Isolation and characterization of native Pseudomonas

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CHIDANAND A. RABINAL, NARAYAN MOGER, N. SHASHI KUMARA, RAMESH METHRE and. K.N. CHANDRASHEKARA. Alleviation of oxidative damage to ...

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GREEN FARMING

(International Journal of Applied Agricultural & Horticultural Sciences) (Abbreviation : Green Farming Int. J.)

Volume 6

Number 1

Bimonthly

January-February 2015 CONTENTS

Research Papers

Previous issue : Vol. 5, No. 6, pp. 946-1165

Evaluation for early seedling vigour of hybrids and varieties of pigeonpea [Cajanus cajan (L.) Millsp.] ? M.G. MULA, D.P. THAKARE, S.P. MEHTRE and A. RATHORE

......... 1

Combined & comparative analysis of RAPD, ISSR & microsatellite markers for phylogenetic studies in tobacco ? DHARMENDRA PATIDAR, R.S. FOUGAT, A.D. PATEL and VISHAL R. PATIL

......... 5

Polyclonal antibody selection against Cry1Ac protein using Tomlinson library ? CHIDANAND A. RABINAL, NARAYAN MOGER, N. SHASHI KUMARA, RAMESH METHRE and

K.N. CHANDRASHEKARA

......... 9

Alleviation of oxidative damage to mungbean (Vigna radiata) by salicylic acid under induced salinity ? Z.M. DAR, A. HEMANTARANJAN, A. MASOOD, F.A. PARRY and M.A. CHATTOO

......... 13

Saponin, amylase inhibitor and trypsin inhibitor of pigeon pea [Cajanus cajan (L.) Millsp.] hybrids as affected by different soaking treatments ? REENA DEVI, SHILPA CHAWLA, A.K. SAXENA and CHARUL CHAUDHARY

......... 17

Generation mean analysis in chilli x bell pepper ? A.K. JOSHI and AMBREEN NABI

......... 21

Combining ability through diallel analysis in okra [Abelmoschus esculentus (L.) Moench] ? ABHISHEK KATAGI, SHANTAPPA TIRAKANNANVAR and R.C. JAGADEESHA

......... 26

Genetic variability & correlation studies in cherry tomato (Solanum lycopersicum L. var. Cerasiforme Mill) ? PARVATI PUJER, R.C. JAGADEESHA and SURESH

......... 30

Combining ability in maize (Zea mays L.) through diallel analysis ? H.V. PATIL, S.K. CHAVAN, V.S. PATIL and A.A. CHAVAN

......... 33

Genetic variability and stress susceptibility index under limited moisture condition in fenugreek ? NEMICHAND SHARMA, A.S. SHEKHAWAT, SURESH MALAV and C.P. MEENA

......... 37

Evaluation of capsicum (Capsicum annuum L. var. Grossum Sendt.) varieties and hybrids for growth and yield attributes under shade net ? G. NARAYANA SWAMY, B. SRINIVASULU, C. MADHUMATHI and M. PARATPARA RAO

......... 41

Genetic divergence in sweet corn (Zea mays L. Saccharata) ? G. SESHU, M.V. BRAHMESWARA RAO and M.R. SUDARSHAN

......... 44

Genetic potential of some elite lines of pearl millet [Pennisetum glaucum (L.) R. Br.] in arid zone of Rajasthan P.C. GUPTA, D.K. GARG and SHIV NARAYAN ?

........ 47

Effect of INM on yield, nutrient uptake, protein content & economic performance of rice (Oryza sativa) and soil fertility in a Vertisol ? G.D. SHARMA, R.K. THAKUR, N. CHOUHAN, B.L. SHARMA and D.K. TIWARI

........ 50

Yield response to bed sowing of soybean and wheat crop in zero tillage with crop residue ? R.C. SINGH, DASHRATH SINGH, C.D. SINGH and VIKAS GONJARI

........ 55

Yield, nutrient uptake & soil moisture extraction under different crops & intercropping systems in rainfed alfisols ? T. SONIYA, G. VEERANNA and P. RAGHU RAMI REDDY

........ 59

Long-term organic manuring & fertilization enhancing growth attributes, productivity & quality of soybean & safflower ? N.A. MESHRAM, SYED ISMAIL and A.S. DHAWAN

........ 63 Contd. ....

