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BIOCONTROL POTENTIAL AND PLANT GROWTH PROMOTIONAL ACTIVITY OF FLUORESCENT PSEUDOMONADS OF WESTERN GHATS

Thesis submitted to the University of Agricultural Sciences, Dharwad in partial fulfillment of the requirements for the Degree of

MASTER OF SCIENCE (AGRICULTURE) In AGRICULTURAL MICROBIOLOGY

By SHIVAKUMAR B.

DEPARTMENT OF AGRICULTURAL MICROBIOLOGY COLLEGE OF AGRICULTURE, DHARWAD UNIVERSITY OF AGRICULTURAL SCIENCES, DHARWAD - 580 005 JUNE, 2007

ADVISORY COMMITTEE
DHARWAD JUNE, 2007 Approved by : Chairman : (A. R. ALAGAWADI) MAJOR ADVISOR _________________________ (A. R. ALAGAWADI)

Members : 1. _______________________ (J. H. KULKARNI) 2. _______________________ (K. S. JAGADEESH) 3. _______________________ (S. LINGARAJU)

CONTENTS

Chapter No.

Title

INTRODUCTION

II

REVIEW OF LITERATURE

III

MATERIAL AND METHODS

IV

EXPERIMENTAL RESULTS

DISCUSSION

VI

SUMMARY

VII

REFERENCES

APPENDICES

ABSTRACT

LIST OF TABLES

Table No. 1. 2.

Title

List of plant pathogens controlled by rhizobacteria Antimicrobial metabolites of fluorescent pseudomonads and the pathogens inhibited Details of fluorescent psuedomonads of western ghats used in the present study In vitro inhibition of different fungal pathogens by fluorescent pseudomonads In vitro inhibition of different bacterial pathogens by fluorescent pseudomonads Effect of culture filtrate and whole cell culture of fluorescent pseudomonads on nematode juveniles HCN production and siderophore production by fluorescent pseudomonads Rf values of the metabolites produced by the fluorescent pseudomonads with high biocontrol potential. Inhibitory activity of eluted metabolites pseudomonads against test pathogens of fluorescent

3.

4.

5.

6.

7.

8.

9.

10.

Performance of effective fluorescent pseudomonads in controlling Sclerotium rolfsii causing collar rot of groundnut Influence of antagonistic fluorescent pseudomonads on plant growth parameters of groundnut Performance of selected isolates of fluorescent pseudomonads in controlling bacterial wilt of tomato

11.

12.

Contd..

Table No. 13.

Title

Influence of antagonistic fluorescent pseudomonads on plant growth parameters of tomato Performance of selected fluorescent pseudomonads in controlling root knot nematode in tomato Influence of antagonistic fluorescent pseudomonads on plant growth parameters of tomato Influence of fluorescent pseudomonads parameters of groundnut at 50 DAS on plant growth

14.

15.

16.

17.

Influence of fluorescent pseudomonads on plant parameters and nutrient uptake by groundnut at 50 DAS Influence of fluorescent pseudomonads parameters of groundnut at harvest on plant

growth

18.

growth

19.

Influence of fluorescent pseudomonads on yield and nutrient uptake by groundnut

LIST OF FIGURES

Figure No.

Title

1.

Number of plant pathogens inhibited in vitro by fluorescent pseudomonads

2.

Control of sclerotial rot and dry matter content in groundnut plants simultaneously inoculated with fluorescent pseudomonads and Sclerotium rolfsii

3.

Control of bacterial wilt and dry matter content in tomato plants simultaneously inoculated with fluorescent pseudomonads and Ralstonia solanacearum

4.

Influence of fluorescent pseudomonads on root gall formation and dry matter content of tomato plants inoculated with Meloidogyne incognita

5.

Dry matter yield and pod yield (g) of groundnut plants as influenced by fluorescent pseudomonads

6.

Nutrient uptake in groundnut plants as influenced by fluorescent pseudomonads

LIST OF PLATES

Plate No.

Title

1.

In vitro inhibition of Rhizoctonia bataticola by fluorescent pseudomonads

2.

In vitro inhibition pseudomonads

of

Sclerotium

rolfsii

by

fluorescent

3.

In vitro inhibition of Ralstonia solanacearum by fluorescent pseudomonads

4.

HCN production by fluorescent pseudomonads

5.

Siderophore production by fluorescent pseudomonads

6.

Control of sclerotial rot of groundnut plants simultaneously inoculated with fluorescent pseudomonads and Sclerotium rolfsii

7.

Control of bacterial wilt of tomato plants simultaneously inoculated with fluorescent pseudomonads and Ralstonia solanacearum

8.

Biocontrol activity of fluorescent pseudomonads Meloidogyne incognita inoculated to tomato plants

against

9.

Plant growth promotional activity of fluorescent pseudomonads in groundnut

I. INTRODUCTION
Modern agriculture apart from improving the overall production and productivity has also caused destruction to the environment. The use of chemical fertilizers has been necessitated due to cultivation of high yielding varieties. This has resulted in degradation of soil health (Cook, 1991). Hence the alternative methods are being envisaged in an ecofriendly approach aimed at sustainable agriculture. While organic manures like FYM, compost, vermicompost, green manure etc. are satisfactory sources for the supply of plant nutrients, we are yet to find suitable alternatives to pesticides for the control of insect pests and diseases of crop plants. Improvement in agricultural sustainability requires optimal use and management of soil fertility and soil physical properties both of which rely on soil biological processes and soil biodiversity. Biological methods offer an excellent alternate strategy for effective control of various diseases and plant growth promotional activity. Several soil microorganisms are known to improve the plant growth directly through nutrient mobilization and production of plant hormones and indirectly through suppression of plant pathogens or by inducing systemic resistance in plants. The soil microorganisms that enhance nutrient availability to plants include nitrogen fixers, P solubilizers, IAA, and GA producers, which have been used to develop biofertilizers. Other soil microorganisms, which possess the ability to improve plant growth indirectly through suppression of disease causing organisms or by inducing systemic resistance in plants, have also been developed as bioinoculants. Many soil microorganisms possess multiple beneficial traits of nutrient mobilization, production of plant growth promoting substances (PGPS) and biocontrol ability. Such organisms have a greater role in sustaining agricultural production. Plant growth promoting rhizobacteria (PGPR), a group of root associated bacteria, intimately interact with the plant roots and consequently influence plant health and soil fertility. They offer an excellent combination of traits useful in disease control and plant growth promotion. Amongst the PGPRs, fluorescent pseudomonads have emerged as the largest and potentially the most promising group of PGPR with their rapid growth, simple nutritional requirements, ability to utilize diverse organic substrates and mobility. Fluorescent pseudomonads produce highly potent broad spectrum antifungal molecules against various phytopathogens, thus acting as effective biocontrol agents. They are well equipped as primary root colonizers. Through several mechanisms viz., production of antibiotics (Gutterson et al., 1988), siderophores (Kloepper et al., 1980), HCN (Defago et al., 1990) and competition for space and nutrients (Elad et al., 1987), they inhibit soil borne plant pathogens. They could serve as promising bioinoculants for agricultural system to increase productivity since the action of such bacteria is highly specific, ecofriendly and cost-effective. However, selecting a potential strain with multiple beneficial traits for production of inoculants from natural biodiversity hot spots assumes significance. Western Ghats is one of the 18 biodiversity hot spots in the world. It hosts for many diversified animals, plants and microorganisms. In an attempt to study the diversity of bacteria in Western Ghats of Uttar Kannada district, a large number of fluorescent pseudomonads were isolated from the forest soils, characterized and maintained in culture library of the Department of Agricultural Microbiology, University of Agricultural Sciences, Dharwad. Several of these isolates have been screened for their beneficial traits under in vitro conditions by the previous workers (Suneesh, 2004; Megha, 2006). Some of them were found to posses in vitro biocontrol potential against one or two pathogens and were also capable of producing plant growth promoting substances in culture medium in addition to solubilizing tricalcium phosphate in Pikovskyas agar. Using a few of these efficient strains an attempt was made to investigate their biocontrol potential against different pathogens as well as their plant growth promotional activity in the present study with the following objectives:

