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Chapter 7

The Role of Indigenous Knowledge in Biological Control of Plant Pathogens: Logistics of New Research Initiatives Arun Kumar and A.K. Purohit

Abstract The Indigenous knowledge (IK) and Bio control exist in a synergy in the strategic repertory of sustainable agriculture. The IK is the systematic body of knowledge acquired by local people through the accumulation of experiences, informal experiments, and intimate understanding of the environment in a given culture. These cultural practices have sustained the farmers from the ancient times, which are often validated as modern conceptual shifts in the agricultural science. The age old farmers’ practice of planting number of different crop combinations is currently recognized as crop diversification- successful in averting diseases, and surviving during drought periods besides other advantages. Increasing use of fungi as mycoinsecticides and biocontrol agents for managing insect pests and plant diseases has opened a vast field of knowledge for studying this huge unexploited fungal resource. Many naturally occurring microorganisms have been used to control diseases. Besides fungi, induced resistance has emerged as a new strategy for managing plant diseases using Plant Growth Promoting Rhizobacteria (PGPR), leaf proteins, botanicals, animal products, organic manures and other IK materials such as ash, butter and milk. The present paper has extensively reviewed the researches made on documentation and validation of indigenous knowledge worldwide during the last decade with some convincing success stories. Attempts have also been made to reckon the logistics of new research initiatives in the wake of plateaus in agricultural production and spot marching researches on underlying mechanisms of IK and bio-control. The new insights being generated as plant neurobiology, plant intelligence, A. Kumar (*) Division of Plant Sciences and Biotechnology, Central Arid Zone Research Institute, Jodhpur, Rajasthan 342003, India e-mail: [email protected] A.K. Purohit Transcience Transactions, Jodhpur, Rajasthan 342001, India e-mail: [email protected]

J.M. Mérillon and K.G. Ramawat (eds.), Plant Defence: Biological Control, Progress in Biological Control 12, DOI 10.1007/978-94-007-1933-0_7, © Springer Science+Business Media B.V. 2012

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A. Kumar and A.K. Purohit

consciousness and genopsych have been amalgamated to underline the knock of a yet another paradigm shift in offing- the shift from integration to convergence, where simultaneous to intra- and inter-disciplinary research, trans-disciplinary research at the interface of advents in nano-technology and emergent situations of system biology, is poised to trigger a fresh sigmoid growth in research outcome pushing IDM at the threshold of converging technologies for sustainable agricultural production.

7.1

Introduction

Domestication of plants was probably the most significant turning point in the human history as agriculture replaced hunting and food collection from the purview of human activity. It is presumed that sustainable disease control practices must have evolved with the development of agriculture [1]. Indigenous knowledge (IK) is the systematic body of knowledge acquired by local people through the accumulation of experiences, informal experiments, and intimate understanding of the environment in a given culture [2]. Local people, including farmers, landless labourers, women, rural artisans, and cattle rearers, are the custodians of indigenous knowledge systems. Moreover, these people are well informed about their own situations and resources. In other words IK is tuned to the needs of local people and the quality and quantity of available resources, along with a natural system of preserving ecological balance [3]. Diversified agro-ecosystems have emerged over centuries of biological evolution, and represent the experience of farmers interacting with their environment without access to external inputs, capital, or scientific knowledge [4]. The efficiency of indigenous practices lies in the capacity to adapt to changing circumstances and recycling of natural resources. The IK is a product of experience followed by informal experimentation. It relies strongly on intuition, directly perceivable evidence, and an accumulation of historical experiences [5]. In scientific colloquium, IK is analogous to technology generation as conceived in on farm trials. The formal experiments are required for the function of technology validation. In view of the fast changing agricultural scenario, a drift has resulted from sustenance to commercial farming. To maximize the yield, farmers are using high yielding varieties and hybrids with higher inputs of chemical fertilizers and pesticides to a large extent. The newly released hybrids, indiscriminate and injudicious use of fertilizers and pesticides have resulted in susceptibility to various diseases and pests. Besides this, a number of other problems such as soil, water and air pollution, residual toxicity in fruit and vegetables, resistance to insects and pathogens, mortality of parasites, predators and pollinators, and resurgence with outbreaks of secondary pests have also cropped up. With increasing environmental awareness, the focus has now shifted towards search for viable alternatives of disease control methods. At the Earth Summit in Rio, Brazil in June 1992 and the International Movement for Ecological Agriculture meeting held in Penang, Malaysia during January 10–13, 1990 also called for natural

7 The Role of Indigenous Knowledge in Biological Control of Plant Pathogens…

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farming based on traditional experiences [6]. Amidst a web of high throughput technologies, low external input sustainable agricultural (LEISA) technologies are in great demand. Apparently, it sounds logical to once again look for traditional practices, which are ecologically non-disruptive and stable. Sustainability is a new paradigm for modern agriculture. Answering this challenge will take the form of dialectic between our understanding of available practices and our expanding knowledge of ecological relationship in agro-ecosystems. Traditional farming systems are one of the sources of ‘non-chemical’ disease management strategies. The other two sources are biotechnology and biological control [7]. The simple cultural practices such as increasing the seed rate to compensate for pest damage, adjusting the time of sowing to avoid pest damage, intercropping, trap cropping and crop rotation have been found to provide adequate protection from pest damage with no additional cost and without harmful effects on the environment. Traditional farming practices are developed by agrarian societies and traditional farming systems in particular ecological setting. Sustainability in these systems has been derived after a long tenure through trial and error with crops and their cultivation practices [8, 9]. Despite plethora of evidences on field efficacy of IK, a great resurgence of interest in biological control and inclusion of practices in Integrated Disease and Pest Management; the relative volume of literature on technology validation, especially through understanding mechanism of action is very meager. The present review, besides documenting the global scenario of interest in IK and biocontrol, attempts to anticipate, logistics of new research needs.

7.2

Disease Management in Traditional Farming Systems

Traditional farming systems have been in existence in India since the time of “Vedas” (2500–1500 B.C.) and “Upanishads” (1500–600 B.C.). The “Vrikshayurveda,” “Agnipurana,” “Brihat Smahitha” and “Arthasastra” (fourth Century B.C.) contain separate sections on Indian agriculture. These texts reveal that the ancient science of agriculture dealt with the collection and selection of seeds, germination, grafting, cutting, sowing, planting, nursing, soil selection, manuring, pest and disease management, nomenclature and taxonomy. The land and its proper utilization occupied an important position in agriculture and traditional farmers were conscious of the nature of soil and its relation to the production of specific crops [10]. However, an interesting observation has been made by Bentley [11] that farmers generally know more about plants, less about insects and still less about plant pathology. Traditional farming techniques have been widely practiced in Korea, Japan, India and China for centuries and are still in use in aboriginal communities. It is heartening to find that the United States, Holland and Australia are pioneers among the developed nations which are gradually turning to ecological agriculture. Sustainable agriculture should combine the wisdom of traditional and natural farming practices with modern technologies. Most of the practices of traditional

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farmers for disease management in developing countries consist of cultural controls, yet little information is available and utilized. Some practices of traditional farmers are altering of plant and crop architecture, biological control, burning, depth or time of planning, fallowing, flooding, mulching, multiple cropping, planting without tillage, using organic amendments, crop rotation and sanitation [12, 13]. The disease resistance of traditional cultivars selected over millennia is also most important. Landraces are usually genetically diverse and in balance with their environment and endemic pathogens. Though, not necessarily high yielding, landraces to yield some harvest even under the worst conditions. As far as sustainability of traditional practices is concerned it has been reported by Thurston [14] that most of the traditional practices are sustainable.

7.3

Traditional Farmers’ Practices for Managing Plant Diseases

Farmers’ knowledge is very broad, practical and comprehensive. Most of the cultural practices are sustaining resource-poor farmers from ancient times. There are number of examples given by Thurston [14] to illustrate this. Conklin [15] has reviewed the agricultural knowledge of mountain tribe of Mindoro in the Philippines. Likewise, Mayan Indians in Mexico and traditional farmers of Honduras have impressive knowledge of soil types; plant classification system, local agro-ecosystem and general information about plants (see [14]). India, being a very old civilization, has rich knowledge about agricultural practices and different farming systems. Efforts are being made to document that information lying in ancient literatures. Some of the dedicated groups are striving to bring out farmers’ wisdom in the form of books, journal articles and in the form of English translations of literature available in Sanskrit, Arabian, Persian and other regional languages [16–22].