Long-term effect of soil test-based fertilizer use with & without organic manure on pearlmillet –wheat crop sequence ? SEEMA SHARMA and RAMNIWAS CHOUDHARY

........ 67

Effect of varieties and nutrient management on growth analysis parameters and nutrient uptake of clusterbean (Cyamopsis tetragonoloba L.) ? UPAMA RAWAT, R.L. RAJPUT, G.S. RAWAT and S.K. GARG

........ 70

Influence of micronutrients and biofertilizers on growth, yield & quality attributes of tomato (Lycopersicon esculentum M.) ? I.A. KADARI, R.M. DHEWARE, B.D. WAGHMARE, S.S. MINGIRE and K.N. DHAWALE

........ 74

Fruit yield and soil fertility in organically managed tomato under rainfed alfisols of southern transition zone of Karnataka ? GANAPATHI, K.S. NIRANJANA, H.M. CHIDANANDAPPA and VISHWANATHSHETTY

........ 77

Effect of different levels of sulphur and boron on yield, nutrient contents and uptake in garden pea ? F.A. PARRY, M.A. CHATTOO, G.R. NAJAR, S.H. KHAN, Z.M. DAR and A. MASOOD

........ 80

Organic fertilizers influencing quality attributes & economics of strawberry (Fragaria x ananassa duch.) cv. Chandler ? B.A. PANDIT, I. UMAR, M.M. MIR, S.A. BANDAY and TALAT MAJID

........ 84

Effect of boron and iron on flowering in gladiolus (Gadiolus grandiflorus L.) cultivars ? I. VIJAY, M.B. NAGESWARA RAO, M. MADHURI and G. VENU GOPAL

........ 88

Effect of INM practices on growth & yield attributes of guinea grass cv. Panicum maximum under rainfed condition in Kerala ? V. JYOTHISH KUMAR, S. LAKSHMI and M.R. ANITA

........ 92

Regulation of pink berries in Thompson Seedless (Vitis vinifera L.) white coloured grape, during ripening by the use of plant growth regulators J.M. KHILARI, J.N. KALBHOR, T.S. MUNGARE and P.B. TAWARE ?

........ 95

Effect of hydropriming technique for the improvement of seed quality and yield of pigeonpea ? PRITI A. SONKAMBLE, ARUNA S. KATOLE and MADHURI M. SADAFALE

........ 100

Effect of alternative growing systems and media on the vegetative growth & yield of tomato in aggregate hydroponics ? ASHA JOSEPH and I. MUTHUCHAMY

Natural resource management through different agro-forestry land use system in central dry zone (IV) of Karnataka ? SHARANAPPA JANGANDI, J. NARAYANA and MUDALAGIRIYAPPA

........ 103 ........ 108

Depth wise distribution of heavy metals in soils irrigated with sewage and industrial effluents ? RAMAPPA JAKANUR, V.R. RAMAKRISHNAPARAM and GANAPATHI

Productivity and economics of mentha (Mentha arvensis) okra-potato cropping system as influenced by irrigation and fertility levels under drip irrigation ? S.K. SINGH, AJAY KUMAR and MANI BHUSHAN

........ 111

........ 114

Genotypic differences against poly ethylene glycol (PEG) simulated drought stress in rice ? SRIHIMA GAMPALA, VIKRAM JEET SINGH, S.K. CHAKRABORTI, K.P. VISHWAJITH and G.R. MANJUNATH ........ 117

Effect of seedling age and transplant density on growth and yield of onion ? DEEPIKA CHANDRAVANSHI, VIJAY KUMAR, PRAVIN SHARMA and CHETNA BANJARE

........ 122

Isolation and characterization of native Pseudomonas fluorescens for biocontrol of Fusarium wilt in greengram ? D.G. PANPATTE, H.N. SHELAT, Y.K. JHALA, V.B. DARJI, PARVEZ NOUSHAD and PATHAK LEENA

and K.J. KHATRI

........ 127

Biology and life-table of Spodoptera litura (Fab.) on sunflower ? S. GEETHA and K.S. JAGADISH

........ 133

Life-table characteristics of arrhenotokous and thelytokous T. pretiosum Riley on eggs of C. cephalonica S. ? T. PRABHULINGA, N.E. THYAGARAJ, K.K. KALAVATHI and S.K. JALALI

........ 137

Seasonal incidence of gram pod borer, Helicoverpa armigera (Hub.) and natural enemies in pigeonpea ? U.A. PAWAR, A.S. MAHALE and K.G. AMBHURE

........ 140 Contd. ....