1.

To evaluate the biocontrol activity of selected fluorescent pseudomonads isolated from Western Ghats against plant pathogenic fungi, bacteria and a nematode under in vitro condition.

2. 3.

Evaluation of biocontrol potential of selected efficient fluorescent pseudomonads in soil. Evaluation of plant growth promotional activity of selected efficient fluorescent pseudomonads.

II. REVIEW OF LITERATURE


Growth and yield of crop plants are influenced by a myriad of abiotic and biotic factors. While growers routinely use physical and chemical approaches to manage the soil environment to improve crop yields, the application of microbial inoculants for this purpose has gained significance in the recent past. The microbial inoculants that are used in agriculture include biofertilizers, biocontrol agents, plant growth promoting rhizobacteria etc. While the biofertilizer organisms make the nutrients available to plants, biocontrol agents protect the plants against the pathogenic organisms and insect pests, where as the growth promoting rhizobacteria enhance the plant growth by various mechanisms. These beneficial microorganisms can be a significant component of management practices to achieve sustainable yields. Literatures pertaining to the plant growth promotion and biocontrol activity of PGPRs is reviewed here under. Plant growth promoting rhizobacteria (PGPR) were first defined by Kloepper and Schroth (1978) as the soil bacteria that colonize the roots of plants by following inoculation on to seed and that enhance plant growth. PGPRs enhance plant growth by direct and indirect means, but the specific mechanisms involved have not been well characterized (Glick, 1995 and Kloepper, 1993). Direct mechanisms of plant growth promotion by PGPR can be demonstrated in the absence of plant pathogens or other rhizosphere microorganisms, while indirect mechanisms involve the ability of PGPRs to reduce the deleterious effects of plant pathogens on crop yield. PGPRs have been reported to directly enhance plant growth by a variety of mechanisms, viz., fixation of atmospheric nitrogen that is transferred to the plants, production of siderophores that chelate iron and make it available to the plant roots, solubilization of minerals such as phosphorous and synthesis of phytohormones (Glick, 1995). Direct enhancement of mineral uptake due to increase in specific ion fluxes at the root surface in the presence of PGPR has also been reported (Bashan and Levanony, 1991; Bertrand et al., 2000). PGPR strains may use one or more of these mechanisms in the rhizosphere. Molecular approaches using microbial and plant mutants altered in their ability to synthesize or respond to specific phytohormones have increased our understanding of the role of phytohormone synthesis as a direct mechanism of plant growth enhancement by PGPRs (Glick, 1995; Persello-Carticaux et al., 2003). PGPRs that synthesize auxins and cytokinins or those that interface with plant ethylene synthesis have been identified (Garcia et al., 2001; Glick, 1995; Percello-Carticaux et al., 2003). The indirect means by which PGPRs enhance plant growth is through suppression of phytopathogens by a variety of mechanisms. These include the ability to produce siderophores that chelate iron, making it unavailable to pathogens; the ability to synthesize anti-fungal metabolites such as antibiotics, fungal cell wall lysing enzymes or hydrogen cyanide, which suppress the growth of fungal pathogens. The ability to successfully compete with pathogens for nutrients or to exclude specific niches on the root and the ability to induce systemic resistance in plants are the other mechanisms (Bloemberg and Lugtenberg, 2001; Glick, 1995 and Persello-Carticaux et al., 2003).

2.1 Biocontrol of plant pathogens


The concept of biocontrol of plant diseases includes disease reduction or decrease in inoculum potential of a pathogen brought about directly or indirectly by other biological agencies (Johnson and Carl, 1972). Outside the host, the biocontrol agent may be antagonistic and there by reduce the activity, efficiency and inoculum density of the pathogen through antibiosis, competition and predation/hyper parasitism. This leads to a reduction in inoculum potential of the pathogens (Baker, 1977). The biocontrol agent may operate primarily in the host tissue, there by indicating a resistance response in the host, by transmitting factors that render the pathogens avirulent (Cook and Baker, 1983). These interactions are mediated by environment and may have an overriding impact in determining whether biocontrol operates in a system or not.

2.2 Rhizobacteria as biocontrol agents


The PGPRs that enhance plant growth by controlling deleterious microorganisms can also exhibit biocontrol of parasitic pathogens. A list of pathogens controlled by rhizobacteria is given in Table 1.

Table 1. List of plant pathogens controlled by rhizobacteria

Rhizobacteria Pseudomonas fluorescens P. putida P. putida A-12

Pathogens controlled Erwinia carotovora Thielaviopsis basicola Erwinia sp. Fusarium oxysporum f.sp. cucumerinum Sclerotium rolfsii Rhizoctonia solani

Reference Kloepper (1983) Ahl et al. (1986) Colyer and Mount (1984) Simeoni et al. (1987)

Upadhyay and Jayaswal (1992) Fridlender et al. (1993) ; Sanchez et al. (1994)

P. cepacia Sclerotium rolfsii Pythium ultimum P. cepacia RB-425 P. cepacia RB-425 P. cepacia 5.5 B P. aeruginosa 7 NSK2 Rhizoctonia solani Pyricularia oryzae Verticillium dahliae Rhizoctonia solani Pythium sp Penicillium sp. Phomopsis sp. Alternaria citri Bacillus subtilis Geotrichum cardidum Rhizoctonia solani Fusarium sp. Alternaria radicina

Homma et al. (1991) Homma et al. (1989)

Cartwright et al. (1995) Buysens et al. (1993) Cubeta et al. (1985)

Singh and Deverall (1985)

Haral and Konde (1986) Obieglo et al. (1990) Hentschel (1991)

2.3 Fluorescent pseudomonads as biocontrol agents


The fluorescent pseudomonads in addition to their ability to aid plant growth promotion are also good biocontrol agents.

