improve the efficiency of selections of simple traits in conventional plant breeding programs (Knapp, 1998; Podlich et al., 2004). Broadly, molecular markers are applied in plant breeding in the
63 following areas: 1. To screen for useful single gene traits e.g. disease resistance. This may facilitate the introgression of new genes from a non-adapted parent and in pyramiding desired alleles into enhanced lines of candidate cultivars. 2. To accelerate backcross breeding programs through identification of the gene of interest and to eliminate the undesirable genome of the donor parent. Unlike conventional backcrossing, this method reduces linkage drag and requires few numbers of repeated backcrosses to recover the genotype of the recurrent parent. 3. To characterize diverse germplasm and establish heterotic patterns. Markers are useful to determine the magnitude of genetic diversity for crop improvement and to assign exotic (or non-adapted) germplasm into an appropriate breeding pool. In inbred lines markers assist in establishing heterotic patterns in order to guide the selection of parents for use in a hybrid breeding program. Marker information may be used in combination with phenotypic and pedigree analyses to ascertain genetic differences between lines of different heterotic groups to enable the breeder to predict the performance of hybrids to be developed from different intergroup crosses (Xiao et al., 1996) 4. To identify and protect commercial cultivars through fingerprinting. The MAS approach is not only a tool of speeding up the process of gene transfer, but also allows pyramiding of desirable genes and QTLs from different genetic back grounds. Gene pyramiding refers to the introgression of several characters at one time, a method that may enhance the durability or degree of pest and disease resistances. When a cultivar is protected by one major gene it is difficult to intro gress additional genes conferring resistance to the same disease because of the difficulty of their discrimination in phenotypic screenings (since the plant already shows resistance). However, by tagging several genes with closely linked molecular markers, MAS strategies facilitate the development of lines with stacked resistance genes, giving the cultivar more durable protection than that afforded by a single resistance gene. Also, genes controlling resistance to different races or biotypes of a pest or pathogen, or genes contributing to agronomic or seed quality traits can be pyramided together to maximize the benefit of MAS through simultaneous introgression (Dwivedi et al. 2007).
64 2.3 Transgenics approach Genetic transformation offers direct access to a vast pool of useful genes not previously accessible to plant breeders. Current genetic engineering techniques allow the simultaneous use of several desirable genes in a single event, thus allowing coordinated approaches to the introduction of novel genes/traits into the elite background. The priorities for applied transgenic research are similar to those of conventional plant breeding, aiming to selectively alter, add or remove a specific character in order to address regional con straints to productivity. Genetic engineering also offers the possibility of introducing a desirable character from closelyrelated plants without associated deleterious genes or from related species, which do not readily cross with the crop of interest or from completely unrelated species even in other taxonomic phyla. In many species, the development of rapid, highly efficient, and routine transformation systems is still in progress and thus represents a bottleneck in the development of stable high yielding transgenic plants. Development and deployment of transgenic plants in an effective manner is an important pre-requisite for sustainable and economic use of biotechnology for crop improvement. As a result of advances in genetic transformation and gene expression during the last decade, there has been rapid progress in using genetic engineering for crop improvement in terms of herbicide tolerance, pest resistance, and male-sterility systems. The potential of this technology has now been widely recognized and extensively adopted in the plant breeding of temperate crops. Various DNA delivery methods can be used to re-introduce a plasmid DNA with a desirable gene into a target line. Double-stranded DNA is a good substrate for recom binase of a site-specific recombination system. With the use of PEG-mediated protoplast transformation (Mathusr and Koncz, 1998) and biolistic-mediated transformation (Srivastava et al. 2004), where a large amount of double-stranded DNA is introduced into a plant cell, the successful production of targeted transgenic plants has been reported. Meanwhile, with the use of Agrobacterium-mediated transformation (Vergunst and Hooykaas 1998; Vergunst et al. 1998), where a small amount of linear single stranded T-DNA is introduced into a plant cell, very few targeted transgenic plants have been reported. Thus, T-DNA was believed to be a poor substrate for recombinase in a site-specific recombination system. With the current transformation methods, a desired gene is randomly integrated into a plant genome. Since the proportion of targeted
65 transgenic plants to randomly into grated ones is extremely low, the development of highly efficient selection methods has become a major issue. To overcome this problem, a strategy in which a selection marker gene can be activated by introducing a desirable gene into a target site is widely used (Albert et al. 1995; Vergunst and Hooykaas 1998; Vergunst et al. 1998; Day et al. 2000; Choi et al. 2000; Srivastava and Ow 2002; Srivastava et al. 2004; Chawla et al. 2006; Louwerse et al. 2007). In sitespecific integration methods, the target line has a transgene where a recognition sequence is placed between the promoter and coding sequences.
Strategies for development of disease resistance pulses through molecular approaches 1. Identification of disease resistance genes through molecular markers in pulses DNA based markers have shown great promises in expediting plant breeding methods. The identification of molecular markers closely linked with resistance genes would facilitate expeditious pyramiding of major genes into elite background, making it more cost effective. Once the resistance genes are tagged with molecular marker the selection of resistant plant in the segregating generations becomes easy (Datta et al., 2011). Major success of identification of disease resistance genes is observed in chickpea and pigeonpea due to availability of genomic resources but still minor pulses like lentil, mungbean and Urad bean needs an attention. A chickpea linkage map was established with help of 354 molecular surveyed among 130 recombinant inbred lines derived from a C. arietinum × C. reticulatum (Winter et al. 2000). DNA markers associated with two closely linked genes for resistance to fusarium wilt race 4 and 5 in chickpea were also identified from a population of 131 recombinant inbred lines derived from a wide cross between Cicer arietinum and Cicer reticulatum (BenkoIseppon et al. 2003). These markers will pave the way for MAS and searching other useful genes. Gowda et al. (2009) identified flanking markers for chickpea fusarium wilt resistance genes in a RIL population. Reddy et al., (2009) performed bulk segregant analysis on a segregating population of ICPL 7035 x ICPL 8863 for identification of RAPD markers associated with pigeonpea sterility mosaic disease resistance. Dhanasekhar et al. (2010) identified two RAPD markers OPF04700 and OPA091375 were linked with the open and tall plant type gene in pigeonpea F2 population of the cross between TT44- 4 and TDI2004-1through bulk
66 segregant analyses . Kotresh et al. (2006) used bulk segregant analysis with 39 RAPD primers which led to identification of two markers (OPM03 704 and OPAC11 500) that were associated with Fusarium wilt susceptibility allele in a pigeonpea F2 population derived from GS1 x ICPL87119. Saxena (2010) assessed the DNA polymorphism in a set of 32 pigeonpea lines screened with 30 SSR markers. Based on polymorphism of marker alleles, higher genetic dissimilarity coefficient and phenotypic diversity for Fusarium wilt and sterility mosaic disease resistance data, five parental combinations were identified for developing genetically diverse mapping populations suitable for the development tightly linked markers for Fusarium wilt and sterility mosaic disease resistance. Tullu et al. (2003) tagged anthracnose resistance gene LCt-2 of lentil cultivar PI 320937 with RAPD and AFLP markers. Taran et al. (2003) identified two molecular markers associated with Ascochyta blight resistance in lentil viz., UBC 2271290 linked with ral1 gene and RB18680 linked with AbR1 and a marker (OPO61250) linked with Anthracnose resistance gene were utilized for identifying lines that possessed pyramided genes in a population of 156 RILs developed from a cross between ‘CDC Robin’ and a breeding line ‘964a-46’. These markers can be converted into more robust SCAR markers for routine use in marker assisted selection. Basak et al. (2004) developed molecular marker linked to yellow mosaic virus (YMV) resistance gene in Vigna sp. from a population segregating for YMV disease resistance. Maiti et al. (2010) identified molecular markers CYR1and YR4 in a F2 population for screening of MYMIV resistance genes. CYR1 cosegregated with MYMV resistance gene in F2 plants and F3 progenies. These two markers can be used simultaneously with the help of a multiplex PCR reaction. Nguyen et al. (2001) converted a RAPD marker into a SCAR (SCARW19) for selecting ascochyta blight resistance gene of lentil accession ILL5588. Rubeena et al. (2003) identified QTLs for ascochyta blight resistance in lentil. Further validation is required to use these markers for MAS. Hamwieh et al. (2005) mapped microsatellite markers identified from a genomic library of lentil. The linkage spanning about 751cM, consisting of 283 marker loci was derived from 86 recombinant inbred lines derived from the cross ILL 5588 × L 692-16-1(s) using 41 microsatellite and 45 amplified fragment length polymorphism markers. The average marker distance was 2.6 cM. Two flanking markers (SSR marker SSR59-2B at 8.0 cM and AFLP marker p17m30710 at 3.5 cM) were linked with fusarium resistance (Table 11).
67 Table 11- Trait mapping in pulses for biotic stress Trait
Gene/QTL
Source Chickpea
Fusarium wilt
foc-0,foc-1,foc-2,foc-3,foc-4, foc-5,QTL
Varshney et al., 2015
Ascochyta blight
QTL
Varshney et al., 2015
Rust
QTL
Varshney et al., 2015
Botrytis gray mold
QTL
Varshney et al., 2015 Pigeonpea
Fusarium wilt
BSA
Varshney et al., 2015
SMD resistance
QTL, BSA
Varshney et al., 2015
Fusarium wilt
QTL
Hamweih et al., 2005
Aschochyta Blight
QTL
Rubeena et al., 2006
Lentil
2. Marker assisted backcross breeding A backcross breeding programme is aimed at gene introgression from a “donor” line into the genomic background of a “recipient” line. The potential utilization of molecular markers in such programmes has received considerable attention in the recent past. Markers can be used to assess the presence of the introgressed gene (“foreground selection”) when direct phenotypic evaluation is not possible, or too expensive, or only possible late in the development. Markers can also be used to accelerate the return to the recipient parent genotype at other loci (“background selection”). It is assumed that the introgressed gene can be detected without ambiguity, and the theoretical study was restricted to background selection only. The use of molecular markers for background selection in backcross programmes has been tested experimentally and proved to be very efficient. Introgressing the favourable allele of QTL by recurrent backcrossing can be a powerful mean to improve the economic value of a line, provided the expression of the gene is not reduced in the recipient genomic background. Yet, recent results show that for many traits of economic importance QTLs have rather small effects. In this case, the economic improvement resulting from the introgression of the favourable allele at a single QTL may not be competitive when compared with the improvement resulting from conventional breeding methods over the same duration. Marker assisted introgression of superior QTL alleles can then compete with classical
68 phenotypic selection only if several QTLs could be manipulated (Datta et al., 2011). Efforts are being made to introgress resistance to different races independently as well as pyramiding of resistance to two races for Fusarium Wilt in some elite varieties in India. ICRISAT (India) is pyramiding resistances for Foc1 and Foc3 from WR 315 and 2 QTLs for Ascochyta blight (AB) resistance from ILC 3279 line into C 214 (Varshney et al., 2013). 3. Genomics Assisted Breeding Approach The advent of markers based on simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) and the availability of highthroughput (HTP) genotyping platforms have further accelerated the generation of dense genetic linkage maps and the routine use of the markers for marker-assisted breeding in several crops (Collard and Mackill, 2008). However, despite the routine use of markers for genome-wide profiling and trait-specific marker-assisted selection (MAS), breeding of crops with many traits of interest such as yield, improved nutritive value and resistance to several biotic and abiotic stresses is still a challenge due to complex inheritance of these traits. Plant genomics has enormous potential to revolutionize crop improvement by providing extensive knowledge from the analysis of genomes which in turn can be used for rapid and efficient plant breeding towards crop improvement (Kumptala et al., 2012). The advent of NGS technologies has changed the dynamics and the pace of genomic research in pulses against biotic stresses because of their rapid, inexpensive and highly accurate sequencing capabilities. Unlike Sanger sequencing method which depends upon capillary electrophoresis, these NGS technologies are highly dependent on massive parallel sequencing, high resolution imaging, and complex algorithms to deconvolute the signal data to generate sequence data. NGS technologies offer a wide variety of applications such as whole genome de novo and resequencing, transcriptome sequencing (RNA-seq), microRNA sequencing, amplicon sequencing, targeted sequencing, chromatin immune precipitated DNA sequencing (ChIP-seq), methylome sequencing etc (Varshney et al., 2015). To facilitate crop improvement, NGS and other accessory technologies can be used for whole genome sequencing, transcriptome sequencing, genome wide and candidate gene marker development, targeted enrichment and sequencing and other applications. These NGS technologies even hold promise for a methodological leap towards genotyping by sequencing (GBS) and genetic mapping applications. Analysis of NGS
69 data from genome wide association studies, transcriptomics and epigenomics in combination with data from proteomics, metabolomics and other ‘omics’ can provide an integrative systems biology approach to understand the regulation of complex traits (Table 12). Table 12- Availability of molecular markers in pulses (Varshney et al., 2013) Chickpea
Pigeonpea
Lentil
Mung bean
Urad bean
Species name Cicerarietinum
Cajanuscajan
Lensculinaris
Vigna Radiata
Vigna Mungo
Ploidy level
2n=2×=22
2n=2×=20
2n=2× =22
2n=2× =22
Genome size 740 Mbp
833.07 Mbp
4063Mbp
579 Mbp
574Mbp
SSR markers ~2000
~4000
~200
~300
~100
SNP markers 9000
10000
—
—
—
2n=2×=16
4. Transgenics Approaches Challenges of food security has directed scientific communnity towards gene revolution which involves direct modification of traits in an organism by transferring desired genes using transgenic approach/ genetic transformation’. In contrast to classical breeding, genetic engineering offers an excellent tool for incorporating gene(s) of unrelated organisms into plant cells. These processes take less time thus accelerating the process of genetic improvement of crop plants. In addition, this exciting technology also allows access to an unlimited gene pool without the constraint of sexual compatibility (Ortiz, 1998). Pulse crops engineered to suit the environment better through incorporation of genes for tolerance to biotic and abiotic stresses have been suggested to represent an improvement in crop production. Over the past few decades, breeding possibilities have been broadened by genetic engineering and gene transfer technologies, as well as by gene mapping and identification of the genome sequences of model plants and crops which resulted in efficient transformation and generation of transgenic lines in a number of crop species (Sanghera et al., 2011; Gosal et al. 2009). The presence of very rigid and thick cell walls coupled with efficient DNA repair systems has made the process of genetic transformation of some legume crops quite challenging. Despite these difficulties, it is possible to transform many pulse crops, such as chickpea and pigeonpea, provided that an efficient protocol of in-vitro regeneration is available.
70 Legume transformation has been mainly performed by Agrobacterium tumefaciens but in some cases, especially in common bean, micro-particle bombardment has been used to deliver exogenous DNA to embryogenic or organogenic tissues (Chandra and Pental, 2003). All these efforts toward improving in vitro regeneration and transformation procedures allow the creation of transgenic lines with improved resistance to pests and diseases mainly in chickpea and pigeonpea. One of the main transformation strategies was the introduction of derivatives of cry1 genes from Bacillus thuringiensis conferring resistance to many pod borer insects. To improve the efficiency of resistance, the strategy has moved from the transfer of a single cry1 gene to constructs containing cry genes with different modes of action. This strategy has worked well for corn and cotton to increase the durability of the resistance and is especially important for insect resistance management (Acharjee and Sarmah, 2011 ). The first successful genetic transformation of nuclear genome of chickpea was reported in 1997 using the cry1Ac gene (Kar et al., 1997). Subsequently, various research groups within India initiated genetic transformation of chickpea using Cry1Ac gene and reported generation of transgenic chickpeas (Sanyal et al., 2005, Indurker et al., 2007; Mehrotra et al., 2011). A second gene, Cry2Aa, was also introduced in chickpea to facilitate pyramiding with existing Cry1Ac lines (Acharjee et al., 2010). Mehrotra et al. (2011) generated pyramided Cry1Ac and Cry1Ab gene chickpea; however, pyramiding two or more genes with different mode of action is preferred. Transgenic pigeon pea consisting chimeric cry1AcF (encoding cry1Ac and cry1F domains) gene and its resistance towards Helicoverpa armigera were also observed by Ramu et al. (2011). Conclusions India needs around 32 million tons of pulses by 2030, to feed the estimated population of about 1.68 billion. Global supply of pulses is limited, as India happens to be the largest producer and consumer of pulses. Hence, India needs to produce the required quantity, but also remain competitive to protect indigenous pulses production. Improved technologies (improved, high yielding varieties and appropriate crop management practices) are available. However, a concerted effort by farmers, researchers, development agencies, and government are needed to ensure that India becomes self-sufficient in pulses in the next 5-10 years. The recent efforts and programs initiated by the government are bearing fruits, and it is hoped that this momentum is sustained and strengthened to make India self-
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78 Rubyogo, J.C., Magreta, R., Kambewa, D., Chirwa, R. and Mazuma, E. (2011). Private Public Partnership in Bean Seed Delivery: Experience from Malawi. African Crop Science Journal (in press). Rubyogo, J.C., Sperling, L. and Assefa, T. (2007). A new Approach for facilitating farmers access to bean seed. LEISA Magazine 23: 27-29. Sanghera, G.S., Wani, S.H.K.B. and Nicolas, R. (2015). Achievements and Challenges in Legume Breeding for Pest and Disease Resistance, Critical Reviews in Plant Sciences, 34:1-3, 195-236, DOI: 10.1080, Singh G., Kashyap P. L. and Singh N. B., 2011, Designing Crop Plants for Biotic Stresses using Transgenic Approach. Vegetos, 24 (1) : 1-25. Sanyal, I., Singh, A.K., Kaushik, M. and Amla, D.V. (2005). Agrobacterium mediated transformation of Chickpea (Cicer arietinum l.) with Bacillus thuringiensis cry1Ac gene for resistance against podborer insect, Helicoverpa armigera. Plant Sci., 168:1135-1146. Saxena, R.K., Saxena, K.B., Kumar, R.V., Hoisington, D.A. and Varshney, R.K. (2010). SSR-based diversity in elite pigeonpea genotypes for developing mapping populations to map resistance to Fusarium wilt and sterility mosaic disease, Plant Breed 129: 135–141. Saxena, K.B., Srivastava, D.P. and Tikka, S.B.S. (2000). “Breaking Yield Barriers in Pigeonpea through Hybrid Breeding” in M Ali, A N Asthana, Y S Rathore, S N Gurha, S K Chaturvedi and S Gupta (ed.), Advances in Management of Biotic and Abiotic Stresses in Pulse Crops, pp 211-19. Saxena, N.P. and Johansen, C. (1990). “Realised Yield Potential in Chickpea and Physiological Considerations for Further Genetic Improvement” in S K Sinha, P V Sane, S C Bhargava and P K Aggarwal (ed.), Proceedings of the International Congress of Plant Physiology, 1: 279-88. Sheridan, G.E.C., Masters, C.I., Shallcross, J.A. and Mackey, B.M. (1998). Detection of mRNA by reverse transcription-PCR as an indicator of viability in Escherichia coli cells. Applied Environmental Microbiology, 64(4): 1313–1318. Singh, A.K., Bhatt, B.P., Sundaram, P.K., Chndra, N., Bharati, R.C. and Patel, S.K. (2012a). Faba bean (Viciafaba L.) phenology and performance in response to its seed size class and planting depth. Int. J. of Agril.& Stat. Sci. 8 (1): 97-109. Singh, A.K., Kumar, P. and Chandra, N. (2013e). Studies on yield production of mung bean (Vigna radiate) sown at different dates. J. Environ. Biol. 34: 1007-11. Singh, D., Kumar, A., Singh, A.K. and Tripathi, H.S. (2013g). Severity of chickpea wilt in north Bihar and nutritional studies on fusarium oxysporum f.sp. Ciceri J.. Plant Disease Sci. 8(2):137-40.6
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3 DEVELOPMENT OF A UNIQUE RHIZOSPHERIC SOLID ORGANIC SUPPLEMENT FOR BETTER CROP PRODUCTIVITY AND BIO-CONTROL OF PATHOGENS Noyonika Mukherjee, Anjali Yadav, Anindita Bhattacharya, Arup Kumar Mitra and Fr. S Xavier S.J. P.G. Department of Microbiology, St. Xavier’s College (Autonomous), Kolkata - 700 016
ABSTRACT The deleterious effects of chemical fertilizers and pesticides used in agriculture calls for immediate change in focus to organic means of farming. The present study deals with the production of a novel solid carrier-based bio-fertilizer using saw dust and charcoal as carriers, and wheat bran and oil cake as nutritive sources for the production of a potent bioinoculant. Nitrogen fixing Azotobacter sp., phosphate and potassium solubilizing Bacillus subtilis, phosphate solubilizing Bacillus megaterium, and bio-control properties of Pseudomonas fluorescens makes the consortium a perfect mixture catering to all needs of the crop. The biofertilizer was applied into pots containing 3 weeks old saplings of Chili plants, and plant growth and yield parameters were compared to plants growing in control soil and soil supplemented with vermicompost, one of the most common organic supplement used in agriculture. Plants supplemented with the biofertilizer showed 46% increase in shoot length, 52% increase in internodal length, 32% increase in leaf lamina length and 37% increase in total chlorophyll content compared to the control plants. Also, the yield of the biofertilizer-supplemented plants were greatly improved; 1.5 fold increase in number of buds and 2.3 fold increase in number of chilies were recorded. Moreover, the biofertilizer supplemented plants showed increased lateral growth, increased tolerance to adverse
80 weather conditions and resistance to soft rot causing microorganisms. In addition, they appeared more sturdy with stronger stems compared to the control plants. The viability of the bioinoculants in the prepared biofertilizer was checked upto 4 months and all the bioinoculants were found to be viable. Thus, the solid biofertilizer was found to have a high shelf life and itnot only improves plant growth and yield by maintaining the NPK ratio of soil and producing several growth-promoting hormones, but can also combat the common problem of pests associated with most biofertilizers, owing to its unique biocontrol properties. Keywords: Biofertilizer, biocontrol agent, Bacillus subtilis, Bacillus megaterium, Azotobacter sp., Pseudomonas fluorescens, saw dust, charcoal, wheat bran, oil cake.
1. INTRODUCTION: Biofertilizersare not true fertilizers, in the sense that they do not directly supply nutrition to plants. They are carrier-based ready-to-use microbial inoculants (bacteria or fungi), which on application to plants or soil, promotes plant growth by increasing availability of various essential nutrients to host plants by their unique biological activities. Biofertilizers are becoming increasingly popular in recent times to counter the negative impact of indiscriminate use of chemical fertilizers on environment. The immediate action and low cost of chemical fertilizers resulted in their widespread acceptance and use in agriculture. However, long term use of chemical fertilizers can lead to harmful consequences like mineral depletion and acidification of soil, destruction of beneficial microbial ecosystems, decline in soil fertility, and also groundwater contamination, and thus pose a serious threat to human health and environment. This led to concern for reviving the soil health and use of alternate sources of fertilizers. Thus came the concept of biofertilizers, a eco-friendly approach to improve soil fertility and crop yields. The microorganisms in biofertilizers restore the natural nutrient cycle of soil and promote production of soil organic matter. Therefore, they can be used to improve plant growth and yields, while enhancing the sustainability and health of soil. Biofertilizers act to improve crop yields in several ways. They fix atmospheric nitrogen in the soil and also solubilize the insoluble forms of phosphates like tricalcium, iron and aluminium phosphates into available forms. They also decompose organic matter and help in mineralization in soil. They produce plant growth hormones and also may produce antimicrobial substances to inhibit the growth of potential plant pathogens. They are also known to increase resistance of plants against deleterious plant pathogens through induction of induced systemic resistance (ISR).
81 However, there are several drawbacks of biofertilizers, which limit their widespread acceptance in agriculture. The major limitations are short shelf lives and contamination of the carrier medium by undesirable microorganisms, which hampers its effectiveness. Moreover, they are not effective if the moisture content, pH and temperature of the soil is not adequate. Also, their slow response is the reason why biofertilizers are not readily accepted in agriculture. 2. OBJECTIVE: The objective of the present study was to demonstrate the production and efficacy of a solid carrier-based biofertilizer using saw dust and charcoal as carriers, and wheat bran and oil cake as nutritive sources for the production of a potent bioinoculant consisting of nitrogen fixing Azotobacter sp., phosphate and potassium solubilising Bacillus subtilis, phosphate solubilising Bacillus megaterium, and bio-control properties of Pseudomonas fluorescens. Also, the purpose was to monitor and evaluate the shelf life andeffect of the prepared solid biofertilizer on Chili plants and to draw a comparison between the various plant growth and yeild parameters of control plants, plants supplemented with commonly used vermicompost and plants supplemented with the prepared solid biofertilizer. 3. MATERIALS AND METHODS: 3.1. Isolation and purification of microorganisms having biofertilizing properties: Bacillus subtulis and Bacillus megaterium cultures were already maintained in theslants and were revived from the slants itself. Pseudomonas fluorescens and Azotobacter sp, both were procured from commercially available powdered form. 1gram of powder was taken in 10ml of sterile water (stock) in separate test tubes. From this stock solution, serial dilution was done for 10-1(1ml of stock + 9ml of sterile water) and 10-2 (1ml of 10-1 + 9ml of sterile water) for both the bacteria. In order to grow Pseudomonas fluorescens and Azotobactersp., King’s B agar and Jensen’s media agar were prepared respectively. 1ml of each diluted solution (10-1 and 10-2) for both the bacteria was pipetted out in sterile petri-plates. Thereafter, 20ml of the respective media cooled to 45°C were poured onto these plates and gently swirled and allowed to solidify. The plates were then incubated at 37°C for 24 hours. Once growth appeared in the plates, six slants were prepared for each bacteria using the respective media. From the plate, a loop full of inoculum was taken and continuous streaking was done on all the slants for the purification of the respective organism. These
82 slants contained the pure culture of Pseudomonas fluorescens and Azotobacter sp.. 3.2. Characterization of microorganisms: All the four concerned bacteria were thoroughly characterized macroscopically, microscopically and biochemically. 3.2.1. C.F.U. count of bacterial cultures: AC.F.U. count of the plates in which the dilutions of the powder of each organism was poured was made using a colony counter. 3.2.2. Morphological characteristics of bacterial colonies: The morphological characteristics of the bacterial colonies including shape, size, colour, texture, margin and elevation were observed to identify the bacteria. 3.2.3. Microscopic characterization: Gram Staining: To study the microscopic characteristics of bacteria, a Gram Staining was done to determine the Gram character and shape of the bacteria. 3.2.4. Biochemical characterization: Methyl Red (MR) test - Sterilized Glucose peptone broth was inoculated with bacteria and incubated for 48 hrs at 37oC. After incubation, a few drops of methyl red solution was added and the colour change was observed. Appearance of a red colour indicates positive result. Voges-Proskauer (VP) test - Sterilized Glucose peptone broth was inoculated with bacteria and incubated for 48 hrs at 37oC. After incubation, 6% á-naphhtol and 6% sodium hydroxide were added to about 1 ml of broth culture. Positive result is indicated by formation of a red colour within 30 minutes. Citrate test - Simmons citrate agar slants were prepared and bacterial cultures were streaked and incubated for 48 hours at 37oC. A change from green to blue colour indicates positive result. 3.2.5. Interaction studies between bacteria used in the biofertilizer: To study the interaction between Bacillus subtilis and Bacillus megaterium, Pseudomonasfluorescens and Bacillus megaterium, Bacillus subtilis and Pseudomonas fluorescens, each of the organisms of the above pairs was streaked as small horizontal continuous streak in separate
83 petriplates containing nutrient agar, followed by incubation of 48 hours. After 48 hours, the other organism of each pair was streaked as small vertical continuous streak on the same plate and again incubated for 48 hours to check for positive or negative interaction between them. This type of streaking is known as T-streaking. To study interaction between Bacillus subtilis and Azotobacter sp., Pseudomonasfluorescens and Azotobacter sp., Azotobacter sp. and Bacillus megaterium, a specialized media (consisting of yeast and mannitol in agar) was used. This media is used instead of nutrient agar because Azotobacter sp. does not grow without mannitol while the other organisms need protein for their growth which is provided by yeast. A similar T-streaking was performed to check for positive or negative interaction between the organisms. 3.3. Soil Characterization: Four sets of soils were used to grow Chili plants.
Control soil (Base soil without any added supplements).
Vermicompost-supplemented soil
Solid Biofertilizer supplemented soil
Soil supplemented with both vermicompost and solid biofertilizer.
The control soil and biofertilizer-supplemented soil are studied to determine their pH, water-holding capacity and electrical conductivity. 3.3.1. Determination of pH of soil: 0.5gm of soil was mixed with 10ml of water to make a suspension. A drop of water from this suspension was put on a pH paper and colour change of the paper was matched with the standard colour index to determine the pH of the soil. 3.3.2. Determination of water holding capacity of soil: Oven-dry method was used to determine soil water holding capacity. 1 gram soil was weighed. The sample was dried in an oven at a temperature of 60° centigrade overnight. After drying, the sample was weighed again. The moisture loss was determined by subtracting the ovendry weight from the moist weight. Moisture content is expressed as a percentage of the oven-dry weight of the soil.
84 3.3.3. Determination of electrical conductivity (EC) of soil: Soil suspension was prepared by mixing 1 gm soil in 50 ml water in a 100ml beaker. The soil suspension was allowed to settle in the beaker overnight. After the calibration, the electrical conductivity cell was dipped in the supernatant of the soil suspension and the conductivity of test solution was read. EC is expressed as µS/cm. 3.4. Production of solid biofertilizer: Saw Dust, Wheat Bran, Oil cake and Charcoal were weighed, mixed in the ratio of 5:3:2:1 and autoclaved in autoclave proof packets. To this autoclaved mixture, 50ml of each bacterial culture in their respective broth were inoculated and this inoculated mixture was kept in the incubator for at least 10 days to allow the bacteria to grow. 3.5. Application of biofertizer on Chili seedlings: The solid biofertilizer was applied to potted chili plants after they attained a height of 3cm, after 3 weeks of seed sowing. The biofertilizer was added to the rhizosphere of the plants, after every 4 weeks and the results were recorded upto 4 months. 3.6. Determination of effect of biofertilizer on plant growth and yield: To study the effect of the solid biofertilizer on Chili plants, plant growth parameters including shoot length, internodal length and leaf lamina length of 10 representative plants of each set were recorded after every 4 weeks. Also, the plant yield parameters including number of buds and chilies were noted after every 4 weeks. 3.7. Chlorophyll content of leaves: Fresh chili leaves were cut into small pieces. 1 gram of leaves were weighed and made into pulp using mortar and pestle by adding 10 ml of 80% (w/v) acetone. The samples were then centrifuged at 5000 r.p.m. for 20 minutes and the supernatant was decanted into a 100 ml beaker and the volume was brought to 100ml using 80% acetone. The absorbance of the solution was read at 645 nm (for chlorophyll b) and 663 nm (for chlorophyll a) against solvent (80% acetone) blank and the chlorophyll content was calculated by standard method (Arnon, 1949). The amount of chlorophyll (mg chlorophyll per g tissue) was calculated using the following equations:
mg chlorophyll a/ g tissue =
[12.7(A663)”2.69(A645)]xV/(1000×W )
85
mg chlorophyll b/ g tissue =
[22.9(A645)”4.68(A663)]xV/(1000×W)
mg total chlorophyll /g tissue =
[20.2(A645)+8.02(A663)]×V/(1000×W) where, A = absorbance at specific wavelengths, V = final volume of chlorophyll extract and W = fresh weigh of tissue extracted. 4. RESULTS: 4.1. Characterization of microorganisms: 4.1.1. C.F.U. count of bacterial cultures: The number of visible colonies of the bacteria isolated from powdered form after overnight incubation were counted and the C.F.U. counts are presented in Table 1. Table 1: C.F.U. counts of bacteria isolated from powdered form: Organism Pseudomonas fluorescens
Azotobacter sp.
Dilution
C.F.U. count
10
-1
650 ± 10
10
-2
275 ± 6.8
10 -1
840 ± 7.5
10
-2
348±9.6
4.1.2. Morphological characteristics of bacterial colonies: The colony morphology, including shape, size, colour, texture, margin and elevation, of all the four bacteria were observed carefully and the results are presented in Table 2. 4.1.3. Microscopic and biochemical characterization of bacteria: All the bacteria used in the preparation of the biofertilizer were characterized microscopically and biochemically, and the observations are presented in Table 3. 4.1.4.Interaction studies between bacteria used in the biofertilizer: Interaction studies between bacteria, as analyzed by T-streaking method, revealed that all the concerned bacteria were interacting positively, indicating that they are compatible with each other and if used together in the biofertilizer, they will co-exist and synergistically act to improve plant
86 growth and yield. 4.2. Soil characteristics: 4.2.1. pH of soil: pH of the control as well as the biofertilizer-supplemented soil was found to be around 6-7. Thus, the pH of both the soil was suitable for the growth of Chili plants. 4.2.2. Water-holding capacity of soil: The water-holding capacity of control soil was found to be 30.4% and that of biofertilizer-supplemented soil was found to be 39.3%. Thus, the application of the biofertilizer enhanced the water-holding capacity of soil by about 9% as compared to control soil, thus making more water available for utilization by plants. 4.2.3. Electrical conductivity of soil: Electrical conductivity (EC) of control soil and biofertilizersupplemented soil was found to be 96.57 µS/cm and 263.5 µS/cm respectively. Thus, the biofertilizer was found to increase the EC of the soil, which corresponds to the increase in the amounts of soluble salts in the soil, such as salts of potassium, magnesium, phosphates, nitrates, making them available for utilization by plants. 4.3. Effect of solid biofertilizer on plant growth and yield: The effect of the prepared solid biofertilizer was recorded on the growth and yield parameters of chili plants. Remarkably, biofertilizer combined with vermicompost, and biofertilizer alone, led to significant increase in plant parameters under study, as compared to the control plants and plants supplemented solely with vermicompost. All the plant parameters under study including shoot length, internodal length, leaf lamina length, number of buds and chilies were recorded after every 4 weeks for 16 weeks. The results showing positive effect of the biofertilizer on shoot length and internodal length are presented in Figure (1a) and (1b) respectively. Figure (2 a) shows the enhancement of leaf lamina lengths in the plants supplemented with the solid biofertilizer. At the end of the sixteenth week, the plants growing in biofertilizer-supplemented soil showed 46% increase in shoot length, 52% increase in internodal length and 32% increase in leaf lamina length compared to control plants. Also, an assay to determine chlorophyll content was done using chili leaves from all four set of plants under study. Increased chlorophyll content was found in plants
87 supplemented with biofertilizer, as compared to the control and vermicompost-supplemented ones. The plants supplemented with biofertilizer showed 25% increase in chlorophyll a content, 53% increase in chlorophyll b content and 37% increase in total chlorophyll content, as Table 2: Morphological characteristics of bacterial colonies: Organisms
Shape
Size (mm)
Colour
Texture
Margin
B. subtilis
Irregular
2-7
Dull white
Dry
Undulated Umbonate
B. megaterium
Circular
2-4
White
Opaque
Entire
P. fluorescens
Circular
1.5-5
Creamy white Slimy
Undulated Convex
3-8
Creamy white Opaque
Entire
Azotobacter sp. Irregular
Elevation
Convex
Raised
Table 3: Microscopic and biochemical characteristics of bacteria: Organisms
Microscopic characteristics Biochemical characteristics Gram character
Shape
Methyl Red Test
VogesProskauer test
Citrate test
B. subtilis
+
rod-shaped
-
+
+
B. megaterium
+
rod-shaped
-
-
+
P. fluorescens
-
rod-shaped
-
-
+
Azotobactersp.
-
oval-shaped
+
-
+
Figure 1: Variation in (a) shoot length and (b) internodal length of plants growing in control soil (cyan), vermicompost-supplemented soil (red), solid biofertilizer supplemented soil (green) and soil supplemented with both vermicompost and solid biofertilizer (blue).
88
Figure 2: Variation in (a) leaf lamina length and (b) chlorophyll content of leaves of plants growing in control soil (cyan), vermicompost-supplemented soil (red), solid biofertilizer supplemented soil (green) and soil supplemented with both vermicompost and solid biofertilizer (blue).
Figure 3:Variation in appearance of (a) no. of buds and (b) no. of chiliesin plants growing in control soil (cyan), vermicompost-supplemented soil (red), solid biofertilizer supplemented soil (green) and soil supplemented with both vermicompost and solid biofertilizer (blue).
89
Figure 4: Pictures of representative plants growing in control soil, bio-fertilizer supplemented soil and soil supplemented with both vermicompost and biofertilizer.
shown in Figure (2b). Also, the biofertilizer-supplemented plants showed enhancement in their yield; the number of buds and chilies appearing on these plants were 1.5 fold and 2.3 fold higher respectively, as compared to the control plants, as presented in Figure (3a) and (3 b) respectively. Representative pictures of plants growing in control soil, bio-fertilizer supplemented soil and soil supplemented with both vermicompost and biofertilizer are presented in Figure (4). Moreover, higher number of leaves and lateral growth was found in biofertilizer-supplemented plants. In addition, these plants appeared much sturdy with stronger stems and were more tolerant to adverse conditions compared to the control plants. Also, most of the control plants were affected by soft rot, mostly caused by species of gram-negative bacteria, Erwinia, Pectobacterium, and Pseudomonas, but none of the plants supplemented with the solid
90 biofertilizer were affected with the disease, indicating that the biofertilizer also has biocontrol properties. 5.DISCUSSION: The present study summarizes the effect of the novel solid carrierbased bifertilizer on the growth and yield of Chili plants. The positive effects of the biofertilizer are reflected in terms of increase in shoot length, internodal length, leaf lamina length, chlorophyll content as well as the overall yield of the plants. The enhanced growth and yield of the biofertilizer-supplemented plants may be attributed to several direct and indirect mechanisms of the bacterial inoculants. Some bacterial inoculants promote plant growth by direct mechanisms including atmospheric nitrogen fixation, phosphate solubilization, phytohormone (like auxins, cytokinins, gibberellins) production, siderophore (iron chelating agents) production,etc., where as some bacterial inoculants promote plant growth by indirect mechanisms including biocontrol of deleterious plant pathogens by antibiosis, production of antimicrobial substances like lytic enzymes or biocidal volatiles or by inducing resistance against plant pathogens (Chaurasia et al. 2005; Compant et al. 2005; Loon et al. 2004). The bacterial inoculants used in the solid biofertilizer under study promote plant growth by several of the above-mentioned mechanisms. Phosphate is one of the most essential plant nutrients and is often present in limited amounts in soil. Bacillus subtilis has been reported to have the ability to solubilize insoluble phosphates. Also, it has been reported that these bacteria produce auxins, siderophores and also antifungal âglucanase (Jong-Hui Limet al.2010). In addition, these bacteria are also claimed to be potential free-living nitrogen fixers (Sataputeet al. 2012) and potassium solubilizers (H.S. Han, 2005). Thus B. subtilis plays an essential role in maintaining the NPK ratio of the soil, which is responsible for enhanced plant growth. B. subtilis has also been reported to produce volatile compounds that play an important role in plant growth promotion and activation of plant defence mechanism by triggering the induced systemic resistance (ISR) in plants (Compant et al. 2005). Another bacteria possessing the ability to solubilize phosphate is Bacillus megaterium, considered one of the mechanisms for observed plant growth promotion (U. Chakrabortyet al. 2012). B. megaterium has also been reported to increase total chlorophyll content of leaves (Marulanda-Aguirreet al.2008). Also, these bacteria have been reported to produce antibiotics against several fungal pathogens (Jung et al.2003). Application of B. megaterium
91 in bioformulations of saw dust, rice husk and tea waste led to significant increase in growth of tea seedlings(U.Chakraborty et al. 2012). Azotobacter sp. is well-known as a nitrogen fixer. Also, these bacteria synthesize auxins, cytokinins and gibberilic acid-like substances responsible for enhanced plant growth (Sartaj A. Wani et al. 2013). Pseudomonas fluorescens are known to extensively colonize the rhizosphere, and so competition with root pathogens for nutrients and root surface colonization is proposed as one of the means of its biocontrol activities (Haas and Défago 2005). Also, these bacteria are well-known for their ability to produce antimicrobial compounds, such as, 2,4diacetylphloroglucinol (DAPG), hydrogen cyanide, phenazines, etc. In addition, P. fluorescens can act as potent elicitors of plant defence. They trigger ISR response in plants which help them to combat against broad spectrum of pathogens (Van Peer et al. 1991). Such activation of defense genes by prior application of a beneficial bioinoculant is a novel strategy of plant protection against several deleterious pathogens (Paul et al. 2003). It has been reported that P. fluorescens can also produce cytokinins (Garcia et al. 2001). These bacteria have also been reported to enhance plant biomass, leaf water potential and relative water content of plants (Sandhya et al. 2010). P. fluorescens has also been reported to possess a potent antioxidative peroxidase enzyme system, which protects plant cells from oxidative damage, thereby delaying senescence (C.A. Jaleel, 2010). Thus, these bioinoculants interact positively with each other and also with the host plants to improve crop growth and yields by their unique mechanisms, as observed in the present study. The viability of the bioinoculants in the biofertilizer was checked upto 4 months, and all the bacteria were found to be viable. The long shelf life of the biofertilizer is owing to the effective carriers and nutritive sources used in the biofertilizer. According to Moses Kolet, 2014, saw dust had been considered an excellent carrier followed by charcoal. In the present study, both sawdust and charcoal has been used as carriers to increase the viability of the microorganisms and the shelf life of the product.( Also, the use of saw dust increases the water-retention capacity of soil which prevents wilting of plants under unfavourable water conditions (A.A.Abd El Halim et al. 2014). Also, the use of wheat bran as a nutrition source serves as an alternative for peptone, decreasing the risk of fungal contamination. Thus, the bioinoculants used in the solid biofertilizer together act to maintain and enhance the NPK ratio of the soil, which is one of the
92 main reasons for improved plant growth and yield. Also, they produce several plant growth promoting substances and also antimicrobial substances against potential plant pathogens, which together act to enhance the growth of healthy plants. In addition, they have been reported to strengthen plant immunity by induction of ISR. Therefore, the solid biofertilizer under study, besides acting as an efficient biofertilizer for improving growth and yields of Chili plants, also can serve as a potent biocontrol agent. 6. CONCLUSION: The present study demonstrates the production of a unique solid biofertilizer. The interactions between the bioinoculants used in the biofertilizer were checked and was found to be positive, which ensured that they will grow together and improve plant growth and yield by virtue of their unique mechanisms in a synergistic manner. The efficacy of the biofertilizer was tested on Chili plants, and the results showed higher plant growth and yields compared to plants growing in control soil, as well as the plants growing in soil supplemented with vermicompost, one of the most commonly used supplement in organic farming. Therefore, it can be concluded that the multi-dimensional utility of the solid biofertilizer could be exploited in the field of agriculture, and hence, can be an effective option for improving crop growth and yield in a sustainable and eco-friendly manner. References:Abd El Halim, A.A. and El Baroudy, A.A. (2014). Influence addition of Fine Sawdust on the Physical Properties of Expansive Soil in the Middle Nile Delta, Egypt, Journal of Soil Science and Plant Nutrition. Chaurasia, B., Pandey, A., Palni, L.M.S., Trivedi, P., Kumar, B. and Colvin, N. (2005). Diffusible and volatile compounds produced by an antagonistic Bacillus subtilis strain cause structural deformities in pathogenic fungi in vitro. Microbiol. Res. 160, 75-81. Cheruth, A.J., Mohammed, A., Salem, M.H. and Kamrun, N. (2010). Plant growth regulator interactions results enhancement of antioxidant enzymes in Catharanthus roseus, Journal of Plant Interactions, 5:2, 135-145. Compant, S., Duffy, B., Nowak, J., Clement, C. and Barka, E.A. (2005). Use of plant growth-promoting bacteria for biocontrol of plant diseases: Principles, mechanisms of action, and future prospects. Appl. Environ. Microbiol. 4951-4959. Garcia de Salamone, I.E., Hynes, R.K. and Nelson, L.M. (2001) Cytokinin production by plant growth promoting rhizobacteria and selected mutants. Can J Microbiol 47:404–411. Haas, D. and Défago, G. (2005). Biological control of soil borne pathogens by fluorescent
93 pseudomonads. Nat Rev Microbiol 3, 307–319. Han, H.S. and Lee, K.D. (2005). Phosphate and Potassium Solubilizing Bacteria Effect on Mineral Uptake, Soil Availability and Growth of Eggplant Research Journal of Agriculture and Biological Sciences 1(2): 176-180, 2005. Mishra, J. and Arora, N. K. (2016). Bioformulations for Plant Growth Promotion and Combating Phytopathogens: A Sustainable Approach. Bioformulations: for Sustainable Agriculture, p 4-24. Jong-Hui, L. and Sang-Dal, K. (2010). Biocontrol of phytophthora blight of red pepper caused by Phytophthora capsici using Bacillus subtilis AH18 and B.licheniformis K11 formulations. Journal of the Korean Society for Applied Biological ChemistryDecember 2010, Volume 53, Issue 6, pp 766–773. Jung, H.K. and Kim, S.D. (2003). Purification and characterization of an antifungal antibiotic from Bacillus megaterium KL 39, a biocontrol agent of red-pepper phytophthora blight disease. Korean. J. Microbiol. Biotech., 31: 235-241. Loon, V.L.C. (2000). Systemic induced resistance. In: Slusarenko AJ, Fraser RSS, Van Loon LC, editors. Mechanisms of resistance to plant diseases. Dordrecht, The Netherlands: Kluwer Academic Publishers. pp 521 574. Marulanda-Aguirre, A., Azcon, R., Ruiz-Lozanoand, J.M., Aroca, R. (2008). Differential effects of a Bacillus megaterium strain on Lactuca sativa plant growth depending on the origin of the arbuscular mycorrhizal fungus coinoculated: physiologic and biochemical traits. J. Plant. Growth. Regul., 27: 10-18. Moses K. (2014). Assessment of Sawdust as Carrier Material for Fungal Inoculum Intended For Faster Composting. ISSN: 2319-7706 Volume 3 Number 6 (2014) pp. 608613. Paul, D., Srinivasan, V., Anandaraj, M. and Sharma, Y.R. (2003). Pseudomonas fluorescens mediated nutrient flux in the black pepper rhizosphere microcosm and enhanced plant growth. In: 6th International PGPR workshop, Calicut, India. pp 1823. Satapute, P., Shetti, A. and Hiremath, G. (2012). Isolation and characterization of nitrogen fixing Bacillus subtilis strain as-4 from agricultural soil. Sandhya, V., Ali, S.Z., Grover, M., Reddy, G. and Venkateswarlu, B. (2010). Effect of plant growth promoting Pseudomonas spp. on compatible solutes, antioxidant status and plant growth of maize under drought stress. Plant Growth Regul 62:21–30. Wani, S.A., Chand, S., Wani, M.A., Ramzan, M. and Hakeem, K.R. (2016). Azotobacter chroococcum – A Potential Biofertilizer in Agriculture: An Overview. Soil Science: Agricultural and Environmental Prospectives, pp.333-348. Chakraborty, U., Chakraborty, B.N. and Chakraborty, A.P. (2012). Induction of Plant Growth Promotion in Camellia sinensis by Bacillus megaterium and its Bioformulations. World Journal of Agricultural Sciences 8 (1): 104-112. Van Peer, R., Niemann, G.J. and Schippers, B. (1991). Induced resistance and phytoalexin accumulation in biological control of fusarium wilt of carnation by Pseudomonas sp. strain WCS417r. Phytopathology 91:728–734.
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4 FUNGAL DIVERSITY IN RHIZOSPHERE SOIL OF DIFFERENT VARIETIES OF PIGEONPEA [Cajanus cajan (L.) Millsp.] B.D. Gachande and V. Jalander Deapartment of Botany, Telangana University, Nizamabad (T.S.) Post Graduate Department of Botany, N.E.S. Science College, Nanded431 605 (M.S.) Email:
[email protected]
ABSTRACT The rhizosphere is the zone of soil surrounding a plant root where the biology and chemistry of the soil are influenced by the root. It is an area of intense biological and chemical activityinfluenced by compounds exuded by the root, and by microorganisms feeding on the compounds. Total 58 fungal species were isolated from the nonrhizosphere and rhizosphere of ten different varieties of pigeonpea [Cajanus cajan (L.) Millsp] i.e. PUSA-992, BDN-2, BDN-708, BSMR-853, BSMR-736, BSMR-175, ICP- 8863, ICPL-8S7119, ICP2376 and AKT-9913 at three different stages of plant growth viz. vegetative, flowering and fruiting stages.Fungal population was higherat flowering stage than at vegetative (non-flowering) and at fruiting stage. Altogether, 29 fungal species belonging to 14 genera was recorded throughout the study from non-rhizosphere and 58 fungal species belongs to 23 genera was from rhizosphere. Comparatively higher fungal population was recorded in rhizosphere than nonrhizosphere. Maximum number of fungal species was recorded from variety PUSA-992 and BDN-708.The population of ascomycetous fungi was higher than that of Deuteromycetous and Phycomyetous fungi. The dominance of Ascomycetous fungi varies according to different stages of plant growth. Aspergillus flavus, A. niger and A. nidulans these three fungal species were dominant in all the stages of
96 plant growth, in all varieties of pigeonpea during the study. Key words: Rhizosphere fungi, Pigeonpea varieties, quantitative and qualitative soil analysis.
INTRODUCTION Pigeonpea [Cajanus cajan (L.) Millsp., Family- Fabaceae], is one of themajor pulse crops of the tropics and sub-tropics, grown in approximately 50countries in Asia, Africa and the Americas, mostly as an intercrop (mixed crop) withcereals. India is the largestproducer with 3.4 million ha. About 95% production of pigeonpea isfrom south Asia. 90% of which belongs to India (Kadam et. al., 2005). Pigeonpea being a leguminous plant is capable of fixing atmospheric nitrogenand thereby restore lot of nitrogen in the soil (Yadav, 1992). The term “rhizosphere” was introduced by Hiltner (1904) to designate that portion of the soil which is subject to the specific influence of the plant root system, and noted that this soil supported greater microbial activity than soil more distant from the roots. The area of the soil influenced by root varies with the type of the plant, age of the plant, soil conditions, and pH of the soil, environmental conditions and moisture content of the soil. Sadasivan (1960) has given an excellent review of the importance of the study of rhizosphere microflora. Microorganisms which can grow in the rhizosphere have great potential as biocontrol agents (Weller, 1988a). It is true that the soil fungi colonize on the dead and decaying plant residues in soil.The ability to decompose organic residues, fungi is the most versatile group. In affecting the processes of humus formation and aggregate stabilization, molds are more important than bacteria. When a crop is grown in a soil year after year, the soil inhabiting pathogens may increases. In such conditions the suppression of soil inhabiting pathogens becomes the first need of cultivars. Soil treatment with certain chemicals also becomes dangerous for the health of human beings, health of useful microorganisms and texture of the soil. In such conditions suppression of soil inhabiting pathogens by using eco-friendly biological control becomes essential. Keeping the above facts in mind, it is now known that the various aspect of soil fungi affected by the role of many factors such as moisture contents, soil temperature, CO2/O2 ratio, depth of the soil, cultural practices and antagonism etc. The justified approach is the relevant information is obtained from our own area, as the soil conditions and cultivation practices, which are vary from place to place. Plant root exudates are a complex mixture of chemicals and organic
97 compounds secreted into the soil by the roots (Bais, 2004). As plant roots grow through soil they release water soluble compounds such as amino acids, sugars and organic acids that supply food for the microorganisms. The food supply means microbiological activity in the rhizosphere is much greater than in soil away from plant roots. In return, the microorganisms provide nutrients for the plants. All these activities make the rhizosphere most dynamic environment in the soil.Most soil microorganisms do not interact with plant roots, possibly due to the constant and diverse secretion of antimicrobial root exudates. However, there are some microorganisms that do interact with specific plants. These interactions can be pathogenic, symbiotic,harmful, saprophytic or neutral. Interactions that are beneficial to agriculture include mycorrhizae, legume nodulationand production of antimicrobial compounds that inhibit the growth of pathogens. MATERIALS AND METHODS Pigeonpea varieties selected:PUSA-992, BDN-2, BDN-708, BSMR-853, BSMR-736, BSMR-175, ICP- 8863, ICPL-87119, ICP-2376 and AKT-9913. Collection of soil samples and isolation of mycoflora: The rhizosphere and non rhizosphere soil samples of these above varieties were collected by shaking up-rooted plants at non-flowering, flowering and fruiting stages of plant growth in sterile paper bags. Non-rhizosphere soil was sampled from trenches away from root zone effect and nearly at the same depth travelled by pigeonpea plants from Pulses ResearchCentre, Badnapur District Jalna (M.S). Isolation of rhizosphere and non rhizosphere mycoflora was done on Martin’s Rose Bengal agar medium containing streptomycin (Martin, 1950), by dilution plate method (Waksman, 1927) at different stages of plant growth. After inoculation plates were incubated at room temperature, and the developing colonies were identifiedon the basis of vegetative and reproductive chasracters as described by Barnett and Hunter (1972), Gilman (1957) and Subramanian (1971). The pure cultures were maintained on PDA slants.Number of colonies of each species as well as total number of colonies in each plate was recorded. Number of fungi per gram of moisture free soil in rhizosphere and non-rhizosphere were also recorded and R: S ratio (Aneja, 2007). Chemical analysis of rhizosphere and non-rhizosphere soil:Simultaneously, the experiment for soil pH, water holding capacity wascarried out (Subramanyam and Sambamurthy, 2000). Organic carbon, organic mattercontent,was calculated by rapid titration method (Walkey and Black, 1934). Total nitrogen by micro Kjeldhal distillation method (Jakson, 1958), Potassium (by flame photometry) and phosphorus (Rao,
98 1993) was analyzed. RESULT AND DISCUSSION Quantitative variation in fungal species: It was observed that higher fungal population harbored in the rhizosphere than the non-rhizosphere. At flowering stage of the plant growth gave higher fungal population than the vegetative and fruiting stages inallvarieties of pigeonpea. There was a great variation in fungal population in non-rhizosphere and rhizosphere soil.Altogether, 58 species representing 23 genera was recorded. Out of 58 species 3 belongs to phycomycetes, 36 ascomycetes, 14 deuteromycetes and 5 of mycelia sterilia (Table 1). Maximum number of species belong to the genus Aspergillus followed by Penicillium. Total 14 numbersoffungal isolates were recorded of Aspergillus, 10 number of Penicillium from the rhizosphere and non-rhizosphere soil of pigeonpea. Table 1: Taxonomic account of rhizosphere and non-rhizosphere soil fungi of different varieties of pigeonpea (Cajanus cajan (L.) Millsp.)
Sr. No. Class, Order and Family I. PHYCOMYCETES 1
Species
Mucorales Mucoraceae
2
Genera
2
2
1
1
3
31
1
1
Hypocreaceae
1
2
Claviciptaceae
1
1
1
1
Dematia
6
11
Tuberculariaceae
2
3
Mycelia sterilia
5
5
Total
23
58
Perenosporiales Pythiaceae II. ASCOMYCETES
1
Eurotiales Eurotiace Trichocomacea
2
3
Hypocreales
Chaetomiales Chaetomiaceae III. DEUTEROMYCETES
1
2
Moniliales
99 Variation in taxonomic groups: Fungal species were assigned to their respective groups like phycomycetes, ascomycetes and deuteromycetes. The population of ascomycetes fungi was higher than phycomycetes and deuteromycetes at three different stages of plant growth. The fungal population was more in rhizosphere than non-rhizosphere soil. The population of the mycoflora was increased from the vegetative (non-flowering stage) to flowering stage and then it was declined at pod formation or maturity of crop in all varieties. Least number of deuteromycetes were recorded throughout the growthstages.The number of phycomycetes were few as compared to other two groups.These variations in fungal population are probably due to amount of rainfall, humidity, age of plant and the biochemical nature of the plant (roots).The highest rhizosphere fungal population (number of fungi per gram of dry soil) at the time of non-flowering stage, flowering stage was found in var. BDN-708 and var. BSMR during three successive years. Qualitative variation in Fungal Species: Altogether 29 fungal species belonging to 14 genera was recorded from non-rhizosphere soil. Out of which, 03 beelongs to phycomycetes, 16 from ascomycetes, and 07 from deuteromycetes and 3 of mycelia sterilia. Maximum number of species was recorded at flowering stages during three successive years (Tables 2, 3 & 4). Altogether 58 fungal species belonging to 23 genera were recorded from rhizosphere soil of different varieties of pigeonpea. Out which, 03 belongs to phycomycetes, 36 from ascomycetes, 14 from deuteromycetes and 05 are of mycelica sterilica. Maximum number of fungal species was recorded during the first year of study. Maximum number of species was recorded at flowering stages of the plant growth duringthe entire study period.(Tables 5,6 & 7) It was observed that, three species belonging to the genera Aspergillusie A. flavus, A. nidulans and A. nigerwere isolated at vegetative, flowering and fruiting stages of plant growth. Out of 58 fungal species, isolated from rhizosphere of ten different varieties of pigeonpea, few of them were common. Totally 38 fungal species were isolated from ten differentvarieties of pigeonpea during first year of study. Out of which,29 fungal specieswere isolated at non-flowering stage, 38 at flowering stage and 31 at fruiting stage. Total 27 fungal species from var. PUSA-992, 26 from BDN-2, 25 from BSMR-175, 24 from BSMR-853 and ICPL-87119, 23 from BDN708 & ICP-8863, 21 from BSMR-736 & ICP-2376 and 20 from AKT-9913 were isolated. At non-flowering stage, total 7 fungal species were isolated
100
101
102
103
104
105 from varieties BDN-2, 10 from BSMR-175, ICPL-87119, 8 from ICP8863,ICP-2376 & AKT-9913, 7 from BSMR-853 & BSMR-736 and PUSA992.At flowering stage, 23 from var.PUSA-992, 20from BDN-2, 16 from var. BSMR-175 & ICPL-87119, 15 from var. BSMR-853 & ICPL-8863, 14 from var. BDN-708, 13 from var.ICP-2376 & AKT-9913 and 12 from var. BSMR-736.At fruiting stage, 11 fungal species were isolated from var. BSMR-853, 10 from var.BDN-708, ICPL-87119, ICP-2376 & AKT9913, 9 from var. BDN-2, BSMR-175, 8 from var. BSMR-736, 7 from var. PUSA-992 & ICP-8863 were isolated. (Table -2). Totally, 28 fungal species were isolated from 10 different varieties of pigeonpea during second year. Out of which, 26 were isolated at nonflowering stage, 28 at flowering stage and 27 at fruiting stage of plant growth during the year. Total 27 fungal speciesisolated from var. BDN708, 23 from var. BSMR-853 & AKT-9913, 22 from var.BDN-2, 21 from var. ICPL-87119, 20 from var. BSMR-736, ICP-8863 & ICP-2376 and 19 from two varieties PUSA-992 & BSM-175 during the year 2008-09.At non-flowering stage, 14 fungal species were isolated from var. BDN-708, 13 from var. BSMR-736, 12 from var. ICP-2376 & AKT-9913, 11 from var. BSMR-853 & ICPL-87119 and 08 from var. PUSA-992 & BSMR175. At flowering stage, 14 fungal species were isolated from the varieties BDN-708, 17 from var. AKT-9913, 16 from var. BDN-2, ICP-2376, ICPL87119, 15 from var. BSMR-736, 14 from var. BSMR-853& ICP-8863 and 08 from var. PUSA-992.At fruiting stage, 12 fungal species were isolated from variety BSMR-853, 11 from var. BSMR-736 & ICPL-87119, 10 from var. PUSA-992, BSMR-175 & ICP-2376, 09 from var. BDN-2 & 708 and 08 from var. ICP-8863 & var. AKT-9913 (Table-3). Totally, 31 fungal species were isolated from 10 different varieties of pigeonpea during third year of study. Out of which, 25 were isolated at non-flowering stage, 29 at flowering stage and 28 at fruiting stage of plant growth. At non-flowering stage of plant growth, total 08 fungal species from each var. PUSA-992, BDN-708, ICP-2376, BDN-2, ICP-8863 and ICPL-87119 were isolated and 10 from var. BSMR-853, 12 from var. BSMR-736, & 175 and var. AKT-9913.At flowering stage, total 17 fungal species from each var. PUSA-992, BDN-2 and BSMR-853, 19 from var. BDN-708, 15 from BSMR-736, 16 from each BSMR-175, AKT-9913, 18 from ICP-8863, 22 from ICPL-87119, 20 from ICP-2376 were isolated.At the fruiting stage, 10 from each var. PUSA-992 and ICPL-87119, 9 from var. BDN-708, BSMR-853, ICP-8863, 8 from var. BSMR-175, 11 from var. BSMR-736, 6 from var. BDN-2 and 7 from AKT-9913 were isolated during third year (Table -4).
106 Changes in pH and moisture content in rhizosphere and nonrhizosphere soils at different stages of plant growth in different varieties of pigeonpea plants during the three years of study depected in table (Table5). It is clear from the table,that the soils had an acid to alkaline reaction with pH ranging from 6.0 to 8.0 and the pH of rhizosphere soils varies slightly during the different stages of plant growth. In all cases drecrease in pH was recorded with gradual aging of the plant and also accompanying drier and warmer weather conditions. Moisture content also decreased with aging of the plant. Organic carbon, organic matter, available phosphorus and potassium content in rhizosphere and non-rhizosphere soils at different stages of plant growth of different varieties of pigeonpea is presented in table (Table-5). It is clear from the results that all the parameterswere higher at flowering stage than non-flowering and fruiting stages. Only total nitrogen content was reducedat non-flowering stage but increases with increasing in age of plant. The total number of fungi is listed in table (Table6) shows the occurrence of fungi in the rhizosphere and non-rhizosphere soils of pigeonpea in varieties at different stages of plant growth. It is clear from the table-6 that the number of fungi was higher in the rhizosphere than non-rhizosphere. More number of fungi was observed at flowering stge in all varieties of pigeonpea. DISCUSSION Studies onsoil fungi from pigeonpea field wascarried out by Joshi and Chavan (1981) and on rhizosphere by Bhowmick and Gupta, 2000; Subhedar et al., 2006; Wahegaonkar, 2009. However, the information is still insufficient to understand the complete biology of these organisms withnon-rhizosphere and rhizosphere of pigeonpea. Hence a comparison of non-rhizosphere and rhizosphere fungi and their interaction under soil in different varieties of pigeonpea was undertaken. The pigeonpea has unique character and occupied an important place in various intercropping systems of cereals,oil seeds, legumes, cotton etc. It is able to reduce the soil erosion in slopy lands and enrich the soil by adding the organic matter through leaf fall. About 40 kg N/ha is fixed through nitrogen fixation (Agarwal, 2003).This crop has good capacity to grow in poor land and provides more stability in rain fed situations than other crops. Different varieties of pigeonpea are under cultivation in different parts of the country and these varieties are varying in their duration, grain quality, and response to fertilizers, resistant to drought, alkalinity and salinity. The cultivation practices adopted in different parts of the country also vary depending upon the climatic conditions, soil, availability of water
107 and crop variety. All these factors including cultivation practices, greatly influences the susceptibility of a given crop variety to one or more diseases (Agarwal, 2003).The initial aim of many studies on soil fungi was to obtain on accurate picture of the distribution of active forms in the many soil microhabitats. Microorganisms present in soil plays an important role in the development of root rot diseases. In the present study, greater numbers of fungi were isolated from the rhizosphere soil than from non-rhizosphere soil of pigeonpea plants at different stages of plant growth. Such similar observations have been made on cowpea (Odunfa and Oso, 1979), cauliflower (Singh and Saxsena, 1991), and tomato (Chandra and Raizada, 1982). In India, from rhizosphere soil of pigeonpea, 51 fungal species were recorded (Wahegaonkar et. al., 2009). Higher numbers of fungal species were present in soil of the root zone (rhizosphere) than in root free soil (non-rhizosphere). The study reveals the distribution pattern of fungal population in the soil during growth stages of pigeonpea.A total 37 fungal species were recorded from pigeonpea rhizosphere soil and a group of unidentified species (Pandey, 2011). Kamal and Verma (1976) isolated several fungal species from pigeonpea field of which, 31 were from rhizosphere, 23 from non-rhizosphere. Presumably the micr obial population is stimulated by rhizodeposition and is almost in variably higher in the rhizosphere than in root free soil as also reported by Edward and Shrivastava (1962), Sundrarao et al. (1962) and Manoharachary et al. (1977). Age of the plant also influences the activities of microflora due to change in qualities of root exudates. A gradual increase in microbial population was observed with age of plant which attained a peak when the plants were in the flowering stage and the fungal population declined when plants reached the senescent ie. fruiting stage. It could presumably be due to abundant exudation of sugars and amino acids from roots at flowering stage and due to carbon and nitrogen sources provided by the breakdown of root hairs and cortical cells during fruiting stage. Such similar observation have been made by Odunfa (1975) and Odunfa and Oso (1979) where a greater number of fungi were observed in the rhizosphere of cowpea in the seedling and flowering stages. Of the fungi isolated, a predominant population of species of Aspergillus and Penicillium was found throughout the growing season which could presumably be due to their high sporulating ability. Presumably the rhizosphere of pigeonpea stimulated the growth of A. niger. Such similar
108 observation have been made in the rhizosphere of barley and oat (Kyrylenko, 1967), berseem (Edward and Srivastava, 1982) and Soybean (Farzana Ali, 1997). Among the pigeonpea varieties, the number of rhizosphere fungi varied during three years. Higher number of fungal species was found during 2007-08 in variety BDN-708 and the dominant population of A. flavus, A. niger and A. nidulanswas observed. There are reports where different lines of wheat and potatoes have also shown different rhizosphere microflora (Azad et. al., 1985; Weller, 1988a). Such similar observation has also been made by Patil and Thitte (1988) in fababean rhizosphere. It would suggest that, not only there are differences in the root microflora of different plant species but also between varieties of single species. This may be attributed to the alteration in the pattern of root exudates among ten different varieties of pigeonpea. There are reports where root exudates have direct influence on the micro population in the rhizosphere (Sadasivan, 1960; Schroth and Hilderbrand, 1964; Rovira, 1965). Both, the type and the amount of exudation are influenced by environmental conditions, cultural factors and the presence of microorganisms but are largely under genetic control (Hale et al., 1978). Differences in the exudates composition between resistant and susceptible varieties have been found and such differences may lead to change in the composition of the microflora in rhizosphere where resistant varieties have been noted to support greater population of antagonistic microorganisms than susceptible varieties (Funck-Jenson and Hocken-hall, 1984). The resistantance or susceptibility of a variety determines the type of microflora supported by the rhizosphere. Agnihothrudu (1955) isolated Streptomyces graseolus and S. erythrochromogenes from the rhizosphere of wilted resistant variety of pigeonpea. They were inhibitory to F.udum and the inhibitory effect was more than tested on medium containing root exudates from the susceptible variety. The total population was also higher in rhizosphere of wilt susceptible plant than resistant varieties of pigeonpea (Agnihothrudu, 1957). There is also report; no potent antagonistic microorganism could be isolated from the rhizosphere of wilt resistant pigeonpea variety C-11-6 (Murthy and Bhagyaraj, 1978).However, it has been concluded that, F.udum and other Fusarium sp. are predominant in the rhizosphere of susceptible varieties of pigeonpea while antagonistic fungi like Trichoderma, Aspergillus niger and certain actinomycetes were isolated from rhizosphere of resistant variety (Shaik and Nusrath, 1987; Rao et. al., 1987).Most of the fungi isolated in the present study belong to ascomycetes (62.06%) than that of the deuteromycetes (32.75%) and
109 phycomycetes (5.08%). The dominance of the ascomycetous fungi also variesfrom variety to variety and age of the plant. Rain and humidity has more influence on the population of these taxonomic groups (Rane, 2008). It was interesting to note that, the number of fungi belonging to ascomycetes washigherduring the entire study. Similiar findings were also reported by Farzana Ali (1997). It was also pointed out from the results during three years that, the rhizosphere and non-rhizosphere fungi was increased with increasing age of the plant from vegetative stage (non-flowering stage) to flowering stage, then it was declined at fruiting stage . The similar results were reported by many workers. Agnihothrudu (1957) reported the age of the plants, the season of the year and the nature of the soil also influenced the rhizosphere mycoflora. Joshi (1983) was reported that, the decline of rhizosphere fungal flora of pigeonpea at the time of senescent stage. Wahegaonkar (2009) also reported the rhizosphere fungi of pigeonpea were increased with increasing the age of the plant. In the present investigation total 58 rhizosphere fungi were isolated from different varieties of pigeonpea and tested for their antagonistic activity against Fusarium oxysporum f.sp. udum Butler a pathogen of wilt of pigeonpea (Patil and Patil, 2012). REFERENCES Agarwal, S.C. (2003). Diseases of pigeonpea. Concept Publishing Company, New Delhi, India. Agnihothrudu, V. (1957).The density of the rhizosphere microflora of pigeonpea (Cajanus cajan (L.) Millsp.) in relation to the wilt caused by Fusarium udum Butler Naturwissenschaften, 44 (18): 497. Aneja, K. R. (2007). Experiments in microbiology, Plant Pathology and Biotechnology. New Age International Publishers, New Delhi. Pp. 603 Azad, H.R., J.R. Davis, W.C. Schnathost and C.I. Kado (1985). Relationship between rhizosphere and rhizosphere bacteria and Verticillium wilt resistance in potato. Arch. Microbiol., 140:347-351. Bais, H.P. (2004). How plants communicate using the underground information Trends in superhighway? Plants Science. 9 (1): 26-32. Barnett, H.L. and B.B. Hunter (1972). Illustrated genera of imperfect fungi. Third Edition Burgess publishing company, Minneapolis, Minnesota. Bhowmick, N. and C. S. Gupta (2000). Cytopathological effect of the legume rhizosphere fungi on Rhizobium sp. Environment and Ecology.18(1):139-142. Edward, T.C. and R.N. Srivastava (1982). Microflora of soils and rhizosphere microorganisms of various field crops of the Allahabad Agriculture Institute. The Allahabad Farmer.36:1-414.
110 Farzana Ali (1997). Study of rhizosphere and rhizoplane mycoflora of sobean. Ph.D. Thesis, University of Karachi, Pakisthan. Pp. 578. Funck-Jenson, D. and J. Hocken-hall (1984). Root exudation rhizosphere microorganisms and disease control. Vaxtskyddsnotiser. 48:49-54. Gilmam. J.C. (1957). A manual of soil fungi. Sec. Eds. Iowa State Univ. Oxford IBM Publishing Co. Calcutta press. Pp. 410. Hale, M.G., L.D. Moore and G.J. Griffin (1978). Root exudates and exudation. In: Interactions between non-pathogenic soil microorganisms and plant. (Eds.) Y.R. Dommerques and S.V. Kuper, Elsevier, North Holland Biomedical Press. Amsterdam. Pp. 260. Jakson, R.M. (1958). An investigation of fungistasis of soil microorganisms. In: Ecology and Soil Borne Plant Pathogens Eds. K.F. Bakel & W. C. Synder. Univ. California Press, Berkeley. Joshi, I.J. (1983). Studies on rhizosphere and rhizoplane mycoflora of Cajanus cajan (L.) Millsp. during pre-harvest and post-harvest periods. Microbiologia Espanola.36(12):1-8 Joshi, I.J. and R.K.S. Chavan (1981). Ecological studies on fungal flora of Cajanus cajan (L.) Mill.sp. Proc. Nat. Acad. Sci. India. 51(B) III:233-239. Kadam, J.R., G.N. Patil, A.P. Chavan, D.B. Kadam and B.M. Mhaske (2005). Efficacy of some insecticide sequences against pod borer complex in pigeonpea. Indian J. Environ. Ecoplan. 10(3): 731-734. Kamaland A.K. Verma (1976). Microfungal flora in the root region of arhar (Cajanus cajan (L.) Millsp.). Fertilizer Technology. 13:155-157. Kyreylenko, T.S. (1967). Aspergillus fungi found in barley and oat rhizosphere in the Polesse districts of the Ukranian. S.S.R. Biol. Abstract. 48:240. Lynch, J. M. (1990). Introduction: Some consequences of microbe rhizosphere for plantand soil. In: Cur, E.A.; Truelove, B. The rhizosphere. Springer-Verlag. NewYork, 1986, Manoharachary, C., K. Venkateshwarulu and P. Ramarao (1977). Studies on mycoflora of rhizosphere and non-rhizosphere soils. Geobios. 4:67-78. Martin, J.P. (1950). Use of acid Rose Bengal and streptomycin in the plate method for estimating soil fungi. Soil Sci. 69:215-232. Murthy, G.S. and D.J. Bagyaraj (1978). Rhizosphere microflora of Cajanus cajan in relation to Fusarium wilt resistance. Plant and Soil. 50: 485-487. Odunfa, V.S.A. (1975). Studies on rhizosphere microorganisms of cowpea and guinea corn. Ph. D. thesis. University of Ibadan, Nigeria. Pp. 305. Odunfa, V.S.A and B.A. Oso (1979). Fungal populations in the rhizosphere of coepea. Trans. Brit. Mycol. Soc.73:21-26. Olutiola, P.O. (1979).Cellulolytic Enzymes in Culture Filtrates of Aspergillus clavatus. Journal of General Microbiology.102: 27-3 1
111 Pandey, V. (2011). Fungal diversity in pigeonpea(Cajanus cajan (L.) Millspaugh) cropping system of calcareous soil.Archives of Phytopath.& Plant Protec.44 (9): 832-839 Patel, S. and R.L. Patel (2012). Wilt of pigeonpea caused byFusarium udum Butler. Lambert Academic Publishing. Pp. 148. Rane, V. (2008). Studies on soil, rhizosphere and rhizosphere mycoflora of rice (Oryza sativa) grown in Goregaon of East Vidarbha. Ph. D. Thesis, RPMN Univ. Nagpur Pp. 145. Rao, K. S. (1993). Practical Ecology. Anmol Publications, New Delhi. Pp. 190. Rao, V.K., A.G.R. Reddy and K. Satyanarayana (1987). Quantitative changes in the rhizosphere microflora of wilt resistant and susceptible varieties of pigeonpea. Indn. J. Mycol. & Plant Path.17(1):59-61. Rovira, A.D. (1965). Plant root exudates and their influence upon soil microorganisms. Pp. 170-186. In: Ecology of Soil-borne Plant Pathogens. (Eds.) K.F. Baker & W.C. Snyder, University of California Press, Berkeley & Los Angeles. Sadasivan, T.S. (1960). The problem of rhizosphere microfloras. Proc. Nat. Inst. Sci., India. 26 Schroth, M.N. and D.C. Hilderbrand (1964). Influence of plant exudates on root infecting fungi. Ann. Rev. Phytopathol., 2:101-132. Shaik, Imam and M. Nusrath (1987). Varietal variation in the rhizosphere and rhizoplane mycoflora of Cajanus cajan with special reference to wilt disease. Indian J. Bot. 10:126-129. Singh, R. and V.C. Saxsena(1991). Wilt of califlower rhizosphere and rhizoplane studies. Internat. J. Trop. Dis.9:71-81 Srivastava, V. B. (1969).Investigations into rhizosphere microflora of certain crop plants. Ph. D. Thesis, Univ. Gorakhpur, (India). Subhedar, A., D. Hande and N. Dharkar (2006). Effect of somerhizosphere fungal flora on the productivity of some crop plants. J. Agronomy, 5 (2): 239-243. Subramanian, C.V. (1971). Hyphomycetes. “An account of Indian species, except Cercosporae.” ICAR publ. New Delhi. Pp.930. Subramanyam, N.S. and A.V.S.S. Sambamurthy (2000).Ecology. Narosa Publishing House, Delhi. Pp. 616. Sundrarao, M.V.B., M.V. Chayanulu, A. Sankaram and K.V. Vankatram (1962). Rhizosphere effects on microorganisms. 9th Int. Cong. Microbiology, Montreal, Qubec, Canada. Pp. 380. Wahegaonkar, N., S.Y. Shinde, S.M. Salunkhe and P.L. Palsingankar (2009). Diversity of rhizosphere and rhizoplane mycoflora of Cajanus cajan (Linn.). Bioinfolet. 6(3):186-192. Waksman, S.A. (1927). Principals of soil microbiology. Bailliere Tindal & Co., London. Walkley, A. and I.A. Black (1934). Rapid titration method. Soil Sci.,37:29-38.
112 Weindling, R. (1934). Studies on a lethal principle effective in the parasitic action of Trichoderma lignorum on Rhizoctonia solani and other soil fungi. Phytopath.62: 442 447. Yadav D.S. (1992). Pulse Crop (Production Technology), Kalyani Publishers, New Delhi Ludhiana.
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5 IN VITRO ANTIMYCOTIC EFFICACY OF PLANT LATEX AGAINST SEED-BORNE STORAGE FUNGI OF SUNFLOWER AND SAFFLOWER Gachande B. D., ManoorkarV. B. and N.F. Shaikh Botany Research Laboratory and Plant Disease Clinic N.E.S. Science College, Nanded-431602 (MS) Email-
[email protected]
ABSTRACT Seed-borne fungi were isolated from sunflower and safflower seeds. Ten dominant fungal species were subjected to solvent and aqueous latex extracts of Jatrophacurcas and Thevetia peruviana for their antimycotic efficacy by agar well diffusion method. Development of clear zone around the well indicated the efficacy of latex extract. Jatrophacurcas latex showed inhibitory effect against all test fungi except A. parasiticus and F. moniliforme while the latex extract of Thevetia peruviana inhibitory to A. parasiticus. Key words: Seed-borne fungi, oil seeds, sunflower, safflower, plant latex.
INTRODUCTION Seed is the plant part associated with either propagation of plant for its continuous existence or serves as food for human beings and it act as a catalyst in agricultural production. Seed plays a vital role for the production of healthy crop and about 90 % of all the world’s food crops are grown from seeds (Schwinn, 1994). Seeds are generally associated with certain saprophytic or parasitic microbes which perpetuate in the seed
114 lots on the advent of favorable conditions. Pathogen free healthy seeds are essential for desired plant populations and good harvest. The viable and vigorous seeds during planting time are very important for achieving the target of agricultural production. (Yadav et al., 2014). Of the 16% annual crop losses due to plant diseases, at least 10% loss occurs due to seed-borne diseases (Fakir, 1983). Coincidentally important or devastating crop diseases are seed borne and caused by fungi. Cultivated crops are infected by one or more seed-borne pathogens. After harvesting, seeds are stored indifferent storage conditions and if these storage conditions arenot proper various microorganisms like fungi, bacteria and viruses are interacted with these seeds. Among these microorganisms’ fungi plays a significant role in infection, altering quality and longevity of seeds during the storage (Christensen and Kaufman,1969).Such infected seeds are not good for human consumption and also rejected at industrial level. Several biological agents, mainly fungi damages oil-seeds from the early stage of their formation on growing plants until their use and consumption. Fats and oils are important ingredients of human food. Vegetable oil is extracted from different oil seed crops (Butt & Ali, 2005). Sunflower (Helianthus annuus L.), an important members of the family Asteraceae and considered a commercial oil seed crop all over the world(Anon., 2007). Sunflower is particularly used for production of edible oil as well as for seed consumption (Shahda et al., 1991; Anonymous, 2007; Afzal et al., 2010). Safflower (Carthamus tinctorius L.) belonging to the same family Asteraceae and is one of the major rabi oilseed crops in India, it is an important source of oil and proteins. Due to high nutritive value of these oil seeds, fungi get interacted with seeds and transmit the diseases. This take place either in field or in improper storage conditions. Improper storage conditions are supportive for fungal attack on seeds (Bhajbhuje, 2014). Various environmental factors like high relative humidity, moderate temperature etc. favors growth of seed- borne mycoflora on stored seeds (Bhajbhuje, 2014).This ultimately affect on the yield and economy of the country. Chemical fungicides play a vital role in the stabilization and increase of agricultural yield. On the other hand, it is being a possible source of environmental pollution, with residual toxicity to mammals and wildlife. The knowledge of extent and mode of inhibition of specific
115 compounds which are present in plant latex, may contribute to the successful application of such natural compounds for treatment of fungal infection and diseases. Latex is a complex mixture of secondary metabolites (Santos et al., 2011), contains various biologically active compounds and antimicrobial activities (Siritaperawee et al., 2012, Kanokwiroon et al., 2008). Biologically active compounds present in the latex of plants have always been of great interest to scientists working in this field. In recent years, this interest to evaluate plants possessing antimicrobial activity against various common pathogens is increasing. In this regard, aqueous, ethanol and methanol latex extract of Jatropha curcas L. (Euphorbiaceae) and Thevetia peruviana (Pers.) K. Schum. (Apocynaceae) were screened for the antifungal activity against storage seed-borne fungi of sunflower and safflower. MATERIALS AND METHODS a) Collection of oil seeds Seeds of Sunflower (Helianthus annuus L.), Safflower (Carthamus tinctorius L.) were collected from local market of Nanded in pre-sterilized polythene bags, brought to the laboratory and a composite sample was prepared. b) Isolation and Identification of oilseed –borne mycoflora Two standard methods i.e. moist blotter paper and Agar plate method (ISTA, 1996) were used for the isolation of seed-borne mycoflora from sunflower and safflower seeds. In the blotter paper method, seeds were surface sterilized using with 0.1% HgCl2 (mercuric chloride) solution and placed aseptically on a pair of moistened blotter papers at equidistance. In Agar plate method, 20 ml of sterilized PDA was poured in pre sterilized glass petriplates of 10 cm diameter under aseptic conditions. In each case, ten treated and untreated seeds were placed (1+9) at equidistance under aseptic conditions. The plates were incubated at room temperature (28 ± 2ÚC) for seven days. The petriplates were observed periodically for any fungal growth. The identification of fungi were done on the basis of morphological and reproductive characters by using binocular compound microscope and confirmed with standard literature (Barnett 1960; Ellies, 1971; Mukadam et al., 2006). The pure cultures of the dominant seedborne fungi were maintained on PDA slants until used.
116 c) Test microorganisms The dominant storage seed-borne fungi of sunflower and safflower viz. Aspergillus niger, A. flavus, A. terreus A. fumigatus, A. parasiticus, Fusarium moniliforme, F. oxysporum, Alternaria alternata, Curvularia lunata and Penicillum chrysogenum were selected and used. d) Collection of plant Latex: The fresh latex of Jatropha curcasand Thevetia peruviana was aseptically collected early in the morning from the cut stalk of leaves and young stem in clean and sterilized amber coloured bottles. The samples were brought to the laboratory, kept in refrigerator at 4oC until use. To obtain the powdered latex, it was spread on thin layers over clean glass sheets and kept in a dark cupboard to dry overnight. The latex sample was also oven dried at 42 . The dried latex was subsequently scrapped off the glass sheet with a sharp razor blade. This was pulverized and packed in amber-coloured bottles. Dried latex was powdered using mortar and pestle. e) Extraction of plant Latex The extraction was carried out by using water and solvents like ethanol and methanol. 10gm of dried latex powder was weighed accurately and dissolved in 100 ml of appropriate solvent in an air tight cork bottles and labeled accordingly. The suspended solutions were kept in rotary shaker for 24 hours and the supernatant was concentrated by drying using water bath. Dried extract was used for bioassays and stored at 4ºC until use (Parekh, 2007). f) Determination of antimycotic activity of plant latex Antimycotic activity of Jatropha curcasand Thevetia peruvianalatexextracts against test oil seed-borne fungi was determined, using agar-well diffusion method. (Perez et al., 1990). The extracts were dissolved in DMSO to obtain final concentration of 100 mg/ml. 100 ìl of test compound was introduced into the well and plates were incubated at 28°C ± 2°C for 48-72 hours. Dimethyl sulfoxide (DMSO) was used as a negative control. Carbendazim is used as standard fungicide. RESULTS & DISCUSSION Moist blotter and agar plate methods were employed for isolation of seed –borne fungi. Sunflower seeds yielded total 19 fungal sp. from eight genera and safflower seeds yielded total 19 fungal sp. from six genera. Plant derived compounds (phytochemicals) have been attracting much
117 interest as natural alternatives to synthetic compounds. These natural products provide clues to synthesize new structural types of antimicrobial and antifungal chemicals that are relatively safe to man and it can help to meet expensive and limited supply of synthetic chemicals. Natural fungicides are free from environmental toxicity as compared to synthetic compound (Hooda and Srivastava, 1998). Naturalcompounds are less phytotoxic, easily biodegradable and more systematic (Saxena et al., 2005). The extensive use of agrochemicals especially fungicides, resulted more carcinogenic risk than other pesticides which may give rise to undesirable biological effects on animals and human beings (Osman and Abdulrahaman 2003). Therefore, the development of biofungicides has been focused as a viable fungus control strategy in recent years. In this regard, aqueous, ethanol and methanol latex extract of Jatropha curcas and Thevetia peruviana were screened for their antimycotic efficacy against storage seed borne fungi (Table 1 and 2).The results summerised in table indicate that the latex extracts from the plants showed inhibition of growth against test fungi with to various degrees. Table 1: Efficacy of Jatropha curcas latex extracts on seed -borne fungi of Sunflower and Safflower. (Agar well diffusion method) Sr. No. Test fungi
Latex extracts Aqueous 100mg/ml
Ethanol 100mg/ml
Methanol 100mg/ml
Control
Carbendanzin(50 µg/ml) 1.
Aspergillus niger
-
18
-
30
2.
A. flavus
-
10
10
28
3.
A. fumigatus
-
13
12
20
4.
A. terreus
-
12
-
29
5.
A. parasiticus
-
-
-
25
6.
F. moniliforme
-
-
-
28
7.
F. oxysporum
-
23
-
37
8.
Alternaria alternata
09
11
10
25
9.
Curvularia lunata
09
13
11
32
10.
Penicillium chrysogenum
10
13
11
35
Ethanol latex extracts of J. curcas showed more or less antimycotic activity against test fungi followed by methanol and aqueous extrct. Ethanol
118 latex extracts of J. curcas showed maximum zone of inhibition against F. oxysporum (23mm) and minimum zone of inhibition was observed in A. flavus (10mm). Methanol latex extracts of J. curcas showed maximum zone of inhibition in against A. fumigatus (12mm) and minimum zone of inhibition was found in A. flavus and A. alternata (10mm). Table 2: Effect of Thevetia peruviana latex extracts on seed borne fungi of sunflower and Safflower (Agar well diffusion method). Sr. No. Test fungi
Latex extracts Aqueous 100mg/ml
Ethanol 100mg/ml
Methanol 100mg/ml
Control
Carbendanzin (50 µg/ml) 1.
Aspergillus niger
10
12
10
30
2.
A. flavus
09
14
10
28
3.
A. fumigatus
11
14
13
20
4.
A. terreus
-
14
11
29
5.
A. parasiticus
-
-
-
25
6.
F. moniliforme
10
16
10
28
7.
F. oxysporum
11
21
17
37
8.
Alternaria alternata
11
15
13
25
9.
Curvularia lunata
11
14
11
32
10.
Penicillium chrysogenum
10
15
13
35
Ethanol and methanol latex extracts of T. peruviana latex extracts showed more or less antimycotic activity against all test fungi except A. parasiticus. Ethanol latex extracts of T. peruviana showed maximum zone of inhibition against F. oxysporum (21mm) and minimum zone of inhibition was observed in A. niger (12mm). Methanol latex extracts of T. peruviana showed maximum zone of inhibition in against F. oxysporum (17mm) and minimum zone of inhibition was found in A. niger A. flavus and F. moniliforme (10mm). Aqueous latex extracts showed least antimycotic efficacy as compared to ethanol and methanol. Latex has antimicrobial properties against many species (Thomas et al., 1989). Oyi et al., (2007) studied antimicrobial activity of the latex of J. curcas and reported a broad spectrum of antimicrobial activity against Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Stapylococcus aureus, Streptocococcus pyogenes, Candida albicans and
119 clinical isolates of Trichophyton sp., using agar and broth dilution methods. Mohamad et al., (2012) found that J. curcas latex inhibited the growth of Trametes versicolor, Gleophyllum trabeum, Fusariumoxysporum and Fusarium solani but negative to A. niger which indicate the powdered latex has limited inhibition effect against different type of organisms. Raghanvendra and Mahadevan (2011) also found the same results in case of antimicrobial activities of petroleum ether extracts of different latex namely; C. Papaya, C. Procera, A. heterophyllus fruits, J. curcas, T. peruviana were screened in vitro against two fungal strains, Aspergillus niger and Candida albicans. Upadhyay et al., (2016) reported the hexanoic, methanolic, petroleum ether, chloroform and water latex of Thevetia nerifolia posses antifungal activity against C. albicans, A. niger, R. stolonifer. CONCLUSION Latex extracts of J. curcas, and T. peruviana having potent antimycotic activity against test fungi and can be used for seed treatment agents for seed storage. REFERENCES Afzal, R., S.M.Mughal, Munir, M., Sultana, K., Qureshi, R., Arshad, M. and Laghari, A.K. (2010). Mycoflora associated with seeds of different sunflower cultivars and its management. Pak. J.Bot., 42(1): 435-445. Anonymous. (2007). Sunflower (Helianthus annuus L.). Pakistan Agric. Res.Council. Islamabad, Pakistan. Barnett, H.L. (1960). “Illustrated genera of imperfect fungi” (second ed.) Burgess Publs. Co. Bhajbhuje, M.N. (2014). Biodiversity of seed mycoflora in storage of Brassica campestris L. Int. J. of Life Sciences, 2(4): 289-303. Butt, A.M. and Ali. M. (2005). Implications of increased oil-seed productions on cropping patterns. Proc. Natl Conf. Pakistan, March 15-17. pp. 31-38. Cristensen, C.M. and Kaufman, H.H.(1969). Grain storage. The role of fungi in quality losses. Univ. Minnesota, Press Minneapolis. Ellies, M.B. (1971). Dematiaceous Hyphomycetes. (1st ed.). CAB International, Wallingford Oxon OX10 8DE, UK. Fakir, G.A (1983). Teaching, research and training activities on seed pathology in Bangladesh. Seed Sci Technol. 11:1345-1352. Hooda, K.S. and Srivastava, M.P. (1998). Biochemical response of scented rice as influenced by fungitoxicant and neem products in relation to rice blast. Indian J. Pl. Pathol.16: 64-66. ISTA, (1996). Seed Science and Technology 21(Suppl.): 1B288.
120 Kanokwiroon K, Teanpaisan, R., Witistuwannakul, D., Kooper, A.B. and Witistuwannakul, R. (2008). Antimicrobial activity of protein purified from the latex of Hevea brasilensis on oral microorganism. Mycoses, 51: 301- 307. Mohamad Syakir, M.S., Ismail, J., Zaini, A., Diyana, I.N. (2012). Bioactivities of jatropha curcas linn latex. 4 th Regional Conference on Natural Resources in the Tropics (NTrop4). Mukadam, D.S., Patil, M.S., Chavan, A.M. and Patil, A.R. (2006). The Illustrations of Fungi. (1st ed.). AksharGanga Prakashan, Aurangabad. Osman, K.A. and Abdulrahman, H.T. (2003). Risk assessment of pesticide to human and the environment. Saudi J. Biol. Sci. 10: 81-106. Oyi, A.R., Onaolapo, J.A., Haruna, A.K. and Morah, C.O. (2007). Antimicrobial screening and stability studies of the crude extract of jatropha curcas linn latex (Euphorbiaceae)Nig. Journ. Pharm. Sci.,6 (2)14–20. Parekh, J. and Chanda, S.V. (2007). In-vitro antimicrobaial activity and phytochemical analysis of some Indian medicinal plants. Turk. J. Biol., 31: 53-58. Perez, C., Pauli, M. and Bazevque, P. (1990). An antibiotic assay by the agar well diffusion method. Acta Biol Med Exp., 15: 113-115. Raghanvendra, R. and G. D. Mahadevan (2011). In vitro antimicrobial activity of various plant latex against resistant human pathogens. Int. J. Pharm. Pharma. Sci., 3:7072. Santos, A. and Van Ree, R. (2011) Profilins: Mimickers of allergy or relevant allergens? Int. Arch AllergyImmunol, 155:191-204. Saxena, A.R., Sahni, R.K., Yadav, H.L., Upadhyay, S.K. and Saxena, M. (2005). Antifungal activity of some higher plants against Fusarium oxysporum f.sp. pisi. J. Liv. World. 12: 32-39. Schwinn, F.J. (1994). Seed treatment - A pancea for plant protection? In: Seedtreatment: Progress and Prospects Mono. 57, BCPC, Thornton Health, UK, pp.3-14. Shahda W.T., Tarabeih A.M., Michail S.H. and Hemeda A.A.H. (1991). Fungi associated with sunflower seeds in Egypt with reference to chemical control measure. J. King Saud Univ. Agric. Sci. 3:287–299. Siritaperawee J,T. and Samasoonsuk, W. (2012) Antimicribial activity of a 48-Kda protease (AM p48) from Atrocarpus hererophylllus latex. Eur. Rev. Med. Pharmacol. Sci, 16: 132-137. Thomas, O.O. (1989).Re-examination of the antimicrobial activity of Xylopla aethiopica, Carica papaya, Ocimum gratissimum, and Jatropha curcas. Filoterapia., 60(2): 147-155. Upadhyay, R.K., Prajapati, K.K. and Gupta, R.K. (2016). Evaluation of antibacterial and antifungal activity of latexes from selected indian plant species. World Journal of Pharmaceutical Research, 5(9),1286-1310. Yadav, V. B., Bharud, R.W. and Nagawade, D.R. (2014). Biochemical changesassociated with storage of summer groundnut (Arachis hypogaea L.) SeedsJournal of Crop Science, 5(1)112-115.
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6 SURVEY IN PLANT PATHOLOGY: NEED, PLANNING, PREPARATION, PERIOD, PROCUREMENTS, PRECAUTIONS, SAMPLING AND EFFICIENCY P.K. Shukla ICAR-Central Institute for Subtropical Horticulture, Rehmankhera, PO:Kakori, Lucknow-226101 E-mail:
[email protected]
INTRODUCTION Survey is a common practice to update the status of incidence and severity of diseases and other plant pathology related subjects. It may be conducted in fixed plots for monitoring the dynamism of diseases on selected crops, or roving but region specific to understand area specific problems or to cover larger geographical locations with wider targets. It may be for assessment of population of plant parasitic nematodes associated with particular crop or for distribution of species in different agro-ecological zones. It may also target incidence and severity of diseases in crops. It may also be done for collection of mycological samples, viz. mushrooms. The articles is written after gaining experience from the survey of plant parasitic nematodes with Menthaspp. during 1990 to 2000 in Udham Singh Nagar, Rampur, Bareilly, Moradabad, Badaun and Bulandshahar; incidence of wilt and association of parasitic nematodes with chickpea, pigeon pea, tomato, chilli and brinjal during 2000 to 2004 in Bulandshahar, Aligarh, Hathras, Agra and Mathura; incidence of khaira, blast, leaf blight of paddy, wheat and potato during 2004 to 2005 in Maunath Bhanjan; mushrooms during 2005 to 2009 in Faizabad, Sultanpur and Mirzapur;
122 incidence of wilt, decline and other diseases of mango and guava during 2011 to 2018 in Bihar, Gujarat, Himachal Pradesh, Jharkhand, Karnataka, Kerala, Madhya Pradesh, Maharashtra, Odisha, Punjab, Rajasthan, Tamil Nadu, Telangana, Uttar Pradesh and West Bengal (Shukla and Haseeb1991, 2002, 2004; Haseeb and Shukla 1994; Haseeb et al. 2002; Ahmad and Shukla 2003; Shukla et al. 2006, 2015, 2016, 2017a,b, 2018; Mishra et al. 2008). More than 75000 km roadside crop survey was carried out in about 27 years. The author has learnt new from every survey and it is going on. Looking into difference in approach during initial period of survey and later, it was thought necessary to share the experience. Need of survey Incidence, severity and distribution of diseases and associated pathogens are ever changing. Cropping patterns, cropping intensity, cultivars, soil types, crop production practices, weather and climate play great role in determining the extent of disease. Major diseases become minor and minor becomes major, and even new diseases emerge as great threat. All these components compel plant pathology workers to regularly keep an eye on incidence and severity of diseases to optimize management strategies, quarantine recommendations and for forewarning the growers. India has vast diversity in geography, climatic conditions, vegetation and mushroom wealth. Several naturally growing mushrooms have been collected and made available in local markets. Few of those were domesticated, others could not be and many more remain still unexplored. Target setting Targets of the survey should be clear e.g. crop, disease(s), data to be recorded, area to be covered, number of locations per day, distance between two nearest locations, number of samples from one location, total number of samples to be collected in one trip and interval of surveys in fixed plot survey. After fixing the targets, planning is the most important component for achieving the targets. Planning should be made for every smallest point related to survey and these points should fulfil the needs of the set targets. Period of survey is also an important component. If annual crop is to be surveyed, it must be at the stage of maximum severity of targeted disease. In case of perennial crop, it should be at critical stage according to the target disease. If mushroom has to be collected, it should be done during specific period of incidence and critical stages. Collection of mushrooms can only be done during the specific period of their
123 occurrence, which varies from species to species and from location to location. The actual period of their natural occurrence and critical factors contributing their production must be known. Preparations necessary Preparation for survey is very important. Dressing should be done according to work. A wide variety of weeds, including thorny ones; sharp stubbles of previous crops and animals like snakes may be encountered while moving to field or inside the field. Therefore, tough cloths and strong long water resistant boots should be preferred. Thick cap or hat protects from direct Sun heat as well as direct cooling of air conditioner in vehicle. Cap protects from alternate cooling and heating of head, which may cause cold and fever. One shoulder bag/ survey kit is also necessary to put necessary materials and samples collected from a field. Planning The survey must be well planned according to the fixed targets and objectives. All aspects of survey, such as crop, disease, methodology, data to be recorded, proforma, sample collection, area, period, etc., must be thoroughly worked out and defined before starting the work. Survey plan is also important, particularly if it is to be carried out at distant places. One may have to travel by plane, train or bus before hiring vehicle at location of survey. Accordingly, tour programme should be planned well in advance to book confirmed tickets. The estimates for per day road journey, availability of roads and location of night halt should be pre decided with alternate options with the help of Google maps. The political and social aspects of targeted area should also be taken into consideration. Several regions of our country have Naxalite problems. While deciding the locations of survey; such risks should also be avoided. Data bases for crops should also be taken into consideration to pin point cropped area for survey. In accordance with the objective of the survey, minimum distance between two fields for sample collection should also be decided. Each survey targeting specific problem ofa particular crop require unique preparations and execution. Some examples are being discussed for creating best possible understanding: 1. Collection of soil and root samples for assessment of plant parasitic nematodes associated with crops or their diversity in geographical region. 2. Assessment of incidence and severity of diseases on aerial portion
124 of crops. 3. Assessment of incidence and severity of soil-borne diseases of crops. 4. Collection of mushrooms from their wild habitat. 5. Collection of soil inhabiting fungi for bio-control research. 1. Collection of soil and root samples for assessment of plant parasitic nematodes associated with crops or their diversity in geographical region Since majority of plant parasitic nematodes are soil inhabitant, soil and root samples are necessarily collected during survey. Therefore, first of all materials like GPS, perforated polypropylenebags of appropriate size, aluminium tags, glass marker pen, thread or rubber bands, scud, garden spade, auger/ cylindrical tube type soil sampler and aerated containers should be procured for collection of samples.Since plant parasitic nematodes are delicate, thin and short lived microbes, especial care must be taken to save the samples from pressure on samples, direct Sun, temperature above 30 oC and shortage of moisture in samples. Collected samples should be brought to the laboratory and be processed at the earliest possible, not later than 4-5 days after collection. Therefore, duration for each survey should not be for more than 4 days. Period of survey is very important. Annual crops are grown during specific periods. To survey a rainy season crop, one has to wait for at least 7 days rain-free period,otherwise it may be difficult to enter the field and collect samples. Collection of samples from crops like paddy, or in flooded fields is further difficult. During winters, different types of problemsmay be encountered, viz. less sunshine hours; heavy dews caused wetness on crop in morning hours, foggy weather and unseasonal rains or irrigated fields. When survey is done for perennial crops, relatively fewer difficulties are faced. Survey can be planned during moderate weather. If any person is looking nearby the field selected for sampling, he should be contacted for collecting information of location, cropping history, input use, cultivar, etc. and most important is the permission to enter the field and dig around plants. Some farmers do not allow. If one has not taken permission and farmer comes, he may go to any extent.In case of cucurbits, majority of farmers do not allow to enter the field wearing shoes. Method of collection of samples should also be appropriate and scientific enough to represent the field (Fig. 1). The size of sampling area and number
125 of spots of sample collection vary according to the purpose of survey (Barker, 1985). The size of sampling area also depends on the size of cropped area and area to be covered. Different sampling techniques and patterns have been described (Barker, 1985), therefore, according to the purpose of sampling appropriate decisions should be made. Since nematodes feed on growing young roots, therefore, sample should be taken from feeder root zone.