Seasonal incidence of insect pests, natural enemies and pollinators in drumstick (Moringa oleifera L.) ecosystem

........ 144

? MAHESH MATH and Y.K. KOTIKAL

Screening of sorghum varieties for susceptibility against Rhyzopertha dominica (Fab.) under South Gujarat condition

........ 149

? MANISHA MEENA, G.G. RADADIA and C.U. SHINDE

Disease dynamics of maize & effect of different temperature levels on radial growth and dry mycelial weight of Trichoderma spp. from maize-based conservation agriculture ecosystem

........ 154

? N.M. PRABHAVATHI, Y.S. AMARESH, M.K. NAIK, P.H. KUCHANUR and S.B. MALLESH

Germinated rice paddy as a bait base for zinc phosphide & bromadiolone against rodent pests of selected crops

........ 157

? P. SAKTHIVEL and P. NEELANARAYANAN

Weed suppression in maize with legume intercrops and sowing pattern under organic conditions in NW Himalayas

........ 161

? RAMESHWAR KUMAR, J.P. SAINI, SANJAY CHADHA and RAJ KUMAR

Gram price movement across major markets of Maharashtra

........ 164

? V.K. PATIL and A.S. TINGRE

Economic impacts of system of rice intensification (SRI) in India ? B. JOHNSON, K. VIJAYARAGAVAN and T. KINGSLY IMMANUEL RAJ

........ 168

Price spread, market margins & marketing problems along the value chain of maize in Rangareddy & Mahaboobnagar districts

........ 172

? I. SHAKUNTALA DEVI, K. SUHASINI and N. VASUDEV

Growth, instability and acreage response function in production of coriander in Rajasthan

........ 177

? V.K. BOYAL, D.C. PANT, S.S. BURARK and J. MEHRA

Effect of chemical solutions on vase life, quality & biochemical parameters of gerbera cv Red Star

........ 181

? PREETI CHOUHAN, VIDHYA SANKAR M. and S.N. MISHRA

Effect of pre-harvest treatments on oxidative enzymes during developmental stages in tomato ? POONAM CHOUDHARY and JITENDRA DHRUVE

........ 186

Effect of foliar application of zinc on vegetative growth, flowering, corm and cormel production in gladiolus

........ 189

? G.V. SUBBA REDDY, M.B. NAGESWARA RAO and R. CHANDHRA SEKHAR

Physico-chemical characters, sensory quality and storage behavior of rose apple RTS blended with jamun

........ 193

? RAVISHANKAR M. PATIL, V. CHIKKASUBBANNA and ABHISHEK KATAGI

Acceptability of stevia as low calorie alternative to sugar in mango RTS juice

........ 196

? GOVIND B. YENGE, HARI G. MORE and SHUBHANGI D. SHELAR

Effect of gibberellic acid on growth, flowering and cut flower yield of gerbera under protected condition

........ 199

? R.V. CHAUHAN, D.K. VARU, K.P. KAVA and S.K. BHUVA

Different recipe for ready-to-serve beverage from karonda and its storage

........ 202

? PRAJAKTA K. KADU and V.U. RAUT

Physico-chemical characteristics of overburden dump materials and native soil of opencast coal mine in Chhattisgarh

........ 205

? G.K. NIGAM, R.K. SAHU, JITENDRA SINHA and N.K. YADAV

Optimization of operational parameters for development of tractor operated rotary weeder ? K. MANJUNATHA, M. ANANTACHAR, VIJAYAKUMAR PALLED, SUSHILENDRA, K.V. PRAKASH and

........ 211

SUNIL SHIRWAL

Solar pump operated drip irrigation on yield and economics of potato to enhance water use efficiency ? N.K. RAJESH KUMAR, G.V. SRINIVASA REDDY and M. CHOWDE GOWDA

........ 215

Short Communication Study of growth, yield and quality of ashwagandha as influenced by sowing dates and seed rates ? S.C. VILHEKAR, R.S. ZADODE, Y.R. PATIL and V.V. TAPRE

........ 218

Seasonal impact of rations on milk yield of Rathi cows in Western Rajasthan ? UPENDRA KUMAR, R.S. SHEKHAWAT and S.P. SINGH

........ 220

Strategic Vision : 25 Food processing sector in India : Overview ? D.M. VYAS and D.K. GOJIYA

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Research Paper

Green Farming Vol. 6 (1) : 127-132 ; January-February, 2015

Isolation and characterization of native Pseudomonas fluorescens for biocontrol of Fusarium wilt in greengram 2

1*

3

4

5

D.G. PANPATTE , H.N. SHELAT , Y.K. JHALA , V.B. DARJI , PARVEZ NOUSHAD , 6

7

PATHAK LEENA and K.J. KHATRI

Department of Microbiology, Anand Agricultural University, Anand - 388 110 (Gujarat) Received : 12 April 2014

;