2.3.1 Inhibition of fungal pathogens by fluorescent pseudomonads


A strain of Pseudomonas fluorescens showed antagonistic property against Rhizoctonia solani (Howell and Stipanovic, 1979). A number of Pseudomonas fluorescens strains isolated from the rhizosphere of potato plants were reported to be antagonistic to Rhizoctonia solani in vitro and effectively reduced stem canker under laboratory conditions (Chand and Logan, 1984). Several strains of siderophore producing P. fluorescens have been shown to inhibit Fusarium oxysporum f.sp. cubense, Fusarium oxysporum f.sp. vasinfectum, Rhizoctonia solani, Acrocylindrium oryzae, Xanthomonas campestris pv oryzae and P. syringiae pv phaseolicola (Sakthivel et al., 1986). Sedra and Maslouty (1994) studied six antagonists from 420 samples obtained from conducive and suppressive soils, for their inhibitory activity against Fusarium oxysporum f.sp albedinis. The antagonists included four Pseudomonas fluorescens isolates (M-B1, M-B2, M-B3 and M-B4), Stachybotrys sp. and an unidentified actinomycete. They suppressed the development of F. oxysporum f sp albedinis in vitro by 24-47 per cent and its sporulation by 70-99 per cent. Sitther and Gnamanickam (1996) screened six strains of fluorescent pseudomonads belonging to Pseudomonas fluorescens and P. putida for their ability to inhibit ragi blast fungus, Pyricularia grisea, by dual plate assays in the laboratory and found them to inhibit the fungal pathogen. Gupta et al. (1999) isolated P. aeruginosa from potato rhizosphere that displayed the strong antagonistic activity against important fungal pathogens viz., Macrophomina phaseolina and Fusarium oxysporum. Rangeshwaran and Prasad (2000) reported that Pseudomonas putida completely inhibited S. rolfsii in dual culture. Petri plate assay revealed that the Pseudomonas sp. exhibited antifungal activity against the plant pathogens, Pythium ultimum, Rhizoctonia solani, Phytophthora capsici, Botrytis cinerea and Fusarium oxysporum (Kim et al., 2000). Pseudomonas fluorescens was reported to be antagonistic to Aspergillus flavus, Sclerotium rolfsii and Aspergillus niger in vitro (Pal et al., 2001). Tripathi and Johri (2002) studied in vitro biocontrol potential of fluorescent pseudomonads recovered from rhizoplane and rhizosphere of pea and wheat against Colletotrichum dematium, Rhizoctonia solani and Sclerotium rolfsii and found them to restrict the growth of all the three pathogens but were most effective against Rhizoctonia solani. Fluorescent pseudomonad isolates tested against Phytophthora capsici on potato dextrose agar (PDA) and carrot agar (CA) by dual culture technique was found to inhibit the growth of pathogen in 5-7 days (Anith et al., 2002). Pseudomonads suppress soil-borne fungal pathogens by producing antifungal metabolites such as pyoluteorin, pyrrolnitrin, phenazines, and 2,4-di-acetyl phloroglucinol (Deepti and Johri, 2003). Pseudomonas aeruginosa RS B29 is reported to inhibit the growth of Fusarium oxysporum f.sp. ciceri, Fusarium udam, Fusarium solani, Rhizoctonia solani and Macrophomina phaseolina under in vitro conditions by producing an antifungal metabolite (Saikia et al., 2004). The culture of pseudomonads cultures showing large variations (0 - 130%) in inhibiting Aspergillus niger, Curvularia lunata, Rhizoctonia solani, Fusarium oxysporum and Pythium aphanidermatum under in vitro condition has been reported (Srivastava et al., 2004). Pseudomonas sp. RSB29 showing significant inhibition of fungal pathogens viz., Fusarium oxysporum f. sp. ciceri RS1, Macrophomina phaseolina RSB9, Fusarium udum RSB19, Fusarium solani RSB38 and Rhizoctonia solani BH49 has been reported by Saikia et al. (2004). Out of 45 isolates of fluorescent pseudomonads isolated from the rhizosphere of sunflower, 26 had antagonistic effects on the sclerotia of S. sclerotiorum (Behboudi et al., 2005). Fluorescent pseudomonads were selectively isolated from black pepper (Piper nigrum) roots and screened for volatile and non-volatile metabolite production and inhibition of growth of Phytophthora capsici, the causal organism of foot rot disease. Among the isolates tested, the inhibition of P. capsici varied from 36.3 to 70.0% by non-volatile metabolites and from 2 to 23% by volatile metabolites. Isolate IISR-51 caused maximum inhibition of P. capsici by production of non-volatile and volatile metabolites (Diby et al., 2005). Fluorescent pseudomonads are also known to benefit mushroom production by promoting formation of primordia and enhanced the development of the basidiome of Pleorotus ostreatus (Cho et al., 2003).

Kaiser et al. (1989) reported seven effective strains of fluorescent pseudomonads to suppress seed rot and pre-emergence damping off in chickpea caused by Phythium ultimum, the most significant causal agent of damping off of sugar beet was suppressed by fluorescent pseudomonads (Martin and Loper, 1999; Gill and Waren, 1988). Tripathi and Johri (2002) studied the biocontrol potential of fluorescent pseudomonads isolated from rhizoplane and rhizosphere of pea and wheat using in vivo biocontrol action in maize against sheath blight caused by Rhizoctonia solani. They found some isolates to possess multiple disease control potential, while some others exhibited biocontrol potential against specific pathogens which indicating that fluorescent pseudomonads are diverse with respect to their biocontrol potential. Six effective Pseudomonas fluorescens cultures isolated from onion rhizosphere were screened for their ability to suppress the onion wilt in vivo by soil and seed treatments. In soil treatment, the isolates Pf22 and Pf52 showed 56% and 51% reduction of the Fusarium wilt of onion where as in seed treatment the isolate Pf22 recorded 41% disease reduction (Tehrani and Ramezani, 2003). About 600 plant-associated bacterial isolates obtained by different methods were screened for suppressive effects in wheat against infection of Fusarium culmorum. Although most of the isolates tested had a neutral effect on test plants and disease development, a few were synergistic to the pathogen and about one-fifth showed >80% disease suppression (Johansson et al., 2003). Ahmadzadeh et al. (2003) reported that antagonistic rhizobacteria, more specifically fluorescent pseudomonads and certain Bacillus species possessed the ability to control fungal and bacterial root diseases of agronomic crops. Seed treatment with P. aeruginosa RS B29 controlled Fusarium wilt and charcoal rot disease of chickpea in both green house and field conditions (Saikia et al., 2004). Six isolates of fluorescent pseudomonads obtained from pathogen suppressive soil of a pigeonpea (Cajanus cajan) field were reported to have biocontrol potential against wilt disease complex in both laboratory and screen house (Siddiqui et al., 2005). Manoranjitham and Prakasam (2000) showed that seed treatment with talc-based formulations of Trichoderma viride and Pseudomonas fluorescens effectively reduced the pre and post emergence damping off of chillies caused by Pythium aphanidermatum. The effect on damping off was more pronounced when two biocontrol agents were used simultaneously compared to their individual effect. T. viride at 4 g/kg + P. fluorescens at 5 g/kg recorded 31.65, 66.66 and 37.58% increase in shoot length, root length and dry matter production over the uninoculated control respectively. P. fluorescens isolated from rhizosphere of rape seedlings has been reported to inhibit Fusarium roseum and Pythium ultimum (Dahiya et al., 1988). Dileep Kumar and Dubey (1992) reported that bacterization of chickpea and soybean seeds with a siderophore producing fluorescent pseudomonad (RBT13) isolated from the tomato rhizoplane, resulted in reduced wilt disease by 52 per cent in wilt sick soil. Gupta et al. (2001) isolated a fluorescent pseudomonad (GRC2) from potato rhizosphere and showed antibiosis against two major plant pathogens viz., Macrophomina phaseolina and Sclerotium sclerotiorum. Bare root dip treatment or soil drench with Pseudomonas aeruginosa strain IE-6S and Pseudomonas fluorescens CHA0 significantly suppressed Rhizoctonia solani of tomato (Siddiqui and Shaukat, 2002). Fusarium wilts were suppressed by the activity of species and non pathogenic strains of Fusarium oxysporum (Boer et al., 2003). Senthil et al. (2003) investigated the efficacy of talc formulated plant growth promoting rhizobacteria (PGPR) Pseudomonas spp. (5 strains; Pf1, VPT4, EP1, KKMI and VPT10) against red rot of sugercane (Colletotrichum falcatum [Glomerella tucumanensis]) in endemic locations of Coimbatore, Tamil Nadu, India, and found all of them to induce systemic resistance against the fungi in sugarcane plants. P. fluorescens strains isolated from rhizosphere of rice, wheat, pigeon pea, groundnut and chilli crops produced extra cellular siderophores which were antagonistic to fungal pathogens like Fusarium oxysporum, Alternaria sp and Colletotrichum capsicii (Suryakala et al., 2004). Pseudomonas fluorescens strain A 506 is a commercially available biological control agent (Available in the name of blight Ban 506; N. farm Americas Inco., Sugar Land, TX) used for the suppression of fire blight on pear and apple trees (Temple et al., 2004).