Fig. 1.Systematic sampling pattern for nematodes.
2. Assessment of incidence and severity of diseases on aerial portion of crops So many diseases of economic significance occur on above-ground parts of crops. The incidence and severity of such diseases vary among the locations and cultivars. Collection of diseased samples during survey is necessary for confirmation of associated pathogens. Therefore, materials like GPS, perforated polypropylene or paper bags of appropriate size, glass marker pen, thread, rubber bands, stapler, secateurs, knife and aerated containers should be procured for collection of samples. Since plant samples may have spores of saprophytic fungi on their surface, either these should be surface sterilized during survey or should be kept in paper bags with limited aeration to avoid moist chamber effect and also drying. If woody samples, twigs, are collected from perennial plants, these can also be kept in paper bags. Collected samples should be properly tagged or labelled, brought to the laboratory and be processed at the earliest, not later than 45 days after collection for getting best results. Period of survey varies according to the targets. In annual crops, expression of symptoms of some of the diseases may be within one month
126 after germination, may be at flowering stage or towards maturity. For assessment of incidence and severity of disease and resulting losses, specific crop stages for recording of data are defined for different diseases. In perennial crops, critical stages for each disease are also known. Therefore, dates of survey should always be decided according to critical stages of crop. Assessment of disease incidence and severity is not an easy task under field conditions unless appropriate method for collection of data is followed systematically. Care should be taken to cover whole field or orchard and sufficient number of plants should be taken into account. 3. Assessment of incidence and severity of soil-borne diseases of crops There are many similarities in survey for plant parasitic nematodes and the soil borne diseases caused by fungi. Therefore, many points viz. procurement of materials, period of survey, collection of samples and care for safe transit are similar as discussed in survey of nematodes. Since, presence of soil borne fungi is mostly confirmed by infection into roots through isolation in laboratory, it is not necessary to collect soil samples. However, when dealing with perennial crops, chances of getting infection in older roots is more than the fine roots. Therefore, one has to dig, find primary roots,scrap to find symptoms and collect cut pieces from several points of healthy, infected and damaged root portions. After collection from field, such root pieces should be washed, surface sterilized and rinsed thoroughly every day after completing the survey work. 4. Collection of mushrooms from their wild habitat On account of saprophytic nutritional habit, mushrooms in nature are generally found to grow in places where dead remains of plants, such as leaves, straw, logs, etc. are decaying; hence in forests, fields and meadows, mushrooms grow in abundance. Although, majority of mushrooms are saprophytes, yet some of them either grow upon living plants as parasite or in symbiotic association with living plants. The saprophytic mushrooms grow upon dung, soil, wood, straw, leaf-litter, insect, etc. Established woods and forests containing a wide variety of species provide the best places for mushroom collection and it is in such areas that the vast majority of species are to be found. Many of these fungi have a symbiotic relationship with trees and their roots. Soil type is also important. As some fungi will only grow with a particular tree on a particular soil, rather than across the whole range of soils. Since mushrooms grow well under high relative humidity
127 conditions, their abundance can be observed during rainy season. At places with dense vegetation and continuous availability of water, mushrooms can grow round the year. Collection from wild growths is still limited to the few known types and that too is an art restricted to the natives and tribes, where this knowledge has descended from generation to generation. In areas, where people do not have this kind of a background, vast amounts of most delicious mushrooms can be seen rotting in the fields and woods. While going for collection of mushrooms, one should have mushroom field guide, note book, hand basket, sharp knife, soft and tough brushes, polythene bags, disposable gloves, tissue paper, strong stick, tough cloths, strong long boots, cap, etc.Be careful of the surrounding countryside and its animals. Mushrooms should be searched, recognized and collected with proper care. If collection is only for edible mushrooms, never collect doubtful species, it is better to cut them from substrate surface without disturbing their under substrate portion. If collection is being done for new search and identification, dig out the specimen with its under substrate portion and keep in separate polythene bag, tag with a serial number and note down the relevant information in note book. Collected mushrooms should be kept in basket and be saved from physical damage. The specimens should be processed at an earliest possible because some of the field characters like colour, shape, size and aroma of stipe, pileus and lamellae change after drying, hence field characters should be described with fresh specimen. 5. Collection of soil inhabiting fungi for bio-control research The soil is a living system containing minerals, organic compounds and living organisms (Allan et al. 1995). The soil is a dynamic system that serves as natural medium of growth of land plants and is inhabited by a wide range of microorganisms. The microbial inhabitants consist of autotrophs, saprophytes, mutualists, parasites of plants and antagonists of other soil microbes. Growth of the soil microorganisms may be influenced by both direct stimulation and indirectly through a variety of interactions. Successful biological control of soil inhabiting plant pathogens requires complete understanding of the interactive functions of microflora and fauna in the specific soil ecology (Lartey and Conway 2004).The success of crops is influenced by direct relationships such as disease induction and growth stimulation and indirectly through a variety of interactions among the microbial populations. Under optimal conditions, the pathogenic populations cause various diseases which lead to reduced plant health and yield loss. Antagonists of pathogenic populations reduce inoculum potential
128 and suppress diseases, thereby enhancing plant growth and yield (Lartey 2006). With this brief background, biological disease management has become popular throughout the world. However, one isolate successful at one location may not be effective at different location. Due to this, preference is given to find local isolates of efficient bio-control agents. Survey for collection of antagonists can be carried out in target location at any time. It should preferably be done in cropped area. If it is targeted to find antagonists for a particular disease of a particular crop, the samples should also be collected from rhizosphere of the same crop. The isolation may be done according to the target species of antagonist on selective medium. Salient Points to enhance efficiency during survey The maximum output is necessary in stipulated time during survey. To achieve it, one should take care on the following points: 1. Timings Survey work should be started before sunrise and be continued even after sunset till visibility is proper. However, calculation of number of fields targeted for the day, number of fields done, distance covered, remaining number of fields and distance should be reviewed at regular intervals throughout the day and adjustments should be made to reach the place of night halt before dark. 2. Food If one follows above-mentioned timings, breakfast cannot be taken before starting survey and if time has to be saved never take lunch in a restaurant. Since, survey areas are mostly remote ones where hygienic food availability is rare and sometimes it becomes impossible to get any food at roadside, therefore, best to keep fruits and food of your choice and drinking water stored in vehicle. Eat only when vehicle is running to save time. 3. Movement in field Use maximum efficiency while moving from one spot to other spot in field and while coming back after collection of data and sampling. Distribute the work among the team members e.g. recording of data, collection of samples and interaction with farmers (if available). Team members should always be visible to each other and be able to communicate necessary information. It is risky and problematic if members are dispersed and are out of site.
129 4. Vehicle speed Before starting survey, driver should be informed with the targets for the day. He must know which crop is targeted, what is the minimum distance to observe next field after recording observations in one field, what speed is proper to observe and identify suitable field for recording observations. If driver is deviating from instructions, time to time remind him. Also take care of drivers’ health, food and sleep. Literature for distribution to farmers After all, our research is for the benefit of farmers. Always keep relevant literature in the form of folders for distribution to farmers. Try to satisfy farmers’ queries in brief without losing your time and targets. Acknowledgements Author is thankful to Indian Council of Agricultural Research, New Delhi for funding under AP Cess Fund Scheme during 1990 to 1996; NATP during 2000 to 2004; AICMIP during 2005 to 2009; NICRA (Pest), AICRP (Fruits) and ERP during 2011 to till date for carrying out survey on diseases caused by nematodes, bacteria and fungi to mints, pulse, vegetable and fruit crops, and for collection of mushrooms from their natural habitat. Thanks are also due to Council of Scientific & Industrial Research for funding during 1996 to 2000 research and survey on nematode diseases of Mentha spp. Author is also thankful to the Directors of CSIR-CIMAP and ICARCISH, Lucknow; Vice Chancellors of AMU, Aligarh and NDUAT, Kumarganj, Faizabad; and Dr. Akhtar Haseeb, Scientist, CSIR-CIMAP and Professor, AMU, Aligarh for their kind guidance and support. Contribution of team members (Dr. R.M. Khan, Dr.Gundappa, Dr.Dushyant Mishra, Mr. R.P. Shankhwar, Mr. Vipin Kumar, Ms. Kahkashan Perveen, Dr. Abrar Ahmad, Mr. Gyan Prakashash Gautam, Mr. Vijay Kant, Dr. SavitaV arma, Mr. Chandrasen, Mr. Surya Kant Yadav, Mr. Ravi Kumar Kushwaha, Dr.Tahseen Fatima, Mr. Rajesh Kumar Gautam, Mr. Shobhit Verma, Mr. Harish Pal) is also acknowledged who has given wide variety experiences during survey. Thanks are also due to Dr. S.K. Shukla, Principal Scientist, ICAR-CISH for providing inputs in the preparation of manuscript and to the most respected farmers throughout the country for their kind help, supportand experience sharing. References Ahmad, A. and Shukla, P.K. (2003). Community analysis of the nematodes associated
130 with vegetable crops in Aligarh, Uttar Pradesh, India. Current Nematology 14(1,2): 27-29. Allan, D.L., Adriano, D.C., Bezdicek, D.F., Cline, R.G. and Coleman D.C. (1995). Statement on Soil Quality. In: Agronomy News, Doran, J.W. and J. Haberen (Eds.). Soil Science Society of America, Madison, WI, p. 7. Barker, K.R. (1985). Sampling nematode communities. In: An Advance Treatize onMeloidogyne. Volume II Methodology.Department of Plant Pathology and the United States Agency for International development Publication, pp. 3-17. Haseeb, A. and Shukla, P.K. (1994). Studies on vertical distribution of plant parasitic nematodes associated with MenthapiperitaL. Proceedings of the SecondAfro-Asian Nematology symposium (18 th-22 nd December 1994) Menoufiya, Cairo, Egypt, pp.121-124. Haseeb, A., Shukla, P.K., Ahmad, A. and Kumar, V. (2002). Survey of farmer’s fields for the association of root-knot nematodes and wilt fungi with eggplant and quantification of losses.Current Nematology 13(1,2): 69-71. Lartey, R.T. (2006). Dynamics of soil flora and fauna in biological control of soil inhabiting plant pathogens. Plant Pathology Journal 5: 125-142. Lartey, R.T. and Conway, K.E. (2004). Novel considerations in biological control of plant pathogens: microbial interactions. In: Emerging Concepts in Plant Health Management, Lartey, R.T. and A.J. Ceasar (Eds.). Research Signpost, Trivandrum, India, pp: 141-157. Mishra, R.S., Singh, R.V. and Shukla, P.K. (2008). Isolation of mycoflora from compost and casing soil of white button mushroom.Journal of Mycology and Plant Pathology 37: 424-425. Shukla P.K., Adak, T., Gundappa and Misra A.K. (2016). Weather based models in predicting powdery mildew of mango for Malihabad mango belt. Journal of Ecofriendly Agriculture 11(2): 142-148. Shukla, P.K. and Haseeb, A. (1991). Studies on the vertical distribution of plant parasitic nematodes under Menthacitrata Ehrh. Indian Journal of Plant Pathology 9: 1923. Shukla, P.K. and Haseeb, A. (2002). Survey of farmer’s fields for the association of plant parasitic nematodes and wilt fungi with pigeonpea and quantification of losses.Indian Journal of Nematology 32: 162-164. Shukla, P.K. and Haseeb, A. (2004). Studies on occurrence, pathogenic potential and management of reniform nematode on spearmint. Indian Journal of Nematology 34(2): 140-146. Shukla, P.K., Adak, T. and Gundappa (2017). Anthracnose disease dynamics of mango orchards in relation to humid thermal index under subtropical climatic condition. Journal of Agrometeorology 19(1): 56-61. Shukla, P.K., Adak, T., Misra, A.K. and Achal Singh (2016). Appraisal of shoulder browning disease of mango (Mangiferaindica L.) in subtropical region of India.Journal of Mycology & Plant Pathology 46(1): 38-46.
131 Shukla, P.K., Gundappa and Adak, T. (2017). Development of sooty moulds in mango orchards in relation to weather parameters and major sucking pests.Journal of Environmental Biology 38: 1293-1300. Shukla, P.K., Pandey, M.K., Pandey, P.S. and Singh, C.M. (2006). Occurrence of leaf blight of wheat in reclaimed usar soil. Indian Phytopathology 59(2): 240-242. Shukla, P.K., Varma, S., Fatima, T., Mishra, R., Misra, A.K., Bajpai, A., Gundappa and Muthukumar, M. (2018). First report on wilt disease of mango caused by Ceratocystisfimbriatain Uttar Pradesh. Indian Phytopathology 71(1): 135-142.
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7 INTEGRATED MANAGEMENT OF TEMPERATE PERENNIAL FODDER GRASS DISEASES - A BIOTECHNOLOGICAL APPROACH Dilip Kumar Verma1, Suheel Ahmad2, Nazim Hamid Mir3, Siraj Ahmad Bhat4 1.
Principal Scientist (Genetics & Plant Breeding), IARI Regional Station, Indore – 452001, E-mail:
[email protected] 2.
Sr Scientist (Hortipasture), IGFRI RRS Srinagar, J&K – 190032 3.
4.
Scientist (Agronomy), IGFRI RRS Srinagar, J&K – 190032
Sr Scientist (Hortipasture), IGFRI RRS Srinagar, J&K – 190032
ABSTRACT Livestock husbandry plays a significant role in the economy of the Himalayan people with dependence largely on grasslands and pastures. Undoubtedly, almond-based hortipasture is a sustainable option for enhancing farmer’s income. The volatility in price especially almonds negatively impacted the hortipasture farmers’ income in the region. The price stabilization of almonds is a necessary condition for enhancement of farmers’ income. The performance of growing fodder crops viz., tall fescue (IC-0615892), orchard grass (IC-0615914), Harding grass (Phalaris-29), timothy grass (IC-0615761), makhan malai grass (IC-0615872) and two legumes viz., white clover (IC0615818) and red clover (IC-0615581) were tested under the freshly established almond orchard of IGFRI, RRS, Srinagar, J&K, India. Growth of the tested fodder crops was very good and significant in all the cases. It is better suited to the shallow soils and low rainfall situations. The crude protein and crude fibre content of the grass and legume had range from 6.25 to 25.20 and 27.30 to 40.20%, respectively.
134 It had better in vitro digestibility and the cattle acceptance was excellent. The varieties were good performers in the North Western Himalayan region with high nutritive value. It could be raised both by direct sowing or transplanting for which 62,500 seedling/rooted slips/ rooted stolons preferably or 4–6 kg seed was required per hectare. Herbage yield can be increased from 50.5 q/ha to 267.0 q/ha with the application of 80kg N and 40kg P205/ha which in turn will double the farmers income. Although fodder cropsare comparatively less prone to pests and diseases, but we should be cautious enough to tackle the coming problems in future as well as create the more balanced nutritional fodder through the use of biotechnological tools are also imperative. Key words: Almond based hortipasture, Farmers’ income, North Western Himalaya
INTRODUCTION Almonds are the healthiest and most nutritious nuts of all, considered a well-balanced cholesterol free food (Ahmed and Verma, 2009). In India it was 1st introduced to Kashmir during 16th century by Persian settlers but in spite of its great potential in the region, the crop could not be developed on commercial scale as that of apple. The North Western Himalaya region spreads across 3.8 million ha in Jammu & Kashmir, Himachal Pradesh, Uttaranchal, Punjab and Haryana States Chauhan (2003) considered the lap of the Western Himalayas. Katoch (2012) stressed upon livestock rearing plays a significant role in the economy of the Himalayan people with dependence largely on grasslands and pastures. Anonymous (2016) called for development of pastures on scientific lines to boost livestock industry in J and K. Speaking at a review meeting of Agrostology, it was being told, “Since our natural resources have been subjected to tremendous biotic pressure by livestock population, there is urgent need to ensure natural regeneration of pastures besides production of quality fodder.” Yatoo et al (2011) had stressed upon various farming practices which were the pre-dominant in the Himalayan region. Horticulture accounts for 18% of the gross cropped area and the livestock husbandry plays a significant role in the economy of the Himalayan people. The shortage of fodder in the North Western Himalaya, India can be mitigated through agroforestric intervention of almond based hortipasture. The increasing trend in the live stock population has resulted in sharp increase in density of live stock per unit grazing land. This almond based hortipasture models will add to the economy of Kashmir valley. District wise total area and it’s classification was given in table 1.
135 Table 1: District wise total area and it’s classification 2012-13 S.No.
District
Total (land put to non-agricultural uses, barren & uncultivable land, permanent pastures & other grazing lands, land under miscellaneous tree crops not included in area sown, culturable waste land, fallow land other fallows than current and current Fruits & Fodder crops fallows (2012-13) vegetables (2011-12) (2011-12)
1.
Srinagar
1,693
9,782
1,121
2.
Ganderbal
23,367
9,627
113
3.
Budgam
32,346
10,681
1,946
4.
Anantnag
20,869
18,507
167
5.
Kulgam
16,773
4,336
412
6.
Pulwama
27,663
2,484
746
7.
Shopian
17,007
7,955
199
8.
Baramulla
43,780
19,788
333
9.
Bandipora
11,906
2,935
1,075
10.
Kupwara
16,653
3,842
-
11.
Leh
38,432
390
2,082
12.
Kargil
9,379
389
-
13.
Jammu
1,09,787
1,730
9,195
14.
Samba
44,285
75
2,160
15.
Reasi
2,71,093
507
109
16.
Poonch
52,714
28
492
17.
Rajouri
1,05,906
515
451
18.
Udhampur
1,10,229
109
249
19.
Kathua
13,145
14
-
20.
Doda
57,003
379
4,018
21.
Kishtwar
33,537
198
375
22.
Ramban
23,250
28
1,895
Total
9,58,106
94,301
28,859
Data Source: Digest of Statistics 2012 – 13
136 Land use changes may have a major influence on soil quality that is important in sustaining ecosystem productivity. Yet, the effects of these changes on soil quality are not well understood, particularly in poor heterogeneous pastures overgrazed by sheep. The short-term response of soil quality indicators were determined to changes in land use from permanent pastures overgrazed to almond orchards. Soil quality indicators were evaluated in land use systems viz., permanent pastures (PP) overgrazed by sheep, pastures converted to almond orchards (AO) for more than 45 years. Composite soil samples from each random replicates of the land use systems were sampled to a depth of 30 cm, and analyzed for soil bulk density, total N, total organic C, wet aggregate stability, C mineralization, microbial biomass C, and phosphatase activities.Land use changes (i.e. conversion of pastures to almond orchards) resulted in significant increases in soil bulk density, and total organic C and N contents. Similarly, total organic C and N pools. The soil porosity and aggregate stability (MWD) in PP and AO land use systems were significantly greater. Carbon mineralization and microbial biomass C and the activity of acid and alkaline phosphatases were substantially higher in overgrazed pastures and almond orchards. The microbial metabolic quotient (qCO2) in almond fields were marginal. It is also concluded that improvement in soil quality and enhanced biological activity in almond fields demonstrated the gradual resilience of the poor and overgrazed pastures once converted to a fast growing and N2 fixing legume crop (Raiesi, 2007).The physical indicators of the soils in all land use systems are presented in Table 2. Table 2. Some physical characteristics of the topsoil layer (0–30 cm) in different land use systems Soil properties
Land use
Some physical characteristics
permanent pasture
almond orchard
Clay (g kg-1)
347
334
Silt (g kg-1)
405
423
248
243
Clay loam
Clay loam
1.45
1.56
Porosity (cm3 cm 3)
0.454
0.412
wet aggregate stability (mm)
0.572
0.405
7.93
7.70
-
Sand (g kg 1) Texture -
bulk density(Mg m 3) -
Some chemical characteristics pH
137
total organic carbon
7.1
7.4
total nitrogen
0.42
0.47
C/N ratio
17.2
15.6
18.2
21.5
270
309
75.6
115.7
Cmin/Corg (g C kg 1 C)
10.8
16.1
Cmic (mg C kg-1 soil)
115.4
147.6
12.2
10.9
BR (mg C kg 1 soil day 1)
2.14
2.32
qCO2 (mg CO2-C mg-1 Cmic day-1)
18.2
17.3
Acid phosphatase activity (mg pnitrophenol g-1 soil h-1)
0.46
1.05
alkaline phosphatase (mg p-nitrophenol g-1 soil h-1)
09.7
16.6
-
Pa (mg kg 1) available P -
K (mg kg 1) Some biological characteristics Cmin (mg C kg-1 soil) -
-
Cmic/Corg (g C kg 1 C) -
-
Each value represents means ± S.E. (n = 4).
Azhar-Hewitt (1999) reported that in a Karakorum-Himalaya valley, women go to high pastures with the livestock. The soils of sub humid ecosystem of Kashmir region are generally slightly acidic to neutral in reaction, associated with high level of organic matter and leaching of bases where as higher pH reported in some areas due to calcareousness. There are five main categories that the Almond Board (2009) says an almond tree needs to grow efficiently. •
Sunlight: The light from the sun gives almond trees energy which aids in the photosynthesis process.
•
Water: Trees need lots of water to keep growing; they also use water when converting nutrients.
•
Air: Plants breathe, but they breathe through their leaves and roots. This process is called transpiration.
•
Nutrients: Many nutrients are already naturally occurring in the soil. However, growers must add fertilizers and compost to assist the tree in helping produce a good crop.
138 •
Soil: Healthy, loosely-packed, nutrient-rich soil is important for plant health.
Figure 1. Agro Climatic Region: Western Himalayas Region
Figure 2. Ahmed, a young Kashmiri shepherd boy carries a lamb on his back in Dudhpathri, some 55 Kilometers from Srinagar, March 25, 2016
Figure 3. Almond horti-pasture blockIGFRI RRS, Srinagar
Figure 4. Fodder harvesting in almond horti-pasture block-IGFRI RRS, Srinagar
Figure 5. Fodder harvesting in almond horti-pasture block-IGFRI RRS, Srinagar
Figure 6. Fodder harvesting in almond horti-pasture block-IGFRI RRS, Srinagar
139
Figure 7. Fodder in almond horti-pasture block-IGFRI RRS, Srinagar
Figure 8. Fodder in almond horti-pasture block-IGFRI RRS, Srinagar
Figure 9. Fodder in almond horti-pasture block-IGFRI RRS, Srinagar
Figure 10. Fodder in almond hortipasture block-IGFRI RRS, Srinagar
Keeping proper augmentation of fodder availability as an urgent need during 2014-2015 and 2015-2016, the present investigation was made to evaluate the performance of tall fescue (Festuca arundinacea (Schreb) Hack, orchard grass/cocksfoot (Dactylis glomerata L), Harding grass (Phalaris aquatica L), timothy grass (Phleum pratense L), makhan malai grass (Lolium perenne L), white clover (Trifolium repens L) and red clover (Trifolium pratense L.) under almond (Prunus dulcis) -fodder plant combination for horti-pastoraly. When pasture is limited and supplies of fodder were reduced. Many alternative sources were thought to feed to dairy stock, although, they vary widely in nutritive value, digestibility, effective fibre value, and may present risks such as ruminal acidosis, mycotoxins and chemical residues. Almond orchards capture and store a significant amount of carbon both above and below the orchard’s surface. Balancing carbon sequestering agricultural systems such as almond production could reduce the effect of urban heat islands and support farmland production on urban boundaries.”Almond can be grown on different types of soils, but deep, well-drained, loamy soils are ideal. Soils with hard pan, waterlogging and high water table, should be avoided. Almond requires a fairly warm dry weather during ripening of the fruit. Cool and foggy summer does not suit it. Due to early flowering it was very
140 much susceptible to spring frosts. Blossoms with petals exposed but not yet opened are known to withstand cold at –2.2°C. The blossoms can often withstand temperature from –2.2° to –3.3°C for a short time and thus almond based hortipasture will pave a new way for fodder security and fodder availability in North Western India. The hortipasture system is recognized as an important integrated farming practice since time immemorial to fulfil the necessity of food, fodder, fuel wood, fibre and timber along with aesthetic and environmental services. This system is supported by government due to its role in improvement in soil-health, nutrient cycling, carbon sequestration and better economic return to existing cropping systems with less use of natural resources. However, fruit trees were reduced gradually due to more demand of food grains on limited land resources in entire world in general and India in particular. The National Commission on Agriculture, 1976 has suggested for implementation of social forestry programme, which covered farm forestry, extension forestry, reforestation in degraded forests and recreation forestry. A field experiment was conducted on almond (Prunus dulcis) to study the effect of N&K fertigation on growth, yields and leaf nutrient status over two seasons (2011 and 2012) in Srinagar, Jammu and Kashmir, India. The results indicated that the maximum tree height (3.21 m and 3.56 m), nut weight (2.73 g and 1.94 g), nut yield (2.41 kg/tree and 5.98 kg/tree; 2.67 t/ha and 6.64 t/ha), and leaf nutrient content (2.34 and 2.38% N; 0.14 and 0.17% P; 1.37 and 1.41% K) were recorded in RDF through fertigation (split application), whereas the highest TCSA of main trunk, primary, secondary, and tertiary branches (72.67 and 90.28 cm2; 16.75 and 24.26 cm2; 3.83 and 7.49 cm2; 0.47 and 1.23 cm2), canopy volume (7.15 and 8.11 m3), and fruit number (990 and 3083/tree) were recorded in 100% RDF through fertigations in almond variety Waris (Kumar and Ahmed, 2013). Indian Farming To promote hortipasture, various major policy initiatives such as ‘National Agriculture Policy 2000’, ‘Planning Commission Task Force on Greening India 2001’, ‘National Bamboo Mission 2002’, ‘National Policy for Farmers 2007’, ‘Green India Mission 2010’ and finally, a dedicated ‘National Hortipasture Policy’ was approved by Government of India in 2014. This policy has recommended for setting up of a Mission or Board to address development of hortipasture sector in an organized manner. The Sub-Mission on Agroforestry (SMAF) under ‘National Mission on Sustainable Agriculture’ (NMSA) is an initiative to this end. The NMSA,
141 one of the eight mission under ‘National Action Plan on Climate Change’ (NAPCC) seeks to address issues related to climate change through adaptation and mitigation strategies. The aim of the submission is to expand the tree coverage on farmland in complementary with agricultural crops. Farmers allocate their resources in production of various commodities on the basis of signals they receive from markets. The allocation of area under crops is influenced by the perceived relative profitability of different crops. The growing of tree on farms for market seemed to farmers in many regions of India a more profitable option than field crops. However, in spite of relative higher profitability to farmers and several concerted efforts made by government, the adoption of hortipasture has not yet reached the expected desired level. In common parlance, two factors determine the adoption of hortipasture in any specific region, viz. relative profitability with other crops and price volatility of wood. Ahmed, et al. (2012) analysed genetic diversity in walnut genotypes from Jammu and Kashmir, India. The field site was located in the Karewa land of the Doodhganga tributary of Jhelum river close to (North Western Himalaya; Budgam district lies between 33.9349° N latitude and 74.6400° E longitude). The region around Karewa Damodar was characterized by temperate climate. The experiment was performed in three replications keeping the plot size 15m x 15m; spacing between plant to plant for apple was kept at 5m. The experimental grass viz., tall fescue (Festuca arundinacea (Schreb) Hack, orchard grass/cocksfoot (Dactylis glomerata L), Harding grass (Phalaris aquatica L), timothy grass (Phleum pratense L), makhan malai grass (Lolium perenne L), white clover (Trifolium repens L) and red clover (Trifolium pratense L) (Table 3 and Table 4.) was kept 90cm for row to row and 30cm for plant to plant under freshly planted almond orchard at IGFRI RRS Srinagar, J&K. In total, 36 large plots, 15 × 15 m in size, were established crossing the experimental factors richness in a near-orthogonal design. All plots were regularly weeded. Plots were mown twice a year in early June and September to mimic the traditional management of extensive hay meadows in the region. Table3. Layout plan of almond based hortipasture experiment at IGFRI RRS Srinagar, J&K Treat-1 Treat-2 Treat-3 Treat-4 Treat-5 Treat-6 Treat-7 Treat-8 Treat-9 Treat- Treat- Treat10 11 12
TF
TF
Ti
Ti
HG
HG
MMG MMG OG
OG
RC
RC
OG
OG
TF
TF
RC
RC
RC
RC
HG
HG
TF
TF
HG
HG
RC
RC
OG
OG
OG
OG
RC
RC
OG
OG
142
Ti
Ti
OG
OG
Ti
Ti
RC
RC
Ti
Ti
HG
HG
Treat-1: G1: Tall fescue [Festuca arundinacea (Schreb) Hack] Treat-2: G2: Orchard grass [Dactylis glomerata L] Treat-3: G3: Harding grass [Phalaris aquatica L] Treat-4: G4: Timothy grass [Phleum pratense L] Treat-5: L: Red Clover [Trifolium pratense L] Treat-6: G1 + L: Tall fescue [Festuca arundinacea (Schreb) Hack] + Red Clover [Trifolium pratense L] Treat-7: G2 + L: Orchard grass [Dactylis glomerata L] + Red Clover [Trifolium pratense L] Treat-8: G3 + L: Harding grass [Phalaris aquatica L] + Red Clover [Trifolium pratense L] Treat-9: G4 + L: Timothy grass [Phleum pratense L] + Red Clover [Trifolium pratense L] Treat-10: G1 + G2 + G3 + G4 + L: Tall fescue [Festuca arundinacea (Schreb) Hack] + Orchard grass [Dactylis glomerata L] + Harding grass [Phalaris aquatica L] + Timothy [Phleum pratense L] + Red Clover [Trifolium pratense L] Treat-11: Control plot without Grasses and Legumes Treat-12: G5: Makhan malai grass [Lolium perenne L] Table 4. Details of tall fescue, orchard grass, Harding grass, timothy grass, white clover and red clover under experiment. Grasses and legumes
Collector No
IGFRI RRS No
NBPGR, N Delhi Accession No
Tall fescue (Festuca arundinacea (Schreb) Hack
Verma-14
Festuca-14
IC-0615892
Orchard grass/cocksfoot (Dactylis glomerata L)
Verma-36
Dactylis-11
IC-0615914
Harding grass (Phalaris aquatica L) Verma-80
Phalaris-29
-
Timothy (Phleum pratense L]
Verma-146
Phleum-23
IC-0615761
Makhan malai grass (Lolium perenne L]
Verma-191
Lolium-38
IC-0615872
White clover (Trifolium repens L.)
Verma-220
Tr-23
IC-0615818
Red clover (Trifolium pratense L.)
Verma-238
Tp-13
IC-0615781
The accessions viz., IC-0615892 of tall fescue, IC-0615914 of orchard grass, Phalaris-29 of Harding grass, IC-0615761 of timothy grass,
143 makhan malai grass, IC-0615818 of white clover and IC-0615781 of red clover were further evaluated for different important fodder traits at K D Farm during 2014-2015 and 2015-2016 for two consecutive years in the Randomized Block Design in three replications. Accessions were selected randomly at full grown stage from IGFRI RRS Srinagar, J&K pastoraleum block of the earmarked aluminium labels. Relative profitability of hortipasture system in North Western Himalaya, India The specific feature of this region is comparatively high land productivity level and delicate water balance as exploitation of ground water has already surpassed 100% of utilizable balance. The yield level of rice and wheat crops experienced stagnancy and net profit showed diminishing trend and created many complications in agro-ecosystem in the region. In such a scenario, farmers of this region need to diversify rotation through appropriate and sustainable land use system to enhance their income. The almond based hortipasture had proved highly remunerative as economically viable option for crop diversification in the region. Almond shells are available in profusion as a local waste material in most parts of Jammu and Kashmir. It is a woody fibrous material having very good value if used as a fuel and in this study an attempt has been made to use this as a stabilizing agent for geotechnical applications. Since it is a biodegradable material and its strength can be affected by ageing and termite action, hence a diminutive percentage of lime was added to optimize its age and binding capacity with soil. An extensive experimental program on the natural and amended soil after adding different percentages by weight of soil, of the stabilizer was undertaken and its physio-chemical properties were determined in the form of Atterberg limits. To understand its strength behavior, Optimum Moisture Content (OMC), Maximum Dry Density (MDD) and Unconfined Compressive Strength (UCS) tests were performed for all the design mixes at different curing periods. It was found that the OMC and MDD were lowest for a mixture of Soil + 2% powder + 0.5% lime. The maximum value of UCS was found to be 0.076N/mm2 for this mixture after 28 days of curing. Scanning Electron Microscopy (SEM) and Powdered X – Ray Diffraction (PXRD) was also conducted on these design mixes to understand their quantitative as well as qualitative behaviour. It was found that the experimental results matched well with the microstructural characteristics for all these design mixes (Wafa et al, 2018)
144 At this time plants were adequately developed to exclude establishment effects. The single shoots were the basic unit for all measurements. Five to seven shoots per accessions and plot were randomly selected along a line transect divided into fixed sections, excluding the outer 50cm from the plot margin. To avoid sampling from a single genet twice, the minimum distance between sampled plants was 1m. For tall fescue, orchard grass, timothy grass, makhan malai grass, the observations were days to 50% flowering i.e. days from cut to flowering,maturity duration i.e. days from cut to 1st picking, plant height at 50% flowering (cm), number of total tillers/meter row length, spike length, spike weight, number of spikelets/spike, 1000-seed weight, fresh fodder yield and dry fodder yield while for white clover and red clover, the observations were perennial herb length,petiole length, flowering head length (condensed spikes), number of flowers/flower, peduncle length, leaflets/pinnae,number of pinnae/leaf (pairs), 1000-seed weight, fresh fodder yield and dry fodder yield, were main parameters for identifying the genotypes. But in case of Harding grass the identifying parameters were plant height at 50% flowering, number of vegetative tillers plant-1, number of reproductive tillers plant-1, tussock diameter, culm length, leaf blade length, leaf blade breadth, leaf area, leaf weight, stem weight, leaf/stem ratio, fresh fodder yield, dry fodder yield, spike/inflorescence/panicle length, spike or inflorescence weight, glume length, lemma length and spikelet length. Rooted slips of identified accessions were collected and the chemical composition viz., crude protein %, crude fibre %, ether extract %, nitrogen free extract %, ash content %, dry matter digestibility % were estimated in the Plant Animal Relationship Division, IGFRI Laboratory, Jhansi following the standard methods. Trait values for almond trees (Table 10) were averaged per sampling unit and plot for each harvest campaign. The quantitative data collected were subjected to analysis of variance procedure of the Statistical Analysis System package (SAS 1988) and means were separated and compared using the Least Significant Difference (LSD) procedure at a 5% level of significance. Table 5. Averaged trait values for almond trees per sampling unit and plot Almond block treatments
Almond tree growth variables (Data recorded on 1st January, 2016) Plant height
Stem diameter
No of Branches
Treatment-1
130.47
5.80
17.53
Treatment-2
128.87
5.13
16.93
145
Treatment-3:
135.13
5.73
14.67
Treatment-4:
118.33
4.33
10.73
Treatment-5
127.20
4.60
13.40
Treatment-6
115.27
4.53
8.00
Treatment-7
121.47
4.67
11.93
Treatment-8
136.47
4.40
14.47
Treatment-9
120.00
4.47
10.80
Treatment-10
118.80
4.40
12.87
Treatment-11
115.60
4.87
11.27
Treatment-12
121.00
5.27
11.33
Mean ± SE
124.05±2.11
4.85±0.15
12.83±0.79
Range
115.27-136.47
4.33-5.80
8.00-17.53
SD
7.34
0.52
2.74
To account for this design imbalance, data were analysed with mixed-effects models. The effects of richness and the presence/absence of particular were assessed. The tree growth variables for almond trees was recorded (date of observation: 01.01.2016) in the experiment for plant height, stem diameter and number of branches on the one year old almond orchard, which were significant. Averaged trait values for almond trees per sampling unit and plot (Table 10) were superior over treatment means for treatment-1 (G1: tall fescue [Festuca arundinacea (Schreb) Hack]), treatment-2 (G2: orchard grass [Dactylis glomerata L]) and treatment-3 (G3: Harding grass [Phalaris aquatica L]). The chemical composition for crude protein, crude fiber, ether extract, nitrogen free extract, ash content and dry matter digestibility of tall fescue, orchard grass/cocksfoot, white clover and red clover as determined at Plant Animal Relationship Laboratory, IGFRI, Jhansi, Uttar Pradesh, India has been presented in table 11. The CP concentrations in the grass components were generally lower than this critical level of 7%. The higher CP values indicate that the legume probably had a positive effect on the quality of the associated species.
NBPGR, N Delhi Accession No
IC-0615892 IC-0615914 Phalaris-29 IC-0615761 IC-0615872 IC-0615818 IC-0615781
Grasses and legumes
Tall fescue (Festuca arundinacea (Schreb) Hack
Orchard grass/ cocksfoot (Dactylis glomerata L)
Harding grass (Phalaris aquatica L)
Timothy (Phleum pratense L)
Makhan malai grass (Lolium perenne L)
White clover (Trifolium repens L.)
Red clover (Trifolium pratense L.)
18.8
19.6
16.2
8.1
25.2
16.3
6.25
CP%
35.6
35.2
27.3
30.3
40.2
30.7
40.00
CF%
2.8
3.6
4.4
1.4
4.9
4.0
1.02
EE%
33.2
32.2
42.5
51.4
16.4
39.33
57.10
NFE%
Chemical composition
Table 6. The chemical composition of tall fescue, orchard grass/cocksfoot, white clover and red clover
9.6
9.4
9.6
8.8
13.3
9.7
8.85
Ash%
60-70
60-65
65-70
55-60
65-70
60-70
50-55
DMD%
146
147 The range and mean of the quantitative traits over the period (2015 and 2016) for tall fescue (Festuca arundinacea (Schreb), Harding grass, timothy grass (Phleum pratense L), makhan malai grass (Lolium perenne L), white clover, red clover (Trifolium pratense L.)were being supported by the findings viz., Progress in breeding perennial clovers for temperate agriculture (Abberton and Marshall, 2005); Anonymous (2013-14, 201415, 2015-16); designer pasture plants: from single cells to the field (Mouradov, 2010); biomass productivity of mixtures (Trenbath, 1974) and the genotype (Verma et al, 2016a,b,c,d,e,f). Table 7. The quantitative traits of tall fescue (Festuca arundinacea (Schreb) over the period (2015 and 2016) Phenological traits
Range
SD
Mean± SE
Days to 50% flowering i.e. days from cut to flowering
58.33-72.66
12.14
67.33±3.23
Maturity duration i.e. days from cut to 1st picking
92.33-104.77
15.12
98.44±3.26
Plant height at 50% flowering (cm)
92.04-129.34
15.16
102.44±3.25
Number of total tillers/meter row length
1.20-8.50
2.22
3.74±0.56
Fresh fodder yield
5.92-7.95
0.60
6.68±0.13
Dry fodder yield
3.29-4.42
0.31
3.68±0.67
Table 8. The quantitative traits of orchard grass/ cocksfoot (Dactylis glomerata L)over the period (2015 and 2016) Phenological traits
Range
SD
Mean± SE
Days to 50% flowering i.e. days from cut to flowering
53.33-78.66
4.21
67.33±1.11
Maturity duration i.e. days from cut to 1st picking
93.77-105.33
18.28
118.56±6.82
Plant height at 50% flowering (cm)
92.36-126.44
4.11
6.30±0.89
Number of total tillers/meter row length
1.00-7.00
1.57
3.80±0.32
Fresh fodder yield
3.34-6.78
0.87
4.96±0.19
Dry fodder yield
2.93-6.39
0.87
2.86±0.19
148 Table 9. The quantitative traits of Harding grass (Phalaris aquatica L)over the period (2015 and 2016) Phenological traits
Range
SD
Mean± SE
Days to 50% flowering i.e. days from cut to flowering
53.33-78.66
4.21
67.33±1.11
Maturity duration i.e. days from cut to 1st picking
93.77-105.33
18.28
118.56±6.82
Plant height at 50% flowering (cm)
92.36-126.44
4.11
6.30±0.89
Number of total tillers/meter row length
1.00-7.00
1.57
3.80±0.32
Fresh fodder yield
3.34-6.78
0.87
4.76±0.21
Table 10. The quantitative traits of timothy grass (Phleum pratense L)over the period (2015 and 2016) Phenological traits
Range
SD
Mean± SE
Days to 50% flowering i.e. days from cut to flowering
53.33-78.66
4.21
67.33±1.11
Maturity duration i.e. days from cut to 1st picking
93.77-105.33
18.28
118.56±6.82
Plant height at 50% flowering (cm)
92.36-126.44
4.11
6.30±0.89
Number of total tillers/meter row length
1.00-7.00
1.57
3.80±0.32
Fresh fodder yield
3.34-6.78
0.87
5.68±0.42
Dry fodder yield
2.93-6.39
0.87
3.88±0.19
Table 11. The quantitative traits of makhan malai grass (Lolium perenne L)over the period (2015 and 2016) Phenological traits
Range
SD
Mean± SE
Days to 50% flowering i.e. days from cut to flowering
53.33-78.66
4.21
67.33±1.11
Maturity duration i.e. days from cut to 1st picking
93.77-105.33
18.28
118.56±6.82
Plant height at 50% flowering (cm)
92.36-126.44
4.11
6.30±0.89
Number of total tillers/meter row length
1.00-7.00
1.57
3.80±0.32
Fresh fodder yield
3.34-6.78
0.87
5.86±0.22
Dry fodder yield
2.93-6.39
0.87
3.78±0.19
149 Table 12. The quantitative traits of white clover (Trifolium repens L.) over the period (2014, 2015 and 2016) Phenological traits
Range
SD
Mean± SE
Perennial herb length
40.00-77.25
12.02
56.06±2.27
Petiole length
13.78-17.11
2.48
14.52±0.46
Flowering head length (condensed spikes)
0.51-0.97
0.12
0.67±0.02
Fresh fodder yield
1.21-2.96
0.57
3.87±0.18
Table 13. The quantitative traits of red clover (Trifolium pratense L.) over the period (2014, 2015 and 2016) Phenological traits
Range
SD
Mean± SE
Perennial herb length
36.07-73.32
11.78
52.02±2.18
Petiole length
10.37-18.93
2.45
14.30±0.45
Flowering head length (condensed spikes)
0.51-0.92
0.12
0.63±0.02
Fresh fodder yield
3.24-5.91
0.81
4.80±0.15
Dry fodder yield
2.53-5.39
0.80
2.95±0.14
The fodder revolution was the need of hour and the farmer has to grow high fodder yielding varieties. To grow the fodder under almond orchard paves a new way of orchard floor management. The scope of profitable production with such quality crops along with environmental concerns make the evaluation of new species desirable. The aim of our study was to determine the most appropriate fodder cropsfor small-scale commercial production in the temperate climate of North Western Himalayan regions. Seven fodder crops viz., tall fescue, orchard grass, Harding grass, timothy grass, makhan malai grass, white clover and red clover were field-grown during 2015 and 2016 in the almond orchard of IGFRI RRS, Srinagar, J&K, India, to determine the most appropriate fodder crops/combination thereof suitable for a temperate climate. The results were very much impressive (Table 7,8,9,10,11,12,13 and 14)) and overall tall fescue, timothy grass and makhan malai grass had given excellent performance and can be recommended to be the best tree-crop combinations for almond-based hortipasture system in North Western Himalaya.
150 Table 14. Comparative Fresh fodder yield and Dry fodder yield of seven temperate fodder crops during 2014-2015 and 2015-2016 at IGFRI RRS Srinagar, J&K, India Temperate fodder crops
Fresh fodder yield
Dry fodder yield
Tall fescue (Festuca arundinacea (Schreb)
6.68±0.13
3.68±0.67
Orchard grass (Dactylis glomerata L)
4.96±0.19
2.86±0.19
Harding grass (Phalaris aquatica L)
4.76±0.21
2.86±0.19
Timothy grass (Phleum pratense L)
5.68±0.42
3.88±0.19
Makhan malai grass (Lolium perenne L)
5.86±0.22
3.78±0.19
White clover (Trifolium repens L.)
3.87±0.18
1.95±0.10
Red clover (Trifolium pratense L.)
4.80±0.15
2.95±0.14
The comparative economic viability of almond-based hortipasture system showed that hortipasture system is able to enhance farmers’ income more than double in seven years and triple, if farmer get value of their carbon sequestered by hortipasture system. If we consider time value of money with discount @ 12%, the estimated net income as 166,145, 364,142 and 518,131 per ha respectively from rice-wheat system, almond-based hortipasture without and with reward of carbon sequestration. Price volatility of almond and price crash since 2013-15 However, the past trend in volatility of almond price not yet proved as sustainable option to enhance the hortipasture farmers’ income. The rosy picture of hortipasture farmers’ income faded away due to various known and unknown factors. The price volatility in almond impacted farmers’ income and discourage the investment in new plantations. The trend in nominal price of almond shows continuous upward movement during 2013-14 to 2015-16 and sharp declining afterward. There has been growing interest in the inclusion of almond hulls in the diet of dairy cows during summer when pasture availability is limited and digestibility is low. However, there is little information on the resulting milk production response. Thirty-two Holstein Friesian cows were offered one of three diets (lucerne hay, lucerne hay plus almond hulls, lucerne hay plus citrus pulp). Cows fed almond hulls had a lower mean milk yield than those on the other diets. Mean fat yields from cows did not differ between diets. Mean protein yields of cows fed almond hulls were lower than those of cows on the other diets. Methane yield (g/kg dry matter intake) from the cows did not differ between diets. Both almond hulls and citrus pulp can
151 be fed to dairy cows as a partial replacement for high-cost purchased forage, but inclusion of almond hulls may be less desirable than citrus pulp due to reduced yields of milk and milk protein. Neither almond hulls nor citrus pulp exhibited any methane mitigation effect in dairy cows (Williams et al, 2014). Impact of almond hulls fed to dairy cows Almond hulls (Aguilar et al. 1984, Singer et al. 2008) when fed to dairy cows, inhibits methane production in vitro (Durmic et al. 2013). The performance has been presented in Table 15. Table 15: Feed intake, milk yield and milk composition of cows fed a control (CON), almond hull (ALH) diet. Parameter
Diet Control (CON),
Almond hull (ALH)
Total (kg/day)
22.4
22.6
Almond hulls (% of total)
0
17
Milk yield (kg/day)
27.4
24.6
Fat (kg/day)
1.04
1.00
Protein (kg/day)
0.87
0.78
Lactose (kg/day)
1.36
1.19
Fat (g/kg)
38.1
41.4
Protein (g/kg)
32.2
32.0
Lactose (g/kg)
49.9
48.8
Feed intake
Milk composition
The comparative economic viability of almond-based hortipasture system with rice-wheat system showed that hortipasture system is able to enhanced farmers’ income more than double in seven years and triple, if farmer get value of their carbon sequestered by hortipasture system. If we consider time value of money with discount rate of 12%, the estimated net income was much higherthan rice-wheat system as a reward of carbon sequestration. However, the past trend in volatility of almond price not yet proved as sustainable option to enhance the hortipasture farmers’ income. The price volatility in almond impacted farmers’ income and discourage the investment in new plantations. Policy framework at multiple scales
152 play seminal role in the multi-scaled nature of hortipasture social-ecological system, for example opening the opportunities for markets in ecosystem services, price policy for hortipasture products, trade policy, credit policy, subsidy policy, energy policy and forest policy are substantially effect the development of hortipasture. Research in this area is needed to address the effects of these policies on hortipasture and other sectors to evaluate how these policy measures can reduce negative inter-sectoral effects and enhance positive inter-sectoral effects. Integrated Management of Temperate Perennial Fodder Grass diseases - A Biotechnological Approach Tremendous technological development in the recent past has equipped the plantscientists with enormous options to tailor the plants according to need. The improvement of forage cr ops thr ough biotechnological approach has started in late eighties has made remarkable headway at the global level. The various biotechnological tools include molecular techniques for understanding the genetic structure of the plants, inserting foreign genes directly into the plant genome, in-vitro regeneration of plants from any plant part. A number of techniques such as embryo rescue, micro-propagation, androgenic haploid plant production and creation of novel variations help at one or more steps involved in conventional breeding methods. These techniques save time and energy required for conventional methods. Further, the plants developed through these techniques do not attract attention of those who are against the genetically modified organisms. The embryo-rescue technique has well been exploited in Lolium-Festuca complex for production of hybrids. Lolium, Dactylis hybrids and many interspecific hybrids in Trifolium have also been developed by this method. Regeneration of the plantlets from reproductive parts such as anther results in haploid plant production. Hence, the process is very effective in developing plants with double set of genome in the otherwise tetraploid tropical grasses. Biotechnological approach offers opportunities for creation of novel variations in forages which as such are not possible through conventional methods. The various means of creating variation in forage grasses and achievement are somaclonal variation, somatic hybridization, genetic transformation, etc. The genetic transformation by chemical, electrical, physical or micro-projectile transfer. In grasses, the success achieved so far has been limited and successful transformation has been reported only in a few perennial grasses, viz. Lolium, Festuca, Agrostis, Dactylis, Paspalum and Dichanthium species. The several biotechnological techniques, viz. restriction fragment
153 length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD) and isozymes were used from time to time for germplasm characterization, cultivar identification, hybrid detection and genetic mapping, quality trait loci (QTL) identification and gene tagging. The molecular characterization of forage crops is equally important. There is a need to classify forage germplasm for the twin objectives viz., firstly for developing DNA fingerprints and secondly for identifying the duplicates. It is desirable that the forage crop varieties are also subjected to molecular characterization in order to avoid any dispute regarding use of germplasm in the comingyears. The major problem encountered with these molecular markers of the forage species is that most of the cultivars are synthetic populations and variability exists within the population. Genetic mapping and gene tagging in forage species have not been attempted much. There are also opportunities for improving the amino acid balance of the plants used in intensive feeding production systems. Another important area is the development of stress tolerance, both biotic and abiotic, in fodder crops through gene pyramiding of identified QTLs. The germplasm can be screened in vitro at two levels: (i) seedling level and (ii) tissue or cell level. In-vitro studies have shown significant interspecific and intraspecific variation among legumeand clover species for salt tolerance. Intracultivar variation had been identified in lucerne (Medicago sativa) and white clover (T. repens). In case of postfertilization incompatibility barriers, embryo rescue is most effective and successful technique. In grass breeding, identification of genes controlling apomixis is an area that can pay good dividends. Identification and cloning of these genes can well be patented and can also be used in transferring in other cross-pollinated crops for fixing heterosis and thus save on the cost on account of producing hybrid seeds every year. Another important aspect is the identification of sexual lines in grasses. The plants with better agronomic traits and apomixis can be selected and advanced for developing better varieties. Crop is comparatively less prone to pests and diseases. However, downy mildew among diseases, shoot fly and root grub among pests are prevalent in many states. Choice of diseases resistant variety is an important step in effectively managing the diseases. The main reasons for low seed production in forage crops include: (i) low priority of crops leading to poor allocation of resources and area, (ii) non-availability of quality/ certified seeds, (iii) indeterminate growth habit coupled with non-synchronous maturity resulting in higher cost in collection of seeds, (iv) low seed productivity due to crops being basically
154 bred for herbage and the crop being harvested at vegetative stage and not allowing to reach to seed stage, (v) low seed production due to seed dormancy, seed shattering, blank/empty seeds, (vi) low multiplication ratio in most of the fodder crops which is often not remunerative to the producing agency, (vii) lack of organized seed outlets for marketing and uncertainty of seed demand, (viii) poor extension machinery responsible for inadequate popularization of improved varieties, and (ix) lack of policy support. SUMMARY It may be concluded that the forage production situation in the region is very alarming and corrective measures have to be taken to improve the same. Delineation of the area for various agricultural activities should be created and adhered. A comprehensive policy needs to be formulated for the entire zone. Both grazing and forage cultivation has to be considered complementary to each other and simultaneous efforts are required to improve the both. Horti-pasture involving agroforestric programmes for higher fodder production have to be initiated. In order to improve the fodder scenario, the horti-pasture involving agroforestric management needs to be considered holistically promoting the interaction of the region. In order to keep the farmers fully engaged and thus to enhance their livelihood opportunities, it is imperative to accelerate growth in the agricultural sector by promoting “agro-horti-pasture” developments. The fertile land can be used for growing almond trees and developing “agro-horti-pasture”. Thus tall fescue, timothy grass and makhan malai grass can be excellent performer and can be recommended to be the best tree-crop combinations for almondbased hortipasture system in North Western Himalaya. The Government of India has already planned to take many measures to increase farm income, stabilize production and, consequently, improve small farm productivity. Integrated farming system approach involving synergic blending of crops, horticulture, dairy, fisheries, poultry, etc. seems viable option to provide regular income and at site employment to small-land holder, decreasing cultivation cost through multiple use of resources and providing much needed resilience for predicted climate change scenario high-yielding varieties and hybrid seeds are very essential for a successful crop production and increasing the yield by 15 to 20% depending upon the crop and it can be further raised up to 45% with efficient management of other inputs. Micro-irrigation along with the nutrient application can be highly efficient and priority should be given to empower farmers with microirrigation. The government is aggressively promoting a new crop insurance (PMFBY) given increased challenges by farmers due
155 to frequent climatic disturbances. Although fodder cropsare comparatively less prone to pests and diseases, but we should be cautious enough to tackle the coming problems in future as well as create the more balanced nutritional fodder through the use of biotechnological tools are also imperative. ACKNOWLEDGEMENTS The Programme 4 under Dr Ramvinod Kumar, Head, Division of Grassland and Silviculture Management supported this research. The authors would like to thank Dr Probir Kumar Ghosh, Director for his untiring help during the course of investigation. We thank for unconditional help rendered during the course of investigation and the facility and guidance provided by ICAR-CITH, Srinagar, J&K, India and Dr Jai Prakash Singh, Coordinator, Indian Grassland and Fodder Research Institute, Jhansi, UP, India is duly acknowledged. We also want to express our acknowledgement to anonymous reviewers because their comments greatly helped to improve the quality of the manuscript. REFERENCES Almond Board of Australia 3 (2008). “All About Almonds.” Almond Board of Australia. July 2008. Web. 22 Oct. 2009. . Abberton, M.T. and Marshall, A.H. (2005). Progress in breeding perennial clovers for temperate agriculture. Journal of Agriculture Science 143:117–135. Aguilar, A.A., Smith, N.E., Baldwin, R.L. (1984). Nutritional value of almond hulls for dairy cows. Journal of Dairy Science 67, 97-103. Ahmed, N. and Verma, M.K., (2009). Scientific Almond Cultivation for Higher Returns. Central Institute of Temperate Horticulture, Srinagar, J&K Ahmed, N., Mir, J. I., Mir, R.R., Rather, N.A., Rashid, R., Wani, S.H., Shafi, W., Mir, H. and Sheikh, M.A. (2012). SSR and RAPD analysis of genetic diversity in walnut (Juglans regia L.) genotypes from Jammu and Kashmir, India, PhysiolMolBiol Plants18:149–160. Annon. (2012) Guidelines for the Conduct of Test for Distinctiveness, Uniformity and Stability On Almond (Prunus dulcis). Protection of Plant Varieties and Farmer’s Rights Authority (PPV & FRA), MOA, Government of India, New Delhi Anonymous. (2012-13). Annual Report, IGFRI, Pahuj Dam, Gwalior Road, Jhansi, U P, India Anonymous. (2013-14). Annual Report, IGFRI, Pahuj Dam, Gwalior Road, Jhansi, U P, India Anonymous. (2014-15). Annual Report, IGFRI, Pahuj Dam, Gwalior Road, Jhansi, U P, India
156 Anonymous. (2015-16). Annual Report, IGFRI, Pahuj Dam, Gwalior Road, Jhansi, U P, India Anonymous. (2016). Altaf for pasture development, quality fodder production, http:// www.greaterkashmir.com/news/2012/Jul/4/altaf-for-pasture-development-qualityfodder-production-25.as dt 3rd July, 2012 Durmic, Z., Moate, P.J., Eckard, R., Revell, D.K., Williams, S.R.O., Vercoe, P.E. (2013). In vitro screening of selected feed additives, plant essential oils and plant extracts for rumen methane mitigation. Journal of the Science of Food and Agriculture 94, 1191-1196. Katoch, R., Thakur, M., Kumar, N. and Bandhari, J.C. (2012). Golden Timothy – Present status and future perspectives in North-West Himalayas, Range Management and Agroforestry, 33:1-7. Kumar, D. and Ahmed, N. (2013). Response of Nitrogen and Potassium Fertigation to “Waris” Almond (Prunusdulcis) under Northwestern Himalayan Region of India. The Scientific World Journal, http://dx.doi.org/10.1155/2014/141328. Mouradov, A.Z. (2010). Designer pasture plants: from single cells to the field. Proceedings of the National Academy of Sciences of Azerbaijan Republic: Biological Sciences 65: 205–211. Raiesi, F. (2007). The conversion of overgrazed pastures to almond orchards and alfalfa cropping systems may favor microbial indicators of soil quality in Central Iran. Agriculture, Ecosystems and Environment 121 (2007) 309–318. Singer, M.D., Robinson, P.H., Salem, A.Z.M., De Peters, E.J. (2008). Impacts of ruminal fluid modified by feeding Yucca schidigera to lactating dairy cows on in vitro gas production of 11 common dairy feedstuffs, as well as animal performance. Animal Feed Science and Technology 146, 242- 258. Trenbath, B.R. (1974). Biomass productivity of mixture, Advance in Agronomy, 26:177210. Verma, D.K., Ahmad, S. and Sultan, S.M. (2016a). Analysis of genetic diversity in orchard grass/cocksfoot/Canary grass (Dactylis glomerata L) and its conservation in North Western Himalaya, Indian Journal of Genetic and Plant Breeding, Communicated dt 10.08.2016. pp 22. Verma, D.K., Ahmad, S. and Sultan, S.M. (2016b). Assessment of makhan malai grass or perennial ryegrass (Lolium perenne L) diversity collected from North Western Himalaya, India, India, Theoretical and Applied Genetics, Communicated dt 15.08.2016. pp 37. Verma, D.K., Ahmad, S. and Sultan, S.M. (2016c). Biodiversity in red clover (Trifolium pratense L.) collected from North Western Himalaya, India, Euphytica, Communicated dt 16.08.2016. pp 26. Verma, D.K., Ahmad, S. and Sultan, S.M. (2016d). Differential aspectsof morphological and phenomenal investigations in white clover (Trifolium repens L) diversity collected from North Western Himalaya, India, Annals of Botany, Communicated dt 16.08.2016. pp 38.
157 Verma, D.K., Ahmad, S. and Sultan, S.M. (2016e). Genetic diversity of tall fescue (Festuca arundinacea (Schreb) Hack., syn. Loliumarundinaceum(Schreb.) Darbysh) in North Western Himalaya, Indian Journal of Plant Genetic Resources, Communicated dt 07.08.2016. pp 22. Verma, D.K., Ahmad, S. and Sultan, S.M. (2016f). Phenotypic diversity in Harding grass (Phalaris aquatica L) collected from North Western Himalaya, India, Plant Genetic Resources, Communicated dt 10.08.2016. pp 53. Verma, D.K., Ahmad, S. and Sultan, S.M. (2016g). Phenotypic diversity in timothy grass (Phleum pratense L) collected from North Western Himalaya, India, Cereal Research Communication, Communicated dt 11.08.2016. pp 29. Verma, T.P., Mahapatra, S.K., Rao, R.V.S., Lal, T., Sidhu, G.S. and Rana, K.P.C. (1999). Change in soil characteristics in relation to physiography and land use in higher Himalayan region of Himachal Pradesh. Journal of soil and Water Conservation, 43:8. Wafa, B., Bashir, A., Nabi, S.Z. and Illahi U. (2018). An experimental investigation of soil stabilized with almond shells: a tenable solution. International Journal of Advance Research in Science and Engineering, 7(4):528-544. Williams, S.R.O., Deighton, M.H., Jacobs, J.L., Wales W.J. and Moate P.J. (2014). Almond hulls and citrus pulp can be used as supplementary feeds for dairy cows, but neither has any methane mitigation potential. Procs of the 5th Australasian Dairy Science Symposium 2014. 273-276. Yatoo, M.I., Devi, S., Kumar, P., Tiwari, R. and Sharma, M.C. (2011). Soil-plant-animal micromineral status and their interrelation in Kashmir valley. Indian J. Anim. Sci., 81: 68-70.
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8 MYCORRHIZA HELPER BACTERIA (MHB) AND THEIR INTERACTIONS WITH MYCORRHIZAL FUNGI AND HOST PLANT Sikha Dutta, Avishek Sarkar, Abhinanda Ghosh and Sunanda Dutta Department of Botany (UGC- CAS), The University of Burdwan, Golapbag, Purba –Bardhaman, West Bengal, India E mail:
[email protected]
INTRODUCTION The association between soil fungi and plant roots is called mycorrhiza. The establishment of mycorrhiza causes morphological and physiological changes in the root, which operates in an integrated manner with the fungus, thus promoting adaptability and survival of symbionts (Costa et al., 2002). According to Wang and Qiu (1999), out of a total of 3,617 species belonging to 263 families of terrestrial plants, 80% of the species and 92% of the families are associated with mycorrhizae. Among the angiosperms, 94% of the families are mycorrhizal. The association of mycorrhiza is affected by other microorganisms of the rhizosphere, specifically by bacteria. Bowen and Theodorou (1979) demonstrated in vitro that some bacteria are able to affect the growth of the ectomycorrhizal fungi Rhizopogon luteoliis in symbiosis with Pinus radiata, positively or negatively, depending on the bacterial strain present. Although most of the interactions are described as competition, some may benefit the process of plant infection by the mycobiont. The roots of most temperate forest trees form symbiotic relationship
160 with ectomycorrhizal fungi. It has been shown that the bacteria present in soil, rhizosphere and mycorrhia strongly help for the establishment of ectomycorrhizal colony. These types of bacteria are called as Mycorrhiza helper bacteria (MHB). Similar result have also been obtained with vesicular arbuscular endomycorrhiza (Mosses, 1962; Meyer and Linderman, 1986; Pakovski, 1989; Duponnois and Garbaye, 1990). This concept is revised and the distinction is made between the helper bacteria, which assist mycorrhiza formation and those that interact positively with the functioning of the symbiosis. Here, the main focus is on the ecological and evolutionary effect of the MHB and mycorrhizal fungus interaction. From the studies of isolation and identification of bacterial species found in mycorrhizal fungi and analyzing the action of bacteria on symbiosis, Duponnois and Garbaye (1991) proposed first the term ‘MHB’. Here they only refer those bacteria that promote the establishment of the root fungus symbiosis. After this much progress was made in the research of this interaction among bacteria fungus and plants. According to FreyKlett et al, (2007), there are 2 functional MHB categories on the basis of bacterial action: the first, mycorrhiza helper bacteria, strictly referring to those that enhance the process of mycorrhiza formation; and the second, mycorrhiza helper bacteria, referring to those that interact positively with the functioning of the already established mycorrhizal symbiosis. Here both the categories can be represented as different group or overlapping groups of bacteria, and the term MHB is used to represent both groups. It is thus timely to carry on the particular field of research at the interface of plant science, mycology, bacteriology and rhizosphere ecology, which is more generally related to the major research topic of fungal– bacterial interactions in ecosystems (de Boer et al., 2005; Artursson et al., 2006). The main issue here is the generalization of MHB and the role of MHB in the mycorrhizal symbiosis from evolutionary, functional and ecological points of view. Objectives of present review MHB are ubiquitous in their distribution (Poole et al., 2001) and they definitely help the mycorrhizal fungi for their establishment as endosymbiont in the root which in turn facilitate plant growth and vigor and consequently enhance soil fertility. Mycorrhiza helper bacteria (MHB) are known to increase host root colonization by mycorrhizal fungi but the molecular mechanisms involved for successful establishment of this potential tripartite interactions are poorly understood. So the aim of the study is focusing to the molecular mechanism involved, by which MHB
161 positively interfere with the establishment of the mycorrhizal fungi as root endosymbiont in the host plant. The present study dealt with an in-depth review on the Mycorrhizal Helper Bacteria (MHB), the members of this group, the role of MHB in the mycorrhizal symbiosis from evolutionary, functional and ecological points of view, in relation to other thriving research fields such as the taxonomy, phylogenetics and functional diversity of mycorrhizal fungi. Examples and Paradigm of MHB The MHB concept is generic. It depends neither on the type of the mycorrhizal symbiosis nor on the taxonomy of the MHB strains. To date, many bacterial strains have been reported to be able to promote either arbuscular or ectomycorrhizal symbioses (Garbaye, 1994; Barea et al., 2002; Johansson et al., 2004; Artursson et al., 2006; Duponnois, 2006). In the case of the arbuscular mycorrhizal fungi, many examples of MHB have been described in the literature since the first mention by Mosse (1962) in the genus Glomus. Of the ectomycorrhizal fungi, to date only Basidiomycetes have been reported to positively interact with MHB. Nevertheless, in the case of the ectomycorrhizal Ascomycete Tuber melanosporum, Mamoun and Olivier (1992) reported an indirect helper effect of soil pseudomonads on the T. melanosporum symbiosis. This could result from a protective effect of the pseudomonads against soil-borne competitors in the T. melanosporum mycorrhizosphere. The MHB strains that have been identified to date belong to many bacterial groups and genera. They are mainly Gram positive and some are Protobacteria. Such asProteobacteria- Agrobacterium, Azospirillum, Azotobacter, Burkholderia, Bradyrhizobium, Enterobacter, Pseudomonas, Klebsiella and Rhizobium. Gram-positive Firmicutes- Bacillus, Brevibacillus and Paenibacillu. Gram-positive actinomycetes- Rhodococcus, Streptomyces, and Arthrobacter (Frey-Klett, 2007). Further research is needed to determine if MHB also exist in non easily culturable bacterial groups that are known to inhabit the mycorrhizosphere such as the Acidobacterium group (Burke et al., 2006). Many plant models have been used to study the MHB effect, including herbaceous and woody plant species, mainly from temperate ecosystems. Only a few studies have focused on tropical plant species.
162 Specificity of MHB MHB show a degrees of specificity in the fungal species which they promote (Aspray, 2006a,b) and this may play a key role in directing the community composition and dynamics of ectomycorrhizal fungi. They could be used to improve the establishment of specific ectomycorrhizal inocula in forest nurseries (Garbaye and Bowen, 1989). MHB are fungus-specific but not plant-specific (Garbey, 1994). Many studies have been found to explore the specificity of the interaction between MHB and the fungi and between MHB and the host plant, and diverse results have been obtained (Aspray, 2006b; Bending, 2007; Duponnois and Plenchette, 2003; Frey-Klett, 2007; Garbaye, 1994). According to Garbaye (1994), the MHB are not plant-specific, but are selective about the fungal species, so they can be called as fungal specific. Among ectomycorrhizal fungi, only basidiomycetes can interact with mycorriza helper bacteria. Studies have shown that the ectomycorrhizal symbiosis has an indirect positive effect on the selective pressure of bacterial communities. The specificity of the helper effect appears to vary with the bacterial strains. Indeed, whereas, Garbaye and Duponnois (1992) and Dunstan et al. (1998) demonstrated a fungus specificity in the interactions between the ectomycorrhizal fungus Laccaria and different mycorrhiza helper bacteria isolates. Frey-Klett et al., (2007) reported that the MHB Streptomyces sp. AcH505 is capable of promoting growth of Amanita muscaria and Suillus bovinus and increase the formation of ectomycorrhizae between A. muscaria and Picea abies, but the growth of Hebeloma cylindrosporum and pathogenic fungi is inhibited. Bending (2007) observed that the production of the metabolite auxofuran by Streptomyces sp. AcH505 and its selective effect on the growth of A. muscaria may support the hypothesis of specificity between some MHB and mycorrhizal fungi, since it is a specific interaction between these microorganisms. The results of Duponnois and Plenchette (2003) support the evidence from Garbaye (1994) that the effect of the MHB is not plantspecific. This was demonstrated in an experiment in which Pseudomonas fluorescens BBc6 promoted the formation of mycorrhiza by Laccaria laccata in four species of conifers (Picea abies, Pinus nigra, Pinus sylvestris and Pseudotsuga menziesii) and in the angiosperm Quercus robur. However, Duponnois and Plenchette (2003) concluded that the effect of the MHB was not fungus-specific, as Pseudomonas monteilii HR13 isolated from
163 Pisolithus alba stimulated the development of mycorrhiza in Acacia holosericea with two species of Scleroderma and more surprisingly, with the arbuscular mycorrhizal fungus Glomus intraradices. According to Freitas and Vildoso (2004), strains of fluorescent Pseudomonas, Bacillus and other rhizospheric bacteria may act as growth promoters of citrus plants. Then, the question arises of whether the MHB are rhizobacteria occasionally acting as auxiliary to mycorrhization, if present by chance near a symbiotic fungus, or are dependent on the fungus and persist in its stages of development. Garbaye (1994) suggested that the second hypothesis is supported by the fact that sporocarp of some ectomycorrhizal fungi, as Laccaria, Tuber, Suillus, Hymenogaster and Cantharellus are usually inhabited by large bacterial populations. Furthermore, many isolates of MHB described in the literature have been collected from mycorrhizospheres, fructification bodies of ectomycorrhizal fungi and the spores of arbuscular mycorrhizal fungi. MHB could also reduce concentrations of antifungal metabolites in the mycorrhizosphere by direct antagonism against microbes which are hazardous to mycorrhizal fungi. The fungus specific MHB could act in such a way, inhibiting microbial strains that act against them. It needs to be will be checked which factors other than water-soluble antibiotics are responsible for fungus specificity. By growing the MHB and ectomycorrhizal fungi separately in two compartments communicating only through the atmosphere, Garbaye and Duponnois (1992) revealed that fungus specificity factors include gaseous compounds. Using solid-phase micro extraction and gas chromatography–mass spectrometry, Barbieri et al. (2005) were able to analyse 65 volatile compounds produced by Staphylococcus pasteuri, which were strongly antagonistic towards mycelium of Tuber borchii in cocultures without liquid contact. Similar methods could perhaps be used to clarify the gaseous factor responsible for fungus specificity indicated by the study of Garbaye and Duponnois (1992). Because of their selectivity, Duponnois et al. (1993) suggested that MHB could become an alternative to soil fumigation; for example, they could be simultaneously used for controlled mycorrhization and for antagonism against competitive symbiotic and/or phytopathogenic fungi. In the light of ‘fungal isolate specificity’, however, the data on the phytopathogen Heterobasidion annosum and Streptomyces sp. AcH 505 are of concern (Lehr et al., 2007). Now, the observation was that, while H. annosum strains tested were suppressed by AcH 505, root infection with
164 one fungal isolate was promoted by the MHB strain. This suggests that some MHB behave as helpers of both symbiotic and pathogenic fungi. Such a hypothesis is consistent with the recent observation of the helper effect of the MHB P. fluorescens BBc6R8 on the wheat pathogen Gaeumannomyces graminis. MHB as PGPR Many MHB are considered nowadays as Plant Growth Promoting Rhizobacteria (PGPR), such as Pseudomonas sp. As reported by Fitter and Garbaye (1994), these classifications may overlap, due to the prominence of Pseudomonas and Bacillus in both the groups of PGPR and MHB. Another factor that complicates the distinction of these two terms (PGPR and MHB) is that studies with PGPR generally exclude the evaluation of mycorrhization. However, it is interesting to note that some fungal signaling pathways are mutually regulated by different rhizobacteria, while others are specific to some MHB. The Effect of MHB on Formation and Mycorrhizal Associations Five possible ways of action of MHB on mycorrhiza were proposed by Garbaye (1994): in the receptivity of the root to the mycobiont, in rootfungus recognition, in fungal growth, in the modification of the rhizospheric soil and in germination of fungal propagules. In the ectomycorrhizae studied so far, the stimulus to fungal growth appears to be the primary MHB effect. The germination of spores and the mycelial growth can be stimulated by MHB through the production of growth factors, detoxification of antagonistic substances or inhibition of competitors and antagonists. The stimulus to growth represents an adaptive advantage to the fungus, which becomes heavily associated to the host plant and acquires more competitive capacity against other mycobionts in the planting area. Currently, the contribution of each of these effects has not been fully established, and further studies are needed to elucidate these issues. Aspray et al., (2006a,b) demonstrated that the contact between MHB cells and the symbionts is necessary for the helper effect to be exerted. The MHB can improve the nutrition of the fungus, for example, through the provision of nitrogen in the case of diazotrophic bacteria, or contribute to the solubilization of minerals by the secretion of protons and complexing agents, such as organic anions of low molecular weight or siderophores. It is possible that the MHB stimulate the production of phenolic compounds by the fungus, such as hypaphorine, and thus enhance the aggressiveness of the mycobiont.
165 One of the features also observed in MHB is the stimulus to the formation of lateral roots in mycorrhizal plants. This fact, associated to the stimulus to fungal growth, could lead to an increase in the number of possible interaction sites between the plant and the fungus (Schrey, 2005) and, consequently, promote greater plant mycorrhization by the mycobiont. Furthermore, apparently, different MHB may develop different helper mechanisms, even for the same pair of mycorrhizal symbionts. For example, Poole et al., (2001) observed that the MHB, Burkholderia sp. EJP67 isolated from Pinus sylvestris-Lactarius rufus ectomycorrhizae stimulated both first and second-order mycorrhizal roots, while Paenibacillus sp. EJP73 isolated from the same ectomycorrhizae only promoted the formation of secondorder mycorrhizal roots. Some strains of MHB are capable of competing with bacteria that inhibit mycorrhization (Frey-Klett, 2007) and, consequently, reduce the concentration of anti-fungal metabolites in mycorrhizosphere. The fungus favors the MHB by releasing exudates that serve as nutrients for the bacteria. An interesting fact is that the fungus Amanita muscaria secret substances (organic acids or proteins) that can modulate the spectrum of antibiotics production by MHB. Keller et al. (2007) reported that the metabolite auxofuran, produced by Streptomyces sp. AcH505, seems to stimulate the pre-symbiotic growth of A. muscaria but inhibit the growth of pathogenic fungi. The researches available so far suggest that MHB may have developed selective mechanisms of interaction with surrounding microorganisms, with neutral or positive effects on mycorrhizal associations, but negative effects on the root pathogens that threaten its habitat (Frey-Klett, 2007). However, there are data concerning MHB stimulating phytopathogenic fungi and this should be considered in the biotechnological applications of MHB, for instance, as inoculum for plants. Further researches are necessary to determine whether MHB could promote the colonization of the roots by pathogenic fungi and development of disease. MHB Induced Morphological Changes Appeared in Mycorrhizal Fungus Deveau et al. (2007) compared Pseudomonas fluorescens BBc6R8 to six other rhizobacteria (Collimonas fungivorans Ter331, Paenibacillus sp. EJP73, Pseudomonas fluorescens Pf29A, Bacillus subtilis MB3, Burkholderia sp. EJP67 and Paenibacillus sp. F2001L) and found that P. fluorescens BBc6R8 was the only one that induced increase in survival, in
166 the apex density of the hyphae, in the branching angle and radial growth of the ectomycorrhizal fungus Laccaria bicolor S238N. The morphological modifications were associated with changes in the transcriptome of L. bicolor that varied throughout the interaction. The authors reported that some responsive genes were partially specific to the interaction with P. fluorescens BBc6R8, which provides evidence of the specificity of the relationship between MHB and the mycobiont. In general, the data suggest that the effect of MHB involves changes in the fungal anabolism and catabolism of lipids that could cause increased lipids synthesis, required for higher growth rates. According to Deveau et al. (2007), the morphological changes of the mycelium in vitro may be beneficial to the host root infection by the fungus, representing a transition from the saprophytic to the pre-symbiotic state. It is interesting to note that not all bacterial strains are able to promote such changes in the fungus. Results suggest that additional mechanisms, not limited to the increase of growth rate, are involved in stimulation of mycorrhization. According to Deveau et al. (2007), the morphological changes of the mycelium in vitro may induce changes in growth and morphology of L. bicolor S238N, although only P. fluorescens BBc6R8 increased the diametric growth of the colony, the density of the hyphal apex and angle of branching at the pre-contact stage. Much remains to be clarified about the consequences of the interaction between MHB and associated fungi. Interesting information that suggests the intensity of interaction is that the MHB Streptomyces sp. ACH505 is capable of altering the regulation of the acting cytoskeleton organization in Amanita muscaria. Mechanism underlying the mycorrhiza helper bacterial effect Spore germination- A focus on fungal gene regulation by helper bacteria Garbaye (1994) reviewed the possible mechanism underlying the mycorrhizal helper bacterial effect. A direct effect of helper bacteria in root receptivity to mycorrhizal fungi has been frequently evoked in different papers that deal with the mechanism of mycorrhiza helper bacterial effect (Schrey et al., 2004). However, the main mechanism favored so far in all these studies is the direct effect of helper bacteria on the pre-symbiotic survival and growth of the mycorrhizal fungi in the soil (Brule et al., 2001; Founoune et al., 2002; Schrey et al., 2002). At the molecular level, the mechanism likely relies on the modification of the fungal nutrients use
167 efficiency and or on the regulation of the fungal cell cycle (i.e. hyphal proliferation) by the helper bacteria. To date, little is known about the signal molecule produced by the helper bacteria, the fungal factor that recognize the bacterial signal molecule as well as the fungal gene networks underlying the fungal–bacterial interactions. Xie et al. (1995) demonstrated the involvement of bacterial Nod factor in the helper effect of Bradyrhizobium japonicum on the Glomus mosseae-soybean endomycorrhizal symbiosis. Moreover, by different RNA display, Requena et al. (1999) were able to identify a cDNA fragment coding a mRNA that was five-fold down-regulated when sporocarps from the endomycorrhizal fungus Glomus mosseae where inoculated with a rhizobacterial strain of Bacillus subtilis that was proved to promote the hyphal growth of Glomus mosseae. This transcript corresponded to a highly conserved gene encoding a multifunctional protein of the peroxisomal â- oxidation that might play a role in the catabolism of long-chain fatty acids during the fungal presymbiotic growth. Even less is known about the interactions between mycorrhiza helper bacteria and ectomycorrhizal fungi: for these fungi, no differentially regulated gene have been identified, although changes in gene profiles and protein patterns have been reported in the presence of helper bacteria. Indeed, Becker et al. (1999) described the effect of two ectomycorrhiza association stereptomycete isolates on global gene expression and protein synthesis of the ectomycorrhizal fungus Laccaria bicolour in vitro assays where liquid cultures of the fungus were incubated over 1-3 days with bacterial culture supernatant. So, the two bacterial isolates significantly, but differently, altered the fungal gene expression and protein patterns.
Fig 1: The sites of action of MHB, After Garbaye, 1994
168 Promoted mycelial growth- Co-culture with MHB may influence the interacting fungal hyphae in various ways, depending on the MHB– mycorrhizal fungus pair. Comparing the impact of different MHB on the growth and morphology of L. bicolor mycelium, they have demonstrated that, of the tested strains, P. fluorescens BBc6R8 is the only MHB that simultaneously enhances significant growth, the branching angle and the branching density of the mycelium, as well as the number of apices (Deveau et al., 2007). In the case of the MHB Streptomyces sp. AcH 505, although it promotes mycelial extension of A. muscaria, it sharply reduces hyphal biomass/colony area ratio as a result of a reduction in mycelial density (Schrey et al., in press, 2005). Moreover, it reduces the thickness of the fungal hyphae (Maier, 2003). The recent analysis on the structural background of this hyphal thinning: bacterial inoculation leads to a change in the organization of the actin cytoskeleton in A. muscaria (Schrey et al., 2005). As expected in the light of these results, the A. muscaria– Streptomyces sp. AcH 505 interaction is accompanied by altered fungal gene expression levels. Fungal genes related to signal transduction pathways, cell stress and cell growth, metabolism and cell structure were found to be up-regulated in mycelium cocultured with AcH 505 (Schrey et al., 2005; Tarkka et al., 2006). In the case of the P. fluorescens BBc6R8– L. bicolor S238N pair, a gene profiling approach has also recently revealed that morphological modifications of the fungal mycelium induced by this MHB strain are coupled to pleiotropic alterations of the fungal transcriptome. The latter are partly specific to the interaction with the strain BBc6R8 and suggest that the strain BBc6R8 induced a shift from the saprotrophic stage of the fungus to the infectious stage (Deveau et al., 2007). The expression level of the A. muscaria cyclophilin gene AmCyp40, which encodes a prolyl isomerase that is involved in cell growth and the cell stress response, is 9-fold up-regulated in A. muscaria–Streptomyces sp. AcH505 coculture (Schrey et al., 2005). To analyse the specificity of A. muscaria–MHB Streptomyces interactions, the modifications of the expression of the cyclophilin gene AmCyp40 were monitored in the presence of culture supernatants of AcH 505 and of another MHB, Streptomyces setonii AcH 1003. Inoculation with culture supernatants of both MHB led to a large increase in AmCyp40 expression, but the application of the culture supernatant of Streptomyces sp. AcH 504, a bacterium that is not a MHB, did not lead to altered AmCyp40 expression (Schrey et al., 2005). Later observation was that the antibiotic WS-5995 B produced by AcH 505 induces AmCyp40 expression (Riedlinger et al., 2006), but it is currently unknown which factor(s) from AcH 1003 is responsible for the induction of AmCyp40.
169 Increased branching of root system and promoting colonization-A direct contact between MHB and plant roots may be required for the promotion of mycorrhizal symbiosis, as demonstrated by Aspray et al. (2006b) for the MHB Paenibacillus sp. strain EJP73. This recent study indicates that the strain EJP73 exudes substances related to the MHB effect only when in contact with the roots; that these substances are attached to the bacterial cell wall; and/or that these substances are short-lived and therefore have to be produced continuously (Aspray et al., 2006b). MHB effectors that facilitate root colonization could be plant cell wall-digesting enzymes, which would enhance the penetration and the spreading of the fungus within the root tissues (Mosse, 1962), or suppressors of the plant defense response (Lehr et al., 2007). The impact of MHB on plant gene expression levels can now be examined on a larger scale because the genome sequences of many plants are available or their annotation is in progress, and large databases of expressed plant genes exist. Stimulation of lateral root formation is a frequently observed characteristic of MHB (Duponnois, 1992; reviewed in Garbaye, 1994; Poole et al., 2001; Schrey et al., 2005), which essentially leads to an increase in potential points at which plant and fungus can interact. As well as increasing the nnumber of lateral roots, the Bacillus strain isolated by Bending et al. (2002) increased the formation of only first-order ectomycorrhiza roots, and Burkholderia and Rhodococcus strains isolated by Poole et al. (2001) increased the formation of only second-order ectomycorrhiza roots in Scots pine (Pinus sylvestris). The mechanisms underlying these spatially organized root ramifications did not relate to bacterial colonization patterns (Poole et al., 2001). Phytohormones, including auxins and ethylene, have been implicated in producing morphological changes in roots during mycorrhiza formation (Kaska et al., 1999), including the formation of lateral roots and dichotomous branching of short roots (Barker and Tagu, 2000). However, no link has yet been established between stimulated root branching and auxin or ethylene production by MHB. Two non-indol-3acetic acid (IAA)-producing strains isolated from Lactarius rufus mycorrhizas, Paenibacillus sp. EJP73 and Burkholderia sp. EJP67, were found to promote dichotomous root branching in Scots pine seedlings (Aspray et al., 2006a). This suggests that other growth factors are produced by EJP67 and EJP73, such as ethylene which has been implicated in dichotomous root branching (Kaska et al., 1999), or that the two bacteria modulate hormone production or transport in Scots pine. Plants produce chemotropic signals to direct mycelia growth towards the fine roots. In both arbuscular mycorrhizas and ectomycorrhizas
170 it has been shown that these substances include flavonoids (Lagrange et al., 2001; Akiyama et al., 2002). MHB could indirectly facilitate root colonization by mycorrhizal fungi, by inducing the release of plant flavonoids. MHB reduce soil-mediated stresses certain fungus–plant combinations may produce an observable MHB effect only when fungal growth is inhibited by the soil substrate. Pseudomonas fluorescens BBc6R8 had a significant positive influence on fungal biomass only when the nursery soil was autoclaved before the bacterial and fungal inoculums were added (Brulé et al., 2001). The authors suggested that toxic metabolites are released by autoclaving, inhibiting mycelial development. The bacterium would then detoxify the soil, restoring soil conduciveness. In accordance with the data obtained by Brulé et al. (2001) with an ectomycorrhizal model, a Bacillus sp. strain had a stronger positive effect on the intensity of root cortex colonization and arbuscule formation by Glomus intraradices when the plants were subjected to drought stress than in a normally watered soil (Vivas et al., 2003). MHB may also release plants from stress caused by heavy metal pollution. It was recently demonstrated that bacteria isolated from heavy metal-contaminated soils had a strong positive impact on spore germination and on pre-symbiotic fungal growth under toxic concentrations of heavy metals: bacterial inoculation not only reduced damage to Glomus mossae hyphae but even resulted in increased mycelial growth and mycorrhiza formation (Vivas et al., 2005). Also, under natural conditions soil solution commonly contains substances that inhibit mycelial growth, and at least some MHB are able to detoxify these molecules. For instance, Duponnois and Garbaye (1990) showed that MHB reduced the concentrations of phenolic antagonistic substances produced by mycorrhizal fungi. Izumi et al. (2006a, b) classified Pseudomonas as an endobacterium. In this context, the term endobacterium is defined as bacteria that exist within the compartments of the fungus or the host of mycorrhiza, or still within the cells of one of the symbionts. The study covered four morphotypes of ectomycorrhizae of Pinus sylvestris, Suillus flavidus, Suillus variegatus, Russula sp. and Russula paludosa. After superficial sterilization of mycorrhizal roots, the culturable bacteria were analyzed by RFLP (Restriction Fragment Length Polymorphism) of the rDNA intergenic spacer regions and 16S rRNA genes. The results showed the presence of Pseudomonas in more than one ectomycorrhizal morphotype and about 50% of the isolates belonged to the genera Pseudomonas and Paenibacillus, suggesting that these two genera should be widely distributed
171 in different ectomycorrhizae of Pinus sylvestris. One of the suggested mediators of the attraction between Pseudomonas fluorescens BBc6R8 and the fungus is the disaccharide trehalose, produced by fungi from the carbon compounds received from the phytobionts. This bacterial strain presents a chemical attraction both to the pure disaccharide and to the hyphae of L. bicolor, which accumulate trehalose. The presence of the low molecular weight fraction from the supernatant of Pseudomonas putida cultures promoted a significant increase in the rates of fungal growth and mycorrhization by Glomus fistulosum, similarly to that caused by the co-inoculation of the cells. This fact suggests that effective substances were present in this fraction . The physical and chemical interactions between ectomycorrhizal fungi and soil can significantly change the structure of P. fluorescens populations, selecting strains potentially beneficial to the symbiosis and to the plant, as described by Frey-Klett et al. (1997). This study showed that populations of Pseudomonas are quantitative and qualitatively regulated in the symbiosis bacteria-fungus, as the genetic diversity of cultivable P. fluorescens was higher in mycorrhizosphere of L. bicolorPseudotsuga menziesii than in bulk soil. Most of the Pseudomonas isolated from the mycorrhizosphere was able to solubilize inorganic phosphate, and this characteristic was not found in the majority of soil bacteria. This ability probably favors the growth of plants in symbiosis. The mycorrhizosphere also contained isolates of P. fluorescens presenting a greater spectrum of antagonism against phytopathogens than other isolates from the rest of the soil. The proportion of P. fluorescens capable of fixing nitrogen did not differ significantly between the mycorrhizosphere and bulk soil, indicating that the symbiosis L. bicolor-P. menziesii did not select this feature. This fact contrasts with the study of Rózycki et al. (1994) which showed an increase in nitrogen fixing bacteria, mainly Pseudomonas, in the mycorrhizosphere of pine and oak. Frey-Klett et al. (2005) suggested that the presence of nitrogen fixing bacteria in various ectomycorrhizal interactions indicating the potential of MHB to assist the nutrition of the associated plant. MHB against pathogenic attack Schey et al., (2004) showed the potential application of Streptomyces isolates, which had previously also showed an inhibitory effect towards two pathogenic fungi. Heterobasidion annosum and Armillaria obscura (Maier et al., 2004). Such a mycorrhiza helper bacterial strain could be used in forest nurseries to promote mycorrhization of tree seedlings
172 and prevent phytopathogen attack. Of course, the results of Maier et al. (2004) and Schrey et al. (2004) should be confirmed by field experiment because the significance of in vitro tests is often controversial (Whipps, 1987). In the recent study by Jessy Labbe, et al., (2014) an effort to Populus microbiome was made, here they isolated 21 Pseudomonas strains from native Populus deltoides roots. These bacterial isolates were characterized and screened for MHB effectiveness on the Populus-Laccaria system. Two additional Pseudomonas strains (i.e., Pf-5 and BBc6R8) from existing collections were included for comparative purposes. They analyzed the effect of co-cultivation of these 23 individual Pseudomonas strains on Laccaria bicolor “S238N” growth rate, mycelial architecture and transcriptional changes. Nineteen of the 23 Pseudomonas strains tested had positive effects on L. bicolor S238N growth, as well as on mycelial architecture, with strains GM41 and GM18 having the most significant effect. Four of seven L. bicolor reporter genes, Tra1, Tectonin2, Gcn5, and Cipc1, thought to be regulated during the interaction with MHB strain BBc6R8, were induced or repressed, while interacting with Pseudomonas strains GM17, GM33, GM41, GM48, Pf-5, and BBc6R8. Strain GM41 promoted the highest roots colonization across three Populus species but most notably in P. deltoides, which is otherwise poorly colonized by L. bicolor. Here they report novel MHB strains isolated from native Populus that improve L. bicolor root colonization on Populus. This tripartite relationship could be exploited for Populus species/genotypes nursery production as a means of improving establishment and survival in marginal lands. Last but not least, the principles and practices of controlled mycorrhization in agriculture, horticulture and forestry should be revisited. Helper bacteria may improve the efficiency of fungal inocula with a low extra cost, because bacteria are easier to grow in commercial quantities than most mycorrhizal fungi. This means that more MHB work should be dedicated to model mycorrhizal fungi that are of obvious commercial interest as well as being of use as research laboratory models. Such fungi include G. intraradices for arbuscular mycorrhizas and Pisolithus spp. and L. bicolor for ectomycorrhizas. In addition, growing concern about the pollution of soils, and the resulting trend towards reducing the input of chemicals in plant production, should promote more environment friendly practices such as controlled mycorrhization or microbial bioremediation, for instance by using mycorrhizal fungi as carriers of de-polluting bacteria (Sarand et al., 1998). This converging of scientific and practical interests,
173 supported by the development of genomics, may represent a unique opportunity to place MHB at the forefront of future mycorrhiza research and to boost the more general field of fungal–bacterial interactions in ecosystems.
Fig 2: Illustration of some interactions established in the rhizosphere among plants, mycorrhizal fungi, and bacteria, after Paola and Iulia-Andra, 2009
Conclusion So, the mycorrhiza helper bacteria promotes the association of mycorrhizal fungi and the plant roots by stimulating the spore germination, hyphal growth and cell division of mycorrhizal fungi. Also it had been reported that the MHB promotes the gene expression, signal transduction, primary metabolism etc. They stimulate the transcription to a highly conserved gene encoding a multifunctional protein of the peroxisomal betaoxidation that might play a key role in the catabolism of long chain fatty acids during the fungal pre-symbiotic growth. Although, the contribution of mycorrhiza-associated bacteria (the mycorrhiza helper bacteria) to mycorrhizal functions such as nutrient uptake, protection of the roots against phytopathogens and provision of the plant with growth factors should be investigated. This would undoubtedly provide a new dimension of the physiology, ecology and evolutionary biology of mycorrhizal symbioses. The studies concerning the action of MHB on the establishment and development of
174 ectomycorrhizae may generate an interesting comprehension about the interaction between these microorganisms and the other components of the environment. More specifically, the study of MHB is essential in promoting the knowledge of how mixed microbial communities stimulate the formation and establishment of mycorrhizae. A deeper study on MHB could generate a model for genomic analysis of bacteria-fungus interactions that may benefit other research areas in which these interactions have a central role, such as protection of plant species. This converging of scientific and practical interests, supported by the development of genomics, may represent a unique opportunity to place MHB at the forefront of future mycorrhiza research and to boost the more general field of fungal–bacterial interactions in ecosystems. References Abdel-Fattah GM, Mohamedin AH. 2000. Interactions between a vesicular-arbuscular mycorrhizal fungus (Glomus intraradices) and Streptomyces coelicolor and their effects on sorghum plants grown in soil amended with chitin of brawn scales. Biology and Fertility of Soils 32:401–409. Akiyama K, Matsuoka H, Hayashi H. 2002. Isolation and identification of a phosphate deficiency-induced C-glycosylflavonoid that stimulates arbuscular mycorrhiza formation in melon roots. Molecular Plant–Microbe Interactions 15: 334–340. von Alten H, Lindemann A, Schönbeck F. 1993. Stimulation of vesicular-arbuscular mycorrhiza by fungicides or rhizosphere bacteria. Mycorrhiza 2: 167–173. Artursson V. 2005. Bacterial–fungal interactions highlighted using microbiomics: potential application for plant growth enhancement. PhD thesis, University of Uppsala, Uppsala, Sweden. Artursson V, Finlay RD, Jansson JK. 2006. Interactions between arbuscular mycorrhizal fungi and bacteria and their potential for stimulating plant growth. Environmental Microbiology 8: 1–10. Aspray TJ, Frey-Klett P, Jones JE, Whipps JM, Garbaye J, Bending GD. 2006a. Mycorrhization helper bacteria: a case of specificity for altering ectomycorrhiza architecture but not ectomycorrhiza formation. Mycorrhiza 16: 533–541. Aspray TJ, Jones E, Whipps JM, Bending GD. 2006b. Importance of mycorrhization helper bacteria cell density and metabolite localization for the Pinus sylvestris–Lactarius rufus symbiosis. FEMS Microbiology and Ecology 56: 25–33. Azcón R, Rubio R, Barea JM. 1991. Selective interactions between different species of mycorrhizal fungi and Rhizobium meliloti strains, and their effects on growth, N2fixation (15 N) and nutrition of Medicago sativa L. New Phytologist 117: 399– 404.
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9 EFFECT OF SELECTED CHEMICAL AND BIOLOGICAL FUNGICIDES ON IN VITRO POLLEN GERMINATION AND POLLEN TUBE DEVELOPMENT OF TWO CUCURBITACEOUS CROPS Manoranjan Paramanik, Sanjeev Pandey1 and Subrata Raha Department of Botany, Sidho-Kanho-Birsha University, Ranchi Road, Purulia, W.B. 1
Department of Botany, Banwarilal Bhalotia College, Asansol. W.B. Email :
[email protected]
Pollen transfers the male genetic materials which germinate on the stigma and develop into pollen tube. For the growth and development, it requires certain nutrients as respiratory substrate; some are needed for ion transportation, while some others for regulation of different biosynthetic pathways. Successful fertilization and fruit set depends on the viability of the pollen grains and normal development of pollen tube to discharge the male gamete into the receptive stigmas. Studies on in vivo pollen germination are cumbersome due to the complications of pistillate tissues. It is possible to study the germination and tube development of pollen grains using a simple nutrient medium through in vitro techniques. The pollen tubes are considered as the most rapidly growing cells in the plant world since they are capable of attaining considerable length in a short duration under optimum conditions. Thus, pollen germination and growth of pollen tubes are important research materials for morphological, physiological, biotechnological, ecological, biochemical molecular and other biological studies (Ottavia et al., 1992). In recent years to determine
186 the importance of cytoskeleton in cell growth and differentiation, pollen germination and pollen tube development is used as research tool (Ma et. al., 2000). So, the pollen grains, being single celled structure provide a unique system for in vitro studies (Katara, 2013). Brewbaker and Emery (1962) stated that pollen grains of flowering plant are excellent experimental material for radio-biological investigations. These are unicellular structures having haploid condition with lesser number of variations than the seeds. Thus these are relatively simple target for low penetrating radiations. In addition the use of pollen for the induction and study of mutations has many commendable features. It has also been suggested that pollen irradiated with low level of radiation could be used to obtain mutation. Pfahler (1983) emphasized that the gametophytic selection is more effective than sporophytic selection in plant breeding. Our knowledge regarding physiology and biochemistry of pollen germination and tube growth largely comes from in vitro studies. Therefore, it is important to know the physiological and biochemical characteristics of pollen germination. In vitro pollen germination is considered as the best indicator of pollen viability (Shivanna et al., 1991). The required environment for in vitro pollen germination is related to genetic composition and also the quality and quantity of nutrient reserves of pollen (Baker and Baker, 1979). The requirements for successful germination of pollens vary from species to species (Shivanna and Johri, 1985). Apart from moisture they require a carbohydrate source, boron, calcium, magnesium and potassium. Besides, temperature, pH and gelling of the medium also affect pollen germination and pollen tube growth (Johri and Vasil, 1961). Different fungicides are widely used today to control fungal diseases of variety of crops. It is well known that these chemicals repel and harms the friendly pollinators, contaminate water bodies and also responsible for human and animal health hazards. An attempt was made in the present study to understand the physical and nutritional requirements for in vitro pollen germination of Lagenaria siceraria (Molina) standl (Bottle gourd) and Luffa acutangula (L) Roxb. (Ridge gourd) and to study the effects of two selected chemical and one bio-fungicide on the growth and physiology of Pollen grains. These two Cucurbitaceous vegetable crops are nutritionally important having broad health beneficial properties and thus widely cultivated as vegetable crop in India. Pollination and in vitro Pollen germination has been studied by different workers on different wild plants and cultivated plants viz. Moringa
187 oleifera (Bhattacharya and Mandal, 2004), cultivated & wild species of cucurbit (Zaman, 2006), Agave (Diaz & Garay, 2007), Trichosanthes dioica (Kumari et al., 2009), Lawsonia inermis (Mandal and Ghanta, 2012), Abelmoschus esculentus and its wild relatives (Patil et al., 2012), Quercus robur clones (sever et al., 2012), Luffa cylidrica (Biswas and mandal, 2014), Mitragyna parvifolia (Dey et al., 2015), Holmskioldia Sanguinea (Sarkar et al., 2016), etc. but the effect of fungicides and insecticides on pollen physiology of crops has yet to be explored except a report of Zarrabi and Imani, 2011, who studied the effect of fungicides on Almond pollen. Material and Method: Newly opened flowers were collected soon after anther dehiscence and transferred to polythene bags for the in vitro pollen germination study. The fresh pollen sample of Lagenaria siceraria (Bottle gourd) & Luffa acutangula (Ridge gourd) were collected from their anthers with the help of a fine brush. Pollens were sown on several grooved slide containing solution of various concentrations of sucrose (2-10%) & boric acid (25 – 200 ppm) to find out the optimal combination for their germination. Then the optimal media is combined separately with 0.2% VITAVAX (Caboxin, 75% W.P), 0.1% HILZIM (Carbendazim, 50% W.P) and bio-fungicide like SUDOBACT (Pseudomonas fluroscens - 2X109 CFU cells bacteria/ml). Treated & control set of slides were kept in tray lined with moist filter paper and examined under a bright field microscope (Zeiss Stemi 508) at different time intervals to record the germination percentage and pollen tube evelopment following the method of Shivanna and Rangaswamy (1993). A pollen grain is considered as germinated if the length of pollen tube length became twice greater than the diameter of pollen grain (Gupta et al., 1989). Result and Discussion: The bottle gourd pollens showed best germination (69.6±0.34) with tube length (480.2±6.65 µm) at 4% sucrose solution where as Ridge Gourd pollen showed 78.6±2.13% germination with maximum tube length in (520.24 ± 8.64 µm) at 5% solution of sucrose (Table 1 & 2).