Revised accepted : 26 December 2014 ABSTRACT

In present investigation total 10 isolates were obtained from rhizospheric and non-rhizospheric soil sample of Vadodara locality. All the isolates were capable of restricting mycelial growth of soil-borne wilt pathogen Fusarium oxysporum and Fusarium udum. Among all the isolates, PR3 showed maximum antipathogenic activity i.e. 42.23 and 45.80 % against F. oxysporum and F. udum and hence, this isolate was further characterized on the basis of morphological, biochemical and molecular characteristics as P. fluorescens AAU PR3 (NCBI Accn. No.: KJ161327). It also showed siderophore production and cell wall degrading enzyme lipase production which represents a possible mechanism of biocontrol of fungal pathogens. P. fluorescens AAU PR3 was found highly resistant to major antibiotics tested. Isolate P. fluorescens AAU PR3 was also capable of reducing wilt symptoms in greengram and also enhanced root and shoot length, fresh as well as dry biomass of greengram in presence and absence of F. udum under laboratory and pot trial conditions. From the present research, native rhizospheric fluorescent P. fluorescens AAU PR3 (NCBI Accn No: KJ161327) has emerged out as a potential biocontrol strain against soil-borne fungal pathogen F. udum. Key words : Antipathogenic, Biocontrol, Fusarium wilt, Greengram, Rhizosphere, Siderophore, Secondary metabolite.

INTRODUCTION Now a days increased environmental awareness and consumers demand for pesticide free safer food leads to decline in use of chemicals and has promoted the development of eco-friendly biological alternative to crop protection agents. Bacteria belonging to family Pseudomonadales and genus Pseudomonas play an important role in plant growth promotion and disease suppression, among which P. fluorescens have attracted considerable attention on account of their potential for biological control by various mode of action (Defago and Haas, 1990). Moreover, P. fluorescens produces plant growth promoting substances such as auxins and gibberellins which enhance plant growth and yield (Dubeikosky et al., 1993). Keeping in view, present research work was undertaken to isolate and characterize the native strains of P. fluorescens from rhizosphere of different field crops to find out efficient strain of P. fluorescens against soil-borne fungal pathogens F. oxysporium (1072) and F. udum (ICRISAT strain) and confirmed the role of siderophores in the antibiosis. This work may help to unearth some potential native Pseudomonas isolates against fungal diseases and promote the usage of microbial biocontrol agent. 1,5,6,7

2

Student *([email protected]), Assoc. Res. Scientist,

3

MATERIALS AND METHODS Collection of soil samples : For isolation of fluorescent Pseudomonas, suppressive soil samples (rhizospheric and non-rhizosphere soil) were collected from the Model Farm and from Diploma school, AAU, Vadodara. Enrichment & isolation of fluorescent Pseudomonas : For enrichment and isolation of fluorescent Pseudomonas King's B medium supplemented with ampicilin, chloramphenicol and cyclohexamide antibiotics was used and isolated colonies showing characteristic fluorescent pigmentation were further studied.

Screening of fluorescent Pseudomonas isolates for anti-pathogenic activity in laboratory : Screening of predictable fluorescent Pseudomonas isolates for their antipathogenic activity against some soil borne fungal pathogens F. Oxysporum and F. Udum was performed by dual culture method as described by Kaur et al. (2007).

Siderophore production : The selected isolates showing highest biocontrol activity were screened by CAS method (Schwyn and Neilands, 1987) for siderophores production.

4

Res. Associate, Assoc. Professor

132

Jan.-Feb. 2015

Determination of lipase : Lipolytic activity was determined on Tributyrin agar plates (Lawrence et al., 1967) containing Tributyrin as lipid supplement. The bacterial cultures were spot inoculated on the surface of plates and incubated at 28±2°C for 5 days and the plates were observed for zone of lipid degradation. Characterization of selected isolate : Selected isolates was characterized on the basis of morphological, cultural and biochemical characteristics (The prokaryotes, 2006; Halt et al., 1994). Molecular characterization was carried out for identification and phylogenetic relationship of the potential isolates, 16S rDNA sequencing was carried out as described by Waturangi et al., 2011. The sequence data of bacterial isolates were compared to sequence from Gene Bank in National Center for Biotechnology Information using BLASTN program for identification and phylogenetic analysis of the isolates Effect of fluorescent Pseudomonas isolate in presence and absence of wilt pathogen F. udum on greengram in laboratory : A laboratory trial was conducted with six treatments (T1: Pseudomonas native isolate @ 109 CFU/ ml, T2: Pseudomonas fluorescens Pf5 (Std strain) @ 109 CFU/ ml, T3: Pseudomonas native isolate @ 109 CFU/ ml + F udum , T4: Pseudomonas fluorescensPf5 @ 109 CFU/ ml + F. udum, T5: Pathogen alone F. udum , T6: Untreated check) and four replications to study effect of native isolate on greengram cv. Meha in presence and absence of wilt pathogen following CRD Surface sterilized were treated with bacterial isolate's (5x109 cfu /ml) and fungal spore (5 x 108 spores/ml for 30 min as per treatment detail. The seeds were than kept on previously sterilized 1% agar media in petri plates and incubated at 28±2°C in seed germinator. Seed germination was examined at 24, 48, 72, 96 and 120 h interval and germination percentage was calculated. Root length, shoot length, fresh and dry weight of plant was measured at 15 days after inoculation.