7.3.1

Traditional Fungicides

The key to effective plant health management is prevention. This includes doing your homework before planting by carefully matching plants those are appropriate to the type of soil, sunlight levels, and watering conditions of the site. Once the plants are in the ground, successful plant health management relies on proper sanitation, appropriate fertilization, and necessary pruning practices. Small numbers of farmers use chemical pesticides, but a large chunk of farmers cannot afford them due to high price and toxic effects on humans, livestock and the environment, if used injudiciously. Farmers in India have been traditionally using ‘ashes’ from burning coal and wood for their crop fields. This has a number of advantages: (i) it makes the soil loose and arable; (ii) it contains potash (2.5–12%), phosphorus (1.6–4.2%) and


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nitrogen (0.5–1.9%); (iii) its application also reduces the incidence of pests like ‘thrips’ and ‘leaf blight’ bacterial disease[6]. In arid areas of western Rajasthan, India, farmers use to call the ash as ‘lichhmi’means the godess of prosperity. Farmers used to dust the ash (50–60 kg ha−1) on the growing crop of cumin (Cuminum cyminum) to ward off powdery mildew, on lucerne (Medicago sativa) to check the growth of dodder (Cuscuta sp.).

7.3.2

Cultural Practices

1. Adjusting density and spacing: It is known that dense plant populations influence plant disease resulting into epidemics [23]. Due to change in micro-climate relative humidity increases with relative uniformity in temperature. Intercropping is the common practice among the farmers knowing the importance of the role of spacing between plants and rows. Close cropping situations help in disseminating foliar pathogens along with soil-borne fungi, damping-off organisms and transmission of viruses [24, 25]. However, the reverse is true in case of groundnut (peanut) rosette disease in Africa. It has been observed that early planting and close spacing increased yield and reduced the incidence of the virus [13, 26]. 2. Multiple cropping: Planting number of different crop combinations is a common practice in the traditional farming systems. The 50–80% farmers of developing tropical countries used to do intercropping in the rain fed areas [27]. In arid areas, traditional ‘mixture’ sowing is practiced. Seeds of pearl millet are sown with rainy season legumes (mung bean, moth bean and cluster bean) and sesame in the ratio of 7:1. Besides this, some farmers also grow cucurbits (melon, Citrullus spp. etc.) in addition to legumes. This practice is used to minimize the crop losses due to onset of drought or pests and diseases along with the problem of ‘rode’ or soil crusting [28]. 3. Fallowing: This is an age-old tradition. Ancient Sumerians (now Iraq) used to practice fallowing for cereal fields [29]. It is a very successful practice for managing soil-borne fungi and nematodes. It becomes more effective when used in combination with crop rotation. It is an old traditional practice in desert areas where crops are not planted in a fallowed field for 2–4 years. However, the practice of fallowing is gradually becoming less popular among young farmers because of shrinking land resources. 4. Crop rotation: Growing economic plants in recurring succession and in defined sequence on the same land is a common practice among the farmers. This helps in managing both soil- and air-borne pathogens. Though, it is more a location specific practice but it has a lot of advantages. It helps prevent soil depletion, maintains soil fertility and reduces soil erosion and controls weeds. Crop rotation as a means to control to insect pests is most effective when the pests are present before the crop is planted have no wide range of host crops; attack only annual/ biennial crops; and do not have the ability to fly from one field to another. In some irrigated pockets of arid district of Barmer, India, which is a double

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cropped area having facilities of sprinkler irrigation, farmers are rotating the cash crop of cumin with brown mustard or raya (Brassica juncea) and wheat in the winter season and with pearl millet during rainy season to reduce the incidence of fusarial wilt [30]. 5. Flooding: The practice of flooding has been used mainly for insect and weed management [31, 32]. Farmers control leaf miner (Aproaerema modicell) of groundnut by flooding field to submerge the crop overnight and letting out water on the next morning. It has been reported that fungi, bacteria and actinomycetes decline in flooded soils. The anaerobic or near anaerobic conditions produced by flooding help in reducing many soil-borne fungi and nematodes [33]. Thurston [14] has discussed the practice at length with the benefits in suppression of plant diseases, especially in the management of fusarial wilt of bananas and other soil-borne pathogens. Farmers of Pali district in semi-arid western Rajasthan used to flood their fields even during the rains to suppress the attack of sooty mould (Leptoxyphium fumago) and Vermiculariopsiella sp. growing on leaf secretions of some insect. The farmers claimed to have successfully eliminated the disease using this practice [34, 35]. 6. Suppressive soils: The phenomenon of disease suppressive soils has fascinated plant pathologists for decades. Suppressive soils are those in which a specific pathogen does not persist despite favorable environmental conditions, the pathogen establishes but doesn’t cause disease, or disease occurs but diminishes with continuous monoculture of the same crop species. The phenomenon is believed to be biological in nature because fumigation or heat-sterilization of the soil eliminates the suppressive effect, and disease is severe if the pathogen is re-introduced. Suppressive soils are living laboratories where the complex interactions among microorganisms that result in disease suppression might someday be unraveled. Characterization of biological communities in soil has proved to be a formidable challenge, and the nature of disease-suppressive soils remains largely an enigma. Suppressive soils have nevertheless proved to be sources of some important antagonists and they continue to provide clues useful in developing biocontrol strategies [36]. Sulfur containing compounds released during the breakdown of crucifer tissues may act as soil fumigants, resulting in less disease. There are many other examples of biological control involving complex microbial communities where the mechanism of biological control is not understood. This includes the use of green manures to control soil-borne pathogens.

7.4

Biological Control Agents in Disease Management of Plants

Effective management of diseases thus becomes an absolute necessity. So far this has been achieved by the use of chemical, cultural, biological and various other techniques. However, among the various disease management strategies adopted, chemical control has emerged as the dominant strategy, and has been the cause of


a

b

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Pathogen

Pathogen

Host

Environment

Biological Agent(s)

Host Environment

Fig. 7.1 (a) Disease triangle (b) Biological control disease pyramid

mounting concern in recent years. The injudicious application of these chemicals has resulted in development of resistance in pathogens and health hazards in the users. Non-biodegradable pesticides have contaminated soil, food chain and water bodies, and in turn become a major component of environmental pollution. With increasing environmental awareness the focus has now shifted towards search for viable and sustainable alternatives of disease control methods. Amidst a web of high throughput technologies, low external input sustainable agricultural (LEISA) technologies are in demand. A number of cultural practices such as growing genetically similar crop plants in continuous monoculture and plant cultivars susceptible to pathogens, and use of nitrogenous fertilizers at concentrations that promote disease susceptibility have actually enhanced the destructive potential of diseases. In the wake of prevailing situation the concept of biological control has been re-surfaced. The multitude of methods used in biological control can broadly be divided into two groups- (1) antagonists directly introduced into plant tissue and (2) cropping conditions and other factors can be modified in ways to promote the activities of naturally occurring antagonists. The reports of direct introduction of antagonists are less frequent; however, inducing resistance in the host by inoculating non-pathogenic or avirulent strains of a pathogen has been commonly demonstrated. Antibiosis, competition and hyper-parasitism are the recognized ways of operating biological control outside the host and may be used together with existing fungicides in integrated disease management strategies to reduce the risk of building up resistance in pathogen. Occurrence of any plant disease is the consequence of the interaction among the susceptible host, virulent pathogen and the favourable environment referred as the disease triangle (Fig. 7.1a). Biological control agents interact with the components of the disease triangle to reduce the incidence of disease. The concept of biological control pyramid is formed by separating the biological control agents from the environmental component of the disease triangle (Fig. 7.1b). This biological control pyramid helps in conceptualizing the factors and their intricate interactions, which play a major role in disease control strategy.