188 Table- 1 Effect of sucrose on in vitro germination of pollen grains in Lagenaria siceraria % of
After 1 hour
After 2 hours
After 4 hours
sucrose
Germination (%)
Tube length (µm)
Germination Tube length (%) (µm)
Germination Tube length (%) (µm)
2
23.9 ±0.660
170.5 ±2.44 34.2±1.12
220.2±6.34 46.4±0.87
378.8±7.76
4
35.7 ±1.033
400.75±8.21 46.8±0.76
438.8±8.8
480.2±6.65
5
18.2 ±0.433
302.25±3.43 29.2±0.72
370.43±4.67 40.14±1.23 410.24±4.45
6
11.94 ±0.617
210±5.53.
8
9.01 ±0.232
130.2±7.32. 14.4±0.45
10
6.11 ±0.246
70.5±2.76
69.6±0.34
21.78±0.43 250.3±3.44 29.9±1.12 170.2±6.43 20.3±0.56
11.65±0.54 86.0±2.21
16.12±0.23
302±4.45 240.4±8.12 110.2±6.45
Table- 2 Effect of sucrose on in vitro germination of pollen grains in Luffa acutangula % of
After 1 hour
After 2 hours
After 4 hours
sucrose
Germination (%)
Tube length (µm)
Germination Tube length (%) (µm)
Germination Tube length (%) (µm)
2
21.65±0.67
170.5±5.56 30.66±1.34 224.4 ±4.5
4
33.13±1,02
310.75±7.34 40.4±1.54
5
41.89±1.23
325.04±7.14 65.10±2.12 430.75±5.2 78.6±2.13
520.24±8.64
6
20.05±0.56
210.34±4.43 29.45±1.05 240.68 ±5.8 34.12±0.76
30.56±2.65
8
7.29±0.32
130.03±4.13 18.68±0.45 150.50 ±3.6 21.8±0.46
190.5±4.43
10
3.21±0.223
60.5±2.6
120.2±5.56
42.2±1.45
395.45 ±7.8 55.34±1.66
09.99±0.56 90.5±2.45
13.34±0.65
390.44±6.78 480.5±7.8
Pollen grains of both the plants showed maximum germination and tube growth at 100 ppm solutions of boric acid. In Lagenaria siceraria 82.65±3.44% germination with a tube length of 210.7±9.66 µm and in Luffa acutangula 80.09±3.24% germination with pollen tube of 169.8± 5.34 µm length was noticed (Table 3 & 4). Table- 3 Effect of boric acid on in vitro germination of pollen grains in Lagenaria siceraria. Boric acid (ppm)
25
After 1 hour
After 2 hours
After 4 hours
Germination Tube length (%) (µm)
Germination Tube length (%) (µm)
Germination Tube length (%) (µm)
22.4±1.24
32.2±1.65
38.2±2.1
30.5±3.37
44.4±1.4
80.8±4.34
189
50
33.8±0.68
42.25±3.12 40 ± 1.12
80.4±3.56
52.4±2.41
122.2±8.23
100
60.6±0.43
60.5±4,75
125
44.44±0.84 50.6±3.2
150
35.1±0.75
175
30.65±0.24 50.89±1.1
41.87± 0.43 80.7±6.2
48.11±0.65 Pollen tube distorted
200
31.92±0.96 40.122.1
30.19±0.54 70.7±3.21
36.21±0.43 Pollen tube ruptured
80.23 ±2.12 128.7±8.2
82.65±3.44 210.7±9.66
51.56± 1.08 70.12±4.4
54.2±1.19
140.3±7.67
48.25±1.25 46.42±0.99 89.55±5.55 50.04±1.65 110.2±3.98
Table- 4 Effect of boric acid on in vitro germination of pollen grains in Luffa acutangula Boric acid (ppm)
After 1 hour
After 2 hours
After 4 hours
Germination Tube length (%) (µm)
Germination Tube length (%) (µm)
Germination Tube length (%) (µm)
25
21.12±0.67 29.24±0.7
30.04±0.76 67.5±2.1
37.23±1.04 80.4±2.4
50
43.91±1.43 46.4±1.56
35.3±0.98
100
50.05±1.46 58.64±2.32 80.47±3.29 119.06±2.4 80.09±3.24 169.8±5.34
125
52.25±1.97 41.9±2.19
68.06±2.1
150
48.2±0.88
50.42±2.62 90.22±4.43 52.00±1,96 144.12±4.13
175
32.12±0.56 25.23±0.64 41.5±1.4
200
51.5±1.21
34.2±0,89
74.4±3.66
92.65±3.94 43.46± 3.34 133.43±4.31
101.3±2.24 78.65±2.12 160.09±5.38
76.4±2.6
51.16±1.56 Pollen tube deformed
64.06±1.86 82.04±2.96 72.8±2.2
Pollen tube ruptured
To obtain the optimum condition for germination of pollen grains and tube development the test pollen were separately placed in mixture of boric acid and sucrose solutions. In Lagenaria siceraria 80.81±4.56 % germinated pollen grains with 491.1±10.43 µm pollen tube formation observed in 4% sucrose and 100ppm boric acid mixture (Table 5). In Luffa acutangula optimum pollen germination (78.23±2.14 %) with 469.65±7.96 µm tube length observed in 5% sucrose with 100 ppm boric acid (Table 6). The pollen grains of both the plants are now placed in a optimum mixture of sucrose and boric acid mixed with recommended doses of chemical and biological fungicides to observe the effect of these supplements on the germination and tube growth. It was observed that a drastic reduction in pollen germination and tube growth in both the test samples when they are contaminated with chemical fungicides but biofungicides have a little or no effect on their physiology (Table 7 & 8).
4
5
6
8
100
100
100
100
20.43±0.56
18.12±0.34
31.7±0.89
43.4±2.18
26.34±1.12
Germination (%)
95.5±3.2
167.09±5.44
215.01±6.97
320.57±9.12
135.5±4.5
Tube length (µm)
After 1 hour
26.18±1.19
22.43±.88
47.46±1.46
75.5±4.32
35.23±2.9
Germination (%)
110.76±5.45.
210.24±2.45
270. 16±6.65
410.65±10.34
168.4±8.9
Tube length (µm)
After 2 hours
2
4
5
6
8
100
100
100
100
100
Boric acid (ppm) Percentage of sucrose
10.45±0,78
19.56±0.67
55.16±1.23
29.89±0.8
27.42±0.45
Germination (%)
95.25±7.6
147.54±5.45
225.15±6.49
190.06±6.23
135.45±3.4
Tube length (µm)
After 1 hour
16.46±1.12
27.5±0.45
69.09±1.45
40.04±0.9
38.2±1.56
Germination (%)
112.34±3.8
200.10±7.78
320.06±8.6
203.65±5.78
170.34±4.4
Tube length (µm)
After 2 hours
Table- 6 Effect of sucrose and boric acid on in vitro germination of pollen grains in Luffa acutangula
2
100
Boric acid (ppm) Percentage of sucrose
Table- 5 Effect of sucrose and boric acid on in vitro germination of pollen grains in Lagenaria siceraria
150.2±3.98
260.04±6.12
340.6±7.61
491.1±10.43
236.6±6.8
Tube length (µm)
30.3±0.56
33.45±0.66
78.23±2.14
56.98±1.67
45.34±1.45
Germination (%)
Pollen tube ruptured
Pollen tube ruptured
469.65±7.96
300.04±8.86
221.1±5.8
Tube length (µm)
After 4 hours
30.2±0.67
27.76±0.87
50.5±2.16
80.81±4.56
45.3±2.1
Germination (%)
After 4 hours
190
0.2% Vitavax
0.1% Hilzim
Sudobact
4% + 100ppm
4% + 100ppm
4% + 100ppm
64.34±24
23.7±0.78
11.12±0.45
Germination (%)
256.34±4.6
140.06±3.68
100.12±2.69
Tube length (µm)
After 1 hour
72.8±2.1
26.4±0.64
18.1±0.76
Germination (%)
391.16±7.88
160.06±5.34
139.20±4.34
Tube length (µm)
After 2 hours
76.6±2.34
27.04±0.34
18.12±0.65
Germination (%)
After 4 hours
Fungicides
0.2% Vitavax
0.1% Hilzim
Sudobact
Sucrose + Boric acid
4% + 100ppm
4% + 100ppm
4% + 100ppm
40.18±1.02
22.2±.65
12.21±0.23
Germination (%)
298.8±6.56
140.34±3.45
110.8±3.98
Tube length (µm)
After 1 hour
68.02±1.65
26.6±0.65
18.1±0.87
Germination (%)
390.98±6.76
199.5±5.96
140.4±4,92
Tube length (µm)
After 2 hours
72.08±2.1
30.61±0.87
24.2±0.54
Germination (%)
After 4 hours
Table- 8 Effects of sucrose, boric acid and fungicides on in vitro germination of pollen grains of Luffa acutangula.
Fungicides
Sucrose + Boric acid
Table- 7 Effects of sucrose, boric acid and fungicides on in vitro germination of pollen grains of Lagenaria siceraria
432.2±9.12
220.06±7.76
170.2±6.04
Tube length (µm)
462.06±8.98
220.4±6.4
170.42±5.42
Tube length (µm)
191
192 Sucrose used for in vitro pollen germination is suitable carbohydrate source for pollen germination, maintains osmotic pressure of the medium and act as a substrate for pollen metabolism (Johri and Vasil, 1961; Shivanna and Johri, 1985). Boron may enhance the sucrose uptake and stimulate germinating ability. Boron has been determined as a mediator to play a role in the growth of the pollen tube and pollen germination (Lewis, 1980). Boron also provided by the stigmas and styles, facilitates sugar uptake and play a role in pectin production in the pollen tube (Richards, 1986). In this piece of work the authors similarly formulated the optimal media for pollen growth using boric acid and sucrose but the decrease in their physiology occur when it was supplemented with chemical fungicides and Vitavax has more deleterious effect than Hilzim. Comment: The present investigation clearly reflects that the indiscriminate and wide use of chemical fungicides may reduce the germinability and pollen tube formation and consequently fruit set, if come in contact with viable pollen grains. On the other hand the pollen grains of these vegetable crops are unaffected by biological fungicides like Sudobact. Naturally the application of bio-fungicides should be encouragement and more research is required for development of novel bio-fungicides for sustainable agriculture and crop management. References: Barker, H. G. & Barker, I. (1979). Starch in Angiosperm pollen grains and its evolutionary significance. American J. Bot. 66: 591-600. Bhattacharya, A. and Mandal, S. (2004). Pollination, pollen germination and stigma receptivity in Moringa oleifera Lamk. Grana. 43: (1): 48-56. Biswas, P. and Mondal, S. (2014). Impact of sucrose and boric acid on in vitro pollen germination of Ceiba pentandra L. Indian J. Applied & Pure Bio. 29(2): 323-329. Brewbaker, J.L. & Emery, G.C. (1962). Pollen radiobotany, Radiat. Bot. 1: 110-154 Dey, K., Mandal, S. and Mandal, S. (2015). In vitro pollen germination of Mitragyna parvifolia (Roxb.) Korth. Int. J. Curr. Microbiol. App.Sci. 5(1):768-777 Diaz, S.L. and Garay, B.R. (2007). Simple methods for in vitro pollen germination & pollen reservation of selected species of the Genus Agave. e-Gnosis [online] 6, Art. 2. Gupta, S., Bhattacharya, K.N. and Chanda, S. (1989). In vitro pollen germination of Solanum sisymbriifolium Lamk. J. Palynology. 25: 65-72 Johri, B. M. and Vasil, I. K. (1961). Physiology of Pollen. Bot. Rev. 27(3):318-381.
193 Katara, M.M. (2013). In Vitro Pollen Germination of Datura metal L.(5) Effect of Hormones. International Journal of Scientific Research, 2 (12): 51-52. Kumari, A. and Komal, R., Rajesh, R. and Pandey, A.K. (2009). In vitro pollen germination, pollen tube growth and pollen viability in Trichosanthes dioica Roxb. (Cucurbitaceae).Intl. J. Pl. Repd. Biol. 1(2): 147-151. Lewis, D.H. (1980). Boron lignifications and the origin of vascular plants. A unified hypothesis. New Phytol, 84: 209-229. Ma, L. G, Fan, Q. S, Yu, Z. Q, Zhou, H. L, Zhang F Z and Sun D Y, (2000) Aluminum inhibit pollen germination via extracellular calmodulin. Plant Cell Physiol. 41 (3): 372-376. Mondal, S. and Ghanta, R. (2012). Studies on in Vitro Pollen Germination of Lawsonia inermis Linn. Adv. Bio. Res., 3(3):63-66. Ottavio, E. Mulahy, D. Sari Goria, M. and Mulahy, G.B. (1992). Angiosperm Pollen and Ovules, Springer-Verlag, Patil, P., Malik, S.K., Negi, K.S., John, J., Yadav, S., Chaudhari, G. and K.V. Bhat (2013). Pollen germination characteristics, pollen-pistil interaction and reproductive behaviour in interspecific crosses among Abelmoschus esculentus Moench and its wild relatives. Grana 52(1): 1–14. Pfahler PL (1983). Comparative effectiveness of pollen genotype selection in higher plants. In: Mulcahy DL, Ottaviano E (eds) Pollen: biology and implications for plant breeding. Elsevier, New York Amsterdam Oxford. 361–366 Richards, A.J. (1986). Plant breeding systems. George Allen & Unwin, London. 529 pages. ISBN 0-04-581020-6. Sarkar, N.R., Mondal, S. and Mandal, S. (2016). Studies on in vitro pollen germination of Holmskioldia sanguine Retz. Intl. J. Current Adv. Res. 5(7): 10.62-1065 Sever, K., Škvorc, Ž., Bogdan, S., Franjić, J., Krstonošić, D., Alešković, I.I., Kereša, S., Fruk , G. and Jemrić, T. (2012) In vitro pollen germination & pollen tube growth differences among Quercus robur L. clones in response to metrological conditions. Grana. 51(1): 25-34. Shivanna, K. R., Linskens, H.F. and Cresti, M. (1991). Pollen viability and pollen vigour. Theor. Appl. Genet., 81: 38-42. Shivanna, K.R. and Johri, B.M. (1985). The angiosperm pollen structure and function. Wiley Eastern Ltd., New Delhi. Shivanna, K.R. and Rangaswamy, N.S. (1993).Pollen biology - A laboratory manual. Narosa Publishing House, New Delhi. Zaman, M.R. (2006). Pollen germination, viability and tube growth in fourteen species cultivated and wild species of cucurbits grown in Bangladesh. J. Life Earth Sci. 1(2):1-7. Zarrabi, A. and Imani, A. (2011). Effects of fungicides on in vitro pollen germination, tube growth and morphology of Almond (Prunus dulcis). African. J. Agri. Res. 6(25): 5645-5649.
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10 INTEGRATED PLANT DISEASE MANAGEMENT IN DEVELOPING COUNTRIES H.K. Chourasia and Akanksha Raman Microbiology and Plant Pathology Laboratory, Department of Botany, TM Bhagalpur University, Bhagalpur-812007 Email :
[email protected] ABSTRACT Integrated disease management (IDM), which combines biological, cultural, physical and chemical control strategies in a holistic way rather than using a single component strategy proved to be more effective and sustainable. In practice and in the majority of cropping systems today, emphasis is still being placed on a single technology. Nevertheless, the use of IDM strategy is gaining momentum, but in developing countries it often lacks the enabling environment for its successful implementation. Success requires appropriate policies in place that cover a wide range of themes such as plant protection, private sector investment, trade and export, food safety, land use, education and awareness, and agriculture extension. Wide adoption of IDM practices is a pre-requisite for achieving impact at the country level. Experience over the last few decades clearly showed that adoption and support for using participatory approaches help farmers improve their overall field management, including disease management, reducing costs and improving production efficiency. In this paper, we will highlight all the elements that require attention to achieve successful IDM adoption at the national level in developing countries. Key words: IDM, enabling environment, participatory approaches.
INTRODUCTION Plant diseases caused by a variety of causal agents (fungi, bacteria,
196 viruses, phytoplasmas and nematodes)is a group of biotic constraints that reduces crop yields worldwide. In developing countries, from tropical to subtropical to Mediterranean and temperate climates, crop losses are often higher than in the developed countries, mainly because farming communities lack most appropriate solutions and resources devoted to their study. It is estimated that 10-15% of the already low yields in developing countries is lost due to disease attack, and losses can be higher if postharvest diseases are considered. This is a significant loss, considering that in developing countries at present more than 800 million people do not have enough food, and around 1.3 billion live on less than one dollar a day (FAO, 2004). It should be mentioned, however, that reported crop losses due to diseases are often exaggerated and not based on a holistic approach that considers all possible production constraints. A recent study dealing with all production constraints (including diseases) for six major crops (wheat, maize, rice, sorghum, chickpea and cowpea) in 13 Asian and African farming system showed that losses caused by diseases ranged from 3 to 14%, whereas yield losses due to all biotic stresses ranged from 16 to 37% and yield losses to all crop production constraints ranged from 36 to 65% (Waddington et al., 2010). The list of pathogens harmful to crops is large and extremely diverse. Crops can be attacked at different growth stages: at seedling establishment (root and seed rots), young seedlings (root and collar rots, seedling blights, wilts), pre-flowering (wilts, leaf blights, yellowing and mottling of the foliage, stunting), flowering(bud rots, flower blight), post flowering (rusts, blights)and post-harvest (fruit rots). The same disease can induce diverse symptoms at different growth stages. Man made activities such as crop intensification and introduction of new crops or new cultivars of existing crops to new regions as well as changes in cropping practices, including plant breeding led to the development of serious epidemics around the globe, mainly because such activities could disturb the balance, which naturally existed for many generations (Buddenhagen, 1977). Over the past few decades, application of pesticides was the dominant form of disease control in developed and, increasingly, in developing, countries. However, many problems have been associated with such an approach, such as the frequent emergence of fungicide resistance in pathogens and the harmful effects of fungicides to human health and the environment. The concept of Integrated Disease Management (IDM), where diseases are managed by integrating a range of control methods and
197 practices, is becoming more popular among farmers and researchers. IDM calls for minimal use of pesticides, and only if deemed necessary, giving preference to other control methods such as host-plant resistance, cultural practices and biological control. Even though the use of IDM strategy is well established and results obtained are encouraging, in practice and in the majority of cropping systems today, emphasis is still being placed on a single technology, may it be host-plant resistance, pesticide applications or cultural practices and rarely the combination of all (Thomas and Waage, 1996). The outcome of such practices is mostly not sustainable. Implementation of IDM in developing countries is gaining momentum, but still requires more serious efforts to achieve impact at the country or regional level. The implementation of IDM in many developing countries at present still follows a top-down approach, which is mainly linked to the traditional extension system. This does not provide farmers with the needed empowerment that is considered essential for the development of sustainable IDM, nor does it provide the extension personnel, especially with the reduction in extension funding, with appropriate knowledge and skills needed to achieve that objective. This paper intend to summarize the progress made so far in developing IDM strategies and their components for major crops in developing countries, the constraints for their wide use and training needs which are essential for their large scale implementation. COMPONENTS OF DISEASE CONTROL IDM is currently defined as: “a sustainable approach to managing diseases by combining biological, cultural, physical and chemical tools in a way that minimizes economic, health and environmental risks”. This concept evolved from the original IPM definition after responding to today’s call for ecologically based pest management (Overton, 1996). Biological tools in this definition include host plant resistance as well as microbial agents for disease control, but in this paper they will be considered as separate components. Accordingly, the major components of disease management summarized here are: host-plant resistance, cultural practices, biological control and chemical control. Even though these components will be dealt with individually, it should be mentioned that often the different components are complementary to each other with strong interaction among and between them and the environment and that it is essential to break away from relying on a single-technology and to adopt a more ecological
198 approach built around a fundamental understanding of population biology at the local farm level and to rely on the integration of control components which are readily available to the resource-poor farmers (Thomas, 1999). Host-plant resistance. Host plant resistance is an important tool to control diseases of major food crops in developing countries, especially wheat, maize, rice, potato, chickpea, peanuts and cowpea. The use of resistant varieties is very much welcomed by resource poor farmers because it does not require additional cost and it is environment-friendly. Rice varieties resistant to rice blast (Bonman and MacKill, 1988), bacterial blight (Mew et al., 1992), rice tungro (Azzam and Chancellor, 2002) and brown spot (Ou, 1985) are widely used. The resistance is often durable, thanks to proper management of genetic diversity by employing gene rotation, multilines and cultivar mixtures, a strategy that proved effective in reducing disease damage in natural ecosystems (Wolf, 1985).Such success stimulated interest in extending the principles of genetic diversity for disease control to other crops in developing countries (Leung et al., 2003). Late blight is considered as one of the most important biotic constraints for potato production in many developing countries including many from Latin America, which are considered as the center of origin for potato crop. Traditionally this disease is controlled by several fungicide spays, but the emergence of fungicide resistance in many locations and the increasing cost of their application, encouraged the search for other control strategies. The development of high yielding varieties resistant to late blight with acceptable quality was very much welcomed by the farming community of Asian and African countries (Nelson et al., 2001). Rusts have been known to cause serious disease on wheat since its domestication. The use of genetic resistanceis still the most economic and feasible mode of disease control. Genetic resistance is often based on a limited number of major genes that are readily overcome by evolving pathogen races. With the reduction of genetic diversity in the wheat cultivars planted over large are as globally, serious rust epidemics are being recorded whenever new aggressive virulent rust races emerge. Atypical example is the yellow rust epidemics that spread from East Africa to Central and South Asia and North Africa during the 1980’s and 1990’s. Presently the breakdown of Yr 27, a gene used to replace Yr 9, and the emerging stem rust race Ug 99 are threatening 80-90% of commercial wheat varieties grown worldwide (Hodsonand Nazari, 2010).
199 Late leaf spot caused by Phaeoisariopsis personata and rust caused by Puccinia arachidis are two most destructive foliar diseases of peanut worldwide. Host plant resistance has been used recently as one control component and a number of peanut cultivars such as ICGV 89104 and ICGV 91114 are now available. Field trials conducted in India showed that these cultivars yield 55-60% more than local cultivar, and the severity of both diseases is significantly lower in these than in the local cultivar (Pande et al., 2001). Similarly, peanut varieties resistant to peanut rosette virus disease whichc auses serious losses in Africa have been developed(Reddy, 1998). Cultural practices. Cultural practices such as cultivation techniques, mulching, intercropping, plant density, planting date, crop rotation, strip farming, timing ofharvest, barrier crops, crop mixtures, roguing, healthy planting material, soil solarization, soil amendments and fertilizer management, and water management have been used singly and in combination as tools for disease management. For some crops in developing countries, such control practices may be the only economically viable method available (Palti, 1981; Thurston, 1992). Cultural control methods not only serve in promoting the healthy growth of the crop, but are also effective in directly reducing inoculum potential(pruning, roguing, crop rotation, ploughing, etc.) and in enhancing the biological activities of antagonists in the soil (solarization, crop rotation, mulching, etc.). Chemical control. For many decades fungicides played an important role in disease control. In the 1960s, systemic fungicides started gradually to replace the older non-systemic chemicals with more effectiveness and specificity in disease control. Very quickly, triazole fungicides gained 24% of the total fungicides market (Hewitt, 1998). However, the non-systemic fungicides such as mancozeb and chlorothalonil plus copper and sulpher-based products continued to have a good share of the market, especially in developing countries because of their lower cost. More recently, new classes of fungicides were developed with significant impact on disease control. These include anilinopyrimidines, phenoxyquinolines, oxazolidinediones, spiroketalamines, phenylpyrroles, strobilurins and activators of systemic acquired resistance. However, the development of pathogen populations showing reduced sensitivity to many of the newly developed products posed a serious challenge that the traditional fungicides (e.g. sulpher, folpet, etc.) did not face. The availability of a variety of new products, with narrow and broad specificity, offer important disease control options, however, their practical application continues to face the risk of selection of resistant pathogen populations
200 (Gullino et al., 2000).Experience accumulated over the last few decades clearly showed that fungicidal application had a better impact when used within an IDM strategy (De Waard et al., 1993). In addition, public concern has increasingly influenced the fungicide industry in developing effective products with low mammalian toxicity and environmental impact and low residues in food, to meet international health standards and compatibility in integrated pest management programs (Knight et al., 1997). Biological control. Success in using microorganisms against plant pathogens started with the control of crown gall with Agrobacterium radiobacter K84 (Kerr, 1980), and that of seedling blights caused by Pythiumand Rhizoctonia with Trichoderma harizanum (Harmanand Bjorkman, 1998), Gliocaladiumvirens (Lumsdenand Walter, 1995) and Streptomyces griseus (Cook et al., 1996). However, the use of naturally occurring bio-control agents (antagonists) of plant pathogens can be traced back to many centuries through the traditional practice of crop rotations that primarily permit the reduction of pathogens’ inoculum potential in the soil below injury level. This approach is still the most important single component, in both developed and developing countries used to manage root pathogens. This process is often accelerated by adding composts or manures, which enrich the soil with antagonistic microflora (Baker and Cook, 1974). THE ENABELING ENVIRONMENTFOR THE SUCCESSFUL IMPLEMENTATION OF IDM STRATEGIES The ultimate aim of promoting IDM is to empower its users to engage in competitive and sustainable agricultural production with longterm positive impacts on poverty and human and environmental health. The impact of successful implementation of IDM approaches beyond pilot scale cannot be achieved if IDM is not strategically positioned within national policies for agricultural production and protection, and within the broader context of agricultural and rural development, and human and environmental health (SP-IPM, 2008).There are a number of the national policies and related regulations that have direct impact on IDM (and generally IPM) adoption and scaling up, with special emphasis on the developing countries. Plant protection policy. This would cover: (i) pest/disease management policies, (ii) pesticides management policies, (iii) plant quarantine, and (iv) seed certification, and can be summarized as follows: Pest/disease management policies implemented by the Ministries of Agriculture in an attempt to reduce crop losses both in quality and quantity.
201 In several developing countries, however, some of these policies could be more harmful for the implementation of IDM or IPM or may help create more resurgent pest problems and environmental and health hazards to the fragile ecosystems. A typical example would be the application of aerial sprays of pesticides, especially, when done on a regular basis (e.g. wheat rusts, or Dubas bugs in datepalms) or the free non-monitored distribution of pesticides and inputs to farmers upon the occurrence of an epidemic or pest outbreak. Examples of IDM-promoting policies include those supporting disease surveillance, or the establishment of national and regional forecasting and early warning systems or those promoting decision support systems and information sharing. Pesticide management policies. These cover regulations related to pesticide registration and labeling, testing pesticide quality and residue levels on commodities for the local market and in trade, guidelines and regulations related to application methods, authorized personnel, clothing and equipment and pesticide disposal. Most developing countries lack the needed skills, laboratories, equipment, financial means and political will to implement these regulations. Schillhorn van Veen et al.(1997) list some government action or inaction) that may directly promote the use of pesticides. These include cases where the regulation authority is dominated by producer interest, or where the regulation or the implementation of regulation is weak, or where public extension lack information on and access to alternative technologies. Policies of banning or taxing hazardous pesticides should be accompanied by the provision of alternative control measures to avoid illegal trade or increased vulnerability of farmers (Shcillhorn van Veen, 1999). With the aim of promoting agricultural productivity, some countries have implemented policies of pesticide subsidy or elimination of import tax on pesticides (SP-IPM, 2008). Such policies would directly undermine the increased adoption of IDM approaches. Plant quarantine. Quarantine systems are the basis for transboundary pest and disease prevention through control of the sources of pest introductions and incursions. Pest and disease surveys as well as pest risk analysis (PRA) studies are critical for preparing and updating quarantine pest lists. Although most of the developing countries have quarantine regulations in place, their effectiveness is very limited often due to scarce financial, human and infrastructural resources. Field surveys and PRA are sporadic when done and pest lists are commonly based on literature reviews and information from researchers. Early detection and reporting of the introduction or emergence of a new disease through regular surveys is indispensable for checking disease spread before it reaches
202 epidemic levels. Citrus tristezavirus (CTV) which affect citrus production in many countries, including the Mediterranean region is a good example of the spread of a disease where not only the pathogen, but also its vector is covered by quarantine regulation and control (Moreno et al., 2008). Seed certification, including vegetative propagation material. Many of the plant pests and diseases are transmitted through seeds or vegetative material and the use of clean planting material would then be a main component of any IDM programme. National policies related to the quality control, availability and cost of such material for farmers is critical for IDM adoption. This encompasses systems for inspection and certification of seeds and vegetative material and nursery control. Such policies, if not properly assessed and designed in terms of their economic feasibility, practicality and accessibility to farmers could be a hindrance to IDM programmes. This is especially true in developing countries where subsistence farmers may not be able to afford the cost of certified seeds and seedlings or have limited transportation access to sources of seeds. Seed policies allowing for GMO or hybrid seeds that cannot be multiplied by resource poor farmers, a common practice within the informal seed system, could also negatively affect the adoption of IDM programmes. Private sector investment policy. This could have a negative or a positive affect on the adoption of IDM practices. This is usually related to the private sector involved in inputs for IDM or IPM technologies including biopesticides, disease-free seeds/vegetative material (certified and tissue cultured material), pheromones, growth-promoting chemicals, biological control agents, etc. Regulations are critical to promote or hinder the involvement and support that the private sector could provide to IDM promotion. Since IDM or IPM is very location-specific, the scope of activities and market of this private sector are usually limited, and welldesigned government incentives or tax breaks may be needed to allow this sector to survive (SP-IPM, 2008). Trade and export policy. Policies at the global and national levels are increasingly interdependent as they influence pest management through regulation of international trade, food and environmental safety and intellectual property rights (Sorby et al., 2003). IDM policies should accordingly be addressed at the global policy level. The management of trans-boundary diseases travelling across continents by wind or accidentally through travellers (such as the soybean or wheat rusts) should be of global concern and international cooperation would be needed to timely share information on the disease spread or the emergence of new virulent strains
203 of pathogens. Concerted international efforts should be undertaken to support countries in need to monitor and manage the disease at its origin before it gets out of control. Examples of such systems include the global wheat rust monitoring system and the Rust SPORE website established at FAO (http://www.fao.org/agriculture/crops/rust/stem/en/) and the late blight disease European network- Euroblight (http://www.euroblight.net/ EuroBlight.asp) Food safety, public health and environmental policies. Food safety regulations and standards regarding pesticide residues and other food contaminants such asmycotoxins are usually a positive driving force for farmers to implement IDM strategies. Other related public health and environmental health issues include soil and water contamination with pesticides, agro-chemicals and other contaminants. Through regulatory systems for the use of pesticides or GMOs governments minimize environmental and public health hazards and determine a proper balance between the costs and benefits of these technologies (Schillhorn van Veen, 1999). However, the implementation of such a regulatory system is faced with several problems especially in the developing countries and these include: (i) involvement of several ministries and agencies with different/ limited mandates, power or authority and responsibilities; (ii) limited human, financial and infrastructure capacities for testing and action taking, and (iii) lack or limited systems for national coordination and information sharing Land use policy. Land tenure arrangements are often limiting factors in the adoption of sustainable production activities and IDM technologies by farmers. Landownership and land size have direct effects on the risk level that farmers would be ready to assume as well as on the motivation of farmers to invest in new technologies and adopt strategies with longerterm returns on investment. Crop and genetic diversity within the larger landscape, including gene deployment strategies, reduction of the number of varieties cultivated in agro-ecological areas, monoculture in time and space, management of field hedges harboring natural enemies or the management of alternate and secondary hosts of pathogens are all factors that would directly affect the effectiveness and successes of IDM strategies and their scaling up at a regional or national level. One example is the large-scale deployment of the yellow rust resistance gene Yr9 in wheat varieties cultivated over large areas and in several countries thus facilitating global yellow rust epidemics during the 1980’ and 1990’s when a new virulent race evolved. Risk of similar epidemics is presently threatening wheat production since the reporting in 2004 of a new race affecting Yr27
204 resistance. As mentioned above, Yr27 resistance has been deployed in most varieties in developing countries to replace the susceptible varieties overcome by the Yr9 virulence (Hodson and Nazari, 2010). Education and awareness policy. Besides farmers’ education in IDM principles (see section on extension policy), consumers, whether in rural or urban areas area major driving force of the IDM or IPM market and could act as an important lobbying force for promoting food safety-related policies such as control of mycotoxins or the excessive use of pesticides and other agricultural inputs and interventions with potential health and environmental risks. Awareness raising of policy makers by bringing them to the field and exposing them to the successes of IDM programmes and their impact on farmers and consumers has proven to be good strategy to get the national support to IPM and IDM programmes through policies, regulations, and funding(van de Fliert et al., 2000). Agricultural extension policy. Agricultural extension has traditionally been the responsibility of Ministries of Agriculture also in developing countries. Most of the extension programmes have been based on the diffusion of innovation concept, a top-down approach that is based on the assumption that technology and knowledge could be transferred from scientists to farmers via extentionists (Sulaiman and Hall, 2002). The training and visit (T&V) system that was promoted by the World Bank during the 1970s and 1980s in many countries in Africa (Chester, 2005) builds on this approach. The T&V system is based on trained extension staff and subject matter specialists, who regularly, often fortnightly, visit predetermined contact farmers, according to a detailed schedule and work plan. However, and although the contact farmers, selected as progressive farmers, usually adopt new technologies, further secondary transfer of technical messages to community members had not been successful, with very low adoption rates among non-contact farmers. Besides, this extension approach was costly and unsustainable for most governments. Despite some of its advantages, T&V is now widely considered as ineffective (Antholt, 1992,1994; Chester, 2005). In many of the developing countries the void left behind the official public extension system was occupied by the private sector. Among lessons learned from the failure of the top down extension approach was the realization that participatory, farmer-centered approaches, which encourage a holistic perspective focusing attention to the whole farm system enhances technology adoption. There is now a shift in many of the development and donor communities from considering extension as
205 mainly an act of transferring technologies to farmers to a new focus on participation of farmers in the innovation process and the facilitation of experimentation among farmer communities. The building of farmers’ management and problem solving capacity requires joint learning through practical fieldwork (Hagmann et al., 1999; Roling and Pretty, 1997). ADOPTION OF IDM PRACTICES AND SCALING-UP IPM and IDM approaches are knowledge intensive and locationspecific. Accordingly, practitioners, whether farmers or extensionists, would need to understand the agro-ecological processes affecting the disease and, accordingly, make informed decisions on how best to manage crops to avoid/prevent pest infestation or disease occurrence, as well as how to manage these pests and diseases once they become a problem. This will require a high level of analytical skill and intensive training in crop monitoring and ecological principles (Kenmore, 1991; van de Fliert et al., 2000). One of these approaches is the Farmers Field Schools (FFS) model that was originally developed for IPM activities in Asia (Kenmore, 1991) and is now being used for a wide range of agriculture related activities (IPM, IDM, soil conservation, watershed management, marketing, animal production, etc.). The training is founded on informal education principles, emphasizing learning by doing and empowering farmers to identify and solve their own problems. The model through experiential learning process promotes participation, self confidence, collective action and decisionmaking. Training is season-long and involves groups of 20-25 farmers coming together on a regular basis. The traineris a facilitator rather than an instructor. Farmers prepare observation fields for group study where they discover and test field processes. They form smaller mini-groups who in every session monitor the observation field, undertake the agro-ecosystem analysis, present and discuss their findings with the other groups. FFS typically have special sessions to discuss problems of relevance to the farmers that may not be related to agriculture or IPM. Special group activities encourage learning from peers, and strengthen communicative skills and group building(Pontius et al., 2002; van de Fliert, 2003). Although every FFS has clear entry point, such as a particular pest or disease problem critical to the farmers or farming community, the impact of FFS goes beyond solving this problem. Experience has shown that FFS participants improve their overall field management practices, reducing costs and improving production efficiency, diminishing risks to health and environment through pesticide management, improve their financial assets
206 and economic resilience and enhance their individual and community social well-being, particular for women participants(Mancini et al., 2006; van de Fliert, 2003). Scaling up IDM activities for a wider impact remains a challenge. This process, however, remains country and location-specific. IDM projects and programmes would need to identify and establish at an early stage of implementation linkages with organizations or individuals that can provide mechanisms for future scaling up (vande Fliert et al., 2000). Key persons and institutions should be involved in the design, planning and evaluation of the programme, as well as in critical events throughout the implementation of the programme. Analysis of potential organizations (public, private, NGOs, etc.) that are critical for the scaling-up should be done assessing their mandates, roles and responsibilities and capacities. Raising the awareness of decision makers at the national and district levels is also key to ensure the political support for sustainability of activities. Reporting is not enough and decision makers should be able to visit and see an FFS first hand and talk directly with farmers (Pontius et al., 2002). CONCLUSIONS The success and sustainability of IDM strategy, especially with resource poor farmers greatly depends on their involvement in helping generate locally specific techniques and solutions suitable for their particular farming systems and integrating control components that are ecologically sound and readily available to them. Training and awareness raising of farmers, disease survey teams, agricultural development officers, extension agents and policy makers remains to be an important factor for the successful implementation of IDM strategies. All direct stakeholders including farmers, extension workers, and local crop protection technicians should have a practical understanding of the ecology, etiology and epidemiology of the major diseases of the crop. Intensive training using participatory approaches should be used to empower farmers with the appropriate knowledge to become better managers of their own fields translating this knowledge into appropriate decision-making tools and practical-control tactics. Based on experiences from developing countries in implementation activities of IPM and IDM approaches, two strategic elements could be considered critical at the national level (Schillhorn van Veen et al., 1997): 1. Eliminating policies that promote environmental sustainable pest and disease management techniques and strengthening regulatory
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11 SCAR MARKERS: A VERSATILE MOLECULAR TOOL FOR DETECTION OF PLANT GENOME Kumari Rekha1, Ravi S. Singh1*, Dharamsheela Thakur3, Sima Sinha1, Ujjwal Jha2, Ankita Sinha1, Shikha Kumari1 and Prabhash K. Singh1 1
2
Department of Plant Breeding and Genetics, Bihar Agricultural University, Sabour, Bhagalpur-813 210, Bihar, India
P.G. Department of Biotechnology, Tilka Manjhi Bhagalpur University, Bhagalpur-812 007, Bihar 3
Department of Molecular Biology and Genetic Engineering, Bihar Agricultural University, Sabour, Bhagalpur-813 210, Bihar, India Corresponding Author:
[email protected]
ABSTRACT: The recent advances in the field of marker assisted selection for breeding aspects has increased the demand of molecular markers generally Simple Sequence Repeats (SSR) because of its co-dominant nature. One more highly potent and efficient marker which can play a vital role in breeding programs has emerged out that is SCAR (Sequence Characterized Amplified Region) marker. It is also a codominant molecular marker reported by Paran and Michelmore (1993). It is a marker which is derived from RAPD (Random Amplified Polymorphic Regions), AFLP (Amplified Fragment Length Polymorphism) and ISSR (Inter Simple Sequence Repeats) bands after cloning and sequencing. These markers have wide range of applications, for example, it helps in easy screening out tolerant genotypes from the susceptible ones, it can be linked to a particular genome, species, gene or trait; helps in taxonomy, detection of gene
212 introgressions, and hybridity testing. Apart from breeding purpose, SCAR too can be used for other purposes like sex determination, authentication of variety and maintaining the genetic purity from admixtures in industrial purpose. SCAR marker can turn out to be a powerful weapon in near future for combating various pest and pathogens which is a major breeder’s concern. Key words: Molecular marker, SCAR, Molecular breeding
1.0 Introduction SCAR literally means mark left after a healed wound but in the terminology of molecular genetics SCAR is a molecular marker i.e. Sequence Characterized Amplified Regions which is used to amplify a particular sequence. Recently, these markers have emerged as a versatile tool for detection of plant genomes more easily, hastily and efficiently. It is a co-dominant marker reported by Paran and Michelmore (1993). These markers were developed to overcome the drawbacks of dominant marker system. In general, the dominant marker system includes RAPD (Random Amplified Polymorphic DNA), ISSR (Inter Simple Sequence Repeat), and AFLP (Amplified Fragment Length Polymorphism). Sequences derived from RAPD/ISSR/AFLP markers are cloned to synthesize a SCAR marker. SCARs have several advantages over RAPD, ISSR and AFLP marker (Table 1) which makes it more specific, authentic and powerful tool. Table 1 Comparison of ISSR, RAPD, AFLP and SCAR markers Feature/ Characteristics
ISSR
RAPD
Information content
High
High Non-specific
AFLP
SCAR
High
Low
Specific
Specific
Specificity
Specific
Loci detection
Multiple or single Multiple
Multiple
Single
PCR-based
Yes
Yes
Yes
Yes
Reproducibility
Moderate
Low
High
High
Genome Coverage
Whole
Whole
Whole
Partially
Reliability
High
Low
High
High
Nature
Dominant
Dominant
Dominant
Codominant
2.0 DEVELOPMENT Steps for the development of SCAR markers is as follows: (a) RAPD/ISSR/AFLP profiling is done (b) unique bands present are cut and
213 eluted from the gel (c) the eluted DNA is then cloned into E .coli cells (d) Blue-white screening is performed for the selection of transformed cells (e) plasmid is extracted from the transformed colonies and is sent for sequencing (f) sequences obtained are used for primer designing (g) primer designed are then validated. The mentioned steps of SCAR marker has been depicted in the following Fig. 1.
Fig. 1.0 Flow chart of steps involved in SCAR development
3.0. APPLICATION OF SCAR MARKER: 3.1 Abiotic Stress Abiotic stress includes high temperature, drought, salinity etc. Various SCAR markers have been reported to have developed as in tomato, 14 RAPD primers linked with QTL for heat stress were used, of which only two primers were able to generate desired bands of size 300bp and 500bp These two bands were converted to SCAR markers and SCAE1 and SCAE2 primers amplified a reproducible band of 300 base pairs (bp) that is dominant and appeared in heat tolerant tomato only (Damra et al, 2017). Similarly, in sugarcane, Srivastava et al. (2012) developed RAPD- derived SCAR marker (OPAK 12724) that was used for screening tolerant and susceptible genotypes to drought stress. It helps in easy identification, screening and selection of tolerant and susceptible genotypes.
214 3.2 Biotic stress Biotic stress includes various pests and pathogens for which a number of SCAR marker have been reported in various crops. Srivastav et al. in 2012 reported a RAPD-derived SCAR marker ScOPX 04880 to screen tolerant and susceptible genotypes for the resistance gene ‘er1’. In B. juncea, putative source of aphid resistance was reported by Chander (2011) based on the molecular analysis of the identified tolerant accessions to mustard aphid. They screened 34 germplasm with 284 RAPD primers, of which 87 primers showed amplification, and from these four were polymorphic and finally one RAPD converted to SCAR marker for distinguishing tolerant and susceptible cultivar. Similarly Rekha et al. in 2018 developed one RAPD derived SCAR marker for aphid tolerance in B. juncea. Blackleg disease caused in canola by Leptosphaeria maculans pathogenicity group 3, Scar markers have been developed to track Canola resistance against this pathgen (Dusabenyagasani et al., 2008). Xcal Bo gene conferring resistance to black rot disease in cauliflower, SCAR markers linked to it had been developed from RAPD and SCAR marker (Kalia et al., 2017). An AFLP Marker linked to Clubroot Resistance Gene in Chinese cabbage is converted into co-dominant SCAR marker (Piao et al., 2002). Similarly, B1SM-F/B1SM-R SCAR marker is linked with smut resistance trait in sugarcane was developed which will facilitate sugarcane breeding programs for marker-assisted selection in sugarcane (Khan et al., 2017). SCAR markers were identified for screening Sigatoka resistance lines in future banana breeding program (Das et al., 2016). Similarly, species-specific SCAR marker was reported in Meloidogyne enterolobii for rapid identification of this nematode from field samples and as a routine diagnostic test for quarantine devices (Tigano et al., 2010). 3.3 Cultivar identification A total of 400 RAPD primers were used for its screening among four cultivars of Kentucky bluegrass (Poa pratensis) and one specific band of ‘KBG04’ was successfully converted into a SCAR marker. This helped in distinguishing the new line ‘KBG04’ from other Kentucky bluegrass germplasms, and also would be useful for cultivar identification and property rights protection in the future (Yuan et al., 2015). Dasypyrum is of interest as a source of germplasm for several agronomically important traits that can be used to improve wheat (Triticum aestivum L.) including improvement to grain quality as well as resistance to crop diseases such as powdery mildew spindle streak mosaic virus eyespot and stripe rust. Scar marker had been developed in it to distinguish it from its cytotypes (Hu et
215 al., 2016). Development of SCAR markers specific for the P genome of Agropyron cristatum useful in a variety of studies on introgressions of the P-genome chromatin into wheat.(Wu et al., 2010). A unique 1371-bp RAPD band specific for Amaranthus gangeticus (syn. tricolor) of a particular phytogeographic region was converted to a sequenced characterized amplified region (SCAR) marker. The translated marker sequence showed homology with hemagglutinin protein. This SCAR marker is potentially useful for germplasm conservation and identification of amaranth ecotype. (Ray et al., 2009). Zingiber officinale Roscoe (common or culinary ginger) is an official drug in Ayurvedic, Indian herbal, Chinese, Japanese, African and British Pharmacopoeias and SCAR marker was developed for the identification and differentiation of the commercially important plant Z. officinale Roscoe from the closely related species Zingiber zerumbet (pinecone, bitter or ‘shampoo’ ginger) and Zingiber cassumunar [cassumunar or plai (Thai) ginger] (Chavan et al 2008) . 3.4 Taxonomical Classification SCAR markers have been identified to have potential role in the solving the taxonomical problems. These markers developed were highly successful in distinguishing the six cultivated species of Brassica and its sub species of U triangle and prevent mis-classification at subspecies level (Federico et al., 2006). 3.5 Self incompatibility Self-incompatibility is inability to fertilize with functional male gametes. It has vital role to play in hybrid seed production. Development of SCAR markers had helped in easy identification of self-incompatible plants from self-compatible reducing time and labour. It had been reported in B. napus (Zhang et al., 2008). 3.6 Cytoplasmic male sterility Cytoplasmic male sterility too has vital role to play in hybrid seed production for which Scar markers have reported in B. napus linked to the male fertility restorer gene (Zeng et al., 2009). A set of AFLP derived SCAR primers have been reported in the cytoplasmic male sterile (CMS) line of B. juncea for rapid identification of introgression of fertility restorer gene from Moricandia arvensis through somatic hybridization and generated amplicons in male fertile plants (Ashutosh et al., 2007). In B. napus, AFLP derived SCAR marker had been identified linked to the rf gene which helps in easy identification of fertile and sterile plants (Hong et al., 2006). Similarly, SCAR markers linked to male fertility trait in citrus
216 were developed and validated in various citrus cultivars (Chae et al., 2011) 3.7 Morphological trait Various crops though seem to be phenotypically similar initially but have distinct morphological traits at post reproductive stage. Screening of plants for traits, which appear late in reproductive results in loss of time and labour. Development of SCAR markers for these late appearing traits is a great boon and hastens the selection of desirable plants. Negi et al. in 2000 was successful in development of AFLP derived SCAR marker in B. juncea for seed coat colour. Similarly, Scar marker have been developed in Azolla which is a free floating aquatic pteridophyte used as a biofertilizer in paddy field to identify morphologically similar species of Azolla (Abraham et al., 2013). 3.8 Quality test Various nutritional qualities can be easily tested with the developed SCAR markers so far reported in various crops. In Brassica juncea, an ISSR marker was found to be tightly linked to high 2-propenyl glucosinolate so SCAR marker was designed found to be associated with it and was found to be widely applicable in screening of mustard genotypes (Ripley and Roslinsky, 2005). Similarly, a point mutation in the ‘or’ gene of Chinese cabbage (Brassica rapa L. ssp. pekinensis) causes it to accumulate carotenoids. So, Zhang et al. in 2008 developed SCAR markers linked to this gene from RAPD and AFLP. 3.9 Genetic purity test SCAR markers plays important role in authentication of the variety from its substitutes as reported in case of cluster bean one genotype specific SCAR-20 for RGC-1031 (tolerant genotype against Macrophomina phaseolina) (Sharma et al., 2014). SCAR markers had been reported in Rosa centifolia for its distinction from its substitutes (Riaz et al., 2012). Similarly, species-specific SCAR marker have been developed in bamboo for Bambusa balcooa and B. tulda to allow for their proper identification, in order to avoid unintentional adulteration that affects the quality and quantity of paper pulp production. (Das et al., 2005). 3.10 Sex determination SCAR marker plays a vital role in sex determination. It helps in determination of the gender in the early vegetative growth phase, resulting in considerable saving of time and economic resources. Sex- linked markers for male determination have been r eported in Bryonia dioica
217 (Cucurbitaceae) (Oyama et al., 2009), in the dioecious rattan species Calamus simplicifolius (Li et al., 2010), in dioecious Pandanus fascicularis L. (Pandanaceae) (Vinod et al., 2007). Similarly, female specific SCAR marker was developed in Eucommia ulmoides Oliv in which pistillate plants have high economical value resulting in precise and quick identification of plant sex types (Xu et al., 2005). 4.0 CONCLUSION SCAR markers being locus specific and co-dominant in nature are proving out to be a reliable tool for cultivar authentication, quality test, assessing hybrid strains, screening tolerant genotypes for biotic and abiotic stress and etc. These markers reduces time and cost and can be effectively be used in markers assisted breeding for future breeding programs. REFERENCES Abraham, G., Pandey, N., Mishra, V., Chaudhary, A.A., Ahamd, A., Singh, R. and Singh, P.K. (2013). Development of SCAR based molecular marker for identification of different species of Azolla. Indian Journal of Biotechnology 12: 489-492. Ashutosh, Sharma, P.C., Prakash Shyam, Bhat, S.R. (2007). Identification of AFLP markers linked to the male fertility restorer gene of CMS (Moricandia arvensis) Brassica juncea and conversion to SCAR marker. Theoretical and Applied Genetics 114: 385-392 Chae, C.W., Dutt, M., Yun, S.H., Park, J.H. and Lee, D.H. (2011). Development of a SCAR Marker Linked to Male Fertility Traits in ‘Jinkyool’ (Citrus sunki).Journal of Life Science 21(12):1659-1665. Chander, 2011; http://krishikosh.egranth.ac.in/handle/1/83830. Chavan, P., Warude, D., Joshi, K. and Patwardhan, B. (2008). Development of SCAR (Sequence Characterized Amplified Region) markers as a complementary tool for identification of ginger (Zingiber officinale Roscoe) from crude drugs and multicomponent formulations. Biotechnol. Appl. Biochem. 50:61-69. Damra, E.M., Kasrawi, M., Akash, M.W. (2017). Development Of Scar Marker Linked To Heat Stress Tolerance In Tomato. Proceedings of 65th ISERD International Conference, Mecca, Saudi Arabia, 23rd-24th January 2017, ISBN: 978-93-8629192-9 Das, T., Mondal, S., Mishral, D.K. and Bhattacharyya, S. (2016) Development of SCAR marker for screening Sigatoka-leafspot resistance in banana genotypes. Indian Journal of Genetics 76(1): 69-74 Dusabenyagasani, M. and Fernando, W.G.D. (2008). Development of a SCAR marker to track canola resistance against blackleg caused by Leptosphaeria maculans pathogenicity group 3. Plant Disease Journal, 92:903-908. Federico, L., Luy, I., Sass, M.E., Jung, G., Johns, M.A. and Nienhuis, J. (2006). Development of robust SCAR markers that distinguished the six cultivated Brassica
218 species and subspecies of the U-triangle. Journal of the American Society for Horticultural Science, 131(3):424-432. Hong, D., Wan, L., Liu, P., Yang, G. and He, Q. (2006). AFLP and SCAR markers linked to the suppressor gene (Rf ) of a dominant genetic male sterility in rapeseed (Brassica napus L). Euphytica 151:401-409 Kalia, P., Saha, P. and Roy, S. (2017). Development of RAPD and ISSR derived SCAR markers linked to Xca1Bo gene conferring resistance to black rot disease in cauliflower (Brassica oleracea var. botrytis L.). Euphytica, 213:232 Khan, M., Pan, Y.B. and Iqbal, J. (2017). Development of an RAPD-based SCAR marker for smut disease resistance in commercial sugarcane cultivars of Pakistan. Crop Protection 94:166-172 Li, M., Yang, H., Li, F., Yang, F., Yin, G. and Gan, S. (2010). A male-specific SCAR marker in Calamus simplicifolius, a dioecious rattan species endemic to China. Mol Breeding 25:549–551 Li, M., Yang, H., Li, F., Yang, F., Yin, G. and Gan, S. (2010). A malespecific SCAR marker in Calamus simplicifolius, a dioecious rattan species endemic to China. Molecular Breeding 25(3): 549-551. Negi, M.S., Devic, M., Delseny, M. and Lakshmikumaran, M. (2000). Identification of AFLP fragments linked to seed coat colour in Brassica juncea and conversion to a SCAR marker for rapid selection. Theoretical and Applied Genetics, 101(2):146– 152 Oyama, R.K., Volz, S.M. and Renner, S.S. (2009). A sex-linked SCAR marker in Bryonia dioica (Cucurbitaceae), a dioecious species with XY sex-determination and homomorphic sex chromosomes. J Evol Biol. 2009 Jan; 22(1):214-24 Pace, C.D., Snidaro, D., Ciaffi, D., Vittori, A., Ciofo, A., Cenci, O.A., Tanzarella, C.O.,Qualset, G.T. and Scarascia, M. (2001). Introgression of Dasypyrum villosum chromatin into common wheat improves grain protein quality. Euphytica 117: 67– 75. Piao, Z.Y., Park, Y.J., Choi, S.R., Hong, C.P., Park, J.Y., Choi, Y.S. and Lim, Y.P. (2002). Conversion of an AFLP Marker Linked to Clubroot Resistance Gene in Chinese cabbage into a SCAR Marker. Journal Korean Society of Horticultural Science, 43(6):653-659. Rajesh, M.K., Sabana, A.A., Rachana, K.E., Rahman, S., Ananda, K.S. and Karun, A. (2016). Development of a SCoT-derived SCAR marker associated with tall type palm trait in arecanut and its utilization in hybrid (dwarf x tall) authentication. Indian J. Genet.,76 (1): 119-122. Ray, T. and Roy, S.C. (2009). Genetic Diversity of Amaranthus Species from the IndoGangetic Plains Revealed by RAPD Analysis Leading to the Development of Ecotype-Specific SCAR Marker. Journal of Heredity 2009:100(3):338–347. Rekha, K., Singh, R.S., Thakur, D., Sinha, S. and Singh, P.K. (2018). Development of SCAR marker for aphid tolerance in Brassica juncea L. Coss and Czern. Thesis submitted in Bihar Agricultural University, SABOUR.
219 Riaz, S., Sadia, B., Awan, F.S., Khan, I.A., Sadaqat, H.A. and Khan, I.A. Development of a species-specific sequence characterized amplified region marker for roses. Genetics and Molecular Research 11 (1): 440-447. Ripley, V.L. and Roslinsky, V. (2005). Identification of an ISSR marker for 2-propenyl glucosinolate content in Brassica juncea L. and conversion to a SCAR marker. Molecular Breeding, 16:57-66. Sharma, P., Kumar, V., Raman, K.V. and Tiwari, K. (2014). A set of SCAR markers in cluster bean (Cyamopsis tetragonoloba L. Taub) genotypes. Advances in Bioscience and Biotechnology, 5:131-141. Srivastava, M.K., Li, C.N. and Li, Y.R. (2012). Development of sequence characterized amplified region (SCAR) marker for identifying drought tolerant sugarcane genotypes. Australian Journal of Crop Science 6(4):763-767 Srivastava, R.K., Mishra, S.K., Singh, A.K. and Mohapatra, T. (2012). Development of a coupling-phase SCAR marker linked to the powdery mildew resistance gene ‘er1’ in pea (Pisum sativum L.). Euphytica, 186:855–866 Tiganoa, M., Siqueiraa, K., Castagnone-Serenob, P., Muletb, K., Queiroza, P., Santosa, M., Teixeiraa, C., Almeidaa, M., Silvaa, J. and Carneiroa, R. (2010). Genetic diversity of the root-knot nematode Meloidogyne enterolobii and development of a SCAR marker for this guava-damaging species. Plant Pathology 59:1054–1061 Vinod, M.S., Raghavan, P.S., George, S. and Parida, A. (2007). Identification of a sexspecific SCAR marker in dioecious Pandanus fascicularis L. (Pandanaceae). Genome 50: 834-839. Xu, W.J., Wang, W.B. and Cui, K.M. (2005). RAPD and SCAR marker(s) linked to sex determination in Eucommia ulmoides Olive. Euphytica 233-238. Yuan, X., He, Y., Huang, J., Hu, W., Zhou, H., Gao, Q. and Zhou, S. (2015). Development of a SCAR Marker for Rapid Identification of New Kentucky bluegrass Breeding Lines. Bot. Horti. Agrobo., 2015, 43(1):79-85 Zeng, F., Yi, B., Tu, J. and Fu, T. (2009). Identification of AFLP and SCAR markers linked to the male fertility restorer gene of pol CMS (Brassica napus L.). Euphytica 165:363–369 Zhang, X., Chaozhi, M., Tingdong, F., Yuanyuan, L., Tonghua, W., Qingfang, C., Jinxing, T. and Jinxiong, S. (2008). Development of SCAR markers linked to selfincompatibility in Brassica napus L. Molecular Breeding 21:305–315
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12 DEVELOPING INSECT RESISTANT RICE THROUGH MOLECULAR BREEDING Mankesh Kumar*, Satyendra, Manoj Kumar, Anand Kumar, S. P. Singh, Kumar Vaibhav and P.K. Singh Department of Plant Breeding and Genetics, Bihar Agricultural University, Sabour, Bhagalpur -813 210, Bihar *Corresponding Author:
[email protected] ABSTRACT Biotic stress due to insect pest is one of the major production barrier of rice in India. Breeding insect resistant rice varieties is the best option to reduce the loss caused by insect pests. But limited source of resistance in germplasm, evolution of new biotypes of insects, yield penalty in lieu of the resistant gene introgression in a cultivar are some of the important constants in conventional breeding for insect resistance. Several successful attempts have been made through transgenic technology to develop insect resistant genetically modified rice using Bt gene from bacterium Bacillus thuringiensis. But any genetically altered rice variety has been released for commercial cultivation due to other issues associated with transgenics. Marker Assisted Backcross breeding is the most suitable method being widely used for developing the resistant lines either through single transfer or multiple gene introgression, using gene pyramiding. Key words: BPH, Gall Midge, Gene pyramiding, insect resistance, MAS, Rice
INTRODUCTION: Rice is one of the most important cereal crops which feed nearly half of the world population. Due to introduction of semi-dwarfing trait during green revolution in India, the productivity of rice was increased inmany folds. The adoption of modern and high yielding varieties of rice
222 also increased the development of new races of pathogens and pests which affected rice cultivation throughout the country. This lead to the adoption of higher dose of fertilizer, chemicals and pesticide application in the field. The hazardous and non-degradable nature of the agrochemicals for controlling disease and insect pests affected not only the human health, but also deteriorated the soil health conditions. Breeding of resistant cultivars is the most cost-effective and environment-friendly strategy for pest management in rice; however, resistant cultivars are currently hampered by the rapid breakdown of the insect resistance. Thus, there is an urgent need to use more effective BPH resistance genes or pyramiding different resistance genes to develop more durable resistant rice cultivars. Right from the seeding in nursery to the storage of the crop, biotic stresses including pathogens, pests and weeds and abiotic stresses such as drought and periodic cycles of submergence, extreme cold, soil salinity, affects rice cultivation. Crop losses caused by major biotic stressors such as bacterial blight and blast disease, and due to insect pests are quite high (Hasan et al., 2015). In terms of relative importance, stem borer (29%) is the most important insect pest of rice in India followed by brown plant hopper (16%), gall midge (13%), leaf folder (10%), green leaf hopper (9%) and white brown plant hopper (9%) (AICRIP Entomology reports 200010). The occurrence of new races of pathogens and pests necessitates development of biotic stress tolerant varieties of rice. It can be achieved through conventional breeding, mutation breeding, transgenic approach and molecular breeding. In India, over 100,000 accessions of rice germplasm evaluated against major insect pests and about 1000 primary donors have been identified. More than 70 varieties against gall midge, 50 against BPH, 10 against stem borer and 25 multiple pest resistant varieties have been released for cultivation (Bentur et al. 2011). The biotic stresses resistance genes are considered as one of the most important natural resources to sustain agricultural production and to minimize crop losses (Mundt 1994). Breeding for resistance to insects possess some difficulties like evolution of new biotypes of insects to adapt to new situations (Roush and McKenzie 1987). The main challenges in breeding for resistance to insects are dynamic nature of host-insect interaction, loss of effectiveness of chemicals, breakdown of natural or artificial plant resistance, and difficulties in screening and selection of the resistant material under uniform insect infestation across environments (Roush and McKenzie 1987). Crow (1957) reported the development of resistance in insects following several generations of insecticide application.
223 Major insect pest of rice in India Rice stem borer In India there are 3 rice stem borer species viz. the yellow stem borer (YSB, Scirpophagaincertulas), the pink stem borer (PSB, Sesamiainferens) and the dark headed borer (DHB, Chilopolychrysus). YSB infestation is predominant from early tillering to maximum tillering stage and decreased gradually with increasing PSB infestation from the flowering stage. The rice stem borers are generally considered as the most serious pest of rice. They occur regularly and attack plants from seedling to maturity stages. In YSB, there is a lack of high level of resistance and precise knowledge of genetics of resistance which lead to incomplete transfer of resistance. Rice leaf and plant hoppers Some of the economically important species of leaf and plant hoppers infesting rice are the green leafhopper, zigzag leafhopper, the white backed plant hopper and the brown plant hopper (Misra and Israel 1970). They frequently occur in large number enough to cause hopper burn. The green leafhoppers, Nephotettixspp. (Homoptera: Cicadellidae) are most devastating pests of rice throughout the rice growing areas of Asia (Razzaque et al.1985). Both the nymphs and adults feed on the dorsal surface of the leaf blades rather than the ventral surface. They prefer to feed on the lateral leaves rather than the leaf sheaths and the middle leaves. They also prefer rice plants that have been fertilized with large amount of nitrogen. At early infestation points, round yellow patches appear which soon turn brownish due to the drying up of the plants. The brown plant hopper (BPH), caused by Nilaparvatalugens, has been one of the most devastating pests to rice crops in Vietnam and Asia. The BPH is a monophagous rice herbivore, and is a typical vascular feeder. It sucks the sap from the rice phloem using its stylet, and this results in direct damage to rice plants and causes ‘hopper-burn’ in the fields (Sogawa, 1973). The insect is also a vector of viral diseases that cause secondary damage to rice (Ling et al., 1970, 1978). It can also transmit Rice Ragged Stunt and Rice Grassy Stunt diseases. The patches of infestation then may spread out and cover the entire field (Heinrichs et al.1985).These insects are common in rainfed and irrigated wetland environments not prevalent in upland rice. Rice bug Rice bug, Leptocorisaacuta (Thunburg) and Leptocorisaoratoria (Fabricius) are important pests infesting the rice crop at the flowering stage.
224 These are also known as Gandhi bugs because of the peculiar odour they emit. The insects were earlier identified as Leptocorisaacuta from India, but now called as Leptocorisaoratoria (Fabricius). These two closely related species may occur together in rice fields. They are most abundant at 270C to 280C and about 80 % relative humidity. Population usually increases at the end of a rainy season but declines rapidly during dry month.Rice bugs damage rice by sucking out the contents of developing grains from preflowering spikelets to soft dough stage, therefore causing unfilled or empty grains and discoloration. Immature and adult rice bugs both feed on rice grains.High rice bug populations are brought about by factors such as nearby woodlands, extensive weedy areas near rice fields, wild grasses near canals, and staggered rice planting. The insect also becomes active when the monsoonal rains begin. Warm weather, overcast skies, and frequent drizzles favor its population build-up. The population of the rice bug increases at the end of the rainy season. Rice bugs are found in all rice environments. They are more common in rainfed and upland rice and prefer the flowering to milky stages of the rice crop. Adults are active during the late afternoon and early morning. Under bright sunlight, they hide in grassy areas. They are less active during the dry season. In cooler areas, the adults undergo a prolonged development in grasses. They feed on wild hosts for one to two generations before migrating into the rice fields at the flowering stages. The nymphs are found on the rice plant where they blend with the foliage. There, they are often left unnoticed. When disturbed, the nymphs drop to the lower part of the plants and the adults fly within a short distance. Rice leaf folder Rice leaf folder complex consisted of three species Cnaphalocrocismedinalis (Guenee), Marasmiapatnalis Bradley and M. exigua Butler (De Kraker et al. 1999). Its larvae always injure the top 3 leaves of rice with most injury occurring approximately 14 days after the population peaks. The main infestation of rice plants is during the heading to milky stages. Grasshopper There are four species of Grasshopper which infest rice crop; Hieroglyphusbanian (rice grasshopper), Oxyanitidula (small green grasshopper), Chrologonustrachypterus (surface grasshopper), and Aiolopustamulus (A. thalassinus) (small grasshopper). Feeding damage caused by short-horned grasshoppers result to cut out areas on leaves and cut-off panicles. Aquatic environments are suitable for the development of short-horned grasshoppers. The nymphs and adults feed on the leaf by
225 consuming large amounts of leaves. Rice Gall Midge Rice gall midge forms a tubular gall at the base of tillers, causing elongation of leaf sheaths called onion leaf or silver shoot. The Asian rice gall midge is found in irrigated or rainfed wetland environments during the tillering stage of the rice crop. It is also common in upland and deepwater rice. The adults are nocturnal and can easily be collected using light traps. During the dry season, the insect remains dormant in the pupal stage. They become active again when the buds start growing after the rains. The population density of the Asian rice gall midge is favored mainly by cloudy or rainy weather, cultivation of high-tillering varieties, intensive management practices, and low parasitization. Techniques to develop insect resistance in rice Developing resistant rice varieties is the most suitable option to reduce the yield losses caused due to insect infestation. Using conventional breeding techniques,it is not possible to screen the unwanted gene in the segregating generations and the problem of linkage drag persists,even with several backcross generations.In the other side, conventionalapproaches are helpfulingermplasm conservation,wide hybridisation, creating novelgenetic variants, andmutations (Werner et al., 2005). Conventional Breeding Strategy Through conventional breeding, methods like backcrossing,recurrent selection, and mutation breeding have been efficiently used for developing high yielding rice genotypes. However, by conventional phenotypic screening techniques, homozygous and heterozygous plants cannot be distinguished for most traits. For developing insect resistance use of molecular tools help hasedge in several ways such as minimising the number of backcross generations required to break the linkage drag and selection of desired lines at early stage of plant growth (Hasan et al.,2015).Hence, Molecularbreeding techniques are currently widely usedfor the transfer of desired gene in desired Rice variety. Insect resistant genetically modified rice: Genes from bacterium Bacillus thuringiensis (Bt) code for insecticidal Crystal (Cry) proteins which has been engineered in rice to develop insect resistant genetically modified plants for lepidopteran (stem borer) and hemipteran (plant hopper) pests. These prominent pests of rice in India has limited source of resistance in rice germplasm, and hence GM
226 technology is useful to overcome the crossing barrier arise in case of wide hybridisation. In China, GM rice lines expressing insecticidal genes with lepidopteran activity [e.g., cry1Aa, cry1Ab, cry1Ac, cry1Ab/Ac, cry1C, cry2A, CpTI (cowpea trypsin inhibitor)] or hemipteran activity (e.g., Galanthusnivalisagglutinin, gna, and Pinelliaternataagglutinin, pta) under control of various promoters have been developed and tested at various stages based on the regulatory process for agricultural genetically modified organisms (GMOs) (Chen et al. 2011). Besides cry1 and cry2 genes used for insect resistant GM rice, the Btvip gene (vip3H) (Fang J. 2008), plantderived insect-resistant lectin genes (e.g., gna, pta), protease inhibitor genes [e.g., CpTI, pinII (potato inhibitor II) and SbTI (soybean trypsin inhibitor)], and animal-derived insect-resistant gene (e.g., spider toxin gene, SpI) are also being used for insect resistant GM rice. Marker assisted selection (MAS) for developing insect resistance in rice Molecular marker techniques are the most effective method available for the transfer of desired gene in desiredrice variety with required combination. MAS is a process in which a marker is used for indirect selection of a genetic determinant (s) of a trait of interest, i.e., abiotic stress tolerance, disease resistance, productivity, and/or quality (Prabhu et al., 2009). Its success depends on several factors, like number of target genes to be transferred and the distance between the flanking markers and the target gene (Perumalsamy et al., 2010). In MAS, individual plants can be selected on the basis of their genotype during the selection procedure. MAS is useful in selecting the parents, increasing the effectiveness ofbackcross breeding and improving sex-limited traits (Zhou et al., 2007). There are various advantages of using MAS in rice breeding over conventional breeding. As the positive insect resistant progenies are confirmed through molecular markers, it is simpler than cumbersome phenotypic screening and therefore, it can reduce time, effort, and resources. Moreover, MAS selection can be conducted at the seedlingstage and undesirable plant genotypes can quickly be eliminated(Khan et al., 2015). Rice faces severe yield losses both due to biotic [like bacterial blight (BB), gall midge (insect) and Blast (disease)] and abiotic stresses (like submergence and salinity). Gene pyramiding is widely used effective, reliable, economical and environmentally friendly appr oach forcomprehensive management of theses stresses by theenhancementof host resistance. Gene pyramiding for a variety MAS shortens the breeding period and removes the extensive trait assessment involved. Through the application of marker assisted selection (MAS) technique, Das and Rao,
227 2015 pyramided genes/QTLs to confer resistance/tolerance to blast (Pi2,Pi9), gall Midge (Gm1,Gm4), submergence (Sub1), and salinity (Saltol) in a released rice variety Improved Lalat which was already incorporated with three BB resistance genesxa5, xa13, and Xa21 to supplement the Xa4 gene present in Improved Lalat. There are many insect resistance genes in rice for Gall Midge and Hopper which are tightly linked SNPs, SSRs, and STS markers are available (Table 1). Table1: List of selected abiotic and biotic stress resistance and quality genes/QTLs and linked markers: Stress/Disease/ Traits
Resistance genes/ QTLs
Available linked markers
References
Gall midge
Gm1, Gm2, Gm4
RM444, RM316, Biradar et al., 2004; and RM219, RG476, Das and Rao, 2015 RG329,RM547
Hopper burn
Bph-1 and Bph-10 (t), Bph-3, Bph-17, Bph-18, Bph-20, Bph21, Bph25, Bph26
XNpb248 and Singh et al., 2011; RG457, RM589, Kurokawa et al., 2016 RM5953, RM6217, BP-20-2, B121, RM6273, RM6775, RM5479
Till date 11 Gall Midge resistance genes have been reported in various rice varieties, Gm1, Gm2, gm3, Gm4, Gm5, Gm6, Gm7, Gm8, Gm9, Gm10, and Gm11(t) (Dutta et al., 2014; Das and Rao, 2015; Hasan et al., 2015; Bentur et al., 2016). In case of BPH, 24 resistance genes in cultivated O. sativa and wild species of rice including O. australiensis, O. eichingeri, O. latifolia, O. officinalis, O. rufipogon, O. glaberrina, and O. minuta (Jena and Kim, 2010; Huang et al., 2012) have been reported. The loci of Bph25, Bph26, and Bph27 have recently been shown to be the same as these of Bph20, Bph21, and Bph18 (Yara et al., 2010; Huang et al., 2012). Among the BPH resistance genes, the dominant gene include Bph1, Bph3, Bph6, Bph9, Bph10, Bph12, Bph13, Bph14, Bph15, Bph16, Bph17, Bph18, Bph20, Bph21, Bph22, Bph23, and Bph24, while the recessive genes include bph2, bph4, bph5, bph7, bhp8, bhp11, and bph19. It is important to know that these genes are only distributed on six of the 12 chromosomes (2, 3, 4, 6, 11, and 12). Among these, Bph14 has been finally isolated and characterized using map-based cloning, and thus provided a basis for understanding the mechanism for rice resistance to BPHs (Du et al., 2009). The Bph14 gene encodes a coiled-coil, nucleotide-binding, and leucinerich repeat (CC–NB–LRR) protein of the NB–LRR family, which is an
228 immune receptor type similar to R proteins functioning in disease resistance. There is a report of the use of SSR and STS markers in pyramiding two BPH resistance genes Bph14 and Bph15 into three elite japonica varieties Shengdao 15, Shengdao 16, Xudao 3 using marker assisted backcross breeding program (Xu, 2013). The pyramided lines in rice containing two or three BPH resistance genes have generally been found to provide greater resistance than those lines containing a single gene (Sharma et al., 2004; Li et al., 2006). ‘Biotype’ are the insectindividuals in a populations of species that have common biological features (including survival and development on a particular host, or host feeding and/or oviposition characteristics) (Claridge and Den Hollander, 1983). Out of four BPH biotypes in rice, biotypes 1 and 2 are widely distributed in Southeast and East Asia, and usually occur on the rice varieties TN1 and Mudgo, respectively (Claridge and Hollander, 1980; Jena and Kim, 2010). The rice variety ASD7 carry resistance gene bph2 against Biotype 3 of BPH (Panda and Heinrichs, 1983). The most destructive biotype, Biotype 4 occurs predominantly on the Rathuheenati variety. This biotype occurs on the Indian subcontinent and is thus referred to as the South Asian biotype (Claridge and Hollander, 1980; Jena and Kim, 2010). Studies have shown that Bph1 provides resistance to biotypes 1 and 3; bph2 provides resistance to biotypes 1 and 2; any of Bph3, bph4, bph8, and Bph9 provide resistance to biotypes 1, 2, 3, and 4; and bph5, Bph6, and bph7 only provide resistance to biotype 4 (Khush and Brar, 1991; Panda and Khush, 1995). Through monoculture of a single resistance gene resistant variety, new biotypes can be generated, which then becomes susceptible to the new biotype (Cohen et al., 1997; Jing et al., 2012). However, rice varieties carrying pyramided resistance genes or a major resistance gene and QTLs show durable resistance (Alam and Cohen, 1998b). Liu et al. 2016 had introgressed a dominant BPH resistance gene Bph27(t) into a susceptible commercial japonica variety Ningjing3 (NJ3) and indica variety 93-11 using marker-assisted selection (MAS), respectively. Further, they had attempted the pyramiding of Bph27(t) and a durable BPH resistance gene Bph3 by intercrossing single-gene introgressed lines through MAS. The introgression of BPH resistance genes significantly improved the BPH resistance and reduced the yield loss caused by BPH.In the marker assisted breeding program for disease and insect, ‘Linkage Drag’ is a common phenomenon. It can be minimised through repeated backcrossing upto 8 to 9 generations alongwith recombinant and background selection using flanking markers form the gene of interest and
229 polymorphic markers located through the genome, respectively. Marker Assisted Breeding Scheme for insect resistant in rice Huahui938, an elite indica CMS rice restorer line with good combined abilities of yield, good rice quality and high blast resistance but susceptible to BPH, was used as the recurrent parent by Wang et al., 2016. B5, a highly resistant line whose resistance genes Bph14 and Bph15 were obtained from wild rice (Oryzaofficinalis), was used as the donor parent. The figure 1 show a flow diagramme of representative marker assisted backcrossing programme to develop NILs (near isogenic lines) for insect resistance in rice.
Fig. 1 Procedure for the development of NILs with homozygous Bph14 and Bph15 loci in the genetic background of Huahui938 (Wang et al., 2016)
The overall breeding scheme consisted of a recurrent backcrossing procedure including one crossing, three generations of backcrossing and four generations of self-fertilization, combined with MAS in each generation (Fig. 1). A cross was made between the recurrent parent Huahui938 and the donor parent B5. In backcrossing generations from BC1F1 to BC3F1, selected individuals with a phenotype similar to Huahui
230 938 and heterozygous Bph14 and Bph15 loci were backcrossed to Huahui938. Three near-isogenic lines (NILs) bearing both homozygous Bph14 and Bph15 loci were generated from the BC3F2 segregation population. Finally, the RICE6K array was used to verify the background of the improved lines. Conclusion: Severe yield loss due to various insect pest in rice are a serious constraint to the rice productivity throughout the world. The most effective and reliable method of management of the stresses is the enhancement of host resistance, through an economical and environmentally friendly approach.Marker assisted breeding has large potential in developing insect resistant rice varieties. Abundant DNA markers is an efficient tool for analyzing the recurrent parent recovery of progenies and can be used to monitor the donor segments in NILs, thus being extremely important for rice molecular breeding for insect resistance. References: Alam, S.N. and Cohen, M.B. (1998b). Durability of brown planthopper, Nilaparvatalugens, resistance in rice variety IR64 in greenhouse selection studies.Entomol. Exp. Appl. 89: 71–78 Bentur, J.S., Rawat, N., Divya, D., Sinha, D.K., Agarrwal, R., Atray, I., et al. (2016.) Rice-gall midge interactions: battle for survival. J. Insect Physiol. 84: 40–49 Biradar, S.K., Sundaram, R.M., Thirumurugan, T., Bentur, J.S., Amudhan, S., Shenoy, V., et al. (2004). Identification of flanking SSR markers for a major rice gall midge resistance gene Gm1 and their validation. Theor. Appl. Genet. 109: 1468–1473 Chen, M., Shelton, A.M., Ye, G.Y. (2011). Insect-resistant genetically modified rice in china: from research to commercialization. Annu. Rev. Entomol. 56: 81–101 Claridge, M.F. and Den Hollander, J. (1983). The biotype concept and its application to insect pests of agriculture. Crop Prot. 2: 85–95 Cohen, M.B., Alam, S.N., Medina, E.B. and Bernal, C.C. (1997). Brown planthopper, Nilaparvatalugens, resistance in rice cultivar IR64: mechanism and role in successful N. lugensmanagement in Central Luzon, Philippines. Entomol. Exp. Appl. 85: 221–229 Crow, J.F. (1957). Genetics of insect resistance to chemicals.Annu Rev Entomol 2:227– 246 Das, G. and Rao, G.J.N. (2015). Molecular markers assisted gene stacking for biotic and abiotic stress resistance genes in an elite rice cultivar. Frontiers in Plant Science. 6(698) Das, G. and Rao, G.J.N. (2015) Molecular marker assisted gene stacking for biotic and abiotic stress resistance genes in an elite rice cultivar. Front. Plant Sci. 6:698
231 De Kraker, J., van Huis, A., Heong, K.L., Van Lenteren, J.C. and Rabbinge, I. (1999). Population dynamics of rice leaf folders (Lepidoptera: Pyraidae) and their nature in the irrigated rice in the Philippines. Bull. Entomol. Res. 89(5):411-421 Du, B., Zhang, W., Liu, B., Hu, J., Wei, Z., Shi, Z., He, R., Zhu, L., Chen, R. Han, B., et al. (2009), Identification and characterization of Bph14, a gene conferring resistance to brown planthopper in rice. Proc. Natl Acad. Sci. U S A. 106: 22163–22168. Dutta, S.S., Divya, D., Durga, R.C.V., Reddy, D.T., Visalakshmi, V., Cheralu, C., et al. (2014). Characterization of gall midge resistant rice genotypes using resistance gene specific markers. J. Exp. Biol. Agric. Sci. 2:339–446. Hasan, M. M., Rafii, M. Y., Ismail, M. R., Mahmood, M., Rahim, H, A., Alam, M. A., et al. (2015). Marker-assisted backcrossing: a useful method for rice improvement. Biotechnol.Biotechnol. Equip. 29: 237–254 Hasan, M.M., Rafii, M.Y., Ismail, M.R., Mahmood, M., Rahim, H.A., Alam, M.A., et al. (2015) Marker-assisted backcrossing: a useful method for rice improvement. Biotechnol. Biotechnol. Equip. 29: 237–254 Heinrichs, E.A., Reising, W.H., Valencia, S.L. and Chelliah, C. (1985). Rules and effects of resurgence inducing insecticides on population of brown plant hopper, Nilaparvatalugensstal. and its predators. Environ. Entomol. 11: 1269-1273 Jena, K.K. and Kim, S.M. (2010). Current status of brown planthopper (BPH) resistance and genetics. Rice 3:161–171 Jing, S., Liu, B., Peng, L., Peng, X., Zhu, L., Fu, Q. and He, G. (2012). Development and use of EST–SSR markers for assessing genetic diversity in the brown plant hopper (Nilaparvataugens (Stål)). B. Entomol. Res. 102:113–122 Khan, M.H., Dar, Z.A. and Dar, S.A. (2015). Breeding strategies for improving rice yielda review Agric. Sci. 6: 467–478 Khush, G.S. and Brar, D.S. (1991). Genetics of resistance to insects in crop plants. Adv Agron. 45:223–74 Kurokawa, Y., Nodaa, T., Yamagatac, Y., Angeles-Shima, R., Sunoharaf, H., Ueharaa, K., et al. (2016) Construction of a versatile SNP array for pyramiding useful genes of rice. Plant Sci. 242: 131–139 Li, J., Xia, M., Qi, H., He, G., Wan, B. and Zha, Z. (2006) Marker assisted selection for brown planthopper (Nilaparvatalugens Stål) resistance genes Bph14 and Bph15 in rice. Sci. Agric. Sinica. 39: 2132–2137. Liu, Y., Chen, L., Liu, Y., Dai, H., He, J., Kang, H., Pan, G., Huang, J., Qiu, Z., Wang, Q., Hu, J., Liu, L., Chen, Y., Cheng, X., Jiang, L. and Wan, J. (2016). Marker assisted pyramding of two brown planthopper resistance genes, Bph3 and Bph27(t), into elite rice cultivars. Rice 9:27 Misra, D.S. and Israel, P. (1991).The leaf and planthopper problems in high yielding varieties of rice. Oryza.7(2): 127-130 Mundt, C.C. (1994). Techniques to manage pathogen co-evolution with host plants to prolong resistance. In: Teng PS, Heong KL, Moody K (eds) Rice pest science and
232 management. International Rice Research Institute, Los Banos, pp 193–205 Panda, N. and Heinrichs, E.A. (1983) Levels of tolerance and antibiosis in rice varieties having moderate resistance to the brown plant hopper, Nilaparvatalugens (Stal) (Hemiptera: Delphacidae). Environ Entomol. 12:1204–14 Panda, N. and Khush, G.S. (1995) Host plant resistance to insects. Wallingford (UK): CAB International; p. 431. Perumalsamy, S., Bharani, M., Sudha, M., Nagarajan, P., Arul, L., Sarawathi, R., et al. (2010). Functional marker-assisted selection for bacterial leaf blight resistance genes in rice (Oryzasativa L.). Plant Breed. 129: 400–406 Prabhu, A.S., Filippi, M.C., Silva, G.B., Silva-Lobo, V.L. and Morais, O.P. (2009) “An unprecedented outbreak of rice blast on a newly released cultivar BRS Colosso in Brazil,” in Advances in Genetics, Genomics and Control of Rice Blast, eds G. LWang and B. Valent (Dordrecht: Springer Science), 257–267 Razzaque, Q.M.A., Heinriches, E.A. and Rapusas, H.R. (1985). Mass rearing technique for rice green leaf hopper Philippines Entomol. 6(4): 398-404 Roush, R.T. and McKenzie, J.A. (1987). Ecological genetics of insecticide and acaricide resistance. Annu Rev Entomol 32:361–380 Sharma, P.N., Torii, A., Takumi, S., Mori, N. and Nakamura, C. (2004). Marker-assisted pyramiding of brown planthopper (NilaparvatalugensStål) resistance genes Bph1 and Bph2 on rice chromosome 12. Hereditas. 140:61–69 Singh, A.K., Gopalakrishnan, S., Singh, V.P., Prabhu, K.V., Mohapatra, T., Singh, N.K., et al. (2011) Marker assisted selection: a paradigm shift in Basmati breeding. Indian J. Genet. 71:120–128 Sogawa, K. (1973). Feeding of the rice plant- and leafhoppers. Rev. Plant Prot. Res. 6:31– 43 Wang, H., Ye, S. And Mou, T. (2016). Molecular Breeding of Rice Restorer Lines and Hybrids for Brown Planthopper (BPH) Resistance Using the Bph14 and Bph15 Genes Rice 9: 53 Werner, K., Friedt, W. and Ordon, F. (2005). Strategies for pyramiding resistance genes against the barley yellow mosaic virus complex (BaMMV, BaYMV, BaYMV2). Mol. Breed. 16: 45–55 Xu, J. (2013). Pyramiding of two BPH resistance genes and Stv-bi gene using markerassisted selection in japonica rice. Crop Breed. Appl. Biotechnol. 13: 99–106 Zhou, L., Wang, J.K., Yi, Q., Wang, Y.Z., Zhu, Y.G., Zhang, Z.H. (2007). Quantitative trait loci for seedling vigor in rice under field conditions. Field Crop. Res. 100:294– 301
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13 MACROPHOMINA ROOTROT OF GROUNDNUT (Arachis hypogaea L.) Manoj Kumar*, Neha Rani, Anand Kumar, Anil Kumar, Satyendra, Mankesh Kumar, S.P. Singh and Sunita Kumari1 Department of Plant Breeding and Genetics, 1K.V.K. Aurangabad Bihar Agricultural University, Sabour (Bhagalpur)-813210, Bihar Email :
[email protected]
ABSTRACT Macrophomina root rot of groundnut (Arachis hypogaea L.) is an emerging problem in Bihar, where groundnut is extensively grows in large area as a major Kharif and spring oilseed crop. Its incidence varies from 0-32 percent in both the seasons. On isolation from diseased plants, the associated pathogen was identified as Macrophomina phaseolina (Tassi.) Goid and its pathogencity were confirmed on Groundnut variety R-20 under artificially inoculated conditions. The typical symptoms of the disease may appear on roots, collar region, stem and branches of infected plants. The affected portions rotted, shrivelled become darker or blackish in colour and plant collapsed and broke down from the rotted portion. Gradually affected plants showed general yellowing, drooping of leaves and ultimately death of plants before maturity. During varietal screening of 20 genotypes of Groundnut under natural field conditions the cultivar, DH 86 (32.49 %) showed maximum root rot incidence percent during Kharif followed by CHICO (19.01 %). Minimum and no disease root rot percent was recorded in ICGV07214 (0.00 %). The cultivar, ICGV00338 (8.08%) showed maximum root rot incidence during Spring followed by ICGV02005 (5.71%). Minimum root rot disease incidence percent was recorded in the cultivar ICGV07210 (1.97%). The systemic fungicide, Carbendazim was highly effective in inhibiting the growth of the fungus at all the
234 concentrations followed by Hexaconazole, Mancozeb and Tricyclazole. The maximum germination percentage 66% were observed in seed treated with Carbendazim (12%) + Mancozeb (63%) followed by thiram (30%). Minimum germination percentage observed in seed treated with Trichoderma (20%). Maximum disease incidence percent was recorded in seed treated with Trichoderma (90%) followed by thiram and Copper oxychloride. Minimum disease incidence (14%) recorded in seed treated with Carbendazim (12%) + Mancozeb (63%). Key words: Groundnut, Macrophomina phaseolina, cultural characteristics, disease incidence.