Efficacy of fluorescent Pseudomonas isolate on growth enhancement and Fusarium wilt control in greengram under net house conditions : Similar set of treatment was repeated in pot trial with six treatments and four replications following CR Design. Severity of wilt disease, root length, shoot length, fresh and dry weight of plants were recorded at 30 days after sowing.

Statistical analysis : The data collected on different experiments and parameters were subjected to statistical analysis by using CRD (Panse and Sukhatme, 1978). Data were subjected to analysis of variance and means and were compared by DNMRT (Duncan, 1955).

RESULTS AND DISCUSSION Isolation of Pseudomonas from soil samples of different cropping areas (rhizospheric and non133 Green Farming

128

Isolation of native P. fluorescens for biocontrol of Fusarium wilt in greengram

rhizospheric soil) of Vadodara locality : Due to enrichment in the media supplemented with antibiotics the chances of getting specific Pseudomonas cultures have been increased. After 3 days of incubation on solid King's B media, yellow to whitish and green colonies were obtained. Total 10 isolates were obtained from 10 soil samples. Among which 5 cultures were obtained from rhizopheric and 5 isolates were obtained from non rhizospheric soil samples. Similarly, Laha & Verma (1998) isolated six fluorescent Pseudomonas spp. from rhizosphere of healthy cotton seedling using King's B medium. Yeole & Dube (2000) isolated twelve rhizobacterial fluorescent Pseudomonas from chilli (3), cotton (3), groundnut (3), and soybean (3). Morphological

identification

of

the

isolates :

Morphological identification of isolates was done by observing yellow, white and green pigments development on King's B agar medium under normal light and by observing fluorescence under U.V. light. Isolates PR1, PR2 and PR5 showed yellow pigment, PR3 showed green pigment and rest of the isolates showed white pigment on King's B medium in visible light. All the isolates showed fluorescence in U.V. light. These results are in corroboration with the results obtained from the studies of Rekha et al. (2010) who have identified P. fluorescens isolated from rhizospheric soil based on morphological characteristics showing blue pigment under normal light and fluorescence under U.V. light.

Screening of fluorescent bacterial isolates for antipathogenic activity against plant microbial pathogens in laboratory : Data in Table 2 shows that all isolates were found effective to inhibit the mycelial growth F. oxysporum. Isolate PR3 was found significantly superior as compared to other isolates showing maximum inhibition of mycelial growth i.e. 42.23 % followed by isolate PR2 28.67 % inhibition (Table 1). PR3 have also proved its ability to restrict mycelial growth of F. udum (45.80 %) (Fig. 1).

Table 1. Anti-pathogenic activity of native fluorescent bacterial isolates against F. oxysporum and F. udum Name of isolate PR1 PR2 PR3 PR4 PR5 PN1 PN2 PN3 PN4 PN5 Control S.Em.± CV %

% inhibition of growth of mycelial F. oxysporum f

17.63 b 28.67 a 42.23 de 21.67 ef 18.00 cd 22.33 def 18.67 bc 25.67 b 26.33 cd 22.33 g 00.00 01.35 10.53

F. udum cde

24.17 b 28.70 a 45.80 ef 22.23 f 19.23 bcd 26.70 ef 22.07 bcd 26.13 bc 27.77 de 23.43 g 00.00 01.27 9.08

129

Panpatte et al.

Green Farming 6 (1)

enzyme. Iron chelation and utilization by siderophore production is one of the mechanism exerted by biocontrol bacteria. Presence of lipase enzyme helps in biocontrol of pathogen by exerting its effect on cell wall of fungi.

(a) F. udum growth on PDF medium

(b) F. udum growth inhibition by P. fluorescens PR3

Fig. 1. Antipathogenic activity of P. fluorescens PR3 against F. udum Kumar et al. (2010) reported that P. fluorescens strain LPK 2 isolated from disease suppressive soil of tomato rhizosphere was able to arrest mycelial growth of F. udum in dual culture plate assay due to their ability to produce siderophores, HCN and chitinase enzyme. The results suggest that the native fluorescent Pseudomonas isolates can play an important role in biocontrol of soil borne wilt disease. Inspired from the results obtained biocontrol potential of isolate PR3 against F. oxysporum and F. udum was further evaluated and characterization of the selected isolates was done.