2.3.2 Inhibition of bacterial pathogens by fluorescent pseudomonads


Fluorescent pseudomonads are effective against bacterial pathogens also. It is known that the extent of inhibition zone formation is related to the ability of the organism to produce inhibitory metabolites against the test organism (Sivaprasad, 2002). Manmeet and

Thind (2002) evaluated in vitro antagoniststic activity of Bacillus subtilis, Pseudomonas fluorescens, Trichoderma harzianum and Penicillium notatum against Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight of rice, and found B. subtilis, P. fluorescens and T. harzianum to inhibit the pathogen. A foliar isolate of Pseudomonas putida (YLFP 14) was found to significantly reduce disease severity of bacterial spot caused by X. axonopodis pv. vesicatoria in sweet pepper under controlled conditions of growth chamber (Tsai et al., 2004). The fluorescent pseudomonad strain RDV 107 has been reported to be suppress the bacterial wilt of tomato caused by Ralstonia solanacearum (Jagadeesh, 2000). Significant reduction in bacterial wilt disease incidence in tomato was obtained with Pseudomonas fluorescens (Pradeep Kumar and Sood, 2001). Among the 3 native in vitro efficient isolates of fluorescent pseudomonads tested against R. solanacearum in chilli and tomato under green house conditions, isolate P1 was found to be the most effective followed by P13 and P11 (Meenakumari et al., 2003) indicating the potential of native fluorescent pseudomonads in suppressing bacterial wilt of tomato and chilly. Yanqing et al. (2004) observed reduced infection of Ralstonia solanacearum in tomato plants treated with the Nitrous Oxide (NO) overproducing transformants of fluorescent pseudomonad as compared to its wild type, suggesting the possibility of increasing the level of NO production through genetic modification of pseudomonads as a new approach to enhance their biocontrol efficacy. The culture liquid of Pseudomonas fluorescens 41 in a dilution of 1:9 is reported to control major cotton diseases caused by Xanthomonas campestris pv malvaceaum., Rhizoctonia solani, Fusarium vasectum and Verticillium dahliae, and also had the stimulating effect on seedling emergence and early growth and yield of cotton (Safiyazou et al., 1995). Pseudomonas fluorescens pv fcp reduced the incidence of P. solanacearum by 50 per cent in banana, 49 per cent in brinjal and 36 per cent in tomato (Anuratha and Gananamanickam, 1990). Out of 41 strains of fluorescent pseudomonads and Bacillus species tested as potential biological control agents against damping off and root rot fungal diseases caused by Pythium ultimum in common bean [Phaseolus vulgaris], only two strains viz., Pf16 and Pf26 significantly increased the fresh weight of bean plants inoculated with Pythium ultimum under green house condition (Ahmadzadeh et al., 2003).

2.3.3 Inhibitory activity of fluorescent pseudomonads against phytopathogenic nematodes


Biocontrol methods for integrated management of plant parasitic nematodes involving various types of antagonistic organisms have been developed (Jatala, 1986). The production of antibiotics like 2, 4-diacetylchloroglucinol by fluorescent pseudomonads, which reduce juvenile mobility has been reported by Cronin et al. (1997). The culture filtrate of P. fluorescens strain PF1 has also been reported to be toxic to Rotylenchulus reniformis at all tested concentrations (Jayakumar et al., 2002). Nematode suppressive potential of two high phosphate solubilizing Pseudomonas fluorescens strains, SCEP3 and SCEP5 was quantified using mortality of second stage juveniles and hatching inhibition in bacterial culture filtrate. Both the strains showed mortality of juveniles and inhibited egg hatching significantly over control (Somasekhar et al., 2003). Jayakumar et al. (2003) evaluated Pseudomonas fluorescens isolates of cotton rhizosphere against Reniform nematode in cotton and observed significant reduction in soil and root population of all Rotylenchulus reniformis and subsequent increase in plant growth parameters in plants inoculated with Pseudomonas fluorescens. A significant increase in plant growth and reduction in root knot galls was observed due to Pseudomonas fluorescens in okra plants (Devi and Dutta, 2002). Seenivasan and Lakshmanan (2003) also reported significant reduction in the population of rice root nematode Hirschmanniella gracilis in rice plants inoculated with Pseudomonas fluorescens isolates of rice rhizosphere. Jothi et al. (2003) reported tomato plants to give maximum yield (64.3%) and minimum soil population of root knot nematode (Meloidogyne incognita) in plants treated with P. fluorescens. Similar effect of P. fluorescens in tomato and brinjal nurseries against M. incognita has been reported by Santhi and Sivakumar (1995). Shanthi et al. (2003) found soil application of Pseudomonas fluorescens effective in reducing soil population and root population of lesion nematodes viz., Radopholus similis, Pratylenchus coffeae and Helicotylenchus multicinctus as much as the application of standard chemical carbofuron 3G. Fluorescence produced by Pseudomonas

was found to have inhibitory effect on the hatching and penetration of nematodes and colonization of pigeonpea roots (Siddiqui et al., 2005).