Introduction Groundnut (Arachis hypogaea L.) is an important legume crop cultivated for food & its oil use. Groundnut is the 6th most important oilseed crop in the world. It contains 48-50% oil and 26-28% protein. The haulms are utilized as fodder and after extraction of oil, cake is used in the livestock feed industry. Groundnut shells are used as fuel, as filler industry, and in making cardboards. Being a leguminous crop, it enriches the soil with nitrogen and is therefore valuable in cropping system. Groundnut is originated in the Northwest Argentina region in South America and is presently cultivated in 108 countries of the world. It is grown on nearly 21.70 million ha with the production of 41.18 million tons and an average yield of 1667 kg/ha in 2012 (FAOSTAT, 2012). In India during 2015-16 the production of Groundnut was 6.78 million tons from 4.59 million ha and average yield was 1465 kg/ha (Ministry of Agriculture, Govt of India). In Bihar, production of Groundnut was 1030 tons from 1020 ha and with an average yield of 1010 kg/ha. It is estimated that domestic demand of Groundnut by the end of 2020 will be 14 million tons in India Therefore, to meet the required demand of 8.2 million tons, we have to increase production by 2.2 % per year. This can be achieved through increase in yield and partly by regaining the lost area, although not the same area. The area under Groundnut cultivation has declined by over 50% since 1990’s and there is scope to expand Groundnut area in non-traditional areas where the Groundnut cultivation would be profitable. In India Groundnut is cultivated largely in Kharif season & in some states it is cultivated in Rabi and Spring seasons. The yields in Rabi & Spring seasons are high owing to less incidence of disease & continuous availability of moisture through irrigation or other sources. The majority of diseases are caused by fungi and several of them caused reduction in yield varying in different them cause reduction in regions and seasons (Mayee, 1995). The Groundnut in India often suffer from various type of root rot & wilt. The dry root rot caused by Macrophomina phaseolina (Tassi) Goid has been noticed to
235 cause 33.33 per cent seed rotting and 23.80 per cent post emergence mortality (Gupta and Kolte, 1982). The first symptom of disease is yellowing of the leaves which droop in next 2 or 3 days and withers off. The plant may wilt within a week after the appearance of first symptom. When stem is examined closely, dark lesion may be seen on the bark at the ground level. If the plants are pulled from soil the basal stem & main root. May show dry rot symptoms the tissues are weakened and break off easily in advanced cases Sclerotial bodies may be seen scattered on the affected tissues. The causal organism Macrophomina phaseolina belongs to Division-Eumycota, SubdivisionDeuteromycotina, Class-Coleomycetes, Order-Sphaeropsidales, & FamilySphaeropsidaceae. The fungus invades the host both inter & intracellularly, it grows rather fast covering large areas of the host tissue & eventually killing they in short time. It produces numerous sclerotial bodies on host tissue, which measure about 110-130-µ in diameter. Often the conidial or pycnidial stage is produced on the host. The fungus is a facultative parasite capable of living saprophytically on dead organic tissue, particularly on many of its natural hosts producing sclerotial bodies, which produces pycnidia. When atmospheric temperature is above 30ÚC & the pycniospores remain viable for over a year since the fungus attack wide range of plant species .The fungus is mainly a soil dweller and spreads from plant to plant through irrigation water, food and implements and cultural operation. The sclerotia & pycniospore may also become air borne and cause further spread of the pathogen (Rangaswami and Mahadevan 2008). Since there was little information on dry root rot of Groundnut, it was important to study biology of this pathogen and information relating to in vitro studies on effect of different fungicides on the growth of the pathogen will help to device suitable management practices for dry root rot of Groundnut. Since use of resistant cultivars is one of the most effective means of disease management. History and nomenclature of pathogen Macrophomina phaseolina causing root root of Groundnut came in existence after several modifications. Due to presence of sclerotial stage, it was first named as Sclerotium bataticola by Taubenhaus (1913). Later on it was transferred under genus Rhizoctonia bataticola (Taub.) Butler in 1918. The Pycnidial stage was described by Maublanc as Macrophomina phaseolina (Maubl.) in 1905 Ashby (1927) showed that the fungus produces a pycnidial stage corresponding Macrophomina philippinensis Petrak; the type species of the genus Macrophomina. Goidanich suggested on earlier
236 name; Macrophomina phaseolina. Tassi and Goid proposed the combination; Macrophomina phaseolina (Tassi) Goid for same fungus. Occurrence and distribution: The disease is very destructive in nature and has been reported in India (Pearl, 1923), Burma and Ceylon (Small, 1927a and 1927b), Palestine (Reichert, 1930), Cyprus (Nattrass, 1934), Greece (Sarejanni and Cortzas, 1935), Uganda (Hansford, 1940), Turkey (Bremer, 1944), Pakistan (Prasad, 1944), Nigeria (Anon, 1955) Syria (AlAhmad and Saidawi, 1988), Iran (Mahdizadeh et al., 2011). Sundararaman (1932) has reported that the yield from affected plants with 36.6 per cent less than that of the normal. The disease has been recorded mainly from Madhya Pradesh (Pearl, 1923), Bihar (Mc Rae, 1930), Madras (Sundararaman, 1931) and Uttar Pradesh (Mehta, 1951). Patel and Patel (1990) observed that the optimum temperature for growth and Sclerotial formation by Macrophomina phaseolina was 35ºC, both declined at temperature below 15ºC and above 40ºC. Under field conditions disease intensity increased with a progressive rise in temperature and decrease in Relative humidity.Maheswari and Ramakrishnan (2000) reported that Saprophytic survival of M. phaseolina was maximum at low moisture levels of 40% recording a mean of 65.9% against 15.9 and 7.9% survival at 60 and 80%, respectively. At 65 days of incubation, the saprophytic ability was 3.4% at 80% moisture holding capacity (MHC) compared with 73.4% at 40% MHC while, it decreased progressively with increase in incubation period at 60 and 80% MHC in both the inoculum levels. Umamaheswari et al. (2001) reported that Maximum disease incidence was recorded in sterilized soil at 15-45 days after sowing. A significant increase in disease incidence (48.7 and 98.5 per cent) was observed with increasing inoculums level from 500 to 1000 mg/kg of unsterilized and sterilized soil, respectively. In unsterilized soil, 50 per cent reduction in root incidence due to the antagonistic activity of bacteria and actinomycetes was observed compared to sterilized soil. Thakare et al. (2002) reported that root rot incidence in groundnut was more in non-mulch with no seed treatment as compared to polythene mulch. Per cent root rot reduction in mulch was 41% caused due to Sclerotium rolfsii [Corticium rolfsii] and Rhizoctonia bataticola [Macrophomina phaseolina] Khan et al. (2003) showed occurrence of the disease but Sahiwal showed high incidence and severity in 1999. Distribution of the disease in Jaiman et al. (2009) reported storage condition has direct influence on seed mycoflora. 8% seed moisture for 6 months displayed a minimum
237 incidence of M. Phaseolina and maximum seed germination. Storage temperature of 40ÚC and RH of 90% for 6 months of storage restricted for incidence of M. Phaseolina in cluster bean. Kale et al. (2009) reported that TG 51 also had a lower dry root rot (Macrophomina phaseolina) incidence (13.0%) than TAG 24 (43.0%) at Kadiri during the Rabi season of 2003-04. Moradia and Khandar (2011) reported that a field survey was done during 2002-2003 at Main Oilseeds Research Station, Junagadh Agricultural University, Junagadh to study the loss of yield of groundnut due to dry root rot. The maximum plant mortality (root rot) of 29.3 per cent due to Macrophomina phaseolina with highest yield loss of 435 kg/ ha was found in Keshod tehsil of Junagadh district of Saurashtra region. The survey in different locations of Cuddalore district revealed the endemic nature of the root rot disease incidence with the maximum incidence of the disease (31.68%) registered in Vengatakuppam (MP18) location. The disease incidence was more in improved cultivars like, VRI2, JL24; more in sandy loam soils and rainfed conditions (Raja Mohan and Balabaskar (2012). Taliei et al. (2013) reported that in the seasons of 2009-2010 and 2010-2011, disease incidence ranged from 0 to 97% and 3 to 91% with the highest in Gorgan and Aliabad, (valley), respectively. Host range Butler and Bishy (1931) cited many records on Solanum tuberosum L. Gossypium spp., Carchorus capsularis L., C olitorius L. Cajanus cajan L. Millsp., Arachis hypogaea L., Alysicarpus spp., Carica papaya L., Citrullus vulgaris schard. L. Millsp., Crotalaria juncea L. Cucurbita maxima Duchesene, Dolichos biflorus L., D. lablab L., Hibiscus cannabinus L., Lycopersicon esculentum L., Medicago sativa L., Morus alba L., Nicotiana tabacum L., Phaseolus lunatus L., P. aureus Roxb., Solanum melongena L. and Vigna sinensis L. as a root, stem and tuber parasite throughout India. Subramanian (1952) and Ramakrishnan (1955) isolated the pathogen from black cotton soil from Udamalpet, and Vandalur state respectively from Madras. Nema and Agrawal (1960) reported it on roots of Cicer arietinum L. and Pisum sativum L. from Jabalpur (M.P.). Singh and Nene (1990) reported that Rhizoctonia incited diseases in wide range of hosts, especially under high temperature and drought stress conditions. Winter rape, sesame, saffron, squash, lucerne, cotton, potato, sorghum, cucumber, okra and capsicum have been reported as new hosts for M. phaseolina in Romania (Ionita et al., 1995). Jayati-Bhowal et al. (2006) observed the phytopathogenic fungus M. phaseolina infects many plants, e.g. jute (Corchorus capsularis), soybean (Glycine max) and groundnut (Arachis hypogaea). Mahdizadeh et al. (2011) observed on marigold
238 (Tagetes erecta), cantaloupe (Cucumis melo var. cantaloupensis), cumin (Cuminum cyminum), hemp (Cannabis sativa), mung bean (Vigna radiata), okra (Abelmoschus esculentus), tomato (Lycopersicon esculentum), turnip (Brassica rapa), and watermelon (Citrullus lanatus), which was reported as new hosts for M. phaseolina Symptomatology. Thirumalachar (1953) observed grey discoloration in the beginning at the infection site followed by blackening of the stems, which bear numerous sclerotia of the fungus, as characteristic symptoms of the disease. Jain and Kulkarni (1965) observed different types of symptoms at various stages of growth of sesame plant. In seedling stage, the roots may become brown and rot resulting in death of the whole plant. In other plants, the fungus attacks on the collar region of the stem showing brown colouration which later extends upwards from few mm to few inches. Slowly and slowly, the whole plants becomes brown coloured and small dot-like black pycnidial structures containing fungal spores are seen on stem, branches, capsules and seeds. Okwulehie (2002) reported that investigations on the anatomical features of various parts of groundnut (Arachis hypogaea cv. valencia) plants infected with the fungus, Macrophomina phaseolina [M. phaseolina], in Nigeria were made with the aid of a binocular microscope. Anatomically significant changes were observed in the hypocotyl, root and stem. Hypocotyl tissues were completely destroyed by M. phaseolina infection. Internal necrosis extended into the stem tissue. Plugging of vascular tissues, especially xylem vessels, was observed which probably induced wilting. Cells were generally experienced hypertrophy while those of the root cortex were destroyed. Rasheed et al. (2004) observed that in seedling symptom test, M. phaseolina, Fusarium spp., Rhizoctonia solani, A. flavus and A. niger caused pre-emergence and post-emergence rot resulting in root rot and dampingoff in seedlings. Morphology: Brooks (1928) observed smooth, hard and black sclerotia measuring 50-1000µ in diameter which occurred mainly on the infected roots. The pycnidia formed on the stems were variable in size ranging from 16-30 x 510 µ. Thirumalachar (1955) reported that the fungus at 30ºC produced greyish white fluffy mycelia which were progeotrophic in growth response. Deshpande et al. (1969) observed white to brown fluffy mycelium in culture. Mycelium was branched and sparsely septate. Sclerotia connected by short strands or fibrillae with the rest of mycelium and were brown to black in colour and globose to irregular in shape. It measures 66.4 µ to 315 µ in diameter (av 136.12 µ), the irregular ones were 166.4 µ to 498 µ in size. Subramanayam (1971) reported that sclerotia were jet-black, minute smooth, externally composed of
239 anastomosed black hyphae, interior light to dark brown, composed of free thick-walled cells. Sclerotia variable in shape, globose, oval, oblong, elliptical, curved or even forked, varying in size 25x22-152x32 µ, produced abundantly in the infected host tissues. Spores are single celled, hyaline somewhat elongated or cylindrical, 16-30 x 5-10 µ in size. Smits and Noguera (1990) reported that the formation of sclerotia in M. phaseolina began with branching and inter-winning of adjacent hyphal filaments. The pycnidial stage began with the merging of hyphal filaments towards a common point, followed by the development of ringed primodia and finally pycnidia. Matured pycnidia were sugblobose with a reticulate appearance, a short neck and a circular ostiole. Cultural and physiological studies Various aspects of morphology, cultural, and physiological parameteters of Macrophomina phaseolina was studied by many workers on different host species. These studies assisted in characterized of the pathogen as well as its variation. Paracer and Bedi (1962) observed the linear growth rate of the fungus and found that it increased as the nutritive status of the culture medium increased. Sclerotial formation started as early as 48 hours after inoculation on Brown’s agar reinformed with 2 per cent potato (starch), while it took 168 hours or one week to start on the poorest medium, viz., water agar on the other hand, rich media like Richard’s agar and nutrient glucose agar, the formation of sclerotia were delayed considerably. Shanmugam and Govindaswamy (1973) observed that Macrophomina phaseolina grew best on Richard’s medium at pH 5, with dextrose and asparagines as carbon and nitrogen sources. Cultural studied indicated that Rhizoctonia bataticola prefered. Rhizoctonia bataticola grew most rapidly on potato dextrose agar and Richards’ medium and showed profuse growth (Gupta and Kolte,1981). This study was further spported by Jha (1996) where potato dextrose agar and Richard’s agar media supported excellent Sclerotial formation. Okwulehie (2004) reported that the best synthetic medium and inoculation technique for the growth and development of M. phaseoli [M. phaseolina] was investigated. The physiological changes in groundnuts under infection with the pathogen were analysed. The media tested were potato dextrose agar (PDA), peanut leaflet oatmeal agar (POMA), Czapeks-Dox agar (CDA) and corn meal agar (CMA). The best growth was observed on POMA and PDA media.Okwulehie (2005) observed some synthetic media were tested to investigate the best media that would best support the growth of Macrophomina phaseoli [M. phaseolina]. The fungus grew in both solid and liquid synthetic media, but the best growth was recorded in Peanut
240 (groundnut) leaflet oat meal agar (POMA), and potato dextrose agar (PDA). Varietal screening Li-Zheng Chao and Qiu-QingShu (2000) reported that Huayu 16 is resistant to root rot, caused by Macrophomina phaseolina, drought and water logging, and tolerant of peanut stripe virus. This new cultivar is recommended for cultivation in medium or high fertility sand loam soils. Gopal et al. (2006) reported that twenty groundnut genotypes were evaluated in Jagtial, Andhra Pradesh, India, for resistance to pod rot disease (caused by soil borne organisms, such as Rhizoctonia solani, Fusarium solani, Sclerotium rolfsii [Corticium rolfsii], Fusarium oxysporum and Macrophomina phaseolina). Based on percent disease index (PDI), the cultivars were classified as immune (disease-free), resistant (0.10-10.0 PDI), moderately resistant (11.1-30.0 PDI), susceptible (30.1-50.0 PDI), and highly susceptible (>50.0 PDI). PDI, percent incidence (PI), and percentage of podsinfected (PPI) ranged from 38.8 to 65.9, 39.9 to 94.4, and 9.3 to 90.7%, respectively. The lowest PDI values were recorded for INS 9013 and R 8808 (38.8% for each), followed by R 8972 (38.9%) and ICGV 86885 (39.2%). PI was lowest in R 8972 (39.9), followed by ICGS 11 (53.6%), ICGV 86885 (55.9%) and R 8806 (61.2%). R 8972, R 8808, ICGV 86885, R 8806 and ICGS 11 registered the lowest PPI (9.3, 10.4, 11.8, 13.1 and 15.1, respectively). Vijay Mohan et al. (2006) said that twelve chickpea cultivars were evaluated for resistance against dry root rot disease (Macrophomina phaseolina) under natural and artificial conditions. Screening under natural conditions were conducted during the Rabi seasons of 2001-02 and 2002-03 in Jharkhand, India. For artificial conditions, potted soils were inoculated with mycelial bits of the pathogen and seeds were sown at 4 seeds per pot three days after soil inoculation. Under natural conditions, the mean disease incidence was lowest (8.0%) in G-543, followed by BG-360 and GNG-1365 which recorded mean disease incidence of 9.6 and 9.8%, respectively. susceptible. Kale et al. (2007) observed that TG 38 showed resistance to stem rot (Sclerotium rolfsii [Corticium rolfsii]) and dry root rot (Macrophomina phaseolina). Prasanthi (2007) screened sixty-one indian bean (Phaseolus vulgaris) lines collected from different sources were screened for resistance to dry root rot disease (Rhizoctonia bataticola [Macrophomina phaseolina]) under natural field conditions of Tirupati, Andhra Pradesh, India, during kharif, 2005.Moradia (2012) conducted a pot experiment was to assess the varietal resistant of seventy one groundnut varieties against dry root rot disease (Macrophomina phaseolina). Out of seventy one varieties, 28
241 varieties were found resistant which are spreading type, 6 varieties were moderately resistant and rest 37 varieties were found susceptible to M. phaseolina which were bunch type of varieties. Chemical control: Spraying of Derosal, J K Stein and Topsin-M gave the best result against stem blight of cowpea induced by Macrophomina phaseolina and increased the yield (Sharma et al., 1995). Shalaby (1997) studied the effect of benlate , vitavax, Rhizolex T and Tecto TBZ (thia bendazole) on disease severity and as seed treatments on soil fungi causing sesame root rot and concluded that benlate and vitavax were the most effective seed dressings against the Macrophomina phaseolina under both laboratory and green house conditions. Rao et al. (1998) reported that Seed treatments consisting of carbendazim WP, carbendazim SD + captan, carbendazim + thiram, captan, thiram, mancozeb, tolclofos-methyl, pyroquilon, TCMTB, carbedazim-Jkstein, quintozene, captafol, carboxin and triadimenol were tested for the management of these fungi under artificial infection of the seed. The seed treatments were found to control the damage due to these fungi. Among the treatments, carbendazim SD (0.05% + captan 0.125%) was the best in reducing seed rot and pre- and postemergence seedling blight and in increasing pod yields significantly in all the three seasons of field experimentation, followed by treatment with captafol (0.25%). Malathi and Doraisamy (2003) reported that the interaction of T. harzianum (TH5) with different fungicides (captan, thiram and carbendazim) in controlling M. phaseolina infecting groundnut. The antagonist and the fungicides were tested individually and in combination on the pathogen. The toxicity of the fungicides at 5, 10, 50 and 100 micro g/ml was also tested on T. harzianum. Growth of TH-5 and the pathogen were completely inhibited by carbendazim at 5 and 100 micro g/ml, respectively. Choudhary et al. (2004) observed the effectiveness of four fungicides, i.e. Bavistin [carbendazim], Antracol [propineb], Indofil M-45 [mancozeb + thiophanatemethyl] and Ridomil MZ [mancozeb + metalaxyl], applied at 300,400, 500 and 1000 ppm, in inhibiting the mycelial growth of Macrophomina phaseolina, the causal agent of stem and root rot of sesame, was studied in vitro using the poisoned food technique. All fungicides dose-dependently inhibited mycelial growth compared with the untreated control, with Bavistin being the most effective. Rai et al. (2005) observed maximum germination was recorded when seeds were treated with Dividend or T. viride combined with Vitavax. Seed treatments with mancozeb and thiram were the least effective, but were superior to the control. Bainade et al. (2007) observed that the effects of chemical and biological control of
242 Macrophomina phaseolina. Treatments comprised: mancozeb, quintal, carbendazim, Benlate [benomyl], tricyclazole and benomyl, and Trichoderma viride, T. harzianum and T. lignorum [T. viride]. Mancozeb (2.5%), quintal (2%) and tricyclazole (1%) completely inhibited the growth of Macrophomina phaseolina in in vitro conditions. Hegde and Chavhan (2009) reported that effective fungicide for the management of root rot. Drenching with fungicides like carbendazim @ 0.1%, hexaconazole @ 0.1%, mancozeb @ 0.2% and carboxin+thiram @ 0.1%, have managed the root rot effectively. Rajani and Parakhia (2009) observed that soil application of neem cake with was found most effective in reducing the disease and in increasing seed yield (2126 kg/ha). It gave the highest net return (1:7.91). Mustard cake with T. harzianum was the next best treatment (ICBR1:5.05). Soil application of neem or mustard cake @ 500 kg/ha along with talc based preparation of T. harzianum @ 5kg/ha may be applied just before sowing in furrow for an effective management of root rot disease and for improving yield of castor. Jaiman and Jain (2010) reported that efficacy of fungicides viz., bavistin, raxil, topsin M, captan, indofil M-45 and thiram were tested against M. phaseolina causing root rot in cluster bean both in vitro and in vivo. Maximum inhibition of fungal growth was found with bavistin followed by topsin M. the fungicides checked the disease as compared to control. Tandel et al. (2010) observed that for the control of the disease, seven fungicides were tested. Among them Carbendazim + mancozeb (Sixer) was found significantly superior over the rest as it resulted minimum (8.13%) disease intensity. This suggested that leaf blight of mung bean (Macrophomina phaseolina) can be controlled very effectively by spraying of carbendazim + mancozeb (Sixer) and the huge crop loss can be saved if sprayed at the time of disease initiation. Moradia (2011) reported that poisoned food technique was employed to study the efficacy of different nine systemic fungicides at 250, 500and 1000 ppm against Macrophomina phaseolina (groundnut isolate) under in vitro conditions. All the fungicides were capable of inhibiting the growth of the fungus at all the concentrations tried. Difenconazole (Score 25% EC), carboxin (Vitavax 75% WP) and saaf (SAAF 75%) were found to be the best, which caused cent per cent inhibition of growth at all the concentrations tried. Sreedevi et al. (2011) said that five Trichoderma spp. were isolated from the rhizosphere soil of healthy groundnut plants, identified using morphological and microscopic characteristics and were evaluated for in vitro antifungal activity against M. phaseolina by dual culture plate technique and bioassay methods (in vitro antibiosis). Scanning electron microscopy was used to study the
243 conidial surface of T. harzianum. Among the five isolates T. harzianum (T3), T. viride (T1) had maximum antifungal activity against M. phaseolina compared to the other Trichoderma spp. In dual culture technique T. viride and T. harzianum reduced mycelial growth by 61.1% and 64.4%, respectively. Based on the dual culture technique, T. harzianum (T3), T. viride (T1) were selected for further research. Nageswararao et al. (2012) reported that pot culture studies with groundnut cv TCGS 888 under sterilized soil conditions indicated efficacy of mancozeb seed treatment @ 3 g/Kg seed (66.7%) and neem cake soil application @ 0.51/ha (66.7%) applied either alone or in integration with T. virens in decreasing groundnut root rot incidence. Summary Macrophomina root rot of groundnut (Arachis hypogaea L.) is an emerging disease in Bihar, where groundnut is extensively grown in large area as a major Kharif and emerging in spring oilseed crop. Its incidence varies from 0-32 per cent in two season. Present investigations were undertaken to identify the pathogen, to study the sequence of symptoms produced by the pathogen, its morphological and cultural status and to evolve effective management of the disease through seed treatment with different fungicidal treatments. Screening of available cultivars/entries to identify resistant source for use in future breeding programme was also done under natural conditions. The typical symptoms of the disease may appear on roots, collar region, stem and branches of infected plants. In the beginning, dark cortical lesions were formed near the collar region on stem. Rapidly these lesions increase in size from few mm to few inches. In advanced stage, the lesion extended upward and downward, girdling the stem from all sides and gradually the whole plant become brown coloured and ultimately dried prematurely. The affected portions rotted, shrivelled become darker or blackish in colour and plant collapsed and broke down from the rotted portion. Gradually affected plants showed general yellowing, drooping of leaves and ultimately death of plants before maturity. The pathogen was isolated on potato dextrose agar medium and pathogenicity was proved following Koch’s postulates. On the basis of morphological character of mycelium produced in nature as well as in the culture and artificially infected plants, the pathogen was identified as Macrophomina phaseolina (Tassi.) Goid. The fungus was grown on nine solid and five sets of liquid media separately under in vitro conditions. Synthetic and semi- synthetic media were found to be superior over natural media in terms of radial growth and
244 biomass production. Highest radial growth (9mm) was found in glucose asparagines agar medium followed by Czapek’s (dox) agar, Richard’s agar, Asthana and Howker’s agar and glucose peptone agar. no and scanty fungal growth was obtained on oat meal agar medium. All the liquid media behaved more or less similarly as solid media in terms of biomass production Richard’s broth was found superior to other media pertaining to biomass production which yielded 725.8g/flask after 21 days of inoculation, which was followed by Glucose asparagines, Czapek’s (dox) medium, Potato dextrose and Asthana and Howker’s agar medium. The screening of cultivars/entries of groundnut against Macrophomina phaseolina root rot under natural condition revealed that, out of 20 cultivars/entries. The mean root rot incidence ranged from 00.00 to 32.49 percent. The cultivar, DH 86 (32.49 %) showed maximum root rot incidence percent during Kharif followed by CHICO (19.01 %). Minimum and no disease root rot percent ICGV07214 (0.00 %). The cultivar, ICGV00338 (8.08%) showed maximum root rot incidence Spring followed by ICGV02005 (5.71%). Minimum root rot incidence was found in the cultivar ICGV07210 (1.97%). All the four fungicides viz., Carbendazim, Mancozeb, Tricyclazole and Hexaconazole tested in vitro at four different concentration viz 1ppm, 5ppm, 10 ppm, 20 ppm, 30ppm,40ppm, 50 ppm, 100 ppm, 100 ppm and 200ppm, were found to be significantly superior over control, in checking the radial growth of the pathogen. The systemic fungicide carbendazim was highly effective in inhibiting the growth of the fungus at all the concentrations followed by Hexaconazole, Mancozeb and Tricyclazole. Under in vitro condition the ability of Trichoderma isolates to inhibit the mycelial growth of M. phaseolina in dual culture was determined on PDA medium. A clear zone of inhibition noted between the Macrophomina phaseolina and Trichoderma viride was near the pathogen. The inhibition growth of pathogen were recorded after 24 and48 hrs. upto 47. 61% and 35.18%, respectively in dual culture experiments. References Ainsworth, G.C. and Bisby, G. R. (1967). Dictionary of fungi. Common Wealth Mycological Institute, Kew, Surrey, England. Al-Ahmad, M. and Saidawi, A. (1988). Macrophomina (charcoal) root rot of sesame in Syria. Arab J. Pl. Protec.6(2): 88-93. Aliyu, B.S. and Kutama, A. S. ( 2007). Isolation and identification of fungal flora associated with groundnut in different storage facilities. Science World J. 2(2): 34-36. Almomani, F., Alhawatema, M., Hameed, K. (2013). Detection, identification and morphological characteristic of Macrophomina phaseolina: the charcoal rot disease pathogens isolated from infected plants in Northern Jordan. Archives of Phytopath
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14 INCIDENCE OF FUNGAL DISEASES ON MAKHANA IN PREHARVEST AQUATIC CONDITIONS AND THEIR POSSIBLE CONTROL Meenu Sodi1 and Sanjib Kumar2 1 2
Research Scholar, B.N. Mandal University, Madhepura
Principal, B.S.S. College, Supaul-852131, Bihar, India. E-mail:
[email protected]
ABSTRACT Makhana (Euryale ferox Salisb.) is an aquatic cash crop known for its nutritional properties since a long time. In India, Bihar state alone accounts for more than 85% of the makhana. Approximately 80% of total production of processed makhana come from Northern Bihar consisting of Darbhanga, Madhubani, Katihar, Purnea, Sitamani, Saharsa, Supaul, Araria and Kishanganj districts. Like other crops fungal diseases pose a severe threat to cultivation of makhana. In the present investigation, a survey was undertaken to know the incidence of fungal diseases on makhana during pre-harvest period and their possible control. Mainly two fungal diseases i.e. leaf blight or leaf spot and hypertrophy of leaf and flower were recorded in this investigation. The pathogen of leaf blight of makhana was identified to be Alternaria alternate (Fr.) Keissl. whereas hypertrophy was caused by Doassansiopsis nymphaeae (Syd. & P. Syd.) Thirum. The incidence of leaf blight of makhana ranged from 10-31 % whereas hypertrophy ranged from 745% in different ponds of Katihar district. Different types of fungicides were also used to control the leaf blight and hypertrophy of makhana. Out of 6 different fungicides sectin (copper oxychlorate) was found to be the most effective in minimizing the incidence of leaf blight (up to
254 92.9%) and Bavistin was found to be the most effective ( up to 89.3%) against hypertrophy of makhana. Keywords: makhana, fungal diseases, Alternaria alternata, Doassansiopsis nymphaeae, control, fungicides.
INTRODUCTION : Gorgon nut or fox nut commonly known as makhana is an aquatic crop with large floating leaves and bright purple flowers. Makhana primarily serves the purpose of being consumed as a food item for local and religious purposes as non cereal food It belongs to family Nymphaeaceae and botanically known as Euryaleferox Salisb. It is grown in stagnant perennial water bodies like ponds, land depressions, oxbow lakes, swamps and ditches. Makhana seeds are also called as black diamond of wetlands (Ghose and Santra 2003). It is a flowering plant native to Asia. Makhana is a high value commodity, commercially cultivated only in Bihar and certain parts of eastern India. Besides the stagnant water bodies it is also cultivated under field conditions in paddy fields & low lying areas. Bihar is the largest producer of makhana in India accounting about 85% of the total makhana produced in the country (Kumari et al. 2013). Northern part of Bihar constituting districts of Madhubani, Darbhanga, Sitamarhi, Saharsa, Katihar, Purnea, Supaul, Kishanganj and Araria are agroclimatically suitable for makhana cultivation. Approximately 80% of total production of processed makhana comes from North Bihar (Mishra 1998). Darbhanga, Madhubani, Purnea and Katihar districts of Bihar alone produce good quantity of makhana. Makhana is also an unique product of state with an annual production of around 96,000 tonnes in 2014. In Bihar Madhubani district occupies the highest share in total production of makhana pop, which contributes about 20% of total production in state i.e. 3000 MT followed by Katihar 18 %, Purnea 15% and Darbhanga 14% and rest other districts (Business Plan for Makhana Cluster in Bihar 2012)[4]. Since makhana is a cash crop, it is exported from Bihar to different parts of India and even outside the country. Besides other cash crops makhana cultivation is also done in Katihar because of low lying lands. Katihar district is fast emerging as important hub for trading and cultivation of makhana. Being one of the largest producers of makhana in north Bihar Katihar district covers a large water spread area of about 4175.89 hectares of makhana cultivation. The land area of this district is surrounded by the three important rivers viz. The Koshi, the Mahananda and the Ganges. Due to enormous importance of makhana, Euryaleferox Salisb. In Koshi-
255 Mithila region, its various uses in theurapeutic, religious and matrimonial ceremonies, there is a need for its conservation. A regular annual incidence of flood, high rainfall, high humidity and high temperature of this district make the environment highly conducive for the growth and development of several fungal diseases in crop plants causing severe loss in crop yield. A very little work has been done earlier on the fungal diseases of makhana in this area. In preliminary survey of makhana cultivated areas of this district several pests and diseases were also found associated with makhana plants a possible cause in the reduction of crop yield. Hence, a thorough investigation on fungal diseases of makhana plants was thought necessary to know the incidence of different fungal pathogens, their severity and methods of control. MATERIALS AND METHODS A diagnostic survey was conducted during Jan 2012 to Dec 2015 in makhana cultivated fields/ponds situated in wetland areas of different blocks of Katihar district Bihar. The survey involved methods of observation, interaction, interview along with individual and group discussions. The study was undertaken in two different production system field system and pond system. These two systems were taken as strata for selection of sample. 100 Makhana farmers were randomly selected making total sample size 200. Focus group discussions were undertaken in makhana growing areas of Katihar district and interactions were made with fishermen, farmers & other various stock holders including Department of Fisheries, Research institutions, processing units etc. in order to get first hand information. Fungal diseases and their pathopgen were analysed with the help of standard methods (Wallace 1973) and consulting with the Department of Plant Pathology, BAU, Sabour, Bhagalpur. RESULTS & DISCUSSION: Like other crops, fungal diseases also pose a threat to the cultivation of gorgon nut Euryale ferox, commonly called makhana. This important cash crop of north Bihar is facing severe fungal pest problems in its preharvest aquatic condition which take a heavy toll of its harvest by minimizing the yield. The present study on different makhana cultivated ponds of Katihar revealed two types of fungal diseases on makhana plants. Different diseases, their pathogens, affected parts of crop plant and the symptoms of the diseases are depicted in Table 1.1 Frequency of diseases in different makhana cultivated ponds are depicted in Table 1.2. However,
256 Table 1.3 & 1.4 depict the control of fungal diseases by different fungicides. Two types of fungal diseases on makhana plants in their pre-harvest aquatic conditions were identified during the present investigation (Table 1.1) - leaf blight or leaf spots and hypertrophy in leaves and floral parts of makhana. These diseases were recorded more or less from all the makhana cultivated ponds in Katihar district. The incidence of leaf blight or leaf spots of makhana ranged from 14-26% at Site-I (Barari Block), 12-31% at Site-II (Korha Block) and 1021% at Site-III (Katihar Block). Maximum incidence was reported at Phulwaria pond of Site-II. The disease was most severe during MarchApril. Hypertrophy or gall disease of makhana was observed at all the makhana growing ponds of the study area (Table 1.2). The incidence of hypertrophy ranged from 7-45% at Site-I (Barari Block), 9-35% at Site-II (Korha Block) and 11-23% at Site-III (Katihar Block). The incidence of hypertrophy was maximum (45%) in Rounia Railway Dhala Pond of SiteI while the Laxmipur Road of the same site exhibited the minimum incidence (7%) of the disease. The maximum severity of the disease was recorded during June to August. Table: 1.1 Symotoms of Diseases on Makhana Occurring in Different Makhana Cultivated Ponds of Katihar District. Name of Disease
the
Blight disease
Pathogen
Parts Affected
Alternaria alternata Leaves (Fr.) Keissl.
Petiole
Hypertrophy
Doassansiopsis nymphaeae (Syd. & P. Syd.) Thirum.
Symptoms Minute to large dark spots with concentric rings appear on leaves. As the disease progresses, the circular spots grow and usually become grey-grey tan or near black colour. These spots develop in a target board pattern of concentric rings.
Leaf
Dark brown spots/ lesions in patches.
Petiole
Swelling is small in the beginning but later on develops in major dimension with the size
257
Peduncle/Pedicel
Increased in size, swollen in width
Flower
Swellings extend from leaf lamina to petiole or peduncle of flower.
Ovary
Basal part of flower swells and becomes distorted in shape. Ovary greatly reduced in size and the number of viable seeds also reduces.
BLIGHT DISEASE OF MAKHANA CAUSAL ORGANISM The causal organism of the blight disease of makhana was identified as Alternaria alternata. (Fr.) Keissl. However, leaf spots of makhana disease had earlier been reported to be caused by Alternaria tenuis Nees ex Pers. (Prasad and Haider 1968, Haidar and Nath 1987, Mahto et al. 1993, Haidar and Mahto 2003). SYSTEMATIC POSITION Kingdom
:
Mycota
Division
:
Eumycota
Sub-division
:
Deuteromycotina
Form-class
:
Hyphomycetes
Form- order
:
Moniliales
Form- family
:
Dematiaceae
VEGETATIVE STRUCTURE The profusely branched and septate mycelium appears light brown in colour. The cells are multinucleate (Singh et al. 2003). In the early stage of infection the intercellular hyphae of the parasitic species were observed but later on they became intracellular. ETIOLOGY Conidia a mass of specialized dark coloured, multicellular,
15 14 26 16
2. Rounia Rly Dhala
3. Laxmipur Road
4. Gagri Jalkar
5. Rupni Jalkar
Katihar
Korha
18
1.Bisharia Pokhar
Barari
21 19 10
3. Nayatola
4. Garbhaily
5. Jafargang
21
5. Laliya
18
19
4. Dighri
2. Kolasi
16
3.Gorgamma Dhar
19
12
2. Najra Chowki
1. Maniya
31
1.Phulbaria Chowk
April
To
January
April
To
January
April
To
January
Duration
March
April
April
Maximum
Leaf Spot /Blight Disease Incidence %
Ponds
Blocks
15
20
11
23
21
9
28
22
19
35
12
9
7
45
15
Incidence severity
TABLE: 1.2 Frequency of Diseases in Different Makhana Cultivated Ponds of Katihar District.
June
To
May
August
To
May
August
To
May
Duration %
Hypertrophy
August
To
June
August
To
June
June
Maximum severity
258
259 obclavate, obovid or muriform shaped spores, were found singly or in chains at the tips of the branched or unbranched condiophores developing from the inter or intracellular mycelium of the affected leaf areas. The condiophores were undifferentiated and emerged through the stomata of the infected leaf. The conidia were both transversely and longitudinally septate (Plate -3). The number of septa ranged from 5-10 in each conidium. The conidium measured about 30-40µ in length and 15-50 µ in breadth. It was surrounded by a two layered wall where the outer layer appeared pigmented. The conidia are readily disseminated by water. After detaching from condidipphores, they germinate on a suitable substratum and produce the same symptoms. PREDISPOSING FACTORS The disease usually becomes apparent during January to April. The maximum severity of leaf spots was recorded during March-April. The pathogen usually prefers a slightly acidic to neutral pH. The spread of the disease was in high cationic (Ca++ + Mg++, NH4, K, P) and anionic (Cl, HCO-3, SO4 -) concentrations but low Na concentration. Low water temperature 18-25 C helps in the spread of the disease. HYPERTROPHY OF MAKHANA CAUSAL ORGANISM The causal organism of hypertrophy or gall or smut disease of makhana was identified as Doassansiopsisnymphaeae (Syd. & P. Syd.) Thirum. SYSTEMATIC POSITION Kingdom
:
Mycota
Division
:
Eumycota
Sub-division
:
Basidiomycotiona
Class
:
Teliomycetes
Order
:
Ustilaginales
Family
:
Doassansiaceae
ETIOLOGY Enlarged aerenchyma cavities formed due to hypertrophied cells
260 contained scattered spore balls. Each spore ball consisted of a mass of spores grouped around a central mass of pseudoparenchymatous sterile hyaline cells. Angularly elliptical to globose spore balls were densely clustered. The size of spherical spore balls ranged from 124µ x 240µ to 250µ x 260µ in diameter, however, the size of the oblong spore balls ranged from 120µ x 250µ to 140µ x 270µ in diameter. The spore balls were surrounded by 10-15µ thick profusely branched hyaline, septate and a delicate layer of hyphae. The light brown or pale brown spores were arranged peripherally as the outermost layer of the spore balls. The size of the spores measured up to 15-20µ x 110-130µ in size. PREDISPOSING FACTORS Hypertrophy in makhana became apparent at the end of May and prominent galls were seen during June to August. The pathogen preferred a slightly alkaline pH of water for causing luxuriant hypertrophy. However, slightly acidie water showed most infrequent infection. The spread of the disease was more in high cations (Ca++ + Mg++, NH4z , Kz ) and anions (Cl{, HCO{ 3, SO4{ ) and low Naz . BLIGHT DISEASE AND HYPERTROPHY ON MAKHANA PLANT IN KATIHAR DISTRICT
261
CONTROL OF DISEASES Different types of fungicides were used to control the leaf blight (Table 1.3, Fig. 1.1) and hypertrophy (Table1.4, Fig. 1,2) of makhana . The fungicides applied were Manzate, Companion, Hexaconazole, Bavistin, Biltox (50 WP), Indophil M 50 and Sectin. These fungicides are usually applied in makhaha ponds to control the fungal pests of makhana by the farmers. Three sprayings of 0.4% concentration of each fungicide at an interval of 15 days were applied to minimize the incidence of fungal diseases. A control was also set up to compare the results.
262 Out of 6 fungicides Sectin (Copper oxychloride) was found most effective in minimizing the incidence of leaf blights of makhana (up to 92.9%) caused by Alternaria alternata. The other effective fungicides were Bavistin (up to 87.1%), Blitox (83.9%), Hexaconazole (up to 71.3%), Companion (67.8%) and Indofil M-50 (up to 49.8%). Manzate showed minimum decrease over control (up to 35.7%). However Bavistinfungicide (Carbendazim 50% WP) was the most efficient one in reducing the incidence of hypertrophy (up to 89.3%) caused by Dossansiopsis sp. on makhana in ponds. The other fungicides helpful in decrease of the incidence of hypertrophy in makhana were Blitox (80.4%), IndophilM-50 (up to 71%), Sectin (up to 61.8%), Companion (up to 51%), Hexaconazole (up to 33.3%) and Manzate (up to 29.6%).
FIG. 1.1EFFECT OF FUNGICIDES ON THE INCIDENCE AND INTENSITY OF LEAF BLIGHT OF MAKHANA(Euryale ferox Salisb.)
FIG.1.2 EFFECT OF FUNGICIDES ON THE INCIDENCE AND INTENSITY OF HYPERTROPHY OF MAKHANA (Euryale ferox Salisb.)
Active Ingredients of Fungicides
Mancozeb 75% WP
Carbendazim 12% + Mancozeb 63% WP
Hexaconazol technical + Propylene glycol
Carbendazim
Copper oxychloride
Zinc-manganese-ethylene bisdithiocarbonate
Feramidone 10% + Mancozeb 50 % W/W
-
Fungicides treated
1. Manzate
2.Companion
3.Hexaconazole
4.Bavistin
5.Blitox (50 WP)
6.Indofil M-50
7.Sectin
8.Control
-
Spray
Spray
Spray
Spray
Spray
Spray
Spray
Methods of Treatment
-
0.4% (4g/L)
0.4% (4g/L)
0.4% (4g/L)
0.4% (4g/L)
0.4% (4g/L)
0.4% (4g/L)
0.4% (4g/L)
Dose %
-
3 Times @ 15 days interval
3 Times @ 15 days interval
3 Times @ 15 days interval
3 Times @ 15 days interval
3 Times @ 15 days interval
3 Times @ 15 days interval
3 Times @ 15 days interval
-
92.9
4.6 64.5
49.8
83.9
87.1
71.3
67.8
35
Decrease over Control %
32.6
10.4
8.3
18.5
20.8
41.5
Frequency of Treatment Intensity of blight %
TABLE : 1.3 EFFECT OF FUNGICIDES ON THE INCIDENCE AND INTENSITY OF LEAF BLIGHT OF MAKHANA (Euryale ferox Salisb)
263
Active Ingredients of Fungicides
Mancozeb 75% WP
Carbendazim 12% + Mancozeb 63% WP
Hexaconazol technical + Propylene glycol
Carbendazim 50 %WP
Copper oxychloride
Zinc-manganese ethylene bisdithiocarbonate
Feramidone 10%+ Mancozeb 50%W/W
-
Fungicides treated
1. Manzate
2.Companion
3.Hexaconazole
4.Bavistin
5.Blitox 50 (WP)
6.Indofil M-50
7.Sectin
8.Control
-
Spray
Spray
Spray
Spray
Spray
Spray
Spray
Methods of Treatment
-
0.4% (4g/L)
0.4% (4g/L)
0.4% (4g/L)
0.4% (4g/L)
0.4% (4g/L)
0.4% (4g/L)
0.4% (4g/L)
Dose %
-
3 Times @ 15 days interval
3 Times @ 15 days interval
3 Times @ 15 days interval
3 Times @ 15 days interval
3 Times @ 15 days interval
3 Times @ 15 days interval
3 Times @ 15 days interval
-
61.8
26.5 42.9
71.1
80.4
89.3
33.3
51.0
29.6
Decrease over Control %
12.4
21.5
4.6
28.6
21.0
30.2
Frequency of Treatment Intensity of blight %
TABLE 1.4EFFECT OF FUNGICIDES ON THE INCIDENCE AND INTENSITY OF HYPERTROPHY OF MAKHANA (Euryale ferox Salisb.)
264
265 CONCLUSION Present investigations on fungal diseases of makhana in makhana cultivated ponds of different blocks (Site-I : Barari Block, Site –II : Korha Block and Site-III : Katihar Block) revealed that makhana plant in this area is mainly affected by 2 fungal diseases, leaf blight or leaf spot diseases and hypertrophy or gall of smutdisease. The spots become apparent during January and present up to April. The maximum severity of the disease appears after winter in April month. The pathogen of leaf blights of makhana was identified as Alternaria alternata. Severe fungal leaf blight of makhana has also been reported by Prasad and Haidar (1968), Haidar and Nath (1987), Mahto et al. (1993) and Haidar and Mahto (2003). However they identified the pathogen of leaf blight of makhana as Alternaria tenuis . Dwivedi et al. (1995) has reported the ultrastructure of this disease. Leaf blight of makhana is characterized by the presence of minute to large, yellow to brown black concentric rings of dead areas on the leaves known as target board effect (Haidar and Mahto 2003). The leaf spots reduce photosynthesis in affected areas and there by yield of the crop. Conidia of the pathogen were also isolated from the infected leaf measuring about 30-40µ in length and 15-40µ in breadth. Conidia possessed both longitudinal and transverse septa surrounded by a 2- layered outer wall. The conidia are actually responsible for secondary infection. Hypertrophy or gall disease or smut disease of makhana in leaves, petioles, pedicel or peduncle and basal part of flower including ovary become apparent at the end of May. The maximum severity to this disease on makhana can be observed during June to August as also reported by Verma et al. (2003). Hypertrophy is due to abnormal enlargement of cells due to infection of a fungus sp. abnormal increase in size and swelling of the infected organ was observed. The infection is not localized and extends from leaf lamina to petiole, pedicel/peduncle of flower to the base of flower causing great distortion. Abnormal thickness of petiole or peduncle is characterized as one of the identifying features of the disease. Highly enlarged aerenchyma and white incrustation within the inner cavities of the infected portion was observed as important anatomical symptom of the disease. The cavities contain mycelia mats with white powdery mass of spore balls measuring up to 120µ x 250µ to 140µ x 210µ (oblong spore balls) and 124µ x 240µ to 250µ x 260µ spherical spore balls) similarly reported by Verma et al. (2003). The pathogen of hypertrophy of makhana was identified to be Doassansiopsis sp. However, Verma and Jha (1999) identified the pathogen
266 of this disease as Doassansiopsis euryaleae. The study is also confirmed by the investigations of Vermaet al. (2003). However, the causal organism of makhana hypertrophy was earlier identified (vide IMI Ref. no. 335137) as a species of Synchytrium (Jha et al. 1992). But further anatomical studies of the host tissues had confirmed the presence of septate branched mycelia ramifying through the inter as well as intra- cellular spaces. This was later confirmed by Dr. JEM Mordue of the International Mycological Institute, London (UK) as a species of Doassansiopsis sp. A member of Ustilaginales (Verma et al. 2003). Among different fungicides treated for minimizing the incidence of leaf blight of makhana in different ponds Sectin (Fenamidon 10%+ Mancozeb 50%) was the best to reduce the infection up to 92.9% followed by Bavistin (87.1%), Blitox (83.9%), Hexaconazole (71.3%), Companion (67.8%) Indophil M-50 (49.8%) and Manzate (35.7%). However Haidar and Mahto (2003) reported that 3 sprayings of 0.3% Fytolan (Copper oxychleride) at an interval of 15 days minimized the incidence of disease up to 90.5% followed by Indophil Z-78 (74.7%) and Indophil M-45 (49.4%). The hypertrophy or gall disease in makhana showed maximum reduction on application of 3 sprayings of 0.4% Bavistin up to 89.3% followed by Blitox (80.4%), Sectin (61.8%), Companion (51%), Hexaconazole (33.3%) and Manzate (29.6%). However, the effect of different fungicides on hypertrophy of makhana has not been studied earlier. Thus avistin can be used to control both leaf blight and hypertrophy of makhana. However, Sectin was most effective against leaf blight and Bavistin against hypertrophy. ACKNOWLEDGEMENT Authors are very much thankful to the Director JRS, Department of Plant Pathology and Horticulture BAU, (Bihar Agriculture University) for providing laboratory facilities and cooperate me in taking photographs in lyenger Phiology laboratory REFERENCES Ghose, S.K.R. and Santra, S.C. (2003). Past and present distribution records of Makhana and future prospects of its cultivation in West Bengal. In : Makhana, R.K. Mishra, Vidyanath Jha and P.V. Dehadrai (Eds.), ICAR, New Delhi,pp.1-3. Kumari, N., Singh S.R.K., Jha S.K. and Choudhary S.B. (2003). Accelerating socioeconomic condition of women through Makhana cultivation in Bihar,pp.102-104. Mishra, R.L. (1998). Gorgon plant: an aquatic ornamental. India Hort. (Jan-Feb), pp. 2021.
267 Businessplan of Makhana Cluster in Bihar (2012).www.udhygmitra.com/asserts/uploades/ 2012/06/makhana-report.pdf. Wallace, H.R. (1973). Nematode as a cause of disease in plants. In: Nematode Ecology and Plant Disease. Edward Arnold Publisher’s Ltd. 1973;pp 146-178. Prasad, H. and Haidar, M.G. (1968). First report of Alternaria tenuis on Makhana (Euryale ferox). Science and Culture,43 : 485. Haidar, M.G. and Nath, R.P. (1987). Chemical control of Alternaria leaf blight of makhana, (Euryale ferox). National Academy of Sciences Letters,10:301-02. Mahto, A., Haidar M.G., Mishra, R.K. and Jha, V. (1993). Nematode association in ponds growing Euryale ferox Salisbury (makhana) in Dharbhanga (North Bihar), India. Journal of Freshwater Biology,5 (4) : 299-304. Haider, H.G. and Mahto, A. (2003). Fungal leaf blight and nematode diseases of gorgon nut (Euryale ferox) and their management. In : Makhana, R.K. Mishra, Vidyanath Jha and Rai P.V. Dehadrai (Eds.) ICAR, New Delhi, pp .159-162. Singh, Suresh; Goel, A.K. and Sharma, S.C. (2003). Prospects of makhana cultivation in central Uttar Pradesh. In : Makhana, R.K. Mishra, Vidyanath Jha and P.V. Dehadrai(Eds.),ICAR,New Delhi,pp.221-227. Dwivedi, A.K.; Shekha, R. and Sharma, S.C. (1995).Ultrastructural studies of Euryale ferox leaf infected by Alternaria alternata.IndianPhytopatholoogy,48 (1): 61-65. Verma, R.A.B.; Jha, V. and Devi, S. (2003). Leaf and floral hypertrophy of Makhana caused by Doassansiopsis euryaleae sp. nov. In : Makhana, R.K. Mishra, Vidyanath Jha, P.V. Dehadrai (Eds), ICAR, New Delhi, pp.163-168. Jha, V.; Saraswati, K.C.; Kumar, R. and Verma R.A.B. (1992).First record of a gall forming Synchytrium species on E. ferox Salisbury in India. Proceedings of the National Academy of Sciences,62 (B) 4 : 627-28.
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269
15 INTEGRATED PESTS AND DISEASE MANAGEMENT BY ORGANIC FARMING AND NATURAL INSECTICIDES 1
Mir Syeda Yuhannatul Humaria and M.R. Sinha Kolhan University, Chaibasa
Email :
[email protected] /
[email protected]
ABSTRACT Growing public awareness and concern about the adverse effects of pesticides on human health, soil and water resources and development of resistance and resurgence among theinsect-pests have necessitated the need to look for eco-friendly, safer and effective methods of pest management. Biological control of insect pests and diseases through biological means is most important component of IPM. In a broader sense, biocontrol is use of living organisms to control unwanted living organisms (pests).In intensive farming systems, organic agriculture decreases yield. Rotating between various crop types, such as annual, winter annual, perennial, grass and broadleaf crops; each of these plant groups has specific rooting habits, competitive abilities, nutrient and moisture requirements. The use of high-quality seed is especially important in preventing disease. The seed supply should be free of smut, ergot bodies or other sclerotia, and free of kernels showing symptoms of Fusarium head blight infection. Some examples of fungi are different species of Hirsutella, Beauveria, Nomurae and Metarhizium which have been reported to infect and kill large number of insects (upto 90 per cent) in the fields. Among viruses, most important examples are of Nuclear Polyhedrosis Virus (NPV) and Granulosis viruses. Among bacteria, Bacillus thuringiensis (B.t.) and B. popillae are very common examples.The increasing awareness of consumer towards residue free produce necessitated the need of nonchemical methods of insect-pest control. Amongst these, cultural practices are basic and eco-friendly way to minimize the insect-
270 pest population.Itcombine scientific knowledge of ecology and modern technology with traditional farming practices based on naturally occurring biological processes. Organic farming methods are studied in the field of agroecology. The principal methods of organic farming include crop rotation, green manures and compost, biological pest control, and mechanical cultivation. Planning for effective insect and disease management must involve the entire farm operation and use all information available. Keywords: Organic, Pests, Eco-friendly, insecticides, Agroeconomy
INTRODUCTION The indiscriminate and unilateral use of pesticides was the only plant protection tool during sixties and seventies for sustaining of agricultural production potential of the high yielding varieties under the intensive cropping systems. This has led to several ill-effects like human and animal health hazards, ecological imbalance, development of resistance in the pests to pesticides, pests resurgence and environmental pollution as well as destruction of natural enemies (bio-control agents) of pests and increased level of pesticides residues in soil, water, food with the increased use of pesticides. Later on National policy on Agriculture - 2000 and National policy on Farmers - 2007 have also supported the IPM. It was also supported by the Planning Commission document for 12th Plan addressing the negative impact of chemical pesticides (Tamizheniyan, S2001). Under the ambit of IPM programme, the Govt. of India has established 31 Central IPM centres in 28 States and One UT. In 12th Five year plan EFC Memo, a “National mission on Agricultural Extension and Technology (NMAET)” was formed under which a “sub-mission on Plant Protection and Plant Quarantine” was introduced from the year 201415.”Strengthening and Modernization of Pest Management Approach in India has become one of the components of this sub-mission with mandate to popularize adoption of Integrated Pest Management (IPM) through training and demonstration in crops inter-alia promotion of biological control approaches in crop protection technology. List of Central Integrated Pest Management Centres (CIPMCs)
CIPMC
States
Regional CIPMCs 1
Faridabad
Haryana
2
Bangalore
Karnataka
271 3
Guwahati
Assam
4
Gorakhpur
Uttar Pradesh
5
Nagpur
Maharashtra
6
Hyderabad
Telangana
7
Patna
Bihar
8
Raipur
Chhattisgarh
9
Ranchi
Jharkhand
10
Bhubaneswar
Odisha
11
Jalandhar
Punjab
12
Lucknow
Uttar Pradesh
13
Kolkata
West Bengal
14
Vadodara
Gujarat
15
Solan
Himachal Pradesh
16
Jammu
Jammu & Kashmir
17
Ernakulam
Kerala
18
Indore
Madhya Pradesh
19
Trichy
Tamilnadu
20
Sriganganagar
Rajasthan
21
Dehradun
Uttarakhand
22
Port Blair
Andaman and Nicobar
23
Itanagar
Arunachal Pradesh
24
Madgaon
Goa
25
Srinagar
Jammu & Kashmir
26
Shillong
Meghalaya
27
Imphal
Manipur
28
Aizwal
Mizoram
29
Dimapur
Nagaland
30
Gangtok
Sikkim
31
Agartala
Tripura
Major CIPMCs
Minor CIPMCs
272 Objectives:
Maximize crop production with minimum input costs.
Minimize environmental pollution in soil, water and air due to pesticides.
Minimize occupational health hazards due to chemical pesticides.
Conserve ecosystem and maintain ecological equilibrium.
Judicious use of chemical pesticides for reducing pesticide residues.
Activities: The 31 Central Integrated Pest Management Centres (CIPMCs) located in 28 States and one Union Territory undertakes the programme with following activities:
Surveillance and Monitoring of insect-pest and diseases.
Augmentation and Conservation of Natural enemies.
Production and release of bio-control agents.
Human Resource Development (HRD) through Farmers’ Field Schools (FFSs)Season-long training programmes, orientation training programme and refresher courses (Sultani MS, Singh R and Dhankhar. 2011). Survey and Surveillance:
To keep a close watch over a desired period of time in an identified cropped area on build up of pests (insects, vertebrates, diseases, nematodes & weeds etc.) and their natural enemy population so that a prior care can be adopted to control the target pests.
The basic objective of pest surveillance is to detect the early sign of existing and emerging pest and their natural enemies for issuance of timely advisories to the State Govt. and farmers for the adoption of suitable intervention.
Survey, monitoring, field scouting are the major activities of the pest surveillance. Fixed plot and rapid roving survey methodology are adopted for pest surveillance.
e-pest surveillance is required to reduce the lead time from pest detection to adoption of interventions (Srinivasa, D.K..1993).
Biological practices: Biological control of insect pests and diseases
273 through biological means is most important component of IPM. In a broader sense, biocontrol is use of living organisms to control unwanted living organisms (pests) (Srinivasa, D.K..1993). Parasitoids: These are the organisms which lay eggs in or on the bodies of their hosts and complete their life cycles on host bodies as a result of which hosts die. A parasitoid may be of different type depending on the host developmental stage in or on which it completes its life cycle. For example, egg, larval, pupal, adult, egg-larval and larval pupalparasitoids. Examples are different species of Trichogramma, Apanteles, Bracon, Chelonus, Brachemeria, Pseudogonotopus etc. Pathogens: These are micro-organisims which infest and cause diseases in their hosts as a result of which hosts are killed. Important examples of fungi are different species of Hirsutella, Beauveria, Nomurae and Metarhizium which have been reported to infect and kill large number of insects (upto 90 per cent) in the fields. Among viruses, most important examples are of Nuclear Polyhedrosis Virus (NPV) and Granulosis viruses. Among bacteria, Bacillus thuringiensis (B.t.) and B. popillae are very common examples. Diseases of pests can be mass multiplied in the laboratory at a low cost in liquid or powdered formulations that can be sprayed like ordinary chemical pesticides. These formulations are known as biopesticides (Hurd, B.H. 1994, Karanth, N.G.K. 2002, Prasad, S.S. 2001). Eco-friendly practices in IPM: Organic farming methods: Itcombine scientific knowledge ofecology and modern technology with traditional farming practices based onnaturally occurring biological processes. Organic farming methods are studiedin the field of agroecology. Theprincipal methods of organic farming include crop rotation, green manuresand compost, biological pest control, and mechanical cultivation. Thesemeasures use the natural environment to enhance agricultural productivity. legumes are planted to fix nitrogen into the soil, natural insect predators areencouraged, crops are rotated to confuse pests and renew soil, and naturalmaterials such as potassium bicarbonate and mulches are used to controldisease and weeds. Organic farmers are careful in their selection of plantbreeds, and organic researchers produce hardier plants through plantbreeding rather than genetic engineering.In intensive farming systems, organic agriculture decreases yield. This isaccomplished by using, where possible, agronomic, biological and mechanicalmethods, as opposed to using synthetic materials, to fulfil any specificfunction within the system”.
274 Benefits of Organic Farming:Organic farming is beneficial for both the humans and the nature.Some of the known benefits of organic farming are:
In organic farming, no fertilizers and pesticides are used, hence, noharmful synthetic chemicals released into the environment.
Organic farming improves productivity of land by healing it withnatural fertilizers.
Organic farms provide support to the diverse ecosystem byproducing safe and healthy environment for humans, plants, insectsand animals as well.
Organic farming is highly beneficial for soil health. Due to thepractices such as crop rotations, inter-cropping, symbioticassociations, cover crops and minimum tillage, the soil erosion isdecreased, which minimizes nutrient losses and boosts soilproductivity. The beneficial living organisms used in organic farmingalso help to improve the soil health.
It helps to promote sustainability by establishing an ecological balance. If organic farming techniques are used for long time, thefarms tend to conserve energy and protect the environment bymaintaining ecological harmony.
When calculated either per unit area or per unit of yield, organicfarms use less energy and produce less waste.
Organic farming reduces groundwater pollution as no synthetic fertilizers and pesticides are used in this method.
Organic farming also helps to reduce the greenhouse effect andglobal warming because it has the ability to impound carbon in thesoil.
In organic farming method, same crop is not located in the farm, which encourages the build-up of diseases and pests that plaguethat particular crop.
The central activity of organic farming relies on fertilization, pestand disease control. Organic far ming relies heavily on the naturalbreakdown of organic matter, using techniques like green manure andcomposting, to replace nutrients taken from the soil by previous crops. Organic farming tends to tolerate some pest populations while takinga longer-term approach. Organic pest and disease control involves
275 thecumulative effect of many techniques, including:
Allowing for an acceptable level of pest and disease damage.
Encouraging predatory beneficial insects to control pests.
Encouraging beneficial microorganisms and insects.
This by servingthem nursery plants and/or an alternative habitat, usually in a form of a shelterbelt, hedgerow, or beetle bank.
Careful crop selection, choosing disease-resistant varieties.
Planting companion crops that discourage or divert pests.
Using row covers to protect crops during pest migration periods.
Using pest regulating plants and biologic pesticides, fungicides andherbicides.
Using no-till farming, and no-till farming techniques as false seed beds.
Rotating crops to different locations from year to year to interrupt pest/disease reproduction cycles.
Using insect traps to monitor and control insect populations that cause damage as well as transmit diseases.
Each of these techniques also provides other benefits viz., Soilprotection and improvement, fertilization, pollination, water conservation, season extension, etc.and these benefits are both complementary andcumulative in overall effect on farm health. Effective organic pest and diseasecontrol requires a thorough understanding of pest life cycles and interactions.Crop protection in organic agriculture is not a simple matter. Itdepends on a thorough knowledge of the crops grown and their likely pests,pathogens and weeds. Successful organic crop protection strategies also relyon an understanding of the effects which local climate, topography, soils andall aspects of the production system are likely to have on crop performanceand the possible host/pest complexes. Organic agriculture is rapidly expanding and includes novel, edible, fibre and processing crops, diversifiedrotations and large scale stockless farming companies alongside traditionalmixed organic farms. The less mobile pests or those which have a specific ornarrow host range are particularly susceptible to crop rotation. Highly mobile,often non-specific pests such as aphids are less affected, or unaffected byrotation design. Reactive treatments for pest outbreaks, including naturalpesticides, are permitted
276 under regulations for specific situations in organicsystems, but cultural pest prevention techniques including the use of breakcrops within balanced rotations will remain the most important means for pestcontrol in organic systems (Arunakumara, V.K. 1995,Anonymous. 2013). Managing Pests and Diseases: Managing the ecosystem on an organic farm is very challenging. Itis made even more complex when factoring in insect and disease pests. Since the use of synthetic pesticides are prohibited, the organic cropping systemshould be focused on the prevention of pest outbreaks rather than copingwith them after they occur. No single method is likely to be adequate for allpests. Successful pest management depends on the incorporation of anumber of control strategies. Some strategies will target insect and disease separately and others will target them together.Planning for effective insect and disease management must involvethe entire farm operation and use all information available. Any strategy inorganic farming should include methods for:
Insect and disease avoidance
Managing the growth environment
Direct treatment
i. Avoidance Techniques To manage pests and diseases effectively, producers need tounderstand the biology and growth habits of both pest and crop. The typeand concentration of pests are often responses to previous crop history, pestlife cycles, soil conditions and local weather patterns (Ram S and Singh S. 2010). Crop Rotations: Crop rotation is central to all sustainable farming systems. It is anextremely effective way to minimize most pest problems while maintainingand enhancing soil structure and fertility. Diversity is the key to a successful crop rotation program. It involves:
Rotating early-seeded, late-seeded and fall-seeded crops
Rotating between various crop types, such as annual, winter annual,perennial, grass and broadleaf crops; each of these plant groups hasspecific rooting habits, competitive abilities, nutrient and moisturerequirements. (True diversity does not include different specieswithin the same family - for example, wheat, oats and barley are allspecies of annual cereals.)
277
Incorporating green manure crops, into the soil to suppress pests, disrupt their life cycles and to provide the additional benefits of fixing nitrogen and improving soil properties.
Managing the frequency with which a crop is grown within a rotation.
Maintaining the rotation’s diversified habitat, which provide sparasites and predators of pests with alternative sources of food, shelter and breeding sites.
Planting similar crop species as far apart as possible. Insects such aswheat midge and Colorado potato beetle, for example, are drawn to particular host crops and may over winter in or near the previous host crops. Diverse rotations are particularly effective in regulating flea beetles, cabbage butterfly, wheat midge, wheat stem maggot and wheat stem sawfly.
Rotations are also effective in controlling soil- and stubbleborne diseases. The success of rotations in preventing disease depends on manyfactors, including the ability of a pathogen to survive without its host and thepathogen’s host range. Other situations that limit the benefit of crop rotations include: the transmission of pathogens viaseed, the presence of susceptible weeds and volunteer crops that harb our pathogens, and the invasion of pathogens by wind and other means. Rotations should be used with other cultural practices to achieve thegreatest benefit (Bhullar MB and Dhatt AS. 2011, Carson R. 2007). Field Sanitation/Crop Residue Management: Reducing or removing crop residues and alternate host sites can beused to control some insects and many diseases. Incorporating the residue into the soil hastens the destruction of disease pathogens by beneficial fungiand bacteria. Burying diseased plant material in this manner also reduces themovement of spores by wind.Reduced or zero-tillage may also reduce the damage by certain pests, as the crop residue creates a micro-climate less preferred by some insects (for example, flea beetles).It is important to maintain a balance between crop sanitation andsoil conservation. Lighter soils and those prone to wind and water erosionmay require postponing tillage until just before seeding to ensure stubblecover for as long as possible.Left uncontrolled, these insect and disease pests can betran smitted to healthy crop plants. Insects may use these plants as alternatehabitat until an appropriate crop occurs in a nearby field. However, these are as may also host many beneficial insects and predators, therefore the grower must carefully assess the potential threat
278 from pest insects in theseareas before mowing or removing any plants. The ecological importance ofareas such as sloughs, wooded bluffs, road allowances, railroad rights-of-way,abandoned farmyards and schoolyards must also be included in long-rangeplanning. Seed Quality: The use of high-quality seed is especially important in preventingdisease. The seed supply should be free of smut, ergot bodies or other sclerotia, and free of kernels showing symptoms of Fusarium head blightinfection. Seed analysis by a reputable seed testing laboratory will helpdetermine specific diseases in the seed supply.Relatively few diseases are exclusively seed-borne, and it is morecommon for pathogens to be transmitted from soil, stubble, or wind, as wellas with the seed.Planting physically sound seed is also important. In crops such asflax, rye and pulses, a crack in the seed coat may serve as an entry point forsoil-borne microorganisms that rot the seed once it is planted. Weed Management:In many cases, weeds provide food and shelter for beneficialinsects. Parasitic wasps, for example, are attracted to certain weeds withsmall flowers. Insects that are generalist feeders, such as beet webworm, thistlecaterpillars and grasshoppers, may prefer to feed on weeds rather than somecrops, only damaging the crop after the weeds are eaten.Each field situation should be considered separately, as weedcompetition must always be taken into account. Sometimes mowing weeds atthe edge of the field results in beneficial organisms moving into the cropwhere they are needed (Chand, Ramesh and Birthal, P.S. 1997, Chaudhary FK and Patel GM. 2008) Forecasting: Producers should pay attention to the forecasts for various pest anddisease infestations for each crop year. Maps of these forecasts are usuallyavailable for many of the major destructive insects such as grasshoppers andwheat midge, as well as some diseases. Agro meteorological warning andforecast can help in this way. Record-Keeping: Keeping diligent field records can provide very useful information. Acomplete history of each field should include any insect or diseaseinfestations, which management methods worked and which did not, and alist of management techniques to try in the future. ii. Managing the Growth Environment - Giving the Crop a Head Start Any crop management technique that contributes to a vigorous, competitive crop is a tool of insect and disease management. Producers mustalso be mindful that many practices that work well in conventional systemsmay not benefit organic systems. Certain crop species, crop varieties
279 andequipment may work well in one system but not in the other. Healthy Soil: A biologically active soil with good drainage supports vigorouscrop growth, allowing a higher level of crop competition with weeds.Adequate, balanced soil nutrition is essential for crop quality, yieldand moisture-use efficiency. The application of nutrients should be based on asound soil testing program, accompanied by plant tissue analysis whendiagnosing problems. High levels of nitrogen can occur after a highnitrogen blowdown, such as sweet clover. This results in lush leaf tissue and a denseplant canopy that provides an ideal environment for plant pathogens.However, a lush crop may also help disperse the damage by a given numberof insects, so astute observations by the producer are necessary at all times.In contrast, inadequate soil phosphorous can pre-dispose to certainroot diseases. Low levels of nitrogen can reduce the incidence of insectoutbreaks. A shortage of micronutrients such as zinc or copper can result indisease-like symptoms on crops, while too much of any one micronutrientmay be toxic.Field experience has also shown that plants fertilized by the slowrelease of nutrients from compost are more resistant to insects and diseasesthan crops fertilized by highly soluble nutrients. Soil testing becomesimportant when applying compost regularly. An imbalance of nutrients caneasily occur if the soil’s nutrient profile is not continuously monitored (Chopra, K. 199, Garg, D.K.1999). Crop and Variety Selection: Producer awareness of insects and diseases in the proximity of thefarm is very important and should influence the crop and the variety of crop to be grown. The selection of insect- and disease-resistant cultivars can be auseful tool, but under no circumstances can genetically modified varieties beused in organic systems. Wheat varieties with solid stems are more resistantto wheat stem sawfly than hollow-stemmed varieties. Wheat varieties with resistance to wheat midge have been developed and should soon beavailable. These insect-resistant varieties were developed throughconventional plant breeding programs. Certain species may avoid diseases such as Fusarium head blight, but often agronomic factors, such as time ofseeding or choosing winter versus spring wheat, have more of an influence onthe incidence of disease.Plants also vary in their degree of attractiveness to insects, diseasesand vectors transmitting disease. Factors such as leaf and stem toughness, pubescence, nutrient content, plant architecture, growth habit and differences in maturity between crops and varieties can influence pest growth,reproduction and host preference. For example, earlier-maturing cropvarieties may be less attractive to migrating populations of grasshoppers latein the season compared to later-maturing varieties (Hurd, B.H. 1994, Karanth, N.G.K.
280 2002) Intercropping: The practice of intercropping (where two crops are grown at the same time) can reduce pest problems by making it more difficult for the peststo find a host crop. This technique also provides habitat for beneficialorganisms. Strip-cropping row crops with perennial legumes often leads tobetter pest control. In particular, alfalfa attracts many beneficial organisms that can destroy insect pests in neighbouring crops. Seeding Date: Planting should be scheduled so that the most susceptible time ofplant growth does not correspond to the peak in pest cycles. Early seeding reduces crop damage caused by grass hoppers, aphids in cereal crops, wheatmidge in spring wheat, barley yellow dwarf virus in barley and wheat,powdery mildew in peas and pasmo in flax. Delayed seeding can be effective in avoiding wireworms and cutworms in cereal crops, Hessian fly in winter wheat, barley thrips, Ascochyta in lentils and wheat streak mosaic virus in winter wheat. However,experience on the Prairies has generally shown that delaying seeding too longcan reduce a crop’s potential yield (http://knnindia.co). Seeding Rate: Using a higher seeding rate to affect insect or disease infestations may have different results. More plants in a field may reduce the impact of agiven aphid population on individual plants, but they may create a more favourable habitat for insects that prefer a dense canopy, such as truearmy worm. A dense leaf canopy can also create a moist soil surface andelevated humidity within the crop, conditions favourable to certain leaf disease pathogens. Reducing the seeding rate may decrease the severity of take-all inspring wheat, but the reduced canopy may also allow weeds to invade. Inother crops, reduced seeding may also produce more insect damage, as in the case of aphids, flea beetles and leaf hoppers, which are attracted to thecontrast between a green host and a dark soil background. Depth and Timing of Seeding: Optimum seeding depth is also important. Deep seeding in cold soilsmay result in seedling blights and damping-off, especially in pulses and smallseededcrops. Seeding depth should generally be no deeper than required for quick germination and even emergence. Variables include seed size, soil typeand moisture conditions. If the soil is loose before seeding, a packing operation will firm up the soil and bring moisture closer to the surface. For most crops, seeding should ideally be done when the soil iswarm enough for rapid germination. Trap Strips: Seeding trap strips around the edge of a cropped field or
281 along afence row helps lure insect pests to a specific area where they can bemanaged more easily. For example, planting bromegrass near a wheat fieldattracts wheat stem sawflies and their native parasites away from the wheatcrop. Similarly, a trap strip of potatoes planted much earlier than the maincrop would attract Colorado potato beetles to the area. The strip could beworked under along with the adult beetles, eggs and larvae before the secondgeneration of beetles spreads to the main crop.Generally, the insect pests in the trap strips are controlled by mowing or cultivating the strip, or by applying an acceptable organic product, such as Bacillus thuringiensis. Trap strips can also act as a barrier to protectthe crop field. Producers have found that planting yellow sweet clover orSirius field peas repels grasshoppers and prevents them from damaging crops. A thorough knowledge of the crop and insect pests of the area is necessary to prevent this technique from backfiring (https://www.thehindu). Tillage: Tillage can be properly timed before seeding, after harvesting andduring summerfallow to reduce populations of insect pests such as cut worms and grasshoppers that spend part of their life cycles in the soil or stubble. Tillage can help starve insects in the spring or during fallow, prevent adults from laying eggs in the soil and expose over wintering insects to predators and inclement weather. Roguing: Roguing refers to the labour-intensive practice of walking the fieldsto remove diseased or insect-infested plants. Roguing may not be practical forlarge fields, but could be suitable for seed plots or crops having highlyinfectious and destructive diseases (for example, bacterial blackleg inpotatoes and certain viruses in other crops) (Bhullar MB and Dhatt AS. 2011, Carson R. 2007). iii. Direct Treatment At times, the organic producer will find that, despite all the bestefforts, an insect or disease pest will grow to levels that cause substantial crop damage. At this point, direct treatment may be necessary. Monitoring: Insect monitoring traps are useful in determining which insect pestsare present in a field and whether they are at economically important levels.It is imperative that the producer has a positive identification of the insect ordisease causing damage before choosing a method of treatment.Certain types of insect hormones called pheromones may be used as attractants to monitor population levels of insects such as bertha army worm, diamond back moth, cabbage looper and European corn borer, or to simplyattract insects into a trap (https://yardcare.).
282 Biological Control: In a healthy, balanced ecosystem, biological control by natural predators is constantly occurring. The more diverse a cropping systembecomes, the greater the spectrum of insect species and microorganisms within it. This leads to the development of more natural predators within theecosystem.Ladybugs, ambush bugs, hoverfly larvae, lacewings, spiders, birds,frogs, toads and a host of other insects are predators of aphids, berthaarmy worm larvae, sunflower beetles, beet web worms, and both grass hopper eggs and adults. The destructive wheat midge may also be partially controlledby a parasitic wasp, but crop damage may still occur.Various types of fungi are insect parasites and can either kill theirinsect hosts or reduce their ability to reproduce. Very few biological controlsare available to reduce the effects of plant diseases, as most commercial products do not perform well if the disease is already established in the crop. Natural Insecticides: Organic certification standards prohibit the use of synthetic pesticides. Permitted disease-management products include copper (fixed copper and copper sulphate), lime-sulphur mixes, elemental sulphur, vinegar, soap and silica. Bordeaux mixture is considered a restricted substance, andfarmers should contact their certifying body before using it. Although the seproducts are allowed, it may not be cost-effective or feasible to apply them tofield crops. Scientific evidence on product efficacy should be researchedbefore using them. The high risk of phytotoxicity should also be considered when usingthese products on certain plants; often the margin of error between benefitand damage to the plant is very small. In addition, there are environmental and ecological concerns surrounding some of these products. Additional soiltests may be required to monitor copper and sulphur levels in the soil. Aswell, organic certification may be denied to farms that overuse or depend onsuch products.Insecticides permitted in organic agriculture include some microbialinsecticides containing the bacteria Bacillus thuringiensis. Three main strainsof these bacteria are used in insect control. One strain, marketed as Dipel or Thuricide, kills only the larvae of moths or butterflies. Another strain, marketed as Novodor, is for beetle larvae only and can be used to controlColorado potato beetles. The third strain is specifically for mosquito and flylarvae. Botanical insecticides, such as rotenone, are also permitted inorganic agriculture, but they are often too expensive to use on largeacreages (http://pib.nic.in). Other Control Methods: For pest control, beneficial organisms, dormant oil, diatomaceous earth, plant-derived pesticides, soap, natural and synthetic
283 insect pheromones which disrupt the insect’s development, and commercial insect vacuums canbe used. Grain Storage: When stored grain is dry and its temperature is low, problems seldom arise. But if the grain is warm and moist, insects and fungi canmultiply rapidly. A grain temperature of 5°C to 10°C is adequate for long-termstorage. Bin aeration helps dry and cool the grain. If bins are not equipped with aeration systems, grain can be moved to cool it. Cold temperatures canbe used to control insects that exist in stored grain.Before storing new grain, the bin should be thoroughly cleaned witha grain vacuum. The empty bin can be treated with diatomaceous earth to control storedgrain insects. Many organic producers have found it to beeffective when applied as a light coating on the floor of the bin, and aroundthe walls and the bin door. It can also be added to the grain as it is beingbrought into storage. List of inputs permitted for crop protection S.No.
Input
Permitted/ Restricted
1.
Mechanical traps, Neem oil and other preparations, Propalis, Pheromones in traps, plant based repellants, Silicates, Soft soap.
Permitted
2.
Copper salts, Chloride of lime/soda, light mineral oils, permanganate of potash, Sulphur, Viral, fungal and bacterial preparations, release of parasite andpredators of insect pests.
Restricted
Commonly available plants that can be used for making herbal extracts are asfollows S. No.
Common Name
Botanical Name
Useful Plant Parts
1.
Neem
Azadirachtaindica
Neem Cake
2.
Pungam
Pongamiapinnata Pongamiaglabra Leaf & flower
3.
Notchi
Vitexnugunda
Leaf & flower
4.
NithiaKalyani
Catharanusrosea
Whole plant
5.
Unni
Lantana camera
Leaf & flower
6.
Devils Trumpet
Datura metal
Leaf, fruit and flower
7.
Yellow Nelliam
Neriumthevetifolia
Flower, fruit and root
8.
Aruku
Calatropisgigantea
Leaf, tender stem and
284
flower 9.
SiriaNangai
Andrographispaniculata
Whole plant
10.
Parthenium
Partheniumsp
Plant before flowering
11.
Adathoda
Adathodavasica
Leaf
12.
Tobacco
Nicotianatobaccum
Dried leaf, plant waste and stem waste
13.
Tobacco Plant (weed)
Lobiliasp
Whole plant
14.
Chevanthi
Crysanthemumcinerrifolia
Flower
15.
Thumbai
Lucusaspera
Flower, leaf, tender and stem
16.
Ginger
Zingiberofficinale
Rhizome
17.
Etti
Strychnosnuvomica
Seeds
18.
Turmeric
Curcuma longa
Rhizome
19.
Artemesia
Artemesia vulgaris
Tender shoots & leaves
Liquid extracts for disease management Disease
Type of compost
Late blight of potato ,tomato
Horse compost extract
Graymold on beans strawberries
Cattle compost extract
Downy and powdery mildew of grapes
Animal manure-straw compost extract
Powdery mildew on cucumbers
Animal manure-straw compost extract
Graymold on tomato, pepper
Cattle and chicken manure compost extract
Apple scab Spent mushroom
compost extract
(http://www.drishtiias)
Pest/Disease Management Packages for Different Crops Integrated Pest Management package for Organic Rice Ecosystem Rice is essentially a crop of warm, humid environments conducive tothe survival and Proliferation of insects. More than 70 species were recordedas pests of rice and about 20 have major significance. Together, they infestall parts of the plant at all growth stages. The insects act as
285 vectors of virusdiseases, and are a major factor responsible for low rice yields particularly inTropical Asia, the world’s rice bowl. The insect problem is accentuated in multicropping or dormancy but occurs throughout the year in over lapping generations. The yield losses vary from 20 to 50 per cent due to the damagecaused by various insect Pests. Mechanical control methods
Collection and destruction of rice stubbles from field after harvest as they harbour egg, larvae, pupae of stem borer, gall midge, white tipnematode and root knot nematodes.
·
Clipping the tips of the seedlings up to 2 inches prior to transplanting to remove the egg masses of stem borer if any.
Collection of egg masses of stem borer and silver shoots from the nursery seedlings.
Flooding the nursery to make the hiding larvae in the soil to come tothe surface and thus they are picked by the birds (army worm).
A rope may be passed over the young crop for dislodging the larval cases from the tillers and then the water should be drained for eliminating them (case worm).
Providing bird perches of 2-3 ft height in vegetative stage @ 1520 /acre. They should be removed after seed setting to avoid the bird damage to seeds. Drinking pots with water should be provide daround the perches.
Collection and destruction of egg masses of stem borer and ear head bugs in main field.
A thorny hedge may be passed over the crop when it is affected byleaf folder to unfold the leaf folds and to expose the larvae within tonatural enemies and botanical sprays (Puri, S.N. 1998).
Conclusion The increasing cost of plant protection and accelerating pest incidents make agriculture a risky and less profitable enterprise. At the same time the toxic materials generated from chemical farming pollute the environment and harm consumers’ and farmers’ health. A more environmentally friendly and economical alternative for India would be adoption of Integrated Pest Management. Additionally, from the viewpoint of sustainability, attaining growth while maintaining the natural capital
286 intact, IPM is superior compared to conventional farming (Chopra 1993). The increasing awareness of consumer towards residue free produce necessitated the need of nonchemical methods of insect-pest control. Amongst these, cultural practices are basic and eco-friendly way to minimize the insect-pest population. The various kind of traps can be used for monitoring and suppression of initial pest population. Further, selection of insect resistance varieties and need based use of biopesticides which are selective and eco-friendly can be used in ecofriendly pest management programs References Arunakumara, V.K. (1995). Externalities in the use of pesticides: An economic analysis in a Cole crop. MSc Thesis (Unpblished), UAS, Bangalore. Anonymous. (2013). State of Indian Agriculture 2012-13. Department of Agriculture and Cooperation, Ministryof Agriculture, Government of India. 221p. Bhullar, M.B. and Dhatt, A.S. (2011). Spider mite, Tetranychusurticae Koch incidence in relation to culturalpractices in nethouse on brinjal. Journal of Insect Science 24: 121-123. Carson, R. (2007). Pesticides and Health. Newsletter 2(1): 1-6. Chand, R. and Birthal, P.S. (1997). Pesticide use in Indian agriculture in relation to growth in area and production and technological change. Indian Journal of Agricultural Economics, 52(3): 488-498. Chaudhary, F.K. and Patel, G.M. (2008). An integrated approach of male annihilation and bait application technique for fruit fly management in pumpkin. Pest Management and Economic Zoology 16 (1): 57-61. Chopra, K. (1993). Sustainability of agriculture. Indian Journal of Agricultural Economics, 48(3): 527-537 Garg, D.K. (1999). Development of an IPM approach in Basmati rice. Annual Report, NCIPM, p: 13-19. Hurd, B.H. (1994). Yield response and production risk: An analysis of integrated pest management in cotton. Journal of Agricultural and Resource Economics, 19 (2): 313-326. Karanth, N.G.K. (2002). Challenges of Limiting Pesticide Residues in Fresh Vegetables: The Indian Experience. In E. Hanak, E. Boutrif, P. Fabre, and M. Pineiro, (Scientific Editors), Food Safety Management in Developing Countries. Proceedings of the International Workshop, CIRAD-FAO, pp.11-13, December 2000, Montpellier, France. Prasad, S.S. (2001). Country Report – India. Report prepared for the meeting of the Programme Advisory Committee (PAC), Ayutthaya, Thailand, November 2001. Puri, S.N. (1998). Present status of Integrated Pest Management in India. Paper presented
287 at Seminar on IPM, Asian Productivity Organization at Thailand Productivity Institute, Bangkok. Ram, S. and Singh, S. (2010). Effect of intercropping of spices, cereal and root crops on the incidence of Helicoverpaarmigera (Hub.) in tomato. Vegetable Science 37 (2): 164-166. Srinivasa, D.K. (1993). Environment and human health. Environmental problems and prospects in India, Oxford and IBH Publications, New Delhi. Sultani, M.S., Singh, R. and Dhankhar. (2011). Morphological and biochemical basis of resistance in selected okragenotypes against Earisvittella (Fab.). Journal of Insect Science 24 (1): 33-40. Tamizheniyan, S. (2001). Integrated Pest Management in rice farming in Thiruvarur District of Tamil Nadu: A Resource Economic analysis. MSc Thesis (Unpublished), UAS, Bangalore. link:http://knnindia.co.in/news/newsdetails/features/integrated-pest-management-an-ecofriendly-approach-to-control-pests https://www.thehindu.com/thehindu/seta/2002/02/14/stories/2002021400120600.htm https://yardcare.toro.com/features/go_green/integrated-pest-management-ipm-an-ecofriendly-approach/ http://pib.nic.in/newsite/PrintRelease.aspx?relid=110364 h tt p:// w w w. d rish t iias . com / u psc -exam -gs-resou rces-IN T E GR AT E D -P E STMANAGEMENT
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16 IDENTIFYING THE IMPACTS OF PEST ATTACK ON SAL TREE (Shorea robusta) IN THE DINDORI DISTRICT OF MADHYA PRADESH USING REMOTE SENSING AND GIS APPROACH Obaidullah Ehrar1; Shubham Kumar1; Rabindar Kumar1 and Ajay Kumar Srivastava2 1
MSc & Integrated M Tech Geoinformatics, Central University of Jharkhand, 2St.Xavier’s College, Ranchi
[email protected]
ABSTRACT Forest plays a very important role for the balancing the ecosystem on Earth. There exists a wide variety of Biodiversity on Earth of which Sal (Shorearobusta) tree is a prominent tree found also in the forests of Madhya Pradesh. During the years 1996 to 2003 millions of trees fell down due to the pest attack in the Dindori District of Madhya Pradesh.This study aims to identify the areas affected by the pest attack by using the various vegetation indices used for monitoring the health of vegetation applying the Remote Sensing and GIS Approach. The various vegetation indices i.e. NDVI (NormalizedVegetation Condition Index), VCI (Vegetation Condition Index), TCI (Temperature conditionIndex), VHI (Vegetation Health Index) are used for monitoring the health of vegetation using theRemote Sensing and GIS Approach.These indices help in monitoring the changes which areoccurring in vegetation or to identify the stress in vegetation for a wide area. Satellite imageshaving various bands provide the necessary information in optical as well as thermal domain.
290 INTRODUCTION Sal tree (Shorearobusta) belongs to the family Dipterocarpacaeae and is mainly distributed in Sri Lanka, India, Myanmar and Philippines.Sal forests occupying an area of 105 790 km2 in India, are very important from both ecological and economical points of view as they not only harbor maximum biodiversity but are also a source of livelihood for millions of people. Sal forests are well known to harbor maximum biodiversity and being semi-evergreen, they constitute an important base for the ecosystem where they grow, provides cool and calm environment. A wide range of Non Timber Forest Products found in these forests which apart from timber, are source of livelihood for millions of people living in and around forests. Timber, because of its excellent qualities, is used for different purposes. Calorific value of completely dried heartwood is as high as 5433 calories. Given the magnanimity of the tree as a keystone, not surprisingly it also harbors some nasty predators like sal-borer (Hoploceram byxspinicornis). The heartwood borer was first noticed as a pest on sal in 1899. The attack of the borer is considered epidemic when the sal trees affected are more than 1% of the total number of trees. In 1905, the first epidemic was observed in Balaghat district of Central India. In 1923-24, serious attack of sal heartwood borer was observed in Mandla and Dindorisal forests in which 7 million trees were affected. As per the advice of the Forest Entomologist, Forest Research Institute, Dehradun, control operations were carried out. About 428,000 adult insects were killed and 387,500 trees felled and removed from affected sal forests. Table 1.Number of Trees Felled From 1996-2003 Year
Name of Division
No of Trees Felled
No. of Beetles killed (Thousands)
1996-1997
Dindori
13728
2000
Mandla
-
200
Dindori
560718
13900
Mandla
146640
1100
Dindori
368272
31000
Mandla
60690
1600
Dindori
19230
12100
Mandla
9335
1300
1997-1998
1998-1999
1999-2000
291
2000-2001
2001-2002
Dindori
109004
17100
Mandla
19013
100
Dindori
219776
-
Mandla
63659
-
Total
1590065
65000
Remote Sensing and GIS play very important role in observing the phenomenon occurring on the earth surface as well as in the Earth Atmosphere. In this study Remote sensing (Satellite Images) are used for observing the changes which occur due to the pest attack on the forests of Dindori District of Madhya Pradesh. Satellite images provide the wide view of Study area which helps in the observation or determination of impacts on the forest by pest attack. OBJECTIVE OF THE STUDY The main aim of this study was the determination of impacts of pest attack on the sal forest of Dindori district of Madhya Pradesh. Satellite data of Dindori district is used for the study. STUDY AREA Dindori district is among most important sal forest area of Madhya Pradesh. Dindori District lie between 22°14’15" to 23°22’30" North latitude &80°25’15" to 81°47’15" East longitude. The Geographical area of Dindori district is 6128 Sq. Km. out of which forest area is 2307. 45 Sq. Km. DATA USED & METHODOLOGY Satellite Data Used 1. Landsat 7 (ETM+) 2. Landsat 5 (TM ) Software Used 1. Arc Gis10.4 2. QGIS 2.18.12 3. Erdas Imagine 4. Ms Word 5. Ms Excel
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Figure 1. Map showingthe study area of the Dindori District of Madhya Pradesh
METHODOLOGY
Figure 2. Showing Methodology & Procedure
Step 1: Satellite Image of Dindori district of 1990 and 2000 are used for the study of sal forest and pest attack on sal forest.
293 Step 2:NDVI of two periods is calculated inQGIS. NDVI = (NIR- RED)/(NIR + RED) NDVI has been used successfully to identify stressed and damage crops and pastures. Its value range from +1 to -1. Positive value shows the healthy vegetation and negative value shows the stressed vegetation. Step 3:VCI (VEGETATION CONDITION INDEX) of two periods is calculated in QGIS VCI = 100*(NDVI – NDVIMIN)/(NDVIMAX - NDVIMIN) VCI separates the short-term weather-related NDVI fluctuations from the long-term ecosystem changes. Therefore, while NDVI shows seasonal vegetation dynamics, VCI rescales vegetation dynamics in between 0 and 100 to reflect relative changes in the vegetation condition from extremely bad to optimal. Step 4:TCI (TEMPERATURE CONDITION INDEX) is calculated in QGIS. TCI = 100*(BT - BTMIN)/(BTMAX - BTMIN) TCI is based on the thermal band converted to brightness temperature (BT). TCI is used to determine temperature-related vegetation stress and also stress caused by excessive wetness. TCI provides opportunity to identify subtle changes in vegetation health due to thermal effect as drought proliferates if moisture shortage is accompanied by hightemperature. Itranges from 100 to 0.Low TCI values correspond to vegetation stress due to dryness by high temperature. Step 5: VHI (VEGETATION HEALTH INDEX) is calculated in QGIS. VHI = 0.5*(VCI) + 0.5*(TCI) Vegetation health index is calculated with help of vegetation condition index and temperature condition index. Equal priority is given to both VCI and TCI for estimating the Vegetation Health Index. It fluctuates from 0 to 100, reflecting changes in vegetation conditions from extremely bad to optimal. VCI=100*(NDVI-NDVImin)/ (NDVImin-NDVImin) TCI=100*(BTmax-BT)/ (BTmax-BTmin) VHI=a*VCI+ (1-a)*TCI
294 RESULTS AND DISCUSSION In this study, we had mainly focused on the various vegetation indices which are used for the detection of stress in vegetation using the different bands of satellite data of Landsat 7 and Landsat 5.
Figure3. Shows the variation of NDVI in Dindori district in year 1990 and year 2000. The max value of NDVI changes from 0.706897 in year 1990 to 0.545455 in the year 2000.
Figure 4. Shows the VCI difference from year 1990 to 2000.
295 This map shows that the low value has decreased from 2.18869 in year 1990 to 1.01799 in year 2000. Which tell us that vegetation condition has stress in this period in some parts of Dindori district due to the felling of tress. There is increase in the area of stress from year 1990 to 2000. The red colour in the map shows the increases stress in between 1990 to 2000. The VCI is calculated with help of NDVI value.
Figure 5.Shows the Temperature ConditionIndex (TCI) which gives us the information about stress in vegetation due to the increase in the temperature of that area.
Theminimum range of TCI changes from 4.75889 in year 1990 to -0.136435 in year 2000, which is the indication of increase in stress due to the increase in temperature of that area.There is increase of Blue colour area in the map from 1990 to 2000 showing the increase in area of stress due to felling of tress.TCI is calculated with the help of Brightness Temperature which is calculated from the Thermal Band of Landsat 7 and Landsat 5 satellite Image.
296
Figure 6. Shows the Vegetation Health Index (VHI) of Dindori District Of Madhya Pradesh
VHI provides the overall effect on Health of Vegetation. VHI ranges from 0 to 100 in which higher values shows the good health of vegetation while low values show the bad health condition of vegetation. From the figure it is clear that the value area increased from 1990 to 2000 in Dindori District. There is also decrease of low value from 7.83352 to 5.68389 in the legend, which clearly shows the effect of felling of trees due to the pest attack on sal tree (Shorearobusta) forest. CONCLUSION With the help of Remote Sensing we can easily identify the areas that are affected by the pest attack in Sal forest and GIS help in calculating all the indices which are important indicators of stress in vegetation as well as for mapping the forest area of Dindori District. From the above study we can easily conclude that there is lot of variation in all the indices i.e. NDVI, TCI, VCI and VHI from year 1990 to 2000 due to the Pest attack in Dindori District of Madhya Pradesh. REFERENCES Bhuiyan, C. (2004). Various drought indices for monitoring drought condition in Aravalli terrain of India. ISPRS International Journal of Geo-Information, 6. Retrieved from http://www.isprs.org/proceedings/xxxv/congress/comm7/papers/243.pdf
297 Boqer, S., & Science, O. (2009). Use of NDVI and Land Surface Temperature for Drought Assessment/ : Merits and Limitations, 618–633. https://doi.org/10.1175/ 2009JCLI2900.1 Chen, M., & Cihlar, J. (2000). Retrieving Leaf Area Index of Boreal Conifer Forests Using Landsat TM Images, 162(May 1995), 153–162. Cohen, W. B., & Goward, S. N. (2004). Landsat ’ s Role in Ecological Applications of Remote Sensing, 54(6), 535–545. Gao, B. (1996). NDWI A Normalized Difference Water Index for Remote Sensing of Vegetation Liquid Water From Space, 266(April), 257–266. Kogan, F., Gitelson, A., Zakarin, E., Spivak, L., & Lebed, L. (2003). AVHRR-Based Spectral Vegetation Index for Quantitative Assessment of Vegetation State and Productivity/ : Calibration and Validation, 69(8), 899–906. Paula, D. (2005). Shorea robusta – an excellent host tree for lichen growth in India Effect of fig trees on Bhimbetka world heritage site, 89(4), 3–4. Shorea, S. A. L., & Singh, N. (2014). IMPACT OF INFESTATION OF SAL HEARTWOOD BORER ON THE CARBON STOCK OF IMPACT OF INFESTATION OF SAL HEARTWOOD BORER ON THE CARBON STOCK . Singh, R. P., Roy, S., & Kogan, F. (2003). Vegetation and temperature condition indices from NOAA AVHRR data for drought monitoring over India. International Journal of Remote Sensing , 24(22), 4393–4402. https://doi.org/10.1080/ 0143116031000084323 Tah, J., & Mukherjee, A. K. (2018). Ecological Hindrances for Establishment of Mass Population of Sal ( Shorea robusta ) in Forest Gardens Overtaking, 2(2), 857–872. Tripathi, R., Sahoo, R. N., Gupta, V. K., Sehgal, V. K., & Sahoo, P. M. (2013). Developing Vegetation Health Index from biophysical variables derived using MODIS satellite data in the Trans-Gangetic plains of India. Emirates Journal of Food and Agriculture, 25(5), 376–384. https://doi.org/10.9755/ejfa.v25i5.11580 Usmerjena, O., Podatkov, A., & Zaznavanja, D. (n.d.). OBJECT-BASED IMAGE ANALYSIS OF REMOTE SENSING.
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17 IMPACT OF ABIOTIC AND BIOTIC STRESSES ON STEVIOL GLYCOSIDE PRODUCTION BY Stevia rebaudiana Bert. – CURRENT SCENARIO Abhijit Bandyopadhyay Centre for Advanced Studies, Department of Botany, University of Burdwan, Burdwan, West Bengal; India Email :
[email protected]
ABSTRACT Stevia rebaudiana Bert. (Family – Asteraceae), is a shrubby, perennial plant species, popular worldwide for its ability to accumulate considerably high level of several commercially important steviol glycosides (SGs). Among these various SGs, Stevioside and Rebaudioside are the major metabolites and are universally used as sugar alternative especially among diabetics. Recent findings reveal that judicious imposition of both abiotic and biotic stresses provides promising results in production of biomass and elevated synthesis of steviol glycosides. This review underlines the significance spatiotemporal patterns and sequence of molecular signalling during cross interactions among host of transcription factors. Perhaps unraveling these complexities would provide immense potential to understand and manipulate steviol glycoside productions. Keywords: Stevia rebaudiana, Steviol glycosides, Abiotic, Biotic stresses
Introduction Stevia rebaudiana Bert. (Family – Asteraceae), is a shrubby, perennial plant species, popular worldwide for its ability to accumulate considerably high level of several commercially important steviol
300 glycosides (SGs) up to ~ 20% of total dry weight (Singh et al., 2017). SGs are non-caloric, non- cariogenic sweeteners with functional and sensory properties superior to many other high potency sweeteners (Chen et al., 2014). Stevia is also known as ‘sweet leaf (in USA)’, ‘sweet honey leaf (in Australia)’ , ‘sweet herb of Paraguay’ , estimated to be 300 times sweeter than sucrose (Dey et al., 2014). Guarani tribes of Paraguay and Brazil used Stevia species primarily S. rebaudiana as a sweetener and source of medicine (Dey et al., 2014). Despite being native to South America (Paraguay, Argentina and Brazil), Stevia cultivation has been expanded globally to China, Japan, Australia, Canada, USA and India. In India, it is mainly cultivated in Rajasthan, Kerala, Maharashtra, Orissa and Himachal Pradesh, and has been expanded to the other part of country. The SG produced by Stevia is a mixture of at least eight different types, including ST (Stevioside), RA - RF (Rebaudiosides A-F), Rubusoside and Dulcoside A (Yadav et al., 2011). Among these various SGs , Stevioside and Rebaudioside are the major metabolites (Allam et al ., 2001;Mantovaneli et al ., 2004; Debnath, 2008 ), pH stable , heat stable, non- fermentable, (Abedullateef and Osman , 2012 ) and possesses health promoting potential (Figure 1).
Figure1: Structures of major Steviol Glycosides (Carakostas et al., 2008)
301 Along with sweetness, Stevia has some bitter aftertaste due to the presence of some essential oils, tannins and flavonoids (Phillips, 1987). Plant organs contain different amounts of the sweet glycosides , which decline in the following order : leaves, flowers, stem, seeds and roots. Roots are the only organs that do not contain steviosides (Srivastava et al., 2014). Leaves of S. rebaudiana has many medical applications like antimicrobial (Satish Kumar et al., 2008 ), anti-viral (Kedik et al., 2009) , antifungal ( Silva et al., 2008 ) , anti-hypersensitive (Chan et al ., 1998 ; Lee et al., 2001 ; Hsieh et al ., 2003) , anti-hyperglycemic (Jeppesen et al ., 2002 ; Benford et al., 2006) , anti-tumor (Satish kumar et al., 2008 ) antiinflammatory, anti-diarrhoeal, diuretic , anti- human rotavirus activities (Das et al., 1992 ;Takahai et al., 2001 ), Anti-HIV(Takahsi et al., 1998), Hepatoprotective (Mohan and Robert, 2009 ) and immunomodulatory effects (Jaroslav et al, 2006; Chatsudthipong and Muanprasat, 2009 ). Stress induced production of phytometabolites: Plant secondary metabolites are often referred to as compounds that have no fundamental role in the maintenance of life processes in the plants, but they are important for the plant to interact with its environment for adaptation and defence. A wide range of environmental stresses (high and low temperature, drought, alkalinity, salinity, and UV stress and pathogen infection) are potentially harmful to the plants. Elicitation has been widely used to increase the production or to induce de novo synthesis of secondary metabolites under in vitro plant cell cultures. Over a period of time, number of researchers has applied various elicitors for enhancement of secondary metabolite production in cultures of plant cells, tissues and organs. Environmental stresses, such as pathogen attack, UV-irradiation, high light, wounding, nutrient deficiencies, temperature and herbicide treatment often increase the accumulation of phenyl-propanoids. Nutrient stress also has a marked effect on phenolic levels in plant tissues. The concentrations of various secondary plant products are strongly dependent on the growing conditions and have impact on the metabolic pathways responsible for the accumulation of the related natural products. Of late, findings reveal that judicious imposition of both abiotic and biotic stresses provides promising results in production of biomass and elevated synthesis of steviol glycosides. Role of abiotic stress in Stevia biomass and their productivity: a) Influence of water stress Water deficit (commonly known as drought) can be defined as the
302 absence of adequate moisture necessary for normal plant growth and to complete the life cycle (Zhu, 2002). Under severe water stress conditions caused by high drought, plant growth is affected and the plant cells accumulate solutes such as sugars and amino acids for osmotic adjustment. Accumulation of sugars in different parts of the plant is enhanced in response to the variety of environmental stresses (Macleod et al., 1958; Wang et al., 2000) Severe water deficit diminishes leaf chlorophyll content, reduces leaf number, leaf area index and biomass production. On the other hand, moderate water stress increased various morphological parameters and biochemical contents at an early phase of plant growth as has been evidenced by increase in leaf numbers and dry biomass (Figure: 2). So, moderate water stress at an early stage can be used as a marker to overproduce the useful phytochemicals, precisely steviol glycosides, for the better commercial exploitation of Stevia plant.
Figure2: Effect of water stress on leaf number and dry biomass of Stevia rebaudiana: (Life Sciences leaflets, Vol. 49, 2014): 35-43
b) Effect of salinity on Stevia biomass Stevia rebaudiana is reported to be moderately tolerant to salt stress. Expression of UGT76G1 was more than the amount of that for UGT74G1 (Figure 3). It has been revealed that, Stevia can withstand moderate salt stress (80mM NaCl) with slightly higher expression of UGT76G1. This finding offers a guide to cultivation of Stevia in salt stressed soil conditions. Moreover, seed germination decreased significantly with the increasing salinity (Fallah et al., 2017). Role of biotic stress on steviol glycoside metabolism: Stevia is known to be free from attacks by insects, which may be due to its inherent sweetness acting as a repellent. Therefore, insecticides are generally not required at an essential basis as in other crops, which helps in producing organic Steviosides. The fungal diseases Septoria leaf
303
Figure 3: Comparative expression of UGT76G1 and UGT74G1 genes under NaCl stress (Fallah et al., 2017) spot (Septoria steviae), Alterneria leaf spot (Alternaria alternata), stem rot (Sclerotium dephinii Welch.), root rot (Sclerotium rolfsii), powdery mildew (Erysiphe cichoracearum DC), damping-off (Rhizoctonia solani Kuehn.) and Sclerotinia sclerotoirum have been reported (Ishiba et al, 1982; Lovering and Reeleeder 1996; Chang et al, 1997; Thomas 2000; Megeji et al. 2005; Kamalakannan et al, 2007). There is a need to develop and identify resistant sources to develop varieties resistant to or tolerant of these diseases. My lab has already reported the induction of disease resistance mechanism between Stevia rebaudiana and Alternaria alternata. These nature of defense responses get heightened in growth retardant (Chloro Choline Chloride) primed plants in comparison to non-growth retardant primed Stevia plants (Kundu et al., 2014). Apart from the fungal pathogens, there are some endophytic bacteria which inhabit Stevia leaf and they either negatively or positively regulate the accumulation of SGs. Sphingomonas and Methylobacterium constituted an important part of the core endophytic community and were positively correlated with the stevioside content and UGT74G1 gene expression, respectively; while Erwinia, Agrobacterium, and Bacillus were negatively correlated with the stevioside accumulation. (Yu et al, 2015). An appropriate combination of mycorrhizal fungi and PGPR can significantly increase the production of leaf dry mass and concentration of stevioside. The application of dual inoculation with AM fungi and N-fixing bacteria to improve plant growth, plant nutrient uptake, total chlorophyll, and stevioside content seems to be the most effective treatment combination for optimum productivity (Vafadar et al., 2014).
304 Understanding the role of transcriptional activation and crosstalk among transcription factors for enhanced steviol glycoside production In nature, plants are continuously subjected to various stresses from the time of seed germination through growth and maturity (Erpen et al., 2018). Stresses tolerated by plants can be abiotic stresses that are caused by environmental effects or biotic stresses that are caused by plant pathogens (Agarwal et al, 2006a; Atkinson and Urwin 2012; Lobell et al, 2011; Niinemets 2010). Recently, many transcription factor have been identified (DREB/ERF, MYB, NAC, WRKY) that functions to control expression of potential stress related genes. These transcription factors increase the tolerance of plant to abiotic and biotic stresses in interactive patterns (Figure 4) Current findings report that combined of abiotic and biotic stress can have a positive effect on plant performance by reducing the susceptibility to biotic stress. Such an interaction between both types of stresses indicates a crosstalk between their respective signalling pathways. These cross talks may have synergestic and / or antagonistc effects on overall metabolism of steviol glycosides (Rejeb et al., 2014). Deciphering the spatio-temporal patterns and sequence of molecular signalling during such cross interactions among host of transcription factors would surely hold immense potential to understand and manipulate steviol glycoside pathway for meaningful production of these medicinally significant metabolites
Figure 4: Crosstalk among transcription factors for increased stress tolerance and secondary metabolite productions (Erpen et al., 2018.)
305 Future prospects In view of the current research trend, it may be emphasised that profiling global transcriptome of Stevia rebaudiana especially under various abiotic and biotic conditions would be rewarding with a view to find novel transcription factors. Such unique transcription factor(s) if found and located at post-steviol production stage, would be of supreme importance. Modulating such transcription factor(s) would provide leverage for metabolic manipulation of steviol glycosides for bettering commercial prospects. Reference: Agarwal, P.K., Agarwal, P., Reddy, M. and Sopory, S.K. (2006a). Role of DREB transcription factors in abiotic and biotic stress tolerance in plants. Plant Cell Rep 25:1263–1274 Akula, R., Ravishankar, G.A. (2011). Influence of abiotic stress signals on secondary metabolites in plants. Plant signalling & behaviour 6:11, 1720-1731 Atkinson, N.J. and Urwin, P.E. (2012). The interaction of plant biotic and abiotic stresses: from genes to the field. J Exp Bot 63:3523–3543 Carakostas, M.C., Curry, L.L., Boileau, A.C. and Brusick, D.J. (2008). Overview: the history, technical function and safety of rebaudioside A , a naturally occurring stiviol glycoside , for use in food and beverages. Food Chem Toxicol. Chang, K. F., Howard, R.J. and Gaudiel, R.G. (1997). First report on Stevia as a host for Sclerotinia sclerotiorum. Plant Dis. 81: 311. Chen, J., Hou, K., Qin, P., Liu, H., Yi, B., Yang, W. and Wu, W. (2014). RNA-Seq for gene identification and transcript profiling of three Stevia rebaudiana genotypes. BMC Genomics 15:571 Dey, A., Kundu, S., Bandyopadhyay, A. and Bhattacharjee, A. (2013). Efficient micropropagation and chlorocholine chloride induced stevioside production of Stevia rebaudiana Bertoni. Comptes Rendus Biologies 336: 17-28 Erpen, L., Devi, H.S., Grosser, J.W. and Dutt, M. (2018). Potential use of the DREB/ERF, MYB, NAC and WRKY transcription factors to improve abiotic and biotic stress in transgenic plants. Plant Cell Tissue and Organ Culture 132: 1-25 Fallah, F., Nokhasi, F., Ghaheri, M., Kahrizi, D., Beheshti Ale Agha, A., Ghorbani, T., Kazemi, E. and Ansarypour, Z. (2017). Effect of salinity on gene expression, morphological and biochemical characteristics of Stevia rebaudiana Bertoni under in vitro conditios. Cellular and Molecular Biology 63(7): 102-106 Gupta, E., Purwar, S., Sundaram, S. and Rai, G.K. (2013). Nutritional and therapeutic values of Stevia rebaudiana; A review. Journal of Medicinal Plants Research Vol. 7(46): 3343-3353 Ishiba, C.,Yokoyama, T. and Tani, T. (1982). Black spot disease of Steviae caused by Alternaria steviae, a new species. Ann. Phytopathol. Soc. Jpn. 48: 44-49
306 Kamalakannan, A., Valluvaparidasan, V., Chitra, K., Rajeswari, E., Salah Eddin, K., Ladhalakshmi, D. and Chandrasekaran, A. (2007). First report of root rot of stevia caused by Sclerotium rolfsii in India. Plant Pathol. 56: 350-355 Kundu, S., Dey, A. and Bandyopadhyay, A. ( 2014). Chlorocholine chloride mediated resistance mechanism and protection against leaf spot disease of Stevia rebaudiana Bertoni. European Journal of Plant Pathology. 139:511-524 Lobell, D.B., Schlenker, W. and Costa-Roberts, J. (2011). Climate trends and global crop production since 1980. Science 333:616–621 Lovering, N.M. and Reeleeder, R.D. (1996). First report of Septoria steviae on Stevia (Stevia rebaudiana) in North America. Plant Disease 80: 959-962 Macleod, A.M., and Orquodale, M.C, (1958). Water soluble carbohydrates of seeds of the gramineae. New Phytologist, 57, 168–182 Megeji, N.W., Kumar, J.K., Virendra, S., Kaul, V.K. and Ahuja, P.S. (2005). Introducing Stevia rebaudiana, a natural zero-calorie sweetener. Current Sci. 88: 801-804. Niinemets, Ü. (2010). Responses of forest trees to single and multiple environmental stresses from seedlings to mature plants: past stress history, stress interactions, tolerance and acclimation. Forest Ecol. Manag. 260:1623–1639 Ponnamperuma, F.N. (1984). Effect of flooding on soils. Report- International Rice Research Institute. Los Banos, Laguna, Philippines: Page 345 Pordel, R., Esfahani, M., Kafi, M., Nezami, A. (2015). Response of Stevia rebaudiana Bertoni root system to waterlogging and terminal drought stress. Journal of Biodiversity and Environmental sciences Vol. 6, No. 3, 238-247 Rejeb, I.B., Pastor, V. and Mauch-Mani, B. (2014). Plant responses to simultaneous biotic and abiotic stress: Molecular mechanisms. Plants 3,458-475 Singh, G., Singh, G., Singh, P., Parmar, R., Paul, N., Vashist, R., Swarnkar, M.K., Kumar, A., Singh, S., Singh, A.K., Kumar, S. and Sharma, R.K. (2017). Molecular dissection of transcriptional reprogramming of steviol glycosides synthesis in leaf tissue during developmental phase transitions in Stevia rebaudiana Bertoni. Scientific Reports 7: 118-35 Srivastava, S. and Srivastava, M. (2014). Influence of water stress on morpho-physiological and biochemical aspects of medicinal plant Stevia rebaudiana. Life Science Leaflets 49: 35-43 Thomas, S.C. (2000). Medicinal plants: Culture, utilization and Phytopharmacology. Technomic Publishing Co. Inc., Basel, Switzerland. p. 517 Vafadar, F., Amooaghaie, R. and Otroshy, M. (2014). Effects of plant-growth-promoting rhizobacteria and arbuscular mycorrhizal fungus on plant growth, stevioside, NPK, and chlorophyll content of Stevia rebaudiana. Journal of Plant Interactions 9(1): 128-136 Yadav, A.K., Singh, S., Dhyani, D. and Ahuja, P.S. (2011). A review on the improvement of Stevia [Stevia rebaudiana (Bertoni)]. Canadian Journal of Plant Science 91: 1-27
307 Yu, X., Yang, J., Wang, E., Li, B. and Yuan, H. (2015). Effects of growth stage and fulvic acid on the diversity and dynamics of endophytic bacterial community in Stevia rebaudiana Bertoni leaves . Frontiers in Microbiology. 6:867. Zhu, J.K. (2002). Salt and drought stress signal transduction in plants, Annu. Rev. Plant Biol. 53:247–273.