Siderophore production : The inhibitory activity of fluorescent pseudomonads against plant pathogenic organisms is said to be due to production of secondary metabolites (Davison, 1986; Defago and Haas, 1990). Another major mechanism involved in suppressive activity of fluorescent pseudomonads is production of siderophores, which can complex with iron and make it unavailable to plant pathogens. Siderophores chelates iron and contribute to disease suppression by conferring a competitive advantage to biocontrol agents for the limited supply of essential trace minerals in natural habitats (Maleki et al., 2010). Appearance of an orange coloured zone after 48 h of incubation indicated that the isolate PR3 was able to chelate Fe3+ from chrome azurol agar medium. The test isolate produced clear orange zone against dark blue background on CAS agar medium. This color change is based on the principle that the blue color of CAS agar medium is due to the iron-dye complex and when siderophores are produced, the iron is released from the complex by chelating with siderophores resulting in an orange color (Dave & Dube, 2000). Similar observation for siderophore production has been demonstrated by (Gupta et al., 2002).

Production of lipase : Isolate PR3 showed production of lipase enzyme on Tributyrene agar plate. The isolate showed zone of lipid degradation around colony which indicates the presence of lipase enzyme in the near vicinity of bacterial growth that breakdown the lipid present in the media and showed the zone of clearance around colony. The results showed that the isolate was capable of producing iron chelating compound siderophore and lipase

Characterization of native isolate : From morphological and biochemical characterization studies as mentioned in table 2, it was clear that the colony of native isolate showed fluorescens under U.V. light and the organisms was G–ve, highly motile, short rod which is a characteristic feature of fluorescent Pseudomonas sp. The selected isolate was positive for nitrate reductase, citrate utilization, malonate utilization, starch hydrolysis and gelatinase and oxidase production and utilized Arabinose, Xylose, Melibiose and Glucose. It has shown negative response for ONPG, lysine utilization, ornithine utilization, Urease, Phenylalanine deamination, H2S production, Vogesproskauer's, Methyl red, Indole and Esculine hydrolysis test (Table 2). Table 2. Characterization of PR3 Characteristic

PR3

Colonial characteristics Shape Margin Elevation Texture Opacity Pigmentation Pigmentation under U.V. light

Round Entire Slightly raised Smooth Opaque green Fluorescence

Microscopic characteristics Shape Arrangement Gram's reaction Motility

Short rod Single G-ve Highly motile

Biochemical characteristics ONPG Lysine utilization Ornithine utilization Urease Phenylalanine deamination Nitrate reductase H2S production Citrate utilization Vogesproskauer's Methyl red Indole Malonate utilization Esculine hydrolysis Oxidase Starch hydrolysis Gelatin liquefaction Arabinose Xylose Adonitol Rhamnose Cellobiose Melibiose Sachharose Raffinose Trehalose Glucose Lactose

+ + + + + + + + + + -

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Jan.-Feb. 2015

Isolation of native P. fluorescens for biocontrol of Fusarium wilt in greengram

130

Table 3. Antibiotic resistance profile of PR3 Antibiotic tested Isolates

R (5)

A (10)

G (10)

C (10)

S (10)

T (30)

K (30)

V (30)

Se (100)

Cb (100)

P (300)

PR3

HR

HR

R

HR

HR

HR

R

HR

HR

HR

HR

Note: The figures in parentheses indicate units in µg/disc K- Kanamycin R- Rifampicin G- Gentamicin V-Vancomycin A- Ampicillin C- Chloramphenicol

These results are confirmed by the findings of Hagedoron (2001) who characteristised and identified P. fluorescens as strictly aerobic G-ve straight or curved rods, without resting spore and colonies are usually yellow to cream white and green pigmented, many species produce extra cellular fluorescence pigment. Mahesh (2007) also reported that among ten P. fluorescens isolates four isolate hydrolyse starch and six isolates liquefy gelatin and produce H2S gas. The biochemical tests i.e. gelatin liquefication, oxidase test, starch hydrolysis and carbon source utilization pattern of isolate further confirmed them to be closely related to P. fluorescens as per Bergey's manual of systemic bacteriology Vol. 1 and standard literature collected (Kumar et al., 2012; Noori and Saud, 2012; Chinthala and Gundala, 2013). Table 3 showed antibiotic resistance profile of isolate. The selected isolate was found highly resistance to rifampicin (5 µg), ampicillin (10 µg), chloramphenicol (10 µg), streptomycin (10 µg), tetracyclin (30 µg), vancomycin (30 µg), spectinomycin (100 µg), carbenicillin (100 µg) and polymyxin-B (300 µg) and resistance to gentamycin (10 µg) and kanamycin (30 µg). The antibiotic resistance profile showed that the test isolate is resistance to all the tested antibiotics indicating an added advantage of these organisms in terms of overcoming competitive inhibition by antibiotic producing soil inhabitants which aids in overcoming the limitation of restricted performance under field conditions. These observations provide an idea about selective marker to be employed in further molecular analysis of this isolate. Similarly Jagadish (2006) showed that native strain of Pseudomonas sp. B-25 showed resistance to ampicillin at all concentrations tested i.e. 25-250 ppm. The strain did not show any growth in presence of streptomycin and kanamycin even at the lower concentrations of 25 ppm, indicating that it is sensitive to these antibiotics.