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The term ‘Biological control’ is commonly used and includes plant disease resistance, biologically derived pesticides, crop rotation etc. Here we are using the term as disease control-mediated by an additional organism(s), which changes the result of interaction between the environment, pathogen and host. Biotechnology for crop protection, is receiving considerable attention today. Many studies are now under way to improve the crop production through genetic engineering and expression of insect and virus resistant genes and microbial pesticides. An area of agricultural biotechnology in which fungi show considerable potential for the future is the biological control of pathogenic fungi, insect pests and weeds [37]. Potential agents for biocontrol activity are rhizosphere-competent fungi and bacteria, which in addition to their antagonistic activity are capable of inducing growth responses either by controlling minor pathogens or by producing growthstimulating factors. Biological control proves to be very successful economically, and even when the method has been less successful, it still produces a benefit-to-cost ratio of 11:1. The organisms, which can be cultured with ease, have maximum potential as commercial product. Unlike the past studies, more ecologically sound approaches involving a combination of organisms is currently being used by a number of workers [38]. Increasing use of fungi as myco-insecticides and biocontrol agents for managing insect pests and plant diseases has opened a vast field of knowledge for studying this huge unexploited fungal resource [39]. The biocontrol methods, such as compost, seed bacterization, fungal biocontrol agents (Trichoderma), seed treatments, induced systemic resistance, genetic manipulation and induced resistance using pathogens and non-pathogens that seem to be useful in managing the diseases are discussed in this paper.

7.4.1

Competition

Competition occurs between microorganisms when space or nutrients (i.e. carbon, nitrogen and iron) are limiting, and its role in the biocontrol of plant pathogens has been studied for many years, with special emphasis on bacterial biocontrol agents. An important attribute of a successful rhizosphere biocontrol agent would be the ability to remain at high population density on the root surface, providing protection to the whole root for a longer period of time. Mycorrhizal fungi can also be considered to act as a sophisticated form of competition or cross-protection, decreasing the incidence of root disease.

7.4.2

Antibiosis

Antibiosis is defined as inhibition of the growth of one microorganism by another as a result of diffusion of an antibiotic. Antibiotic production is very common among soil-dwelling bacteria and fungi, and in fact many of our most widely used


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medical antibiotics (e.g., streptomycin) are made by soil microorganisms. Antibiotic production appears to be important to the survival of microorganisms through elimination of microbial competition for food sources, which are usually very limited in soil. The production of antibiotics by actinomycetes, bacteria and fungi has been adequately demonstrated in vivo. Numerous agar plate tests have been developed to detect volatile and non-volatile antibiotic production by putative biocontrol agents and to quantify their effects on pathogens. Species of Gliocladium and/or Trichoderma are well-known biological control agents that produce a range of antibiotics that are active against pathogens in vitro [40]. Within bacterial biocontrol agents several species of Pseudomonas produce antibiotics to control plant pathogens.

7.4.3

Mycoparasitism

This is parasitism of a pathogenic fungus by another fungus. It involves direct contact between the fungi resulting in death of the plant pathogen, and nutrient absorption by the parasite. Mycoparasitism occurs when one fungus exists in intimate association with another from which it derives some or all its nutrients while conferring no benefit in return. Biotrophic mycoparasites have a persistent contact with living cells, whereas necrotrophic mycoparasites kill the host cells, often in advance of contact and penetration. Mycoparasitism is a commonly observed phenomenon in vitro and in vivo, and its mode of action and involvement in biological disease control has been reviewed. The most common example of mycoparasitism is that of Trichoderma spp., which attack a great variety of phytopathogenic fungi responsible for the most important diseases, suffered by crops of major economic importance worldwide.

7.4.4

Control of Insect Pest

Over 400 species of fungi attack insects and mites, so there is great potential for the use of these organisms as biological insecticides. As insect biocontrol agents, fungi are markedly superior to other microorganisms because they are generally non-specific in their action and are useful against a wide range of insect pests. Most of these entomopathogenic fungi belong to the classes’ Phycomycetes and Deuteromycetes of division Mycophyta (Table 7.1). Spores of these fungi attack the external or gut cuticle of their insect hosts. Death may result from the production of a toxin secreted by the fungus or following the direct utilization of the body fluids. Insecticidal toxins produced by fungi are non-enzymic in nature having low molecular weight, which can kill insects when present even at low concentrations. The best examples of the use of fungi to control insects are provided by species of Beauveria and Metarhizium. Almost all the acridid pests are highly susceptible to

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Table 7.1 Principal Deuteromycetes fungal species for biocontrol of insects

Species Aschersonia aleyrodis Beauveria bassiana Beauveria brongniartii Hirsutella thompsonii Metarhizium anisopliae Nomuraea rileyi Verticillium lecanii

Target pests Whiteflies Colorado beetle Cockchafers Rust mites Beetles, bugs, grasshoppers, Caterpillars Aphids, whiteflies

the fungus requiring about 1,000 spores or even less to infect and kill 50% of a population in 10 days at 28°C. Metarhizium is promising as a mycoinsecticide for use against locusts and grasshoppers. Constant temperatures between 20°C and 35°C (optimum 28–30°C.) facilitate the fungal infestation of these insects. Fungus is effective under field conditions when sprayed at a rate of 1–5 × 1012 conidia ha−1 using an oil-based ULV spray or an oil/water emulsion using a boom sprayer. There is great commercial interest in developing a product for the biocontrol of locust and grasshopper.

7.4.5

Fungal Metabolites

Biologically active secondary fungal metabolites produced are not only being evaluated as potential pesticides but also for controlling plant growth. These compounds have the advantage over conventional pesticides in being effective at very low concentrations while proving essentially non-persistent and harmless to the environment.

7.4.6

Mycorrhizal

Mycorrhiza are symbiotic associations between soil fungi and higher plants. There are around 150 species in Zygomycotina having obligate symbiotic association with agricultural crops. These associations are known to produce growth promoters and induce resistance to plants against different pathogens. It was soon recognized that mycorrhizal association could often greatly increase the rate of uptake of nutrients such as nitrogen and phosphorus from nutrient-deficient soils. This has led to the view that the inoculation of mycorrhizal fungi in soils should lead to an increase in the uptake of these essential plant nutrients. Two types of mycorrhiza have been recognized, the endotropic or vesicular-arbuscular mycorrhiza (VAM), and the ectotrophic type. In VAM the fungal partner is restricted to the cells of the plant cortex where it grows within and without the cells, invading the host cells at intervals to form a dichotomously branched structure called the arbuscle, thought to be the site of nutrient exchange between plant and fungus. The fungal partner


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appears to have no independent existence is soil. Neither is the interaction specific, since a single species of fungus can infect a wide range of plant, including most crop species. In ectotrophic mycorrhizas, the fungal partner forms a tight sheath around the plant root and from this sheath hyphae grow into the outer cortex to form a network called the Hartig net. Ectotrophic mycorrhizas, unlike VAM, tend to be non-specific. Vesicular-arbuscular mycorrhizas can directly enhance the uptake by plants of essential nutrients such as phosphorus, copper and iron on the other hand; zinc and manganese uptake may be reduced. Therefore, mycorrhizal associations protect some plants from the toxic effects of these elements. Ectotrophic mycorrhizas also show enhanced uptake of phosphorus, and by mineralizing organic nitrogen facilitate availability of nitrogen to the plant. They may also protect their plant hosts from heavy metals and attack by pathogens, and they also help increase the uptake of water from soil to plants.

7.5

Biocontrol of Airborne Diseases

Many naturally occurring microorganisms have been used to control diseases on the aerial surfaces of plants. The most common bacterial species that have been used for the control of diseases in the phylloshpere include Pseudomonas syringae; P. fluorescens, P. cepacia, Erwinia herbicola, and Bacillus subtilis. Fungal genera that have been used for the control of air borne diseases include Trichoderma, Ampelomyces, and the yeasts Tilletiopsis and Sporobolomyces. Wan and Tian [41] studied the effect of ammonium molybdate (NH4Mo) as an additive to improve biocontrol efficacy of antagonistic yeasts Rhodotorula glutinis and the use of NH4Mo is a practical approach to improve the efficacy of R. glutinis for post harvest disease control. Phytopathogenic bacteria possess several genes that encode phenotypes that allow them to parasitize plants and overcome defense responses elicited by the plant. In addition, phytopathogenic bacteria possess pathogenicity genes like hrp. Isogenic avirulent mutants can be produced by insertional inactivation of genes involved in pathogenicity. Antibiosis has been proposed as the mechanism of control of several bacterial and fungal diseases in the phyllosphere. Recently, the advances of plant and plant growth promoting bacterial (PGPB) interaction research focusing on the principles and mechanisms of action of PGPB (both free living and endophytic bacteria) and their potential use in biological control of plant diseases is reviewed by Compant et al. [42]. Molecular biological techniques could be used to enhance the efficacy of biocontrol agents that use antibiosis as a mode of action. Biocontrol agents must normally achieve a high population in the phyllosphere to control other strains, but colonization by the agent may be reduced by competition with the indigenous microflora. Integration of chemical pesticides and biocontrol agents has been reported with Trichoderma spp. and P. syringae. Biocontrol agents tolerant to specific pesticides could be constructed using molecular techniques.