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18 INTEGRATED PLANT DISEASE MANAGEMENT (IDM) – CONCEPT, ADVANTAGES AND IMPORTANCE Raj KamalSahu, P.K. Sah1 and Abha Anand2 University Department of Chemistry, TM Bhagalpur University, Bhagalpur 1
2
Univ. Dept. of Home Sc. – Food & Nutrition, TM Bhagalpur University, Bhagalpur
Department of Economics, Govt. PG College, Rajgarh-Biaora, M.P.
INTRODUCTION Plants are constantly under attack by a variety of pathogens including bacteria, viruses, fungi, insects and nematodes. The percentage of crop losses caused by plant pathogens, insect, pests and weeds worldwide has been estimated to 42 per cent. Recent survey has shown that the world population has almost doubled in past 50 years, while the food production has increased meagrely by 25 per cent per head. To meet the food requirements for 8 billion population by 2020, the production has to be raised by 30 per cent. One possibility to achieve this demand is by striving to prevent damage caused due to pest and diseases. Presently most of the control measures rely on the use of conventional methods including use of chemical pesticides. Biological control measures through deployment of antagonists have been used with limited success to control soil borne diseases. Several exciting approaches and candidates are now available for developing designer plants capable of providing effective resistance against diseases. The present article provides concept, advantages and importance
310 of integrated plant disease management. Integrated plant disease management can be defined as a decision-based process involving coordinated use of multiple tactics for optimizing the control of pathogen in an ecologically and economically. The implications are: i.
Simultaneous management of multiple pathogens
ii. Regular monitoring of pathogen effects, and their natural enemies and antagonists as well iii. Use of economic or treatment thresholds when applying chemicals iv. Integrated use of multiple, suppressive tactics. Principles of Plant Disease Control i.
Avoidance—prevents disease by selecting a time of the year or a site where there is no
ii. inoculum or where the environment is not favorable for infection. iii. Exclusion—prevents the introduction of inoculum. iv. Eradication—eliminates, destroy, or inactivate the inoculum. v. Protection—prevents infection by means of a toxicant or some other barrier to infection. vi. Resistance—utilizes cultivars that are resistant to or tolerant of infection. vii. Therapy—cure plants that are already infected Factors affecting Occurrences Factors which affect plant diseases are micro-organisms, including fungi, bacteria, viruses, mycoplasmas, etc. or may be incited by physiological causes including high or low temperatures, lack or excess of soil moisture and aeration, deficiency or excess of plant nutrients, soil acidity or alkalinity, etc. Factors that limit the rate of disease development are the relatively low amounts of inoculum in the lag stage and the paucity of healthy plants available to the inoculum in the stationary stage. The causative agents of disease in green plants number in a tens of thousands and include almost every form of life. But primary agents of disease may also be inanimate. Thus nonliving (abiotic) agents of disease include mineral deficiencies and excesses, air pollutants, biologically produced toxicants, improperly used pesticidal chemicals, and such other
311 environmental factors as wind, water, temperature, and sunlight. Nonliving things certainly qualify as primary agents of disease; they continuously irritate plant cells and tissues; they are harmful to the physiological processes of the plant; and they evoke pathological responses that manifest as the symptoms characteristic of the several diseases. But the abiotic agents of disease in plants. The abiotic agents of plant disease are termed noninfectious, and the diseases they cause are termed non-infectious diseases. Micro-organisms The micro-organisms obtain their food either by breaking down dead plant and animal remains (saprophytes) or by attacking living plants and animals (parasites). In order to obtain nutrients, the parasitic organisms excrete enzymes or toxins and kill the cells of the tissues of the host plant, as a result of which either the whole plant or a part of it is damaged or killed, or considerable disturbance takes place in its normal metabolic processes. Parasites One of the factors causing plant diseases is parasites, those living organisms that can colonize the tissues of their host-plant victims and can be transmitted from plant to plant. These biotic agents are, therefore, infectious, and the diseases they cause are termed infectious diseases. The infectious agents of plant diseases are treated in the standard textbooks on plant pathology. Ability to produce an Inoculum The parasitic pest must produce an inoculum, some structure that is adapted for transmission to a healthy plant and this can either parasitize the host directly or develop anotherstructure that can establish a parasitic relationship with the host. For example, inocula for viruses are the viral particles (virions); for bacteria, the bacterial cells; for fungi, various kinds of spores or the hyphal threads of mold; for nematodes, eggs or secondstage larvae. Agents/ Media for transportation of Inoculum The inoculum must be transported from its source to a part of a host plant that can be infected. This dispersal of inoculum to susceptible tissue is termed inoculation. Agents of inoculation may be insects (for most viruses and mycoplasma like organisms and for some bacteria and fungi), wind (for many fungi), and splashing rain (for many fungi).
312 Wounds and Natural openings The parasite must enter the host plant, which it can do (depending on the organism) in one or more of three ways; through wounds, through natural openings, or by growing directly through the unbroken protecting surface of the host. Viruses are literally injected into the plant as the homopterous insect carrier probes and feeds within its host. Bacteria depend on wounds or natural openings (for example, stomates, hydathodes, and lenticels) for entrance, but many fungi can penetrate plant parts by growing directly through plant surfaces, exerting enormous mechanical pressure and possibly softening host surfaces by enzymatic action. Availability of Food For occurrence of disease one of the factor affecting is, availability of nourishment to grow within its host. This act of colonizations is termed infection. Certainly the parasite damages the cytomplasmic memberanes of the host cells, making those membranes freely permeable to solutes that would nourish the parasite And parasitism certainly results from enzymatic attacks by the parasite upon carbohydrates, proteins, and lipids inside the host cell. The breakdown products of such complex molecules would diffuse across the damaged host cell membranes and be absorbed by the parasite in the form of sugars, amino acids, and the like. Air-borne parasites of foliage, flower, and fruit. Preventive and Control Measures A. Preventive Measures Cultural Practices Cultural practices usually influence the development of disease in plants by affecting the environment. Such practices are intended to make the atmospheric, edaphic, or biological Surroundings favorable to the crop plant, unfavorable to its parasites. Cultural practices that leads to disease control have little effect on the climate of a region but can exert significant influence on the microclimate of the crop plants in a field. Three stages of parasite’s life cycle namely, Survival between crops, production of inoculum for the primary cycle and inoculation can be control by following preventive measures. Survival between Crops Organisms that survive in the soil can often be controlled by crop rotations with unsusceptible species. Depending on the system, either of
313 two effects results. Catch crops have been used to control certain nematodes and other soil-borne pathogens. Soil-borne plant pathogens can be controlled by biological methods. Plant parasites may be controlled by antagonistic organisms that can be encouraged to grow luxuriantly by such cultural practices as green manuring and the use of appropriate soil additives. The soil-invading parasite thus becomes an amensal in association with its antagonist. Soil-borne plant parasites may also be killed during their over-seasoning stages by such cultural practices as deep ploughing (as for the pathogen of southern leaf blight of corn), flooding (as for the cottony-rot pathogen and some nematodes), and frequent cultivation and fallow (as for the control of weeds that harbor plant viruses). Plant diseases caused by organisms that survive as parasites within perennial hosts or within the seed of annual plants may be controlled therapeutically. Therapeutic treatments of heat and surgery are applicable here; those involving the use of chemicals will be mentioned later. Removal of cankered limbs (surgery) helps control fire blight of pears, and the hot-water treatment of cabbage seed controls the bacterial disease known as black rot. Heat therapy is also used to rid perennial hosts of plant-parasitic nematodes. Production of Inoculum for the Primary Cycle Environmental factors (particularly temperature, water, and organic and inorganic nutrients) significantly affect Inoculum production. Warm temperature usually breaks dormancy of over seasoning structures; rain may leach growth inhibitors from the soil and permit germination of resting spores; and special nutrients may stimulate the growth of over seasoning structures that produce inoculum. Dispersal of Inoculum and Inoculation Cultural practices that exemplify avoidance are sometimes used to prevent effective dissemination. A second hierarchy of regulatory disease control is plant quarantine, the legally enforced stoppage of plant pathogens at points of entry into political subdivisions. The Plant Quarantine Act of the United States governs importation of plant materials into the country and requires the state govt. to enforce particular measures. Also, states make regulations concerning the movement of plant materials into them or within them. E.g., Florida imposes quarantine against the citrus-canker bacterium, which was eliminated from the state earlier by means of cooperative efforts led by the Florida Department of Agriculture. Sample Inspection One of the preventive measures to control the diseases is the use
314 of sample inspection method. Laboratory evaluation of the representative sample drawn by the certification agency for the determination of germination, moisture content, weed seed content, admixture, purity, seed borne pathogens. B. Control Measures Chemical Control The pesticidal chemicals that control plant diseases may be used in very different ways, depending on the parasite to be controlled and on the circumstances it requires for parasitic activities. E.g., a water-soluble eradicative spray is applied once to dormant peach trees to rid them of the overwintering spores of the fungus of peach-leaf curl, whereas relatively insoluble protective fungicides are applied repeatedly to the green leaves of potato plants to safeguard them from penetration by the fungus of late blight. Also, systemic fungicidal chemicals may be used therapeutically. The oxathi in derivatives that kill the smut fungi that infect embryos are therapeutic, as is benomyl (which has systemic action against powdery mildews and other leaf infecting fungi).Volatile fungicides are often useful as soil-fumigating chemicals that have eradicative action. The chemical control of plant diseases is classified in three categories: seed treatments, soil treatments, and protective sprays and dusts. Seed Treatments Chemical treatments of seed may be effective in controlling plant pathogens in, on, and around planted seed. Seed treatment is therapeutic when it kills bacteria or fungi that infect embryos, cotyledons, or endosperms under the seed coat, eradicative when it kills spores of fungi that contaminate seed surfaces, and protective when it prevents penetration of soil-borne fungi in to seedling stems. Certified seed is usually given treatment necessary for the control of certain diseases. Seed treatment is of two types; viz., physical and chemical. Physical treatments include hotwater treatment, solar-heat treatment (loose smut of wheat), and the like. Chemical treatments include use of fungicides and bactericides. These fungicides are applied to seed byd ifferent methods. In one method, the seed in small lots is treated in simple seed-treaters. The seed-dip method involves preparing fungicide suspension in water, often at field rates, and then dipping the seed in it for a specified time.
315 Some chemicals commonly used to control plant diseases Chemical and use
Relative toxicity Oral
Dermal
Chloraneb
Low
Low
Dichlone
Low
High
Thiram
Moderate
High
Carboxin (systemic and therapeutic)
Low
Low
Methyl bromideb (general pesticide)
Very high
Very high
PCNB (fungicide)
Low
Moderate
SMDC [vapam] (fungicide, nematicide)
Moderate
Moderate
MIT [“Vorlex”] (fungicide, nematicide)
Moderate
Moderate
D-D mixture (nematicide)
Moderate
Low
Copper compounds (fungicides, bactericides)
Moderate
Low
Sulfur (fungicide)
Low
Moderate
Maneb (fungicide)
Very low
Low
Zineb (fungicide)
Very low
Low
Captan (fungicide)
Very low
Very low
Dinocap (fungicide for powdery mildews)
Low
Low
Streptomycin (bactericidal antibiotic)
Very low
Low
Cyclohexamideb (fungicidal antibiotic)
Very high
Very high
Benomyl (protective and therapeutic fungicide)
Very low
Very low
Seed treatments (all fungicides)
Soil treatments
Plant-protective treatments
The oxathiins (carboxin, DMOC) used to kill embryo infecting smuts of cereal grains have little effect on other organisms, most eradicative and protective chemicals have a wide range of fungicidal activity; they are effective against most seed-infesting and seedling-blight fungi. But specific seed-treatment chemicals often work best to control a given disease of a single crop-plant species. Moreover, the toxicity of chemicals to seeds varies, and farmers should use only the compounds recommended by the Cooperative Extension Service of their country and state.
316 Copper and mercury-containing compounds were first used as seedtreating chemicals. But copper is toxic to most seeds and seedlings, and mercury has been banned from use in seed treatments because of the danger it poses to humans and animals. Organic compounds now widely used as protective and eradicative seed treatments include thiram, chloraneb, dichlone, dexon, and captan. Soil Treatments Soil-borne plant pathogens greatly increase their populations as soils are cropped continuously, and finally reach such levels that contaminated soils are unfit for crop production. Chemical treatments of soil that eradicate the plant pathogens therein offer the opportunity of rapid reclamation of infested soils for agricultural uses. Pre-planting chemical treatment of fields oils for the control of nematode-induced diseases, and fumigation of seedbed and green house soils (with methyl bromide, for example) is commonly practiced to eradicate weeds, insects, and plant pathogens. Field applications of soil-treatment chemicals for fungus control are usually restricted to treatments of furrows. Formaldehyde or captan applied is effective against sclerotia producing fungi that cause seedling blights, stem rots, and root rots of many field crops. Others oil-treatment fungicides are vapam and “Vorlex.” Soil treatments made at the time of planting are most effective against parasitic attacks that come early in the growing season. Protective Sprays and Dust Protective fungicides prevent germination, growth, and penetration. In order to use protective fungicides effectively, the farmer must not only select the right fungicide for the job, but also apply it in the right amount, at the right times, and in the right way. Too little fungicide fails to control disease; too much may be toxic to the plants to be protected. The farmer and applicator, therefore, must always follow use instructions to the letter. Timing of applications is also critical. Advantages Integrated approach integrates preventive and corrective measures to keep pathogen from causing significant problems, with minimum risk or hazard to human and desirable components of their environment. Some of the benefits of an integrated approach are as follows: i.
Promotes sound structures and healthy plants
317 ii.
Promotes the sustainable bio based disease management alternatives.
iii.
Reduces the environmental risk associated with management by encouraging the adoption of more ecologically benign control tactics
iv.
Reduces the potential for air and ground water contamination
v.
Protects the non-target species through reduced impact of plant disease management activities.
vi.
Reduces the need for pesticides and fungicides by using several management methods
vii.
Reduces or eliminates issues related to pesticide residue
viii. Reduces or eliminates re-entry interval restrictions ix.
Decreases workers, tenants and public exposure to chemicals
x.
Alleviates concern of the public about pest & pesticide related practices.
xi.
Maintains or increases the cost-effectiveness of disease management programs
318
319
19 UTILIZING THE EFFECTIVENESS OF MICROBIAL CONSORTIA AS BIOPESTICIDE Riddhi Basu1, Atmeeya Sarkar1, Bedaprana Roy1, Poushali Mondal1, Amrita Maity1, 1, Sulagna Banerjee1 and Arup Kumar Mitra* Department of Microbiology, *
Associate Professor, Department of Microbiology,
St. Xavier’s College (Autonomous) 30, Mother Teresa Sarani, Kolkata 700016, West Bengal, India *
Corresponding Author. Email –
[email protected]
ABSTRACT Massive use of synthetic formulations is harmful for targeted as well as non-targeted pests and also for humans, which has become a threat for the ecological system. So, it is high time that we focus on selection of the appropriate formulations which are biodegradable with better shelf life, have improved product stability, viability and also cheaper than other synthetic ones and do not alter the fertility of the soil. They are ideally lethal, prevent pests and also influence the plant health and growth promotion. Here in this project, unique bio pesticide: a bactericide and a fungicide (in liquid and solid form), were designed using Bacillus megaterium, Pseud omonas fluorescens and Trichoderma sp., Beauveria bassiana along with ground nut oil (for liquid) and oil cake (for solid) respectively. In order to find out their morphological and biochemical characteristics, isolation and characterization were also performed. Seeds of ladies’ finger (Abelmoschusesculentus) were used as experimental crop. Solid biopesticide was applied in rhizosphere and liquid biopesticide was applied in both rhizosphere and phyllosphere. Consequently to find out positive or negative symbiotic relationship, interaction study was
320 done and according to the results two products i.e. the fungal and the bacterial one were applied separately. Chloprophyll assay was also performed which showed much higher chlorophyll content in the plant applied with the fungal product than the same applied with bacterial one, though the overall result was much higher than the control. Similarly, different experimental setup was established for characterization of five different type of soil. And lastly some nutritional source and carrier components were added in order to check their shelf life which showed that the viable count after 15 days maintained the threshold CFU count. After application of two different biopesticides in two different plants, noticeable increase of root length, shoot length and internode length for both of the plants was observed. After period of 10 days, budding and flowering of the plant, treated with the fungal one, was also observed. Though there was appearance of black spots at the beginning but after 4-5 days, those spots disappeared after application of the fungal product. Thus, the product also helped in plant growth promotion along with prevention of pests Key words: interaction study, systemic resistance, chlorophyll assay, phylosphere, rhizosphere, growth promotion
INTRODUCTION This is high time we focus on the selection and usage of the appropriate formulations which are biodegradable with better shelf life, improved product stability and also cheaper than other synthetic ones and does not alter soil fertility. The term ‘pesticide’ actually covers a wide range of compounds that includes insecticides, herbicides, fungicides, nematicides, plant growth regulators and others. So, the term “biopesticide” actually emphasizes on the usage of several “biological pesticides” depending on parasitic, predatory or chemical relationships. So, in other words, it is the manipulation of living organisms. The significance of using these biopesticides are : They perfectly obey the critical rule that “the pesticide must be lethal to the targeted pest only”, Biodegradable, Cheaper than chemical ones, Better shelf life, Do not alter soil fertility, Maintain soil microflora. Recently, it wasreported that pest related damages result inan estimated Rs 50,000 crore loss annually in agricultural production in the field and storage India. According to the Union Ministry of Agriculture, half of the potential yield of rice may be lost due to pests. So, the Union Ministry of Agriculture is concerned with slow progress in the Integrated Pest Management and thereby the demand for biopesticides in Indian Market is growing day by day. Keeping this in focus, several projects were taken up, but in most of the cases, the attempt was unsuccessful due to several reasons, like – Poor shelf life, Improper identification of organisms,
321 Error in interaction study, Not so much effective for plant growth. Biopesticides can be classified in several ways. Such as-Biofungicide- using Trichoderma sp , Bio-insecticide- using Beauveria bassiana. According to the results of the interaction study, two different products one in solid form and another in liquid form were developed consisting Trichoderma sp, Beauveria bassiana and Bacillus megaterium Pseudomonas fluorescens respectively . 2) OBJECTIVES: This project was taken to develop a unique biopesticide which has a better shelf life, proper identification and interaction study was done to get error free perfect results. Development of biopesticide shows diverse mechanisms involving the suppression of plant pathogens which, in turn , is often indirectly related to enhanced plant growth. Thereby this project was undertaken to develop a biopesticide formulation involving bacterial and fungal genera which not only prevent pests but also influence plant health and plant growth promotion. 3)MATERIALS AND METHODS: 3.1 ISOLATION OF MICROBES Bacteria used for this formulation were Bacillus megaterium and Pseudomonas fluorescens and fungi used were Trichoderma viridae and Beauveria bassiana.1mg of microbial dust was dissolved in 10ml of distilled water. Various dilutions were prepared and they were propagated in suitable medium by pour plate method. The bacterial plates were incubated at370C for 24 to 48 hours. Fungal plates were incubated at 280C for 4 to5 days. Bacillus megaterium was isolated directly from a previously procured slant and streaked on the plate. 3.2 PRESERVATION AND MICROSCOPIC CHARACTERIZATION OF MICROBES Nutrient agar and Kings B agar slants were prepared in test tubes and the cultures procured viz. Bacillus megateriuma nd Pseudomonas fluroscens were streaked onto the respective slants and incubated for 24 to 48 hours. Gram staining was done for the characterization of the microbes. For the procurement of fungal culture loopful of culture was taken from PDA plates and was streaked (block streaking method) into PDA slants. Slants were incubated at 28 0 C for 3-4 days. Microscopic
322 characterization for both the organismsviz. Trichoderma viridae and Beauveria bassiana was done by staining both in lactophenol cotton blue and observing them under 45X. 3.3 CHEMICAL CHARACTERIZATION Chemical characterization of microbes was done by performing Methyl-Red, Voges-Proskauer and Citrate Test. In Methyl-red and VogesProskauer test glucose peptone broth was prepared and then inoculated with the bacterial cultures and incubated for 4-8hrs and 48 hours respectively at 370C. For citrate test Simmons citrate agar slants were prepared and bacterial culture was streaked and incubated for 48hours at 370C. 3.4 HAEMOCYTOMETER COUNT AND MORPHOLOGICAL CHARACTERISATION CFU count of the bacterial cultures was done from the dilutions 10-1 and 10-2.Morphological characterisation was done by observing the colony characteristics. Haemocytometer count for the fungal spores was also performed and their morphological characterization was done based on the presence or absence of mycellial mat. 3.5 Interaction study between isolated organisms:
For checking bacteria-bacteria interactions, the isolated bacterial organisms were streaked in both nutrient agar(NA) and potato dextrose agar (PDA).
For studying bacteria-fungi interactions, the bacterial organisms were streaked using the same technique and the fungal organism was placed in between with the help of cork borer in both NA and PDA plates.
Fungi-fungi interactions were studied by inoculating the isolated fungal organisms in both NA and PDA plates using corkborer (solid to solid transfer technique) as above.
All the inoculated plates were incubated for 48 hours at 27°C.
3.6 Soil characterisation Combinations used were: Control soil: Without any biopesticide and vermicomposts, Sesbania, KSB.
323 Activated soil : consisting of vermicompost , Sesbania and KSB. Fungal biopesticide treated soil and bacterial biopesticide treated soil combinations were also used. Parameters checked for characterisation are: A. pH :
2gm of soil was dissolved properly in 5 ml of distilled water with the help of vortex machine to prepare a homogenized solution.
pH of the above solution was checked using litmus paper.
B. Water holding capacity :
Weight of soil sample was measured in weighing machine and noted.
This soil sample is incubated at 90°C for 8 hours and after incubation the weight of this sample was again measured and noted carefully.
Water holding capacity is calculated by following formula= (Weight of soil - Weight of soil after incubation at 90R”C for 8 hours)
C. Microbial load :
1 gm of soil was dissolved properly in 10 ml of distilled water with the help of vortex machine.
From the above solution dilutions of 10-1and 10-2 were prepared.
1ml from each of the dilution was spread on Nutrient Agar plates and incubated for 24 hours to obtain colonies.
These colonies were counted to determine colony forming unit (CFU) and microbial load was calculated. CFU/ml= (number of colonies × dilution factor)/volume of sample
D. Electrical conductivity:
5 gm soil was dissolved properly in 50 ml of deionised water by the help of vortex machine.
Electrical conductivity of the above solution was checked and noted.
324 3.7) Preparation of liquid formulation:
Inoculums each from B. megaterium and P. fluorescens culture tube was taken and mixed with autoclaved ground nut oil and growth medium consisting of dextrose, peptone, MgSO4, distilled water and mixed well.
This preparation was kept in the shaker for 1 week at 37°C.
3.8) Preparation of solid formulation :
Trichoderma sp. & Beauveria bassiana were inoculated in potato dextrose broth and incubated at 37°C until fungal mat were formed.
The fungal mat was filtered by passing the fungal culture through filter paper lining the funnel and allowed to dry at room temperature.
The above dried mass was mixed well with charcoal and oil cake.
The prepared formulation was packaged in transparent plastic packets and kept in incubator before its direct field application.
3.9) Assessment of pesticidalactivity of bio-formulations Bacterial and fungal formulations were applied to Abelmoschus sp. (Ladies finger) at an interval of 7 days . The biopesticide applied plants were compared with control plants having no biopesticide treatment & judged on the basis of growth, disease progression, suspectibility to phyto-pathogens and yield. The lengths of leaf, shoot, root, node and internode were measured for control and biopesticide treated plants. 3.10)Chlorophyll assay Leaves collected from 4 sets of plants namely control, activated soil grown and bacterial and fungal biopesticide applied plants and were examined to determine its chlorophyll content. 0.5 gram leaves of each set was crushed & dissolved in acetone. The prepared extract was centrifuged at 10,000 rpm for 1 min and the supernatant was taken for spectrophotometric readings . A blank was prepared by adding equivalent amount of acetone. O.D. of the collected supernatant of the 4 sets were taken at 645 nm & 663 nm for chlorophyll b and chlorophyll a respectively.
325 3.11) Checking shelf life Shelf life of the prepared formulations were checked after 7 days by streaking the inoculums from the samples into nutrient agar and incubated for 24hrs at 37oC. 4) RESULTS 4.1) MICROSCOPIC CHARACTERISATION AND COLONY CHARACTERISTICS: Organism
Gram Shape Size in Character of Colony Microand meter Morphology
B.megaterium
Gram Circular positive, short rods in chains or clusters.
P.fluroscens
Gram Circular negative, Short rods in chains.
Colony Color
Margin
Elevation
3.5+0.116 Off-white Opaque
Entire
Raised
1.5+ 0.1471
Undulate Convex
Creamy white
Texture
Slimy
4.2) CFU COUNT OF BACTERIAL CULTURE Or Organism
Dilution
CFU Count
Bacillus megaterium
10 -110 -2
1000+10.571516+6.21
Pseudomonas fluorescens
10 -110 -2
850+6.78378+7.65
4.3 HAEMOCYTOMETRIC COUNT OF SPORESORGANISM
SPORE COUNT (per unit ml of inoculum)
B.bassiana
10 8+ 12.406
T.viridae
2 x 108+ 8.64
4.4)MORPHOLOGICAL CHARACTERIZATIONORGANISM
PRESENCE OF MYCELIAL MAT
BIOMASS (gm.) (weight of mat – weight of filter paper)
B.bassiana
Present (very small amount)
(3.28 - 0.58) = 2.7 + 0.12116
T.viridae
Present
(6.80 – 0.58) = 6.22 + 0.1
326 4.5) OBSERVATION OF INTERACTION STUDY
All bacteria-bacteria, fungi-fungi plates for both nutrient agar and potato dextrose agar showed no interaction, i.e. they do not influence each other’s growth in either of positive or negative way.
But for bacteria-fungi plates for both nutrient agar and potato dextrose agar showed positive interaction, i.e. they inhibit growth of each other.
All bacteria-bacteria, fungi-fungi plates for both nutrient agar and potato dextrose agar showed no interaction, i.e. they do not influence each other’s growth in either of positive or negative way.
But for bacteria-fungi plates for both nutrient agar and potato dextrose agar showed positive interaction, i.e. they inhibit growth of each other.
It is notable that Pseudomonas fluorescens has inhibited the fungal growth more strongly than Bacillus megaterium. COMBINATION
MEDIA
INTERACTION
Bacteria-bacteria
Nutrient agarPotato dextrose agar
NeutralNeutral
Fungi-fungi
Nutrient agarPotato dextrose agar
NeutralNeutral
Bacteria-fungi
Nutrient agarPotato dextrose agar
AntagonisticAntagonistic
4.6) Result of soil characterization: Soil combination
pH
Before incubation (in gm)
After Water incubation holding (in gm) capacity
Microbial Microbial Electrica l load 10-1 load 10-2 conductance (microsecond)
Control soil
7
0.9960+ 0.5497
0.897+ 0.68
0.099+ 0.08
120+12
84+5.5
1.46+ 0.21
Activated soil
7
0.76+0.41 0.6985+ 0.41
0.0705+ 0.11
190+13.5 90+7.8
1.53+ 0.26
Activated soil+ 7 solid formulation
0.689+ 0.38
0.654+ 0.52
0.035+ 0.015
230+14
150+ 6.2 1.76+ 0.41
Activated soil + 7 liquid formulation
0.638+ 0.38
0.588+ 0.41
0.05 + 0.02
84+7.5
126+7.5
1.62+.41
327 4.7)Result after application of formulation No. of days
Type of soil
Shoot length (cm)
Internode Leaf length Root length length (cm) (cm) (cm)
1
ControlP-IP-II
1215.216
2.84.54.8
4.55.46
5.26.87
4
ControlP-IP-II
12.515.818.7
3.14.55.2
4.66.06.6
5.57.25.9
6
ControlP-IP-II
13.71812.2
3.34.55.3
4.17.46.8
9.510.512.1
9
ControlP-IP-II
14.22023
3.75.15.7
4.78.48.9
11.212.37.2
10
ControlP-IP-II
15.323.824.2
4.25.66.7
5.39.16
11.812.814
Observation
After 6 hours of exposure to sunlight on application of the formulation some white patches were observed .
So, the dosage was reduced and interval was increased to 7 days and as a result the patches were reduced and normal growth was observed.
The rate of increase of root length suit length internode length leaf length and color of the leaf was much higher than the control one.
Although there was presence of white patches on the control ones but no further patches were observed in the case of the others.
Buds were observed on day 10 and flowering was also observed.
Although there was presence of white patches on the control ones but no further patches were observed in the case of the others.
Buds were observed on day 10 and flowering was also observed.
4.8) Result of chlorophyll assay:
Concentration of chlorophyll (µg/ml)
The Graph for Chlorophyll assay of the Various Set ups.
328 4.9) Results indicating shelflife: Plating inoculum from both formulations after 28 days showed CFU count 108 which means that formulations have a better shelf life.
Length in (cm)
The Graph For the variation of Different growth parameters with respect to each set up.
DISCUSSION: A study in European countries regarding advancement of biopesticides suggested that one of the main resaons for their failure was improper targeting of pests .i.e. the product non-specifically kills nonpathogenic pests.( PMC3130386) But this product has been observed to be lethal to pathogenic pests only. And a study conducted in Haryana, India, showed due to lack of proper shelf life the results were not so effective (Gatekeeper series no 93). This study showed that both the products have proper shelf life , both the products maintained the CFU count and remained viable for long period.Also a study published by Indian Agriculture Research Institute showed that the most important reason behind the failure of using biopesticide is improper interaction study between microorganisms. This study deals with proper interaction study between organisms and thereby two separate formulations were applied separately. B. megateriumis a phosphate solubilizing soil bacterium, phosphorus being a component of nucleic acid and ATP is involved in the regulation of various pathways in plants. Hence use of this bacterium might have promoted the uptake of nutrients and enhanced plant growth. Bacterium P. fluorescence not only play major role in plant growth promotion but also have induced systemic resistance. Fungi Trichoderma sp affects plant health, it produces a variety of compounds that influence resistance, hence important weapon for
329 managing pest diseases. Moreover root colonization by Trichodermasp might have enhanced root growth and plant development.B. bassiana acted as biological insecticide to control a number of pests such as white flies. Thus formulations have suppressed plant pathogens which in turn has also influenced the plant growth involving several mechanisms. REFERENCES Hoq, M.M., Arafat, A.M., Shishir, M.A Khan, M.M, Rahman, M.N. A. and Khan, S.N. Bioprocess development for eco-friendly microbial products and its impacts on bio-industry establishment in bangladesh Murali C.M., Narasimha, P.R., Devi, U.K., Kongara, R. and Sharma, H.C. Growth and insect assays of Beauveriabassiana with neem to test their compatibility and synergism. Krishnamoorthy, P.N. Saroja, S. and Shivaramu. K. Bio-efficacy of neem products and essential oils against thrips (Scirtothripsdorsalis Hood) in Capsicum Kumar, S. (2012). Biopesticides: a need for food and environmental safety. J Fertil Pestic 3:e107. Nicholson, G.M. (2007). Fighting the global pest problem: preface to the special toxicon issue on insecticidal toxins and their potential for insect pest control. Toxicon 49: 413-422. Al-Zaidi, A.A., Elhag, E.A., Al-Otaibi, S.H. and Baig, M.B. (2011). Negative effects ofpesticides on the environment and the farmers awareness in Saudi Arabia: a case study. J Anim Plant Sci 21: 605-611. Hubbard, M., Hynes, R.K., Erlandson, M. and Bailey, K.L. (2014). The biochemistry behind biopesticide efficacy. Sustainable Chemical Processes 2: 18. Bailey, K.L., Boyetchko, S.M. and Längle, T. (2010). Social and economic drivers shaping the future of biological control: a Canadian perspective on the factors affectingthe development and use of microbial biopesticides. Biol Control 52: 221-229. Kumar, S. and Singh, A. (2014). Biopesticides for integrated crop management: environmental and regulatory aspects. J FertilPestic 5: e121. Fitches, E., Edwards, M.G., Mee, C., Grishin, E., Gatehouse, A.M.R., et al. (2004). Fusion proteins containing insect-specc toxins as pest control agents: snowdrop lectin delivers fused insecticidal spider venom toxin to insect haemolymph following oral ingestion. J Insect Physiol 50: 61-71. Leahy, J., Mendelsohn, M., Kough, J., Jones, R. and Berckes, N. (2014) Biopesticide oversight and registration at the U.S. Environmental Protection Agency.
330
331
20 INTEGRATED DISEASE MANAGEMENT FOR SUCCESSFUL STRAWBERRY CULTIVATION IN SEMIARID SUBTROPICS OF GANGATIC PLAINS OF BIHAR Ruby Rani1*,Pawan Kumar1 and Chanda Kushwaha2 1 2
Department of Horticulture (Fruit& Fruit Technology)
Department of Plant Pathology, Bihar Agricultural University, Sabour *Email:
[email protected]
The cultivated strawberry (Fragariax ananassa Duch.) of family Rosaceae is a monoecious plant, octoploid in nature having basic chromosome number X=7.It is a hybrid plant between two American species, Fragaria chiloensis of western north of South America mainly Chilie and Fragaria virginiana of eastern part of North America. The name strawberry probably comes from ‘strew’ for the runners that produce young plants to stray from the parent. The Anglo-Saxon word “strew” was later converted to the English word “straw. According to Anon (2006b), the name Fragaria is most probably derived from the word “fragrans”, referring to the odorous flesh of the fruit (Anon, 2006b). Botanically strawberry described as an aggregate fruit, wherein many one-seeded achene are fused together to form a large fleshy receptacle. The low creeper, perennial spreading shrubs with rosette crown of strawberry bears small white flowers which eventually develop into small conical, light green, immature fruits. They turn red upon maturity featuring red pulp with tiny, yellow color seeds piercing from within through its surface. The proximal end of fruit carries a green leafy cap (calyx with peduncle) that is adorning as a crown.
332 Strawberry is a very attractive, luscious, tasty and nutritious soft fruit with a distinct and pleasant aroma, and delicate flavor and mainly consumed as fresh in several ways. . Strawberries is rich source of iron, potassium, manganese, magnesium besides higher level of fibre, vit C and vit A. Studies have shown its additional benefits to reduce risk of cancer, enhance memory function. The high antioxidant levels in strawberries can help the body to neutralize the destructive effects of free radicals. The regular consumption of strawberry is found effective to lower the risk of stroke due to high potassium content by regulating the electrolytes in the body.Strawberries are good for diabetics, as they help stabilize the level of blood glucose. It also makes excellent ice cream and jam on account of its rich aroma. Strawberries nutrition (Fragaria X ananassa), ORAC Value 3577, Values per 100 g Principle
Nutrient Value
Percentage of RDA
Energy
32 Kcal
1.5%
Carbohydrates
7.7 g
6%
Protein
0.67 g
0.1%
Total Fat
0.30 g
1%
Cholesterol
0 mg
0%
Dietary Fiber
2.0 g
5%
Folates
24 µg
6%
Niacin
0.386 mg
2.5%
Pantothenic acid
0.125 mg
2.5%
Pyridoxine
0.047 mg
3.5%
Riboflavin
0.022 mg
2%
Vitamin A
12 IU
0.5%
Vitamin C
58.8 mg
98%
Vitamin E
0.29 mg
2%
Vitamin K
2.2 µg
2%
Sodium
1 mg
0%
Potassium
153 mg
3%
Vitamins
Electrolytes
333
Minerals Calcium
16 mg
1.6%
Iron
0.41 mg
5%
Magnesium
13 mg
3%
Manganese
0.386 mg
17%
Zinc
0.14 mg
1%
Carotene-ß
7 µg
—
Lutein-zeaxanthin
26 µg
—
Phytonutrients
(Source: USDA National Nutrient data base)
1. Area and distribution: Strawberry (Fragaria sp.) is a native of temperate regions. Strawberry is commercially grown in throughout Europe, in every state of the United States, Canada, China and South America. The leading strawberry- producing countries are United States, followed by China and Spain In India, it is commercially cultivated in Himachal Pradesh, Uttarakhand, Uttar Pradesh, Maharashtra, West Bengal, Nilgiri Hills, Delhi, Punjab, Haryana and the some parts of Rajasthan. Nainital and Dehradun districts of Uttarakhand, Mahabaleshwar, Pune (Maharashtra), Jammu &Kashmir, Bangalore and Kalipong (West Bengal) are the main centers of strawberry cultivation in India. Its cultivation can be extended to other suitable areas having assured irrigation and transport facilities using plasticulture technology. 2. Climate and soil: Strawberry is a cool seasoned short day plant that requires exposure to about 10 days of less than 8 hours sunshine for initiation of flowering. The exposure to low temperature during this period helps in breaking dormancy of the plant. Although strawberries are grown in full sun, high summer temperatures have a negative effect on fruit size as well as fruit quality. Fruit size and quality is enhanced by cooler temperatures, more pollen is available for pollination, the fruit ripen gradually and better-shaped fruit is produced. The varieties grown in milder subtropical climate do not require chilling and continue to make some growth during winter. From the standpoint of response to length of the light period, strawberries are placed in two groups: (1) varieties that develop flower
334 buds during both long and short light periods, the overbearing varieties and (2) varieties that develop flower buds during the short light periods only. The strawberry can be grown on any type of soil—poor sand to heavy clay—provided proper moisture, organic matter and drainage is present. Strawberry requires a well-drained medium loam soil, rich in organic matter. The soil should be slightly acidic with pH from 5.0 to 6.5. At higher pH root formation is poor. The presence of excessive calcium in the soil causes yellowing of the leaves. In light soils with high organic matter, runner formation is better. Strawberry should not be cultivated in the same land for more than 4 years. It is preferable to plant it in green manured field. Alkaline and water logged infected with nematodes should be avoided.There should be no underlying lime layer up to 15-20 cm, otherwise it causes burning of leaves Owing to wide climatic and soil adaptation and high returns, it has tremendous potential in India. Attractive appearance, unique taste, availability during the period of fresh fruits scarcity in the market, short duration nature, high return per unit area and wider acceptability of its processed products has made the strawberry a choicest crop of growers of sub-tropical areas in India. Now it is being grown commercially around the various states of northern India like Punjab, Haryana, Uttar Pradesh and Bihar. Cultivated area of strawberry has increased significantly during the last few years in sub-tropical areas despite of its temperate nature. 3. Variety: There are number of varieties of strawberry that are grown in India and abroad. Important commercial strawberry varieties grown in different parts of India are Chandler, Grand, Gorella Belrubi, Fern, Albion, Tioga, Torrey, Selva, Belrubi, Fern and Pajaro. Other varieties include Premier, Red cost, Local Jeolikot, Dilpasand, Bangalore, Florida 90,Katrain Sweet, Safari, Senga Sengana, Sabrina, Missionary, Fortuna, Elyana, Douglas, Fortuna, Ofra, Crystal, Pusa Early Dwarf , Blakemore etc. Altogether 18 varieties have been tried to exploit its feasibility in agro climate of Bihar. Amongst the varieties tried, Festival, Sweet Charlie, Chandler, Camarosa, Winter Dawn and Nabila has been found promising for commercial cultivation in Bihar. Winter Dawn and Camarosa are early varieties, Sweet Charlie, Festival, Chandler are mid-season varieties.
335 4. Cultivation Practices: 4.1 Propagation Strawberry is propagated by crown, seeds and runners. Runners are most common propagating material.Now a daydue to more prevalence of different strains of virus tissue-cultured planting material is preferred. The plants are allowed to produce runners after end of fruiting season. A single plant usually produces 12 to 18 runners if proper care is taken. 4.2 PLANTING 4.2.1 Planting techniques The land should be thoroughly prepared by deep ploughing followed by light ploughing and harrowing before planting of strawberry. The application of 50 kg N, 25 kg P2O5 and 30 kg K2O/ acre has shown to produce very high yield (22t/ha). These doses of inorganic fertilizers can be replaced and 25-30 ton FYM should be incorporated in the soil before plating. Strawberry can be planted on flat beds, in the form of hill rows or matted rows, or it can be planted on raised beds. For commercial cultivation it is plantedon raised bed which ensures proper drainage, easy intercultural operations and facilitates installation of micro irrigation system. After land preparation, beds of 25-30 cm height and 90-105 cm width of convenient length should be made at a distance of 50 cm. Planting of runners should be done at 25 cm x 25-35cm spacing depending on growth of the variety with two to four rows of plants per bed. About 60 thousand fresh and healthy runners are required for planting one hectare area. Planting time is considered as one of the mostimportant factors for profitable cultivation of strawberry. In north-India it is usually planted after first week of October till mid-November. However, staggered planting from mid-September to mid-November at weekly/bi-weekly interval is quite remunerative for longer period of availability of fruits (January to April). 4.2.2 Planting Systems 1. Matted Row Systems: In this system, the strawberry is planted 40 to 75 cm apart in rows three to four feet apart. Daughter plants areallowed to root freely to become a matted row not wider than two feet. 2. Spaced-Row Systems: Limited number of daughter plants is allowed to grow from a mother plant. The mother plants are set 40 to 75 cm apart in rows three to four feet apart. The daughter plantsare spaced to root not
336 closer than 10cm apart. Excess runners are pulled or cut from themother plants. More care is needed under this system but higher yields, larger berries can be obtained with fewer disease problems. 3. Hill System This is the best system for growing day-neutral and ever bearing strawberries. In this system allthe runners are removed so only the original mother plant remains. Removing the runners causesthe mother plant to develop more crowns and flower stalks. Multiple rows are arranged in groupsof two, three or four plants with a two foot walkway between each group of rows. Plants are setabout one foot apart in multiple rows. During the first two or three weeks of growth, the plantingshould be weeded; then the bed should be mulched. 4.3 Micro-Irrigation Strawberry has shallow root system, and maximum roots are found within 15 cm of soil depth.Hence, frequent irrigation is one of the most important factors for its cultivation in semiarid subtropics like Bihar. After bed preparation, Micro irrigation system (drip system + micro sprinkler system) should be installed in the field for supplying precise water to the crop according to the stage of growth of the crop. During early vegetative growth, irrigation is given through micro sprinkler system and in reproductive phase (flowering and fruiting), it is replaced by drip system, which provides uniform and timely irrigation and facilitates fertigation. Drip system should be run twice or thrice a week as per the need of plants and fertigation should be given fortnightly for proper growth and development of the crop. 4.4 Mulching and row covers Strawberries are very susceptible to frosts. Mulches and row covers is used to protect the plants from frost , improves plant growth , causing early flowering and fruiting and improves fruit quality, harvesting span and yield in strawberry. Use of protected structures like row covers and black polyethylene mulch with micro irrigation can advance the harvesting season by 15-20 days and protects the crop from winter rains and frost injury. Whereas, straw mulch in combination with sprinkler/ drip irrigation can extend the fruiting season by 20-25 days (Pandey et al, 2012). Plastic mulch and row covers prevent weed growth, frost injury and crop spoiling. It has been found that covering with green colored shade net can save mother and runner plants in areas where temperature goes >
337 420C during summers. The use of 50% and 75% shading net has been found effective for runner formation in subtropical areas depending on early and late varieties.
4.5 Fertigation for Strawberry Cultivation: Initial dose of fertilizers and manures are applied at the time of land preparation. Fertigation scheduled followed in different parts of subtropics in India are as follows in.: 20-30 days after plantation NPK (19:19:19) 50 kg/ acre Up to next 40-60 days 19:19:19 – 1.5 kg & CaNO3 1.2 kg/ acre/ at three days interval Up to next 60-80 days 13:0:45 – 1.5 kg /acre and CaNO3 1.2 kg/ acre three days interval Technology pertaining to fertigation in strawberry has been released after three years of trial at Bihar Agricultural University, Sabour, Bhagalpur. A fertilizer dose of 25g N:P:K (19:19:19) /sqm should be applied after 30 days of planting in four equal splits at 30 days interval. However interval of fertilizer application may be reduced by increasing the number of splits. Strawberry cultivation requires high in initial inputs and gives quick and high return. Higher return and short duration of crop have recently attracted large number of progressive farmers towards its cultivation and profit oriented venture. But the profit of margin is reduced in sub-tropical areas due to lack of runner production as survival of mother plants in these areas caused by high temperature and high light intensity followed by heavy rain(Sharma and Yamdagni, 2000). Thus margin of profit under sub-tropical
338 areas is greatly reduced due to regular annual investment for the procurement of runner plants in every planting season. Besides, varietals suitability, huge annual investment for planting materials, frost injury, production of mid-season crop instead of early and late strawberry, integrated nutrient management, weed management, post-harvest management including handling, transportation, marketing and value addition and integrated pests and diseases management are some of the major problems being faced by the strawberry growers of sub-tropical areas. To address the above constraints, the specific technologies have been refined/ generated, and are being transferred at farmers’ fields for making the strawberry growers to be self-sustainable. 5.6 Managing disease in strawberry: Strawberries are a unique and high value small fruit crops and due to seasonal and perishable nature of crop, nearly all fruits are sold and consumed fresh in the markets. Infestation of insect pests and diseases are the major constraints in obtaining a profitable crop of strawberries. Strawberry is affected by various diseases infected by diverse pathogens such as fungi, bacteria, nematode, viruses and virus like organisms causing considerable loss in yield and quality of fruits. These pests and diseases come at every stages of crop production right from planting to harvesting. Leaf blight, fruit rot and root rot are of commercial importance especially in sub-tropical areas. Major Diseases of Strawberry: 5.6.1 Powdery Mildew This is the most important foliar disease of strawberry caused by fungus Sphaerotheca macularis (Wall ex. Fr.) Jazc., and reported from all strawberry grown areas in world. The fungal infection starts from underside of leaves which develop into white powdery growth. In severe infection leaves become burnt at margins to a greater extent. The white powdery mass also develops on infected flower stalk; flower and fruits. This disease inhibits plant growth, reduces the yield and fruit quality. Warm, wet weather conditions are favourable to the occurrence of fungal diseases and the longer these conditions prevail, the higher the risks of fungal infections. To manage this disease too dense planting should be avoided. Use of resistant cultivar could be one of the best option for managing this disease. Some cultivars like Milsei-Tudla showed high resistance while cultivars Camarosa, Aromas, Oso Grande, Sweet Charlie and Campinas exhibited moderate resistance. Hukkanen et al. (2007) reported that Benzothiadiazole can
339 enhance the accumulation of phenolics in strawberry plants which may involve in the BTH-induced resistance to powdery mildew. Foliar sprays of Calcium chloride and potassium silicate reduced powdery mildew symptoms in cultivars ‘Aromas’ and ‘Selva’ to significant levels, (Palmer et al., 2006). Copper-containing chemicals, such as copper oxychloride control fungal diseases to a reasonable extent, if applied as prophylactic measures 5.6.2 Anthracnose: It is caused by Colletrotichum fragariae Brooks and was reported from Florida for the first time (1926-29) by Brooks in 1931. In China Dai et al. (2006) reported Colletotrichum acutatum as causal organism of anthracnose fruit rot of strawberry. In India it was first reported by Singh (1974) from Bangalore. Anthracnose disease in Strawberry iscaused by pathogen complex consisting of Colletotricum species, C. acutatum, C. fragariae and C. gloeosporioides, that results in a variety of symptoms (Yamaguchi, 2006). Primarily the crown of the plants is affected and causes crown rot and wilt. Symptoms appear as sunken, dark brown, circular lesions on petioles, Symptoms on stolon (runners) results in in wilting and death of the leaf or of the daughter plant beyond the lesion on the stolon. The fungus often spreads into the crowns of young plants, causes rotting and death of plants in the nursery and is prominent in field after transplanting. Contaminated plant weed hosts and infected plant debris are the main source of infection.Use of straw mulch (Maiden et al., 1993) and use of disease free planting stock could be an important means of managing this disease. As far as possible minimum fertilizers should be used in nursery as increased fertilizer application many lead to enhance disease levels (Agrios, 2005).The twelve resistant varieties of strawberry against anthracnose were reported by Denoyes and Guerin (1996). ‘Honeoye’ and ‘Pandora’ cultivars showed a useful level of resistance, even against the most aggressive isolates (Simpson et al., 2006). ‘Sweet Charlie’, ‘Carmine’, and ‘Earlibrite’ were the most resistant cultivars. ‘Strawberry Festival’ exhibited moderate levels of susceptibility; while ‘Camarosa’ and ‘Treasure’ were highly susceptible to anthracnose (Chandler et al., 2006). Fungicides like Prochloraz and difenoconazole were found most effective fungicides for control of strawberry black flower (Domingues et al., 2001). Octave Reg. (462 g/kg prochloraz as the MnCl2 complex) was highly effective in reducing the incidence of Colletotrichum crown and stolon rot in runner production (Hutton, 2006).
340 5.6.3: Leaf spots: Strawberry is affected by various leaf spot disease. 5.6.3.1 Leafspot: Also called chocolate spot, is the most common fungal disease of strawberry leaves. The first signs of this disease appear as small, brown spots on the upper leaf surfaces of older leaves. The number of spots is usually an indication of the extent of the disease. The most effective principle for ptimum control of this disease is proper sanitation. Infected and old leaves should be removed and destroyed. Good air flow through proper plant spacing will minimise the spread of the disease. The volume of free water around the plants should be reduced. Hot, humid conditions promotes development of fungal infections and rapid spread of the disease. 5.6.3.2 Mycosphaerella leaf spot Caused by Mycosphaerella fragariae Tul. (= Ramulariatulasnii Sacc, imperfect stage). In India this disease was first reported from Niglar, Bhowadi hill of Uttar Pradesh during October 1952 (bosh, 1970). In initial stage, small circular purple scattered spots appear at upper surface of young leaves with an average size of 2-6 mm in diameter. The leaf spots enlarge, then turn to white and are surrounded by dark purple margins. Theunder surface of the leafbecome dark purple to reddish and eventually almost white. In severe infection the pulps of berry becomes discolored and render the fruits unmarketable. Cultural practices like proper spacing between plants, well-drained soil and weed free plots reduce disease incidence. Excessive use of nitrogenous fertilizers increases the incidence, (Harnendo and Casada, 1976). whereas the spray of micronutrients like manganese, copper and boron can reduce the infection t (Dorozhkin and Grisanovich, 1972). Copper fungicides have been found to be effective against this disease.
341 5.6.3.3Angular Leaf spot This is an important bacterial disease of strawberry caused by Xanthomonas fragariae reported from various strawberry growing countries. The water soaked spots appear on upper surface of leaves in initial stages of disease development, that later on becomes dark brown and angular in shape.In severe condition of disease the affected plants may be die. Disease can be effectively control by spraying of streptomycin or streptocyclene, starting from the first appearance of symptoms of the disease (Alipi et al., 1989). 5.6.3.4 Pestaliopsis leaf spot Caused by fungus Pestaliopsis disseminate (Theum) stey. The disease is found in all strawberry grown areas in India and first time recorded by Singh et al. (1975) from Bangalore on cv. Pusa early dwarf. Symptoms are characterized by circular, dark brown to chocolate coloured spots surrounded by reddish brown or yellowish margins that shows patchy appearance later. The infection starts from the leaf margin and extends towards midrib.The infected areas become brittle and get detached from the healthy ones. Severely infected leaves get eventually defoliated. 5.6.3.5Leaf blight This disease was first reported by Lele and Pathak (1965) from India. The disease is caused by Rhizoctonia bataticola (Taub). Bull. [= Macrophomina phaseolina (Tassi.) Goid]. The initial symptom of this disease isfound on all plant parts during August to October months. More or less circular spots with ash grey centre and purplish dark margins were found on leaves which become oval to irregular in shape in advanced stages. The lesion starts spreading from margin of the leaves towards center. Infected runners and stalks turned dark brown or black with irregular lesions and new root and plant growth is suppressed. The disease is most prominent in drier and warmer conditions. 5.6.3.6 Leaf Scorch The disease leaf scorch was first reported by Wolf (1924) worldwide and in India recorded by Bose (1970) from Kumaon hills of Uttar Pradesh. This disease is caused by fungus Marssonina fragariae (Sacc.) Kleb [(Diplocarponcarliana) (E & E) Wolf]. The disease mostly occurred in month of April or May and symptoms like irregular, oval or angular and purple colored spots surrounded by halo appear on leaves, petioles, peduncle and runners. Disease can be effectively managed by the
342 spraying of carbendazim. 5.6.4 Botrytis grey mould which is most probably the biggest enemy of strawberries. This disease can only infect damaged dead plant tissue. Most of the infection occurs during the flowering period, especially during hot, humid weather conditions. Infections leads to symptoms on petals, stalk, fruit caps and the fruit appears as soft, light brown, infected berries that dries up and remain on the plant. Fruit which is damaged by insects or other means is usually get infected. The infected fruit starts rotting and covered with a dense grey blanket of mycelium. Botrytis can be controlled by fungicides and should be applied from flowering until harvest. Plants should not be spaced too closely to promote adequate aeration. Excessive application of nitrogen fertilizer should be avoided that leads to enhanced susceptibility. The disease spread rapidly on fruit which is in direct contact with warm, wet soil or on wet organic material. The use of plastic mulching could be a better means to minimize this disease. 5.6.5 Black Root Rot Black root rot is a serious and common problem of strawberries and generally referred as a root rot complex disease.It is caused by a number of soil inhabiting fungi i.e. Laptosphaeriaconiothyruim (Fckl). Sacc. Fusariumortocereras App. and Wollenw, Pezizellalytheri Shear and Dodge, Ramullaria species, Corticiumvagum Berk and Cut., Packybasidiumcadium Sacc., Pythium species Trow, P. irrigulare Buis, Pyrenochaeta spp., Cylindrocarpondestrctans and Ceratobasidium species. (Bhardwas and Sharma, 1999). Besides the fungal pathogens certain nematodes, adverse environmental effect such as winter injury, fertilizer burn, edaphic factors like soil compaction, water stress or water logging , high soil pH are also involved in this disease complex. Prominent symptoms include the affected plants show much small root. The main roots become brown and small, fibrous rootlets are completely killed.Lesions extend and the entire root is blackened. The disease is more prevalent in heavy and water logged soil Continuous cultivation of strawberries on a same land should be avoided. Soil treatment with chemicals can reduce the population of damaging nematodes and fungi in the soil. 5.6.6 Verticillium Wilt Verticillium wilt disease is caused by fungus Verticillium spp. Mainly Verticillium, V. dahlia and V. albo-atrum .Mitra (1993) reported from India that the symptoms of Verticillium wilt in strawberry characterized by browning of wood at the base of crown and reddening of
343
petiole and stolon late in season. This disease causes total collapse of stawberry plants during peak growth. Both the species produce short lived conidia, but the mycelium and microsclerotia of fungus overwinters in the soil and can survive up to 15 years. V. albo-atrum grows best at 20 to 25ºC, whereas V. dahliaep refers slightly higher temperatures (25-28ºC) and sometime more common in warmer regions (Agrios, 2005).It is difficult to control by chemical means. It can be controlled by adoption of long crop rotation, use of disease free planting material in disease free soil, use of soil disinfectant and adoption of resistant cultivars to combat the disease (Ercole, 1976 and Agrios, 2005). The thermal inactivation via soil solerization is providing useful for the control of Verticillium wilt in regions with high summer temperature and low rainfall. Use black mulch with low dose of nitrogenous fertilizers has also been found effective..
Integrated management of disease in Strawberry The success of integrated pests and diseases management in strawberry depends upon a combination of sanitation, cultural, physical, biological and chemical control measures.These are more ecofriendly and promote sustainability. There are many new molecules available which are substantially less toxic, selective and more eco-friendly than the conventional pesticides.Experiment was conducted to study “the efficacy
344 of bio- control agents and chemical treatments on minimizing disease incidence in strawberry” at Bihar agricultural University, Sabour and incidence of different diseases round the year was recorded. Month
Dec
January
Fortnight
2nd
1st
Wilt
+
+
February
March
2nd
1st
1st
2nd
+
+
++
++
Leaf spot Leaf blight Root rot Fruit rot/ botrytis grey mould Bact. blight
+
2nd
April
May
June
JulyAug
+
+
+
+
+
+
++
++
+
++
++
++
++
++
+++
+++
+
+
+
+
++
+++
+++
+++
++
+
++
++
++
++ +
+
+= Minor (1-10%), ++= medium (11-25 %), +++= major (26% and above)
The different bio control agents and commonly available chemicals were used as different treatments as T1-Root dip of carbendazim and foliar spray of carbendazim (T1), Soil application of Trichoderma and foliar spray of Trichodermaviridae (T2) , Soil drench and foliar spray of Metalaxyl +mancozeb(T3) , root dip of carbendazim+ soil application of Trichoderma + foliar application of Metalaxyl+mancozeb (T4),root dip of carbendazim+ soil application of Trichoderma + foliar application of Metalaxyl +mancozeb+ mulch (black plastic) (T5) and No treatment (T6) as control were applied to strawberry plants inorder to find an effective way of managing disease under warm humid conditions of Bihar. The finding of the experiment indicated that major disease under warm humid climatic condition of Bihar was root rot, leaf blight and wilt. On fruits Botrytis grey mould was pre-dominant and occurs mostly during 2nd fort night of February to first fortnight of march. Soil drenching with metalaxyl and mancozeb had showed good plant stand. The minimum plant mortality of 17.5 per cent during fruiting stage from planting to harvesting was recorded with the application of Soil drench and foliar spray of Metalaxyl + mancozeb. Trichoderma treated plants showed better survival ability of 61.33 per cent after harvest during runner production. The maximum yield of 205.35 g per plant was recorded under integrated disease management i.e. root dip of carbendazim+ soil application of Trichoderma + foliar application of Metalaxyl + mancozeb + mulch (black plastic) (T5)
345 indicating it to be the best option for management of root rot, leaf blight and wilts together in strawberry. The different bio control agents and chemicals used as treatments were T1-Root dip of carbendazim and foliar spray of carbendazim (T1), Soil application of Trichoderma and foliar spray of Trichodermaviridae (T2) , Soil drench and foliar spray of Metalaxyl + mancozeb (T3) , root dip of carbendazim + soil application of Trichoderma + foliar application of Metalaxyl + mancozeb (T4),root dip of carbendazim+ soil application of Trichoderma + foliar application of Metalaxyl + mancozeb + mulch (black plastic) (T5) and No treatment (T6) as control. The finding of the experiment was the major disease of strawberry observed in climatic condition of Bihar was root rot, leaf blight and wilt. On fruits Botrytis grey mould was pre-dominant. Soil drenching with metalaxyl and mancozeb had showed good plant stand with reduced root rot incidence. The minimum plant mortality of 17.5 per cent upto fruiting stage was recorded with the application of soil drench and foliar spray of Metalaxyl + mancozeb.Trichoderma treated plants showed better survival ability of 61.33 per cent after harvest during runner production. The maximum yield of 205.35 g per plant was recorded under integrated disease management schedule i.eroot dip of carbendazim+ soil application of Trichoderma + foliar application of Metalaxyl + mancozeb + mulch (black plastic) (T5). The results implies that a integrated approach is best the management option for the management of major diseases like root rot, leaf blight and wilts in strawberry under condition of Bihar. Harvesting and yield: In agro climate of Bihar strawberry starts to mature by end of December and continued fruiting till mid-April. Strawberry is a nonclimacteric fruit and harvested when it is fully ripe and turn bright red stem and cap intact. Harvesting should be done at 2-3 days interval.Harvesting is done either during the early morning or late in the afternoon when temperatures are low. Overripe or damaged fruit must be removed. After harvesting it should be kept in cool place protect it from direct sunlight, warm winds and dirt .It should be marketed after proper grading and packing. The yield varies according to variety, season and climate. A yield of 12 to 20 tons per hectare is good, though yields up to 40 tons per hectare have been reported under ideal conditions.
346 Non-climacteric nature, soft pulp and high rate of catabolic activities make this fruit highly perishable, and thus requires special attention for its post-harvest handling, transportation and value addition. Improper shaped, small, damaged, poor colored low grade fruits should be discarded during packing. Unmarketable fruits can be successfully processed in to a variety of products like jam, chutney, squash, pickles etc. References: Agrios, G.N. ( 2005). Plant Pathology (fifth edition). Elsevier Academic Press, UK, pp. 922. Alipi, A.M., Ronco, B.L., Carrainza, M.R. (1989). Angular leaf spot of strawberry, a new disease in Argentina.Comparative control with antibiotics and fungicides. Advances Hortic. Sci., 1: 3-6. ANON, (2006b). Strawberry. Wikipedia – The free encyclopedia. Wikimedia Foundation. Inc. http://en.wikipedia.org/wiki/Strawberry, 16 October 2006, 12:00. Bhardwaj, L.N. and Sharma, S.K. (1999). Diseases of strawberry, Gooseberry and raspberry. Diseases of horticultural crops-fruits.Indus Pub. Com., New Delhi, 316-336. Bose, S.K. (1970). diseases of valley fruits in Kumaon (III) leaf spot disease of strawberry. Prog. Hortic., 2: 33-53. Chandler, C.K., Mertely, J.C. and Peres, N. (2006). Resistance of selected strawberry cultivars to anthracnose fruit rot and Botrytis fruit rot. Acta Horticulturae, 708: 123-126. Dai, F.M., Ren, X.J. and Lu, J.P. (2006). First report of anthracnose fruit rot of strawberry caused by Colletotrichumacutatum in China. Plant Disease, 90(11): 1460 Denoyes, R.B. and Guerin, G. (1996). Comparison of six inoculation techniques with bacteria Colletotrichumacutatum on cold stored strawberry plants and screening for resistance to this fungus in French strawberry collections. Eur. J. Plant Pathol., 102: 615-621. Domingues, R.J., Tofoli, J.G., Oliveira, SH.F. and Garcia, Junior O. (2001). Chemical control of strawberry black flower rots (Colletotrichumacutatum Simmonds) under field conditions. Arquivos do InstitutoBiologico Sao Paulo, 68(2): 37-42. Dorozhkin, N.A. and Grisanovich, A.K. (1972). Effect of micro elements on susceptibility of strawberry to white spot and grey rot. Khimiyav Sel’ Shom Khozyaistve, 10:5051. Harnendo, V. and Casada, M. (1976). The relationship between Mycosphaerellafragariae infection and the nutritional status of strawberry plants. Anales de Edafologia-yAgrobiologia, 35: 1-2. Hukkanen, A.T., Kokko, H.I., Buchala, A.J., McDougall, G.J., Stewart, D., Karenlampi,, S.O. and Karjalainen, R.O. (2007). Benzothiadiazole induces the accumulation of phenolic and improves resistance to powdery mildew in strawberries. Journal-ofAgricultural-and-Food-Chemistry, 55(5): 1862-1870
347 Hutton, D. (2006). Successful management of Colletotrichumcrown and stolon rot in runner production in sub-tropical Australia. Acta Hort., 708: 293-298. Madden, L.V., Wilson, L.L. and Ellis, M.A. (1993). Field spread of anthracnose fruit rot of strawberry in relation to ground cover and ambient weather conditions. Plant Dis., 77: 861-866. Palmer, S., Scott, E., Stangoulis, J. and Able, A.J. (2006). The effect of foliar-applied Ca and Si on the severity of powdery mildew in two strawberry cultivars. Acta Horticulturae., (708): 135-139.
Pandey, M.K., Shankar, U. and Sharma, R.M. (2012). Sustainable Strawberry Production in Sub-Tropical Plains Ecologically Based Integrated Pest Management, D.P. Abrol and Uma Shankar (eds.), pp. 787-820New India Publishing Agency, New Delhi (India) Simpson, D., Hammond, K., Lesemann, S. and Whitehouse, A. (2006). Pathogenicity of UK isolates of olletotrichumacutatum and relative resistance among a range of strawberry cultivars. Acta-Horticulturae, 708: 281-285. Singh, S.J. (1974). A ripe fruit rot of strawberry caused by Colletotrichumfragariae. Indian Phytopath., 27: 433-434. Singh, S.J., Sastry, K.S.M. and Sastry, K.S. (1975) Investigations on mosaic disease of cape gooseberry. Curr. Sci., 44: 95-96.
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21 POST-HARVEST PATHOLOGY OF ONION AND GARLIC Sangeeta Shree*1, Md. Ansar2, Vijay Kumar Singh3 and Ramesh Sharma4 134
Department of Horticulture (Vegetable and Floriculture), BAU, Sabour-813210 2
Department of Plant Pathology, BAU, Sabour-813210 *Email:
[email protected]
INTRODUCTION Onion and garlic are important bulb vegetables which are produced underground. They have vertical shoots that have modified leaves (or thickened leaf bases) which are used as food storage organs by the dormant plants.These vegetables belong to the genus Allium. Among vegetables, Alliums are perhaps the most important genus and are consumed throughout the world.In genus Allium, garlic (A. sativum) and onion (A. cepa) are mostly consumed throughout the globe. Apart from being used as a prime vegetable, garlic also has numerous medicinal properties. In India, references about cultivation of onion and garlic are found from ancient times onwards as is evidenced by mention in Charaka-Samhita, a famous early medical treatise of India. Garlic (Allium sativum) is an annual, vegetatively grown crop, which can be sown in mild and cold climates. There are different types or subspecies of garlic, and are most notably hard neck garlic and soft neck garlic (Volk, 2004).Right kind of garlic has to be recommended for a given latitude, since it can be day-length sensitive. In general, hard neck garlic is generally grown in cool climates, while the soft neck garlic is grown close
350 to the equator. Garlic is a monocot species, having flat leaves, slender scape, long-beaked spathes and heads bearing bulbils. The small bulbs are always mixed with the flowers of the inflorescence and the flowers usually abort at the bud stage. Commonly, garlic is propagated through cloves. Cloves are the sole organs of storage and these are the modified axillary buds of the foliage leaves. At maturity, the main stem of the bulb, roots and leaves attached to it all die. Only the cloves remain to carry the plant on to the next generation (Brewster, 2008).Garlic is completely sterile i e. garlic has no complete flower for performing pollination and unable to set seed, therefore propagated asexually only from cloves (Kamenetsky, 2007; Shemesh, 2008). Onion (Allium cepa) is a vegetable and is the most widely cultivated species of the genus Allium (Block, 2010). Onion is normally a biennial or a perennial plant, but for commercial purposes it is taken usually as an annual crop. Onion plant has a bluish-green leaves which are tubular and hollow and the underground bulb is used for vegetable and salad purposes. Bulb formation starts when a certain day-length is reached. Onion with multiple bulbs is also known like shallots and potato onions. At maturity the foliage dies down and the outer layers of the bulb become dry and brittle. The crop is harvested and cured and the onions are ready for use or storage. Both onion and garlic crop is attacked by several pests and diseases, like, thrips, mites nematodes and various fungi cause rotting.Several post harvest diseases also spoil the post harvest quality of these vegetables and also reduce the storage life. Both abiotic and biotic causes responsible for post harvest losses in vegetable.Physiological changes occur after harvesting.Physiological changes can be caused by high temperature, low atmospheric humidity and physical injury which lead produce losses.Onion and garlic are highly susceptible to mechanical injury which is generally received by the use of poor harvesting practices such as the use of rusted equipments for harvesting, unsuitable containers used at harvest time or during the marketing process, e.g. containers that can be easily squashed or have splintered wood, sharp edges or poor nailing, over packing or under packing of containers and careless handling of containers etc. This results in damage of bulbs, internal bruising and crushing of skin of produce. Onion and garlic being succulent in nature contain a wide range of organic substances and high water content, and thus are good substrates for microbial spoilage. The most common pathogens causing decays in fruits and vegetables are several species of the fungi, namely, Alternaria, Botrytis, Botryosphaeria, Collectotrichum, Diplodia, Monilinia, Penicillium,
351 Phomopsis, Rhizophus and Sclerotinia and of the bacteria Erwinia and Pseudomonas (Wills et al., 2007). A wide range of fungal and bacterial pathogens feed on and damage bulbs of onion, garlic and other member of Allium species. Usually these are organisms which are commonly found in the field or may be seedtransmitted, as was found with Botrytis allii and certain bacterial pathogens. Each pathogen has its own temperature requirements for its growth and development, while all are encouraged to grow by high humidity (>80% RH) during storage. They include pathogens such as those causing neck rot, and most of the bacteria, black mould or Aspergillus niger and some other fungi. The cultivation and usage of bulb crop is gradually becoming popular among the growers as well as the consumers. The growers prefer to store the crop until the next planting season and the consumers also like the crop having long shelf life. Therefore it becomes imperative to have proper information and understanding about the pests and pathogens causing damage of the crop on storage. DISEASE AND DISORDERS Some of the more serious post harvest diseases and disorders of onion and garlic which cause greater loss of produce are the following :i) Fusarium basal rot of onion and garlic Fusarium basal rot disease affects onion, garlic, leek, shallots and also some other Allium members and are caused by Fusarium oxysporumcepae. The fungus persists in the soil debris and infects the bulbs which are injuredeither by insects or by faulty handling. During later half of the crop season the symptoms appear on the standing crop as yellowing or tanning of leaves, wilting and dieback. In onion rotting and discolouration of the basal disc appears in the beginning which gradually advances up into the spongy scale of bulb. The affected bulbs when cut appear brown and watery. Bulbs appear soft spongy or sunken and the rot progresses on storage. In garlic white or light pink or reddish fungal growth may be noticed on clove and even shattering of the clove of affected bulbs has been seen. Such cloves are small, reduced in size and somewhat dry. Management : For control of this disease long crop rotation of not less than four years duration should be followed with non-host crop such as cowpea, barley or wheat. Resistant onion cultivars (Arka Pitamber and ArkaL alima) should be used. Plants should be protected from all types of damages due to insects, improper cultural practices and various types of
352 mechanical injuries. Bulbs should be cured properly and should be dried enough before topping. Proper handling and curing is essentially required for safe storage of the bulbs. Storage of damaged and injured bulb must be avoided. Bulb should only be stored in dry conditions with proper ventilations. ii) Botrytis neck rot of onion and garlic Botrytis neck rot is a serious storage disease of onion and garlic of fungal origin, mainly Botrytis allii which grows optimally at 21oC and is therefore a problem in temperate climates, such as Northern Europe and Canada. The pathogen is soil borne as well as seed borne. The infection survives in dormant stage on plant debris, in diseased bulb and also as sclerotia in soil. The Botrytis neck rot on storage is caused by different species than the Botrytis leaf spot, seen on the standing crop. Infections of Botrytis neck rot disease spread through neck tissue or wounds in bulbs. Bulbs which do not have very clear and visible symptoms of this disease become infected and symptomatic during topping of the necks, when the fungus directly enters the neck via airborne spores. The neck rot disease in onion is more common and clear and conspicuous after harvest which aggravates and progresses rapidly on storage. Symptoms occur after 8-10 weeks of storage, with a softening and rotting of neck tissues. Numerous small and small black sclerotia develop beneath the outer dry skins (Hayden and Maude, 1997). As the infection advances the rotting and discoloration moves in to the inner scales and finally bulb ends up into a soft mass In garlic, the disease generally becomes visible at initial stage on necks near the soil surface. The fungus travels swiftly into the neck region of succulent garlic bulb, producing water soaked appearance. A grey mould develops on the surface of the bulb or between garlic scales. Later black bodies called sclerotia develop around the neck. The infected plant may not survive at all or infected bulb may be reduced into a soft mass and saprophytes may destroy it further. Neck rot is more severe if infection occurs early in the growing season and after this artificial curing may not be effective (De Visser et al., 1994). Moist, cool and humid conditions favour disease development. There is evidence to show that B. allii conidia produced at low temperatures cause more rapid and destructive rots than the conidia produced at higher temperatures (Bertrolini and Tian, 1997). Management : Onion and garlic bulbs tops should be allowed to mature well before lifting them. In dry weather, bulbs should be cured on the
353 ground for about 6 to 12 days. Only mature, dry and bulbs with tight feel and appearance should be harvested. Bacterial diseases and Botrytis neck rot fungus do not move in dry tissue. Hence, proper curing and drying prevents infection of these diseases. Care should be taken to ensure minimum bruising and least mechanical injury in topping and storing. They should be stored in well ventilated houses at 32°F or slightly higher. They can be stored at higher temperatures if humidity cannot be held below 75%. The other means of discouraging pathogen development include postharvest treatment by heated-air drying at 30-32 o C during the early stage of storage. Crop rotation should be adopted. Frequent and excessive irrigation which creates moist condition should be avoided. iii) Purple Blotch of onion and garlic Purple Blotch of onion and garlic is caused by a fungus Alteraria porri. The pathogens of this fungus survive on infected bulbs and debris in the field and also in seeds. This disease appear on the leaves as large bleached lesions with purple centre, that rapidly gets enlarged, eventually leading to rot of infected bulbs. The most favourable temperature is 28oC30oCwith 80- 90 % relative humidity. Another fungus, Stemphylium vesicarium attack both onion and garlic. This is a major fungal disease on leaves and seed crop in Northern India. It can also cause purple blotch in onion. Both these diseases are the most common as leaf diseases, but can also affect bulbs in storage. Secondary infection often follows as injury caused either by other fungi, bacteria, viruses, insects or by sand or dust particles on windy days. Mature leaves and plants are more susceptible than young and juvenile plants. Spores require moist and humid conditions continual dew to cause infection. Optimum temperature to induce disease epidemics is ranged between 25 °C and 27°C (Cavanagh and Hazzard 2013.) Infection does not occur below 13°C. Symptoms appear as small yellow to orange spots or streaks in the middle of leaves and on flower stalks on one side. In moist weather condition, these spots become covered with a brownish black, powdery fungus growth. Leaves with large spots turn yellow and are blown over by the wind. Bulbs may decompose or grow mouldy during or after harvest. At first watery rot around the neck appears which further progresses into e yellowish to wine red discoloration in the neck region and eventually the whole bulb dies. Management : Cultural control like crop rotations, lesser hours of leaf wetness, wider plant spacing and other good cultural control should be practiced to avoid and evade such fungal infections. Old onion culls should be destroyed and debris must be buried or burnt. Wounded, bruised and
354 damaged bulbs should be removed. Proper curing of bulbs in the field is recommended. Optimum drying of bulbs before lifting and topping is advised. Tolerant or resistant varieties must be planted. Eexcessive irrigation is often the culprit. Fungicides and some insecticides in case of secondary infections can be used to fight this disease. iv) White rot of onion and garlic White rot of onion and garlic is caused by a fungus, Sclerotium cepivorum which is soil- borne. All members of Allium family such as onion, leek, garlic and shallot can be infected by this fungus but onion and garlic are the most susceptible. White rot disease spreads through sclerotia which live in the soil for a long period and even one sclerotia can infect a large number of adjoining plants. Sclerotia can infect plants from 25-30 cm below the soil surface and symptoms appear as watery rot of bulbs and roots with some fuzzy white material, which is nothing but the fungal mycelium. Above the ground symptoms first appear on the outer leaves which proceed inward as the disease advances usually anytime from midseason to harvest. Even hundred percent plants can die due to this disease. Infected bulbs become unmarketable or fetch very poor price. Once the disease comes in a field, it is very not an easy task to grow onion and garlic productively. Disease spreads with infected seeds, sets or transplants, water, equipment, shoes, grazing cattle ( movement of infested soil) and in the wind. Fungal activity is favoured by cool weather and is restricted above 75°F. High humidity also favours the disease. Decay of infected bulbs due to white rot in storage can persist if humidity continues to be high. Management : White rot thrives under the same climatic condition that are conducive for onions and garlic (cool weather and moist soil). Hence, it is difficult to evade the pathogens by altering planting time. Only disease free seeds or sets should be planted in disease free soil free from any infection. Rouging for infected or the diseased plants from field of the healthy plants must be practiced. Soil solarization practices in infected areas can reduce the number of sclerotia. All equipment, boots etc. must be washed with water so that all soil is washed off. Safe disposal of the material should be practiced. Diallyl disulfide (DADS), can be applied artificially in the field in the absence of Alliums, so that sclerotia germinate and in the absence of Allium host, they die, rather than lying dormant (Ehn et al., 2012). Besides, three fungicides, tebuconazole, fludioxonil and boscalid which are currently registered for white rot control can be useful.
355 v) Blue mold of garlic Blue mold of garlic is caused by Penicillium spp. fungi. It is also known as Penicillium mold. Common species of Penicillium that cause blue mold in garlic are P. hirsutum (syn. P. corymbiferum), P. aurantiogriseum (syn. P. cyclopium), P. citrinum, P. digitatum, P. expansum, and P. funiculosum. Infection first occurs on wounds or bruises that occur when cloves when separated from the parent bulb. The fungus is transferred from soil to the bulbs and the cloves. The symptoms become prominent on storage of bulbs. At start yellowish lesions appear which are soon covered with the characteristic blue-green spores. In extreme cases, the entire clove may be reduced to a mass of spores. The primary source of inoculums is the infected planting material. Air borne spores contaminate the cloves when the bulbs are broken for planting. Wounded and bruised cloves are very much susceptible to this disease. Cloves are often invaded by saprophytes which further aggravate the disease. Spore germination and disease development is favoured during warm weather conditions. Management : Planting garlic early when soil temperatures are still high (above 25°C)) favors disease incidence and augments disease severity. Bulb should be harvested carefully. Care should be taken to maintain minimum bruising and wounding while harvesting the bulbs. The bulbs must be cured and dried promptly. Hot water seed should be done before planting of the cloves else the germination would be adversely affected. Bulb should be stored at 4-5°C with low relative humidity for safe and longer storage. vi) Black mold Black mold, caused by Aspergillus spp. fungi, is a postharvest disease under hot climate which cause significant losses. The disease is common in onion and garlic stored in hot climates where the temperature ranges between 30 and 40oC. Symptomatically, bulbs show black tint at the neck and streaks of black mycelium and conidia beneath the outer dry scales. The black discoloration is due to black dust of spore clusters of fungus. The bulb has cheap market value because of the black appearance on the outer as well as the inner scales of the bulb. When the garlic scales are thin these spores mass is usually visible through the scales. In severe stage of disease development all the cloves get infected and the bulbs wither. Management : For management of the disease, the bulb should be stored only after proper drying. Onion should be stored at 1-15°C (Chand et al., 2014) Bruising should be avoided when bulbs are harvested, stored or transported. The crop should be sprayed with fungicides 10-15 days before
356 harvesting. Before planting garlic cloves should be treated with suitable fungicides. vii) Bulb canker/ skin blotch Bulb canker/ skin blotch is a one of the major problems of onion garlic on storage caused by Embellisiaalli (Campan). The symptoms include the manifestation of greyish spots on the outer scales of the bulb in the initial stage and slowly the entire bulb is coated with dark blackish color. Management : To evade this problem bulb should be stored essentially only after proper curing and drying with forced heated air at 27-35oC. Application of suitable fungicide during the period of growth reduces the severity in the symptoms of stored bulbs of onion and garlic. Irrigation should be strictly discontinued and tops should be allowed to dry down as the time of harvest comes nearer. viii) Bacterial brown rot Bacterial brown rot is caused by bacterium Pseudomna saeruginosa. It is a serious storage disease and infection is spread through the oozing of the neck. Dark brown discoloration in bulb scale is the typical feature of this disease. Browning and rotting of inner scale is an important symptom of this disease. The inner scales are first affected and show rotting symptoms which is soon passed on to the outer scales. Affected bulb should not be stored along with the healthy bulbs. Rain at maturity aggravates the problem. Management : Streptocyclin 200 ppm is recommended at weekly interval, if rain occurs at maturity stage, to minimise diseases incidence percentage during storage. Neck cutting is about 2.5-3.0 cm. long above the bulb must be practiced to reduce the bacterial infection incidence. Light irrigation should be provided during entire cropping period. ix) Waxy breakdown: Waxy breakdown is a physiological disorder that affects garlic during later stages of growth particularly near harvest or afterwards in storage and is usually associated with high temperature during these periods. Poor ventilation and lack of oxygen supply during storage results in waxy breakdown in garlic. Early symptoms are small, light-yellow sunken areas in the clove flesh that darken to amber colour. Later, the clove becomes translucent, soft, rubbery gummy and waxy. The outer scale covering of the bulb is not affected and it often obscures the inner symptoms until very advance stage is reached and cloves begin to shrink and shrivel and the
357 yellow waxy cloves becomes noticeable through the outer flimsy scales. Waxy breakdown in garlic normally occurs in storage and during long distance and hardly ever in the field. Proper aeration and sufficient oxygen levels during storage may reduce the cause of waxy breakdown in garlic. Table 1: Important post harvest diseases and pests of onion and garlic Disease
Causal Agent
Class
Bulbs Bacterial soft rot
Erwiniacaratovora
Bacterium
Bacterial Brown Rot
Pseudomnasaeruginosa
Bacterium
Black rot
Aspergillusniger
Hyphomycete
Blue mold rot
Penicilliumspp.
Hyphomycete
Fusarium basal rot
Fusariumoxysporum
Hyphomycete
Neck rot
Botrytis spp.
Hyphomycete
Purple blotch
Alternariaporri
Hyphomycete
Sclerotium rot
Sclerotiumrolfsii
Agonomycete
Smudge
Colletotrichumcircinans
Coelomycete
REFERENCES Bertrolini, P. and Tian, S.P. (1997). Effect of temperature of production of B. allii conidia on their pathogenicity to harvested white onion bulbs. Plant pathology, 46 432438 Block, E. (2010). Garlic and Other Alliums: The Lore and the Science.Royal Society of Chemistry. ISBN 0-85404-190-7 Brewster, J. L. (2008). Onions and Other Alliums.CABI Publishing. ISBN 978-1-84593399-9 Cavanagh A. and Hazzard, R. (2013). information & images from Oregon State Extension: http://ipmnet.org/plantdisease/ Chand, P., ýNair, B., ýSingh, K.P. (2014). Fundamentals Of Vegetable Crop Production. https://books.google.co.in/books?isbn=9386237350 De Visser, C.L.M., Hoekstra, L. and Hoek, D. (1994). Research into effective chemical control of leaf spot and neck rot and into methods to predict neck rot in onions. Verslag Proefstationvoor de Akkerbouwende Groenteteelt in de Vollegrond No. 178, Proefstationvoor de Akkerbouwende Groenteteelt in de Vollegrond, Lelystad, The Netherlands, 85pp (in Dutch) Ehn, B., Ferry, A., Turini, T. and Fred Crowefile: ///C:/Users/welcome/Downloads/ Fall%202012%20Newsletter-insert.pd Hayden, N.J. and Maude, R.B. (1997). The use of integrated pre and post harvest strategies
358 for the control of fungal pathogens of stored temperate onions. Acta Horticulturae., 433, 475–479. Kamenetsky, R. (2007). Garlic: Botany and horticulture. Hort. Rev. (Amer. Soc. Hort. Sci.) 33:123–171 Shemesh, E., Scholten, O., Rabinowitch, H.D. and Kamenetsky, R. (2008). Unlocking variability: Inherent variation and developmental traits of garlic plants originated from sexual reproduction. Planta 227:1013–1024. Volk, G.M., Henk, A.D. and Richards, C.M. (2004). Genetic diversity among US garlic clones as detected using AFLP methods. J. Amer. Soc. Hort. Sci.129:559–569. Wills, R. B. H., W.B. McGlasson, D. Graham, and D. C. Joyce. (2007). Postharvest – An Introduction to the Physiology and Handling of Fruits, Vegetables and Ornamentals (5th ed.). CAB International, Oxfordshire, UK. 227 pp.
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22 THE ANAEROBIC FERMENTATION OF WASTES FOR PRODUCTION OF BIOGAS Santosh Kumar singh* and Niranjan Kumar Mandal *Deptt. of Chemistry, S.P.Mahila Mahavidyalaya Dumka, Jharkhand P.G. Deptt. Of Chemistry, Sido Kanhu Murmu University, Dumka, Jharkhand
ABSTRACT In the age of rapid urbanization and population growth have put a challenging task before the scientists and researchers for the necessity of adequate solid waste management throughout the world to minimize the risk to the environment and human health, economically feasible solution for the treatment of solid waste, particularly in urban areas of low- and middle-income countries. Urban solid waste management in general and inadequate disposal in particular are considered to be of the most immediate and serious environmental problems in urban areas of developing countries. Inadequate management like uncontrolled dumping bears several adverse consequences. It can lead to uglification of the living areas, high risk of polluting surface and groundwater through leachate and furthermore promotes the breeding of flies, mosquitoes, rats and other disease vectors and also it emits unpleasant odours and methane, a major greenhouse gas contributing to global warming. Anaerobic digestion is a promising method to treat the particular fraction of organic solid waste, besides composting and direct animal feeding. Anaerobic digestion can be used to treat faeces, grey water and other organic waste with the aim to produce biogas and a fertilizer. A significant reduction of pathogens can be achieved through this at raised temperatures. It can be used Household toilets, but can also include showers, bath tubs, sinks, treatment for faeces and grey water. Substrates on which anaerobic treatment processes can be used are high-strength grey water (as a pre-treatment step), approx. BOD >
360 400 mg/L, black water with or without urine (black water: faeces, urine, small amount of water) as a pre-treatment step, human excreta together with animal excreta and grey water, followed by reuse in agriculture. Anaerobic treatment works with organic input materials, such as: liquid organic material, solid organic material (provided it is has a water content of ~ 50% or more), i.e. Slurries/sludge, organic kitchen waste, grey water together with excreta. Methane is the main gaseous product which is a dangerous (potent) greenhouse gas with a global warming potential over 100 years of 23 i.e. when averaged over 100 years each kg of CH4 warms the earth 23 times as much as the same mass of CO2. Methane is the major component of “natural gas”, about 97% by volume. As a gas it is flammable only over a narrow range of concentrations (5–15%) in air. Methane has a calorific value of 10 kWh/Nm3 or 35,900 kJ/Nm3. Hence, biogas with 65% methane has a calorific value 6.5 kWh/m3 (23,300 kJ/m3). Key words: Anaerobic digestion, solid waste, grey water, BOD.
Introduction: Rapid urbanisation and population growth have magnified the necessity for adequate solid waste management throughout the world. In order to minimise the risk to the environment and human health, economically feasible solutions are sought for the treatment of solid waste, particularly in urban areas of low- and middle-income countries. Particularly urban- and per urban areas of low and middle income countries are confronted with great challenges concerning appropriate Solid Waste Management (SWM) in order to minimise the risk to human health and avoid environmental degradation. However, most municipalities struggle to provide sufficient and adequate SWM services (Kassim & Ali, 2006). Considering the fact that the largest fraction of waste in developing countries is of organic nature and therefore amendable to anaerobic digestion, it makes environmental and economic sense to survey this option (Mbuligwe & Kassenga, 2004). Urban solid waste management in general and inadequate disposal in particular areconsidered to be of the most immediate and serious environmental problems in urban areas of developing countries (Zurbrügg, 2002). In most cities of low- and middle - income countries the physical composition of solid waste consists mostly of organic, hence biodegradable matter (Troschinetz et al., 2008), yet less than50% of the total waste generated is collected and disposed of in sanitary manner (Parrot et al, 2008). Inadequate management like uncontrolled dumping bears severaladverse consequences: It not only leads to an uglification of the living area, but also to a high risk of polluting surface and groundwater through leachate and further more promotes the breeding off lies,
361 mosquitoes, rats and other disease vectors. In addition, it emits unpleasant odours and methane, a major greenhouse gas contributing to global warming (Yhdego, 1995). Besides composting and direct animal feeding, anaerobic digestion is a promising method to treat the particular fraction of organic solid waste. While anaerobic digestion for the treatment of animal dung is fairly common in rural areas of developing countries, information on technical and operational feasibilities concerning the treatment of organic solid waste is limited. Anaerobic digestion can be used to treat faeces, greywater and other organic waste with the aim to produce biogas and a fertiliser. A significant reduction of pathogens can be achieved through this at raised temperatures It can be used Household toilets, but can also include showers, bath tubs, sinks, treatment for faeces and grey water. Regarding the enormous waste problems in the urban areas of these countries, the question arises whether AD could be an appropriate and sustainable method to treat organic house hold waste as well as wastes from markets and restaurants. Sale of fertiliser (sanitised human excreta); irrigation with treated grey water to non-edible plans, production of clean energy in the form of methane and to perform a number of mechanical works are the added advantages of this. Substrates on which anaerobic treatment processes can be used are high-strength grey water (as a pre-treatment step), approx. BOD > 400 mg/L, black water with or without urine (black water: faeces, urine, small amount of water) as a pre-treatment step, human excreta together with animal excreta and grey water, followed by reuse in agriculture. Anaerobic treatment works with organic input materials, such as: liquid organic material, solid organic material (provided it is has a water content of ~ 50% or more), i.e. slurries/sludges, organic kitchen waste, grey water together with excreta. Methane is the main gaseous product which is a dangerous (potent) greenhouse gaswith a global warming potential over 100 years of 23 i.e. when averaged over 100 years each kg of CH4 warms the earth 23 times as much as the same mass of CO2. Methane is the major component of “natural gas”, about 97% by volume. As a gas it is flammable only over a narrow range of concentrations (5–15%) in air. Methane has a calorific value of 10 kWh/Nm3 or 35,900 kJ/Nm3. Hence, biogas with 65% methane has a calorific value 6.5 kWh/m3 (23,300 kJ/m3). Anaerobic Digestion Anaerobic Digestion (AD), also referred to as biomethanization, is a natural process that takes place in the absence of oxygen. It involves the biochemical decomposition of complex organic material by various
362 bacterial processes with the release of an energy rich biogas and the production of a nutritious effluent. the methanogens are a type of microorganism, but do not belong to the group of bacteria. Natural gas is a gaseous fossil fuel consisting primarily of methane but including significant quantities of ethane, butane, propane, carbon dioxide, nitrogen, and helium and hydrogen sulphide. Volatile fatty acids (VFAs) are an intermediate product of anaerobic fermentation, which cannot accumulate in normal conditions as it will significantly drop down the pH and digestion process will cease. Digesters or reactors are physical structures that facilitate anaerobic digestion by providing an anaerobic environment for the organisms responsible for digestion. The biological conversion of organic material under anaerobic conditions can be described by the following four stages: 1. Hydrolysis The first step involves the extracellular enzyme-mediated transformation of higher molecular mass organic polymers and lipids into basic structural building blocks such as fatty acids, monosaccharide, amino acids, and related compounds which are suitable for use as a source of energy and cell tissue. This stage consists of microorganisms attacking the organic matter where complex organic compounds such as cellulose and starch are converted to less complex soluble organic compounds. Higher molecular mass organic polymers are transformed into soluble monomers through enzymatic hydrolysis as shown below:n(C6H10O5) + nH2O
n(C6H12O6)
(1)
2. Acidification These monomers of sugars and amino acids, become substrates for the microorganisms in the second stage where they are converted into organic volatile fatty acids like propionic, butyric and veleric acids, acetates, molecular hydrogen and carbon dioxide by a group of by a group of fermentative bacteria. Ammonia is also produced by the degradation of amino acids . n(C6H12O6) acid forming bacteria
3n(CH3COOH)
(2)
3. Acidogenesis These organic acids primarily acetic acid form the substrate for the third-stage. Both long chain fatty acids and volatile fatty acids (VFA) are degraded generating acetate, carbon dioxide and hydrogen.
363 4. Methanogenesis The forth and last step involves the bacterial conversion of hydrogen and acetic acid formed by the acid formers to methane gas and carbon dioxide. CH3COOH methane forming bacteria
CH4+CO2
(3)
In this step, methanogenic bacteria generate methane by two routes, (I) by fermenting acetic acid to methane (CH4) and CO2 and (II) by reducing CO2 via hydrogen gas or formate generated by other bacterial species. CO2+4H2 reduction
CH4+ 2H2O
(4)
Similarly CO2 can be hydrolysed to carbonic acid and to methane as given below:CO2+H2O Hydrolysis
H2CO3
(5)
4H2+H2CO3 reduction
CH4+3H2O
(6)
The carbon dioxide and hydrogen sulphide in the biogas are undesirable. They may be removed for optimum performance of biogas as fuel. Carbon dioxide may be removed by passing the gas into lime water which turns milky due to formation of calcium carbonate. Ca(OH)2(aq) + CO-2(g)
CaCO3 + H2O
(7)
H2S may be removed by passing the gas through a lead acetate solution. (CH3COO)2Pb (aq) + H2S(g)
2CH3COOH (aq) + PbS (s)
(8)
The bacteria responsible for this conversion are strict anaerobes, called methanogenic. Due to their very slow growth rates, their metabolism is usually considered rate limiting in the anaerobic treatment of organic waste (Mata-Alvarez, 2003). In one-stage systems, the above described reactions occur simultaneously in a single reactor, whereas in two- or multistage systems, the reactions take place sequentially in at least two reactors with the second stage consisting of the conversion to methane. Another differentiation in systems is related to its dry matter content: Wet - systems: TS375
Death through intoxication (after several hours)
>750
Unconsciousness and death through still stand of breathing in
30-40 min>1000
Rapid death through respiratory paralysis in few minutes
367 Effluent Due to the decomposition and breakdown of its organic content, the residue of the biomethanization process, also called slurry, normally gets rid of smell and provides fast acting nutrients (mainly NH4-N) which easily enter into the soil solution, thus becoming immediately available to the plants. Hence digested sludge can increase agricultural yields according to its nutrients. The pathogenic organisms present in the digesters raw influent (or inoculums) from animal faeces get eliminated during the mesophilic digester process at 35°C. Tests showed a complete elimination after 3 months of all harmful pathogens like enteric virus, Salmonella, Shigellas, Vibrio cholera, Pathogenic Escheria coli, Trichuris and Hookworms (Costech, 2006). Even more important for agricultural use is the fact that all plant-pathogenic germs are completely destroyed during the anaerobic digestion process (Wellingeretal ,1991 ) . Factors affecting biogas production: Production of biogas depends largely on temperature as it is a microbiological reaction. Microorganisms are sensitive to pH changes. Buffering is necessary for pH. In addition to temperature other parameters are also equally important to be controlled to ensure proper operation of biogas digester. The important of them are being listed below: pH of influent and effluent the nature of the substrate temperature organic loading rate toxicity nutrients slurry concentration digester type and size of the digester Solid and hydraulic retention time Carbon to nitrogen ratio Total solids of feed material Total volatile solids Chemical oxygen demand
368 Biochemical oxygen demand Mixing of different feed materials etc. Anderson (1979) has reported that the concentration of water soluble substances such as sugar, amino acids, proteins and minerals decrease with increase in the age of plants and water insoluble substances such as lignin, cellulose, hemicelluloses and polyamides increase in content with increase in the age of the plants. This means that vegetable matter from younger plants produce more biogas compared to those from the older plants. For waste products from animals, the type and age of animal, its feeding and living conditions, the age and storage of the waste product are factors affecting the quality and quantity of the gas produced. In general finely ground waste products produce more biogas due to large surface area of contact with bacteria. (Maarshishwari and Vasidevan, 1981) References: Kassim, S.M., Ali, M. (2006). Solid waste collection by the private sector: Households’ perspective Findings from a study in Dares Salaam city, Tanzania; Habitat International 30(2006) 769-780 Mbuligwe, S.E., Kassenga, G. (2004). Feasibility and strategies for anaerobic digestion ofsolid waste for energy production in Dares Salaam city, Tanzania; Resources, Conservation and Recycling 42 (2006), 183-203 Zurbrügg, C. (2002). Solid Waste Management in developing countries, Sandec, Dübendorf Troschinetz, A.M., Mihelcic, J.R. (2008). Sustainable recycling of municipal solid waste in developing countries; Waste Management (2008),doi :10.1016/j wasman.2008.04.016 Parrot, L., Sotamenou J., Dia, B.K. (2008). Municipal solid waste management in Africa: Strategies and livelihoods in Yaoundé, Cameroon; Waste Management (2008) doi:10.1016/ j .wasman.2008.05.005 Yhdego, M. (1995). Urban solid waste management in Tanzania: Issues, concepts and challenges; Resources, Conservation and Recycling 14 (1995) 1-10 Mata-Alvarez, J. (2003). Biomethanization of the organic fraction of municipal solid wastes; IWA Publishing, Cornwal Koottatep, S., Ompont, M., Hwa, T.J (). Biogas: Green Productivity Option for Community Development Deublein, D., Steinhauser, A. (2008). Biogas from waste and renewable resources: An introduction; Wiley-VCH-Verlag, Weinheim Eder, B., Schulz, H. (2006). Biogas Praxis; Oekobuch Magnum, Staufen Costech, (2006). Energy resources in Tanzania, Volume 1; Tanzania Commission for Scienceand Technolgy
369 Wellinger, A., Baserga, U. Edelmann, W. Egger, K. and Seiler, B. (1991). Biogas Handbuch; Verlag Wirz, Aarau Schmitz, T.D., (2007). Feasibility study for a national domestic biogas programme in Tanzania; published by GTZ, Eschborn
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23 BLAST: THE MOST DEADLY DISEASE OF RICE Satyendra*1, Mankesh Kumar1, Anand Kumar1, Manoj Kumar1, S. P. Singh1, Rahul Singh1, Surabhi Sinha1 and P. K. Singh1 Department of Plant Breeding and Genetics, Bihar Agricultural College, Bihar Agricultural University, Sabour-813210 Bhagalpur *corresponding author:
[email protected]
ABSTRACT Increase in rice production is still a difficult task to feed the ever increasing population. Development of resistant varieties is the most economic and environment friendly method for achievement of the production targets. Several minor pathogens have become major in context of the changing disease scenario. Rice blast is the most threatening disease of rice not in India but in the world. It is also one of the oldest known diseases and also known as ‘rotten neck’ and ‘rice fever’. The pathogen can survive in wide range of environmental conditions due to which it has wide distribution throughout the country. It can affect all the Arial parts of rice plant specially leaves, neck and nodes. Breeding varieties resistant to blast will have direct impact on the production of rice crop. Several conventional breeding techniques have been used by the breeders. But with the availability of modern tools and techniques, it has become more easy, effective, and precise to develop varieties having one or more resistant genes/ QTLs. In this article all these issues have been discussed in context with the rice blast. Key words: Oryza sativa, blast, disease resistance, MAS, QTLs, DNA markers
INTRODUCTION Rice is one of the most important crops in India covering
372 approximately 44 million hectare land with a production and productivity of 110 million tons and 24.20 quintals per hectare, respectively(Directorate of Economics and Statistics, 2016). Definitely, tremendous growth have been achieved in rice production, a lot is to be done to fulfil the requirement of the country’s increasing population. By 2030, India will require 135 million tons of milled rice for which per hectare productivity must be 31.2 quintals per hectare (Fig.-1). This figure will increase up to 180 million tons by 2050 which can be achieved by the average productivity of 40.6 quintals per hectare. Achievement of targets becomes more complex in the context of so called ‘climate change’ scenario. We all know about the positive impacts of the ‘green revolution’ but rarely we discuss about its negative impacts on crops and human being.