2.4 Mechanisms of biocontrol by fluorescent pseudomonads


Fluorescent pseudomonads fit into one of the three categories viz., pathogens, biodegraders and root colonizers or biocontrol agents. The last category exerts a protective effect on the roots through antagonism against phytopathogenic fungi and bacteria (Dwivedi and Johri, 2003). Fluorescent pseudomonads exhibit diverse mechanisms of biocontrol which include antibiosis, HCN production, siderophore production, competition for space and nutrients and induced systemic resistance.

2.4.1 Cyanide production


Cyanide ion is metabolized mainly to thiocyanate. The cyanide ion is exhaled as HCN and metabolized to lesser degree to other compounds. HCN inhibits the electron transport thereby energy supply to the cells is disrupted leading to the death of the organism. It affects the proper functioning of the enzymes and natural receptors by reversible mechanisms of inhibition (Corbett, 1974). It is also known to inhibit the action of cytochrome oxidase (Gehring et al., 1993). Hydrocyanic acid (HCN) is produced by many rhizobacteria and is postulated to play a role in biological control of pathogens (Defago et al., 1990). Voisard et al. (1989) presented evidence that HCN is involved in biological control by Pseudomonas flourescens strain CHA0. Cyanide producing strain CHA0 stimulated root hair formation, indicating that the strain induced altered plant physiological activities. Keel et al. (1989) developed a disease assay for Thielaviopsis basicola on tobacco using Fe rich clays, which were conducive to biocontrol by strain CHA0. The Tn5 generated mutant strain CHA5, which lacked HCN production gave significantly less control than CHA0. They opened two possible actions of HCN for the observed biocontrol, which include direct inhibition of Thielaviopsis basicola on roots, without damaging the plants or induce plant defense mechanisms. Ramette et al. (2003) reported that hydrogen cyanide (HCN) is a broad spectrum antimicrobial compound involved in biological control of root diseases by many plant associated fluorescent pseudomonads. Further, they noted that the enzyme HCN synthase is encoded by three biosynthetic genes (henA, henB and henC).

2.4.2 Siderophore mediated biocontrol


Siderophores are low molecular weight ferric iron chelating compounds that are secreted extracellularly under iron limiting conditions and whose main function is to supply iron to the iron starved cells. Some PGPR strains produce siderophores that bind Fe3+, making it less available to certain members of native microflora (Kloepper et al., 1980). The strains of rhozobacteria that produce siderophore under Fe limiting conditions in the rhizosphere chelate Fe3+, the form that is insoluble in water, hence not available to bacteria (Neilands, 1981; Leong, 1986). Isolates belonging to Pseudomonas fluoresens were reported to produce extracellular siderophores when grown in CAS under iron deficiency (Suryakala et al., 2004). Instant golden yellow colour is a positive test for siderophore production on succinate medium and casamino acid medium (CAA). Manwar et al. (2000) reported that the amount of siderophores produced by Pseudomonas (SC1 and SC4) on different media were in the range of 50 to 57 (per cent of siderophore units). Most evidences to support the siderophore theory of biological control by rhizobacteria comes from the work with pyoverdin, a class of siderophores that comprise the fluorescent pigment of fluorescent pseudomonads (Demange et al., 1987). Hofte et al. (1991) reported pyoverdin production by Pseudomonas aeruginosa 7 NSK 2 that was able to increase the yield of barley, wheat, maize, cucumber and spinach. Suryakala et al. (2004) suggested that tri-hyobroxamate siderophores might be exploited as potent biocontrol compounds against plant pathogens. Becker and Cook (1988) suggested the role of siderophores in suppression of Pythium species and increased growth response of wheat by fluorescent pseudomonads. Mutants of Pseudomonas flourescens 3551, deficient in siderophore production showed reduced potential to control damping off of cotton by Pythium ultimum than the parent strain (Loper, 1988). However, Kloepper (1993) is of the opinion that siderophores are not always

the prime mode of action for biological control by this group of bacteria. Manwar et al. (2000) reported in vitro suppression of plant pathogens through siderophore production by fluorescent pseudomonads. Suryakala et al. (2004) have reported that siderophores exerted maximum impact on Fusarium oxysporum than on Alternaria sp. and Colletotrichum capsici. Kurek and JaroszukScire (2003) reported that two P. fluorescens strains (resistant to streptomycin and kanamycin) produced Fe3+ chelating compounds (including siderophores) and inhibited the in vitro growth of F. culmorum (cycoheximide resistant strain) strain by competition for Fe3+. Through HPLC analysis, Hultberg et al. (2000) identified production of 2 pseudobactin/pyoverdine type siderophores by Pseudomonas fluorescens 5.014 where as its mutant 5-2/4 showed 47 and 33 % less production of the two siderophores.

2.4.3 Antibiosis
Antibiosis has been postulated to play an important role in disease suppression by rhizobacteria (Gutterson et al., 1986). A variety of secondary metabolites produced by fluorescent pseudomonads that exhibit antimicrobial properties are given in Table 2. The role of antimicrobial compounds in biocontrol has been studied by the generation of mutants that do not produce antibiotics. Pseudomonas fluorescens HV37a controls damping off in cotton caused by Pythium ultimum whereas one of its isogenic mutant deficient in the antifungal compound was significantly less effective in protecting cotton against P. ultimum (Gutterson et al., 1988). Phenazin deficient mutants of P. fluorescens 2-79 failed to inhibit Gaeumannomyces graminis pv triticii (Ggt) on media supportive of antibiotic production and were significantly less suppressive than the wild strain to take all of wheat (Thomashow and Weller, 1988). Similarly, the antibiotic-minus mutant of P. fluorescens NERLB-15135 exhibited reduced suppression of G. graminis pv triticii (Ggt) in green house experiments (Poplawsky et al., 1988). In field trials, the anti-fungal antibiotic minus mutants of P. fluorescens Pf 7-14 failed to control rice blast and sheath blight caused by Pyricularia sp and Rhizoctonia solani respectively (Chatterjee et al., 1996). P. fluorescens A1163 effective against P. ultimum is reported to produce an antimicrobial metabolite which was missing in the mutant (Rajendran et al., 1998). Antimicrobial compounds produced by Pseudomonas cepacia were reported to inhibit the radial growth of some important soil borne pathogens viz., Fusarium oxysporum, Macrophomina phaseolina, Sclerotium rolfsii, Rhizoctonia solani, and Pythium ultimum (Mansour et al., 1999). An antifungal antibiotic isolated from the culture filtrate of Pseudomonas sp. was identified as N-butylbenzenesulphonamide. The ED50 values of the Nbutylbenzenesulphonamide against P. ultimum, Phytophthora capsici, Rhizoctonia solani, and Botrytis cinerea were 73, 41, 33 and 102 ppm, respectively (Kim et al., 2000). Ahmadzadeh et al. (2004) reported that antimicrobial metabolites produced by antagonistic rhizobacteria play an important role in reducing most root diseases. Hydrogen cyanide, proteinase, siderophore and some antibiotic compounds produced by fluorescent pseudomonads have been identified and structurally characterized among which 2,4-diacetylphloroglucinol is the most important antibiotic. The mutants of biocontrol strain IC1270 of Serratia plymothica deficient in pyrrolnitrin production, developed through replacement of GrrA, GrrS and rpoS genes, were markedly less capable of suppressing Rhizoctonia solani and Pythium aphanidermatum under green house condition (Ovadis et al., 2004). Haas and Defago (2005) reported that during root colonization, fluorescent pseudomonads produce antifungal antibiotics, elicit induced systemic resistance in the host plant or interfere specifically with fungal pathogenicity factors.