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7.6


Biocontrol of Soil-Borne Diseases

Chemical control of soil-borne plant diseases is frequently ineffective because of the physical and chemical heterogeneity of the soil, which may prevent effective concentrations of the chemical from reaching the pathogen. Enrichment, conservation and management of microorganisms have been extensively used for the biological control of soil borne plant diseases as well as for promoting plant growth. Fluorescent pseudomonades are the most frequently used bacteria for biological control and plant growth promotion, but the species of Bacillus and Streptomyces have also been used commonly. Competition as a mechanism of biological control has been exploited with soil borne plant pathogens as with the pathogens on the phylloplane. Naturally occurring nonpathogenic strains of Fusarium oxysporum have been used to control wilt diseases caused by pathogenic Fusarium spp. Molecular techniques have been used to remove various deleterious traits of soil borne phytopathogenic bacteria to construct a competitive antagonist of the pathogen. Molecular techniques have also facilitated the introduction of beneficial traits into rhizosphere competent organisms to produce potential biocontrol agents. Chitin and E – (1, 3)-glucan are the two major structural components of many plant pathogenic fungi, except by oomycetes, which contain cellulose in their cell wall and no appreciable levels of chitin. Biological control of some soil borne fungal diseases has been correlated with chitinase production, bacteria producing chitinases or glucanases exhibit antagonism in vitro against fungi. A recombinant Escherichia coli expressing the chi A gene from Sclerotium marcescens was effective in reducing disease incidence caused by Sclerotium rolfsii and Rhizoctonia solani. In other studies, chitinase genes from S. marcescens have been expressed in Pseudomonas spp. and the plant symbiont Rhizobium meliloti. Shahnaz et al. [43] have reported biological control of soil borne, root-infecting fungi (Fusarium spp., Macrophomina phaseolina and Rhizoctonia solani) on mung bean and okra using strains of Rhizobium and Bradyrhizobium spp. All rhizobial treatments were effective in controlling the soil borne fungi on these plants. The rhizobial strains also increased nodulation as well as shoot and root growth of treated plants. The effectiveness of certain on-farm weeds as soil amendments was ascertained against Macrophomina phaseolina, a soil-borne pathogen causing dry root rot of crops grown under rainfed conditions in arid regions. Mawar and Lodha [44] have reported significant reductions in the population of M. phaseolina with the weed residues. Accordingly, Celosia and Euphorbia residues completely eradicated viable propagules of M. phaseolina. Bio-agents and neem based seed treatment for management of root-rot complex in cluster bean has been studied by Jatav and Mathur [45]. They have observed maximum suppression of Fusarium solani by Bacillus subtilis. However, Rhizoctonia solani was successfully managed by Trichoderma harzianum. It was noted that for R. solani fungal biological control agents were more effective, whereas bacterial antagonists were effective against F. solani.


7.6.1

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Soil Solarization

This seems to be one of the field technologies directly related to the field of biological control of soil borne pathogens. A number of studies have been carried out towards managing soil borne pathogens. The effects of soil solarization, residue incorporation, summer irrigation and biocontrol agents on survival of Macrophomina phaseolina have been worked out in the past. Results suggest that in hot arid regions use of Brassica residues can be a practical and feasible substitute for polyethylene mulching in managing soil-borne diseases [46].

7.6.2

Trichoderma – An Environment-Friendly Biocontrol Agent

Species of Trichoderma are one of the small groups of beneficial fungi, which have proven commercially viable as a biological control agent. This micro-organism is now registered as bio-fungicide in India, France, UK, Switzerland, Sweden, Belgium, Chile, New Zealand and the USA, and regulations are pending in several other countries. Trichoderma is completely safe for humans and livestock. Although, it is commonly considered as a contaminant that may cause infections in presence of certain predisposing factors, but in 55 years of research there has been no account of recorded adverse reaction. The predatory qualities of Trichoderma are a big part of the appeal of this fungus along with other associated benefits for commercial applications. The thought of biological control of plant pathogens by mycoparasites (hyperparasites) dates back to Weindling [47]. He discovered that Trichoderma lignorum would parasitize a number of soil borne fungi in culture and suggested controlling certain pathogenic fungi by augmenting soil with an abundance of this mycoparasite. Comprehensive reviews on the subject have been published in the past showing the production of chitinases to break down the mycelial cell walls of fungal plant pathogens as a major cause of biocontrol activity [48–51]. Description and Natural Habitats – Trichoderma is a filamentous fungus that is widely distributed in the soil, plant material, decaying vegetation, and wood. Hypocrea spp. are the teleomorph of some of the Trichoderma species. Trichoderma thrives in the leaf litter or mulch, and it requires a minimum organic carbon level of 1% to ensure proliferation in cropping locations. This species is a myco-parasite or saprophyte, which feeds on pathogenic fungi. There are large number of photographic evidences highlighting this phenomenon where Trichoderma are seen actively parasitizing several group of plant pathogens. Species – The genus Trichoderma has five major species utilized in biocontrol of plant diseases viz. T. harzianum, T. koningii, T. longibrachiatum, T. pseudokoningii, and T. viride. Morphological features of the conidia and phialides help in differentiation of these species from each other.

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Cultural Features – Colonies of Trichoderma grow rapidly and mature within 5 days at 25°C. Its colonies develop as wooly and compact mycelium on potato dextrose agar (PDA) medium. At the time of sporulation scattered blue-green or yellow-green patches are formed. These patches may form concentric rings. They are more readily visible on PDA in comparison to Sabouraud dextrose agar. The fungal growth is pale, tan, or yellowish in colour on the reverse side of cultures. Microscopic Features – Septate, hyaline hyphae, conidiophores, phialides, and conidia are observed. Some of the species like Trichoderma longibrachiatum and T. viride also produce chlamydospores. Conidiophores are hyaline, branched, and occasionally display a pyramidal arrangement. Phialides are hyaline, flaskshaped, and inflated at the base. They are attached to the conidiophores at right angles. The phialides may be solitary or arranged in clusters. Conidia (3 Pm in diameter) are one-celled and round or ellipsoidal in shape. They are smooth- or rough-walled and grouped in sticky heads at the tips of the phialides. These clusters frequently get disrupted during routine slide preparation procedure for microscopic examination. The color of the conidia is mostly green. Screening of strains can be conducted in four ways: (1) selection of active strains in relation to plant pathogens (2) screening isolate/s which have high biotechnological indexes (3) analysis of pathogen properties for plant, useful insects, animals and peoples (4) search of low economic value substrates which are convenient for cultivation and saving of spores’ activities. For developing effective biocontrol agent to combat damping-off in nurseries, we investigated fungal strains in the genus Trichoderma that was isolated from soil and fruiting bodies of Ganoderma lucidum [51, 52]. Pathogen Interaction – Mycoparasitism is a complex process, which include several successive steps. The interaction of Trichoderma with its host is specific. Trichoderma spp. have been extensively studied as biocontrol agents [53]. The first detectable interaction shows that the hyphae of the mycoparasite grow directly towards its host. This phenomenon appears a chemotropic growth of Trichoderma in response to some stimuli in the host’s hyphae or toward a gradient of chemicals produces by the host. When the mycoparasite reaches the host, its hyphae often coil around it or are attached to it by forming hook like structures. In this respect, production of appressoria at the tips of short branches has been described for T. hamatum and T. harzianum. The possible role of agglutinins in the recognition process determining the fungal specificity has been recently examined. Indeed, recognition between T. harzianum and two of its major hosts, R. solani and S. rolfsii, was controlled by two different lectins present on the host hyphae. R. solani carries a lectin that binds to galactose and fructose residues on the Trichoderma cell walls. This lectin agglutinates conidia of a mycoparasitic strain of T. harzianum, but did not agglutinate the non-parasitic strains. This agglutinin may play a role in prey recognition by the predator Moreover, because it does not distinguish among biological variants of the pathogen, it enables the Trichoderma species to attack