Fig.-1: Future demand projection of rice in India
CHANGING RICE DISEASE SCENARIO IN INDIA There are several points out of which one is the changing scenario of diseases which affect rice at different phases of the crop. In 1945, only one (brown spot) disease was considered to be major to rice in India. The number reached as much as 6 in 1990 and now (2015) there are 10 diseases which have been reported to be ‘major’ for rice (Laha et al., 2016) (Fig.2).
373
Fig.-2: Rice disease scenario in India 1945: Brown Spot; 1960: Brown Spot, Blast; 1975: Brown Spot, Blast, Bacterial Leaf Blight; 1990: Brown Spot, Blast, Bacterial Leaf Blight, Rice Tungro Virus, Sheath Blight; 2005: Brown Spot, Blast, Bacterial Leaf Blight, Rice Tungro Virus, Sheath Blight, False Smut, Sheath Rot; 2015: Brown Spot, Blast, Bacterial Leaf Blight, Rice Tungro Virus, Sheath Blight, False Smut, Grain Discoloration, Bakane, Sheath Rot, Stem Rot
RICE BLAST Blast disease caused by fungus pathogen Mangnaporthe oryzae (Pyricularia oryzae) is the most deadly biotic stress of rice (Miah et al., 2013).It is also one of the oldest known diseases and also known as ‘rotten neck’ and ‘rice fever ’. The pathogen can survive in wide range of environmental conditions due to which it has wide distribution throughout the country. It can affect all the Arial parts of rice plant specially leaves, neck and nodes. However, leaves and neck are the most affected parts based on which the disease is classified as leaf blast and neck blast. Its presence has been reported in 80 rice growing countries of the world. It was first reported in India during 1918. It can cause as much as 70-80% losses in grain yield (http://agritech.tnau.ac.in). SYMPTOMS OF BLAST As typical symptoms in case of leaf blast, spindle shaped lesions occur on leaves which remain pointed at both the ends with brown margin and grayish centre. In severe cases of infection, entire crop give a blasted or burnt appearance and hence it is called ‘blast’. Crop lodges after ear emergence. In case of neck blast, dark and necrotic lesions develop around the panicle base (neck) due to which panicle loses its strength and breaks before maturity which leads to complete (chaffy) or partial grain filling. In
374 case of node blast, infected nodes become black having necrotic brown to black lesions which leads to breaking of culms (Fig.-3).
(a)
(b)
(c)
(d)
(e)
(f)
375
(g)
(h)
(i) Fig.-3: Symptoms of blast disease in rice-Leaf Blast (a, b and c), Neck Blast (d, e, and f) and Node Blast (g, h and i)
SPREAD OF THE RICE BLAST Blast as discussed earlier, is the problem of all the rice growing states of the country. Table-1 elaborates about the endemic places in different districts and the period found to be favorable for spread of the disease. Table-1:State wise endemic area and favorable period of spread State
Endemic Districts/Area
Favorable Period
Andhra Pradesh/ Telangana
Srikakulam, Vishakapatnam, Guntur, Nellore, Chittoor, Nizamabad, Medak, Ranga Reddy, Mahboobnagar and East & West Godavari
September – February
Arunachal Pradesh
Whole state
April – July
Assam
Karimganj, Tinsukia, Nowgong, Kamrup,
376
Goalpara and North Lakhimpur
August – October
Bihar/ Jharkhand
Ranchi and Hazaribagh
August – October
Chhattisgarh
Northern hill regions
September – October
Gujarat
Kheda
September – October
Haryana
Hisar and Karnal
August – October
Himachal Pradesh
Kangra valley (Malan, Palampur), Kulu and Mandi
August – October
Jammu & Kashmir
Hill zones of Anantnag, Rajouri, Jammu, Udampur and Larnoo
July – September
Karnataka
Mandya, Kodagu, Shimoga and Dharwad
September – October
Kerala
Palghat and Kuttanad
September – February
Madhya Pradesh
Bastar region, Rewa and Bilaspur
September – October
Maharashtra
Pune, Ratnagiri, Kolaba, Parbhani and Kolhapur
September – October
Manipur
Manipur central valley
July – October
Meghalaya
West Khasi hills
June – October
Mizoram
Mizoram
August – October
Orissa
Cuttack, Ganjam and Koraput
July – August
Punjab
Amritsar, Bhatinda, Patiala, Ferozpur, Ropar and Hoshiarpur
August – October
Tamil Nadu
Tanjavur, Coimbatore, Chengalpat, Vellore, Erode, Madurai, Pudukkotai and Thirunalvelli
October – February
Tripura
West & South Tripura
July – October
Uttaranchal
Almora, Nainital and other hill areas
August – October
Uttar Pradesh
Faizabad and Balia
August – October
West Bengal
Darjeeling and Cooch Behar
September
(Source: Laha et al., 2016)
377 BREEDING STRATEGIES Among the several management practices, use of resistance varieties seems to be effective, economic and environment friendly (Khush and Jena, 2009).Several attempts have been taken to develop resistant varieties using conventional breeding techniques and mutation breeding and a significant number of resistant varieties have been developed. The development of high yielding improved cultivars led to the shrink in the genetic pool by replacement of the landraces and traditional varieties. This has resulted in decline of the available genetic diversity. But, breeders need to combine other good traits related to grain and cooking quality along with the resistance to the disease. Development of a new variety by crossing two genetically diverse genotypes is a very common and popular method of creation of genetic variation. Several methods like pedigree method, back-crossing, recurrent selection and mutation breeding have been used. When the resistance is governed by the major genes, pedigree method is highly suitable and has been most widely used (Khush, 1978). But the disadvantage with this is that it takes long time to develop a resistant cultivar. But where resistance is governed to be polygenic, diallel selective mating design is suitable (Jensen, 1970). Back-cross technique is the most widely used for transfer/ introgression of the genes from donor parent to recipient parent i.e. popular mega cultivars showing susceptibility to the blast. Several popular varieties including basmati have been improved using the back cross technique. Recurrent selection is another popular conventional breeding technique which shortens the breeding cycles and has relatively more precise and defined genetic gains (Fujimaki, 1979). Mutation breeding is also very effective for improvement in the major traits including disease resistance. With the advancement of the system, it is now possible not only to identify and tag the mutated gene but such genes can also be pyramided into a single genotype (Shu, 2009). Through mutation breeding, several mutant line of popular varieties like Samba Mahsuri have been developed. Several major genes like Pib, Pita, Pia, Pi1, Pikh, Pi2 and Pi4 have been introduced into rice varieties for blast resistance using conventional breeding programs (Kiyosava, 1982). Identifying key genomic regions associated with blast resistance against a broad spectrum of isolates in backcross introgression lines have been developed through conventional breeding program (Korinsaka et al., 2011). By using conventional and molecular breeding many blast-resistant varieties have been developed (Liu et al., 2008).
378 Marker Assisted Selection (MAS) is comparatively simple, more efficient and precise technique than conventional approaches of breeding for disease resistance. In case of blast resistance, phenotype is often simple or governed by major genes therefore, marker assisted selection is extremely powerful in breeding. Therefore, efficiency of the conventional breeding may be increased through MAS. As far as the gene discovery is concern, more than 86 candidate R genes and 350 QTLs have been identified related to resistance against blast. Out of these genes 23 namely pb1, Pi-a, Pi-b, Pi-d2,Pi-d3, Pi-k, Pik-h/Pi-54, Pik-m, Pik-p, Pi-sh, Pi-t,Pi-ta, Piz-t, Pi-1, Pi-2/Piz-5, Pi5, Pi-9, pi-21, Pi-25,Pi-36, Pi-37, Pi-35 and Pi-64 have been characterized (Liang et al., 2017). Most of them have been identified from primitive cultivars, landraces and wild relatives (Miah et al., 2013). To increase precision of introgression, tagging of the molecular marker to a particular R gene is used. This approach is very effective and convenient (Shanti et al., 2001).As discussed earlier, several of R genes have been characterized and their gene specific or tightly linked SSR markers have been developed for application in the breeding programs. Using MAS several resistant genes like Pita, Piz, Pi37, Pi35 and Pi1, Pi9 have been introduced to new varieties. Through MAS, using DNA markers, it is possible to transfer more than one gene without phenotypic selection of plants. Plants having multiple R genes can be easily selected on the basis of closely linked DNA markers. Continuous discovery and generation of informationon new DNA markers may be quite helpful for the breeders working blast resistance and accelerate the improvement. Further, information related to DNA markers associated with QTL for a particular trait may increase the rate of genetic improvement through MAS. Ultimately, MAS in combination with the precise phenotypic selection techniques seems to be the best way to develop resistant varieties for blast. CONCLUSION In view of the ever increasing population, increasing the production and productivity of rice is still the most important. One of the measures to increase production is to reduce losses due to abiotic and biotic stresses. Changing disease scenario in India has made the task more difficult. Among biotic stresses, rice blast is the most deadly disease of rice having a very large distribution and affecting the different parts of the plant. However, several varieties have been developed by the breeders using conventional plant breeding techniques; recent advances in genomics have provided breeders additional tools. It will be helpful for the breeder to make use of these molecular techniques in even more simple way. Need is to adopt a
379 perfect mixture of the conventional and modern breeding tools to develop cultivars resistant to blast disease. REFERENCES Fujimaki, H. (1979). Recurrent selection by using male sterility for rice improvement. Jpn Agric Res Q 13(3):153–156. Jensen, N.F. (1970). A diallel selective mating system for cereal breeding. Crop Sci 10(6):629–635. Khush, G.S, Jena, K.K. (2009). Current status and future prospects for research on blast resistance in rice (Oryza sativa L.). In: Wang GL, Valent B. Advances in Genetics, Genomics and Control of Rice Blast Disease. New York, USA: Springer: 1–10. Khush, G.S. (1978). Breeding methods and procedures employed at IRRI for developing rice germ plasm with multiple resistance to diseases and insects. In: Symposium on methods of crop breeding. Tropical Agricultural Research Series, vol 11, pp 69–76. Kiyosawa, S. (1982). Gene analysis for blast resistance. Oryza18:196–203. Korinsaka, S., Sirithunyab, P., Meakwatanakarnd, P., Sarkarunge, S., Vanavichitc, A. and Toojindaa, T. (2011). Changing allele frequencies associated with specific resistance genes to leaf blast in back cross introgression lines of Khao Dawk Mali 105 developed from a conventional selection program. Field Crops Res 122:32–39. doi:10.1016/j.fcr.2011.02.005. Laha, G.S., Sailaja, B., Srinivas, Prasad, M., Ladhalaksmi, D., Krishnaveni, D., Singh, R., Prakasam, V., Yugander, A., Kannan, C., Valarmathi, P. and Ravindra Babu, V. (2016). Book: Changes in Rice Disease Scenario in India: An analysis from Production Oriented Survey. Indian Institute of Rice Research, Hyderabad. Liu, W.G., Jin, S.J., Zhu, X.Y., Wang, F., Li, J.H., Liu, Z.R., Liao, Y.L., Zhu, M.S., Huang, H.J., Liu, Y.B. (2008). Improving blast resistance of athermo-sensitive genic male sterile rice line GD-8S by molecular marker-assisted selection. Rice Sci 15(3):179– 185. doi:10.1016/S1672-6308(08)60040-2 Liyang, Y., Yan, B., Peng, Y., Ji, Z., Zeng, Y., Wu, H. and Yang, C. (2017). Molecular screening of blast resistance genes in rice germ plasms resistant to Magnapor the oryzae. Rice Science. 24(1): 41-47. Miah, G., Rafii, M., Ismail, M.R., Puteh, A.B., Rahim, H.A., Asfaliza, R. and Lafif, M.A. (2013). Blast resistance in rice: a review of conventional breeding to molecular approaches. Molecular Biology Reports. 40: 2369-2388. DOI 10.1007/s11033012-2318-0 Shanti, M.L., George, M.L.C., Cruz, C.M.V., Bernardo, M.A., Nelson, R.J., Leung, H., Reddy, J.N. and Sridhar, R. (2001). Identification of resistance genes effective against rice bacterial blight pathogen in eastern India. Plant Dis 85(5):506–512. doi:10.1094/PDIS.2001.85.5.506 Shu, Q.Y. (2009). Induced plant mutations in the genomics era. Food and Agriculture Organization of the United Nations, Rome, pp 425–427.
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24 INNATE IMMUNITY IN PLANTS Surabhi Sinha1 and Satyendra2 1
Post graduate in plant breeding and genetics department, BAU, Sabour Email :
[email protected]
2
Asstt. Prof. Cum Jr. Scientist, Plant Breeding And Genetics Department, BAU, Sabour Email :
[email protected]
ABSTRACT Recognition between self and non self molecule is the characteristic of all living organisms. Plants have evolved a two tier system for the recognition of pathogen molecules. First is the pattern recognition receptors located on the plasma membrane, which is involved in recognition of pathogen associated molecular patterns (PAMPs) and triggers pattern triggered immunity (PTI). This provides the first line of defence. As soon as the plant recognises the pathogen molecules, there is rapid elevation in intracellular Ca+2 level, production of reactive oxygen species, cyclin dependent kinases (CDKs) and mitogen activated protein kinases (MAPKs) (Zvereva et al,2012). The second one is the resistance(R) protein. They are involved in the recognition of effector molecules. And hence, trigger effector triggered immunity (ETI). This provides the second line of defence. A zig-zag model proposed originally by Jones and Dangl, clearly shows the plantpathogen interaction. For better understanding of plant-pathogen interaction, several concepts have been proposed i.e. gene-for-gene hypothesis, guard model, decoy model. Pathogen recognition and initiation of defence mechanism involves hierarchies of R genes namely plant receptor genes like receptor like proteins and receptor like kinase, MAPKs, phytohormones, transcription factors, resistance related proteins, resistance related metabolites.
382 Introduction The ability of an organism to resist infections caused by pathogens or state of protection against foreign substances is called immunity. Immunity is broadly classified as innate immunity and acquired immunity. Innate immunity provides the first line of defence and is present since birth. It is not pathogen specific rather acts against all the foreign molecules. It does not rely on previous exposure to pathogens and triggers new defence response each time a pathogen attack the organism. Whereas, acquired immunity is not present since birth and relies on previous exposure to pathogens. Acquired immunity is also called adaptive immunity. Plants unlike mammals, does not have mobile defender cells and a somatic adaptive immune system and has to rely on innate immune system for protection against different pathogens like bacteria, viruses, fungi, oomycetes etc. Also, there are several microbes which acquire nutrients in similar manner to pathogens but are important for plants. It is therefore, necessary for the plants to make a clear cut distinction between the harmful pathogens and useful microbes. To serve this purpose, plants have evolved an efficient defence mechanism which involves an efficient monitoring system. Monitoring includes the recognition of entry of pathogen molecules in the host cell and triggering of suitable defence mechanism against pathogens. Entry of pathogen into host cell Pathogen may enter the host cell directly through stomata, hydathodes or wonded tissues or may enter the hosts by forming a fungal hyphae thus exerting pressure on the cell wall to invaginate into the plasma membrane and release their molecules into the apoplast/cytoplasm. For example- Many bacteria such as P. syringae swim towards openings like stomata and hydathodes and enter the apoplastic space of plant tissue. From there, bacterial effectors can be injected into the cytoplasm via various secretion systems, like the type III secretion system in Pseudomonas and the type IV secretion system found in Agrobacterium. Recognition of pathogen Pathogen molecules such as Pathogen Associated Molecular Patterns(PAMPs) and elicitors/effectors are recognised by a large group of Pathogen Recognition Receptors(PRRs) and Resistant(R) proteins/genes. There may be a direct or indirect recognition of pathogen molecules. Physical barriers such as cell wall, cell membrane, lignin, chitin etc.
383 provides the first line of defence to pathogens. But, this does not provide enough strength to plants to defend pathogens. Hence, plants have evolved an intracellular defence mechanism which consists of PRRs[includes receptor like protein (RLPs) and receptor like kinase (RLKs)] and regulation of hierarchies of R proteins/genes. Most of the R proteins belongs to Nucleotide Binding Site-Leucine Rich Repeat (NBS-LRR) family. That means, R proteins consists of 2 domains i.e NBS domain which acts as a molecular switch for signal transduction and LRR domain which acts as a platform for protein-protein interaction. Several molecular models have been proposed for the recognition of pathogen and initiation of defence responses. Molecular models f or pathogen recognition 1. Gene for gene model Gene for gene model was proposed by H. H. Flor in 1947. According to this model, for each avirulence gene from pathogen there is a resistance (R) gene in the host, having compatibility reaction which leads to disease resistance. There is a direct recognition of effector molecules (fig.-1a). But in some cases, there is a need of an additional protein to identify the effector molecules. This also leads to the fact that being small number of R genes in the host, large numbers of pathogen effectors can be recognised and defence response can be elicited. 2. Guard hypothesis Guard hypothesis was proposed by Van Der Biezen and Jones in 1998. This model is an extension of gene for gene model. According to this model, the target protein of the pathogen effector (guardee) is guarded by suitable guard protein namely NBS-LRR. Thus there is an indirect recognition of effect molecules (fig.-1b). Presence of guardee does not affect the fitness of host (Glowacki et al, 2011). 3. Decoy model This model is a modification of guard hypothesis. According to this model, specific proteins, that are similar to targeted proteins, are synthesised by the plant in some plant-pathogen interaction. There only function is to bind to the effectors and act as a mediator in interaction with R proteins (Glowacki et al, 2011) (fig.-1c)
384
Fig.-1 molecular models for plant pathogen interaction. A. Gene for genemodel B. Guard model C. Decoy model
Phases of plant immunity A. PAMPs/ pattern triggered immunity Pathogens produce elicitors/effectors called Pathogen/Microbe Associated Molecular Patterns (PAMP/MAMP )(Kushalappa et al, 2016) upon attack to suppress the plant defence system. PAMPs are microbial signatures conserved throughout the whole classes of pathogens. These conserved sequences are highly indispensable for the survival of the
385 pathogens. PAMPs are not present in the host cell. Bacterial flagellin (flg2, flg22), Pep 13, lipopolysaccharides etc are the examples of PAMPs. These PAMPs are recognised by pattern recognition receptors located on plasma membrane. These PRRs are synthesised in endoplasmic reticulum and transported to plasma membrane. Flagellin sensing2 (FLS2), BAK1(brassinosteroid insensitive-1 associated receptor kinase1) are some examples of PRRs. Recognition of elicitors/PAMPs by PRRs triggers immunity and is termed as PAMPs/pattern triggered immunity. This provides the first line of defence. For example-the bacterial flagellin-derived peptide flg22, is recognized by a complex composed of the RLK FLS2 (flagellin sensing 2) and the regulatory kinase BAK1 (Brassinosteroid 1associated kinase 1). Specific binding of flg22 to FLS2 activates the FLS2BAK1 complex eliciting PTI. Likewise, BAK1 is required for PTI upon specific recognition of the bacterial translation elongation factor Tu (EFTu)-derived peptide elf18 by the RLK EFR (Elongation Factor Receptor). As soon as PAMPs are recognised by PRRs, pathogen-related response is generated, resulting in an increase in intracellular Ca+2 levels, production of ROS, activation of various kinsase metabolic pathway including Cyclin Dependent Kinase (CDKs) and Mitogen Activated Protein Kinase (MAPKs) (Zvereva et al, 2012). B. Effector triggered immunity(ETI) Successful pathogens counteract to PTI and produce race specific effectors/elicitors, which modifies the host to establish robust infection in susceptible host. This is called Effector Triggered Susceptibility (ETS). These effectors are specific to biotrophs, hemi-biotrophs and necrotrophs (Kushalappa et al, 2016). These effectors are recognised by plant produced specific R genes, which suppresses the effector molecules from pathogens and results in Effector Triggered Immunity (ETI). This provides the second line of defence. This is an amplified version of PTI. ETI leads to hypersensitive response of which ultimate result is Programmed Cell Death (PCD). NBS-LRR domains of R genes are involved in either direct or indirect recognition of effector molecules (Muthamilarasan et al, 2013). C. Zig-Zag model of plant-pathogen interaction Widely accepted zig-zag model of plant- pathogen interaction was originally proposed by Jones and Dangl in 2006. This model illustrates the perception of PAMPs and initiation of primary defence response as PTI. To counteract this defence, pathogens release effector molecules to suppress PTI and cause susceptibility i.e ETS. NBS-LRR based recognition of modified self- byproducts of ETS triggers ETI (fig.-2)
386
Fig.-2. Zig-Zag model of plant- pathogen interaction
Hierarchies of R genes involved in resistance The resistance in plants against pathogen stress is governed by hierarchies of R genes mainly Resistance Related Metabolite (RRM) and Resistance Related Protein(RRP). Hierarchies of R genes include plant receptor genes mainly RLKs and RLPs, mitogen activated protein kinase genes, phytohormones, transcription factors, RRPs and RRMs. Plant receptor genes are located on plasma membrane i.e RLPs and RLKs which are involved in direct recognition of the pathogen effector molecules and thus triggering the PTI. For example- FLS2 and BAK1. Mitogen activated protein kinase cascade consists of MAPKKK, MAPKK and MAPK. Phosphorylation of MAPKKK activates MAPKK and phosphorylation of MAPKK activates MAPK which in turn activates several phytohormones and transcription factors to produce different RRPs and RRMs which ultimately activates defence response against various pathogens. For example-A. thaliana RAF like MAPKKK, Constitutive Triple Response 1 (CTR1) and Enhanced Disease Resistance1 (EDR1), are known to participate in ethylene-mediated signalling and defense responses(Rodriguez et al, 2010). After perception of pathogen effectors by plant receptor genes, it activates phytohormones that binds to nuclear proteins with specific domains to activate downstream genes. Salicylic acid (SA) and Jasmonic Acid (JA) are the most common phytohormones being activated upon pathogen perception. SA is most effective against biotrophic (Koornneef
387 et al, 2008, Bari et al, 2009)pathogens and hemi-biotrophic pathogens whereas JA/ET is effective against necrotrophic and herbivore insects. JA-SA interaction is antagonistic to each other. For example-silver leaf whitefly Bemisia tabaci activates SA signalling and suppresses JA signaling in Arabidopsis ( Bari et al, 2009). Transcription factor also play a crucial role in pathogen defence by regulating downstream resistance metabolite genes. For example- overexpression on OsWRKY13 in Minghui63 resulted in enhanced resistance to bacterial blight and rice blast by activating SA biosysnthesis and suppressing JA signalling (Pandey et al, 2009). CASE STUDY 1. Active oxygen species (AOS) as mediators of immunity A study was conducted using antisense catalase 1 transgenic tobacco with regard to the defence activation and enhanced resistance against P. syringae or tobacco mosaic virus under high light conditions. Transgenic plants were illuminated with high light intensity which resulted in generation of AOS which led to local expression of acidic and basic PRproteins in the absence of necrosis. Strong local induction and systemic induction of PR-proteins were accompanied by necrosis. Peaks of induced salicylic acid and of ethylene occurred 2 – 5 h after onset of highlight only in the antisense plants. 48 hours later, macroscopic lesions developed all over the plant as a result of PR1 transcript accumulation which persisted for at least 2 weeks. Transcripts for glutathione peroxidase and basic glucanase were also induced in the antisense plants. Tobacco with catalse 1-antisense High light intensity Excess H2O2, tissue lesions, induced free and conjugated salicylic Acid, ethylene, ACC, transcript for PR1, acidic glucanase and Chitinase, basis glucanase, GSH peroxidase Protection against P. syringae or tobacco mosaic virus Fig. 3 Pathogen Tolerance of Transgenic Antisense Plants with Lowered Activity of Catalase1.
388 2. Over-expression of OsWRKY13 in the genetic background without R genes, enhanced resistance to bacterial blight and blast in rice. An experiment was conducted on Minghui63 which is partially resistant to X. oryzae pv oryzae as it contains 2 genes for bactreial blight resistance and Madanijang8 which is highly susceptible to X. Oryzae pv oryzae without unknown R genes. OsWRKY was over-expressed in Madanijang8. At booting stage, out of 47 palnts, 13 plants showed significantly enhanced resistance to PXO61 with lesion area ranging from 24.4 to 48.6% versus 61.8 and 32.2% for the controls of Mudanjiang 8 and Minghui 63 respectively. The reduced lesion area of leaf was associated with accumulation of OsWRKY13 transcripts. This result suggested that over-expression of OsWRKY13 improves resistance to bacterial blight at both seedling and adult stages. In second case, Madanijang8 was infected with M. grisea in a natural infection field, the T1 plants from OsWRKY13-overexpressing T0 plant D11UM18 segregated into two groups, a highly susceptible group having numerous lesions on the leaves and a partly resistant group having few lesions on the leaves. The enhanced resistance of the plants cosegregated with both the over-expression of OsWRKY13 and the expression of â- glucuronidase, which represented the presence of the transgene. Results suggested that OsWRKY was also involved in blast resistance at both seedling and adult stage. Conclusion Immunity of plants is important for reducing the losses of feed and fibre. Changing scenario of pathogens indicates that most of the minor pathogens are becoming major pests in different crops which can make the situation more complex in future as consequences of climate change scenario. In order to manage this, available information regarding genes, like PRRs molecules which play an important role in perception of conserved molecular patterns and induce defence mechanism, can be effectively employed using modern tools and technologies. Likewise, study of R-proteins can also help us to design defence strategy in different plant- pathogen interacting systems. Hormonal crosstalk is also a beneficial way of minimising the energy use for tuning the defence response against various pathogens.
389 References Kushalappa, A.C., Yogendra, K.N., Karre, S. (2016). Plant Innate Immune Response: Qualitative and Quantitative Resistance. Critical Reviews in Plant Sciences, 35:1, 38-55. Koornneef, A., Pieterse, C.M.J., (2008). Cross Talk in Defense Signaling. Plant Physiology, Vol. 146, pp. 839–844. Shigenaga, A.M., Berens, M.L., Tsuda, K., Argueso, C.T., (2017). Towards engineering of hormonal crosstalk in plant immunity. Current Opinion in Plant Biolgy, 38:164– 172. Zvereva, A.S., Pooggin, M.M., (2012). Silencing and Innate Immunity in Plant Defense Against Viral and Non-Viral Pathogens. Viruses,4(11), 2578-259. Spoel, S.H., Dong, X., (2008). Making Sense of Hormone Crosstalk during Plant Immune Responses. Cell Host & Microbe 3, 348-351. G³owacki, S., Macioszek, V.K., Kononowicz, A.K. (2011). R Proteins As Fundamentals Of Plant Innate Immunity. Cellular & Molecular Biology Letters. Volume 16 pp 124. Qiu, Q., Xiao, J., Ding, X., Xiong, M., Cai, M., Cao, Y., Li, X., Xu, C., Wang, S. (2007). OsWRKY13 Mediates Rice Disease Resistance by Regulating Defense-Related Genes in Salicylate- and Jasmonate-Dependent Signaling. Molecular Plant-Microbe Interactions. Vol. 20, No. 5, pp. 492–499. Sandermann, H., (2000). Active Oxygen Species as Mediators of Plant Immunity: Three Case Studies. Biological Chemistry, Vol. 381, pp. 649 – 653. Bari, R., Jonathan, E., Jones, J.D.G., (2009). Role of plant hormones in plant defence responses. Plant Molecular Biology 69:473–488. Rodriguez, M.C.S., Petersen, M. and Mundy, J., (2010). Mitogen-Activated Protein Kinase Signaling in Plants.Annual Review in Plant Biology. 61:621–49
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25 STUDY OF SYMPTOMS EXPRESSION OF ORANGE SOFT ROT INFECTED BY Aspergillus niger. Dara Singh Gupta1 , Bably Sarkar2 and Ashok Kumar3 1
University Dept. of Botany, K. U. Chaibasa-833202 Email:
[email protected]
2
M.Sc. student, Univ. Dept. of Botany, K U Chaibasa 3
Dept. of Botany, A.S. College, Deoghar
ABSTRACT Aspergillus Niger is a member of the genus Aspergillus which includes a set of fungi that aregenerally consider asexual, although perfect forms (i.e., reproduce sexually) have been found. Aspergilli are ubiquitous in nature. They are geographically widely distributed and have been observed in a broad range of habitats because they can colonize a wide variety of substrates. Aspergillus niger is commonly found as a saprophyte growing on dead leaves, stored grain, compost piles and other decaying vegetation. Microspically, its conidiophores are smooth walled, hyaline or turning dark towards the vesicle. Conidial heads are biseriate with the phialides borne on brown, often septate metulae. Conidia are globose to subglobose (3.5 -5.0 micron meter in dm), dark brown to black and rough – walled. It is known to create increased amount of pathogenicity in various species of plants, which can be treated by antibiotics, chemicals and antibiosis. Biological control however is the best and most effective. Orange is the most common and easily available fruit of India. But during storage it is affected by few diseases. Such as post-harvest diseases during storage or transportation especially caused by Aspergillus Niger. The fungus is slightly harmful for living being and
392 commercial lose. So, this research is indicated to show the disease cycle and control management. KEY WORDS: Orange fruit, Aspergilius niger, conidiophores, pathogenicity, control management.
INTRODUCTION Post Harvest Fungal Diseases of Orange (Citrus sinensis (L.) Osbeck) Orange is a popular citrus fruit is India. It grows first in Asia but now grows in many parts of the world. Orange is a very good source of vitamins, especially vitamin C. It is transported to Kerala markets from various parts of India, very popular and consumed by Keralities. Post harvest fungal diseases of this fruit are not reported earlier from the state. This disease recorded during the investigation was black mould rot (Aspergillus niger) and soft rot (Geotrichumcandidum). Both these disease were severe in nature and produced extensive damage of the fruits under storage. Black mould rot was observed in all the samples are collected from Jamshedpur, Tatanagar, Jharkhand, markets during post winter months. Soft rot was prevalent in Kadma and Sakchi markets during the same period. But the plants are affected by a number of diseases especially fungal diseases. Post harvest diseases are common in Oranges. MATERIALS AND METHODS A] Material for pure culture: potato dextrose agar medium, culture tube, conical flask, Inoculation chamber, spirit, spirit lamp. B] Material for inoculation of pathogen: Distilled water, freshly washed test tube, spirit lamp, inoculation chamber, spirit, inoculation needle. Methods of sterilization:Sterilization is the process of destroying of life from the object being sterilized by physical methods and chemical agents it as refer to the complete absence, destruction of microorganisms. Sterility can be achieved by exposing object or substance to lethal agents which may be chemical, physical or ionic in nature or in case of method depends upon the desired efficiency, its applicability, its toxicity, availability and cost, and effect properties of the object to sterilize. The definition of sterilization is the complete destruction or removal of all living organism from the object being sterized. The development of methods of sterilization was mainly a consequence of the controversy over spontaneous generation culminating
393 in the work of Pasteur. Experiment designed prove or disprove spontaneous generation depend upon two general principles:1. The complete sterilization of a suitable growth medium so that no living organisms exist at the start of the experiment. 2. The design of the vessel of the type that is impossible for microbes to enter from outside this was necessary following the realization of the existence of microbes floating around in the air. These two principle are strictly, followed and conditions are otherwise, suitable for multiplication of microbes, any growth occurring must be the result of spontaneous generation. Thus key question was how good methods were for:1. Attaining 2. Maintaining sterility The Attainment of Sterility:The method depends upon the heat treatment, but soon, it was realized that microorganism are very widely in their resistance to heating. Bacteria on higher temperature produce heat stable spores. So, at the normal pressure boiling was not sufficient to kill these spores and thus autoclave was design to increase the pressure and there by temperature sterilization sealing of kind of prevent the entry of microbes, but not of air, this led to the division of cotton wool plug that was soon adopted university by microbiologist. By the ends of 19th century most of the methods currently used for sterilization had been developed. These are briefly summarized as below:Heat: It is usual methods of sterilizing, one of the most effective reliable and economic sterilizing agents. For general sterilization at a time and temperature that kill organisms including heat resistant spores is used. The methods are generally adopted as follows:a) Wet heat in autoclave:- The usual method is a time of 30 min at a pressure of 1.05 kg/cm that will give a temperature of 121°C This is the best methods if predictable. b) Tyndalization:-This is a course of their periods of boiling at 100°C for 30 min, at daily intervals.
394 c) Dry heat: -This is done in a dry over, where a temperature of 160° C for two hours is usually required. Filtration:Oxygen is needed for many forms of life. So sealing of flask was not proper method as oxygen will no longer enter the vessel. It was necessary therefore, to include some kind of life to prevent the entry of microbes but not of air. The liquid or gas to be sterilized is passed through a filter with a porosity sufficient to remove any micro filtration organism is suspension. The cotton wool is used for gases. For liquid, variety of filter are available, made of material such as cellulose nitrate Millipore filters. This method is very useful for sterilization of liquid contains heat liable component. 1.) Radiation:For sterilization of air ultraviolet light is very effective. 2.) Chemicals:Many chemicals are lethal to microbes hypo chloride solution and phenolic derivative are used as general laboratory disinfectants . Similar chemical is gaseous ethylene oxide. However, these may not cause sterilization under some conditions. Temperature and Humidity Chart DAYS
DATE
TEMPERATURE 0
HUMIOITY/RAIN 0
1
18-02-2018
Max 26 CMin 14 C
Max 84%/00 mm, Min 24%
2
20-02-2018
Max 260 CMin 140 C
Max 84%/00 mm, Min 24%
4
22-02-2018
Max 260 CMin 140 C
Max 84%/00 mm, Min 24%
0
0
6
24-02-2018
Max 26 CMin 14 C
Max 84%/00 mm, Min 24%
7
26-02-2018
Max 260 CMin 140 C
Max 84%/00 mm, Min 24%
8
28-02-2018
Max 260 CMin 140 C
Max 84%/00 mm, Min 24%
0
0
10
02-03-2018
Max 26 CMin 14 C
Max 84%/00 mm, Min 24%
12
04-03-2018
Max 260 CMin 140 C
Max 84%/00 mm ,Min 24%
Observation table for Orange Fruit 1 No.of observation st
Symptoms
18.02.18 (1 day)
The surface area of the orange is 18.5cm. The area of the spot size is 0.0 cm. This time spot is not appeared.
20.02.18 (2nd day)
The surface area of the orange is 18.5cm. The powder mass
395
increase in size of 1.5cm. th
22.02.18( 4 day)
The surface area of the orange is 18.5cm. The orange shows green powdery mass is increase in size. The of the spot is 2.2 cm.
24.02.18( 6th day)
The surface area of the orange is 18.5cm. The orange shows green powdery mass is increase in size. The of the spot is 3.0 cm.
26.02.18( 8th day)
The surface area of the orange is 18.5cm. The orange shows dark green powdery mass is increase in size. The of the spot is 4.5 cm
28.02.18( 10th day)
The surface area of the orange is 18.5cm. The size of the spot is 5.0 cm. The spot is become deep green in colour.
02.02.18( 12th day)
The surface area of the orange is 18.5cm. The dark greenish powdery masses appeared and cover the surface of orange. The size of the spot is 5.3 cm.
04.02.18( 14th day)
The surface area of the orange is 18.5cm .The powdery mass is covered less half surface of the orange. The spot size is 5.5 cm.
Observation table for Orange Fruit 2 No.of observation
Symptoms
18.02.18( 1st day)
The surface area of the orange is 15.8cm. The area of the spot size is 0.0 cm. This time spot is not appearing.
20.02.18( 2nd day)
The surface area of the orange is 18.5cm .The powder mass increase in size of 1.5cm and having few light green powdery mass is appearing.
22.02.18( 4th day)
The surface area of the orange is 15.8cm. The area of the spot size is increase in 2.9 cm.
24.02.18( 6th day)
The surface area of the orange is 15.8cm. The orange shows light green powdery mass is increase in size .The size of the spot is 4.1 cm.
26.02.18( 8th day)
The surface area of the orange is 15.8cm. The orange shows dark green powdery mass is increase in size. The of the spot is 5.3 cm
28.02.18( 10th day)
The surface area of the orange is 15.8 cm .The size of the spot is 6.4 cm. The spot is become deep green in colour.
02.02.18( 12th day)
The surface area of the orange is 15.8cm. The dark blackish green powdery masses appeared and cover the surface of orange. The size of the spot is 7.1 cm.
396
04.02.18( 14th day)
The surface area of the orange is 15.8 cm .The greenish powdery mass is very dark blackish in colour. The powdery mass is covered almost half surface of the orange and rotting is started in water substance. The spot size is 8.9cm.
Observation table for Orange Fruit 3 No.of observation st
Symptoms
18.02.18( 1 day)
The surface area of the orange is 20.6 cm. The area of the spot size is 0.0 cm. This time spot is not appearing.
20.02.18( 2nd day)
The surface area of the orange is 20.6 cm .The area of the spot size is 1.0 cm and having few light green powdery masses is appearing.
22.02.18( 4th day)
The surface area of the orange is 20.6 cm. The area of the spot size is increase in 2.0 cm.
24.02.18( 6th day)
The surface area of the orange is 20.6 cm. The orange show light green powdery mass is increase in size .The size of the spot is 3.4cm.
26.02.18( 8th day)
The surface area of the orange is 20.6 cm. The powdery mass is increase in size. The of the spot is 4.8 cm
28.02.18( 10th day)
The surface area of the orange is 20.6 cm. The size of the spot is 5..4 cm. The spot is become deep greenish in colour.
02.02.18( 12th day)
The surface area of the orange is 20.6 cm. The dark blackish green powdery mass appeared and covers the surface of orange. The size of the spot is 6.1 cm.
04.02.18( 14th day)
The surface area of the orange is 20.6 cm .The greenish powdery mass is very dark blackish in colour. The powdery mass is covered almost half surface of the orange and rotting is started in water substance. The spot size is 6.9cm.
Observation table for Orange Fruit 4 No.of observation
Symptoms
18.02.18( 1st day)
The surface area of the orange is 14.6cm. The area of the spot size is 0.0 cm. This time spot is not appearing.
20.02.18( 2nd day)
The surface area of the orange is 14.6 cm .The area of the spot size is 0.5 cm and having few light green powdery masses is appearing.
22.02.18( 4th day)
The surface area of the orange is 14.6 cm. The area of the spot size is increase in 1.0 cm.
24.02.18( 6th day)
The surface area of the orange is 14.6cm. The orange shows
397
light green powdery mass is increase in size .The size of the spot is 2.1 cm. 26.02.18( 8th day)
The surface area of the orange is 14.6 cm. The orange shows dark green powdery mass is increase in size. The of the spot is 1.5 cm
28.02.18( 10th day)
The surface area of the orange is 14.6cm .The spot is deep greenish in colour.The spot size is 1.9 cm.
02.02.18( 12th day)
The surface area of the orange is 14.6cm. The dark blackish green powdery mass appeared. The size of the spot is 2.2 cm.
04.02.18( 14th day)
The surface area of the orange is 14.6 cm .The greenish powdery mass is very dark blackish in colour. The powdery mass is covered almost half surface of the orange and rotting is started in water substance. The spot size is 2.7 cm.
Disease index of both orange inoculated Aspergillus niger Disease index
Days After Inoculation
Symptoms
0
Immediately Both orange are healthy, no. of spot were forms. The after weights of the oranges are 115gm, 105gm, 130gm & inoculation 95gm. The entire surface of the orange is 18.5cm.
1
2
Green powdery mass is appearing in the orange. No change in shape and size.
2
4
Green powdery mass is increased and looking like a dark in colour.
3
6
Blackish green powdery mass increase in outer surface area and few water drops are found.
4
8
Powdery mass is increased and rotting is started.
5
10
Powdery mass become thick and rotted area increased.
6
12
Powdery mass further increase in size and some smells are found.
7
14
Powdery mass increased to about more than half surface on the orange and more water drops are found and smell is odourly.
8
18
Complete rotting the orange is burst like and excess water are found. The shape is deformed and shrinked. The both orange wt. are 90, 79, 105 & 85 gms.
398
399 RESULT AND DISCUSSION During my work I found gradual increase in disease on inoculated orange fruits by Aspergillus Niger post harvest storage condition. In first two days the diseased developed about 11.1%, 18.3%, 9.7% and 6.8% in inoculated fruit. Then after two days the disease symptoms increases 24.3%, 33.5%, 23.3% and 10.2% respectively. After 14 th days the fruit were observed almost completely rotted (wet) about more than 50% to 60%. It is clear that the pathogen cause severe damage to orange fruit in store condition specially in tropical areas and it also shows rapid destruction. CONCLUSION In this project, I concluded that orange fruit is infected by Aspergillus niger. It is very important fruit in all over India. Huge loss takes place because of these infections. So, I suggest some precautioinary measure to prevent this disease. Some of them are follows:1. Remove the infected orange from the content of healthy orange. 2. By using turmeric in the storage place disease can be prevented. 3. By using dry red chilli in the storage place disease can be prevented. 4. Since the orange get infected in field also therefore, field sanitation is necessary. 5. By using Tulsa pest in the storage place disease can be prevented. 6. Burning of the diseased crop to protect the field. AKNOWLEDGEMENT Authors are very thankful to all the persons who help to provide all laboratory facilities to complete the research work especially the HOD, University Deptt. Of Botany, Kolhan University, Chaibasa, Jharkhand, and the supervisor Dr. Dara Singh Gupta for providing correct direction and valuable guidance, without them this research project wark whould not be have been completed. REFERENCES Bernet, H.L. (1960). I illustrate Genera of imperfect fungi, Burgess Publishing company, II Edition West Bengal. Bilgram, K.S., Jamaluddin, S, and Rizwi, M.A. (1981). Fungi of India part II list and Reference Today and Tomorrow’s. Printers and Publishers, NewDelhi.
400 Dorby, S. (2006). Improving quality and safety of fresh fruits and vegetables after harvest by the use of biocontrol agents and natural material, Acta Horticul 709:95-51. Kanaujia, R.S. (1979). A new Aspergillus storage of mandarian orange Indian phytopath 32:620-621. Mukherji, K.G. and Bhasin, J. (1986). Plant disease of India. Tata MC Graw Hill publishing company Ltd, New Delhi. Philip, S. (2002). Fruits diseases in the field of storage , Kalani Publishers, New Delhi. Ratnam, C.V. and Neelam, K.G. (1967). Studied on market disease of Fruits and vegetables , Andhra agricultural J, 14:60-65
401
26 FUNCTIONAL BEVERAGES OF WHEATGRASS JUICE: BROAD SPECTRUM APPLICATION IN THE TREATMENT OF VARIOUS DISEASES Amresh kumar1, M. Dutta Choudhury1, Patha Pallit2 1
2
Assam University Biotech-Hub, Assam University, Silchar, Assam-788011
Department of Pharmaceutical Sciences, Assam University, Silchar, Assam-788011 E-mail :
[email protected]
INTRODUCTION Functional beverage is the quickest developing section in the functional food category (Gruenwald, 2009). In Thailand, functional drink market grew from Bt1.8 billion in 2009 to Bt6.6 billion in 2014 (Ketnil, 2014). The record predicts the world purposeful beverage market to develop with a CAGR of 6.3% over the forecast period of 2018-2024. The study conducted on functional beverage market covers the evaluation of the leading geographies such as North America, Europe, Asia-Pacific and RoW from the period of 2016 to 2024. The consumer interest in natural functional drinks, with anti-aging, Energy supplying, relaxing, or beauty enhancing effects is increasing. To avoid intake of chemical substances, natural substances from plant, which are favored than animal sources, have been an increasing number of used as functional phyto-components in beverages. The cereal grasses (young leaves of grain-bearing plants), which include wheat, barley, alfalfa, rye, oat, and kamut, are interesting ingredients
402 for functional drinks. They comprise huge concentrations of phytochemicals and vitamins (Gruenwald, 2009). Especially wheatgrass juice, received from younger wheat plant was once first used for promoting human fitness by Ann Wigmore, founder of the Hippocrates Health Institute in Boston (Wigmore, 1985). It is frequently known as the “green blood” due to its high chlorophyll content material (Padalia et al., 2010). Chlorophyll bears structural similarity to hemoglobin and has been discovered to regenerate or act as a substitution for hemoglobin in hemoglobin deficiency conditions. This may be the reason in the back of the utility of wheatgrass in medical conditions like thalassemia and hemolytic anemia (Padalia et al., 2010; Marawaha et al., 2004). In addition, chlorophyll collectively contains essential enzymes like superoxide dismutase, the plant hormone Abscisic acid or dormin, and its alkalinity play their roles in the anticancer properties (Padalia et al., 2010). Antioxidants content in wheatgrass juice such as (pro) nutritional vitamins C, E, beta-carotene and zinc are accountable for anti-allergic and anti-asthmatic treatment, while bioflavonoids account for many medical utilities such as management of inflammatory bowel disorder and as a general detoxifier. Wheatgrass juice is protected and the incidence of facet outcomes is very low. In case of barley grass, there is a document on the contents of diet, complete polyphenols, ferulic acid, monosaccharides and amino acids indicating that it is a precious plant fabric (Paulíèková et al., 2007). In this present proceeding we are trying to assess the value of wheatgrass therapeutically in various ailments and may help researcher to step forward to explore more potential unidentified bioactive of this grass. Chemical composition of wheatgrass: Rich source of Vitamins A, C, E and B complex. It contains a plethora of minerals like calcium, phosphorus, magnesium, alkaline earth metals, potassium, zinc, boron, and molybdenum. Pharmacological actions are protease, amylase, lipase, cytochrome oxidase, transhydrogenase, super oxide dismutase (SOD). High content of bioflavonoids like apigenin, quercitin and luteolin and other therapeutically active are indole compounds and laetrile. Mode of action of constituent in wheatgrass juice Quercitin, an important bioflavonoids constituent of wheatgrass juice which initiate apoptosis via the mitochondrial pathway which involves
403 the activation of caspase-3 and caspase-9 followed by the release of cytochrome c (Cyt c) and cleavage of poly-ADP-ribose polymerase (PARP). This action of inhibiting tumor progression is reported in a variety of human cell lines, including breast cancer MCF-7 cells, nasopharyngeal carcinoma CNE2 and HK1 cells, leukemia HL-60 cells, thymus-derived HPBALL, and oral squamous carcinoma SCC-9 cells (Lautraite et al., 2002; Haghiac et al., 2005; Russo et al., 2014). It also inhibits the oxidative harm to DNA molecule of cells. Antiangioactivity: - Angiogenesis, characterized by the formation of incipient vessels from a presubsisting microvascular network, is a crucial step in the magnification and metastasis of cancer and replenishes the growing tumor cells with oxygen and nutrients (Tuli et al., 2014 ; Tuli et al., 2015). This flavonol was found to inhibit several steps of angiogenesis including proliferation, migration, and tube formation of human microvascular dermal endothelial cells in a dose-dependent manner (Chien et al., 2009). A decrease in the expression and activity of MMP-2 and MMP-9 was additionally observed in a variety of cancer cell lines. In the other hand, Luteolin activates JNK, which inhibits TNF-á mediated NFkB translocation, promoting TNF-á induced apoptosis in cancer cells. However luteolin can mediate autophagy as cell death mechanism by triggering intracellular acidic lysosomal vacuolization and accumulation of microtubule-associated LC3II protein, which in turn enhances autophagy flux.
404 Therapeutic activity of wheatgrass juice: A. Blood building exercise in Thalassemia major: - Beta-thalassemia is a genetically inherited disease that arises due to peculiar beta globin chains which are required for the synthesis of grownup hemoglobin (HbA). Individuals with thalassemia might also continue to produce gamma globin chains in an effort to enlarge the amount of fetal hemoglobin (HbF) and compensate for the deficiency of HbA. 3-5 fold amplifies in the production of HbF on consumption of wheatgrass has been documented with the use of a cellular assay (Fibach et al., 1993). This has now been confirmed via the development of a unique assay approach for HbF, which is based on detecting its production in human erythroleukemia cells the usage of a fluorescent protein gene that replaces the genes for HbF (Reynolds, 2005). The superior anti-oxidative ability of the RBCs can also extend the survival time of now not only the newly formed cells, however additionally of the transfused RBCs (Fernandes et al., 2005). B. Anticancer activity of wheatgrass juice:- Excessive antioxidant content material chlorophyll, laetrile and antioxidant enzyme super oxide dismutase (SOD) which converts unsafe free radical reactive oxygen species (ROS) into hydrogen peroxides (having greater oxygen molecule to kill cancer cells) and an oxygen molecule (Mates et al., 2000). Another constituent of wheatgrass associated as an anticancer agent is the plant hormone abscisic acid (ABA). This hormone is forty fold more potent 4 hours after cutting the wheatgrass plant. ABA can neutralize the effect of the hormone chorionic Gonadotropin and a compound comparable to this hormone has been discovered to be produced through the most cancers cells (Livingston, 1976). Other postulated mechanisms by using which wheatgrass juice seems beneficial include antioxidant activity in preventing oxidative harm to deoxyribonucleic acid (DNA) and lipid peroxidation, stimulation of gap junction communication, consequences on cell transformation and differentiation, inhibition of cells proliferation and oncogene (cancer causing gene) expression, outcomes on immune feature and inhibition of endogenous formation of carcinogens. (Wheat et al., 2008). The clinical studies performed on human breast cancers cells have proven that chlorophyllin, a compound similar to chlorophyll produced synthetically, has functionality to reduce the probability of breast cancer (Chiu et al., 2005).
405 C. Adjuvant therapy in haemolytic anemia: - The effects of the wheatgrass juice therapy may additionally be due to the action of herbal antioxidants of red blood cell (RBC) antioxidant characteristic and corresponding outcomes on cellular enzyme function and membrane integrity. This thought is supported by means of studies that show lowered antioxidant capacities of RBCs of sufferers with hemolytic problems as nicely as recommended effects on RBC life-span via supplementation of antioxidants in vivo (Shyam et al., 2007). It may additionally suggest that the natural antioxidants contained in the wheatgrass juice are better capable to avert cellular harm than to restore RBC enzymes/membranes once damaged. Therefore, wheatgrass juice and other dietary remedies may additionally be viewed as an adjuvant to drug therapy. D. Anti-ulcer activity Wheat grass: In a randomized, double-blind, placebo-controlled learn about on WGJ (Ben-Arye et al., 2002) observed that the use of wheat grass (Triticumaestivum) juice is very superb and secure as a single or adjuvant treatment of lively distal Ulcerative colitis (UC). Clinical research endorse that chlorophyll may be high-quality agent known for use in the therapy of supportive diseases, indolent ulcers or at any place stimulation of tissue repair is favored (Bowers, 1947; Singh et al., 2012). Which are believed to possess each anti-inflammatory and antioxidant residences as it is prosperous in bioflavonoid. One of this bioflavonoid, apigenin, has been shown to inhibit tumour necrosis issue triggered transactivation (Ben-Arye et al., 2002; Shah, 2007). E. Anti-arthritic activity of wheat grass:- In a study conducted to see the impact of raw vegetarian diet enriched with lactobacilli, in rheumatoid patients randomized into weight-reduction plan and manipulate groups, it has been discovered that and raw vegetarian diet, riched in lactobacilli, decreased subjective signs of rheumatoid arthritis. The research indicated that the following crew of dietary elements used to be in part (48%) accountable for the found minimize in the diseases activity index: fermented wheat drink, wheat grass drink, dietary fiber and iron. The research confirmed considerable response in arthritic sufferers (Nenonen et al., 1998; Kumar et al., 2011). F. Digestive System Disorders: Wheat grass juice used as an enema helps detoxify the walls of the colon. This enema is very beneficial in disorders of the colon, mucous and ulcerative colitis, persistent constipation and bleeding piles (Hvatum et al., 2006).
406 Conclusion Widespread information from a range of studies has made recognised the multitude results of Wheatgrass juice is recognized to help cut back fatigue, enhance sleep, expand strength, naturally regulate blood strain and blood sugar, support weight loss, enhance digestion and elimination, assist wholesome skin, eyes, muscles and joints, improve the characteristic of our heart-lungs and reproductive organs, heal ulcers and pores and skin sores, slow cell aging, improve intellectual function, and is recommended in arthritis and muscle cramping, Thalassemia, Hemo-lytic anemia, cancer, asthma, allergy, inflammatory bowel disorder and detoxification. Thus, it should be made part of day by day dietary intake of wheatgrass juice in order to discover its utmost benefits. The structural homology of chlorophyll with hemoglobin shows the role of chlorophyll as a blood builder in a variety of clinical stipulations involving hemoglobin deficiency. Thus it is called “green blood”. To conclude wheatgrass looks to be very promising natural drug and sizeable lookup work is wished in order to discover its therapeutic potential in a variety of diseases in future. References Ben-Arye, E., Goldin, E., Wengrower, D., Stamper, A., Kohn, R. and Berry, E. (2002). Wheatgrass juice in the treatment of active distal ulcerative colitis: A randomized double-blind placebo-controlled trial. Scand J Gastroenterol, 37(4):444-9. Bowers, W. (1947). Chlorophyll in wound healing and suppurative disease. Am J Surg, 73:37-50. Chiu, L.C., Kong, C.K. and Ooi, V.E. (2005). The Chlorophyllin induced cell cycle arrest and apoptosis in human breast cancer MCF 7 cells is associated with ERK deactivation and Cyclin D1 depletion. Int J Mol Med, 16(4):735-740. Chien, S.Y., Wu, Y.C., Chung, J.G., Yang, J.S., Lu, H.F., Tsou, M.F., Wood, W.G., Kuo, S.J. and Chen, D.R. (2009). Quercetin-induced apoptosis acts through mitochondrial and caspase-3-dependent pathways in human breast cancer MDAMB-231 cells. Hum Exp Toxicol, 28:493–503 Fibach, E., Burke, L.P., Schechter, A.N., Noguchi, C.T. and Rodgers, G.P. (1993). Hydroxyurea increases fetalhemoglobin in cultured erythroid cells derived from normal individuals and patients with sickle cell anemia or bete-thalassemia. Journal of American Society of Hematology. 81(6):1630-1635. Fernandes, C.J. and O’Donovan, D.J. (2005). Natural antioxidant therapy for patients with haemolytic anemia. Indian Pediatrics; 42:618-619. Gruenwald, J. (2009). Novel botanical ingredients for beverages. Clin Dermatol, 27:210– 216.
407 Hvatum, M., Kanerud, L., Hällgren, R. and Brandtzaeg, P. (2006). Hallgren R, Brandtzaeg P. The gut–joint axis: cross-reactive food antibodies in rheumatoid arthritis. Gut, 55:1240–1247. Haghiac, M. and Walle, T. (2005). Quercetin induces necrosis and apoptosis in SCC-9 oral cancer cells. Nutr Cancer, 53:220–31 Ketnil, N. (2014). Thailand food market report. National Food Institute. Kumar, P., Yadava, R.K., Gollen, B., Kumar, S., Verma, R.K. and Yadav, S. (2011). Nutritional Contents and Medicinal Properties of Wheat. Life Sciences and Medicine Research, 22 Livingston, (1976). Abscisic acid tablets and process, United States Patent, 395-8025. Lautraite, S., Musonda, A.C., Doehmer, J., Edwards, G.O. and Chipman, J.K. (2002). Flavonoids inhibit genetic toxicity produced by carcinogens in cells expressing CYP1A2 and CYP1A1. Mutagenesis, 17: 45–53. Marawaha, R.K., Bansal, D., Kaur, S. and Trehan, A. (2004). Wheat grass juice reduces transfusion requirement in patients with thalassemia major: a pilot study. Indian Pediatr. 41:716–720. Matés, J.M. and Sánchez-Jiménez, F.M. (2000). Fransisca M. Role of reactive oxygen species in apoptosis: implication for cancer therapy. The International Journal of Biochemistry and Cell Biology 32(2):157-170. Paulíèková, I. and Ehrenbergerová, J. (2007). Evaluation of barley grass as a potential source of some nutritional substances. Czech J Food Sci. 25:65–72. Padalia, S., Drabu, S., Raheja, I., Gupta, A. and Dhamija, M. (2010). Multitude potential of wheatgrass juice (green blood): an overview. Chron Young Sci.1 (2):23–28. Russo, M., Spagnuolo, C., Bilotto, S., Tedesco, I., Maiani, G. and Russo, G. (2014). Inhibition of protein kinase CK2 by quercetin enhances CD95-mediated apoptosis in a human thymus-derived T cell line. Food Res Int, 63:244–51. Shyam, R., Singh, S.N., Vats, P., Singh, V.K., Bajaj, R., Singh, S.B. and Banerjee, P.K. (2007). Wheat Grass Supplementation Decreases Oxidative Stress in Healthy Subjects: A Comparative Study with Spirulina. Journal of Alternative and Complementary Medicine 13(8):789-792. Reynolds, C.A. (2005). DNA technology-based cellular assay used to measure specific biological activity in a wheatgrass extract. Journal of Australasian Integrative Medicine Association, 1-3. Singh, N., Verma, P. and Pandey, B.R.. (2012). Therapeutic Potential of Organic Triticum aestivum Linn. (Wheat Grass) in Prevention and Treatment of Chronic Diseases. International Journal of Pharmaceutical Sciences and Drug Research, 4(1):10-14. Shah, S. (2007). Dietary Factors in the Modulation of Inflammatory Bowel Disease Activity Medscape General Medicine, 9(1):60. Tuli, H.S., Sandhu, S.S., Sharma, A.K and Gandhi, P. (2014). Anti-angiogenic activity of the extracted fermentation broth of an entomopathogenic fungus, Cordyceps
408 militaris 3936. Int J Pharm Pharm Sci, 6(7):581–3. 102. Tuli, H.S., Kashyap, D., Bedi, S.K., Kumar, P., Kumar, G. and Sandhu, S.S. (2015). Molecular aspects of metal oxide nanoparticle (MO-NPs) mediated pharmacological effects. Life Sci, 143:71–9. Wigmore, A. (1985). The wheatgrass book. New Jersey: Avery Publishing; 1985. Wheat, J. and Currie, G. (2008). Herbal medicine for cancer patients: An evidence based review. Internet Journal of Alternative Medicine, 5(2).
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27 SOYABEAN CULTIVATION AND ITS INTEGRATED PEST MANAGEMENT IS A BOON IN GANGATIC PLAIN ON PLACE OF RICE CULTIVATION IN RAINY SEASON H.C. Chaudhary1, A.K. Singh2, Ranju Kumari3, Anupama Kumari4 1,2,4
KVK, Saraiya,Muzaffarpur under Dr. R.P.C.A.U, Pusa, Samastipur. 3
NCOH under BAU, Sabaur, Bhagalpur
Soyabean (Glycine max) is a highest miracle crop of 20th century because its contains 60% vegetable protein and 30% of the oils content. It is cultivated for both as a pulse and oilseed crop. it can be fixes enormous amount of atmospheric nitrogen in soil. Soyabean got second position in vegetable oil economy of India after ground nut. Soybean can be grown successfully as an alternate crop in place of paddy in Bihar under changing climatic scenario for sustainable agriculture. Sandy loam soil suitable for its cultivation. The growth rates of rice and wheat yields are either stagnating or declining due to erratic and week mansoon. The productivity of paddy crops has already ceased to increase in most parts of Bihar and in most of the district which are depend on Rice-wheat cropping system, it has shown declining trends. Now cultivation of Paddy crop has become less profitable and is considered to be more prone to climate than wheat in context to sustainable agriculture production because: 1. Rice is a high water-demanding crop. To meet its water requirement lot of ground water is pumped out, with the result water table is going deep (Jeevandas et al., 2008). There are fears that Punjab, the most forward state in agriculture in the
410 country, may become desert due to continuous pumping out of large volume of water. 2. Week and erratic rainfall during sowing and transplanting. 3. Before transplanting of paddy, puddling is done, which results in creation of a hard pan in the soil. This hard pan is not broken with normal cultivation, with the result waterlogging takes place in low area in succeeding wheat crop, thereby decreasing its yield. 4. Cultivation of rice makes conducive conditions for the multiplication of insect pests and diseases. Rice cultivation has also other negative roles such as;i.
Increased population of mosquitoes due to stagnating water which helps in their breeding.
ii. Increased incidences of deficiencies of micronutrients in the crops and iii. Gluts in the market due to over-production of Paddy, thereby causing market problems. Above reasons, it is felt that for sustainable agriculture production Paddy must be replaced by other crops. Soybean (Glycine max) offers a good alternative crop in place of paddy because being a legume crop, it will not only meet its own nitrogen requirement to a great extent through biological nitrogen fixation but it will also leave considerable amounts of nitrogen in soil and in crop residues for utilization for the succeeding crops (Herridge et al., 2008). India imports vegetable oil, so soybean production in the country will not only help in meeting vegetable oil requirements but also save huge amount of foreign exchange. It is necessary to make any crop successful in any new area, it is a must to have good genotypes and improved production technology for realizing good yields. Our gangatic belt of Bihar and other parts of country are suitable for cultivation of Soyabean. Climate and soil: Soyabean needs about 15 to 320C temperature for germination but for rapid growth the crop needs higher temperature. The crop requires about 60-65 cm annual rainfall drought at flowering or just before flowering results in flower and pod drops, while rains during maturity impairs the grain quality of soyabean. It grows well in sandy loam to clay soil soils.
411 The best soil type is sandy loam having good organic matter content..Soil having pH of 7 and a fair degree of water retention capacity are best suited for its cultivation.. The crop is highly sensitive to latitude because of its photosensitivity. The optimum temperature for germination of soyabean is approximately 300 . It can be grown rainy, spring and summer seasons. Crop rotation: Crop rotation is important in all crops to break disease and insect cycles and increase yield, and soybean is no exception. Diseases such as soybean cyst nematode, white mold, brown stem rot and sudden death syndrome survive in the soil or in crop residue, and readily attack a successive soybean crop. Most soybean diseases survive more than one or two years in the soil, so rotation does not eliminate the problem. But time away from soybeans diminishes the amount of disease inoculum available to infect the next crop, and thereby lessens its severity. For this reason, two or more years away from soybeans is preferable to just one, in terms of disease impact on the crop. Rotation studies in Minnesota and Wisconsin showed that soybeans in a corn/soybean rotation yielded 8% more than continuous soybeans. These studies were conducted in good growing environments where moisture was not severely limiting. Soybeans following five years of continuous corn yielded 15 to 17% more than continuous soybeans. Soyabean cultivation fitted in different types of cropping system which are namely Soyabean-Wheat, Soyabean-Potato-Green gram, SoyabeanChickpea, Soyabean-Mustard-green gram etc. Lant preparation: Tillage has long been used to bury crop residue, prepare a seedbed and control weeds. Current planting equipment and herbicides now allow growers to achieve excellent soybean stand establishment and weed control with little or no tillage. No-till or reduced till practices can help minimize soil loss and increase organic matter levels that contribute to long-term productivity. Research studies have demonstrated that soybeans yields are similar across conventional till, minimum till and no-till. For this reason, growers can choose a tillage system that makes sense economically, environmentally and logistically, and focus on optimizing other management practices within that tillage system. being a leguminous crop it requires fine seed bed and deep ploughing followed by 2-3 cross harrowing should be done. To avoid water stagnation.
412 Variety:PK-416,PK472,VLS-21,Pusa-16,PS-1024,PS1042,PS-1347,PS1029,PS-1092,SL-295, PS 1241,SL-525,Pusa-9814,Pusa-9712,SL-688,PS1225. Time of sowing: Soyabean is a mostly rainy season crop, sown in June last week to first week of July and harvested in October. Seed rate, spacing and plant population: Seed rate depend upon the viability of seed, size of the seed and variety. Generally 70 -80 kg seed /ha is recommended in kharif season. Row to row 30 to 45 cm and plant to plant 5 to 10 cm require for maintain plant population 0.4 to 0.6 million / ha. depth of sowing require 3-4 cm for sandy loam. Sowing must be done with the help of seed drill or multi -crop planter. Seed treatment and seed inoculum: for prevention of seed born disease seed must be treated with Thirum 75 WP + Carbendazim 50WP (2:1) @ 3g/kg of seed or Trichoderma viride 5-10 g/kg of seed. For getting highest yield seed should be inoculated with Bradyrhizobium japonicum and PSB/PSM @ 500 g. each /70 to 80 kg of seed . Mannuring and Fertilization: It depends on expected yield, soil-test based, productivity potential, cropping system etc. In addition to 10 tones FYM/ha 20 kg N,60-80kg P2O5, 20 kg K2O and Sulphur recommended for its cultivation.FYM must be applied at the time of first ploughing/land preparation. Fertilizer must be applied at the time of final land preparation / at the time of sowing. In northern plain application of 5 kg Zinc sulphate monohydrated is recommended in addition to recommended dose of NPK and S. Weed management: 25 to 30 days after sowing the soyabean field must be kept weed free by manual hand weeding. Pre emergence application of Fluchloralin or Pendimetalin @ 1 kg a.i./ha as pre plant incorporation or Imazetapyr @ 100g a.i./ha or Clorimuron-ethyl @10 g a.i. or quizalophop ethyl @50 g a.i./ha as post emergence (15-20 DAS) in 700 to 800 lt. of water/ha. Water management: Avoid flooding in any stage of the crop. In case of Kharif crop generally water is not needed otherwise crop needs water at critical growth stage (seed emergence, flowering , pod initiation, grain filling) light irrigation is required. Irrigate the crop before pod development and filling stage.
413 Harvesting and threshing: When pod are golden yellow, the crop are ready for harvest. After harvest moisture content of seed should be 15-17%. Soyabean can be harvested by using reapers oor manually whean the crop are 90 to 140 days old depending upon the varieties. It can be threshed by using wheat thresher with change in sieve, reduction in cylinder speed 300 to 400 rpm and increase in fan speed. Moisture cntent of seed is 13-14 % ideal for threshing with thresher. Threshing should be done carefully and any kind of severe seed coat damage which results poor germination. Yield potential: Under rainfed condition seed yield varies from 16 to 20 q/ha nad irrigated condition it varies from 20 to 25 q/ha. Drying and storage: for maintaining of longer shelf life and avoid fungal infestation during storage, drying to 10% seed moisture content is essential. Suggestion: 1. Viability of soyabean seed is vary low ( 1 year). So use less than one year old seeds for sowing. 2. Harvest the crop immediately after maturity, otherwise pods will split/shelter and seed fall on the ground. 3. Recycle the previous crop residues back the soil which on decomposition acts as an organic manure. 4. Organic content of the soil should be more than 2%, so that nitrogen fixing bacteria can thrive well. 5. Ferrus sulphate should be apply @ 10 to 12 kg/ha for getting highest yield. Insect pest and disease: Soybean being luxuriant crop, having lush green, soft, succulent and nutritive dense foliage is attacked by over 273 types of insects. Out of the whole range, however, only about two dozens of insects are of significant importance. 1. Bihar hairy caterpiller: Spilosoma Obliqua (Wal.) (Lepidoptera: Arctidae)
414 Nature of damage: Early instars skelatonise the leaves gregariously. Young larvae feed gregariously on the under surface of the leaves and cause loss by way of defoliation and the leaves of the plant give an appearance of net and web Some times after defoliated the crop larvae feed on the pods also. Pupation takes place in the soil, dry foliage and plant debris. Soyabean stem fly: Melanagromyza sojae (Diptera: Agromyzidae) Symptoms of damage : The adult flies are shining black and about 2 mm long. The damaging stage is maggot which is white in colour and remains inside the stem. Female adults lay eggs inside the cotyledon or leaves. After hatching from eggs, the maggots mine through the leaf and reach the petiole. From petiole, they enter the branch and stem, and feed on xylum and phloem. This results in non availability of food material to the plants. When grown up plants are attacked, they do not show any external symptoms of damage except the exit holes made by the maggots for emergence of adults. Infestation due to stem flies reduce the grain yield by 16 to 20%. With proper control measures at least 12% yield loss can be avoided. Girdle beetle: Oberopsis brevis Symptoms of damage The initial damage is caused by the female which makes two parallel girdles with its mandibles either on petiole, branch or main stem, for egg laying. These girdles reduce the water supply and thus ensure successful hatching. A female can make 4-6 pairs of girdles on a single plant; eventually the entire portion above the girdles dries. This insect remains active from July to October damaging most severely during August-September.
415 The grubs after hatching from eggs, move downwards by tunnelling the petiole or branch or the stem as the case may be, finally reaching upto the ground level. It has been found that about 75% of the plants get totally damaged when the insect attacks 15-17 days old crop. When the crop is attacked between one and a half to two months age, the plants will appear healthy from outside, but there may be a reduction upto 53% in pod number, 65.5% in pod weight, 56.5% in grain number and 66.6^ in grain weight. This loss is further increased when the grubs cut the stem of maturing plants from 5 to 25 cms above the ground level causing a net loss of 5.43 kg of grains for every per cent of infestation. In general, if 25% of the total plant population is infested by girdle beetle, an inevitable loss of about 136 kg/ha is observed. Early sowing is more prone to attack. The grubs of this insect which tend to fail to complete their development within the same season, enter into diapause during winter. The grubs cut small portion of the stem and crawl along with it by remaining inside and closing one end of it. Finding a suitable place in the crevices they close the other end also and remain till the onset of monsoon (July) when the grubs develop into pupae from which adults emerge out in 8-11 days and again start infesting the crop. Leaf folder : Hedylepta indicate Symptoms of damage : Larvae are defoliators, feeding only on leaf tissue of legumes. Initially the larva cuts a small, triangular patch at the edge of the leaf, folds over the flap, and takes up residence within this shelter. The larva leaves the shelter to feed, and lines the shelter with silk. These flaps are used until the third or fourth instar, when the larva constructs a larger shelter formed by folding over a large section of the leaf by webbing together two separate leaves. Again, the leaf fold is used for shelter, the larva leaving to feed. Larvae feed nocturnally.
416 Leaf minor: Aproerema modicella Nature of Damages: Newly hatched larvae mine into the leaves and feed between the epidermis. One leaf may contain 2-4 mines, Later on the larvae stick the adjacent leaves together and feed by remaining inside the folded cup-shaped structure. One account of heavy infestation, plant growth retards, pod number reduces and gr ains become shrivelled, consequently yield losses up to 50% may occur. Control measures: Soybean leaf miner is a rare pest in soybean and therefore an threshold has not been developed. However, in areas with persistent defoliation or bean pod mottle virus, monitor fields closely throughout the season. Foliar spray of Quinolphos 25 EC @ 1.0 lt./ha. Green semilooper : Chrysodeixis acuta Nature of Damage: Caterpillars first feed on leaves by scratching the green matter. Later on, grown up larvae consume entire leaf leaving behind only the midribs and veins. When not controlled, the infestation can result into 30% undeveloped pods and about 50% yield loss. In case of heavy attack, the caterpillars are also found to feed on flowers and pods. The caterpillars of this insect are found to suffer from a fungal disease. Pod borer : Heliothis armigera Symptoms of damage: On Hatching, the larvae feed for a short time on the tender leaflets by scrapping green tissueand then shift to flower buds and tender shoots. Slowly it enters and feeds on the seeds inside the pods. The half portion of larvae remains inside pod while feeding n the developing seeds. They can cut hole on one to another locule and feed 20-25 pods in its lifetime.
417 Tobacco Caterpillar: Spodoptera litura Nature of Damages: Newly hatched larvae have characteristic gregarious feeding behaviour. They feed only on the chlorophyll leaving behind the white thin papery leaves. Late r instars gradually disperse and feed on the leaves eating away the entire portion. After eating the leaves, they also start feeding on young pods, consequently damaging 30-50% of the pods. It has been observed that soybean crop with higher dose of nitrogen are more prone to attack. Green Jassids Nature of Damage: Adults and nymphs are light green in colour and suck the sap from leaves and stem. Infested leaves start yellowing from the margins. In case of severe attack, all the leaves become yellow and eventually fall off the plants. Whitefly Nature of Damages: Both adults and nymphs suck the plant sap from tender leaves and stem, and transmit YMV disease. Plants suffering from this disease have reduced pod formation and yield. It is reported that if infection occurs when plants are 30 days old, reduction in yield is upto 19% compared to healthy plants. Thrips Nature of Damage: Both nymphs and adults scratch the leaf surface and lick the sap coming out of the scratches. This results into brownish spots the leaves. Important Disease of Soyabean Charcoal rot : Rhizoctonia bataticola Symptoms: Symptoms usually begin during the reproductive stages of soybean development and are first evident in the driest areas of the field. Earliest symptom s include smaller than normal leaves, reduced vigor, premature yellowing of top leaves and plants wilting during the midday heat. A light gray
418 discoloration develops on the surface tissues of the roots and lower stem Control measure: Treat the seed with
[email protected] + Carbendazim @ 1.5 g/kg of seed before sowing. Rhizoctonia aerial blight : Rhizoctonia solani Symtoms: The infection is more common during vegetative stage. Round to irregular grayish to green water soaked spot developed on the leaves. Spots grow into brown necrotic lesion with reddish brown margin. Lesion can spread to he petiole, stem and leading to the pod also. Leaves can woven together with cottony growth of the fungus. The colonization to the plant by the fungus lading to defoliation. Severe infestation cause stem and pod blight. Alternaria leaf spot : Alternaria alternata Symptoms: Alternaria leaf spot of soybean, caused by a fungal species called Alternaria spp., is usually a secondary invader following mechanical injury, insect damage, or another disease. Alternaria leaf spot occasionally appears in seedlings but generally attacks leaves and pods of plants approaching maturity. Because the disease usually occurs late in the growing season, yield losses typically are minimal. Diseased lesions are round or restricted by a major vein or merge with another lesion. Some have brown concentric rings with a well-defined border. The lesions expand and may combine to yield larger dead areas on the leaves. Infected leaves eventually dry out and fall. Anthracnose: Colletotrichum truncatum Symptoms: When infected seeds are planted pre-emergence and postemergence damping-off may occur. Sunken, dark brown lesions develop on the cotyledons of seedlings. Seedling lesions may expand to the stem and kill young plants. Plants may become infected at any stage of development, but in Nebraska, chances of infection tend to increase with
419 maturity. The most common symptoms are brown, irregularly shaped spots on stem, pods and petioles. The girding of petioles by large lesions results in premature defoliation. When pods are infected, mycelium may completely fill the cavity and no seeds are produced (pod blanking) or fewer and/or smaller seed form. Seed that does form may appear brown, moldy and shriveled or may look normal. Dark acervuli develop in lesions on all host tissue areas. Leaf infections result in leaf rolling, necrosis of laminar veins, petiole cankers and premature defoliation. This disease is commonly observed on soybean stems at maturity. Bacterial Pastules : Xanthomonas phaseoli Symptoms: Early symptoms consist of small, pale green spots with raised centers on leaves in the mid- to upper canopy. As the disease progresses, small brown-colored pustules form in the middle of the spots and the spots turn yellow. The spots may merge, forming large irregularly yellowing lesions. Bacterial pustule symptoms are easily confused with soybean rust pustules. Soybean rustpustules have circular openings at the top as gateways for spore spread. Bacterial pustules do not have this feature although under microscope bacterial pustules may appear to have crack openings on their surfaces. Bacterial pustule lesions are sometimes confused with the lesions caused by bacterial leaf blight. Bacterial leaf blight lesions appear water-soaked while the lesions of bacterial pustule do not. As in other bacterial diseases, if a soybean leaf with lesions due to bacterial pustule is cut and submerged in water, bacteria will stream out of the infected tissue. Control Measure: Scouting is recommended between R1 through R6 of soybean developmental stages. Resistant varieties and pathogen-free seeds should be used to manage bacterial pustule. Crop rotation and plowing the field before planting also help to reduce field inoculum level of the pathogenic bacterium. Try to avoid cultivation when the leaves are wet. Spray Streptocycline / Bacterinashak 200/ Streptomycin sulphate and copper oxychloride have been found superior for reducing the infestation. Bacterial blight (Pseudomonas glycinea) Symptoms: Bacterial blight can be identified by small, angular, translucent, water-soaked, yellow to light-brown spots on the leaves and petioles. As bacterial blight progresses, affected leaf tissues dry out, turn reddish-brown to black, and become surrounded by water-soaked margins bordered by
420 yellowish-green halos (Figure 1). In advanced stages, lesions enlarge and their interiors tend to produce large, irregularly shaped dead areas. Frequently, the leaves are badly shredded after strong winds and/ or hard rains. This gives affected leaves a very ragged appearance. Infected young leaves frequently are distorted, stunted, and chlorotic. Control Measure: Soak the seeds with a solution of plantomycin 10gms or streptocyclin 1.5gms and copper oxychloride 25gms in 10 litres of water. Spray the affected crop with the same chemicals at 500 litres /ha at 7-10 days intervals 2-3 times on need basis. Streptomycin sulphate and copper oxychloride have been found superior for reducing the infestation . Soyabean Mosaic Virus: Symptoms: Symptoms of plants infected with soybean mosaic virus can range from no apparent symptoms to severely mottled and deformed leaves. Mottling appears as light and dark green patches on individual leaves. Symptoms are most obvious on young, rapidly growing leaves. Infected leaf blades can become puckered along the veins and curled downward. Soybean mosaic virus can cause plant stunting, reduced seed size, and reduced pod number per plant. The disease is one of several factors associated with discoloration of seeds, causing a dark discoloration at the hilum. Symptoms of SMV may not apparent when temperatures are above 900F. Symptoms are often confused with growth regulator herbicide damage where the leaves will be elongated and which usually occurs in a pattern such as along a field edge. SMV can interact with bean pod mottle virus (BPMV) to create severe symptoms in plants infected with both viruses. Soyabean Rust: Symptoms begin on leaves in the lower plant canopy. Tan or reddish-brown lesions (spots) develop first on the underside of leaves. Small pustules (blisters) develop in the lesions, which break open and release masses of tan
421 spores. The lesions and pustules, which can be seen with a 20X hand lens, may also appear on pods and stems. Early symptoms of soybean rust (before pustules develop) are difficult to distinguish from other common leaf diseases on soybean. Synopsis of Integrated Pest management in Soyabean: Mechanical Practices: 1. Deep summer ploughing during summer month should be done. 2. Soil solarisation with polythene sheet during summer month should be done. 3. Collection and destruction of Infected plant from the field. 4. 10 to 12 bird percher should be established in cultivated land 5. For management of Tobacco caterpillar and Pod borer 10 to 12 pheromone trap should be installed. 6. Use of castor as trap crop of tobacco caterpillar and dhaincha for girdle beetle. 7. Use diseases and insect-pest resistant variety . Biological Control: 1. Conserve Spiders, Coccinellid beetle, Tachinid fly, Praying mantidis, Dragon fly, Chrysoperla and Meadow grass hopper through minimal use of broad spectrum pesticides. 2. Spray Bacillus thuringenesis var. kurstaki, Serotype H-39, 3b,strain Z-52 @ 0.75 to 1.0 kg for the management of semilooper complex . 3. Spray SL NPV for Tobacco caterpillar and HA NPV for Pod borer. 4. Spray Neem oil 300 ppm @ 3 ml/Lt of water. 5. Spray NSKE @ 5% for management of early stage of larvae and sucking pest. 6. Seed treatment of Trichoderma viridi @ 5-10 g /kg .
422 Chemical Control: For insect –pest: Insect
Scientific name
Insecticides
Dose
Tobacco caterpillar
Spodoptera Litura
Chlorantraniliprole 18.5% SC
150 ml/ha
Indexacard15.8 % EC
333ml/ha
Quinolphos 25EC
1000ml/ha
Lambda Cyholothrin 5%EC
500 ml/ha
Chlorantraniliprole 18.5% SC
150 ml/ha
Indexacard15.8 % EC
333ml/ha
Quinolphos 25EC
1000ml/ha
Lambda Cyholothrin 5%EC
500 ml/ha
Thiomethoxan 30% FS
10 kg /ha (Soil) 500ml/ ha (Spray)
Acetamiprid20 % SP
75-100 g/ha
Pod Borer
Hiliothis armigera
White fly, Jassid Thrips Bemissia Tabaci
Stem fly
Girdle beetle
Melanogromyza sojae Thiomethoxan 30% FS
Obereopsis brevis
Bihar hairy catterpiller Spilosoma Obliqua
10 kg /ha (Soil) 500ml/ ha (Spray)
Chlorantranilipore 10.5% SC
150ml/ha
Triazophos 40EC
625 ml/ha
Chlorantraniliprole 18.5% SC
150 ml/ha
Chlorantraniliprole 18.5% SC Quinalphos 25EC Chlorpyriphos 20EC
150 ml/ha 1.0 lt/ha1.0 lt/ha
Leaf Minor
Aproerema modicella Quinolphos 25EC Methyldemeton 25 EC Dimethoate 30 EC
1000ml/ha 750 ml/ha 1000ml/lt
Leaf Folder
Hedylepta indicate
Quinalphos 25EC Chlorpyriphos 20EC
1.0 lt/ha1.0 lt/ ha
Green Semilooper
Chrysodeixis acuata
Chlorantraniliprole 18.5% SC Quinalphos 25EC Chlorpyriphos 20EC
150 ml/ha 1.0 lt/ha1.0 lt/ha
423 For Disease: Name of the disease
Chemical
Charcoal rot
Treat the seed with
[email protected] + Carbendazim @ 1.5 g/kg of seed before sowing. Spray Thiophanate methyl @ 0.5 kg /ha when the disease appears,second spray be given 15-20 days after first spray.
Rhizoctonia aerial blight
Treat the seed with
[email protected] + Carbendazim @ 1.5 g/kg of seed before sowing. Spray Thiophanate methyl @ 0.5 kg /ha when the disease appears, second spray be given 15-20 days after first spray.
Alternaria leaf spot
Treat the seed with
[email protected] + Carbendazim @ 1.5 g/kg of seed before sowing. Spray Mancozeb 63% and Cabendazim 12% or @ 2g /lt of water when the disease appears, second spray be given 15-20 days after first spray.
Anthracnose
Spray one of the following: Difenoconazole @30 ml/100 litre wate Thiophenate methyl @200 gm/100 litre water Chlorothalonil + metalaxyl @250 gm/100 litre water Fostyl aluminium @ 500 gm/100 litre of water
Bacterial Pastules
Soak the seeds with a solution of plantomycin 10gms or streptocyclin 1.5gms and copper oxychloride 25gms in 10 litres of water Spray with Streptomycin sulphate Copper oxychloride.
Bacterial Blight
Soak the seeds with a solution of plantomycin 10gms or streptocyclin 1.5gms and copper oxychloride 25gms in 10 litres of water Spray with Streptomycin sulphate Copper oxychloride.
Soyabean Mosaic Virus:
Spray with Thiomethoxan 30% FS@ 500ml/ha and Acetamiprid20 % SP @ 100g/ha
Soyabean rust
Give prophylactic spray of Hexaconazole or propiconazole or Tridimefon of oxycarboxyn @ 0.8 lyt./ ha.
References: Hand book of Agriculture, ICAR, New Delhi. John Capinera, university of florida, Charudatta D Thipse. Krishi vigyan Kendra, Udegaon
424
425
28 GENETIC ENGINEERING APPROACHES IN PLANT DISEASE MANAGEMENT: CURRENT AND PROSPECT Satish Kumar1*, Pawan Kumar2, Sardar Sunil Singh3, Alok Kumar4 and Vinay Kumar5 1.
2.
Asstt. Prof.cum-Jr-Scientist, Deptt. Vegetable, NCOH , Noorsarai, Nalanda
Asstt . Prof.cum-Jr-Scientist, Deptt. Fruit and Fruit Technology, BAU , Sabour, Bhagalpur 3.
4.
Asstt . Prof.cum-Jr-Scientist, Deptt. PBG NCOH , Noorsarai, Nalanda
Asstt . Prof.cum-Jr-Scientist, Deptt. Basic science & Humanities – Genetics, NCOH , Noorsarai, Nalanda 5.
Asstt . Prof.cum-Jr-Scientist, Deptt. Plant Pathology NCOH, Noorsarai, Nalanda E.mail:
[email protected]
INTRODUCTION Agricultural crops are being threatened by a wide range of diseases and insect-pests which is lowering of production and quality even some time leading to wipe out entire harvest. About 42% of the world’s total agricultural crops are destroyed yearly by diseases and pests. Farmers often must contend with more than one pest or diseases and new pesticideresistant pathogenic strains attacking the same crop. However, crop losses can be minimized and specific treatments can be tailored to combat specific pathogens if plant diseases are correct diagnosed and identified early. These
426 need-based treatments also translate to economic and environmental gain. The traditional method of identified plant pathogens is through visual examination. But pathogens capable of causing systemic infections on their host plants are usually transmitted through vegetative propagation from infected mother plants to the young plants. Infected seedlings or planting materials can easily be transported to new areas, resulting in the invasion and establishment of new diseases. In area where the pathogens already exit, infected seedlings or planting materials serve as inoculums sources of pathogens that can be transmitted by insects and others, causing outbreaks of diseases. Because these pathogens are always located inside the cells and systemically spread throughout the plant, the diseases they cause are often very difficult to control by conventional measure such as pesticide sprays. Diseases causing organisms involved in plant defence and in limiting the spread of infection. Pathogens also produce protein and toxins to facilitate their infection, before diseases symptoms appear. The possibilities of genetic engineering in disease management are enormous. Modern biotechnology offers many applications to diagnose diseases caused by pathogens as well as diseases caused by intrinsic genetic disorders of an organism. This technique helps in sustainable agriculture and precision farming. Molecular technique helps us to rapid screen large populations of plants for several plant diseases simultaneously with high precision. That probes can detect pathogens even occurring in low quantities as well as in plants with no apparent symptoms. Molecular technique is to develop plants resistant to diseases and insect pests. The molecules play vital role in the development of plant diagnostic kits. Advances in molecular biology, plant pathology and biotechnology have made the development of molecular tools to detect plant diseases early, either by identifying the presence of the pathogen in the plant (by testing for the presence of pathogens DNA) or molecules( protein) produced by either the pathogen or plant during infection. The techniques require minimal processing time and are more accurate in identifying pathogens. Using Real Time PCR, it is possible not only to detect the presence or absence of the target pathogen, but also possible to quantify the number of the pathogen in the sample. Agricultural crops can be attacked by many pathogens which, in addition, often occur in complexes. Therefore, many diseases diagnostic applications requite simultaneous detection and quantification of several targets. DNA Microassay technology, originally designed to study gene expression and generate single nucleotide polymorphism (SNP) profiles, is currently a new and emerging pathogen diagnostic technology, which in there, offer a platform for unlimited multiplexing capability. It is viewed as a technology that fundamentally alters molecular diagnostic. The fast growing databases
427 generated by genomics and biosystematics research provides unique opportunity for the design of more versatile, high-through out, sensitive and specific molecular assays which will address the major limitations of the current technology and benefit plant pathology. An attempt has been made in this paper to introduce the plant pathologist to the basics and major techniques of genetic engineering relevant to diseases management and to assess the potential of such techniques in devicing novel strategies of management. Principles and techniques of genetic engineering in disease management 1. Diseases management through molecular diagnostics 2. Developing transgenic plants resistant to diseases. Table: 01 Cause
Description
How they reproduce
Fungi
Grow as tiny Spores, cell thread like division filaments, large fruiting structures may develop from these filaments
Bacteria
Tiny single- Cell division Microscope 400-1,000 cell organism
Bacterial blights, fire blight, canker, bacterial wilt
Viruses
Very tiny rod Cause host to Electron shaped or manufacture microscope spherical virus particles, composed of RNA/DNA with a protein coat
20,000100,000
Tomato leaf curl, papaya ring spot virus, mosaics
20,00050,000
Aster yellows, little leaf, big bud (purple top in potato and tomato
Phytoplasmas Very tiny Division organisms without a cell wall, no definite shape
Equipment
Magnification Examples required
Microscope 20-250
Electron microscope
Rust, smuts, leaf spots, wilt, powdery mildew , scabs
428
Nematodes
Tiny Round worm
Eggs
Microscope 1-60 (naked eye)
Disease caused by for larger forms) organism in India
#The number of times pathogen must be magnified to be visible. For example, a pathogen 1/1,000 inch in size when magnified 100 times would appear to be 1/10 inch in size; a pathogen 1/1000000 inch in size when magnified 100000 times would appear to be 1/10 inch in size.
Steps of general methods used for identification of plant diseases: 1. Check for the symptoms 2. Look for signs of diseases causal agents 3. Identify plant part affect 4. Check distribution of symptoms 5. Check for host specificity 6. Then recommend control measure Table: 02 - Difference between general methods and molecular method used for identification of plant diseases: S.No
Identification of plant diseases: General methods
Molecular method
1.
Identification is time consuming
Identification in very less time
2
Lesser accuracy than molecular method
Highly accuracy
3.
Not suitable for analysis of a large number of sample
Suitable for analysis of a large number of sample
4.
Not always sensitive and specific enough
Always sensitive and specific enough
(C) Molecular technique for fungal diseases management: Fungi represent decomposers in ecosystems. It is conservatively estimated that 1.5 million species of fungi exit. This is the greatest eukaryotic diversity on earth. Many species of fungi cause diseases in plants, animals, and humans. Accurate and robust detection and quantification of fungi is essential for diagnostic, modelling and surveillance and implementing a diseases management strategy. Therefore, molecular techniques are required when attempting to detect fungi in the environment.
429 Various strategies have been suggested to engineer fungus resistant transgenic plants (Herrera- Estrella et al. 1996) when a plant is attacked by an invading pathogen, it responds by inducing a set of protein know as pathogenesis related (PR) proteins. These protein are coded by at least nine gene families which includes basic and acidic chitinase, 1,3 beta D glucanases, hevein, thaumatin, osmotin-like protein etc. Transfer of genes encoding PR proteins is therefore an important strategy to impart resistance in plants Chitinase abd glucanase destroy fungal cell wall containing glucan and chitin and thereby inhibit fungal growth. Zhu et al. (1994) have expressed rice chitinase and alfalfa glucanase under a constitutive promoter in tobacco plants and found enhanced protection against fungal resistance. (D) Molecular technique for bacterial diseases management: Significant progress has been made in engineering bacterial and fungal resistance in plants. The most promising strategy for resistance c against bacterial diseases is to transfer into crop plant genes that detoxify the bacterial toxin. In this approach, transgenic tobacco plants resistant to the bacterial pathogen Pseudomonas syringae, which that causes wildfire in tobacco, have been obtained by expressing a tabtoxin resistant gene (ttr) this gene encodes a tabtoxin inhibiting acetylase which inactivates tabtoxin by acetylation. Transgenic plants expressing ttr gene were less susceptible to the wildfire diseases than control plants .Bacterial genes encoding S enzymes degrading the bacterial cell wall have been introduced into plants e.g. the lysozyme gene from hen eggwhgite has been transferred into tobacco And potatos. Sebsequent infection with pathogenic Erwinia carotovora sp. Atroseptica showed protection against this bacterium ( Trudel et al. 1992) (E) Molecular technique for viral diseases management: Plant viruses major losses to several agricultural and horticultural crops around the world. Unlike other plant pathogens, there are direct methods available yet to control viruses and, consequently, the current measures rely on indirect tactics to manage the viral diseases. Therefore, methods for detection and identification of viruses, both in plants and vectors, play a critical role to virus diseases management. Expression of the coat protein gene in transgenic plants provides protection to crops against viruses. The possible mechanism of protection due to coat protein could be their interference with the uncoating of incoming virus particles in the cell or quick coating of the incoming viral nucleic acid thus preventing its replication and expression. Powell-Abel et al. (1986) have produced the first transgenic tobacco plants containing the protein gene of TMV
430 under the control of the constitutive 35S promoter. Similarity has also been achieved by transferring the genes of satellite RNA and antisense RNA in a number of crops (Harrison et al. 1987) Vegetable crops for viral resistance engineered through various approaches are listed below Table 03 Virus –resistant transgenic vegetable crops Crops
Gene
virus
Cucumber
Coat protein
Cucumber mosaic , melon mosaic 2, zucchini yellow mosaic
Lettuce
Nucleocapsid
Tomato spotted wilt
Melon
Coat protein
Cucumber mosaic , , zucchini yellow mosaic, papaya ring spot, watermelon mosaic 2
Potato
Antisense coat protein Potato leaf roll, potato virus , potato virus x Coat protein
Tobacco rattle , tobacco vein mottling
Coat protein
Barley yellow dwarf, bean yellow mosaic
17KD
Potato leaf roll
replicase
Potato leaf roll
protease
Potato leaf roll
Squash
Coat protein
papaya ring spot, Cucumber mosaic, zucchini yellow mosaic, watermelon mosaic 2
tomato
CBI Nucleocapsid
Coat protein papaya ring spot, Cucumber mosaic, zucchini yellow mosaic
Coat protein
Beet curly top, tomato yellow leaf curly, tomato mosaic
Coat protein Replicase Cucumber mosaic ,tobacco mosaic watermelon
Coat protein
watermelon mosaic 2, zucchini yellow mosaic
(Source: Malik1990)
(F) Molecular technique for insect resistance: Crops are infected by various insect pests which seriously affect productivity. Agricultural productivity is highly influenced by insect pests and diseases; known as the most harmful factors concerning growth and productivity of crops worldwide (Karthikeyan et al. 2011) , causing the
431 loss of 15% worldwide (Maxmen, 2013) .Chemical insecticides that have been used to control these pests pose a serious threat to environment and human health . An alternative approach to control pests is genetic engineering of crop plants with genes encoding insecticidal proteins. A numbers of protein from various microorganisms and plant are known to be toxic to important pests of crop plants. Genes encoding these insecticidal proteins have been cloned and transferred to crop plant to enable protection against insect attack (Schuler et al. 1998). Of the several insecticidal proteins, the crystal proteins of Bacillus thuringiensis are being currently exploited to engineer crop plants. Genes of plant origin such as lectins , protease inhibitors, amylase etc., are also being used for production of transgenic crops. The advances in genetic engineering have emerged a powerful modality against the important insect pests. Conventional breeding methods are being used to develop the varieties more resistant to biotic stresses, at the same time are time taking, resource consuming and germplasm dependent. Besides, it requires evaluation at hot spot area and does not give consistent results, but improves flavour and nutrition. More often, the traditional crop breeders are focused on disease and insect pest resistance, these efforts are regional in nature (Andrew, 2004). On the other hand, insect pest management by chemicals obviously has brought about considerable protection to the crop yields over the past few decades. Regrettably, extensive and very often indiscriminate usage of chemical pesticides has resulted in the eradications of beneficial insects and development of pest resistant insects. At this situation, tool of the genetic engineering has provided human kind with unprecedented power to manipulate and develop the novel crop genotypes towards a safe and sustainable agriculture in the 21 stcentury. One of the best modern agricultural defences against plant-eating insects is Bacillus thuringiensis (Ibrahim and Shawer, 2004). Cry genes from Bacillus thuringiensis Transgenic crops modified by cry (Bt) genes obtained from bacterium Bacillus thuringiensis, are the firstly used insecticidal genes for plant transformation (Table 04). B. thuringiensis is a Gram-positive bacterium producing highly insecticidal protein crystals toxins during sporulation. Digestive system of the insect is the first target of this toxin. After ingestion by susceptible insects, toxins bind to specific receptors in the gut and are solubilized and activated by proteinases in the insect midgut epithelium. More than 400 genes encoding toxins from wide range of B. thuringiensis have been identified so far. Many of the identified cry genes (e.g, cry1Aa, cry1Ab, cry1Ac, cry1Ba, cry1Ca, cry1H, cry2Aa, cry3A, cry6A, cry9C, cry1F) have been engineered into plants against insect pests. Most cry proteins, even within cry1A subfamily have a distinctive
432 insecticidal spectrum. While most crystal toxins are specific to larvae of Lepidopteran pests, some others are toxic to Coleopteran, or Dipteran pests. The level of expression of wild-type Bt toxins in transgenic plants is low compared to many other heterogonous genes, but become sufficient to cause high mortality of target pests in the field. Transgenic crops are used worldwide to control major pests of the cotton, corn and soyabean. The first planted cultivars were corn producing Bt toxin Cry1Ab and cotton producing Bt toxin Cry1Ac (Tabashnik, 2009). Table 04: Successful examples of B. thuringiensis genes integration for insect pest resistance in plants S.no
Gene
Target pest
Reference
1
1 cry 1 a (b)
yellow stem borer & pink borer
Prashantya et al.
1
1 cry 1 a (b)
yellow stem borer & striped stem orer
Wunn et al.
2
2 cry 1a(b)
yellow stem borer & striped stem borer Ghareyazie et al.
3
cry 1a(b)
yellow stem borer
Datta et al.
4
cry1a(b)/ cry1a(c)
leaf folder & yellow stem borer
Tu et al.
5
cry 1a(b)/ cry1a(c
yellow stem borer
Ramesh et al.
6
cry 1a(c)
yellow stem borer
Nayak et al.
7
cry 1a(c)
yellow stem borer
Khanna and Raina
8
cry 1a(c)
striped stem borer
Liu et al
9
cry 2a
10
leaf folder & yellow stem borer
Maqbool et al
cry 2a/ cry 1a(c)
leaf folder & yellow stem borer
Maqbool et al.
11
cry 1le
1le corn borer
Liu et al.