Molecular characterization of isolate : From 16S rRNA partial gene sequence isolate PR 3 was identified as Pseudomonas fluorescens AAU PR 3 with 98 % similarity and 99 % query coverage Pseudomonas fluorescens Pf0-1. The phylogenetic tree (Fig. 2) indicated PR3 belongs to Gracilicutes and grouped with other Pseudomonas sp. Manjunatha and Naik (2013) characterized fluorescent Pseudomonas isolates RPF-13 and RPF-81 obtained from the rhizosphere soil of brinjal and chilli crop respectively, on the basis of 16S rRNA amplification profile. A 1316 bp fragment of 135 Green Farming

S- Streptomycin T- Tetracycline

Se- Spectinomycin Cb- Carbenicillin

P-Polymyxin-B

Fig. 2. Phylogenetic tree based on 16S rRNA sequence of isolate PR 3 the 16S rRNA gene of isolates were amplified using primers fD1 and rP2, PCR products were eluted and sequenced. The Strains RPF-13 and RPF-81 were identified as P. Putida and P. Fluorescens respectively on the basis of 16S rRNA sequence homology. The phylogenetic tree was constructed based on the 16S rRNA sequence showed the similarity with closely related strains P. alcaligenes (Z76653) and P. aeruginosa (EU915713) using Multiple Sequence Alignment Clustal W software (GenomeNet).

Effect of P. fluorescens AAU PR3 in presence and absence of wilt pathogen F. udum on green gram cv. Meha : Germination was visualized at 24 h, 48, 72 and 120 h after inoculation. At 24 h after inoculation T1 inoculated with P. fluorescens AAU PR3 showed 100% germination followed by T2 (98%). Treatment receiving pathogen + inoculation of P. fluorescens AAU PR3 (T3) showed 98% germination which was significantly superior over T5 receiving pathogen alone showing 83% germination. The inoculation of standard strain of P. fluroscens Pf5 in combination with pathogen showed 93% germination at 24 h. Similar trend was observed at 48 h, 96 h and 120 h (Table 5). Moreover,T3 inoculated with F. udum in presence of native P. fluorescens AAU PR3 showed significant increase in root and shoot length (8.20 and 12.07 cm) as compared to T5 receiving F. udum alone showing 4.45 cm and 5.06 cm root and shoot length. T4 showing 7.82 and 11.32 cm root and shoot length stands at par with T3. Similar trend was observed for fresh and dry biomass (Table 4, Figure 3).

Panpatte et al.

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Green Farming 6 (1)

Table 4. Effect of P. fluorescens AAU PR3 on greengram in presence and absence of F. udum in laboratory Germination percentage

Treatments

T1 : P. fluorescens AAU PR3

24 h

48 h

72 h

120 h

a

a

a

a

100

T2 : P. fluorescens AAU PR3

98a

T3 : P. fluorescens AAU PR3

98

T4 : Pseudomonas fluorescens

93

T5 : Pathogen only

83

T6 : Uninoculated control

88

S.Em.± C.V. %

b

ab bc d cd

2.28 4.91

100

98 a 100 93b 85 90

b

a c

d cd

1.86 3.96

100 100 100 98 88 95

a a

a b a

1.86 3.86

100 100 100 100 88 95

Root length

Shoot length

Fresh biomass

Dry biomass

(cm)

(cm)

(gm)

(gm)

9.15

a

9.01

a

8.20

a

7.82

c

4.45

b

6.48

1.56 3.21

a

12.76

a

12.31

ab b

b

11.32 5.06

c

7.07

0.38 10.18

a ab

12.07

d

a

d c

a

2.72 2.61 2.49 2.41

ab ab bc

1.90 2.18

0.30 5.92

d c

0.08 7.04

1.88 1.72 1.68 1.63 1.06 1.59

a b b b c b

0.05 6.66

Note : P. fluorescens AAU PR3 @ 5 ml (109 cfu/ml)/kg seed before sowing and F. udum @ 108 spores/ml 15 DBS and at the time of sowing

inhibit F. udum growth. Fukhouri and Buchenaur (2002) screened 80 strains of P.fluorescens against Fusrium wilt of tomato, out of which nine isolates viz., 8 P. fluroescens (W21, W34, WB 15, WBG 52, GL 54, GL 307, G308 and GB09) and one Pseudomonas spp. (W2) were capable of reducing the Fusarium wilt disease of tomato.