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different R. solani isolates. D-glucose mannose residues inhibited the activity of a second lectin isolated from S. rolfsii, apparently present on the cell walls of T. harzianum. Following these interactions the mycoparasite sometime penetrates into the host mycelium, apparently by partially degrading its cell wall. Microscopic observations led to the suggestion that Trichoderma spp. produce and secrete mycolytic enzymes responsible for the partial degradation of the host’s cell wall. The complexion and diversity of the chitinolitic system of T. harzianum involves the complementary modes of action of six enzymes, all of which might be required for maximum efficiency against a broad spectrum of chitin-containing plant pathogenic fungi. The level of hydrolytic enzymes produced differs from host-parasite interaction analyzed. This phenomenon correlates with the ability of each Trichoderma isolate to control a specific pathogen. It is considered that Mycoparasitism is one of the main mechanisms involved in the antagonism of Trichoderma as a biocontrol agent along with chemotropic growth, secretion of extra cellular enzymes and lyses of host. Thus, the biocontrol ability of Trichoderma is most likely conferred by a number of mechanisms [40]. Efficacy of the culture filtrates of different species of Trichoderma against the powdery mildew (Leveillula taurica) of cluster bean has revealed that T. viride effectively managed powdery mildew, while T. harzianum recorded the highest yield with percent increase in yield over the control [54, 55].

7.7

Induced Resistance for Plant Disease Control

Induced resistance (IR) is a new strategy for managing plant diseases. It is an alternative procedure to protect plants against disease by activating plants’ own defense mechanisms using specific biotic or abiotic elicitors [56]. The basic tenet of IR lies in enhancing resistance in response to an extrinsic stimulus without altering the genome. The protection is based on the stimulation of defense mechanisms by metabolic changes that enable the plants to defend themselves more efficiently. A number of publications with different host-parasite systems have proven the efficacy of IR against fungi, bacteria and viruses through the manipulation of the host plant’s physical and biochemical properties [57–59]. The elicitors secreted through bio-agents are non-specific and therefore, can be effective against a wide range of pathogens. These elicitors work by bringing about certain metabolic changes in plants to fight against infections. The landmark studies on the development of the classic Systemic Acquired Resistance (SAR) models were conducted during the 1980s in plants, such as common bean (Phaseolus vulgaris L.) and Arabidopsis thaliana (L.) Heynh, demonstrating that SAR was conserved across diverse plant families and was effective against a broad range of viral, bacterial, and fungal pathogens [60]. Additional interests in the biological control of soil borne diseases of plants led to the unexpected discovery of another form of induced

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resistance associated with the colonization of plant roots by certain plant growth promoting rhizobacteria (PGPR), referred to as induced systemic resistance (ISR) [58]. ISR is distinct from SAR in several types of physiological and biochemical phenotypes that are best defined in A. thaliana. Results of laboratory and field studies show that, like SAR, ISR is effective against a broad range of diseases caused by viruses, bacteria, and fungi [61–65]. It is likely that other forms of induced resistance exist that vary in their reliance on salicylic acid, ethylene, and Jasmonic acid and other as yet discovered plant regulators. However, it is the availability of chemical inducers of SAR, such as BTH, and the characterization of numerous PGPR strains, that makes the applied use of induced resistance in conventional agriculture a reality. Besides these agents, integration of these bio-agents with indigenous knowledge is also developing in modern times as a logical strategy to manage plant diseases [66]. Milk has been demonstrated to effectively control powdery mildew [67], downy mildew [66] and leaf curl virus [59, 66]. Field experiments involving the effects of INA (2, 6-dichloro-isonicotinic acid) and BTH (benzo-1, 2, 3 thiadiazole7-carbothioic acid) on diseases of legumes have been reported [68–71]. Reduced densities of uredinia of the rust fungus, Uromyces appendiculatus, on trifoliolates of common bean were obtained when INA was applied at least 7 days before inoculation, but not at 2 h before inoculation [71]. An additional application of INA during pod-set did not improve resistance of common bean plants to U. appendiculatus, as opposed to a single application to the first trifoliolate [71]. Repeated applications of INA to field-grown soybean (Glycine max) partially reduced symptoms of white mold caused by Sclerotinia sclerotiorum in field trials. The INA was most efficacious in suppressing white mold on the susceptible cultivars.

7.8

The Use of Composts in Plant Disease Control

Research on natural suppression of fungal plant pathogens has significantly increased worldwide during the last decade. The use of complex organic substrates has been shown to be effective in protecting plant health. Composted organic material such as plant debris and animal manure has been used from a very long time to improve fertility. It is known that there is a close connection between soil borne plant disease occurrences and the organic matter content in the soil. The importance of composted organic material in suppressing soil borne pathogens has often been documented. Stimulation of antagonistic microorganisms in the rhizosphere or induced defense reactions in the host plant tissue is considered responsible for the beneficial effects. In general, three approaches have been taken to use organic amendments for biological control: (1) compost amendments added to the soil to suppress powdery mildews; (2) seed treatment to suppress damping-off of seedlings; and (3) foliar application of liquid extracts from compost to suppress foliar diseases. Lodha and Burman [72] applied soil amendment of pearl millet and weed composts for higher seed yield of cluster bean and cowpea.


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Trichoderma is more commonly found living in the soil rather than in plant tissues. More than 200 strains of the organism have been identified to date, the majority of which are soil-dwellers. The species produce spores at a tremendous rate, rapidly colonizing the growing areas. Maintenance of adequate organic carbon levels is necessary as carbon is the home base for all beneficial microbes. Commercial products can simply be boom-sprayed or irrigated into the soil, but the best carrier is the compost. Good compost contains high humus and billions of microorganisms, some of which provide metabolites necessary for the proliferation of Trichoderma. The Trichoderma-inoculated compost provides huge numbers of thriving fungal protectors, set up in an organic carbon home-base, which help in ensuring their successful colonization.

7.9

Validating People’s Knowledge: Some Case Studies

Practically sound and encouraging results were recorded when validation of the use of milk against major viral and fungal diseases of arid zone crops were made at Central Arid Zone Research Institute (CAZRI), Jodhpur. These results are discussed here as case studies of using IK practice of raw cow milk with species of farmer-friendly fungi Trichoderma spp. and Gliocladium virens.

7.9.1

Effect of Raw Cow Milk and Gliocladium virens Against Downy Mildew of Pearl Millet

Downy mildew (DM) of pearl millet is the most important disease caused by Sclerospora graminicola (Sacc.) Shroet. occurring in all the millet cultivating tracts of India. Symptoms of the disease appear on ear head with all possible degrees of proliferations and malformations. In malformation the florets are converted into leafy structures of diverse appearance (Fig. 7.2). Systemic symptoms generally appear on the second leaf in the form of chlorosis at the base of infected leaves followed by production of sporulation on the lower side of leaves known as the ‘half-leaf’ symptom (Fig. 7.3). The disease has caused considerable yield losses, and several single-cross F1 hybrid cultivars of pearl millet have been withdrawn during last 35 years because of high susceptibility to DM [73]. There seems to be a continuous struggle between millet breeders and rapidly evolving races or pathotypes of DM pathogen. In a recent field survey conducted in Rajasthan, the higher DM incidence (up to 78%) was recorded on pearl millet hybrids [74]. Pre-sowing treatment of seed with systemic fungicides are commonly used technologies to manage the disease [75]. However, the lack of durable resistance, existence of pathogenic variability, and concerns about fungicide resistance has limited the potential of such strategies for managing the disease. With increasing concern regarding environmental protection