12
cry 1a(b)
striped stem borer & leaf folder
Fujimoto et al
The closest to the commercial used cry genes against insect pests are the VIP genes from B. thuringiensis and B. cereus. Vip3Aa1, is highly insecticidal to several major lepidopteran pests of maize and cotton. A highly effective protein (cholesterol-oxidase) that killed the larvae of the boll weevil (A. grandis) was discovered in Streptomyces culture filtrate. Morphological changes induced by ingestion of cholesterol oxidase suggest that enzyme has a direct effect on the midgut tissue of boll weevil larvae (Table, 2), and disrupted the midgut epithelium at low doses and lysed its cells at higher doses.
433 Different techniques used in molecular diagnosis (A) DNA-Based Diagnostic Kits DNA diagnostic kits are based on the ability of single stranded nucleic acids to bind to other single stranded nucleic acids that are complementary in sequence (referred to as homologous). The tool used in DNA diagnostic kits is the Polymerase Chain Reaction (PCR). There are 3 steps involved in PCR. The DNA is first unwound, and its strands separated by high temperatures. As the temperature is lowered, short, single-stranded DNA sequences called primers are free to bind to the DNA strands at regions of homology, allowing the (Taq) polymerase enzyme to make a new copy of the molecule. This cycle of denaturation-annealing-elongation is repeated 30-40 times, yielding millions of identical copies of the segment. The primers in PCR diagnostic kits are very specific for the genes of a pathogen, and amplification will occur only in diseased plants.
Figure 1: PCR-based Diagnostic Methods Source: Alberts, et. al., 1994.Photos courtesy of http://www.msu.edu
434 The primers in PCR diagnostic kits are very specific for the genes of a pathogen, and DNA amplification will occur only in diseased plants. (Figure 1) Several PCR-based methods have successfully been adapted for plant pathogen detection. Real-time PCR (RT PCR) follows the general principle of polymerase chain reaction; its key feature is that the amplified DNA is quantified, using fluorescent dyes, as it accumulates in the reaction mixture after each cycle. It offers several advantages over normal PCR, including: reduced risk of sample contamination, provision of data in real time and simultaneous testing for multiple pathogens. Real-time PCR protocols are among the most rapid species-specific detection techniques currently available. Since the development of microarray technology for gene expression studies new approaches are extending their application to the detection of pathogens. Microarrays are generally composed of thousand of specific probes spotted onto a solid surface (usually nylon or glass). Each probe is complementary to a specific DNA sequence provides a signal that can be detected and analysed. Although there is great potential for microarray technology in the diagnosis of plant diseases, the practical development of this application is still in progress. This is important, as plants are often infected with several pathogens, some of which may act together to cause a disease complex. Microarrays consist of pathogenspecific DNA sequences immobilized onto a solid surface. Sample DNA is amplified by PCR, labeled with fluorescent dyes, and then hybridized to the array (Figure 2). PCR-based diagnostics is very sensitive compared to other techniques; detection of a small amount of DNA is possible. PCR can also help farmers detect the presence of pathogens that have long latent periods between infection and symptom development. Moreover, it can quantify pathogen biomass in host tissue and environmental samples, and at the same time detect fungicide resistance. PCR-based detection, however, is expensive compared to protein-based diagnostic methods, and also requires costly equipments. So far, PCR kits have been developed to detect black Sigatoka disease in bananas, Phytophthora infestations in potatoes, and Fusarium infection in cotton. Protein-Based Diagnostic Kits The first step in a defense response reaction is the recognition of an invader by a host’s immune system. This recognition is due to the ability of specific host proteins, called antibodies, to recognize and bind proteins
435
Figure 2: DNA Microarray Source: Alberts, et. al., 1994. Photos courtesy of http://www.msu.edu
that are unique to a pathogen (antigens) and to trigger an immune reaction (Figure 3a).
Figure 3: Antibody-Antigen Interaction
Protein-based diagnostic kits for plant diseases contain an antibody (the primary antibody) that can either recognize a protein from either the pathogen or the diseased plant. Because the antibody-antigen complex cannot be seen by the naked eye, diagnostic kits also contain a secondary
436 antibody, which is joined to an enzyme. This enzyme will catalyze a chemical reaction that will result in a color change only when the primary antibody is bound to the antigen. Therefore, if a color change occurs in the kit’s reaction mixture, then the plant pathogen is present, (Figure 3b). The enzyme-linked immunosorbent assay (ELISA) method makes use of this detection system, and forms the basis of some protein-based diagnostic kits. ELISA kits are very easy to use because test takes only a few minutes to perform. Some of them detect diseases of root crops (e.g. cassava, beet, potato), ornamentals (e.g. lilies, orchids), fruits (e.g. banana, apple, grapes), grains (e.g. wheat, rice), and vegetables. ELISA techniques can detect ratoon stunting disease of sugarcane, tomato mosaic virus, papaya ringspot virus, banana bract mosaic virus, banana bunchy top virus, watermelon mosaic virus, and rice tungro virus. One of the first ELISA kits developed to diagnose plant disease was by the International Potato Center (CIP). It can detect the presence of all races, biovars, and serotypes of Ralstonia solanacearum, the pathogen that causes bacterial wilt or brown rot in potato. They also developed a kit that samples for the presence of any of the following sweet potato viruses: SPFMV (sweet potato feathery mottle virus), SPCSV (sweet potato chlorotic stunt crinivirus), SPMSV (Sweet potato mild speckling virus), SPMMV (Sweet potato mild mottle virus), SwPLV (Sweet potato latent virus), SPCFV (Sweet potatochlorotic fleck virus), SPCaLV (Sweet potato caulimovirus), and C-6 (new flexuous rod virus). Conclusion Genetic engineering has made it possible to development diagnostic techniques to identify pathogen with an unprecedented accuracy and speed and to tap genes from as diverse sources as microbes, plants, and animal to enable the researcher to develop plants resistant to diseases and insects. In the year to come a gene revolution will contribute to green revolution. This revolution help in green agriculture cultivation. References Alberts, et.al. (1994). Photos courtesy of http://www.msu.edu Control, 2014 Andrew, K. The failure of biotechnology to increase crop yield. Control freaks Ibrahim, R.A. and Shawer, D.M. (2014). Transgenic Bt-Plants and the Future of Crop Protection. International Journal of Agricultural and Food Research.; 3(1):1440 ISSN, 1929-0969 Karthikeyan, A., Sudha, M., Pandiyan, M., Senthil, N., Shobana, V.G. and Nagarajan,
437 P. (2011). Screening of MYMV resistant Mungbean (Vigna radiata(L) wilczek) progenies through Agroinoculation. International Journal of Plant Patholology.; 2:115-125 Harrison, B. Maya, M. and Baulombe, D. (1987). Virus resistance in transgenic plants that express cumber mosaic virus satellite RNA. Nature , 328: 799-802. Herrera- Estrella, A., Van Montagu, M. and Wang, K. (1983). Chimeric genes – a dominant selectable markers in plant cells. EMBO J., 2: 987-95. Hawksworth, D.L. (1991). The fungal dimension of biodiversity: magnitude, significance, and conservation. Mycological Research95: 641-655 Malik, V.S. (1999). Biotechnology: multi-billion dollar. In: Chpra VL, Mallik VS & Bhat SR (Ed.) Applied plant Biotechnology, pp.1-69, Oxford & IBH Publishing Co. New Delhi. Maxmen, A. (2013). Crop pests: under attack. Nature, 501:15-17 Powell-Abel, P.A., Nelson, R.S. De Hoffman, N., Roger, S.G., Fraley, R.T. and Beachy, R.N. (1986). Delay of diseases development in transgenic plants that express the tobacco mosaic virus coat protein gene. Science, 232: 738-43 Schuler, T.H., Poppy, G.M., Kerry, B.R. and Denholm, I. (1998). Isect resistant transgenic plants. Trends Biotech., 16: 168-75 Tabashnik, B.E., Van, R.J.B.J., Carriere, Y. (2009). Field-Evolved Insect Resistance to Bt Crops: Definition, Theory, and Data. Journal of Economic Entomology. 102:2011-2025 Trudel, J., Potvin, C. and Asselin, A. (1992). expression of active egg white lysozume in transgenic tobacco, Pl. Sci.,87:55-67. Zhu, Q., Maher, E.A., Masoud, S., Dixon, R.A. and Lamb, C. (1994). Enhanced protection against fungal attack by constitutive coexpression of chitinase and glucanase genes in transgenic tobacco. Bio/Technology. 12: 807-12
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29 GENOMIC DATABASES, GENOMIC RESOURCESAND AGROINFORMATICS ASA BIOTECHNOLOGICAL PERSPECTIVE Dilip Kumar Verma1, H. K. Chourasia2, Somya Verma3and Heena Verma4 1
Principal Scientist (Genetics & Plant Breeding), IARI Regional Station, Indore – 452001, For correspondence. E-mail:
[email protected] 2
University Deptt of Botany, TM Bhagalpur University, Bhagalpur – 812007, E-mail:
[email protected] 3
Research Scholar (Pharmacy), Ganpat University, Mehsana, Gujarat E-mail:
[email protected] 4
Engineer (Electronic and Communication Engineering) H No 47, Sanjana Park, Behind Agrawal Public School, Pipliyahana, Indore – 452001, M P. E-mail:
[email protected]
ABSTRACT Genomic Databases and Resources play a primary public repository of genomic sequence data, collects and maintains enormous amounts of heterogeneous data. Data for genomes, genes, gene expressions, gene variation, gene families, proteins, and protein domains are integrated with the analytical, search, and retrieval resources through the Web site. Many text-based search and retrieval system, provides a fast and easy way to navigate across diverse biological databases. Customized genomic BLAST enables sequence similarity searches against a special collection of organism-specific sequence data and viewing the resulting alignments within a genomic context using genome browser, Map Viewer. These tools lead to further understanding
440 of evolutionary processes, quickening the pace of discovery. The agroinformatic system should be integrated into a global concept for a computer based information extension system. It is now being demonstrated that new information, communication and educational technologies - agroinformatics can be used in efforts to upgrade the skills of practicing agricultural professionals. Key words: Genomic Databases, agroinformatics, database, data management system.
Introduction The Information and Communication Technology (ICT) in this era of globalization has accentuated new modes of knowledge transformation and communication patterns. ICT has opened up uncommon opportunities for developing countries in terms of providing low cost access to information. This is the fastest growing tool of communication. India has 70% of its population, which is dependent on Agriculture for its livelihood. Considering this, use of ICT in Agriculture is of strategic importance in a country like India. ICT have tremendous potential in timely collection of data and distributing it to the potential users even in developing countries. Advances in biotechnology, bioinformatics and agrinformatics led to a flood of genomic data and tremendous growth in the number of associated databases. The agroinformatic system should be integrated into a global concept for a computer based information extension system. It is now being demonstrated that new information, communication and educational technologies - agroinformatics can be used in efforts to upgrade the skills of practicing agricultural professionals. A key factor in the success of use of agroinformatics is designing culturally appropriate agroinformatics. Additionally, it is maintained that institutions working in isolation cannot efficiently and effectively design and deliver national and regional elearning efforts. New partnerships and innovative partnership arrangements must be cultivated and supported. The use of computers in agriculture as well as its possible impact was evaluated. Nobel Peace Prize winner Dr. Norman Borlaug points out that “Ways must be found to improve access to information by less-educated farmers-because of equity reasons and also to facilitate accelerated adoption of the newer knowledge-intensive technologies.” (Borlaug and Dowswell, 2001). Word processing, statistical analysis, graphics, data banks and spreadsheets are the main uses. In industrialized countries, information technology (IT) is widely used in agriculture where governmental organizations provide information and databases through computer networks. Recently, participative
441 approaches are emerging, where organizations, communities and individuals have started to work together in providing IT services. The approaches became more feasible with the introduction of the Internet. The Internet is truly a democratic network, has the potential power to improve the cooperative activities in agriculture, and to alter the directions of the flow of information. The object-oriented software (OOS) presents some basic concepts related to hypertext and hypermedia systems. The development and use of WWW Resources for training is very useful with tremendous potential for teaching and training applications. However, WWW and the hypertext mark-up language (HTML) are very new technologies and very few teaching applications have been developed. The effort should be to explore the potential and effectiveness of WWW-based hypermedia for teaching. Most of these exploit text, colour photographs, diagrams, charts, hyperlinks and interaction. The WWW server can be programmed and used to support the automatic logging and/or validation of user inputs. The WWW can be an effective technology as a platform for computer aided instruction applications but that the authoring, rendering and document management tools are still very primitive and constitute a barrier to the widespread and nontrivial use of WWW technology in education. Hamilton et al (2002) emphasized that a “hit” is one recorded access event of a World Wide Web site. The implementation of an effective site on the World Wide Web can be enhanced by setting appropriate standards. World Wide Web (WWW) site is not very difficult. With gopher technology back issues were archived. Subsequently, the newsletter became key word searchable, and finally, developed into a full-blown World Wide Web site. Information and Communication Technologies (ICTs) are an excellent way to deliver knowledge-intensive management to the agricultural sector because this management strategy is information-based and dynamic. The e-learning, a form of distance education that uses ICTs, is an appropriate medium for the delivery of knowledge-intensive management strategies to agricultural professionals. e-learning offers many advantages for delivering knowledge-intensive management strategies to agricultural professionals. Of the many e-learning tools available, simulations hold particular promise. ICT infrastructure and human resource development improves farmers direct access to e-learning tools, and simulations will be at the forefront of delivering knowledge-intensive management skills directly to farmers. Information on complete and ongoing genome projects is also available in Genomes OnLine Database (GOLD) (Liolios, et al., 2007), a community-supported World Wide Web resource. Hundreds of thousands
442 of genomic sequences for viruses, organelles, and plasmids are available in the three public databases of the International Nucleotide Sequence Database Collaboration [INSDC, www.insdc.org] – EMBL, GenBank (Benson, et al., 2008), and the DNA Data Bank of Japan (Sugawara, et al., 2008). Additional information on biomedical data is stored in an increasing number of various databases. Navigating through the large number of genomic and other related ‘‘omic’’ resources becomes a great challenge to the average researcher. Understanding the basics of data management systems developed for the maintenance, search, and retrieval of the large volume of genomic sequences will provide necessary assistance in travelling through the information space. This is focused on the infrastructure developed so far. NCBI, as a primary public repository of genomic sequence data, collects and maintains enormous amounts of heterogeneous data. The databases vary in size, data types, design, and implementation. They cover most of the genomic biology data types including the project description, project sequence data (genomic, transcript, protein sequences), raw sequence reads, and related bibliographical data (Wheeler, et al., 2008). More recently, NCBI started to collect the results of studies that have investigated the interaction of genotype and phenotype. Such studies include genome-wide association studies, sequencing, molecular diagnostic assays, as well as association between genotype and nonclinical traits (Mailman, et al., 2007). All these databases are integrated in a single Entrez system and use a common engine for data search and retrieval, which provides researchers with a common interface and simplifies navigation through the large information space. There are many different ways of accessing genomic data. Depending on the focus and the goal of the research or the level of interest, the user would select a particular route for accessing the genomic databases and resources. These are (1) text searches, (2) direct genome browsing, and (3) searches by sequence similarity. All of these search types enable navigation through pre-computed links to resources. This chapter describes the details of text searching and the retrieval system of the major genomic databases and also illustrates methods of accessing the genomic data. Data Flow and Processing The National Center for Biotechnology Information was established in order to develop computerized processing methods for biomedical research data. As a national resource for molecular biology information, it’s mission is to develop automated systems for storing and analyzing knowledge about molecular biology, biochemistry, and genetics;
443 facilitate the use of such databases and software by the research and community; coordinate efforts to gather biotechnology information both nationally and internationally; and perform research into advanced methods of computer-based information processing for analyzing the structure and function of biologically important molecules. The fundamental sequence data resources consist of both primary databases and derived or curated databases. Primary databases such as GenBank (Pruitt, et al., 2007) archive the original submissions that come from large sequencing centers or individual experimentalists. The database staff organizes the data but do not add additional information. Curated databases such as Reference Sequence Collection (Pruitt, et al., 2007) provide a curated/expert view by compilation and correction of the data. Records in the primary database are analogous to research articles in a journal, and curated databases to review articles. This difference is not always well understood by the users of sequence data. In response to the users’ inquiries, and more specifically to a request on microbial genomes, the differences between GenBank, Ref Seq, and TPA databases. In the same way an expert view or provide a result of computational analysis, the databases can be manually curated and/or computationally derived. For more detailed information on all NCBI and database resources see also (Wheeler, et al., 2007).The biological sequence information that builds the foundation of NCBI primary databases and curated resources comes from many sources. This section discusses the flow of sequence data, from the management of data submission to the generation of publicly available data products. An information management system that consists of two major components, the ID database and the IQ database, underlies the submission, storage, and access of GenBank (Benson, et al., 2007), BLAST (Pruitt, et al., 1997), and other curated data resources (Benson, et al., 2007) or Entrez Gene. Whereas ID handles incoming sequences and feeds other databases with subsets to suit different needs, IQ holds links between sequences stored in ID and between these sequences and other resources. In the ID database, blobs (binary large objects) are added into a single column of a relational database and are stored and processed as a unit. Although the columns behave as in a relational database, the information that makes each blob, such as biological features, raw sequence data, and author information, is neither parsed nor split out. In this sense, the ID database can be considered as a hybrid database that stores complex objects. The IQ database is a Sybase data-warehousing product that
444 preserves its SQL language interface, but which inverts its data by storing them by column, not by row. Its strength is in its ability to increase speed of searches based on anticipated indexing. This non-relational database holds links between many different objects. Agroinformatics for Agriculture which is becoming a KnowledgeIntensive Industry As Moe (2000) states, “Technology is the driver of the New Economy, and human capital is its fuel. In today’s world, not only does knowledge make the difference in how an individual performs, but it also makes the difference in how well a company performs and, for that matter, how well a country performs.” Numerous recent authors echo this view (Skyrme, 1997; Abell and Oxbrow, 1999; Atkinson and Randolph, 1998). It talks about the growth and development of a global knowledge society where “individuals who are well-educated, self-motivated, and linked into information networks, are the most likely to live prosperous and fulfilling lives. Verma and Singh (2003) had made a detailed review of agroinformatics and its strategic role in agricultural research. They had summarised the informations under the sub-headings viz., Agro-informatics in planning and development of sustainable agro-ecosystems, remote sensing and GIS in resource management, precision farming technologies, crop modelling and farming systems, DBMS as a tool in agricultural services, decision support systems and expert systems, socio-economic issues of agricultural information technology, network based transfer of technology and E-Agribusiness. Text Search and Retrieval System: Entrez Organizing Principles Entrez is the text-based search and retrieval system used for all of the major databases, and it provides an organizing principle for biomedical information. Entrez integrates data from a large number of sources, formats, and databases into a uniform information model and retrieval system. The actual databases from which records are retrieved and on which the Entrez indexes are based have different designs, based on the type of data, and reside on different machines. These will be referred to as the ‘‘source databases.’’ A common theme in the implementation of Entrez is that some functions are unique to each source database, whereas others are common to all Entrez databases. An Entrez ‘‘node’’ is a collection of data that is grouped and indexed together. Some of the common routines and formats for every Entrez node
445 include the term lists and posting files (i.e., the retrieval engine) used for Boolean queries, the links within and between nodes, and the summary format used for listing search results in which each record is called a DocSum. Generally, an Entrez query is a Boolean expression that is evaluated by the common Entrez engine and yields a list of unique ID numbers (UIDs), which identify records in an Entrez node. Given one or more UIDs, Entrez can retrieve the DocSum(s) very quickly. Query Examples Each Entrez database (‘‘node’’) can be searched independently by selecting the database from the main Entrez Web page (http://www.ncbi.nlm.nih.gov/sites/gquery). Typing a query into a text box provided at the top of the Web page and clicking the ‘‘Go’’ button will return a list of DocSum records that match the query in each Entrez category. These include nucleotides, proteins, genomes, publications (PubMed), taxonomy, and many other databases. The numbers of results returned in each category are provided on a single summary page and provide the user with an easily visible view of the results in each databases. The results are presented differently in each database but within the same framework, which includes the common elements such as search bar, display options, page formatting, and links. In processing a query, Entrez parses the query string into a series of tokens separated by spaces and Boolean operators (AND, NOT, OR). An independent search is performed for each term, and the results are then combined according to the Boolean operators. The main goals of the information system are reliable data storage and maintenance, and efficient access to the information. The retrieval is considered reliable if the same information that was deposited can be successfully retrieved. The Entrez system goes beyond that by providing the links between the nodes and precomputing links within the nodes. The links made within or between Entrez nodes from one or more UIDs (Unique IDentifier) are also a function across all Entrez source databases. There are three different linking mechanisms described below. Links Between the Nodes The power of Entrez organization lies in the connections between the individual nodes that increase the information space. These links, created during indexing, are reciprocal and stored in a special database, for example, links between genome sequence records and the corresponding genome project. Links can also be provided by the original submitters, for example, links between a nucleotide sequence and a publication (PMID). Links
446 between nucleotide and protein sequences (conceptual translation) of the annotated coding region can also be provided by the original submitters. Links Within the Nodes Entrez data can be also integrated by calculating the relationships between the records in a single database. For example, nucleotide and protein sequences can be linked by sequence similarity. The similarity is calculated using BLAST (Altschul, et al., 1997), stored in a special database, and made readily available in Entrez via the ‘‘Related Sequences’’ link. In PubMed, the inter-database links are calculated by comparing the frequency terms of the document. The similarity between two documents is based on the number of the weighted terms the two documents have in common. The highest scoring documents can be viewed for each document by selecting Related Articles from the Links menu. Links Outside the Nodes Links to outside resources are available through LinkOut, a special service of the Entrez system. It allows relevant outside online resources to link directly to the records in Entrez system. The outside users provide a URL, a resource name, the UID of the record they wish to link to, and a brief description of their Web site in a simple XML format. The request is processed automatically and links are added to the corresponding records in Entrez. This resource gives the end user a central place to look for the information available at NCBI and easily explore the relevant resources. Tools for Advanced Users The Entrez Programming Utilities (eUtils) are a set of eight server side programs that provide a stable interface to the Entrez query and database system. The eUtils use a fixed URL syntax that translates a standard set of input parameters into the values necessary for various NCBI software components to search for and retrieve data and represent a structured interface to the Entrez system databases. The software can thus use any computer language that can send a URL to the eUtils server and interpret the XML response, such as Perl, Python, Java, and C++. Combining eUtils components to form customized data pipelines within these applications is a powerful approach to data manipulation. Genomic Databases Genomic studies of model organisms give insights into understanding of the biology enabling better prevention and treatment of diseases. The genome sequencing era that started about 20 years ago has
447 brought into being a range of genome resources. Comparative genome analysis leads to further understanding of fundamental concepts of evolutionary biology and genetics. A review on genome resources (Hillary, et al., 2006) reports on a selection of genomes of model species – from microbes to human. Species-specific genomic databases comprise a lot of invaluable information on genome biology, phenotype, and genetics. However, primary genomic sequences for all the species are archived in public repositories that provide reliable, free, and stable access to sequence information. In addition, NCBI provides several genomic biology tools and online resources, including group-specific and organism-specific pages that contain links to many relevant Web sites and databases. Trace Repositories Most of the data generated in genome sequencing projects is produced by whole genome shotgun sequencing, resulting in random short fragments (traces).For many years, the traces (raw sequence reads) remained out of the public domain because the scientific community has focused its attention primarily on the end product: the fully assembled final genome sequence. As the analysis of genomic data progressed, it became necessary to go back to the experimental evidence that underlies the genome sequence to see if there is any ambiguity or uncertainty about the sequence. Trace Archive To meet these needs, NCBI and The Welcome Trust Genome Campus, a repository of the raw sequence traces generated by large sequencing projects that allows retrieval of both the sequence file and the underlying data that generated the file, including the quality scores. The Assembly Archive (Salzberg, et al., 2004) links the raw sequence information found in the Trace Archive with consensus genomic sequence. Short Read Archive (SRA) Trace Archive has successfully served as a repository for the data produced by capillary-based sequencing technologies for many years. New parallel sequencing technologies (e.g., 454, Solexa, Illumina, ABI Solid, Helicos) have started to produce massive amounts of short sequence reads (20–100 kb). Due to the structure and volume of this data, it is clear that it does not efficiently and effectively fit in the current Trace Archive design, so NCBI has constructed a more appropriate repository, the Short Read Archive. The SRA project is well underway and is being built in collaboration with Ensembl, sequencing centers, and the vendors themselves.
448 GenBank – Primary Sequence Archive GenBank is the NIH genetic sequence database, an archival collection of all publicly available DNA sequences (Benson, et al., 2008). GenBank is part of the International Nucleotide Sequence Database Collaboration, which comprises the DNA DataBank of Japan (DDBJ) (Sugawara, et al., 2008), the European Molecular Biology Laboratory (EMBL) (Cochrane, et al., 2008), and GenBank at NCBI. These three organizations exchange data on a daily basis. The data come from the large sequencing centers as well as from small experimentalists. These sequences are accessible via Web interface by text queries using Entrez or by sequence queries using BLAST. Entrez Databases A family of Entrez databases comprise an integrated information system that links together heterogeneous information on biomedical and bibliographical data. The three examples of Entrez databases containing information are genome projects, genomic sequences, and protein sequence encoded by complete microbial genomes. Entrez Genome Entrez Genome (Tatusova, et al., 1999), the integrated database of genomic information at the NCBI, includes the types of records and formats for major taxonomic groups, as well as the precomputed data and online analytical programs developed to aid investigation. The database was created for large-scale genome sequencing projects. It was motivated by the release of the first complete microbial genome of Haemophilus influenzae sequenced at TIGR (Fleischmann, et al., 1995). Entrez Genome displays data from small viral and organelle genomes, complete and nearly complete genomes from bacteria, and eukaryotes. An entry in Genomes database represents a single replicon such as a chromosome, organelle, or plasmid. As Entrez Genome houses a collection of 7,850 entries organized in six large taxonomic groups: Archaea, Bacteria, Eukaryota, Viroids, Viruses, and Plasmids. It presents the tools and views at various levels of detail. For each record, Entrez Genome provides a graphical overview of the chromosome with genes colour coded by COG (clusters of orthologous groups) (Tatusov, et al., 2003) functional categories as well as other types of text views including flat file, ASN.1, XML, and many others that can be user-selected from a menu. The table provides additional genome information and access to analysis tools. The available tools include multiple alignments of complete genomes for viruses, precomputed protein clusters from microbial genomes,
449 GenePlot (a genome-scale dotplot generator), TaxPlot (for three-way genome comparisons), gMap, and many others. Some of these tools are described in Section 5 of this chapter. More detailed description of microbial genome resources at NCBI can be found in ‘‘In Silico Genomics and Proteomics’’ (Klimke and Tatusova, 2003). Plant genome resources at NCBI have been recently published in a chapter of ‘‘Plant Bioinformatics’’ (Tatusova, et al., 2006). Microbial genome sequencing has come a long way since the first H. influenzae project. The collection represents a very diverse set of organisms; ranging from small (160 kb) endosymbiont Carsonella (Nakabachi, et al., 2006) to the 13-Mb genome of myxobacterium Sorangium cellulosum (Schneiker, et al., 2007). There are organisms isolated from extreme environments such as Hyperthermus butylicus (Bru¨gger, et al., 2007), an extreme hyperthermophilic, anaerobic archeon, and bacterial species representing deeply branching taxa such as Rhodopirellula baltica (Teeling, et al., 2004). On the other hand, many projects are aimed toward the comparative analysis of pathogenic bacteria and sequencing multiple strains and isolates of the same organism. For example, H. influenza bacterium is represented in the database by 16 entries including chromosomes and plasmids from different isolated strains. Entrez provides tools that facilitate comparative genome analysis leading into new insights to be gained from genome sequences. Entrez Genome Project The NCBI Genome Project database is a collection of complete and incomplete large-scale sequencing, assembly, annotation, and mapping projects for cellular organisms. A project is defined by a unique combination of organism name (or metagenomic project name), sequencing center, and sequencing method. Currently, the database is comprised of projects that have submitted data toNCBI, intend to submit data, or have received public funding. A large eukaryotic genome project usually consists of several components. In the database, projects are organized in a hierarchical, parent–child relationship. A top-level project represents an organism-specific overview and links together all relevant child projects. Each project has its own unique identifier, the Project ID. The International Nucleotide Sequence Databases Consortium (INSDC) has acknowledged the need to organize genomic and metagenomic data and to capture project metadata. ‘‘A project is defined as a collection
450 of INSDC database records originating from a single organization, or from a consortium of coordinated organizations. The collective database records from a project make up a complete genome or metagenome and may contain genomic sequence, EST libraries and any other sequences that contribute to the assembly and annotation of the genome or metagenome. Projects group records either from single organism studies or from metagenomic studies comprising communities of organisms.’’ NCBI has developed a SOAP (simple object access protocol) compliant Web service, supporting the functions of inserting, updating, deleting, and retrieving of the documents which are used by INSDC collaborators to access/edit the Genome Project database, which in turn controls ProjectIDs and Locus-tag prefixes as well as other project information. The NCBI Entrez Genome Project database (GenomePrj) is organized into organism-specific overviews that function as portals from which all projects pertaining to that organism can be browsed and retrieved. GenomePrj is integrated into the Entrez search and retrieval system, enabling the use of the same search terms and query structure used in other Entrez databases. GenomePrj is a companion database to Entrez Genome. Sequence data are stored in Entrez Genome (as complete chromosomes, plasmids, organelles, and viruses) and Entrez Nucleotide (as chromosome or genomic fragments such as contigs). While Entrez Genome does not collect all data for a given organism, GenomePrj provides an umbrella view of the status of each genome project, links to project data in the other Entrez databases and a variety of other NCBI and external resources associated with a given genome project. Sequences associated with a given organism can also be retrieved in the taxonomy browser. However, no distinction is made between GenBank (non-curated) and RefSeq (curated) sequences. There is also no distinction based on which sequencing center submitted the data. Entrez Genome Project also lists projects that are in progress or for which NCBI has not yet received any data. See Table 1 for a comparison of all three databases. The database entry contains brief project description, listing of all related subprojects, and project data which include links to genomic data, publication, and Trace data. NCBI Resource Links include an option to BLAST against this particular collection as well as an option to BLAST against all available environmental sequences.
451 Table 1. Comparison of entrez databases Entrez databases
Organism specific sequences
Project specific sequences
Submitter specific sequences
Complete and in progress
GenBank and RefSeq sequences
Genome
Yes
No
Yes
No
Separated
Taxonomy
Yes
No
No
No
Together
Genome project
Yes
No
Yes
Yes
Separated
Entrez Protein Clusters Protein Clusters database is a rich collection of related protein sequences from complete prokaryotic and organelle Reference Sequence (RefSeq) genomes. Proteins from all complete microbial genomes and plasmids (and separately all chloroplasts) are compared using BLAST allagainst-all. Protein clusters are created using a modified BLAST score that takes into account the length of the hit (alignment) versus both the query and the subject. The modified score is then sorted, and all proteins that are contained within the top hits are clustered together. Automatically constructed clusters are then evaluated manually by curators. Based on the sequence alignment information and biological expertise, curators can join or split clusters and add annotation information (protein name, gene name, description) and publication links. The Entrez Protein Clusters database uses all of the features of other Entrez databases. There are numerous ways to query protein clusters, either with search terms in Entrez or with a protein or nucleotide sequence. The display for each cluster provides information on cluster accession, cluster name, and gene name, as well as links to protein display tools, external databases, and publications. Protein Clusters database can be queried with a protein or nucleotide sequence by using Concise Protein BLAST, a new Web resource developed at NCBI. Concise BLAST is an efficient alternative to standard BLAST. The searchable database is comprised of only one randomly chosen protein from each cluster, and also proteins which are not included in any cluster to assure completeness. This allows rapid searching of the smaller database, but still assures an accurate identification of the query while providing a broader taxonomic view. gMap is one of the tools available in Entrez Genome that allows to view and analyze the regions of similarity in closely related genomes.
452 Analysis of Prokaryotic Genome Data gMap – Compare Genomes by Genomic Sequence Similarity Genomic sequences are compared using BLAST and the resultant hits are filtered out to find the largest syntenic regions. Similar regions are shown colour-coded and numbered in each genome with an arrow denoting the 50–30 direction of the hit with respect to similar segments in other genomes. Additional sequences can be added by inputting the accession number. The tool allows navigating from the general overview of domains of life (e.g., bacteria or viruses) down to genome sets with different degrees of mutual similarity. It allows more detailed views of every similarity segment, including the ability to view sequence alignment of two selected similarity regions. Zooming in can be accomplished by clicking on a syntenic group to expand all similar segments. Alternately, a user can click on a hit bar just below the segments to zoom into the surrounding region of the current sequence; this action also displays homologous syntenies from other organisms. After zooming in, all segments are recalculated, recoloured, and renumbered, providing a truly dynamic and interactive system with each calculated view presented as a standalone display which is visually easy to comprehend. Pairs of genomic sequences can be selected for output to BLAST, GenePlot, or HitPlot and any number of sequences can be removed from the list by the user to customize the final view to be most appropriate for the user’s project. HitPlot shows a dotplot of the two genomes selected based on the magnification level. Precomputed results are available for two categories, one for genomes from the same genus and one for genomes based on the coverage of BLAST hits. Genomes of two or more species from the same genus may not display high levels of synteny, but similar segments in their two genomes can be found at different levels of hit coverage. An example of this would be the Mycoplasma genomes. The converse is that organisms from different genera have large syntenic blocks in their genomes such as is found in Escherichia, Salmonella, and Shigella, which are all members of the Enterobacteriaceae family (Darling, et al., 2004). Genomes in both categories are grouped together based on single linkage clustering of coverage level. For example, if genome A has 75% coverage to genome B and genome B has 75% coverage to genome C, then they will all be included in a cluster at the 75% level even though the coverage between A and C may not reach the 75% level.
453 Genome ProtMap –Compare Genomes by Protein Sequence Similarity Genome ProtMap is a comparative display of the genome neighbourhoods linked by the orthologous protein sequences. It displays a 10-kb region surrounding either all the proteins in the cluster or, alternately, all the proteins that have the same Cluster of Orthologous Group – COG (Tatusov, et al., 2004) – or in the case of viruses, VOGs. In the Genome ProtMap display, the organism groups are collapsed; clicking the + will expand the group. Clicking the accession number will link to the RefSeq nucleotide record. Mouse over the proteins gives detailed information such as name, cluster ID, and genome location. Clicking on any protein brings up a pop-up menu with links to protein, gene, or cluster. The list of taxa in the ProtMap can be collapsed or expanded by clicking the + or _ next to the taxon. ‘‘Show Legends’’ gives the colour-coded functional category for the proteins while ‘‘Show Cluster Colors’’ lists all the clusters in the ProtMap coloured by COG functional category and the name of the cluster. Concise BLAST Concise protein BLAST uses the BLAST engine to allow searching of protein clusters’ data sets with a protein or nucleotide sequence query. The database represents protein sequences from complete microbial (prokaryotic) genomes. It uses pre-calculated clusters of similar proteins at the genus level to represent proteins by groups of related sequences. One representative from each cluster is chosen in order to reduce the data set. The result is reduced search times through the elimination of redundant proteins while providing a broader taxonomic view. Browsing Eukaryotic Genome Data The main NCBI genome browser Map Viewer provides special browsing capabilities for a subset of organisms in Entrez Genome. The list of organisms available for Map Viewer browsing can be found on the Map Viewer home page (http://www.ncbi.nlm.nih.gov/projects/mapview/). Map Viewer can display a collection of aligned genetic, physical, or sequence-based maps, with an adjustable focus ranging from that of a complete chromosome to that of a portion of a gene. The maps displayed in Map Viewer may be derived from a single organism or from multiple organisms; map alignments are performed on the basis of shared markers. The availability of whole genome sequences means that objects such as genes, markers, clones, sites of variation, and clone boundaries can be positioned by aligning defining sequences from these objects against the genomic sequence. This positional information can then be compared to
454 information on order obtained by other means, such as genetic or physical mapping. The results of sequence-based queries (e.g., BLAST) can also be viewed in genomic context as described in the next section. Any text search term can be used as a query at the top of the Map Viewer home page. These include, but are not limited to, a GenBank accession number or other sequence-based identifier, a gene symbol or alias, or the name of a genetic marker. For more complex queries, any query can be combined with one of three Boolean operator terms (AND, OR, and NOT). Wild cards, which are denoted by placing a * to the right of the search term, are also supported. Map Viewer uses the Entrez query search engine, described in section 3, to analyze a complex query and perform a search. Another way of getting to a particular section of agenomeis tousea range of positions as a query. First it is necessary to select a particular chromosome for display from a genome-specific Map Viewer page. Once a single chromosome is displayed, position-based queries can be defined by (Liolios, et al., 2007) entering a value into the Region Shown box. This could be a numerical range (base pairs are the default if no units are entered), the names of clones, genes, markers, SNPs, or any combination. The screen will be refreshed with only that region shown. Map Viewer provides an option to simultaneously search physical, genetic, and sequence maps for multiple organisms. This option is currently available for plant and fungal genomes. Since the early 1990s several researchers have shown that large scale genome structure is conserved in blocks across the grasses (Ahn and Tanksley, 1993); Devos, et al., 1994; Kurata, et al., 1994; van Deynze, et al., 1995). Locus nomenclature is organism-specific and is unreliable as a query method between species; however, the regular nomenclature of plasmids (Lederburg, 1986) is not influenced by how the plasmid or insert is used. The data for the plant maps available through Map Viewer include the probe–locus relationship for each locus where the allelic state is identified by the probe. This information enables the rendering of the visual connection between those mapped loci in adjacently displayed maps that were identified by the same probe. This locus–probe relationship allows a cross-species text search using the probe name as the query string. Figure 2.10 shows the result of the search across all plants using ‘‘cdo718’’ as a query. ‘‘cdo718’’ is the name of a plasmid with an oat cDNA insert. This probe was used to map loci in nine maps available in Map Viewer: the AaXAh-92 map in Avena sativa, the Cons95 map in Hordeum vulgare, the
455 RC94,RW99,R, RC00, andRC01maps in Oryza sativa, the E-01 map in Secale cereale, the S-0 map in Triticum aestivum, JKxC map in Triticum turgidum, and theRW99mapinZeamays. The dark grey lines between each map connect the loci identified by the probe. The light gray lines connect the other loci in adjacent maps that have been identified by the same probe. Searching Data by Sequence Similarity (BLAST) The Basic Local Alignment Search Tool (BLAST) (Altschul, et al., 1990) finds regions of local similarity between sequences. By finding similarities between sequences, scientists can infer the function of newly sequenced genes, predict new members of gene families, and explore evolutionary relationships. Organism-Specific Genomic BLAST Genome-specific BLAST pages that restrict a search to a specific genome are provided for several organisms and allow the results of the search to be displayed in a genomic context (provided by Map Viewer). Query sequence (protein or nucleotide) can be compared to genomic, transcript, or protein coded by the genome. Not all databases are always available; some projects provide additional data sets such as SNP, traces, and alternative assemblies. If the reference genome (the default) is selected as the database to be searched, the Genome View button will appear on a diagram showing the chromosomal location of the hits. Each hit links to a Map Viewer display of the region encompassing the sequence alignment. Multi-organism Genomic BLAST Microbial Genomic BLAST provides access to complete genomes and genome assemblies of Bacteria and Archaea and Eukaryota. Genomic BLAST has been recently extended to include data sets for insects, fungi, nematodes, protozoa, and metagenomes. The genomes can be viewed in taxonomic groups or in alphabetical order. A flexible user friendly interface allows to construct virtual blast databases for the specific searches. FTP Resources for Genome Data The source genome records can be accessed from the Gen-Bank directory; these are the records that were initially deposited by the original
456 submitters. The reference genomes, assemblies, and associated genes and proteins can be downloaded from the Genomes and RefSeq directories. Recently, Govt. of India has sanctioned the AGRISNET programme to provide the different kind of services to the farmers of the state. Reference Services E-Library facility using CAB Database, J-Gate, Science Direct Personalizing Research, Annual Reviews, Springer Link and Consortium for e-Resources in Agriculture services has been put on intranet. This enables the end users to get quick information through computer on any reference related to Agricultural research published from 1972 to 2090 through Network. This is unique kind of service not found anywhere in India which is provided free of cost to the scientists and the student of the University. This has made the reference services very fast and has saved a lot of time of the scientists and the students. DVAR Technology DVAR technology is implemented as a pilot project at Navali Village of Anand district. DVAR technology is developed to address the need of an easier audio-visual message (grievances, suggestions, feedback etc.) submission without the knowledge of the computer and reading-writing skill. Feature DVAR is an easy to operate technology; even illiterate person can operate it and submit a message in fraction of minute; Innovative two button human interface (green/red); Audio/ visual messages from authority can be played before submitting a grievances/ complaints/ suggestions; As a proof of message submission, automatic printed receipt can be delivered; Audio/ visual interaction is also possible through numeric key pad. National Knowledge Network (NKN): Convergence of IT and communication (often referred to as ICT), and exponential growth in the communication capacity opens up new vistas for application of these technologies. ICT, when properly deployed in a country, has the potential of solving major challenges faced by human race, such as, Climate Change, Energy Security, Green Design, and so on. Besides, ICT coupled with processing power is a potent instrument for any planning process. ICT, when properly harnessed, can render the country flat (devoid of social inequities) and convert it to knowledge society. The National Knowledge Network is an initiative in that direction to enable India leapfrog into Knowledge Society and Knowledge Economy. Features are high capacity, highly scalable backbone; provide Quality of Service (QoS) and security; wide geographical coverage; common standard
457 platform; bandwidth from many nld’s; highly reliable & available by design; test beds (for various implementation); dedicated and owned. The main objective of NKN is to interconnect libraries, laboratories, agricultural institutions enabling nationwide sharing of data and resources. The important objective of NKN service is to establish connectivity among member institutions to enable collaborative research in emerging areas such as climate modelling, bio-informatics and agriculture. Moreover, the network will facilitate distance education in specialised fields such as medicine and other departments which depend heavily on wide-ranging research. Gyandoot The community-owned ‘Gyandoot’ project in Dhar district of Madhya Pradesh provide information through kiosks (Suchanalaya) on agriculture produce, auction rates, land records etc. It is a low cost usercharge-based-service and the expense of running it is being borne by panchayats and the communities. Gyandoot creates a cost-effective, replicable, economically self reliant and financially viable model for taking the benefits of Information and Communication Technology (ICT) to the rural masses. Gyandoot is managed by a society called ‘Gyandoot Samiti’ registered under Madhya Pradesh Societies Registration Act with District Collector as the President of the Samiti. E-choupal Traditionally, choupals are community gathering places in village where local people meet to discuss issues and solve out their problems. In this era of globalization, e-choupals are gradually revolutionalising the way Indian farmers do business. Farmers can use the kiosks to check the current market prices of their commodities, access market data, information on local and global weather and best farming practices. The system consists of an internet enabled kiosk in a village, which is manned by a prominent local leader who is familiar with computers, known as the ‘choupal sanchalak’. Today ITC-IBD is buying agriculture products such as soybeans, coffee, shrimp, wheat, rice and pulses, all through e-choupals. Shashank Joshi, a soya farmer in Mendki village, M. P. has a new status as a ‘Sanchalak’ in ITC’s Soya e-choupal. The days of hanging around the mandi, waiting for the agents to examine their stock and dictate prices are over. Prices of major ‘mandis’ are transparently provided on the computer screen, giving the farmer the option of selling his stock to ITC or a mandi of his choice.
458 Greenstar This is a new consortium of companies from India and the United States which launched its first solar powered internet community centre in Parvatpur village, 150 kms from Hyderabad, providing e- commerce services and offering agricultural information through fax, e-mail and voice mail. Kisan.com is a website conceptualized and developed by Nagarjuna group which offers weather forecasts, commodity news, product availability, online loan facilities, chat rooms and discussion forums. It enables the farmers to communicate with other farmers, suppliers and consumers across the world. Farmerbazzar.com allows farmers to sell their produce through auctioning. The biggest advantage of this site is that you know the best price before clinching a deal. Aquachoupal.com provides shrimp farmers information on world shrimp prices and technological information on shrimp farming technology. The process of market liberalization, the demise of state marketing boards and the lifting of price controls have created a vacuum for the farmers in many developing countries in terms of information on the pricing of their produce and farm inputs, commodity markets and export channels. Just as nature abhors a vacuum, so opportunistic middleman are rushing in to take advantage of farmers’ lack of information. ICT services could bring much needed price and product information directly to farmers. Other examples of areas where ICT could play a major role in the lives of farmers are sound decision making due to timely information, knowledge of market outlook, creating employment through the establishment of rural information centres etc. Challenges ahead India cannot truly advantage itself of its growing strength in the field of ICT without servicing its own domestic needs first, particularly those in rural areas. Many food security and agriculture experts are anguished by the amount of money being pumped in to make it feasible. While farmers struggle to procure one decent meal, millions are being spent on building new synergies between industry and the farmers. ICTs offer both challenges and promises for political, social, economic and environmental development and that is nowhere more apparent than in the world’s poorest countries. t is true that in spite of all the efforts made by
459 the various agriculture and technology scientists, there is still a long way for an average farmer to enjoy the fruits of Information and Communication Technology (ICT) and relish its ultimate use. Some of the challenges associated with the use of new ICT for farmers are listed below: Policy considerations: In most developing countries, the regulations are rigid and telecommunication tariffs and import duties on ICT equipment are high. The situation is compounded by lack of political goodwill. High telecommunication costs: The cost of basic Internet remains a strong deterrent in many developing countries including India. First, it was the television, then telephones and now computers. What one expect from a farmer having less than 2 hectares of land holding? These are the only tactics to help the country grow. Infrastructure: The telecommunication and electricity infrastructure in developing countries is lacking or is poorly developed in rural areas. Satellite and wireless technologies are now used in some developing countries, but these are largely developed around urban areas. In Indian context the infrastructure problems can be very frustrating. The country has along way to go before its telecommunication infrastructure can provide easy access to a majority of its people. ICT has to contend with poor rural infrastructure and unreliable web connectivity – the prime requisites for the success of this project. Lack of local content and language barrier: Information available through new ICTs is mostly in English, which the majority of developing country rural communities cannot read. There is a marked shortage of relevant material in local languages that responds to their needs and this calls for “significant investment and support for local content”. No doubt some efforts in making websites in Hindi in India such as www. Webdunia.com have been done. Also Chennai based company has launched e-mail in 12 Indian languages but all these are still in their infancy. High rate of Illiteracy: Illiteracy is a fundamental barrier to participation in knowledge societies. A large portion of rural population of developing countries are illiterate i.e. they do not possess the basic skills required to harness the benefits of new ICTs. Moreover in a society like India where 75% of the population including the urban poor is out of the mainstream, could ICT make a difference without access to the technology? Only an elite segment might stand benefited in an ICT based environment, while for the poor, everyday survival is a bigger problem, thus failing the basic motto of education for all.
460 Inadequate human resources; It is often seen that most of the staff managing new ICT based projects lack adequate training which would enable them to take full advantage of the new technologies. So, in order to ensure more meaningful participation in rural development and to pave the way for the creation of a critical mass of people that effectively harness ICTs in developing countries, training and capacity building must be an integral part of all ICT projects. Gender differences: Women produce half the world’s food (Anon, 1999). Still when new technologies are introduced, women are seen as a domain of men and they are often left out of the initiatives associated with new ICTs. No doubt, farmwomen often have wisdom and indigenous knowledge that is rooted on culture, traditions, values and experience. E.g.: The Self Employed Women’s Association (SEWA) in India has trained rural women in production and use of video to generate income, disseminate new skills and to advocate for changes in policy. Information-based technologies in agriculture (including ICTs) need to be used within a framework that gathers, synthesizes, analyses, interprets, and applies the information. Knowledge-intensive management provides such a framework. (Price and Balasubramanian (1996), “Knowledge-intensive resource management and technology is an approach to fine-tuning farmer management to enhance profitability and environmental integrity in high-productivity systems.” Knowledge-intensive management brings together disparate pieces of information (e.g. cold weather, 10 days till harvest, 12 harmful insects per rice plant) to make intelligent decisions (e.g. the insects would not multiply before harvest-time due to cold weather therefore no need to spray). Meeting the Production Challenge& Delivering Knowledge-Intensive Management The “yield gap” is a commonly acknowledged phenomenon in agriculture and is defined in two ways. One is to describe the difference between the attainable yields that agricultural scientists at research stations achieve, and the actual yields obtained by farmers using the same seeds and inputs. Given the same seeds, fertilizers, pesticides, irrigation access, and labour, a farmer who uses knowledge-intensive management to make production decisions will likely harvest a larger or higher-value crop than one who does not. Productivity gains accrue to farmers from differences in the way inputs are used; that is, the timing and method of input use (Byerlee, 1987). The farmer makes more profit, while at the same time
461 producing more food to help meet increasing demand. ICTs are an obvious and appropriate medium for information delivery and even expanding farmers’ knowledge - information with understanding. Knowledge-intensive management has been delivered through numerous modalities, including farmer participatory research, farmer field schools, mass media campaigns, and traditional distance education. Although many of these were successful, their impact and coverage have been limited by high-costs, small program sizes, or other factors. The emergence and expansion of cost-effective ICT networks offer the potential to deliver information and the skills needed to apply it to agriculture via a medium that is itself information based. This idea of delivering knowledge-intensive management over ICTs has excited many in the agricultural sector. e-learning for Agricultural Professionals ICTs promise to play an important role in the delivery of information and knowledge-intensive management skills to agricultural professionals. Perhaps the most exciting aspect of the application of these technologies for agricultural education is the emerging field of e-learning. e-learning is the most recent evolution of distance learning - a learning situation where instructors and learners are separated by distance, time or both. 1. e-learning (sometimes also defined as ‘Internet-enabled learning’), uses network technologies to create, foster, deliver, and facilitate learning, anytime and anywhere. E-learners use a variety of tools while learning. For example, email, e-mail newsletters, listservs, discussion groups, chat, instant messaging, and internet broadcasts can be used for communication (White, 2001), while hyper linked web pages, downloadable documents, multimedia, interactive forms, and simulations are used to engage and involve learners with content. Whether to use and how to use these different tools is an important consideration of instructional design for e-learning. The characteristics of one type of tool, simulations, with reference to the applicability of this tool in delivering knowledge-intensive management strategies to agricultural professionals should be discussed. INTERNET& Digital Literacy The Internet and its associated information and communication technologies have given us the tools to bridge the farm technology gap and educate agricultural professionals. e-Learning is beginning to show tremendous potential as a learning tool. While there are many barriers to
462 successful implementation of e-Learning in Asia there are also numerous ways of countering these barriers. Bringing together the diverse stakeholders that have the vision and skills to use these new tools will play a very important role. Another key factor for the success of online learning for the importance of developing e-Learning programs. Digital literacy is “The ability to access and take advantage of networked computer resources and to use and understand information as presented by computers” (Raab, 1999). The specific skills associated with digital literacy include the capacity to take advantage of such varied Internet communication resources as email, listservs, and online conferences. It includes being able to use a range of online search strategies and tools to locate and access important information. It involves using these tools to collaborate with others and also includes the skills required to learn online and take advantage of Internet-based learning opportunities. Acquisition of such skills is vital in today’s world where the Internet is transforming business and education. As Fontaine (2001) states, “The worst case scenario is that the digital divide will grow, economic inequality will increase both within and between countries, entrepreneurs not plugged into the global network will be unable to reach markets, and nations not online will fail to attract international investment, leaving regions with large populations facing an economic crisis of unparalleled proportions.” Agroinformatics: Static entities or a dynamic flow? Agroinformatics is now internationally responsibility to conserve and disseminate agricultural informations worldwide from all parts of the world. One of the first Agriculturists has to continue this activity to ensure that the irreplaceable time and resources could be conserved for all people, everywhere. The general diversity in agriculture everywhere has enabled the world agriculture and partner organizations to develop such a system so that informations can be assessed and disseminated easily. A widely held misconception about agroinformatics is that what we find in remote areas of today is essentially the same as found in the same location 100 years ago. But what actually happens in the real fields? It is critical to remember that varieties grown by farmers, including smallholders, are subject to both environmental selection and human management practices, which greatly influence whether a gene (and trait) is lost or fixed and at what frequency it occurs. Incomplete knowledge of smallholder farmer management and seed selection practices poses a major constraint to determining what factors
463 influence the diffusion of genes (including transgenes) into maize agroinformatics and what the potential impacts might be. There is an urgent need to address this gap in our understanding with further research. Other key related questions should also be addressed: How may this process of diffusion affect the livelihoods of small-scale maize farmers? Can this process and its impacts be managed? And if so, how? There is an urgent need for a centralized database on the maize agroinformatics of Mexico and the rest of the world. This database should contain information on the agroinformatics’ agronomic and quality traits, and when feasible, genetic information. Aside from serving as a “baseline” for diversity and being useful in breeding programs, this database would have other practical applications? In the dispute on patenting high oilcontent maize, for example, no data were readily available to show that Mexican agroinformatics with high oil content were being cultivated prior to the patent applications. If we do not have access to this kind of information, it can reduce the value of biodiversity. The Potential Impact of Agroinformtics at the Global Level It has been more than 31 years since the nutritional importance of the Opaque2 gene was discovered at Purdue University, nevertheless, the benefits of this discovery are only now-three decades later-disgustingly and belatedly beginning to reach commercial production in several countries. This implies we must explore the reasons for the long lag time between the time of initiation of the breeding programs designed to develop commercially acceptable QPM varieties and hybrids, and the time of release and wide-spread commercial utilization of QPM varieties and hybrids. In particular, I see no good or acceptable reason why it has taken three decades since the discovery of the nutritional value of the Opaque2 gene to produce commercially acceptable and competitive QPM varieties and hybrids. Even more inexcusable, it has taken 15 years since it became apparent-to any imaginative visionary aggressive plant breeder-that the genetic variability and methods were available to convert the inferior-yielding soft Opaque2 type endosperm to dent and flint grain types, and while doing this, increase grain yield and disease and insect resistance to the levels of the best conventional varieties and hybrids. These comments do not imply that the only reason for the slow development of commercially acceptable varieties and hybrids lies primarily with the plant breeders. My curiosity and interest in the development of maize varieties and hybrids with improved nutritional properties dates back to the 1964 publication by Dr. E. T. Mertz and colleagues in which they clearly showed
464 the nutritional benefits that were associated with the Opaque2 gene. I recall the enthusiasm of that period, when maize scientists in nearly all hybrid corn companies around the world, as well as in most university programs in countries where maize is an important crop, modified their research and incorporated the Opaque2 gene into their breeding program with the aim of increasing the levels of lysine and tryptophan. Since both lysine and tryptophan levels were known to be tightly linked and both controlled by the Opaque2 gene, this goal appeared to be rapidly and inexpensively achievable. Unfortunately, it proved to be otherwise. Conclusion The tremendous increase in genomic data in the last 20 years has greatly expanded our understanding of biology. Genome sequencing projects now span from draft assemblies, complete genomes, large-scale comparative genomic projects, to the new field of metagenomics where genetic material is recovered directly from environmental samples and the entire complement of DNA from a given ecological niche is sequenced. Although these provide an ever greater resource for studying biology, there is still a long way to go from the initial submission of sequence data to the understanding of biological processes. By integrating different types of biological and bibliographical data, NCBI is building a discovery system that enables the researcher to discover more than would be possible from just the original data. By making links between different databases and computing associations within the same database, Entrez is designed to infer relationships between different data that may suggest future experiments or assist in interpretation of the available information. In addition, NCBI is developing the tools that provide users with extra layers of information leading to further discoveries. Genomics is a very rapidly evolving field. The advance in sequencing technologies has lead to new data types which require different approaches to data management and presentation. NCBI continues to add new databases and develop new tools to address the issue of ever-increasing amounts of information. New ICTs have a major role to play in the life of farmers as they provide them with latest know-how on agriculture, on – line selling and buying i.e. e – commerce, problem solving through internet. They can also access daily weather forecasts, information on cropping patterns, soil conservation, and government schemes. These developments have also opened the door to a whole new generation of ‘multi- modal’ market information services, which bring much needed price and product
465 information directly to the farmers. India’s advantage lies in its population density, its network, economies of scale, understanding of rural poor needs and its cultural and geographic diversity. By addressing these challenges and connecting the rural communities, India can boost its efficiency and economic competitiveness. The world economy is moving very fast and the last thing India can afford is to leave most of its population disconnected from the high- speed global network. India would benefit from approaching the issue by using ICT in Agriculture by focusing on providing broadband connectivity and a centric development approach. References Abell, A. and Oxbrow, N. (1999). Skills for the knowledge economy. Library and Information Commission Bull 1:7-9, LIC, London, UK. Ahn, S. N. and Tanksley, S. D. (1993). Comparative linkage maps of the rice and maize genomes. Proc Natl Acad Sci USA 90, 7980–7984. Altschul, S. F., Gish, W., Miller, W., et al. (1990). Basic local alignment search tool.J Mol Biol 215(3), 403–410. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D. J. (1997). Gapped BLAST and PSIBLAST: a new generation of protein database search programs. Nucleic Acids Res 25(17), 3389–3402. Review. Anonymous. (2001). E-Learning can Level the Playing Field. sa.internet.com, Internet news, 26 February 2001. Atkinson, R.D. and Court, R.H. (1998). The New Economy Index: Understanding America’s Economic Transformation. Progressive Policy Institute, Washington, DC. Benson, D.A., Karsch-Mizrachi, I., Lipman, D.J. and Wheeler, D.L. (2008). GenBank. Nucleic Acids Res 36(Database issue), D25–D30. Borlaug, N.E. and Dowswell, C. (2001). Agriculture in the 21st Century: Vision for Research and Development. Bru¨gger, K., et al. (2007). The genome of Hyperthermus butylicus: a sulfur-reducing, peptide fermenting, neutrophilic Crenarchaeote growing up to 108 degrees C. Archaea 2(2), 127–135. Byerlee, D. (1987). maintaining the momentum in post-green revolution agriculture: a micro-level perspective for Asia. Internl Development Paper No. 10. Michigan State Univ, East Lansing, Michigan, 57pp. Devos, K.M., Chao, S., Li, Q.Y., Simonetti, M.C. and Gale, M.D. (1994). Relationship between chromosome 9 of maize and wheat homeologous group 7 chromosomes.Genetics 138, 1287–1292. Fleischmann, R.D., et al. (1995). Whole-genome random sequencing and assembly of Haemophilus influenza Rd. Science 269(5223), 496–512. Fontaine, M. (2001). How Information Technology Can Help Development: Opportunities
466 and Obstacles TechKnowLogia, May/June. Hamilton, D. P. (1994). Distant Vision: In Asia, Electronic Communication Is Growing— But It Still Has a Long Way to Go. Asian Wall Street Journal. 18 November. S2. Hillary, E.S. and Maria, A.S., eds. (2006). Genomes (Cold Spring Harbor Monograph Series, 46). Cold Spring Harbor, New York. Klimke, W. and Tatusova, T. (2006). Microbial genomes at NCBI in (Mulder, N., Apweiler, R., eds.) In Silico Genomics And Proteomics: Functional Annotation of Genomes And Proteins, Nova Science Publishers; 1st ed., pp. 157–183. Kurata, N., Moore, G., Nagamura, Y., Foote, T., Yano, M., Minobe, Y. and Gale, M.D. (1994). Conservation of genome structure between rice and wheat.Biotechnology (NY) 12, 276–278. Lederburg, E.M. (1986). Plasmid prefix designations registered by the Plasmid Reference Center 1977–1985. Plasmid 1, 57–92. Liolios, K., Mavrommatis, K., Tavernarakis, N. and Kyrpides, N.C. (2007). The Genomes On Line Database (GOLD) in 2007: status of genomic and metagenomic projects and their associated metadata. Nucleic Acids Res. 36(Database issue), D475–D479. Mailman, M.D., Feolo, M., Jin, Y., Kimura, M., Tryka, K., Bagoutdinov, R., Hao, L., Kiang, A., Paschall, J., Phan, L., Popova, N., Pretel, S., Ziyabari, L., Lee, M., Shao, Y., Wang, Z. Y., Sirotkin, K., Ward, M., Kholodov, M., Zbicz, K., Beck, J., Kimelman, M., Shevelev, S., Preuss, D., Yaschenko, E., Graeff, A., Ostell, J., Sherry, S. T. Benson, et al., (2007). The NCBI dbGaP database of genotypes and phenotypes. Nat Genet 39(10), 1181–1186. Moe, M.T., (2000). E-learning in the New Economy. e-learning Magazine, Advanstar Communications. Nakabachi, A., Yamashita, A., Toh, H., Ishikawa, H., Dunbar, H. E., Moran, N. A. and Hattori, M. (2006). The 160-kilobase genome of the bacterial endosymbiont. Carsonella Sci 314(5797), 267. Price, L.M.L. and V. Balasubramanian (1996). Securing the future of intensive rice systems: a knowledge intensive resource management and technology approach. Chapter 6 in: Rice Production Systems in the Asian Region Volume I: Challenges for Rice Research in Asia, Ken S. Fischer, ed. The Pacific basin Study Center, San Francisco. Pruitt, K. D., Tatusova, T. and Maglott, D. R. (2007). NCBI reference sequences (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins. Nucleic Acids Res 35(Database issue), D61–D65. Raab, R.T. (1999). Singapore to Philippines Video-Conferencing Session on Bioinformatics delivered over APAN via SingAREN, Schneiker, S., et al. (2007). Complete genome sequence of the myxobacterium Sorangium cellulosum.Nat Biotechnol 25(11), 1281–1289. Skyrme, D.J. (1997). The global knowledge economy. Insights 21, David Skyrme
467 Associates. Sugawara, H., Ogasawara, O., Okubo, K., Gojobori, T. and Tateno, Y. (2008) DDBJ with new system and face.Nucleic Acids Res 36(Database issue), D22–D24. Tatusov, R.L., Fedorova, N.D., Jackson, J.D., Jacobs, A.R., Kiryutin, B., Koonin, E.V., Krylov, D.M., Mazumder, R., Mekhedov, S.L., Nikolskaya, A.N., Rao, B.S., Smirnov, S., Sverdlov, A.V., Vasudevan, S., Wolf, Y.I., Yin, J.J. and Natale, D.A. (2003). The COG database: an updated version includes eukaryotes. BMC Bioinformatics 4, 41. Tatusova, T.A., Karsch-Mizrachi, I., Ostell, J. A. (1999). Complete genomes in WWW Entrez: data representation and analysis. Bioinformatics 15(7–8), 536–543. Tatusova, T., Smith-White, B., Ostell, J. A. (2006). Collection of plant-specific genomic data and resources at the National Center for Biotechnology Information, in (David, E., ed.), Plant Bioinformatics: Methods and Protocols (Methods in Molecular Biology), Humana Press, 1st ed., pp. 61–87. Teeling, H., Lombardot, T., Bauer, M., Ludwig, W. and Glockner, F.O. (2004). Evaluation of the phylogenetic position of the planctomycete ‘Rhodopirellula baltica’ SH 1 by means of concatenated ribosomal protein sequences, DNA-directed RNA polymerase subunit sequences and whole genome trees. Int J Syst Evol Microbiol 54, 791–801. van Deynze, A.E., Nelson, J.C., O’Donoghue, L.S., Ahn, S.N., Siripoonwiwat, W., Harrington, S.E., Yglesias, E.S., Braga, D.P., McCouch, S.R. and Sorrells, M.E. (1995). Comparative mapping in grasses: oat relationships. Mol Gen Genet 249, 349–356. Verma, D.K. and Singh, N.K. (2003). Agro-informatics and its strategic role in agricultural research.Editors. Irfan Ali Khan and Atiya Khanum, Ukaaz Publications, Hyderabad. Pp110-177. Wheeler, D.L., et al. (2008). Database resources of the National Center for Biotechnology Information.Nucleic Acids Res 36(Database issue), D13–D21. White, N. (2001). The Tools of Online Connection.
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30 ROLE OF ALLELOPATHIC CHEMICAL LIKE GA3 ON NUCLEIC ACID AND PROTEIN CONTENTS OF AFLATOXIN B1 TREATED MAIZE SEEDS (Zea mays L.) Gajendra Prasad, Kumari Nitu and Kumari Ragni Plant physiology and Mycotoxin Laboratory, University Deptt. of Botany, L.N.M.U, Darbhanga – 846004.
ABSTRACT The effect of phytohormone on mycotoxin treated maize seeds was observed to understand the potentiality of hormones on detoxification of mycotoxins. The levels of nucleic acids and protein contents (Qualitative and Quantitative) were observed in maize seeds (Zea mays L. CV Madhuari – 01). The reduction in protein, DNA and RNA contents of maize seeds shown to be inhanced by combine responses of Aft B 1 and GA3 (at 2000 µg/l : v/v) ratios i.e. 1 : 2 and 1 : 3 v/v. Besides variation in the contents of protein, the combined effect of AFT-B 1 and GA3 ratios were also altered the qualitative composition of protein. Protein spectra in disc-electrophoresis revealed nine peaks in the control seeds, of which five peaks were distinct. Peaks were designated by I, III, V & VIII and IX. They were lowered or lost at 3 : 1 combination ratio of AFT-B1 and GA3. Key words : Nucleic Acid, Protein, AFT-B 1, Phytohormone (GA3) and Maize.
Introduction The word allelopathy derives from two separate words i.e. allelon which means “of each other” and pathos which means “to suffer”. Allelopathy referes to the chemical inhibition of one species by another.
470 The “inhibitory” chemical is released into the environment where it effects the development and growth of neighbouring plants. It is a biological phenomenon by which an organism produces one or more biochemicals that influence the germination, growth survival and reproduction of other organism. Plant allelopathy is one of the modes of interact between receptor and donor plants and may exert positive effects (eg – for agricultural management such as weed control, crop protection or crop reestablishment). It is consist of various chemical families and are classified into the following 14-categories based on chemical similarity (Rice, 1974), water soluble organic acids, straight chain of alcohols allipatic aldehydes and ketones, simple unsaturated lactones, long chain fatty acids and polyacetylenes, benzoquinone, anthraquinone and complex quinones, simple phenols, benzoic acid, and its derivatives, cinnamic acid and its derivatives, caumarin, flavonoids, tannins, terpenoids and steroid amino acids and peptides; alkaloids and cynohydrini sulfide and glucosinalates and purines and nucleosides. Plant growth regulators including salicyclic acid, gibberellic acid (GA3) and ethylene are also considered to be allelochemicals i.e. nonnutritive substances mainly produced as plant secondary metabolites or decomposition products of microbes are the active media of allelopathy. Aflatoxin B 1 is known to interfere with the synthesis of phytohormones like GA3 which has been shown to stimulate the activities of lipase and á-amylase activity, which are required primarily for seed germination and seedling growth (Chaterjee, 1988). Literature survey shows that GA3 plays a key role in seed dormancy breaking, seedling growth and induce seed germination in soybean seeds (Tiwari et. al., 1986), Brown et. al. (1986) proposed that GA3 acted the most active group of hormones in the breaking of dormancy in Protea compacta. Phytohormones like GA3/ Kinetin are stimulating the chlorophyll, protein synthesis as well as áamylase activity in cotton seeds (Black and Altschul, 1965; Jones et. al., 1967) Singh et. al. (1991) also revealed that GA3- reversed the inhibition rate caused by the aflatoxin B1 treated seeds of chickpea and pigeon-pea. Maize (Zea mays L.) is an important cereal crops of India and consumed as staple in food in many regions but infected mostly by aflatoxin produced by microbes like Aspergillus flavus (Bilgrami and Sinha, 1984; Prasad and Sahay, 1987; Prasad et. al., 1987; Ahmad, 1999, 2002, 2010, 2012, 2016). During harvesting, aflatoxin have been shown to produce the changing the seed quality, nutritional value, the failure of seed germination
471 ability, inhibit the chlorophyll synthesis etc. (Bilgrami and Sinha, 1992; Sinha et. al., 1993; Prasad et. al., 1993, 1997) ie. ultimately result in severe loses of maize productivity. Since only the few reports are available about the impact of phytohormone on mycotoxins treated seeds, the present work was designated to find out if the additional dose of GA3 can reverse the inhibitory effects of aflatoxin B1 on nucleic acid and protein contents of maize seedlings. Materials and methods Seeds of Zea mays L. CV Apurva seeds hybrid madhuri – 01 were obtained from dayal traders manures and seed storage centre, Kadirabad, Darbhanga, Bihar. Stock solution of Aflatoxin-B1 and GA3 (Sigma, st. Louis, U.S.A) were initially prepared each in 1 cm3 ethnol from which the dilutions (2gm-3 ) made in sterilized distilled water. Solution of these toxin and phytohormone were mixed in different combination ratios like 1 : 1, 1 : 2, 2 : 1, 1 : 3, 3 : 1 (v/v). The seeds were steeped initially in distilled water for 1 hour and subsequently in different combination of AFT-B1 and GA3 for 20 hrs. For each treatment 100 seeds were taken in triplicate. The steeped seeds subsequently germinated on moist blotting papers at 28 ± 2ºC. The quantitative and qualitative estimation of protein in seeds were measured by the spectrophotomatric method of lowry et. al. (1951) and the disc electrophoretic method of Ornstein and Davis (1964), respectively. Gels were scanned by the ultrascan-XL-Enhanced laser Densitometer (LKB, Bromma, Sweden). The nucleic acid contents of the control and treated seeds were estimated by the method of Gottlieb and Tripathi (1968). The least significant differences at 1 and 5% confidence levels (LSD01 and LSD05) were determined following the procedure of Dospekshov (1984) Result Protein Qualitative and Quantitative : High significant fail in the levels of protein of maize seedling was recorded with the treatment of different levels of AFT-B1 (Table : 01). The variations in the control and that of corresponding LSD01 and LSD05 (least significant differences at 1 and 5% levels) values in Table : 01 revealed
472 significant inhibitory effect of AFT-B1 on protein levels. Per cent inhibition in protein were 15.30, 30.24, 48.80, 62.25 and 73.44% at 100, 250, 500, 1000 and 2000µg/l concentration of AFT-B1, respectively. The combined effect of AFT-B1 and GA3 (2000µg/l, v/v) in various combination ratios on protein contents was depicted in Table : 02. The maximum inhibition was 61.79% at 3 : 1 ratio, followed by 53.65, 41.79, 33.05 and 14.73 at 2 : 1, 1 : 1, 1 : 2 and 1 : 3 ratios, respectively. Beside reduction in the concentration of protein contents, AFT-B1 and GA3 ratios also effected their qualitative composition. Protein spectra in disc electrophoresis (Fig. : 1), revealed peaks in the control reeds of which five distinct peaks were designated by I, III, V & VI. They were lowered or lost at 3 : 1 combination of AFT-B1 and GA3 ratios. Nucleic acid : The AFT-B1 and GA3 individually and in combination, exhibited marked inhibition (increase and decrease) in DNA and RNA contents of maize seeds (Table : 03 and 04) Percent inhibition in DNA and RNA contents were 6.23, 10.28, 19.85, 42.99, 58.24% and 5.72, 10.03, 20.18, 42.09, 53.49 % at 100, 250, 500, 1000 and 2000µg/l concentration of AFTB1, respectively. The combined effect of AFT-B1 and GA3 (2000µg/l v/v) in various combination ratios was depicated in Table : 04. The maximum and minimum inhibition in DNA and RNA contents were 46.06, 8.91% as well as 36.09 and 5.27% at 3:1 and 1 : 3 combination ratios, respectively. The variation in control and that of corresponding LSD01 and LSD05 values in table revealed significant valuable effect of AFT-B1 and GA3. Discussion The chemical responsible for the toxicity in Black walnut is juglone (5-hydroxy-1,4, napthoquinone) remains in the soil around the tree and is most potent at the drip line, through the roots can spread out well beyond this and is a respiration inhibitor. These plants, when exposed to the allelotoxin, exhibit symptoms such as wilting, chlorosis (Faliar gellowing) and eventually death. The suppression of protein and nucleic acid synthesis might been due to the inhibition in chromatin bound DNA depended polymerase activity by toxin (Tripathi and Mishra, 1983). Variations in the polyacrylamied Gel electrophoresis pattern of Soluble protein of Aspergillus parasiticus
473 and A. ryzae in peanut seeds have been reported by Ory and Cherry, 1972. The drasting reduction as well as enhancement of protein and nucleic acid contents in Aflatoxin B1 treated seeds was reduces due to stimulating effects of Phytohormones (Table : 2, 4 and Fig. : 01). Reveals that phytohormone have been shown to modify the metabolism of toxin treated seeds by breaking dormancy of seeds. Conclusion The allopathic effects of the plant residuces were similar to those exhibited by some weeds and trees that inhibited the germination of agricultural crops. This study suggests that some plant residues also contained allelochemicals which are released into the soil during decomposition. Previous assertions by Cheema (1988) revealed that nature sorghum contained benzoic acid, P-hydorxybenzoic acid, vanillic acid, m-comadic acid, p-coumaric acid, gallic acid, cafferic acid, ferulic acid and chlorogenic acid while Chou and Lin (1976) asserted that rice husks contained phenolic compounds such as P-hydroxybenzoic, vanillic, ferrulic, p-coumaric, and o-hydrophenyletic acid. All of these compounds constitute allelochemicals to maize. Most of the studies had, however, revealed that the inhibition obtained in the laboratory experiments might differ from the situations in the fields (Houser, 1993; Lisanework and Michelsen, 1993, Tian and Kang, 1994; Mehar et. al., 1995; Hansen Ouartey et. al., 1998). Acknowledgements The authors are grateful to the Prof. & Head, University Deptt. of Botany, L.N.M.U, Darbhanga for providing laboratory facilities and thanks to the CSIR-I, New Delhi for financial assistance as well as thanks to K.K. Sinha for discussion. Table 01: Impact of Aflatoxin B1 on Protein contents of Maize seedlings. Concn. of Aft-B1 (µg/l)
Protein (mg/100mg) Amount ± S.E.
Difference with control
% inhibition
00
8.43 ± 0.10
-
-
100
7.14 ± 0.02
1.29
15.30
250
5.88 ± 0.02
2.55
30.24
500
4.65 ± 0.03
3.78
44.80
474
1000
3.19 ± 0.05
5.24
62.15
2000
2.23 ± 0.08
6.20
73.44
LSD 01
-
0.33
6.23
LSD 05
-
0.23
4.44
Figures in Parenthesis are S.E. All values are significant at both 1 & 5% levels. Table 02: Combined Impact of Aflatoxin B 1 & GA 3 on protein contents of Maize seedlings. Concn. of Aflatoxin (2000) µg/l : GA3 (AFT-B1: GA3) its, v/v
Protein (mg/100 mg) Amount ± S.E
Difference with control
% inhibition
0:0
8.35 ± 0.04
-
-
1:1
4.86 ± 0.06
3.49
41.79
1:2
5.59 ± 0.05
2.76
33.05
2:1
3. 87 ± 0.02
4.48
53.65
1:3
7.12 ± 0.05
1.23
14.73
3:1
3.19 ± 0.01
5.16
61.79
LSD
01
-
0.16
2.88
LSD
05
-
0.08
1.51
Table 03: Impact of Aflatoxin B1 on DNA & RNA contents of maize seedlings. Concn. of Aflatoxin (µg/l)
DNA (µg/100mg)
RNA (µg/100mg)
Amount ± S.E
Differe- % Amount ± S.E nce with inhibition control
Differe- % nce with inhibition control
00
11.28±0.14
-
-
38.25±0.09
-
-
100
10.57±0.09
0.71
6.29
36.06±0.14
2.19
5.72
250
10.12±0.02
1.16
10.28
34.41±0.03
3.84
10.03
500
9.04±0.11
2.24
19.85
30.53±0.22
7.72
20.18
1000
6.43±0.06
4.85
42.99
22.15±0.01
16.10
42.09
2000
4.71±0.15
6.57
58.24
17.79±0.05
20.46
53.49
LSD
01
-
0.26
2.98
-
1.63
5.45
LSD
05
-
0.18
2.12
-
1.16
3.88
Figures in Parentheses are S.E.,
All value are significant at both 1 & 5% level.
Absorption of Peaks (Relative)
475
AFT-B1 : GA3 (1 : 3)
AFT-B1 : GA3 (1 : 2)
0:0 (Control)
AFT-B1 : GA3 (1 : 1)
AFT-B1 : GA3 (3 : 1)
AFT-B1 : GA3 (2 : 1)
Position of Peaks (mm) Fig. – I : Combined effect of Aflatoxin B1 and GA3 in different combinations on protein qualitative in maize seedlings according to gel electrophoresis methods. Table 04: Combined Impact of Aflatoxin B1 & GA3 on DNA & RNA contents of maize seedlings. Concn. of Aflatoxin (µg/l)
DNA (µg/100mg)
RNA (µg/100mg)
Amount ± S.E
Differe- % Amount ± S.E nce with inhibition control
Differe- % nce with inhibition control
0:0
11.44±0.28
-
-
38.26±0.14
-
-
1:1
8.72±0.14
2.72
23.77
28.74±0.24
9.52
24.88
1:2
9.50±0.09
1.94
16.95
32.63±0.08
5.63
14.71
476
2:1
7.32±0.14
4.12
36.01
27.16±0.16
11.10
29.01
1:3
10.42±0.14
1.02
8.91
36.24±0.19
2.02
5.27
3:1
6.17±0.12
5.27
46.06
24.45±0.23
13.81
36.09
LSD
01
-
0.38
6.88
-
3.15
10.18
LSD
05
-
0.41
4.90
-
2.24
7.26
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31 Momordica charantia - PHYTOCHEMICALS, PESTS AND ITS CONTROL Satya Shubhangi and V.N. Singh PG Department of Zoology, TM Bhagalpur University, Bhagalpur E-mail-
[email protected]
ABSTRACT Momordica charantia is a herbal climber belonging to family Cucurbitaceae . All the parts of plant is bitter in taste .It is widely cultivated as vegetables crops in various parts of world. In different parts of karela contains various biological activities. The nutritional and chemical composition of Momordica charantia show various biochemical content ie. lipid, protein, fibre, carbohydrate, different types of element and vitamin . Phytochemical constituents of M. charantia are protein, steroid, alkaloid, triterpene, inorganic, lipid and phenolic compounds. Chemical constituents of Momordica charantia momorcharaside, momorcharin , momordenol, momordicilin, momordicins, momordicinin, momordicosides, momordin, momordolo, charantin, charine, crypotxanthin, cucurbitins, cucurbitacins, cucurbitanes, amino acids. The different pests of bitter gourd has been indetified which effects the fruit and defoliate the crop. Keywords: Momordica charantia, Biological Activities, Biochemical, Phytochemical, Pests.
INTRODUCTION Herbs are plants valued for aromatic properties which are used in medicine, for flavoring food or as fraganaces (D Satish Kumar et.al. 2010). Herbs include a variety i.e culinary, medicinal and in some case spiritual. In medicine or spiritual different part of plants are used while in culinary fruit and flower parts are used (Wikipedia. Herb)
480 Momordica charantia is an herbal climber belonging to family Cucurbitaceae grown in tropical and sub tropical region. It is common food in Indian cusine and in various part world. All the parts of fruits is bitter in taste .It is widely cultivated as vegetables crops in various parts of world. In Ayurveda, the various parts of Momordica charantia has been used in treatment of various diseases. Momordica charantia contains of various phytochemicals ie; alkalodis, peptides, falvoniods , saponins, triterpene, inorganic, lipid and phenolic compounds etc (Hyun Young Kim et. al. 2013) . ORIGIN AND DISTRIBUTION Momordica charantia has been cultivated since ancient times in tropical and sub-tropical regions of the world. It is widely cultivated in grown in Indian sub-continent , South-East Asia, China, Africa, Carribean and South America (Javed et. al. 2011). CULTIVATION Momordica charantia is a genus of annual or perennial climbers found throughout India and is cultivated upto 1500 m. Cultivation during warm season ie; April to July. Seeds are sown in the pit in 2-3 number at a distance of half meters with manures. Flowering begins after 30-35 days of plantation. And the fruit is ready for harvesting after 15-20 days of flowering (K P Sampath Kumar et. al. 2010). BOTANICAL DESCRIPTION: PLANT- Annual, slender climber, 2-4 meter high, scarcely to dense pubescent (tender part woody), monoceious. STEM- Stems prostrate, five edges, dark green, hairy. Sections of main vine buds movement strong and can branch out lateral bine, lateral bine sections buds also could grow outside lateral bine, create a more luxuriant vine leaf system, is one of the melon species which has more lateral bines and slim. In additional, there are sections outsides axillary buds and tendrils, tendrils outside. ROOT- Bitter Gourd plant roots is relatively developed, multi-lateral, mainly grow in the 30-50 cm depth plow layer. The root mass distribution scope more 130 cm wide, 30 cm deep or more, the roots prefer moist, afraid waterlogging. LEAF- Bitter gourd cotyledon unearthed, generally donot carry out photosynthesis. Primary leaves one pair, opposite, shield shape, green. After
481 the leaf is alternate, palmately lobed, green, veins radial, with 5 radial veins, leaf length 16-18 cm and width 18-24 cm, petiole length 9 to 10 cm, yellow green(Sampath et. al. 2010) . FLOWER- Staminate flowers usually solitary on a bracteates scape, hypanthium shallow, calyx 5 lobed, petals 5, usually yellow, distinct, 1-3 with incurved scales at base, usually 3, inserted toward base of hypanthium, filaments distinct, broad, anthers, distinct or coherent, 2 of them dithecal, the other monothecal, cells curved or flexuous; pistillate flowers usually solitary on a bracteates scape, hypanthium ovoid to spindle shape, perianth usually smaller than in staminate flowers, staminodes absent or 3, ovules numerous, horizontal, stigma 3, 2 lobed. Seeds few to numerous, ovate, usually sculptured. Each plants bears separate yellow male and female flowers (www.mdidea.com). FRUIT- Fruit epllisiod, spindle ovoid shaped, usually ridged or warty looking exterior and an oblong shaped. It is hallow in cross- section, with a relatively thin layer of fleshy surrounding a central seed cavity filled with large flat seeds and pith. Seeds in size 8-13 mm, long compressed, corrugate on the margin, both faces are sculptured and grey coloured. Young fruit is emerald green, turning into orange-yellow when ripe. At maturity, the fruit splits into three irregular valves that curl backwards and release numerous reddish- brown in scarlet arils (Sonia et. Al. 2011). NUTRITIONAL VALUE The nutritional and chemical composition of Momordica charantia showed various percentage of moisture, ash, crude lipid, crude fibre, crude protein, and carbohydrate content. Different calorific values for fruit, seed and leaf were 241.66, 176.61 and 213.26 kcal/100 g were observed. The element like potassium (413 ppm), sodium (2200 ppm), calcium (20510 ppm), zinc (120 ppm), magnesium, iron, manganese and copper are present; while different vitamins were observed ie; vitamin A ( â- carotene; 0.03 ppm), vitamin E (á- tocopherol; 800 ppm), vitamin B9 (folic acid; 20600 ppm), vitamin B12 (cyanocobalamin; 5355 ppm) and vitamin C (ascorbic acid). Niacin (B3), Pyridoxin (B6), Thiamin ( B1), Riboflavin (B2), Folic acid (B9), Cholecalciferol (vitamin D), phylloquinone ( vitamin K ) were observed in traces. Nutritional value of Momordica charantia per 100 g: energy 79 KJ (19 Kcal), carbohydrates 4.32 g, sugar 1.95 g, dietary fiber 2 g, fat 0.18 g, protein 0.84 g and water 93.95 g (Nadkarni KM et. al. 1993).
482 PHYTOCHEMICALS Main constituents of M. charantia are protein, steroid, alkaloid, triterpene, inorganic, lipid and phenolic compounds (Bakare RI et. al. 2010). Chemical constituents of Momordica charantia consist of alkaloid, charantin, charine, crypotxanthin, cucurbitins, cucurbitacins, cucurbitanes, cycloartenols, diosgenin, elaeostearic acid, gentistic acid, goyaglycosides, goyasaponins, gunanylatecyclase inhibitors, gypsogenin hydroxytryptamins, karounidiols, lanosterol, lauric acid, linoleic acid, momorcharaside, momorcharin, momordenol, momordicilin, momordicins, momordicinin, momordicosides, momordin, momordolo, multiforenol, myristic acid, nerolidol, oleanolic acid, oleic acid, oxalic acid, pentadecans, peptides petroselinic acid, polypeptides, proteins, ribosomes inactivating proteins, rosmarinic acid, rubixanthnin, stigmasterol, taaxerol, trehalose, trypsin inhibitors, uracil, vacine, v-insulin, verbascoside, vicine, zeatin, zeatin riboside, zeaxanthin, zeinoxanthin,. Amino acids – aspartic acid, serine, glutamic acid, thscinne, alanine, g- amino butyric acid, pipecolic acid, ascorbigen, b-sitosterol-d-glucoside, citrulline, elasterol, flavochrome, lutein, lycopene, pipecolic acid (Rekha et. al. 2010). PHYTOCHEMICAL CONSTITUENTS IN DIFFERENT PARTS OF PLANT BODY Plant Body: Momorchains, momor denol, momordiclin, momordicins, momordicinin, momordin, momordolol, charantin, charine, cryptoxanthin, cucurbitins, cucurbitanes, cycloartenols, diosgenin, elaostearic acids, erythrodiol, galacturonic acids, gentisic acid, goyasaponins, mutifernol, goyaglycosides, goyasaponins, mutliferonal, glycosides, saponins, alkaloids, fixed oils, cucurbitane- type triterpenes, proteins and steroids. Momordicine, charantin, polypeptide- p-insulin, ascorbigen. Fruit: Amino Acid- Aspartic acid, serine, glutamic acid, threonine, glutamic acid, threonine, alanine, g-amino butyric acid, pipecolic acid and luteolin. Fatty Acids: Lauric, myristic, palmitic, palmitoleic, stearic, oleic, linoleic, linolenic acid Seeds: Enzyme- urease Amino Acid- Valine, threonine, methionine, isoleucine, leucine,
483 phenylalanine, glutamic acid. CHEMICAL COMPOSITION Several bioactive compounds have been screened in Momordica charantia, they have been classified as carbohydrate, lipids, protein and more. Momordica charantia saponins, alkaloids, triterpeniods, polypeptides, flavonoids and steroids (Grover et. al. 2003). The phytochemicals showed various bioactives compounds and their related functions. POLYSACCHARIDES Polysaccharides are the bioactive components of Momordica charantia. The polysaccharides of Momordica charantia posses various biological activities, such as antioxidant, antidiabetic, immune enhancing, neuroprotective, antitumour and antimicrobial (Zhang et. al. 2016). Extracts of Momordica charantia (Fan T. et. al. 2015, Shao P. et. al. 2015, Dong Y. et. al 2005) such as hot water, acid or alkai extractions, as well as microwave, ultrasonic and enzymatic assisted extractions, followed by ethanol precipitation have been applied for the separation o crude polysaccharides from Momordica charantia. Polysaccharide makes about 6% of bitter gourd powder, are composed of galactose, glucose, mannose, arabinose, rhamnose (Dong et. al. 2005, Deng et. al. 2014) showed that the contents of polysaccharide contents in cultivated varities range from 5.91% to 10.62% of dry powder. A water-soluble polysaccharide (MBP) is isolated from M. charantia fruits, and is composed of Ara, Xyl, Gal and Rha with a molar ratio of 1.00:1.12:4.07:1.79, and Mw of 1.15x106 Da; it showed a significant hypoglycemic effect (Zhang PP et. al. 2008). M. charantia polysaccharides ameliorate oxidative stress, hyperlipidemia, inflammation and apoptosis during myocardial infarction by inhibiting the NF-êB signaling pathway. M. charantia polysaccharides also had the ability to enhance total volatile fatty acids production, modulate the rumen fermentation pathway and influence the number of cellulolytic bacteria population (Kang J et.al. 2017). PROTEINS AND PEPTIDES Proteins and peptides are also main functional components in the fruit and seeds of Momordica charantia. Many types of proteins and peptides have been isolated from different parts of plants such as ribosomes inactivating protein (RIPs), Momordica charantia lectin (MLC), Momordica anti-HIV protein of 30 kD (MAP 30), -momorcharins (-
484 MMC), - momorcharin (-MMC), -momorcharins, -momorcharins, momorcharins, which posses RNA glycosidase activity, PAG activity, DNase-activity, lipase activity, superoxidase dismutase activity, anti-tumor, anti-cancer, immunosuppressive and anti- microbial activity (Puri M et. al. 2012, Pu Z et. al. 1996, Meng Y et. al 2012, Leung S.O et. al. 1987, Jabeen U et. al 2014, Fang E.F et. al. 2012). RIPs are a kind of RNA glycosylases that cleave an adenine–ribose glycosidic bond; it is a type of alkaline protein, which can inhibit the process of protein synthesis by inactivating ribosomes. They can be further divided into three classes; RIPs with only a RIP chain are classified as type I, and the structure of type II RIPs generally has two chains, A and B, which are interconnected by disulfide bonds (Peumans et. al. 2001). And the structure of B chain allow them binding with galactose residues on the oligosaccharide chain. There are also atypical type I RIPs (on the basis of their structure) which are classified as type III RIP (Fang E.F et. al. 2011). M. charantia lectin (Type II RIP) and -MMC have been isolated from M. charantia seeds; it can significantly inhibit human nasopharyngeal cancer cells and xenograft tumors in vitro (Fang et. al. 2012). MCL is atype II RIP, known to be particularly toxic, and has been used as an antitumor agent (Wang et. al. 1998). Momordicin is also a type II (singlestranded) RIP that has been successfully isolated from M. charantia together with other factors. MAP30 is a single chain RIP, named for its molecular mass of 30 kD; it has been found to have strong anti-tumor potential similar to MCL (Fang et.al. 2012, Peunams WJ 2001). The protein also significantly inhibits proliferation and causes apoptosis in a panel of cancer cells from prostate, breast, lung, hepatocellular and brain glioblastoma (Fang EF et.al. 2012). The MAP30 protein consists of 286 amino acids and the mature protein contains one N-glycosylation site and a glycosylase that aids in the binding of elongation factors (Fang EF et. al. 2012). Like MAP30, both MMC and -MMC are type I RIPs, containing only one enzymatic chain (Fang EF et. al. 2011). -MMC is also a 30-kDa glycoprotein, while MMC is slightly smaller (29-kD) glycoprotein. Both have anti-tumor activity individually. Polypeptide-P, a hypoglycemic peptide, is a kind of carbohydrate binding protein secreted by plant cells; it plays an important role in cell recognition and adhesion reactions. It is isolated from the fruit, seeds and tissues of M. charantia with a Mw of approximately 11 kD; it contains 166 amino acid residues and another polypeptide with a Mw of 3.4 kD has also been isolated from bitter melon (Yuan et. al. 2008). Other proteins and
485 peptides, such as peroxidase (43 kDa), Momordica cyclic peptides (Mahatmanto T et. al. 2015), trypsin inhibitors (McTI-I, -II and -III), cystine knot peptides, RNase MC2 (14 kDa), antifungal protein and MCha-Pr have also been isolated from M. charantia (Zhang B et.al. 2015) SAPONINS AND TERPENOIDS Saponins are a class of glycosides in which the aglycone is a triterpenoid or a spiro-steroid compound. All of the compositions are of sugar and aglycone, and the difference between them lies in the structure of aglycones. Saponins are found in the roots, stems, leaves and fruit of the M. charantia. Research has shown that the major chemical constituents are tetracyclic triterpenoids and their glycosides, most of which are referred to as cucurbitanes, and are well-known for their bitterness and toxicity. The content of total saponins in M. charantia powder is about 0.0432% (Xu B et. al. 2005). The saponins substances are the active ingredients of multiple drugs, widely distributed in a variety of plants (Vinchen et.al. 2007), which contain triterpenoidal saponins (e.g., cucurbitacin alkyl type, oleanane type, ursane type) and steroidal saponins. The cucurbitacins are a group of bitter-tasting, highly-oxygenated, mainly tetracyclic, triterpenic plant substances derived from the cucurbitane skeleton. Many pharmacological studies further indicated that cucurbitanes from M. charantia are responsible for their anti-diabetic and hypoglycaemia activities (Chen JC et. al. 2005) Cucurbitane-type triterpenoids: ,19-epoxy-3, 25-dihydroxycucurbita-6, 23(E)-diene, and 3,7, 25-trihydroxycucurbita-5, 23(E)-dien19-al were isolated from the methanol extract of M. charantia dried gourds, which could lower blood sugar in diabetic mice. Chang JC et. al. 2004) isolated four new cucurbitane-type triterpenes, cucurbita-5, 23(E)-diene3,7, 25-triol, 3-acetoxy-7-methoxycucurbita-5,23(E)-dien-25-ol, cucurbita-5(10), 6, 23(E)-triene-3,25-diol and cucurbita-5, 24-diene-3,7, 23-trione, from the methyl alcohol extract of M. charantia stems. In 2011, five kinds of saponins and cucurbitanetriterpenoids, including 3,7,25trihydroxycucurbita-5,23(E)-dien-19-al, momordicine I, momordicine II, 3-hydroxycucurbita-5,24-dien-19-al-7,23-di-O--glucopyranoside and kuguaglycoside G were isolated from M. charantia. In another study, eight new cucurbitane-type glycosides, kugua saponins A–H and six known compounds, were isolated by the directed fractionation of M. charantia fruits (Zhang et. al. 2014). Zhang et al. 2016 also reported that four new cucurbitane-type triterpenes, (23R)-7-hydroxy-3-Omalonyl-23-methoxycucurbita-5,24-
486 diene-19-al, (23E)-7,25-dihydroxy-3-O-methylmalonylcucurbita-5,23diene-19-al, (23E)-7-hydroxy-3-O-methylmalonyl-25-methoxycucurbita-5,23-diene-19-al, (23E)-7,25-dihydroxy-3-O-crotonylcucurbita5,23-diene-19-al, and one new glycoside 7-hydroxy-3-O-malonylcucurbita-5,24-diene-19-a-23-O--D-glucopyranoside, were isolated fromthe rattans of wild M. charantia FLAVONOIDS AND PHENOLIC COMPOUNDS Flavonoids and phenolic compounds are important components of M. charantia (Shan B et. al. 2012, Tan SP 2014). They include gallic acid, protocatechuic acid, gentistic acid, (+)-catechin, vanillic acid, syringic acid, (-)-epicatchin, p-coumaric acid, benzoic acid, sinapinic acid, ocoumaric acid, chlorogenic acid, t-cinnamic acid and t-ferulic acid. M. charantia flesh, the main phenolic acids were gallic acid, gentisic acid, catechin, chlorogenic acid and epicatechin .Ethyl acetate crude extract of M. charantia contained ascorbic acid (576.5 ng/mg), 3-coumaric acid (528.55 ng/mg), luteolin-7-O-glycoside (725.50 ng/mg), apigenin-7-Oglycoside (1955.55 ng/mg), caffeic acid (215.6 ng/mg) and naringenin-7O-glycoside (181.30 ng/mg) (Kenny O et. al. 2013). The amounts of protocatechuic acid, p-coumaric acid, syringic acid, vanillic acid and benzoic acid ranged from 2.07 to 8.78, 1.83 to 8.23, 1.77 to 3.67 and trace to 2.42 mg/100 g dry material in the flesh of all varieties of the bitter melons, respectively (Horax R et al. 2005). Main phenolic constituents in the extracts were catechin, gallic acid, gentisic acid, chlorogenic acid and epicatechin (Horax R et al. 2010). p-coumaric acid, tannic acid, benzoic acid, ferulic acid, gallic acid, caffeic acid, and (+)-catechin have also been found in aqueous extract fractions of M. charantia. IMPORTANT CHEMICAL CONSTITUENTS: 1. MOMORDICIN I Momordicin I or 3,7,23-trihydroxy-cucurbitan-5,24-dien-19-al; a chemical compound present in leaves of the bitter melon and responsible for its reputed medicinal properties. The compound was characterized of isolated by M. Yasuda and others in 1984. It is found in white crystalline solid with formula C30 H48O4,which extracted from ground dry leaves by dichloromethane in soluble in water and soluble in methane (M Ullah et. al. 2011, NM Pusphawati et.al. 2008) .
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2. MOMORDICIN 28 Momordicin 28 or 13-hydroxy-28-methoxy-urs-11-en-3-one is a triterpene compound. It is found in fresh leaves. The chemical compound formula is C13 H50 O3 with mol. mass 470.74 g/mol. Melting point of soluble in ethyl acetate and chloroform but not in petrol. It crystallizes as fire needles and was isolated by S. Begum and others in 1997 ( Sabira Begum et.al. 1997).
Fig: Momrodicin 28
3. MOMORDICILIN The chemical compound was isolated by S.Begum and other in 1997. Momordicilin or 24-[1’-hydroxy,1’-methyl-2’-pentenyloxy]-ursan3-one. It is found in fresh fruit of momordica charantia with chemical formula C36H60O3. It crystallizes as needle and melts at 170-171°c. It is soluble ethyl acetate and cholorofrom but not in petrol (Wikipedia. Momordicilin).
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Fig: Momordicilin
4.CHARANTIN Charantin was identified by Lolitkar and Rao in 1960. It is a chemical substances obtained from Momordica charantia; cited to be responsible for the hypoglycaemic properties of those plant. Charantia is mixture of 2 steroidal saponins in the ratio of 1:1; ß-sitosteryl glucoside(C 35 H 60 O8) and 5,25-stigmastrely glucoside(C 35 H58 O6 ). It is whitish crystalline substance, tasteless, neutral with melting point of 266268°C. It is sparing soluble in water at highly polar solvents as well as in apolar solvent like hexane; but soluble in ether, methane and ethanol. It can be effectively extracted from the plant by pressurized ethonol or acetone at 100°C (M.M Lolitkar et. al. 1962, Sonal Desai et. al. 2015).
PESTS Pest is a plant or animal detrimental to humans or human concerns including crops, livestock and forestry. A pest is any living organism, whether animal, plant or fungus, which is invasive or troublesome to plants
489 or animals, human or human concerns, livestock, or human structures. CERCOSPORA LEAF SPOT Cercospora leaf spot disease caused by Cercospora citrullina Cooke is one of the most important disease of bitter gourd. Cercospora generally cause leaf spots not only in bitter gourd but in many plants. The fungus usually produces long, slender, colorless, straight to slightly curved, multicellular conidia on short conidiophores which usually arise from the plant surface in clusters through the stomata and form conidia successively on new growing tips. Leaf spot symptoms in many plants caused by infection is highly attributed to their toxin called cercosporin. When the toxin is secreted and activated by light, it becomes toxic to their host by generating singlet oxygen. The singlet oxygen then causes lipid peroxidation in plant membranes and leakage of nutrients thereby killing the plant’s cells and at the same time enhances the virulence of the pathogen. C. citrulline Cooke cause leaf spot disease of bitter gourd shows sensitive against four fungicide benomyl, chlorothalonil, copper oxychloride and mancozeb. These fungicides can be use to cure high incidence and severe infection of the pathogen causing leaf spot in bitter gourd (Dindo King M. Donayre et. al. 2014). YELLOW MOSAIC VIRUS Bitter gourd Yellow Mosaic Virus is a Whitefly transmitted geminivirus. It causes yellow mosaic disease in bitter gourd. Symptoms appear as light and dark green mottling of the leaves, distortion of leaves and stunting of the plant might occur. Sometimes fruit might show sunken concentric circles or a raised marbled pattern. It is transmitted by five different species of aphid vectors and was tentatively named as Bitter gourd mosaic virus. This disease attains significance because the virus can attack the crop of all stages. A severe loss is yield due to the infection of this stages. Bitter gourd plants infection due to yellow mosaic virus treated with Bougainvillea spectabilis challenge inoculate with virual infection and reduced the disease incidence and growth of plant increases ( Nataraj Rajinimala et. al. 2009). DOWNY MILD DEW Downy mildew caused by Pseudoperonospora cubensis is one of the important foliar disease of bitter gourd. It was reported for the first time in 1868 and still are severe in bitter gourd, muskmelon, watermelon, cucumber, sponge gourd and ridge gourd. Symptoms first appear as pale green areas on the upper leaf surfaces and changes to yellow angular spots.