2.4.4 Competition for infection sites and nutrients


Suslow (1982) reported that PGPR prevented deleterious rhizobacteria from colonizing sugar beet to their full potential, presumably because PGPR occupy and exclude deleterious rhizobacteria from the cortical cell junctions, where the exudation of the nutrient is maximal. Weller (1985) was of the opinion that pseudomonads catabolize diverse nutrients and have a fast generation time in the root zone. Hence, they are projected as logical candidates for biocontrol by competition for nutrients, more so against slow growing pathogenic fungi.

Table 2. Antimicrobial metabolites of fluorescent pseudomonads and the pathogens inhibited Metabolites Pseudomonas sp. Pathogen Reference Howell and Stipanovic (1980); Kraus and Loper (1992) Yashihima et al. (1989) Carmi et al. (1994) Hill et al. (1994) Brisbane and Revira (1988) Rosales et al. (1995) Vincent et al. (1991) Rosalel et al. (1995) Bangera and Thumathow (1996) Gutterson et al. (1986) Howie and Suslow (1991)

Pyoluteorin

P. fluorescens Pf-5

Pythium

P. cepacia Pyrrolnitrin P. fluorescens PEM-2 P. fluorescens BL915

R. solani R. solani R. solani Gaeumannomyces R. solani Gaeumannomyces R. solani Gaeumannamyc es Pythium ultimum P. ultimum

Phenazine-1carboxylic acid (PCA)

P. fluorescens 2-79

P. aeruginosa P. aureofaciens 2,4-diacetyl phloroglucinol (DAPG) P. cepacia P. fluorescens Q2-87

P. fluorescens Oomycin A P. fluorescens

Elad and Chat (1987) evaluated the antagonistic mechanisms of rhizobacteria that provided biocontrol against Pythium damping off and noted that the competition for nutrients between germinating oospores of Pythium aphanidermatum and biocontrol rhizobacteria correlated significantly with disease suppression. The dynamics of sugar beet seed colonization by Pythium ultimum and rhizobacterial biocontrol agents was investigated by Osburn et al. (1989) who observed competition as the mechanism of biocontrol where in the biocontrol agents protected the pericarp from occupation by the pathogen. Bell et al. (1990) studied the growth characteristics of five octapine catabolizing pseudomonads under octapine limitation in chemostats and their potential to compete with Agrobacterium tumefaciens. One of the P. fluorescens strain, E 175D was able to produce its peak population in the presence of octapine as nitrogen source. Competition models arrived at, based on pure culture parameters, and indicated that two of the Pseudomonas sp. dominated Agrobacterium tumefaciens B6 and ATCC 15955 when in simple competition for octapine as a limiting substrate. Mohamad and Caunter (1995) observed Pseudomonas fluorescens to inhibit Bipolaris maydis both in vitro and in vivo in infected maize plants but could not detect any inhibitory substances, assayed by a variety of methods, indicating nutrient competition as the operative component of antagonism.

2.4.5 Induced systemic resistance


Induced systemic resistance is broadly defined as activation of latent defense mechanisms in plants prior to pathogenic attack. The mechanism has been hypothesized in recent years to be an operable mechanism in several rhizobacterial systems. Induced systemic resistance is associated with increased synthesis of certain enzymes such as peroxidase (Lagrimini and Ruthstein, 1987), increased levels of certain acid soluble proteins (Zdor and Anderson, 1992) and the accumulation of phytoalexins in the induced plant tissue (Vanpeer et al., 1991). Fusarium wilt of carnation was significantly reduced by Pseudomonas sp. 417r through the mechanism of increased accumulation of phytoalexins that signal the systemic resistance in bacterized plants compared with non bacterized plants (Vanpeer et al., 1991). The seed bacterization of common bean with P. fluorescens S97 suppressed the haloblight caused by P. syringe pv phaseolicola through induced systemic resistance mechanism (Alstrom, 1991). The O-antigenic side chain of lipopolysaccharides (LPS) of P. flourescens WCS 374 also has been shown to induce systemic resistance against Fusarium oxysporum f sp. raphani in radish (Leeman et al., 1995). P. fluorescens induced systemic resistance against Rhizoctonia solani causing sheath blight in rice with a two fold increased activity of pathogenesis related peroxidase and chitinase proteins (Nanda Kumar et al., 1998). Choong et al. (2004) reported that plant growth promoting rhizobacteria in association with plant roots could trigger induced systemic resistance (ISR). Considering that low molecular weight volatile hormone analogues such as methyl jasmonate and methyl salicylate can trigger defense responses in Arabidopsis plants, the signaling pathway activated by volatiles from Bacillus subtilis GB03 is dependent on ethylene, albeit independent of the salicylic acid or jasmonic acid signaling pathways. Haas and Defago (2005) reported that fluorescent pseudomonads during root colonization elicit induced systemic resistance in the host plant or interfere specifically with fungal pathogenicity factors.