178 Fig. 7.2 Green ear affected lower half of the panicle

Fig. 7.3 Leaf showing downy growth on lower surface



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and human health, the use of biological control as an alternative, environment-friendly means for the management of fungal diseases has attracted extensive attention and been considered as a potential strategy for plant disease management in recent years. An alternative procedure to protect plants against disease is to activate their own defense mechanisms by specific biotic or abiotic elicitors [76]. Biocontrol agents have emerged as a new strategy of managing plant diseases by inducing systemic resistance (ISR) in plants against diseases. A number of publications with different host-parasite systems have proven the efficacy of IR against fungi, bacteria and viruses through the manipulation of the host plant’s physical and biochemical properties [58, 77]. The emerging paradigm of sustainability in agriculture strives to amalgamate modern technology with traditional farming wisdom. Reports are available on the effectiveness of milk as abiotic inducer of resistance in susceptible plants [39, 67, 78–80]. Studies undertaken to manage DM in rainfed crop of pearl millet using eco-friendly approach employing biocontrol agents such as raw cow milk (RCM) together with Gliocladium virens as seed and soil treatments provided encouraging results with 72.9% protection over control [66]. In spite of the intriguing capacity of RCM and Trichoderma spp. to confer protection against a gamut of diseases [39, 78], very little information is available. Therefore, our major objective was to explore the ability of RCM and G. virens to protect pearl millet against DM disease. The fact that RCM and Trichoderma successfully protect pearl millet against DM [66] indicated that these agents might facilitate defense response in pearl millet against DM disease. It has been demonstrated that defense related enzymes have been involved in resistance against pearl milletdowny mildew interaction, and that these enzymes act as biochemical markers for induction of pearl millet downy mildew resistance [75, 81]. In the present study the effects of raw cow milk and G. virens were examined on the possible induction of defense-related metabolites and enzymes for their ability to induce downy mildew disease resistance by seed treatment in pearl millet together with application of G. virens mixed with FYM in soil. A number of chemical compounds and microorganisms (Biocontrol agents or BCAs) are reported to induce resistance against plant diseases [82]. However, so far there has been no report on induction of resistance by raw cow milk and Gliocladium against plant diseases. In this study, an attempt was made to analyze changes in a number of key plant biochemical parameters for biocontrol treated and untreated (control) pearl millet plants to correlate those changes with the resistance induced in the treated plants. The chlorophyll a, b, total chlorophyll and carotenoids contents were evaluated. The concentrations of all pigments were reduced in control leaves when compared with the leaves of treated plants. As shown in Table 7.2, the chlorophyll a, b and total chlorophyll in treated plants were observed higher by 22%; 59% and 31%, respectively in healthy leaves of treated plants. Results showed that in the diseased leaves of treated plants the level of chlorophyll a, b and carotenoids was much higher with 76% increase in chlorophyll a; 141% in chlorophyll b, 90% in total chlorophyll and 106% in carotenoids in comparison to the healthy and diseased leaves of control plants (Table 7.2).

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Table 7.2 Effect of biocontrol agents on photosynthetic pigments of healthy and downy mildew diseased pearl millet plant leaves Treated leavesa Untreated leaves −1 Pigments (mg dry wt.) Healthy Diseased Healthy Diseased LSD (P d 0.01) 3.65 1.46 0.231 Chlorophyll a 4.45 (+21.9)b 2.57 (+76.0) Chlorophyll b 1.72 (+59.2) 0.94 (+141.0) 1.08 1.46 0.195 Total chlorophyll 6.18 (30.6) 3.52 (+90.2) 4.73 1.85 0.517 Carotenoids 1.36 (+47.8) 0.70 (+105.8) 0.92 0.34 0.305 a Combination of seed treatment of RCM (1:1, i.e. RCM diluted to 50% by adding water) and G. virens (0.6%) with soil application of G. virens (10 g m−2) b Figures in the parenthesis are % changes in treatment over untreated control

Table 7.3 Effect of biocontrol agents on some metabolite in the treated and downy mildew diseased pearl millet plant leaves Treated leavesa Untreated leaves Metabolite Healthy Diseased Healthy Diseased LSD (P d 0.01) 6.36 (+10.0) 4.65 5.78 0.592 Total phenol 4.73 (+1.72)b Ortho-dihydroxy 0.58 (+13.7) 1.16 (+54.6) 0.51 0.75 0.427 phenol (OD) Free proline 1049.8 (−49.6) 1174.9 (−42.8) 1967.7 2057.2 107.51 Free amino acids 2.08 (−10.7) 2.29 (−18.2) 2.33 2.80 0.192 (Pg g−1 dry wt.) Total soluble sugars 47.10 (−32.1) 60.68 (−4.48) 69.41 63.53 3.128 a Combination of seed treatment of RCM (1:1, i.e. RCM diluted to 50% by adding water) and G. virens (0.6%) with soil application of G. virens (10 g m−2) b Figures in the parenthesis are % changes in treatment over untreated control

Phenolics are substances that are involved in plant-pathogen interactions. Therefore, the contents of total soluble phenols and O-dihydroxy phenol (ODP) were determined in the soluble fraction. The total phenolic content showed increase in healthy (2%) and diseased leaves (10%) of treated plants when compared with that of healthy and diseased leaves of control plants. Likewise, ODP contents exhibited 14% and 55% increase over untreated healthy and diseased leaves, respectively (Table 7.3). Results indicated (Table 7.3) that free amino acids reduced by around 11% in healthy and about 18% in the diseased leaves of treated plants. Similarly, free proline contents were also considerably decreased in treated healthy (47%) and diseased (43%) leaves. Free amino acids are important indicators of the plant conditions, arising as a consequence of protein degradation in tissues under programmed cell death or senescence [83]. Amino acid proline has an important role in physiological and pathological stress in plants [84]. Since little information is available in literature about the role of proline in inducing resistance in plants at the biochemical level [85],


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Table 7.4 Effect of biocontrol agents on defense-related enzymes in the treated and untreated leaves of pearl millet in relation to downy mildew disease Plant part a −1 Leaves (treated)a Leaves (control) Enzyme (OD) min mg protein) LSD (P d 0.01) Healthy Diseased Healthy Diseased Polyphenol Oxidase (PPO) 0.0054 0.0129 0.0019 0.0101 0.0010 Peroxidase (POX) 6.449 8.037 4.591 5.577 0.7159 Catalase (CA) 0.1075 0.3362 0.0583 0.1762 0.0497 Soluble protein (SP) 24.307 19.82 38.089 22.967 2.6910 a Combination of seed treatment of RCM (1:1, i.e. RCM diluted to 50% by adding water) and G. virens (0.6%) with soil application of G. virens (10 g m−2)

evaluation of endogenous proline content in the leaves of treated and control plants revealed that free proline content were reduced by 47% in the healthy and 43% in diseased leaves of treated plants in comparison to the corresponding healthy and diseased leaves of control plants (Table 7.3). This suggests that the leaf tissues in control plants are under senescence. Results revealed that the levels of the enzymes were considerably higher in treated plants than in water-treated control plants. High activity of PPO was recorded in both healthy (184.2%) and diseased (27.72%) leaves of RCM and G. virens (BCAs) treated plants when compared to the corresponding control plants. However, the low PPO activity (58.13%) was recorded in healthy leaves when compared to the diseased ones in treated plants. The same was also found true in case of control plants. Peroxidase (POX) activity was also increased (28.8%) in healthy and diseased (27.7%) leaves of BCAs treated plants. Interestingly, the catalase (CA) activity was higher in healthy and diseased leaves of the BCAs treated plants by 45.7% and 47.5%, respectively. However, soluble proteins were decreased in the treated plants in comparison to the control ones (Table 7.4). Eco-friendly disease resistance strategies are major components of modern, sustainable agriculture. Induced resistance has emerged as a potential alternative and a complementary strategy for crop protection, which signifies the control of pathogens and pests by prior activation of plants’ innate defense pathways. As milk is not a potential environmental or food contaminant; consequently it can be used in organic agriculture. In India, farmers had the tradition of using milk in managing plant diseases. Milk is known to boost immune systems in the plants and the management of several diseases caused by fungi Sphaerotheca fuliginea [32, 78], and effects of RCM seed treatment together with seed and soil treatments with Gliocladium virens on downy mildew disease of pearl millet are also reported [66]. In this study, an attempt was made systematically to analyze changes in a number of key plant biochemical parameters. The key symptom of DM development is the lighter green colour. This colour change of the DM infected leaves could indicate alterations in plastid metabolism. During the disease process, a