490 A fine white to greyish downy growth soon appears on the lower leaf surface. Infected leaves generally die but may remain erect while the edges of the leaf blades curl inward. Usually, the leaves near the center of a hill or row are infected first. The infected area spreads outward, causing defoliation, stunted growth, and poor fruit development and the entire plant may eventually dies. During moist weather the corresponding lower leaf surface is covered with a downy, pale grey to purple mildew. The colour of the mildew varies from white to near black. Infected leaves generally die but may remain erect, while edges of the leaf blade curl inward. Usually, the leaves near the centre of a hill or row are infected first. The infection area spreads, causing defoliation and poor fruit development which reduces yield. In rainy and humid weather entire vein is killed. Early infection of downy mildew can cause reduction in crop yield upto 60% where as late infection is less damaging. In India, it is present all over the country except in high altitude temperate zone in the Himalaya (Vijay Kumar et. al. 2018). REFERENCES: Bakare R I et.al.(2010). Nutritional and Chemical Evalution of Momordica charantia : Journal of Medicinal Plants Research.Vol.4(2).2189-2193. Cai, Y.; Liu, M.; Wu, X.; Wang, Z.; Liang, C. and Yang, Y. (2010). Study on the antitumor and immune-stimulating activity of polysaccharide from Momordica charantia. Pharm. Clin. Res., 18, 131–134. Chang, L.Y.; Tang, L.; Yan, F.;Wang, S. and Chen, F. (2004). The Effect of the Total Saponin Extract from the Shoots of Momordica charantia L. on Anti-virus HSV-II Activity. J. Sichuan Univ., 3, 043 Chen, J.C.; Chiu, M.H.; Nie, R.L.; Cordell, G.A.; Qiu, S.X. (2005) Cucurbitacins and cucurbitane glycosides: Structures and biological activities. Nat. Prod. Rep., 22, 386–399. [CrossRef] [PubMed Deng, Y.; Zhang, M.; Liu, J.; Zhang, Y.; Zhang, R. andWei, Z. (2014). Comparison of the content, antioxidant activity, and_-glucosidase inhibitory effect of polysaccharides from Momordica charantia L. species. Mod. Food Sci. Technol., 30, 102–108 Deng, Y.Y.; Yi, Y.; Zhang, L.F.; Zhang, R.F.; Zhang, Y.;Wei, Z.C. and Zhang, M.W. (2014). Immunomodulatory activity and partial characterization of polysaccharides from Momordica charantia. Molecules, 19, 13432–13447.[CrossRef] [PubMed] Dindo King M. Donayre and Lucille T. Minguez (2014): Sensitivity of Leaf Spot Causing Pathogen of Bitter Gourd (Cercospora citrullina Cooke) to Different Fungicides. Annals of Tropical Research 36[1]: 75-87. Dong, Y.; Xu, B.; Lu, Q. and Zha, Q. (2005). Studies on the Isolation, Purification and Composition of Momordica charantia L. Polysaccharide. Food Sci., 11, 023. Duan, Z.Z.; Zhou, X.L.; Li, Y.H.; Zhang, F.; Li, F.Y.; Su-Hua, Q. (2015). Protection of Momordica charantia polysaccharide against intracerebral hemorrhage-induced
491 brain injury through JNK3 signaling pathway. J. Recept. Signal Transduct., 35, 523–529. [CrossRef] [PubMed] Fan, T.; Hu, J.; Fu, L. and Zhang, L. (2015). Optimization of enzymolysis-ultrasonic assisted extraction of polysaccharides from Momordica charantia L. by response surface methodology. Carbohydr. Polym., 115, 701–706.[CrossRef] [PubMed] Fang, E.F. and Ng, T.B. (2011). Bitter gourd (Momordica charantia) is a cornucopia of health: A review of its credited antidiabetic, anti-HIV, and antitumor properties. Curr. Mol. Med., 11, 417–436. [CrossRef] [PubMed] Fang, E.F.; Zhang, C.Z.Y.; Ng, T.B.; Wong, J.H.; Pan, W.L.; Ye, X.J.; Chan, Y.S. and Fong, W.P. (2012). Momordica charantia lectin, a type II ribosome inactivating protein, exhibits antitumor activity toward human nasopharyngeal carcinoma cells in vitro and in vivo. Cancer Prev. Res., 5, 109–121. [CrossRef] [PubMed Fang, E.F.; Zhang, C.Z.Y.; Wong, J.H.; Shen, J.Y.; Li, C.H. and Ng, T.B. (2012). The MAP30 protein from bitter gourd (Momordica charantia) seeds promotes apoptosis in liver cancer cells in vitro and in vivo. Cancer. Lett., 324, 66–74. [CrossRef] [PubMed] Grover J K et.al.(2004). Pharmocological Action and Potential Uses of Momordica charantia. A Rev. J. Enthopharmocol.93(1):123-132. Horax, R.; Hettiarachchy, N.; Chen, P. (2010). Extraction, quantification, and antioxidant activities of phenolics from pericarp and seeds of bitter melons (Momordica charantia) harvested at three maturity stages (immature, mature, and ripe). J. Agric. Food Chem., 58, 4428–4433. [CrossRef] [PubMed] Horax, R.; Hettiarachchy, N.; Islam, S. (2005). Total Phenolic contents and phenolic acid constituents in 4 varieties of bitter melons (Momordica charantia) and antioxidant activities of their extracts. J. Food Sci., 70. [CrossRef] Hyun Young Kim, So-Youn Mok, Su Hyeong Kwon et.al. (2013). Phytochemical Constituent of Bitter Melon (Momordica charantia). Natural Product Sciences.19(4): 286-289. Jabeen, U. and Khanum, A. (2017). Isolation and characterization of potential food preservative peptide from Momordica charantia L. Arabian J. Chem., 10, S3982– S3989. [CrossRef] K P Sampath Kumar et.al. (2010). Traditional medicinal uses and therapeutic benefits of Momordica charantia Linn. Int. Journal of Pharmaceutical Sciences Review and Research. 4(3): 004. Kenny, O.; Smyth, T.J.; Hewage, C.M.; Brunton, N.P. (2013). Antioxidant properties and quantitative UPLC-MS analysis of phenolic compounds from extracts of fenugreek (Trigonella foenum-graecum) seeds and bitter melon (Momordica charantia) fruit. Food Chem., 141, 4295–4302. [CrossRef] [PubMed] Leung, S.O.; Yeung, H.W. and Leung, K.N. (1987). The immunosuppressive activities of two abortifacient proteins isolated from the seeds of bitter melon (Momordica charantia). Immunopharmarcology, 13, 159–171.[CrossRef] M Ullah et.al.(2011). Nutrient and Phytochemical Analysis of Four Varieties of Bitter
492 Gourd(Momordica charantia) Grown in Chittagong Hill Tracts, Bangladesh. Asain J. Agric. Res. M.M. Lolitkar and M.R. Rajarama Rao (1962). Note on a hypoglyceamic principle isolated from the fruits of Momordica charantia. Journal of University of Bombay: 29: 223-224. Mahatmanto, T. (2015). Review seed biopharmaceutical cyclic peptides: From discovery to applications. Pept. Sci., 104, 804–814. [CrossRef] [PubMed] Meng, Y.; Liu, S.; Li, J.; Meng, Y. and Zhao, X. (2012). Preparation of an antitumor and antivirus agent: Chemical modification of _-MMC and MAP30 from Momordica charantia L. with covalent conjugation of polyethyelene glycol. Int. J. Nanomed., 7, 3133. N M Puspawati (2008). Isolation and identification of Momordicin I fom leaves extract of Momordica charantia L. Jurnal Kimia. Vol. 2(1):52-56. Nadkarni KM (1933). Indian Materia Medica.Vol.1, Popular Prakashan,805-806. Nataraj Rajinimala, Ramalingam Rabindran and Mathan Ramaiah (2009): Management of Bittergourd Yellow Mosaic Virus (BGYMV) by using virus inhibiting chemical, biocontrol agents, antivirual principles (AVP) and insecticide. Archives of Phytopathology and Plant Protection. Vol. 42(8). Peumans, W.J.; Hao, Q. and van Damme, E.J. (2001). Ribosome-inactivating proteins from plants: More than RNA N-glycosidases. FASEB J., 15, 1493–1506. [CrossRef] [PubMed] Pu, Z.; Lu, B.Y.; Liu, W.Y. and Jin, S.W. (1996). Characterization of the enzymatic mechanism of -momorcharin, a novel ribosome-inactivating protein with lower molecular weight of 11,500 purified from the seeds of bitter gourd (Momordica charantia). Biochem. Biophys. Res. Commun., 229, 287–294. [CrossRef] [PubMed] Puri, M.; Kaur, I.; Kanwar, R.K.; Gupta, R.C.; Chauhan, A. and Kanwar, J.R. (2009). Ribosome inactivating proteins (RIPs) from Momordica charantia for anti viral therapy. Curr. Mol. Med., 9, 1080–1094. [CrossRef][PubMed] Raish, M. (2017). Momordica charantia, polysaccharides ameliorate oxidative stress, hyperlipidemia, inflammation, and apoptosis during myocardial infarction by inhibiting the nf-_b signaling pathway. Int. J. Biol. Macromol., 97, 544–551. [CrossRef] [PubMed] Rekha Bhaduari et.al.(2011). Momordica charantia Linn.(karela): Nature’s Silent Healer: Int. Journal of Pharmaceutical Sciences Review And Research:11(1):07. Sabira Begum, Mansour Ahmed, Bina S. Siddiqui, Abdullah Khan et.al.(1997). Triterpenes, a sterol and monocyclic alcohol from Momordica charantia. Phytochemistry. Vol. 44 (7) : 1313-1320. https://en.m.wikipedia.org./wiki/.Momordicilin. Sathish Kumar et.al. (2010). A Medicinal potency of Momordica charantia: Int. Journal of Pharmaceutical Sciences Review and Research; (2): 018 https.// en.m.wikipedia.org›wiki›herb.html Shan, B.; Xie, J.H.; Zhu, J.H. and Peng, Y. (2012). Ethanol modified supercritical carbon
493 dioxide extraction of flavonoids from Momordica charantia L. and its antioxidant activity. Food Bioprod. Process., 90, 579–587. [CrossRef] Sonal Desai, Pratima Tatke (2015). Charantin : an important lead compound from Momordica charantia for the treatment of diabetes. Journal of Pharmocognosy and Phytochemistry: 36(6):361—362. Tan, S.P.; Stathopoulos, C.; Parks, S.; Roach, P. (2014) An optimised aqueous extract of phenolic compounds from bitter melon with high antioxidant capacity. Antioxidants, 3, 814–829. [CrossRef] [PubMed] Vijay Kumar, Anurag Kerketta, Anjani Sahu, Amrotin Teta and CP Khare (2018): Management of prevalent diseases of bitter gourd (Momordica charantia L.). Journal of Pharmacognosy and Phytochemistry. SPI: 26-35. Vincken, J.P.; Heng, L.; de Groot, A.; Gruppen, H. (2007) Saponins, classification and occurrence in the plant kingdom. Phytochemistry, 68, 275–297. [CrossRef] [PubMed] Wang, H. and Ng, T.B. (1998). Ribosome inactivating protein and lectin from bitter melon (Momordica charantia) seeds: Sequence comparison with related proteins. Biochem. Biophys. Res. Commun., 253, 143–146. [CrossRef][PubMed] Xu, B.; Dong, Y. (2005) Determination on total saponins of Momordica charantia L. by spectrophotometry. Food Sci., 10, 165–169. Xu, X.; Shan, B.; Liao, C.H.; Xie, J.H.; Wen, P.W.; Shi, J.Y. (2015). Anti-diabetic properties of Momordica charantia L. polysaccharide in alloxan-induced diabetic mice. Int. J. Biol. Macromol., 81, 538–543. [CrossRef] (PubMed) Yuan, X.; Gu, X. and Tang, J. (2008). Purification and characterisation of a hypoglycemic peptide from Momordica charantia L. Var. abbreviata Ser. Food Chem., 111, 415– 420. [CrossRef] [PubMed] Zhang, B.; Xie, C.; Wei, Y.; Li, J. and Yang, X. (2015). Purification and characterisation of an antifungal protein, M Cha-Pr, from the intercellular fluid of bitter gourd (Momordica charantia) leaves. Protein Expr. Purif., 107, 43–49. [CrossRef] [PubMed] Zhang, F.; Lin, L., and Xie, J.A (2016). Mini-review of chemical and biological properties of polysaccharides from Momordica charantia. Int. J. Biol. Macromol., 92, 246– 253. [CrossRef] [PubMed] Zhang, L.J.; Huang, H.T.; Liaw, C.C.; Huang, S.Y.; Lin, Z.H.; Kuo, Y.H. (2016) Cucurbitane-type triterpenes and glycoside from the rattan of wild Momordica charantia and their anti-inflammatory and cytotoxic activities. Planta Medica, 81, S1–S381. [CrossRef] Zhang, L.J.; Liaw, C.C.; Hsiao, P.C.; Huang, H.C.; Lin, M.J.; Lin, Z.H.; Kuo, Y.H. (2014) Cucurbitane-type glycosides from the fruits of Momordica charantia and their hypoglycaemic and cytotoxic activities. J. Funct. Foods, 6, 564–574. [CrossRef] Zhang, P.P.; Liu, J.F.;Wang, C.L.; Ye, Y.T. and Xie, J.H. (2008). Study on the antimicrobial activities of the extracts from Momordica charantia L. Nat. Prod. Res., 20, 721– 724. [CrossRef]
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32 BIOTECHNOLOGICAL APPLICATIONS OF DETECTION, ESTIMATION, ELECROPHORESIS, ISOZYME ANALYSIS, MOLECULAR CHARACTERIZATION, TRANSMISSION, ELISA AND BUFFERS FOR PLANT DISEASE MANAGEMENT Dilip Kumar Verma1, H. K. Chourasia2 and Somya Verma3 1
Principal Scientist (Genetics & Plant Breeding), IARI Regional Station, Indore – 452001, For correspondence. E-mail:
[email protected] 2
P G Deptt of Biotechnology, TM Bhagalpur University, Bhagalpur – 812007 E-mail:
[email protected]
3
Research Scholar (Pharmacy), Ganpat University, Mehsana, Gujarat E-mail:
[email protected]
The Biotechnological Applications for effective plant disease management requires practical application of Detection, Estimation, Elecrophoresis, Isozyme Analysis, Molecular Character ization, Transmission, DAC-ELISA and use of Buffers. Therefore, an attempt has been made to describe the methodologies of above techniques. The Biotechnological Applications for effective plant disease management requires practical application of Detection, Estimation, Elecrophoresis, Isozyme Analysis, Molecular Character ization, Transmission, DAC-ELISA and use of Buffers. Therefore, an attempt has
496 been made to describe the methodologies of above techniques as reassessment made by Verma et al (2018) for the dynamic confluence of plant systematics and biotechnology. The techniques are1. Detection methods of seed-borne fungi, 2. Detection methods of seed-borne bacteria, 3. Estimation of protein by Lowry’s method, 4. Electrophoresis of Proteins on SDS-PAGE, 5. Isozyme analysis: Detection of Esterase and Peroxidase on Native-PAGE, 6. Isolation and purification of plant and fungal DNA, 7. Molecular characterization of fungal isolates using Random Amplification of Polymorphic DNA (RAPD) Marker- PCR, 8. Transmission of plant virus through whitefly vector, 9. Direct Antigen Coating ELISA (DAC- ELISA), 10. BUFFERS preparation. Jauhar (2006) had also stressed that modern biotechnology should be considered as supplement to plant breeding. 1. Detection Methods of Seed-borne Fungi We have hardly found any field crop free from seed borne diseases. Seed health is of prime importance. Monitoring of seed health in respect of seed borne pathogen is desirable for healthy crop, better yield, quarantine purpose etc. Methods for detection of seed borne fungi described by Bhale et al (2001) are as under: Inspection of dry seed 1. Pour the sample on the purity analysis board on the top left surface. 2. With the help of a spike, separate the pure seeds into the container placed on right side below the board. 3. In other two small containers, separate the other crop seed and inert matter. 4. The inert matter may consists of soil, sand, stones, various types of plant debris, sclerotia, smut balls, seed galls, bunt balls of fungi. 5. The physical abnormalities may appear on seed and include shrivelling of the seed, reduction or increased seed size, discolouration or spots on the seed coat. 6. Abnormal seeds/inert matter may be tested with aid light under stereoscopic microscope. 7. Seeds showing abnormalities and inert matter can further be tested by blotter or agar plate method. 8. Make report on all the three components (pure seed, inert matter and other crop seed) by weight.
497 Standard blotter method Procedure: 1. Keep the cooled pre-sterilized glass petri plates on the clean surface of the working table in required quantity (sterilization of glassware is done in an electric oven for 2 hrs at 180°C). 2. Keep the filter papers near the petri plates, count and make the sets of 3 filter papers for one plate. 3. Disinfect the forceps tips (keeping it over the flame for few seconds, cool it). 4. With the help of forceps, dip one set of filter paper in a glass tray containing distilled water after complete soaking just; lift the set in air over the tray, allowing the extra water to run-off. 5. Place the moist filter paper into lower half, holding the paper with the help of forceps in right hand. Set the papers, turning the plate clockwise. 6. Prepare the plates in the same way. Wipe off and dry the working table. 7. Place the seeds on a plain paper sheet (Number of seeds to be placed in one plate depends on the size of the seed. In a plate 5, 10, 25 or 50 seeds can be placed). Write the accession number and date of examination of the seed sample. 8. Count and make small groups of 10 or 25, seeds for 21 plate. Do not touch the seeds. Use spikes. 9. Arrange the counted seeds on moist blotter (lined in plates) using forceps at equidistance from each other. Close the lid (For plating the 25 seeds, keep one seed in the center, 8 in middle and 16 in outer ring. Whereas for 10 seeds, one is plated in the center, and 9 in the outer ring. 10. Collect the plates in the plastic trays without disturbing the seeds. 11. Incubate at 20-25°c for 7 days in alternate cycles of 12 hr. dark and 12 hr. light (The common source of light used is near ultra violet (NUV)supplied by black light tubes or day light provided by cool, white fluorescent tubes. In either case light is provided by two tubes hanging horizontally, 20 cm apart. Distance between tubes and plates should be 40 cm. Proper care for protection from
498 NUV light must be taken by wearing eye glasses and hand gloves). 12. After seven days of incubation, seeds are examined one by one under stereoscopic binocular microscope (associated mycoflora are identified based on habitat characters. These are also confirmed by examining slides under compound microscope). 13. Count the number of fungi on seeds and enter the observations in data sheer. Also, make comments on symptoms on seed and seedlings. 2,4-D Blotter dip method Blotters (filter papers) are moistened with 0.1-0.2 % solution of sodium salt of 2, 4-dichlorophenoxy acetic acid instead of plain distilled water. Rest of the method is same. Advantage The sodium salt of 2, 4- D retards the seed germination and seedling growth. Hence the seeds are not displaced, remain undisturbed where they were plated. Examination is made easy. Deep freezing blotter method 1. Seeds are plated as in blotter method. 2. Incubate the plates initially for 24 hr under controlled conditions in the growth chamber. 3. Plates are transferred to deep-freezer (-20°C) under complete darkness for 24 hr. 4. Plates are retransferred to growth chamber for remaining 5 days. Standard agar-plate method Procedure 1. Prepare the potato dextrose agar medium (peeled potato slices 200 g boiled in 700 m1 water, agar agar powder 20 g dissolved in 300 m1 with 20g dextrose. Final mixing and making up volume to 1000 ml) 2. Sterilize the media in autoclave (121.6°C for 20 min at 15lb. pressure) and after semi-cooling, pour the melted medium in presterilized petriplates (approximately 17-20m1 per 90 mm petriplate) under aseptic conditions of laminar flow.
499 3. After solidification of medium, invert the plate for 12 hr. 4. Reject the contaminated plates (contamination refers to the development of bacterial, fungal, actinomycete or mix colonies on the medium. This also indicates the improper sterilization of glassware or media or preparation faults). 5. Place 10 surface sterilized seeds (treated with NaOCl) on the media under aseptic conditions. One in center and 9 in outer ring. 6. Incubate the plates containing seeds in the growth chamber for 7 days. 7. Examine the seeds for the developing associated mycoflora by naked eye based on colony colour, and stereoscopic binocular on the basis of habitat characters. Modifications Peptone- PCNB method The basic medium (FDA) can be replaced by Malt-agar or Peptoneagar. Medium is supplemented by antibiotics (e.g. chloro-tetracycline, streptomycin sulphate) and/or fungicide (pCNB-penta-cWoro-nitrobenzene) in different concentrations. Rolled paper towel method 1. Take two sheets of standard germination testing paper (paper towel); enter the number, date and crop on the other side with waterproof ink. 2. Mist first sheet with sterile-distilled water and stretch over clean surface of working table. 3. Arrange 50 seeds in 5 rows of 10 seeds at equidistance, as in germination test on one sheet. Total 400 seeds are placed. 4. Cover the seeds by second pre-soaked sheet carefully without disturbing the already arranged seeds. 5. Roll and tie the sheets with rubber band at both the ends. 6. To avoid water losses, use butter-wax coated paper for rapping the sheets at one side. 7. Place the paper towels, containing seeds in gem1inator with slightly tilting the bunch of towel.
500 8. Incubate in dark. 9. Observe the towels after 7-14 days by opening and removing the cover sheet. 10. Examine the seeds by naked eye and stereo binocular for the presence of mycoflora and seed germination. Seed washing test 1. Take one gram of seed from a working sample in a clean and small conical flask. 2. Add 10 ml of water and a drop of wetting agent (e.g. Tween 20). 3. Make 10 replications. 4. Shake the flasks for 10 minutes with care. 5. Transfer the water in tubes and centrifuge for 10 minutes at 23002500 rpm. 6. Decant the supernatant liquid leaving the sediment at the bottom of the tube. 7. Suspend the sediment in 2 ml of distilled water. 8. Examine the water drops under compound microscope (200 x) for the presence of oospores. The oospores are yellow-brown, spherical, 3 layered thick walled spores. Test tube water agar-seedling symptom test 1. Take clean, rimless glass test tubes of 16-mm diameter. 2. Dissolve 10g agar-agar powder in 1000ml distilled water and autoclave at 15lb, for 15min. 3. Transfer 10ml of water agar into each tube under aseptic condition. 4. After solidification of agar place one seed in each tube. Cover the mouth of tube with a piece of aluminium foil. 5. The tubes are placed vertically in a tray and incubated in growth chamber as in standard blotter method. 6. As the seedling reach the cover, the foil is removed. 7. Symptoms caused by the associated mycoflora on the seed and developing seedling are observed after 14th day.
501 Sodium-hydroxide seed soak method 1. Dissolve 2 g NaOH in 1000 ml (for preparing 0.2% solution). 2. Working seed sample of 4000 seed with 2-replication of 2000 seed each for foundation seed and 800 seeds with 2 replications of 400 each seed for certified wheat seed is prepared. 3. Seeds are soaked in a small conical flask or beaker of 250 ml capacity for 24 hr at 25°C. 4. Solution is decanted after 24 hr and seeds are washed in tap water. 5. Seed are placed on blotter to soak extra water. 6. Spread the soaked seeds over white background and examine by naked eyes. 7. Numbers of such seeds are counted as infected seeds and reported in percentage. 8. Black seeds can be observed under compound microscope for the presence of teliospores. Embryo count method Procedure 1. Soak 100 g (2200-2800 seeds) wheat seeds in 1000 ml of 5 per cent solution 2. of sodium hydroxide (NaOH) and 0.005 per cent trypan blue in a 2000 ml 3. Capacity beaker for 18-22 hr at 22-24°C. 4. Pass the soaked seeds through a sieve set of 10 and 30 mesh and wash the seeds thoroughly with running tap water to separate the embryos. 5. Agitate the soaked seed material to facilitate the separation of embryos. 6. Collect the embryos over 30 mesh size. 7. Wash the separated embryos into a tea strainer and dehydrate them with 8. Methyl spirit for 2-5 minutes.
502 9. Pass the dehydrated embryos in solution of lactic acid + glycerol + water(1:2:1) in small beaker. 10. Take a glass funnel; connect the stem of the funnel with rubber tubing 11. Provided with a stopper. 12. Pass the mixture of lactic acid + glycerol + water through the funnel. The 13. Embryo float at the top of the funnel and chaff sinks. 14. The chaff can be run-off through a tea strainer and collect the solution in a beaker) which can be reused for extracting the embryos for additional 3 samples. 15. Repeat the process 45 times until embryos are separated from chaff. 16. Transfer the separated embryos in 60 ml solution of lactic acid + glycerol (1: 2). 17. Heat the solution with embryos for 2 minutes until boiling. 18. Allow the solution to cool for 30 minutes. 19. Arrange the embryos in petri plates in ring of individual embryo and pour 20. Solution of lactic acid and glycerol to facilitate examination. Evaluate the test by examining under 12-to 15-x magnification under stereoscopic binocular microscope. Infected embryo exhibit blue stained mycelium of loose smut pathogen in scutellum, plumule or whole embryo. 2. Detection Methods of Seed-borne Bacteria Seed Health Testing of Bacteria The bacterium can be detected in the laboratory from seeds by the rolled towel method (seedling symptom test) developed by Singh and Rao (1977) or by plating seed extracts from crushed seeds on specific agar medium. Roll towel method Procedure (for detection of leaf blight pathogen of rice Xanthomonasoryzae pv. oryzae)
503 1. Draw your hundred seeds from the sample make eight replication of 50 seeds each. Plate seeds of each replicate equally spaced between two paper towels 45 X 28 cm size (ordinary blotting paper) which have been previously soaked in tap water. 2. Roll the towels close the ends with rubber bands and place them in polythene bags and finally incubate them in up right position at 30 ± 2ºC. 3. Remove the bands after 9 days, unroll the towels and examine the seedlings carefully. Seedlings having water soaked areas, brown discoloured coleoptile or yellow discoloration in leaves are suspended to be infected with BLB organism. 4. To confirm that the observed symptoms are caused by the BLB pathogen cut small section of the affected tissue on a glass slide and mount in drop of water. The drops must be covered by cover slips and they cut edge of the leaves should be examined for bacterial ooze under compound microscope. Only if bacterial ooze is observed, proceed with the isolation and testing for pathogenecity. 5. Pathogenicity test: take out all seedlings showing infection and cut them into small pieces (except roots) in a beaker having enough sterile water to immerse them. Allow 15 min. for bacteria to ooze out of the tissue. Use this bacterial inoculum suspension to inoculate 30-40 leaves of 6 weeks –old rice plants of a ºC susceptible variety , such as Taichung native -1 (TN1). This can be done by dipping a pair of scissors in the bacterial suspension and then cutting leaf tips (3/4th of an inch below the tip). 6. Cover the inoculated plants for 24 hour with a polythene bag and incubate at 30ºC with a 12 h light cycle. 7. Check plants within 48-72 hour for water soaked area in the inoculated leaves, usually beginning from the inoculated ends as water soaked stripes. Lesions enlarge and may turn yellow within a few days. Milky opaque drops of bacterial exudates may be observed on the stripes. Observed the plants up to 14 days and the stripes should proceed downwards along the lamina if it is caused by the Xanthomonas oryzae pv. oryzae.
504 Gram staining to test Gram-positive or Gram-negative bacteria Principal The morphology of bacteria can be observed better under the light microscope after staining. The cell wall of Gram-negative bacteria is thinner than that of Gram-positive bacteria and contains a higher percentage of lipid content. During the staining of Gram-negative bacteria, the alcohol treatment extracts the lipid. This results in increased porosity or permeability of the cell. The crystal violet-iodine (CV-I) complex, thus can be extracted and the Gram-negative bacteria is decolourized. The cells subsequently take up the colour of the counter stain. The Gram-positive bacteria will retain the crystal violet and appear deep violet in colour and the Gram-negative bacteria lose the crystal violet on decolurization and are counter stained by the safranin appear red in colour. Smear Preparation: The success in bacterial staining depends on the preparation of suitable smear of the organisms. A properly prepared bacterial smear withstands one or more washings during staining without loss of organisms. The bacteriological smear differs according to the source of the organisms. If the organism are froma liquid medium such as broths, milk, saliva, etc., place one or two lopful of the liquid medium directly on the slide. If the culture is from solid media such as nutrient agar, blood agar or from any solid part, place one or two loopful of water on the slide. Using a straight inoculating wire, disperse the organism in the water and allow to spread on the slide to occupy about 1 or 2cm square and than dry by normal evaporation of the water but do not apply heat. Heat Fixation: Heat fixation is a prerequisite for staining. Pass the slide over a burner one to two times so as to coagulate bacterial proteins. Care must be taken to avoid overheating.
505
Fig. 1 The Gram-strain procedure
Procedure: 1. Flood the slide with crystal violet staining reagent for 1 min. 2. Wash the smear by direct stream of tap water for 2 seconds. 3. Flood the slide with iodine mordant for 1 min. 4. Wash the slide by indirect stream of tap water for 2 seconds. 5. Blot the smear dry with absorbent paper. 6. Immerse the smear in 95 % ethanol for 30 seconds. 7. Blot the smear dry with absorbent paper. 8. Immerse the smear for 2 minutes with counter stain. 9. Wash the smear in a gentle and indirect stream of water until no colour appears in the wash water. 10. Blot the smear dry with absorbent paper. 11. Examine under the microscope.
506 Decolourization with ethanol removes crystal violet from Gramnegative cells and not from gram-positive cells. The Gram-negative cell turns pink to red with safranin, the counter stain. 3. Estimation of protein by Lowry’s method Protein can be estimated by different methods as described by Lowry and also by estimating the total nitrogen content. No method is 100 % sensitive. Hydrolysing the protein and estimating the amino acids alone will give the exact quantification. The method developed by Lowry et al. is sensitive enough to give a moderately constant value and hence largely followed. Protein content of enzyme extracts is usually determined by this method. Principle: The blue color developed by the reduction of the phosphomolybdicphospotungstic components in Folin-Ciocalteau reagent by the amino acids tyrosine and tryptophan present in the protein plus the colour developed by the biuret reaction of the protein with the alkaline cupric tartarate are measured in the Lowry’s methods Materials: Reagent A: 2% Sodium Carbonate (Na2.Co3) in 0.1 N NaOH Reagent B: 0.5% Copper Sulphate (CuSO4.5H20) in 1% potassium sodium tartarate Reagent C: Alkaline Copper Solution: Mix 50 ml of A and 1 ml of B prior to use. Reagent D: Folin-Ciocalteau reagent: (1N) Protein standard stock: Weigh 50mg of bovine serum albumin (fraction V) and dissolve in distilled water and make upto 50 ml. Protein standard working: Dilute 10 ml of stock solution to 50 ml with DW. (200 µg/ml) Protocol: Extraction of protein from sample: Weigh 200-300 mg of tissue and grind with 2ml of phosphate buffer in mortar and pestle
507 Estimation of protein: 1. Pipette out 0.2, 0.4, 0.6, 0.8, 1.0 ml of the working standard into a series of test tubes. 2. Pipette out 0.1 and 0.2 ml of the sample extract in two other test tubes. 3. Make up the volume to 1 ml in all test tubes. A tube with 1ml water serves as a blank. 4. Add 5ml of reagent C to each tube including the blank. Mix well and allow to stand for 10 min. 5. Then add 0.5 ml of reagent D, mix well and incubate at RT in the dark for 30 min. Blue color is developed. 6. Take reading at 660nm 7. Draw a standard graph and calculate the amount of protein in the sample. 4. Electrophoresis of Proteins on SDS-PAGE Most biological polymers at electrically charged and hence show differential mobility in an electric field. The transport of particles through a solvent by an electric field is called “Electrophoresis”. A useful way to characterize macromolecules is by their rate of movement in the electric field. This property can be used to determine protein molecular weights, to distinguish molecules by virtue of their net charge or their shape and to separate different molecular species quantitatively. Basically when a particle with charge “q” suspended in an insulating medium, is in an electric field “E”, the particle will move at a constant velocity ‘v’, determined by the balance between the electrical force ‘Eq’ and the viscous drag fv, in which f is the friction coefficient. i.e. Eq = fv ——— velocity V = Eq/f Polyacrylamide gels Electrophoresis in acrylamide gels is frequently referred to as PAGE (Poly Acrylamide Gel Electrophoresis). Cross-linked polyacrylamide gels are formed from the polymerization of acrylamide monomers in the presence of small amounts of N’N’, methylenebisacrylamide (also referred to as “Bis”). Bisacrylamide is made up of two acrylamide molecules linked by a methylene group, which is used as a cross linking agent. Acrylamide monomers are polymerized in a head to tail fashion into long chains and
508 occasionally a bis-acrylamide molecule is introduced into the growing chair. Thus forming a second site for chaixn extension. In this way across linked matrix of fairly well defined structure is formed. CH2 = CHCONH2
CH2(NHCOHC = CH2)2
(Acrylamide)
(Bis-Acrylamide)
Free radicals - CH2 – CH – CH2 – CH – CH2 – CH |
|
|
CO
CO
CO
|
|
|
NH
NH2
n
NH
|
|
CH2
CH2
|
|
NH
NH2
NH
|
|
|
CO
CO
CO
|
|
|
- CH2 - CH – CH2 –
CH - CH2 - CH -
Polyacrylamide Gel Matrix The polymerization of acrylamide is an example of free-radical catalysis, and is initiated by the addition of ammonium per sulphate and the base N,N,N,N-tetra- methylenediamine (TEMED). TEMED initiates the decomposition of the persulphate ion to give a free radical. S2O82- + e- SO42- + SO4If the free radical is represented as R and an acrylamide monomer as M, then the polymerization reaction can be represented as R* + M
RM*
RMM*
M
M
RMM* + M
RMMM*
509 Thus long chains of acrylamide molecules are built up being cross linked by the inclusion of a bis-acrylamide molecule into the growing chain. Oxygen removes free-radicals from the system and therefore all gel preparations should normally be degassed prior to polymerization. Separating and Stacking Gels Normally the polyacrylamide gel Electrophoresis consists of two different types of gels namely Separating Gel and Stacking Gel. The separating gel has a pH of 8.8 units and makes up to 85% of the gel body. This has lower pore size than the stacking gel and is mainly responsible for the separation of different proteins in a given sample, depending on their differential mobility. The concentration of the separating gel usually varies from 7.5% to 15% and is mainly governed by the size of protein(s) being studied. The stacking gel has a pH of 6.8 units and is poured over the separating gel after the polymerization of separating gel. As the name indicates “Stacking Gel” is basically used to stack the proteins into a narrow band before they enter into the separating gel. This is achieved by utilizing the differences in the ionic strength and the pH between the electrophoresis buffer and the stacking gel. SDS-Polyacrylamide Gel Electrophoresis Acrylamide gel electrophoresis is the method of choice for fractionating and characterizing mixtures of proteins. Electrophoretic procedures are rapid and small amounts of protein can be conveniently detected in gels by staining or autoradiography. Electrophoretic migration is inversely proportional to the molecular weight of a protein, if the protein is dissociated and denatured with sodium doecylsulfate (SDS) and a reducing agent, before electrophoresis. SDS is a negatively charged detergent that binds hydrophobically to polypeptide chains. With the addition of reducing agents (viz. 2-merecaptoethanol) and heat all intra and inter chain disulfide bonds break, leaving the denatured protein fully reduced and separated into individual polypeptides. Most proteins bind SDS in a constant ratio i.e. 1.4 gm of SDS gm of protein. In doing so they acquire a fixed charge to mass ratio. Proteins under such conditions move separate only according to their sizes. The distance of electrophoretic migration relative to the buffer front is then inversely proportional to the lost of the molecular weights. The acrylamide polymeralse acts as a kind of molecular sieve through which each molecule must pass. The smaller the molecule (the) more easily it can find its way through the tortuous passage of the gel.
510 Hence, the mobility increases with decreasing molecular weight. Therefore, it a protein of unknown molecular weight is electrophoresed with two or more proteins of known molecular weights, then the Unknown can be calculated to an accuracy of between 5% and 10%. Increasing the number of standards closely related to the unknown sample can increase the accuracy. Requirements Apparatus: Electrophoretic apparatus, Comb(s), Spacer (x2), Glass plates (x2), Plastic trays (x4), Conical flasks, Measuring cylinder(s) Reagents: Acrylamide, N,N-methylenebisacrylamide, SDS, Tris buffer (salt), Glycerol, Ammonium per sulphate, TEMED, Ethanol, Glycine, 2Mercaptoethanol, Bromophenol blue dye, Glacial Acetic Acid, Methanol, Coomassie Brilliant Blue (R250), Sodium thio-sulphate, Silver nitrate, Sodium carbonate Preparation of Reagents 1. 30% Acrylamide-bisacrylamide stock Acrylamide 30% (w/v) Bis-acrylamide 0.8% (w/v) 2. (SDS) Sodium dodeocyl sulfate SDS – 10% (w/v) 3. Stacking gel buffer Tris-HCl buffer 0.5 M (pH 6.8) 4. Separating Gel buffer Tris-HCl buffer 1.5 M (pH 8.8) 5. Electrophoresis buffer (For SDS-PAGE) Tris base
25 mM
Glycine
250 mM
SDS
0.1% (w/v)
6. Sample/Loading buffer (For SDS-PAGE) Tris HCl
50 mM (pH 6.8)
SDS
2%
2-Mercaptoethanol
5%
511 Bromophenol Blue
0.1%
Glycerol
10%
7. Comassie staining solution: I a)
b)
CBB (R-250)
0.1%
Methanol
40%
Glacial Acetic acid
8%
Comassie staining solution: II TCA : 6% (w/v)Methanol : 18% GAA : 6%
CBB (R-250) : 0.0250%
(Note : Use either solution I or II for staining) 8. A) Commassie Destaining solution : I Methanol
40%
Glacial acetic acid
8%
B) Commassie Destaining solution : II Glacial acetic acid
5%
Methanol
28%
C) Comassie destining solution : III NaCl
3%
(Use either of the three solution for destining) Table 1: Comparison of separating Gel (12%) for SDS and Native PAGE Component
Volume for SDS-PAGE
Volume required for native PAGE
H2 O
6.6 ml
6.6 ml
Acrylamide stock (30%)
8.0 ml
8.0 ml
Separating buffer
5.0 ml
5.0 ml
SDS (10%)
0.2 ml
-
Glycerol (50%)
-
0.2 ml
(10%) ammonium per sulphate
0.2 ml
0.2 ml
TEMED
0.008 ml
0.008 ml
Final volume
20 ml
20 ml
512 Table 2: Comparison of stacking Gel (5%) for SDS and Native PAGE Component
Volume for SDS-PAGE
Volume required for native PAGE
H2 O
5.5 ml
5.5 ml
Acrylamide stock (30%)
1.3 ml
1.3 ml
Stacking buffer
1.0 ml
1.0 ml
SDS (10%)
0.08 ml
-
Glycerol (50%)
-
0.08 ml
Ammonium per sulphate
0.08 ml
0.08 ml
TEMED
0.008 ml
0.008 ml
Final volume
8 ml
8 ml
Procedure: 1. Carefully clean the glass plates with warm soapy water. Rinse them thoroughly with distilled water and then wipe them dry & clean with ethanol. 2. Assemble the Gel apparatus as per the instructions of the guide. 3. Prepare a 12% separating gel (According to the table given in tables) and pour the separating gel carefully between the glass plates. Overlay it with ethanol and allow it to polymerize for 40 min. after polymerization. 4. Remove the overlay solution and wash the gel three times with distilled water. Prepare a 5% stacking gel (According to the table given in Appendix III) and pour it over the separating gel. Insert the comb and let the gel polymerize for 30 min. 5. Assemble the gel chamber and fill the buffer reservoir with 1x Tris glycine electrophoresis buffer. Remove the comb & clean the wells with electrophoresis buffer. 6. Boil the probe in a sample buffer for five minutes. 7. Load the protein samples and standard probes and run the gel at 15 V/cm, till the dye front reaches 0.5 cm from the lower edge of the gel. Isolation of protein 1. Crush 200 mg of tissue in mortar and pestle with liquid nitrogen
513 2. Add fine tissue powder in centrifuge tube containing 2 ml of extraction buffer (0.05M Triss-Cl, pH–7.2, 5% Glycerol, 0.5% SDS, 0.1% â-Mercaptoethanol) or (1.5 ml of 0.05M Triss-Cl, pH– 7.2, 400 ìl of 2% SDS, 100 ìl of 5% â-Mercaptoethanol) 3. Centrifuge at 10,000 rpm for 15 min. At 4oC 4. Collect supernatant and boiled it at 100oC for 2-4 min. 5. Store sample at 4oC Detection and Staining Procedures for Proteins on Gels Coomassie Brilliant blue-R-250 Staining: After the completion of electrophoresis, the most commonly used stain for detecting the proteins on gel is the dye “Coomassie Brilliant BlueR-250 (CBB-250)”. Staining is usually carried out using a 0.1% (w/v) solution of CBB-250 in methanol, water and glacial acetic acid. This mixture also helps to precipitate and fix the proteins in the gels, which prevents the protein from being washed out while it is being stained. Staining is usually accomplished in 30 min to 120 min time. (Depending on the conc. of the staining soln. and the number of times the staining soln. has been used previously). The gel is usually left in the destaining soln. (methanol, water and glacial acetic acid soln.) overnight with gentle agitation, for proper destaining. The CBB-R-250 stain is fairly sensitive and can detect upto 100 ng (0.1 µg) of protein in 3 litre acrylamide gel. Procedure 1. Take out the gel and immerse in commassie staining soln. for one hour with gentle shaking. 2. Pour off the staining soln. and wash the gel gently in destining soln. Until the background of the gel becomes clear. Silver staining: Although the CBB staining is fairly sensitive but many times we require a higher sensitivity of detection of protein bands because of the low amounts of proteins loaded on the gel. In such cases we use silver staining protocol, wherein free silver ions (Ag2) are reduced to metallic silver on the surface of the protein molecules. The metallic silver is deposited to give a blackish brown band.
514 Silver staining can be used immediately after the completion of electrophoresis or alternatively after staining and complete destaining with CBB-R-250. With the second approach, the major bands on the gel can be visualized with CBB and then the minor bands can be resolved/visualized using silver staining. The silver staining method is approximately 100x times more sensitive than the coomassie staining method and if used/developed properly, can detect even 1ng of protein bands. Procedure: 1. Rinse the gel in (40% methanol + 10% glacial acetic acid) soln. 2. Transfer the gel in 50% ethanol and shake gently for 10 min. 3. Transfer the gel in fresh 50% Ethanol and shake for 20 min. 4. Rinse the gel thoroughly with double distilled deionized water. 5. Soak the gel in freshly prepared 0.02%. Sodium thio sulfate soln. for one min. 6. Wash the gel with double distilled deionized water for 1 min. 7. Soak the gel in 0.2%. Silver nitrate soln. for 20 min with gentle shaking. 8. Transfer and develop the gel in (6% sodium carbonate + 100 µl HCHO) soln. 9. Develop the gel till all the bands are visible and transfer the gel to distilled water as soon as the background of the gel starts turning brown. 10. Store the gel in 50% methanol. Precautions: 1. All chemicals and distilled water should of high quality. 2. Acrylamide as a monomer is highly neurotic, handle with extreme care. 3. Use the gel immediately following polymerization. The separation gel after setting can be stored overnight with four fold diluted separation gel buffer poured in the overlying space. However gels should not be stored after stacking gel has been poured due to the pH difference between both the buffers.
515 4. Degassing of the gel mix should be adequate for easy polymerization. 5. The water layered over the separation gel should be completely removed for quick polymerization of the stacking gel. 6. In 10% polyacrylamide gels, the polypeptides of lower molecular weight i.e. lesser than 10kd will diffuse on migration. For fine resolution use gels of higher concentrations. 7. Sensitivity of coomassie blue stains in visualizing protein band is of 0.1 µg approximately. For lesser concentration use silver staining. 5. Isozyme Analysis: Detection of Esterase and Peroxidase on NativePAGE The method of SDS-PAGE is of little use if our aim is to detect a particular protein (often an enzyme) on the basis of its biological activity, because the protein/ enzyme gets denatured by the SDS-PAGE protocol. In such cases it is essential to run the gels under non-denaturing conditions. In native or non-denaturing gels, polyacrylamide is again used as the matrix but the SDS-detergent is absent and the proteins are not denatured prior to loading. As all the proteins in the sample being analyzed carry their native charge at the pH of the separating gel (pH 8.8), proteins separate according to their differential electrophoretic mobilities and the sieving effects of the gel. It is quite difficult to predict the behaviour of a given protein in a buffer gel, because of the range of different charges and sizes of protein in a given protein mixture. The enzyme of interest can be identified by incubating the gel in an appropriate substrate solution, so that a coloured product is formed at the site of the enzyme. Often duplicate samples are run on a single gel, wherein one is used for the enzymatic activity while the other is used for staining purposes. Principle Gel of Acrylamide-bis Acrylamide is polymerized between two glass plates in a slab form. An electrophoretic potential is applied which causes any charged macro-molecule to migrate through the gel, with an electrophoretic mobility based on Electro-motive strength of the charge and the molecular size. At pH 8.3 (pH of the buffer), all proteins have net negative charge and move towards the anode end. Requirements: Vertical Electrophoretic Apparatus: (Regular model), Tris-Bis-
516 acrylamide, Acrylamide, Glycerol, TEMED, Ammonium per sulphate, Bromophenol blue Preparation of Reagents 1.
Solution A
Distilled water
2.
Solution B
1M Tris (pH 8.8) Tris
36.6 g
1M HCl
4.8 ml
TEMED
0.025 ml
Water
95 ml
Stored at 4°C 3.
Solution C
Acry-bis solution Acrylamide
30 g
Bisacrylamide 0.8 g Water
100 ml
4.
Solution D
50% of v/v glycerol
5.
Solution E
TEMED 20 µl
6.
Solution F
APS 0.25% w/v (Ammonium per sulfate)
7.
Electrode buffer
Tris-Glycine
(pH) 8.3
Tris
6g
Glycine28.8 g Make upto 1000 ml By water Sample preparation: a. Extract protein sample (25 µl) containing 25 µg protein. b. Extraction Buffer (25 µl) containing 1 µl of bromophenol blue dye (0.5%) and 15% glyerol. All reagents are kept in fridge + taken out only ½ an hour before use.
517 Preparation of 7% resolving gel Solution A
3.087 ml
Solution B
11.440 ml
Solution C
7.082 ml
Solution D
2.3606 ml
Solution E
0.036 ml
Solution F
5.992 ml
Total vol. = 30 ml (7%) Preparation of stacking gel Solution A
2.58 ml
Solution B
5.72 ml
Solution C
2.50 ml
Solution D
1.80 ml
Solution E
0.02 ml
Solution F
3.00 ml
The mixture is gently shaken and poured in between the glass plates. The gel took 1 h to polymerize. Procedure: 1. Wash the plates thoroughly with detergent solution. Rinsed with distilled water and then wipe with methanol and filter papers. 2. Fix the dried plate using 0.5 mm spacers with the help of Silicon grease and with clamps. 3. Fill the distilled water between the plates and left for 10 min for checking leakage. 4. Turn out the water by overturning the plates. 5. Prepare 30 ml of 7% acrylamide gel and pour the gel between two plates very carefully to avoid air bubbles. 6. Allow to stand for 30 min. 7. Prepare the 5% stacking gel (10 ml) and pour the gel above
518 separating gel. 8. Insert plastic comb of 12 rectangular teeth in the U shape space in one of the plates, to form wells for the sample application at the top of the gel. 9. Allow to stand for 15 min. remove the bubbles found in the gel by tilting the plates or with the help of a curve syringe. 10. Remove the plastic comb. 11. Keep the plate between two reservoirs (upper cathodal site, lower anodal side) filled with electrode buffer. 12. Load the sample with the help of microsyringe. 13. Apply the voltage 70 V for gel. 14. Remove the plate after dye front had reached to the bottom. 15. Remove the gel and stain. 16. Destain the gel. Isozyme analysis: The isozyme analysis is performed using polyacrylamide gel electrophoresis a described earlier. The gels were recovered and stained for isozymes namely peroxidase and esterase. The composition of gels in terms of Acrybis was slightly varied. Visualization of peroxidase: Requirements: The staining solution consists of the following reagents, prepared fresh each time. 1. 100 ml of 0.01 M Phosphate buffer, pH-6.0 and add freshly just before use 250 µl 6% of H2O2 2. 50 mg of O-Dianisidine dissolved in 1ml of methanol Procedure: 1. Incubate gel in solution (a) for 10 min. 2. Add solution (b) and mix properly. 3. Appearance of blue bands immediately indicates the peroxidase activity.
519 4. (Blue bands change to brown after around half an hour). 5. Transfer the gel to 7% acetic acid for 3 min which ensures fixation of bands in the gel. 6. Wash the gel in the tape water or 3-4 min (washing removes excess acetic acid and stain). 7. Finally transfer the gel to the tray containing distilled water and visualize on an white light illuminator. Visualization of Esterases: Requirements: The staining solution of esterase consists of the following reagents: 1. Sodium di-hydrogen phosphate (NaH2PO4) 2.8 gm 2. di- Sodium hydrogen phosphate (Na2HPO4) 1.1 gm 3. Fast blue RR salt 0.2 gm 4. alpha napthyl acetic acid-0.03 gm in 2 ml of acetone water (60:40). Procedure: 1. Mix all contents in distilled water to make final volume 200 ml and pour over the gel. 2. Keep in dark for half an hour. 3. Fix the gel with 7% acetic acid for 3 min. 4. Then wash the gel with distilled water and observe over the illuminator. Precautions: 1. Acrylamide is neuro-toxic compound so never should be touched by hands. 2. There should be no bubbles (air) during pouring of the gel. 3. High voltage should be avoided for native PAGE for maintaining non-denaturing conditions. 4. For preservation of the gel it should be dried and UV sterilized.
520 Pit falls and trouble shooting guide: Problem
Cause
Remedies
Failure or slow polymerization of the gel
Absence of catalyst Glass plates Stock solution aged Presence of oxygen
Add fresh catalyze. Degrease the plates. The fresh stocks. Degas the Acrylamide stock.
Long duration of Run
Air bubble interference
Flush air bubbles
Staining is poor
Dye absorption is not sufficient.
Dye may be old hence in a strong solution of dye or change to a more sensitive.
6. Isolation and Purification of Plant and Fungal DNA The high molecular plant DNA preparation can be used for digestion with restriction endonucleases that can be integrated to make transgenics. The integration can be confirmed through Polymerase chain reaction (PCR) or southern hybridization. DNA preparations should be nuclease free. Isolation of DNA from plant and fungi poses some problem as one protocol may work with one plant group but may fail with other. Different groups have developed number of isolation methods. Methods developed by Dellaporta et al. (1983) and Murray and Thompson (1980) are being used with slight modifications. The later method can be used for rice, wheat, chick pea and Brassicaand fungal pathogens as described below. Requirements 1. Extraction buffer : 2% CTAB, 10 mM Tris HCl (pH 8.0, 1.4 M NaCl), 20 mM EDTA, 0.2% B-mercapto ethanol, 4% Sarkosyl. 2. TE buffer : 10 mm Tris, 1 MM PDTA, pH 8.0. 3. Phenol: Chloroform: Isoamyl alcohol (25:24:1). 4. Sodium acetate (3M) 5. Ethanol (70%) 6. RNase 7. EDTA 8. Et Br 9. Agarose 10. DNA
521 11. Potassium citrate (5M) 12. SDS-20% Isopropanol Preparation of Reagents 1. 1M Tris buffer pH 8.0 (100 ml) Dissolve 12.11 g Tris base in 80 ml of distilled water. Adjust the pH to 8.0 within HCl. Make up the final volume to 100 ml with distilled water. Autoclave and store at Room temperature. 2. 0.5 M EDTA (100 ml) Dissolve 18.612 g of Na2 EDTA.2H2O in 80 ml of distilled water, adjust the pH to 6.0 with NaOH (by adding 2 g of NaOH pellet). Stir vigorously for several minutes OD a magnetic stirrer. Make up the volume to 100 ml. Note : Na2EDTA will not dissolve in water in absence of NaOH. 3. DNA extraction buffer (100 ml) 1 M Tris buffer
50 ml
0.5M EDTA
50 ml
NaCl
14.6 g
Adjust pH to 8.0 with HCl. Autoclave and store at 4°C. 4. 70% Ethanol (100 ml) Absolute ethyl alcohol
70 ml
H2O
30 ml
Store at 4°C. 5. 5M Potassium acetate (100 ml) Potassium acetate
49.07 g
Glacial acetate acid
11.5 ml
Make up to 100 ml, autoclave and store at room temp. 6. 20% SDS (100 ml) SDS
20 g
H2O
80 ml
522 Store at 60°C to dissolve properly. Make up to 100 ml. Autoclave and store at room temperature. 7. 3M Sodium acetate (100 ml) Sodium acetate
24.6 g
H2O
40 ml
Adjust pH to 5.2 with glacial acetic acid. Make up to 100 ml. Autoclave at store at room temp. 8. TE buffer (100 ml) 1M Tris buffer
1.0 ml
0.5 M EDTA
0.2 ml
Make up to 100 ml. Autoclave and store at room temp. 9. TE Buffer (100 ml) (Tris-10 mm, EDTA-10mM) 1M Tris buffer
5.0 ml
0.5 M EDTA
2 ml
Make up to 100 ml. Autoclave and store at room temp. 10. Isopropanol Store at -20°C in 100 ml dark bottle. 11. CTAB solution CTAB (10%)
2g
NaCl (0.79%)
0.799 g
Add distilled water to make the volume to 20 µl. 12. SDS (10%) SDS
10 g
Distilled water
100 ml
13. NaCl (5M) NaCl
19.62 g
Distilled water
50 ml
523 DNA isolation from rice seedlings 1. Harvest 1.0g soft fresh leaves and grind in liquid nitrogen using pre-chilled mortar and pestle. Don’t allow other liquid to thaw. 2. Remove the contents to 50 ml polypropylene tube and add 15 ml DNA extraction buffer (pre warmed) and 1 ml of 20% SDS. Incubate at 65°C for 10 min. Disperse the material with the help of spatula. 3. Add 10 ml of 5 M potassium acetate and incubate the content on ice for 20 min. 4. Centrifuge at 10,000 rpm at 4°C for 25 min. 5. Collect the supernatant in another 50 ml tube and add 15 ml isopropanol. Mix it gently. 6. Incubate at -20°C for 1 hr or overnight and than spin at 11,000 rpm at 4°C for 15 min to pellet the DNA. Gently pour off the supernatant. 7. Wash the pellet with 70% ethanol and dry the pellet by inverting the tube on a paper towel. 8. Re-dissolve the DNA pellet in 1.4 ml T50 E10 buffer and transfer the solution to an eppendorf tube. 9. Re-precipitate the DNA by adding 0.6 volume of isopropanol and incubate at -20°C for 30 min. 10. Spin the tubes and wash the pellet with 70% ethanol. Air dry the pellet by inverting the tube. Finally re-dissolve the DNA in 100 µl TE buffer and store at -20°C. DNA Isolation from fungi CTAB Method: 1. Grind 0.2 g lyophilized mycelia. 2. Suspend powder is 10 ml extraction buffer. 3. After shaking very well, add 1 ml of 10% SDS. 4. Shake gently at 37°C for 1 hr. 5. Add 1.5 ml of 5M NaCl and mix thoroughly. 6. Add 1.25 ml of CTAB/NaCl solution. Mix and incubate at 65°C
524 for 10-20 min. 7. Extract DNA by adding an equal volume of chloroform: isoamyl alcohol and mix. Spin at 10,000 rpm for 12 min at 10°C. 8. Remove aqueous, viscous supernatant to a fresh tube and precipitate with 0.6 volume of cold isopropanol. Leave inside the freezer for at least an hour. 9. Spin at 10,000 rpm for 10 min at 10°C. Wash pellet with 70% ethyl alcohol and dry. Add 5 ml 1X TE buffer. Leave over night. 10. Add 50 µl RNAse and incubate for 1-2 hrs. Extract with chloroform : isoamyl and spin for 12 min at 10,000 rpm at 10°C. 11. Precipitate with cold 150 propanol and spin at 10,000 rpm for 10 min at 10°C. 12. Wash the pellet with cold 70% ethanol and dry completely. 13. Precipitate with cold isopropanol and spi at 10,000 rpm for 10 min at 10°C. 14. Wash pellet with cold 70% ethanol and dry completely. 15. Dissolve in 1 ml of 1X TE buffer. Precautions 1. Do not subject the plant samples to freezing or thawing during grinding. 2. Do not over dry DNA pellet otherwise it will take long time to dissolve DNA. 3. Do not force DNA into solution. It might cause shearing. 4. Wheat DNA should not be dissolved in TE buffer, instead in 10 nM Tris HCl, pH 8.0, since EDTA activate DNase causing shearing of DNA. 5. Wear gloves while using phenol and work in a fume hood. 6. High molecular weight DNA will float, with air bubbles, after isopropannol precipitation, it could be spooled out by pipette. 7. It is very import to prevent carry over contamination from outside/ hand/ chemicals. 8. Autoclaved solutions must be used, and gloves should be worn by
525 lab personnel. 9. For PCR, DNA preparation must be very clean, as PCR can amplify dirty DNAs easily. Pitfalls and Trouble Shooting Guide Problem
Cause
Remedies
Smear of DNA on gel
1. DNA seems to be degraded.
1. Quicken the isolation and do them at 4°C. 2. Check the purity of chemicals. If necessary autoclave them.
2. Storage is not proper.
1. Store at proper conditions (5to -70°C). 2. Store in high salt Tris (1M) in the presence of Na2 EDTA (10 mM) at pH 8.5.
7. Molecular Characterization of Fungal Isolates using Random Amplification of Polymorphic DNA (RAPD) Marker- PCR Due to advances in molecular biology techniques, large numbers of highly informative DNA markers have been developed for the identification of genetic polymorphism. In the last decade, the random amplified polymorphic DNA (RAPD) technique based on the polymerase chain reaction (PCR) has been one of the most commonly used molecular techniques to develop DNA markers. RAPD markers are amplification products of anonymous DNA sequences using single, short and arbitrary oligonucleotide primers, and thus do not require prior knowledge of a DNA sequence. Low expense, efficiency in developing a large number of DNA markers in a short time and requirement for less sophisticated equipment has made the RAPD technique valuable although reproducibility of the RAPD profile is still the centre of debate. Reagents / chemicals: 1. PCR buffer (10 x) Sigma Aldrich 2. Primer (10 pmoles/ml) 3. dNTPs (10 mM) Sigma Aldrich 4. Taq DNA polymerase (5U/ml), Sigma Aldrich 5. Template DNA (20 ng/ml)
526 Table 1. RAPD PCR reaction mixture PCR buffer (10 x) with 15 mM MgCl2
2.5 ml
Primer (10 pmoles/ml)
1.0 ml
DNTPs mix (10 mM each)
0.5 ml
Taq DNA polymerase (5U/ml)
0.3 ml
Template DNA (20ng/ml)
2.5 ml
Sterile distilled water
18.2 ml
Total
25.0 ml
Table 2. RAPD PCR conditions Step
Temperature (ºC)
Duration
Initial Denaturation
94
2.0 min
Denaturation
94
1.0 min
Annealing
40
2.0 min
Extension
72 (39 times to step 2)
2.0 min
Final extension
72
10.0 min
Hold
4
—
Protocol: 1. Take 200 ml of PCR tube; add 25 ml reaction mixture as per table 1 and mix the content by short spin 2. Carry out amplification with programme given in table 2 3. Analyze result by agarose gel electrophoresis on 1.5% gel 4. Visualization of banding pattern on UV-Transilluminator or gel documentation imaging. 8. Transmission of Plant Virus through Whitefly Vector Whitefly (Bemisia tabaci Genn.) is the vector of more than 23 different plant viral diseases. 1. Whitefly transmitted viruses have the following properties in common
527 2. Whitefly borne viruses are not acquired as rapid as aphid borne viruses. 3. The transmission efficiency of the vector increase with longer feeding periods upto several hours on virus sources. 4. In most cases there is a definite but relative short incubation period. Tools required in transmission study: 1. Aspirator 2. Micro cage 3. Acquisition feeding bottle Collection of whitefly adults: The whitefly adults will be collected from cotton field early in the morning by means of modified aspirator from the undersurface of the cotton leaves and will be released into acquisition feeding bottle. The acquisition feeding bottle will be scaled using cotton plug immediately after releasing the whiteflies. These will be then transferred to cotton plants maintained in an insect proof glass house. Rearing of non viruliferous whiteflies: 1. The nonviruliferous whiteflies will be reared on young seedlings in an insect proof glass house. 2. The whiteflies collected from cotton field will be released on the cotton plants kept in insect proof wooden cages (60 x 60 cm) in glass house released on tomato seedlings. Methodology: Nonviruliferous whiteflies will be collected by aspirator and released in acquisition feeding bottle. An infected twig of diseased plant will be inserted carefully into the acquisition feeding bottle through the open mouth portion and plugged with cotton immediately. Varying acquisition feeding period scheduled will be taken into consideration immediately after landing of whiteflies on the leaf surface. By allowing 30 min., 1 hr., 2 hr., 3 hr., 6 hr. of acquisition feeding, known number of viruliferous whiteflies (20 flies/plant) will be again collected from the feeding bottle by means of an aspirator and transferred to healthy test plants covered with micro cages. Whiteflies will be allowed for 12 hrs. to feed on healthy test plants and thereafter the micro cages will be removed and
528 test plants will be sprayed with triazophos 40 EC @ 0.04 0/10 to kill the viruliferous whiteflies. Transmission of plant virus through aphid vector Aphids (Aphis cracsivora K.) will collected from Indian beans and cowpea and brought to the glasshouse for multiplication and maintained on separately caged cowpea plants. The apterous aphids transferred in petriplate with the help of a camel hair brush (No.1). The brush will moisten with water to make the transfer easier. The aphid will starve for at least 1 hour in petriplate prior to using fine camel hair brush and allow to develop. 3. Keep such aphid-changed plant in a field cage (6.10 m long x 3.0 m wide x 1.8 m height) and cover all sides with plastic shade net. 4. Harvest aphid colonies developed on host plant on sixth day. We can harvest 4000 aphid by inoculating 800 aphids/20 plants after six days. Acquisition feeding period 1. Aphids will starved for 1 hour pre-acquisition then transferred on diseased leaf for 2 hours acquisition feeding period. 2. The virus acquisition-feeding period was schedule from 2 minute to 24 hours. 3. The feeding was watch with a hand lens and timed with a metronome. Inoculation feeding period 1. Viruliferous aphids then transferred (20 aphids/test plant for different inoculation feeding period ranging from 2 minutes to 24 hours on test plants. 2. After each inoculation feeding, aphids will kill by spraying methylo-demeton (0.02%). 3. The test plants were placed in an insect proof glasshouse till the expression symptoms. 9. Direct Antigen Coating ELISA (DAC- ELISA) 1. Samples are collected from the field and symptoms are noted down. 2. Take 1 gm of the leaf sample, crush with the help of mortar & pestle & dilute upto 5-10 % adding the coating buffer. 3. Filter it with the help of four layered muslin cloth or centrifuge in Eppendorf tube for 30 seconds -1 min.
529 4. 100 µl of the antigen (Sample supernatant) is added to each well of the plate & kept for overnight in the refrigerator. 5. Wash with water, then PBS tween – 20 for 3 times at the interval of 3 Min. 6. Blotto (Non fat milk) 1-5 % is prepared in PBS (Phosphate buffer saline). 100 µl of Blotto is loaded in each well & left for 30 min. Again washed 3 times. 7. Dilute the antibody with Blotto (containing PBS), (dilution of the antibody depend upon the titre of antisera), 100 µl is loaded to each well. It is incubated for 45 min – 1 hrs at room temp. 8. Wash with PBS tween-20 for 3 times at the interval of 3 mins. 9. Dilute the second antibody conjugate (HRP / alkaline phosphate / penicillinase) with PBS containing 5% non fat milk (Blotto) in 1:1000 dilution (Goat antirabbit in cash of Polyclonal antibody & Goat antimouse in cash of monoclonal antibody 9.9 % dilution), 100 µl is added to each well & kept for 45 min – 1 hrs at room temp. 10. Wash with PBS Tween – 20, 5 times at the interval of 5 mins. 11. Then prepare substrate solution, use (ABTS) 5 µl/ml in substrate buffer. Add H2O2, 2 µl/ml just before the use. 12. Add 50 µl of the substrate solution to each well. Read the reaction ate O.D 490 nm in ELISA reader after about 10-15 mins 13. For visual observation, HRP will greenish blue colour, Alkaline Phosphatase will give light orange colour and Penicillanse will give light blue colour. Buffers for ELISA 0.1 M Coating buffer (pH- 9.6) 0.1 M Na2Co3
1.50 g
0.1 M NaHCo3
2.93 g
Distilled Water 1.0 L PBS (Phosphate buffer saline) (pH-7.4) KCl
0.2 g
530 NaCl
8.0 g
KH2Po4
0.2 g
Na2HPo4.12 H2O
2.9 g
Distilled Water
1.0 L
PBS- T (PBS- Tween- 20) (pH- 7.4) PBS (1000 ml) + 0.8 ml Tween 20 HRP substrate buffer (C/P) (pH- 4.0) (Citrate / Phosphate buffer, 0.1M) Citric acid (C6H8O7.H2O), M.W= 210.14, 0.1M= 21.9 gm/lit Sodium Phosphate (Na2HPO4.2H2O), M.W= 177.9, 0.2M= 35.59 gm/lit 61.45 ml Citric acid solution (0.1M) + 38.55 ml Phosphate solution (0.2M) OR 1.9 gm Citric acid + 1.37 Phosphate/ 100 ml double distilled water ELISA test – Triple Antibody Sandwich (TAS) based on the method of Thomas et al. (1986) This ELISA employs African Cassava Mosaic (ACMV) polyclonal antiserum as the trapping antibody, and mouse monoclonal antisera (Appendix 1) for detection. Microtitre plates (Nunc Maxisorp Immunoplate) were used. Known infected plants to be used as positive controls together with healthy plants of the same species as the test plants as a negative control. 1. Add the crude ACMV polyclonal antibody at the recommended dilution (10-3) to the coating buffer (Appendix 1). Pipette the solution into the microtitre plates, 100ul per well. Incubate for 3 hours at33°C. 2. Flick out the contents of the wells. Wash the wells three times with PBS –Tween (Appendix 1) with 3-minute soaks between washes. Blot dry on absorbent paper. 3. Add sample homogenate at 100ul per well, using two wells per test sample. Incubate at 4°C overnight.
531 4. Flick out the contents of the wells as before (2.2) but wash 4 times. 5. Add TYLCV/ToMoV monoclonal antibody SCR 23 at the recommended dilution in dried milk buffer (Appendix 1) at 100ul per well. Incubate for 2 hours at 33°C. 6. Flick out the contents of wells and wash as in (2.4). Prepare alkaline phosphatase conjugate at appropriate dilution in dried milk buffer. Add 100ul to each well. Incubate as 2.5. 7. Flick out the contents of the wells and wash as in (2.4). Prepare alkaline phosphatase substrate solution (Appendix 1). Add 100ul to each well. Incubate at ambient temperature for 1 hour. 8. Read absorbance at 405nm. Interpretation of the ELISA test: The ELISA test is negative if the absorbance of the sample is less than 2 times the absorbance of the healthy control. The ELISA test is positive if the absorbance of the sample is equal or greater than 2 times the absorbance of the healthy control. Direct Antigen Coating ELISA (DAC- ELISA) Protocol: 1. Dispense samples -
Grind infected leaf samples into indirect sample extraction buffer at 1:10 ration (Tissue weight in gm: Buffer volume in ml)
-
Centrifuge homogenate for 30 sec. or 1 min
-
Dispense 100 µl of sample extract into sample wells of antigen coated ELISA plate, dispense 100 µl of positive control into positive control wells and negative control into negative control wells
2. Incubate plate -
Incubate the plate for 1 hour at room temperature.
3. Wash plate -
Quick dump of all wells into sink without mixing
-
Fill all wells completely with 1X PBST, and quickly empty again. Repeat 7 times.
them
532 4. Add antibody -
Dispense 100 µl of prepared antibody per well.
5. Incubate plate -
Incubate plate for 2 hours at room temperature or the refrigerator at 4 °C.
overnight in
6. Wash plate -
As before, wash the plate 8 times with 1X PBST.
7. Add enzyme conjugate -
Dispense 100 µl of enzyme conjugate per well.
8. Incubate plate -
Incubate plate for 1 hour at room temperature
9. Wash plate -
As before, wash the plate 8 times with 1X PBST.
10. Add PNP substrate -
Dispense 100 µl of PNP substrate solution per well.
11. Incubate plate - Incubate plate for 1 hour at room temperature 12. Evaluation of results -
Examine the wells by eye, or measure on plate reader at 405 nm. Wells in which colour develops indicates positive result and in which no significant colour development indicates negative result.
Preparation of antibody: Always prepare within 10 minute before use If dilution of antibody concentrates is 1:100, -For 10 ml solution, dissolve 100 µl of antibody to 10 ml of ECI buffer. Preparation of enzyme conjugate: Always prepare within 10 minute before use If dilution of enzyme conjugate concentrates is 1:100,
533 -For 10 ml solution, dissolve 100 µl of enzyme conjugate to 10 ml of ECI buffer. Buffer formulations: Indirect Extraction buffer (1X): Dissolved in distilled water to 1000 ml - Sodium carbonate (anhydrous)- 1.59 gm Sodium bicarbonate – 2.93 gm Sodium azide – 0.2 gm Polyvinyl pyrrolidone (PVP)- 20.0 gm Adjust pH to 9.6. store at 4°C PBST Buffer (Wash buffer, 1X) : Dissolved in distilled water to 1000 ml Sodium chloride- 8.0 gm Sodium phosphate, dibasic (anhydrous)-1.15 gm Potassium phosphate, monobasic (anhydrous)-0.2 gm Potassium chloride- 0.2 gm Tween-20- 0.5 gm Adjust pH to 7.4. ECI Buffer (1X) : - Add to 1000 ml 1X PBST - Bovine serum albumin (BSA)-2.0 gm - Polyvinyl pyrrolidone (PVP)-20.0 gm - Sodium azide-0.2 gm Adjust pH to 7.4. store at 4°C PNP Buffer (1X): Dissolve in 800 ml distilled water Magnesium chloride hexahydrate- 0.1 gm Sodium azide-0.2 gm
534 Diethanolamine- 97.0 ml Adjust pH to 9.8 with hydrochloric acid. Adjust final volume to 1000 ml with distilled water. Store at 4°C Buffer Preparation Inoculation buffer: pH 7.6 KH2PO4 = M.wt.= 136.09 g 0.1 M = 0.136 g for 900 ml dist. water. KH2PO4 = M.wt.= 17.418 g 0.1 M = 15.67 g for 900 ml. Dist water. Note: Buffers of constant ionic strength for use in electrophoresis have been described by Miller and Golder, Arch. B.29, 420 (1950) and by Green, J.A.C.S. 55, 2331 (1933). It should be noted that with many buffers the pH changes appreciably with temperature. In buffers employing Na+ or K+ these may be interchanged with negligible alteration to the pH values. Glycine- HCL buffer (0.05M) pH 2.2-3.6 (Gomoro, methods in enzymology, 1, Academic press Inc., New York (1955), p. 139; after Sorenson) Glycine, M. wt. = 75.07; 0.2Msoln. Contains 15.01 g in 1000 ml. PH
0.2M-glycine (ml)
0.2N-HCL (ml)
2.2
50
44.0
2.4
50
32.4
2.6
50
24.2
2.8
50
16.8
3.0
50
11.4
3.2
50
8.2
3.4
50
6.4
3.6
50
5.0
Dilute to 200 ml with H2O
535 Phthalate – HCl buffer (0.05 M), pH 2.2 – 3.8 (Bower and Bates, J. Res. Nat. Bur. Stand.,55, 197(1955)) KH phthalate, M. wt. = 204.23; 0.2 M – soln. contains 40.85 g in 1000 ml PH at 25°C
0.2M-KH phthalate (ml)
0.2N-HCL (ml)
2.2
5
4.95
Dilute to 20 ml with
2.4
5
4.22
H2 O
2.6
5
3.54
2.8
5
2.89
3.0
5
2.23
3.2
5
1.57
3.4
5
1.04
3.6
5
0.63
3.8
5
0.29
Na2HPO4- Citric acid buffer, pH 2.2 – 8.0 (Mcllvaine, J.B.C. 49, 183 (1921)) Can not be used in the presence of Ca++ or Mg++ Na2HPO4 2H2O, M. wt. = 178.05; 0.2 M-soln. contains 35.61 g in 1000 ml. Citric acid H2O, M. wt. 210.14; 0.1 M-soln. contains 21.01 g in 1000 ml pH
0.2 M Na2HPO4 (ml)
0.1 M Citric acid (ml)
pH
0.2 M Na2HPO4 (ml)
0.1M Citric acid (ml)
2.2
0.40
19.60
5.2
10.72
9.28
2.4
1.24
18.76
5.4
11.15
8.85
2.6
2.18
17.82
5.6
11.60
8.40
2.8
3.17
16.83
5.8
12.09
7.91
3.0
4.11
15.89
6.0
12.63
7.37
3.2
4.94
15.06
6.2
13.22
6.78
3.4
5.70
14.30
6.4
13.85
6.15
3.6
6.44
13.56
6.6
14.55
5.45
3.8
7.10
12.90
6.8
15.45
4.55
4.0
7.71
12.29
7.0
16.47
3.53
4.2
8.28
11.72
7.2
17.39
2.61
536
4.4
8.82
11.18
7.4
18.17
1.83
4.6
9.35
10.65
7.6
18.73
1.27
4.8
9.86
10.14
7.8
19.15
0.85
5.0
10.30
9.70
8.0
19.45
0.55
Citric acid – sodium citrates buffer (0.1 M0, pH 3.0 – 6.2. (N. Hemington and R.M.C. Dawson) Citric acid H2O, M. wt. = 210.14; 0.1 M-soln. contains 21.01 g in 1000 ml Na3 citrate 2H2O M. wt. = 294.12; 0.1 M-soln. contains 29.4 g in 1000 ml pH
0.1 M Citric acid
0.1 M Na3 Citrate
pH
0.1 M Citric acid 0.1M Na3 Citrate
3.0
16.4
3.6
4.8
8.0
12.0
3.2
15.5
4.5
5.0
7.0
13.0
3.4
14.6
5.4
5.2
6.1
13.9
3.6
13.7
6.3
5.4
5.1
14.9
3.8
12.7
7.3
5.6
4.2
15.8
4.0
11.8
8.2
5.8
3.2
16.8
4.2
10.8
9.2
6.0
2.3
17.7
4.4
9.9
10.1
6.2
1.6
18.4
4.6
8.9
11.1
Na2HPO4 – NaH2Po4 buffer (0.1 M0, pH 5.8 – 8.0 (Gomori, Methods in Enzymology, vol. 1, Academic Press Inc., New York (1955), p. 143; after Sorenson) Na2HPO4 2H2O, M. wt. = 178.05; 0.2 M-soln. contains 35.61 g in 1000 ml. Na2HPO4 12H2O, M. wt. = 358.22; 0.2 M-soln. contains 71.64 g in 1000 ml. NaH2PO4 H2O, M. wt. = 138.0; 0.2 M-soln. contains 27.6 g in 1000 ml. NaH2PO4 2H2O, M. wt. = 156.03; 0.2 M-soln. contains 31.21 g in 1000 ml.
537
pH
0.2 M Na2HPO4 (ml)
0.2 NaH2PO 4 (ml)
5.8
8.0
92.0
Dilute to 200 ml with
6.0
12.3
87.7
H2 O
6.2
18.5
81.5
6.4
26.5
73.5
6.6
37.5
62.5
6.8
49.0
51.0
7.0
61.0
39.0
7.2
72.0
28.0
7.4
81.0
19.0
7.6
87.0
13.0
7.8
91.5
8.5
8.0
94.7
5.3
KH2PO4 – NaOH buffer (0.05 M), pH 5.8 – 8.0 (Bower and Bates, J. Res. Nat. Bur. Stand. 55, 197 (1955)) For sodium free buffer use KOH KH2PO4, M. wt. = 1-6.09; 0.2 M soln. contain 27.22 g in 1000 ml pH at 250C
0.2 M KH2PO 4 (ml)
0.2 N-NaOH (ml)
5.8
5
0.36
6.0
5
0.56
6.2
5
0.81
6.4
5
1.61
6.6
5
1.64
6.8
5
2.24
7.0
5
2.61
7.2
5
3.47
7.4
5
3.91
7.6
5
4.24
7.8
5
4.45
8.0
5
4.61
Dilute to 20 ml with H2O
538 Sodium bicarbonate 5 per cent CO2 buffer, pH 6.0 – 8.0 Calculated from the Henderson-Hasselbach equation (HCO3-) pH = pK‘ + log —————CO2 Concentrations of bicarbonate are approximately correct when atmospheric pressure varies between 725 and 760 mm Hg. Temperature: For every degree decrease in temperature down to 200 the bicarbonate conc. Is increased by approximately 1.88% to give the same pH as at 370C. pH at 370C
Conc. Of NaHCO3 (M wt. = 84.02) in equilibrium with gas phase containing 5% CO2
6.0
5.86 x 10-4 M
6.2
9.29 x 10-4 M
6.4
1.47 x 10-3 M
6.6
2.33 x 10-3 M
6.8
3.70 x 10-3 M
7.0
5.86 x 10-3 M
7.2
9.29 x 10-3 M
7.4
1.47 x 10-2 M
7.6
2.33 x 10-2 M
7.8
3.70 x 10-2M
8.0
5.86 x 10-2M
Tris –(hydroxymethyl)- aminomethane- HCl (Tris-HCl‘) buffer (0.05 M), pH 7.2 – 9.1 (Sigma Chemical Co. Bulletin, 106) Tris –(hydroxymethyl)- aminomethane, M. wt. = 121.14; 0.2 M soln. contains 24.23 g in 1000 ml. pH
0.2 M – ‘Tris‘ (ml)
0.1 N-HCl (ml)
Dilute to 100 ml with H2O
23 0
37 0
9.10
8.95
25
5
8.92
8.78
25
7.5
8.74
8.60
25
10.0
539
8.62
8.48
25
12.5
8.50
8.37
25
15.0
8.40
8.27
25
17.5
8.32
8.18
25
20.0
8.23
8.10
25
22.5
8.14
8.00
25
25.0
8.05
7.90
25
27.5
7.96
7.82
25
30.0
7.87
7.73
25
32.5
7.77
7.63
25
35.0
7.66
7.52
25
37.5
7.54
7.40
25
40.0
Boric acid-borax buffer (0.2 M in terms of borate), pH 7.4 – 9.0 (Holmes, Anat. Record 86, 157 (1943)) Borax, Na2B4O7 10H2O, M. wt. = 381.43; 0.05 M soln. (= 0.2 m BORATE) CONTAINS 19.07 G IN 1000 ml. Boric acid, M wt. = 61.84; 0.2 m soln. contains 12.37 g in 1000 ml. Borax, Na2B4O7 10H2O, may lose water of crystallization and it should be kept in a stoppered bottle. Solns. of borax can be prepared also by halfneutralizing solns of boric acid. pH
0.05 M-borax (ml)
0.2 M-boric acid (ml)
7.4
1.0
9.0
7.6
1.5
8.5
7.8
2.0
8.0
8.0
3.0
7.0
8.2
3.5
6.5
8.4
4.5
5.5
8.7
6.0
4.0
9.0
8.0
2.0
540 Glycine-NaOH buffer (0.05 M), pH 8.6 – 10.6 (Gomori, Methods in Enzymology, vol. 1, Academic Press Inc., New York (1955), p.145; after Sorenson) Glycine, M. wt. = 75.07; 0.2 M-soln. contains 15.01 g in 1000 ml. pH
0.2 M-glycine (ml)
0.2 N-NaOH (ml)
8.6
50
4.0
8.8
50
6.0
9.0
50
8.8
9.2
50
12.0
9.4
50
16.8
9.6
50
22.4
9.8
50
27.2
10.0
50
32.0
10.4
50
38.6
10.6
50
45.5
Dilute to 200 ml with H2O
Sodium carbonae-bicarbonate buffer 90.1 M0, pH 9.2 – 10.8 (Delory and King, B.J. 39, 245 (1945)) Can not be used in the presence of Ca++ or M++ NaHCO3 10H2O, M. wt. = 286.2; 0.1 M-soln. contains 28.62 g in 1000 ml NaHCO3, M. wt. = 286.2; 0.1 M-soln. contains 28.62 g in 1000 ml pH
0.1 M Na2CO3 (ml)
0.1 M-NaHCO3 (ml) 20 0
37 0
9.16
8.77
1
9
9.40
9.12
2
8
9.51
9.40
3
7
9.78
9.50
4
6
9.90
9.72
5
5
10.14
9.90
6
4
10.28
10.08
7
3
10.53
10.28
8
2
10.83
10.57
9
1
541 Standard pH Solution The British Solution Standards Institution (Publication B.S. 1647:1950) recommend the following solutions for calibration of glass electrodes: Primary Standard 0.05 M-potassium hydrogen phthalate (10.21g of pure, dry KH phthalate in freshly distilled water, or recently boiled and cooled distilled water, and made upto 1000 ml); the pH of this solution is defined as 4.000 at 15°, 4.005 at 25°, and 4.026 at 38°C. Secondary Standards 0.01 M-potassium tetroxalate* (KH3 (C2O4)2 2H2O, M. wt. = 254.19), pH = 1.48 at 25° and 1.50 at 38°C. 0.01 N-HCl+ 0.09 M-KCl, pH = 2.07 at 25° and 2.08 at 38°C. 0.1 M-CH3COOH and 0.1 M-CH3COOHNa (prepared from acetic acid half neutralized with Na OH, not from sodium acetate). PH = 4.65 at 12°, 4.64 at 25° and 4.65 at 38°C. 0.025 M-KH2PO4 + 0.025 M- Na2HPO4 2H2O, pH – 6.85 at 25° and 6.84 at 38°C. 0.05 M-Na2B4O7 10H2O (m.wt. = 381.43), pH = 9.18 at 25° and 9.07 at 38°C. The National Bureau of Standards (U.S.A.) defines the pH scale in terms of the following solutions (data from Bates, Electrometric pH Determination, Wiley & Sons Inc., New York (1954), p.118). 0.05 M-potassium tetroxalate* (12.71 g KH3 (C2O4)2 2H2O per lit.), pH = 1.68 at 25° and 1.69 at 30°C. A solution potassium hydrogen tetroxalate (standard at 25°C), pH – 3.56 at 25° and 3.55 at 30°C 0.05 M- potassium hydrogen tetroxalate (10.211 g per lit.), pH – 4.00 at 20° and 4.01 at 25-30 °C. 0.025 M-KH2PO4 + 0.025 M- Na2HPO4 (3.402 g KH2PO4 + 4.451g Na2HPO4 2H2O per lit.), pH – 6.88 at 20° and 6.86 at 25 ° and 6.85 at 30 °C. 5.) M- borax (3.814 g Na2B4O7 10H2O per lit.), pH – 9.22 at 20°, 9.18 at 25 ° and 9.14 at 30 °C.
542 References Verma, D.K., Dand, S.A. and Chourasia, H.K, (2018). The Dynamic Confluence of Plant Systematics and Biotechnology: A Reassessment. Editors: HK Chourasia and D P Mishra, Today & Tomorrow’s Printers and Publishers, New Delhi – 110002, India. Pp 275-303. Jauhar, P. (2006). Modern Biotechnology as supplement to plant breeding: the Prospects and Challenges. Crop Sci. 46:1841-1859.
543
Integrated Management of Crop Diseases
Editors Prof. (Dr.) H.K. Chourasia University Department of Botany T.M. Bhagalpur University, Bhagalpur Prof. (Dr.) S.K. Choudhary Principal, TNB College, Bhagalpur T.M. Bhagalpur University, Bhagalpur
TTPP
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PREFACE In view of environmental and health concerns, a determined effort is currently made in almost any agroecosystem in the world, to reduce and rationalize the use of chemicals (pesticides, fungicides, nematocides, etc.) and to manage pests/pathogens more effectively. This consciousness is not only related to the need of nourishing a still growing world population, but also derives from the impact of side effects of farming, like soil, water and environmental contamination, calling for a responsible conservation of renewable resources. There are increasing expectations at the producers and consumers levels, concerning low inputs agriculture and residues-free food. Disciplines like IPM/IDM (integrated pest management / integrated disease management) are now central to the science and technology of crop protection. In the classical version of IPM/IDM, a pesticide/fungicide is applied only when the pathogen population reaches a level that would lead to economic losses in the crop. In other words, classical IPM/IDM concentrates on reducing the numbers of noxious organisms through the application of agrochemicals. However, IPM/IDM actually means “A disease management system that, in the context of the associated environment and the population dynamics of the pest/pathogen species, utilises all suitable techniques and methods in a manner as compatible as possible and maintains the pest/pathogen population at levels below those causing economic injury”. IPM/IDM in the broad sense has been defined as “the optimization of pest/pathogen control in an economically and ecologically sound manner, accomplished by the coordinated use of multiple tactics to assure stable crop production and to maintain pathogen pest damage below the economic injury level, while minimizing hazards to humans, animals, plants and the environment”. Plant health depends on the interaction of a plethora of microorganisms, including pathogens and pests, which give rise to a complex system based on multiple food webs and organisms interactions, including the physical and chemical environment in which plants grow. Thus IPM/IDM moves beyond a one-plant one pathogen/one-pest control view of disease control towards an integrated view of plant health as a result of complex interactions. Moreover, the basic concern of IPM/IDM is with designing and implementing pest/disease management practices that meet the goals of farmers, consumers and governments in reducing pest/disease losses while at the same time safeguarding against the longer term risks of environmental pollution, hazard to human health and reduced
agricultural sustainability. The volume is a compilation of the thoughts from a wide array of experts in the areas of modern plant pathology, epidemiology, disease forecasting and dissemination of plant pathogens, host-parasite interaction, host defence mechanism, conventional and integrated pests / disease management, biotechnological tools for diagnosis of plant pathogens and development of transgenic plants for disease resistance, covering a wide range of problems and solutions proposed. The chapters are contributed by leading experts with several research years’ expertise, investigating and applying advanced tools in their work, and offer several illustrations and graphs, helping the reader in his/her study.
H.K. Chourasia S.K. Choudhary
Contents 1.
INTEGRATED DISEASE MANAGEMENT IN CHICKPEA THROUGH CONVENTIONAL AND BIOTECHNOLOGICAL APPROACHES Sadia Perween, Anand Kumar, S.P. Singh, Manoj Kumar, Anil Kumar, Satyendra, Ravi Ranjan Kumar, Mankesh Kumar and Sanjay Kumar
2.
PRODUCTION CONSTRAINTS AND GENETIC IMPROVEMENT OF PULSE CROPS IN BIHAR Anil Kumar, Anand Kumar, Kumari Rajani, Chandan Kishore, Manoj Kumar, Ravi Ranjan Kumar, Vinod Kumar and Abhijeet Ghatak
3.
DEVELOPMENT OF A UNIQUE RHIZOSPHERIC SOLID ORGANIC SUPPLEMENT FOR BETTER CROP PRODUCTIVITY AND BIO-CONTROL OF PATHOGENS Noyonika Mukherjee, Anjali Yadav, Anindita Bhattacharya, Arup Kumar Mitra and Fr. S Xavier S.J.
4.
FUNGAL DIVERSITY IN RHIZOSPHERE SOIL OF DIFFERENT VARIETIES OF PIGEONPEA [Cajanus cajan (L.) Millsp.] B.D. Gachande and V. Jalander
5.
IN VITRO ANTI-MYCOTIC EFFICACY OF PLANT LATEX AGAINST SEED-BORNE STORAGE FUNGI OF SUNFLOWER AND SAFFLOWER Gachande B. D., Manoorkar V. B. and N.F. Shaikh
6.
SURVEY IN PLANT PATHOLOGY: NEED, PLANNING, PREPARATION, PERIOD, PROCUREMENTS, PRECAUTIONS, SAMPLING AND EFFICIENCY P.K. Shukla
7.
INTEGRATED MANAGEMENT OF TEMPERATE PERENNIAL FODDER GRASS DISEASES - A BIOTECHNOLOGICAL APPROACH Dilip Kumar Verma, Suheel Ahmad, Nazim Hamid Mir, Siraj and Ahmad Bhat
8
MYCORRHIZA HELPER BACTERIA (MHB) AND THEIR INTERACTIONS WITH MYCORRHIZAL FUNGI AND HOST PLANT Sikha Dutta, Avishek Sarkar, Abhinanda Ghosh and Sunanda Dutta
9.
EFFECT OF SELECTED CHEMICAL AND BIOLOGICAL FUNGICIDES ON IN VITRO POLLEN GERMINATION AND POLLEN TUBE DEVELOPMENT OF TWO CUCURBITACEOUS CROPS. Manoranjan Paramanik, Sanjeev Pandey and Subrata Raha
10. INTEGRATED PLANT DISEASE MANAGEMENT IN DEVELOPING COUNTRIES H.K. Chourasia and Akanksha Raman 11.
SCAR MARKERS: A VERSATILE MOLECULAR TOOL FOR DETECTION OF PLANT GENOME Kumari Rekha, Ravi S. Singh, Dharamsheela Thakur, Sima Sinha, Ujjwal Jha, Ankita Sinha, Shikha Kumari and Prabhash K. Singh
12. DEVELOPING INSECT RESISTANT RICE THROUGH MOLECULAR BREEDING Mankesh Kumar, Satyendra, Manoj Kumar, Anand Kumar, S. P. Singh, Kumar Vaibhav and P.K. Singh 13. MACROPHOMINA ROOT ROT OF GROUNDNUT (Archis hypogaea L.) Manoj Kumar, Neha Rani, Anand Kumar, Anil Kumar, Satyendra, Mankesh Kumar, S.P. Singh and Sunita Kumari
14. INCIDENCE OF FUNGAL DISEASES ON MAKHANA IN PREHARVEST AQUATIC CONDITIONS AND THEIR POSSIBLE CONTROL Meenu Sodi and Sanjib Kumar 15. INTEGRATED PESTS AND DISEASE MANAGEMENT BY ORGANIC FARMING AND NATURAL INSECTICIDES Mir Syeda Yuhannatul Humaria and M.R. Sinha 16. IDENTIFYING THE IMPACTS OF PEST ATTACK ON SAL TREE (Shorea robusta) IN THE DINDORI DISTRICT OF MADHYA PRADESH USING REMOTE SENSING AND GIS APPROACH Obaidullah Ehrar, Shubham Kumar, Rabindar Kumar and Ajay Kumar Srivastava 17. IMPACT OF ABIOTIC AND BIOTIC STRESSES ON STEVIOL GLYCOSIDE PRODUCTION BY Stevia rebaudiana Bert. – CURRENT SCENARIO Abhijit Bandyopadhyay 18. INTEGRATED PLANT DISEASE MANAGEMENT (IDM) – CONCEPT, ADVANTAGES AND IMPORTANCE Raj Kamal Sahu, P.K. Sah and Abha Anand 19. UTILIZING THE EFFECTIVENESS OF MICROBIAL CONSORTIA AS BIOPESTICIDE Riddhi Basu, Atmeeya Sarkar, Bedaprana Roy, Poushali Mondal, Amrita Maity, Sulagna Banerjee and Arup Kumar Mitra 20. INTEGRATED DISEASE MANAGEMENT FOR SUCCESSFUL STRAWBERRY CULTIVATION IN SEMIARID SUBTROPICS OF GANGATIC PLAINS OF BIHAR Ruby Rani, Pawan Kumar and Chanda Kushwaha
21. POST-HARVEST PATHOLOGY OF ONION AND GARLIC Sangeeta Shree, Md. Ansar, Vijay Kumar Singh and Ramesh Sharma 22. THE ANAEROBIC FERMENTATION OF WASTES FOR PRODUCTION OF BIOGAS Santosh Kumar Singh and Niranjan Kumar Mandal 23. BLAST: THE MOST DEADLY DISEASE OF RICE Satyendra, Mankesh Kumar, Anand Kumar, Manoj Kumar, S. P. Singh, Rahul Singh, Surabhi Sinha and P. K. Singh 24. INNATE IMMUNITY IN PLANTS Surabhi Sinha and Satyendra 25. STUDY OF SYMPTOMS EXPRESSION OF ORANGE SOFT ROT INFECTED BY Aspergillus niger. Dara Singh Gupta, Bably Sarkar and Ashok Kumar 26. FUNCTIONAL BEVERAGES OF WHEATGRASS JUICE: BROAD SPECTRUM APPLICATION IN THE TREATMENT OF VARIOUS DISEASES. Amresh Kumar, M. Dutta Choudhury and Partha Pallit 27. SOYABEAN CULTIVATION AND ITS INTEGRATED PEST MANAGEMENT IS A BOON IN GANGATIC PLAIN ON PLACE OF RICE CULTIVATION IN RAINY SEASON H.C. Chaudhary, A.K.Singh, Ranju Kumari and Anupama Kumari 28. GENETIC ENGINEERING APPROACHES IN PLANT DISEAS MANAGEMENT: CURRENT AND PROSPECT Satish Kumar, Pawan Kumar, Sardar Sunil Singh, Alok Kumar, Vinay Kumar, M. D. Ojha and P.K. Singh
29. GENOMIC DATABASES, GENOMIC RESOURCESAND AGROINFORMATICS ASA BIOTECHNOLOGICAL PERSPECTIVE Dilip Kumar Verma, H. K. Chourasia, Somya Verma and Heena Verma 30. ROLE OF ALLELOPATHIC CHEMICAL LIKE GA3 ON NUCLEIC ACID AND PROTEIN CONTENTS OF AFLATOXIN B1 TREATED MAIZE SEEDS (Zea mays L.) Gajendra Prasad, Kumari Nitu and Kumari Ragni 31. Momordica charantia - PHYTOCHEMICALS, PESTS AND ITS CONTROL Satya Shubhangi and V.N. Singh 32. BIOTECHNOLOGICAL APPLICATIONS OF DETECTION, ESTIMATION, ELECROPHORESIS, ISOZYME ANALYSIS, MOLECULAR CHARACTERIZATION, TRANSMISSION, ELISA AND BUFFERS FOR PLANT DISEASE MANAGEMENT Dilip Kumar Verma, H. K. Chourasia and Somya Verma
About the Book
Over the last few years, scientists have researched extensively on integrated pest management (IPM) as the viable alternative of pesticides for reduction of damage due to insect pests, pathogens and weeds in crop plants. This disease management strategy is bio-safe, environmental friendly, ecologically compatible and economically sound. Excellent biotechnological developments have taken place and a number of technologies are currently crossed the pilot tests for commercialization. More recently emphasis is given on the development of resistant varieties but sometimes the failure of resistant gene in a variety has been observed and hence the scientists are busy to manage the diseases by developing transgenic varieties in addition to regular hybridization. This proceedings volume of the International Conference will be of great help to solve the problems of crop disease management. The volume consists of 32 review articles / research papers on biotechnological approaches by well-known researchers throughout India on different crops.
About the Authors
Prof. H. K. Chourasia Prof. H. K. Chourasia is University Professor of Botany, T.M. Bhagalpur University, Bhagalpur. He has previously worked as Head, Dept. of Botany, TNB College, Bhagalpur and Course Co-ordinator, P.G. Department of Biotechnology, TMBU, Bhagalpur. He has been awarded Certificate of Merit for Best Research Presentation in XIth IBS Conference, Tiruchirapalli (Tamil Nadu) in 1989; National Scholarship for Study Abroad by Human Resource Development Group, Department of Education, Govt. of India in 1990; Best Poster Award by the Peanut Research Council, Atlanta, USA in 1991; Research Associate by the CSIR, New Delhi in 1992; Scientists’ Pool Officer by the CSIR, New Delhi in 1992; Young Scientist Award by the DST, New Delhi in 1996; D.Sc. (HC) by International University for Complementary Medicines, Colombo, Sri Lanka in 2000; Gold Medal by the Indian Holistic Medical Academy (IHMA) during World Congress on Holistic Medicine, Goa University, Goa in 2003; Merit of Excellence by the IHMA during World Congress on Holistic Medicine at Bangalore in 2008; Guest of Honour by the Zoological Society of India during National Conference of Recent Concept of Biodiversity and Biotechnology at Satna (M.P.) in 2009; Dr. S. R. Bhargava Medal – 2013 in 15th Indian Agricultural Scientists and Farmers Congress at Allahabad; Dr. M. R. Siddiqi Medal – 2014 in 16th Indian Agricultural Scientists and Farmers Congress at Lucknow, Dr. R.S. Paroda Medal-2018 in the 20th Indian Agricultural Scientists & Farmers Congress at Allahabad. Prof. Chourasia has administrative experience of more than 05 years as Course Coordinator, Biotechnology Vocational Course, T.N.B. College, Bhagalpur. He has organized 14 Symposia / Conferences / Seminars / Workshops at National Level. He has completed 08 Minor / Major Research Projects funded by different govt. agencies. He is the Life Member of several Scientific Research Societies. He has edited and written 12 ISBN numbered text and reference books, published 75 original Research Papers and 15 Review Articles in Journals of National and International repute. He has attended 05 International Conferences (France. USA and Sri Lanka) and delivered Invited Lectures in 15 National Conferences and Seminars. He has worked as Chairperson and Co-Chairperson in several National Conferences. He has successfully guided 02 Ph.D. students and 03 others are working for Ph.D. degree.
Prof. Chourasia has been Nominated as Resource Person by the Department of Education, Govt. of Bihar for providing technical support to the colleges under Center of Excellence (CoE) in accreditation process. He is also working as Co-ordinator IQAC, RUSA and Nodal Officer of TNB College, Bhagalpur and Member, NAAC Steering Committee, T. M. Bhagalpur University, Bhagalpur.
Prof. Sanjay Kumar Choudhary Prof. Sanjay Kumar Choudhary has completed his M.Tech in Material Technology from RIT, Jamshedpur under Ranchi University after completing his M.Sc. in Physics. He awarded Ph.D. degree both in Applied Physics and Electronics. He has attended several National Level Seminars, Symposia, Workshops and has large number of quality research articles to his credit. He started his career as a faculty member in the department of Applied Physics in B.I.T. Mesra, Ranchi and was associated with an ISRO Project related to Plasma Science & Technology (Surface Coating). He became Principal, first in the year 2007 under U.P. Technical University and then became permanent Principal in Jai Prakash University, Chhapra in the year 2009. In the year 2015, because of Inter University Transfer his service was transferred to T.M. Bhagalpur University. And at present he is the Principal of T.N.B. College, Bhagalpur