2.5 Fluorescent pseudomonads as PGPR


Fluorescent pseudomonads have emerged as the biggest and potentially the most promising group amongst the PGPRs involved in biocontrol of diseases (Suslow and Schroth, 1982). Fluorescent pseudomonads are gram negative, aerobic rods, motile with polar flagella and have the ability to produce water soluble yellow green pigment (Palleroni et al., 1973). They comprise the species of P. fluorescens (four bio types), P. putida (two bio types), P. aeruginosa, P. chlororaphis, P. aureofaciens and P. syringe (Schippers et al., 1987). They are well adapted to rhizosphere and rhizoplane, have a fast growth rate in the rhizoplane (Bowen and Rovira, 1976) and are able to utilize a large number of organic substrates (Stolp and Godkari, 1981) including root exudates (Rovira and Davey, 1974). The worldwide interest in this group of rhizobacteria was sparked off by the studies initiated at the University of California, Berkeley, USA during the 1970s. In 1978, Burr et al. reported that strains of P. fluorescens and P. putida applied to seed tubers improved the growth of potato. These findings were confirmed and later exemplified in the radish (Kolepper and Schroth, 1978), sugar beet (Suslow and Schroth, 1982), cotton (Sakthivel et al., 1986), vegetables (Elad et al., 1987), soybean (Polonenko et al., 1987), tomato and eggplant (Dileep Kumar and Dubey, 1993), groundnut (Vikram, 1997) and lentil (Rao et al., 1999). Fluorescent pseudomonads are known to produce plant growth promoting substances like, auxins, gibberrelins, cytokinines etc (Benizri et al., 1998; Suneesh, 2004). Thirty isolates of fluorescent pseudomonads from wheat rhizosphere were found to produce 1.1 to 12.1 mg of auxin per liter of medium without tryptophan and 1.8 to 24.8 mg per liter with tryptophan (Khalid et al., 2004). Suneesh (2004) recorded IAA and GA production in the range of 1.63 to 17.0 g and 0.72 to 5.27 g/ 25 ml broth by fluorescent pseudomonads isolated from moist deciduous forest of Western Ghats. IAA and GA production by 52 fluorescent pseudomonads ranged from 80 to 760 g and 24.82 to 262.8 g of GA per liter of broth respectively (Megha, 2006). Pal et al. (2003) reported four PGPRs strains belonging to fluorescent pseudomonads groups increased the root length, shoot length and pod yield of groundnut which were attributed to production of siderophores and IAA like substances. Two strains of fluorescent Pseudomonas sp. isolated from the potato epidermis and roots significantly increased growth

of potato plants up to 500 per cent greater than control in green house assays (Kloepper et al., 1980). Tomato, cucumber, lettuce and potato plants bacterized with plant growth promoting Pseudomonas strain showed increased root and shoot fresh weight and simultaneous suppression of deleterious pathogenic microflora was observed (Vanpeer and Schippers, 1989). Dileep Kumar and Dube (1992) reported that bacterization of chickpea and soybean seeds with a siderophore producing fluorescent pseudomonad RBT13 isolated from the tomato rhizoplane, resulted in increased seed germination, growth and yield of plants. Inoculation of wheat plants with Pseudomonas fluorescens in potted soil naturally infested with Gaeumannomyces graminis var tritici, showed 29 per cent higher grain yield over untreated control (Mroz et al., 1994). Seed treatment of radish with Pseudomonas fluorescens WCS 374 reduced the incidence of Fusarium wilt and increased the yield from 19.5 to 100 per cent in greenhouse conditions (Leeman et al., 1995). Plant growth promoting strains of Pseudomonas fluorescens ANP15 and Pseudomonas aeruginosa 7 NSK-2 were found to protect maize seeds from cold stock damage and significantly increased the seed germination as well as dry matter content of inoculated plants (Hofte et al., 1991). Pierson and Weller (1994) tested the ability of fluorescent pseudomonas strains either alone or in combinations to suppress take all in fields infested with Gaeumannomyces graminis var triticii, and found the combined application of fluorescent pseudomonas strains to increase the yield by 20.4 per cent over untreated control and performed better over singly or individually applied treatments. Pseudomonas species isolated from rhizosphere of fir and spruce plants in forest floor of Kashmir valley have been reported to increase the height, number of leaves, girth and weight of fir and spruce plants significantly in addition to enhancing the nitrogen, phosphorous and potassium contents of plants (Zarger et al., 2005). Significant increase in seedling weight, mature root weight and total sucrose yield of sugar beet has been reported due to seed inoculation of fluorescent Pseudomonas sp. under field condition (Suslow and Schroth, 1982). Vrany and Fiker (1984) recorded 4-30 per cent improvement in plant growth and tuber yield of potato inoculated with P. fluorescens under field conditions. Yuen and Schroth (1986) observed 18 - 41 per cent increase in the fresh top weight of carnation, sunflower, vinca and zinnia due to inoculation of P. fluorescens to the seeds or rooted cuttings. Kaiser et al. (1989) studied effectiveness of seven strains of fluorescent pseudomonads to suppress seed rot and pre emergence damping off in chickpea caused by Pythium ultimum and found two strains of Pseudomonas fluorescens, viz., Q29z 80 and M8z-80, to increase emergence and yield of chickpea as compared to untreated controls. Walley and Germida (1997) observed enhancement of shoot dry weight from 16 to 48 per cent and root dry weight from 82 to 137 per cent when inoculated with fluorescent pseudomonads. Gupta et al. (2002) reported that peanut seeds bacterized with Pseudomonas GRC2 showed a significant increase in germination (83%) under field conditions. Plant growth promoting fluorescent pseudomonad isolates, viz., PGPR1, PGPR2 and PGPR4, significantly enhanced pod yield (23-26%, 24-28% and 18-24%, respectively), haulm yield, nodule dry weight, root length, pod number, 100 kernel mass and shelling out turn over 3 years. The yield of sunflower inoculated with Pseudomonas sp, viz., PSI and PSII was increased by 32.8 per cent and 18.3 per cent, respectively over uninoculated control (Bhatia et al., 2005).

III. MATERIAL AND METHODS


The present study was undertaken at the Department of Agricultural Microbiology, University of Agricultural Sciences, Dharwad on the biocontrol potential and plant growth promotional activity of fluorescent pseudomonads isolated from soils of the Western Ghats in Uttar Kannada district, Karnataka state. The materials used and the methods employed in the investigation are outlined below in detail.

MICROBIAL CULTURES
3.1 Fluorescent pseudomonads
a) Isolates Nineteen fluorescent pseudomonads isolated from the forest soils of Western Ghats of Uttara Kannada district, Karnataka state characterized and maintained as culture bank in the Department of Agricultural Microbiology, University of Agricultural Sciences, Dharwad were used in the present investigations. The details of fluorescent pseudomonads used in present study are given in Table 3. b) Reference strains Pseudomonas fluorescens (NCIM 2099) and Pseudomonas aureofaciens (NCIM2026) obtained from National Collection of Industrial Microorganisms, Pune were used as reference strains for comparison of biocontrol efficiency.

3.2 Plant Pathogens


The following plant pathogenic fungi and bacteria obtained from the Department of Plant Pathology, University of Agricultural Sciences, Dharwad were used in the present investigations. Plant pathogenic fungi: Rhizoctonia bataticola, Sclerotium rolfsii, Fusarium udum and Pythium species Plant pathogenic bacteria: Ralstonia solanacearum, Xanthomonas campestris, Xanthomonas axonopodis.

3.3 In vitro screening of fluorescent pseudomonads for their biocontrol activity


All the nineteen isolates were tested in vitro for their biocontrol activity against plant pathogenic fungi, bacteria and nematode as detailed below. 3.3.1 Against fungal pathogens All the nineteen isolates were tested for their inhibitory activity against four fungal pathogens, viz., Rhizoctonia bataticola, Sclerotium rolfsii, Fusarium udum and Pythium species by dual culture technique (Ganesan and Gnanamanickam, 1987). Each fungal pathogen was grown on a PDA (Appendix) plate till it covered the whole surface of the agar. With the help of a sterile cork borer, a disc of fungal growth from this plate was taken and placed at the center of a fresh PDA plate. Twenty four hour old culture of each bacterial strain was then streaked parallelly on either side of the fungal disc 3 cm away from the disc. o The plates were kept for incubation at 30 C for 96 hours. Visual observations on the inhibition of growth of fungal pathogen were recorded after 96 hours of incubation in comparison with the PDA plate simultaneously inoculated with only the fungal pathogen.