Fig. 3. Effect of P. fluorescens AAU PR3 inoculation on germination of greengram cv. Meha at 48 h Similarly, Hebbar et al. (1991) tested three species of Pseudomonas viz. P. fluorescens putida, P. maltophilia, P. cepacia, Flavobacterium odoratum and Bacillus sp. In soil bioassays P. cepacia strain N24 when used as seed inoculum in the presence of S. rolfsii, increased significantly the percentage of Sunflower seedling emergence. Moreover, only 30 percent of seedlings were diseased with 1 to 3 percent wilting when treated with the antagonistic strains, in the presence of the pathogen as compare to while 60 percent and respectively 14 percent in pathogen alone control.

Efficacy of P. fluorescens AAU PR3 isolate on growth enhancement and Fusarium wilt control in green gram cv. Meha under net house conditions : The native strain of P. fluorescens AAU PR3 effectively controlled wilt disease caused by F. udum in green gram and also enhanced plant growth and development. Pot trial showed that the T4 inoculated with F. udum and native strain of P. fluorescens AAU PR3 showed no wilting of plants at 30 DAI. Similarly, T5 inoculated with standard strain showed no wilting of green gram plants. T6 inoculated with F. udum without any bacterial biocontrol agent showed complete wilting of plant. These results showed that the inoculation of native biocontrol strain showed reduction in disease symptoms in green gram plants which was confirming the results obtained during in-vitro antipathogenic activity testing where the native isolate was found to

Moreover inoculation of native P. fluorescens AAU PR3 showed significantly higher root and shoot length. T3 inoculated with F. udum in the presence of native strain of P. fluorescens AAU PR3 showed significantly higher root and shoot length i.e. 9.95 and 15.83 cm as compared to T5 which do not receive any biocontrol agent showing 7.04 and 11.90 cm root and shoot length, respectively. Similarly, T3 infected with F. udum in the presence of native P. fluorescens AAU PR3 showed significantly higher fresh biomass (0.59 g) as compared to T5 (0.34 g) which do not receive any biocontrol agent. T4 inoculated with standard strain of P. fluorescens Pf5 along with F. udum stands at par with T3 for fresh biomass (0.56 g) and superior over T5 i.e. pathogen control (Table 5).

Table 5. Efficacy of P. fluorescens AAU PR3 isolate on growth enhancement and Fusarium wilt control in green gram cv. Meha under net house conditions Treatment

T1

Root length (cm) 11.08

T2

10.16

T3

09.95

T4

09.36

T5

07.04

T6 S.Em.± C.V. %

08.68 00.49 10.38

a ab abc bc d c

Shoot length (cm) 16.54 16.04 15.83 15.56 11.90 13.69 00.72 09.62

a a ab ab c bc

Fresh biomass (gm) 0.67 0.66 0.59 0.56 0.34 0.48 0.02 8.07

a a b b d c

Dry biomass (gm) 0.25 0.24 0.23 0.23 0.11 0.23 0.01 8.86

a a a a b a

Note : Same dose as used in footnote of Table 4. Treatment means with the letter/letters in common are not significant by Duncan’s New Multiple Range Test at 5% level of significance

136

Jan.-Feb. 2015

Isolation of native P. fluorescens for biocontrol of Fusarium wilt in greengram

Akhtar et al. (2005) studied management of root-knot nematode, Meloidogyne incognita-wilt fungus, F. oxysporum disease complex of greengram, Vigna radiate cv. ML-1108 and reported that greatest suppression of nematode reproduction i.e. reproduction factor (Rf) and root-knot index was achieved by application of carbofuran (Rf=0.46, RKI=0.25) followed by A. Indica seed powder (Rf=0.99, RKI=0.50), T. harzianum (Rf=1.27, RKI=1.50), P. fluorescens (Rf=1.38, RKI=1.75) respectively as compared to controls (Rf=1.74,RKI=3.00). Bavistin was highly effective in suppression of taproot colonization by fungus F. oxysporum (8% root colonized) followed by neem seed powder (30%), T. harzianum (35%), P. fluorescens (65%) and carbofuran (75%) respectively.

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137 Green Farming

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