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decrease in chlorophyll b levels was observed, which was followed by decreases in chlorophyll a and the carotenoids levels. This decrease probably leads to a reduction in photosynthesis, previously reported for T. cacao infected with C. perniciosa [86]. A number of possible biochemical connections for this phenomenon can be visualized. Furthermore, the reduced photosynthesis could be a negative feedback response to the augmented levels of soluble sugars in the infected tissue. In plants, sugars can work directly as gene regulation signals, attenuating the expression of several plastid-localized nuclear genes required for normal chloroplast development [87], and their presence could reduce the need for photosynthesis and, therefore, the need of pigment synthesis [88, 89]. The high levels of sucrose and glucose in the infected tissues have been observed previously in other biotrophic pathosystems [90]. This study corroborates those previous findings and found that diseased control plants had a significant increase in soluble sugar concentrations when compared with the treated ones. Moreover, the decreases in the chlorophyll concentrations during senescence has been demonstrated to be followed by increases in the concentration of soluble sugars and starch [91], which are somewhat similar to characteristics found in this study. Taken together, the observed biochemical alterations associated with the infection suggest that the plant uses unspecific mechanisms to try to eliminate the fungus, such as an increase in phenolics. However, these mechanisms seem not to be sufficient to avoid the disease suggests that a cascade of events has been triggered to cause the death of the infected organ. Induction of resistance has been measured by using biochemical markers in the form of induction of defense related enzymes that are activated upon pathogen infection. In the present study, we report the involvement of PPO, POX and catalase during the pearl millet and downy mildew disease interaction. A number of previous studies have shown that enhanced enzyme content of POX, PPO and catalase along with decreased soluble protein is associated with induced resistance against a broad range of pathogens [92–94]. An increase in POX, PPO and CA with decrease in soluble proteins induced by RCM and G. virens may be facilitating pearl millet seedlings to prevent the invasion by pathogen. Similar results were observed in studies carried out on Norway spruce (Picea abies) upon infection with Pythium dimorphum [95]. They showed an increased peroxidase activity in infected roots. Effective DM management requires a definite reduction in primary inoculum from seed and soil. On this count, Gliocladium virens appeared to have grown readily along with the developing root system of the treated plant and protects the roots from initial infection (Fig. 7.4). There is a long tradition of indigenous innovations involving prophylactic use of milk and its derivative for controlling diseases in plants as well as animals in India. In spite of awareness about the hazardous effects of chemical pesticides in the developed countries, recommendations to use milk in controlling diseases are few. The question arises as to whether simple innovations are to be ignored for the fact that they are uncomplicated. Since pearl millet is a crop of low economic value grown by resource-poor farmers, seed treatment with biocontrol agents is a more viable and less expensive option than spraying of fungicides for control of DM. There is a high risk of the pathogen developing resistance that is associated with the


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Fig. 7.4 Growth of bio-control fungus G. virens emerging out from the isolated root of G. virens treated pearl millet plants on PDA medium

use of chemical fungicides unlike biocontrol agents. As a treatment option RCM and G. virens are very promising for pearl millet downy mildew disease management by seed treatment which is economical and environment-friendly. These treatments, apart from their action against pearl millet downy mildew disease, are good plant growth promoters, which is an added advantage for advantageous cultivation of pearl millet.

7.9.2

Effect of Raw Cow Milk and Trichoderma Induced Protection Against Leaf Curl Disease of Chilli

With increasing concern regarding environmental protection and human health, the use of biological control as an alternative, environmentally friendly means for the management of diseases has attracted extensive attention and been considered as a potential strategy for plant disease management in recent years. Bio-control agents (BCAs) have emerged as a new strategy of managing plant diseases by inducing systemic resistance (ISR) in plants against diseases. The emerging paradigm of sustainability in agriculture strives to amalgamate modern technology with traditional farming wisdom. Reports are available on the effectiveness of milk as abiotic inducer of resistance in susceptible plants [78].

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Fig. 7.5 Leaf curl disease affected chilli plant

Fig. 7.6 RCM and Trichoderma treated plant (Left) and control plant

In order to assess efficacy of a bio-management strategy for leaf curl disease (LCD) of chilli (Fig. 7.5) extensive ‘on-farm’ experiments were conducted in farmers’ fields of Mathania, Narwa and Manai villages of Jodhpur district in western Rajasthan. Chilli seeds were treated with raw cow milk (RCM) for 24 h in 1:1 ratio


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(i.e. RCM diluted to 50% by adding water) at the room temperature (30 ± 2°C) and Trichoderma viride (6 g Kg−1 seed) and T. viride (10 g m−2) in nursery soil followed by dipping of nursery-raised saplings in RCM (15%) for 20 min before transplantation. After 20-days of transplanting the plants were sprayed with RCM (15%) for four times at 15 days interval. The farmers’ practice (FP) was treated as control. Treatment of bio-control agents was found superior over FP in all the trials providing about 17–65% (mean 48.4%) protection over FP (Fig. 7.6). Yield attributes like plant height, root length, number of branches plant−1, number of fruits plant−1, fruit size, fruit weight and fruit yield plot−1 showed an increase when compared to FP. Besides reduced incidence of LCD and yield attributes, the net monitory return was more (Rs. 8,849 ha−1) in the treatment of bio-agents (RCM and T. viride) in comparison to the FP with benefit: cost (B: C) ratio of 1.68: 1.31 in the treatment and FP, respectively [96]. The protection is based on the stimulation of defense mechanisms by metabolic changes along with increase in defense related enzymes such as polyphenol oxidase and peroxidase that enabled the plants to defend themselves more efficiently against LCD virus. Treatment Flow Chart for Managing Leaf Curl Disease in Chilli Seed Treatment ȣ Treated chilli seeds with raw cow milk (RCM) for 24 hrs ȣ After drying in shade ȣ Treated dried seeds with Trichoderma viride (6 g kg−1 seed) ȣ Soil Treatment in Nursery Treated nursery soil with Trichoderma viride (10 g m−2) mixed with FYM ȣ Sowing of treated seed in nursery ȣ Dipping plant roots in RCM (15%) for 10 min before transplanting ȣ RCM (15%) sprayed on the plants after 20 days of transplantation

7.9.3

Rejuvenation of Ganoderma Affected Prosopis Trees Through Biocontrol Agents (BCAs)

Khejri (Prosopis cineraria (L.) Druce) is a drought hardy and multipurpose tree of semi arid areas. This is highly valued tree of desert ecosystem as renewable source of energy and biomass. It is used as food (vegetable, dry fruit), feed and rich source of fuel. It also enriches soil and improves the growth of various arid zone crops.

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It is available in abundance in protected agriculture lands because of excellent eco-friendly nature. It is therefore termed as a backbone of rural economy and has become an integral part of traditional agro-forestry system. It can grow on a variety of soils but preferably on deep sandy loam with availability of moisture. The tree is so hardy that it can survive even under dry (less than 100 mm annual rain fall) and harsh climatic conditions (temperature as high as 48°C). Recently, heavy mortality of these trees has been reported from various parts of Rajasthan and has caused serious concern. Khejri is a ‘State Tree of Rajasthan’ and its sudden death has caused worries among the farmers, environmentalists and scientists. The mortality of adult trees was found as high as (5%) in Nagaur, Jhunjhunu, Jodhpur, Churu, Sikar and Jaipur districts of Rajasthan in India. Pathological investigations revealed that white rot fungus, Ganoderma lucidum impaired the nutrient and water transport system of the grown up trees. Moreover, Ganoderma fungus loves to grow and parasitize the basal portion (roots and stem) of tree. Therefore, the disease is named as basal stem rot. The disease is major limiting factor for survival of age-old Khejri plantation. Several workers reported different management practices to contain the mortality of Khejri, but the results are not consistent. Now, fungal biocontrol agents (BCA) have been proved to be a potent method against soil borne plant pathogenic fungi. A number of technical, economical and environmental factors stimulate the use of biocontrol agents for the control of Ganoderma pathogens. Trichoderma species are reported all over world for its beneficial uses not only in disease control but also in improvement of plant health. In these days BCAs have emerged as modern strategy to manage plant diseases. In view of this development, efforts were made to develop a suitable management practice by using BCAs. To achieve the success the native strains of Trichoderma and Gliocladium were used for the recovery of partial to severely affected trees. To achieve the success native strains of Trichoderma and Gliocladium was successfully used for the recovery of partial to moderately diseased trees. Most of affected trees recovered from drying stage to grow again to green stage (production of new flushes of green leaves) after BCA treatments. The success of recovery is mostly dependent on three factors-1. Use of potential BCA 2. Multiplication of BCA on active medium and 3. Maintenance of proper moisture for keeping viability of BCA during field application (Fig.7.7). Rejuvenation of Gigantic Sacred Tree- “Ram Khejda”: A sudden drying of 256-yearold religiously important “Khejri” tree was observed in Kherapa village (JodhpurNagaur-NH 65) of Jodhpur district in Rajasthan, India. The tree was highly respectable among the devotees due to its religious and historical importance. This blessed tree was healthy until the month of May, 2003, which dried off suddenly and became leafless. On the basis of encouraging results obtained earlier with biocontrol agents isolated from native soil and diverse habitats (such as sick plant, decaying wood and fruiting bodies of Ganoderma lucidum), the affected tree was treated with potential strains of BCAs. In this case, sick soil and affected tree parts along with Ganoderma fungus was removed. A circular ring around the periphery of trunk (4cdeep and 2.5c wide) was prepared. Four holes (2–4 cm wide and 5–10 cm long) were drilled in woody roots and trunk with electric driller and then inoculated with


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Fig. 7.7 Rejuvenation of Khejri (Prosopis cineraria) using biocontrol agents. Sudden Drying and Rejuvenation of “256 Years Old Ram Khejda Tree” Near Ramkund at Kherapa Village: (a) suddenly dried tree is seen in background, (b) A new sapling emerged from within the roots of affected tree after BCAs’ treatment (fenced), (c) Fruiting body of Ganoderma was seen on the trunk of the affected tree much above the ground after the application of BCAs, (d) The priest and other devotees are worshipping the new emergence, the same has grown to become a big tree

potential strains of T. pseudokoningii and G. virens (GTP-7 & GGV-3) in the month of July 2003. The potential strains of Trichoderma and Gliocladium were mixed with Jaggery to pour the slurry inside the drilled holes of trunk and roots. Biocontrol fungi were also mixed with FYM to treat the affected soil in ring basin. The tree started rejuvenating in the month of December 2003 by sprouting new growth from the root zone which was treated with biocontrol agents + other additives. Rejuvenated shoots were protected with wire-net so that it can attain a proper growth and to avoid abiotic and biotic damages (Fig.7.8). Tree was further given follow up treatments of biocontrol agents (GTP-7 & GGV-3) with micronutrients in FYM around the root zone of affected tree and rejuvenated sprouts for further development of “New Emergence” turn into a “Young Tree”. The tree has attained a height 11.5 ft. with 1 ft. collar diameter having 16 branches. The scientific story demonstrates that these native strains have an important role to play in managing plant pathogenic fungi causing root and butt rots. Presently the “New (recovered) tree is young with lush green foliage and true to type”. The rejuvenated tree has reinstated faith among followers and disciples who have again religiously started worshipping this sacred tree.

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Fig. 7.8 In the year 2006 experts visited the site and appreciated the efforts made to rejuvenate the affected tree: (a) Main gate of Kherapa Ramdwara, (b) Expert team visiting the tee and newly grown tree, (c) In the year 2010 the same tree has attained good growth, (d) Ramdwara devotees have inscribed the name of the newly developed tree on a stone slab, (e) Ramdwara Guru Gaadee

7.10

Conclusions

While writing this chapter it reminds us the famous quote of Sir Isaac Newton, who said that “I was like a boy playing on the sea-shore, and diverting myself now and then finding a smoother pebble or a prettier shell than ordinary, whilst the great ocean of truth lay all undiscovered before me”. This chapter is therefore a modest beginning as we are also attempting to a difficult task to summarize findings of indigenous knowledge in a comprehensive manner. Information on traditional practices for managing plant diseases has never been documented. In his only treatise on the subject Prof. H. D. Thurston [14] has stated that “ the study of traditional management of plant diseases should be a rewarding area for future plant pathologists who are not completely seduced by the terms ‘new’ and ‘innovative’ and the prestigious and intellectually appealing basic research in biotechnology”. Disease management in crops is heavily dependent upon the application of synthetic fungicides for pathogen control. However, restrictions on fungicide use


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and widespread emergence of pathogen resistance has increased global demand for more sustainable production systems and driven research towards alternative disease control strategies. Biological control, which includes elicitors of host defense, microbial antagonists and natural products, offers an attractive alternative to synthetic pesticides. Biocontrol strategies exist in different forms viz. natural (organisms and environmental factors), classic (involve an active human role), augmentative (to increase population of biocontrol agents) and inundative. The occurrence of any plant disease essentially depends upon the susceptible host, virulent pathogen and favourable environment forming a disease triangle. Bio-control organisms in fact, interact with components of this triangle to reduce disease. A large number of plant diseases have been managed using biocontrol agents. What is important now is to discover and use the natural biological control mechanisms evolved so far against the plant diseases. Present research trends include the increased use of bio-rational screening processes to identify microorganisms with potential for biocontrol, increased testing under semi-commercial and commercial production conditions, increased emphasis on combining biocontrol strains with other control methods and integrating biocontrol into an overall system. Albeit, intensive activity is currently being geared toward the introduction of an increasing number of biocontrol agents into the market, commercialized systems for the biological control of plant diseases are limited in number. However, some biocontrol agents have been reported to be as effective as fungicide control. In view of awareness toward nature-friendly management of plant diseases, use of biological control measures will be a most promising proposition for disease management.

7.10.1

The Need and Logistics of New Research Initiatives

The plateaus are in vogue, being experienced in agricultural productivity, extent of disease and pest control and understanding underlying mechanisms in biological sciences. The fact is to be reckoned with that with all genuine concern for environment and quality food, the extent of use and support to IK and biological control is way behind the chemical technology. The reasons are many, e.g., lack of proper standardization, product formulation, industrial production and value addition in IK technologies. A new approach to research-extension linkage is needed to address these issues and fill the gaps for a sustainable and environment friendly agriculture. In the context of understanding underlying mechanisms, plant physiological and biochemical studies have hitherto contributed, leading to an interface of plant pathology with molecular biology and biotechnology. Yet, it is being realized that in order to understand better some of the initial questions like- why compost is better? Why do we need an organic matrix for fertilizer application? How biodynamic preparations work? Why bio control is more eco-friendly than chemical control? How things like ash, ghee (butter oil) and butter act to control or inducing resistance? How yajnas like Agnihotra can be useful in enhancing agricultural productivity and as homa therapy? Could there be any significant difference in the milk, urine and

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dung of indigenous cow and exotic cattle or buffalo?-a trans-, disciplinary research including nano technology, quantum physics, genopsych, intramolecular electronics of DNA etc. are needed to break the barrier and surge upward beyond the saturating lines. Moreover, plant adaptations to biotic and abiotic stresses need to be reassessed in the light of new insights being generated as plant neurobiology[97], plant intelligence [98], consciousness [99] and genopsych [100, 101]. Experts used to say that the main reason why grass root innovations were being ignored because peer pressure often forced scientists to focus on high-impact research with wide visibility. The situation is changing with a horizontal emphasis on ecological and quality concerns. Recent patenting of a milk-based product active against a number of fungal diseases in general and mildews in particular from Horticulture and Food Research Institute of New Zealand Limited [102] is, in fact a matter of recognition to the Indigenous Knowledge and farmers’ wisdom. Now it is strongly advocated to strengthen such systems through village based initiatives and actively involving local peasants are considered the keys to successful sustainable agriculture and rural development programs.

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