Table 3. Details of fluorescent psuedomonads of western ghats used in the present study Sl. Isolates Forest No. No. type 1 47(2) MDF 2 433(1) DDF 3 427 MDF 4 76 SEF 5 73 DF 6 139(2) DF 7 440(1) DF 8 521(2) DF 9 494(2) CF 10 518(1) CF 11 327(2) MDF 12 173(3) MDF 13 203(1) MDF 14 227(2) MDF 15 234(1) MDF 16 309(2) MDF 17 313(1) EGF 18 331(2) MDF 19 361(1) MDF DDF Dry deciduous forest MDF Moist deciduous forest EGF Evergreen forest DF Degraded forest CF Coastal forest SEF Semi evergreen forest Location Yellapur Sirsi Caslerock Diggi Gunda - Shivapur Sirsi - Banavasi Sirsi Sorab Siddapur Sorab Caslerock - Diggi Gokarna Yellapur Murudeshwar Gokarana beach Kandra-Kodasalli Kesarolli-Dandeli Gobral near Dandeli Kumbarada Ulavi Ulavi Dandeli Bapoli cross Ramnagar Kadra Kesarolli Kadra Kesarolli Yellapur Satod falls Plant species Tectona grandis Acacia auriculformis Arbcarpus heterophyllus Lagestomea lanceolata Eucalyptus steretcorris Casurina equisetifolia Adna cardifolia Euginea jambulana Grass with sand on seashore Calotropis gigantus Teminalia paniculata Eupatorium odoratum Terminalia chebula Terminalia abla Terminalia paniculata Adna carolifolia Terminalia paniculata Embelca officinalis Dendrocalomus strictus Tentative identification Pseudomonas fluorescens Pseudomonas fluorescens Pseudomonas fluorescens Pseudomonas fluorescens Pseudomonas fluorescens Pseudomonas fluorescens Pseudomonas fluorescens Pseudomonas fluorescens Pseudomonas fluorescens Pseudomonas fluorescens Pseudomonas fluorescens Pseudomonas fluorescens Pseudomonas fluorescens Pseudomonas fluorescens Pseudomonas aureofaciens Pseudomonas fluorescens Pseudomonas fluorescens Pseudomonas fluorescens Pseudomonas fluorescens

3.3.2 Against bacterial pathogens The isolates were also screened for their inhibitory activity against three bacterial pathogens, viz., Ralstonia solanacearum, Xanthomonas campestris, Xanthomonas axonopodis by following the dual inoculation technique (Ganesan and Gnanamanickam, 1987). Overnight grown culture of each pathogen was added to lukewarm molten Kings B agar medium (Appendix) in 1: 10 ratio (pathogen: medium) and then poured into sterile petri plates and allowed to solidify. With the help of a sterile cork borer, the agar was scooped out to make wells in the medium. Ten microlitres of the overnight growth of fluorescent pseudomonads was poured into each well made previously in the plates. Plates were incubated upright at 30oC for 2-4 days. The plates were observed for the zone of inhibition around the wells. The wells receiving sterile distilled water served as control. 3.3.3 Against nematode pathogen The culture filtrate and the whole cell culture of the fluorescent pseudomonads were examined for their ability to immobilize or kill root knot nematode, Meloidogyne incognita, under laboratory condition by following the procedure of Jayakumar et al. (2002) as detailed below. 3.3.3.1 Preparation of culture filtrate of fluorescent pseudomonads The isolates of fluorescent pseudomonads were grown individually in100 ml Kings B broth for 7 days at 282o C. Twenty-five ml of the broth culture was centrifuged at 8000 rpm for 10 min, the supernatant obtained was passed through a bacteriological membrane filter (2 m) and the filtrate was used for bioassay against the root knot nematode. Remaining broth culture was used as whole cell culture for bioassay. 3.3.3.2 Nematode collection The egg masses of root knot nematode, Meloidogyne incognita were collected from the coleus (Coleus forskholii) plants grown in pots and were made to hatch in to juveniles by using modified Baermanns assembly. Then population of juveniles present in water suspension was counted under stereomicroscope and used for bioassay test. Procedure Three ml of the nematode suspension containing about 50 juveniles was taken in each petri plate of 5 cm diameter to which 10 ml of culture filtrate of fluorescent pseudomonad was added and kept for 72 hours for incubation. The plates containing nematode suspension, which received sterile water and autoclaved plain broth served as controls. Each treatment was replicated twice. At 24 hours, 48 hours and 72 hours of incubation, observations for immobilized and killed nematodes were recorded in each plate. In another set of plates containing nematode juveniles, 10 ml of whole cell culture was added. The plates were incubated for 24 hours and observed for immobilized and killed nematodes. The revival test was also conducted by transferring the immobilized nematodes into fresh water to confirm mortality in both the sets of plates.

3.4 Elucidation of mechanisms of biocontrol in isolates of fluorescent pseudomonads


3.4.1 HCN production (Wei et al., 1991) Whatman No. 1 filter paper pads were placed on the lids of Petri plates and the plates were sterilized. Trypticase soy agar (TSA) medium amended with glycine (4.4 g/l) (Appendix) was sterilized and poured in to the sterile plates. Each isolate showing biocontrol potential against different pathogens was streaked on the medium in triplicate plates. The filter padding in each plate was soaked with 2 ml of sterile picric acid solution (Appendix) under aseptic condition and the lids closed. The plates were sealed with parafilm in order to contain gaseous metabolites produced by the antagonists and to allow for chemical reaction with picric acid present in the filter paper padding. After incubation for a week at 30C, the colour change of the filter paper pad was noted and the HCN production potential of the antagonists was assessed as shown below:

No colour change (-) : no HCN production Brown colouration (+) : weak HCN production Brownish to orange (++) : moderate HCN production Complete orange (+++) : strong HCN production 3.4.2 Siderophore production (Schwyn and Neilands, 1987) All the glassware used in the test were soaked in 2N HCl solution for 24 hours to avoid contamination of iron from the glassware. 3.4.2.1 Preparation of CAS solution Dehydrated Chrome Azurol S (60.5mg) was dissolved in 50ml double distilled water and was mixed with 10ml of iron solution (1mM FeCl3. 6H2O in 10mM HCl). The solution was then slowly added with 40 ml aqueous solution containing 72.9 mg Cetyl trimethyl ammonium bromide with continuous stirring and the final solution was autoclaved. 3.4.2.2 Media preparation Kings B agar medium was prepared with PIPES (30.2 g) and Difco agar (18.0 g). The pH of the medium was adjusted to 6.80 by addition of 50% sodium hydroxide (NaOH) solution and autoclaved. After cooling, the CAS solution dye (100ml) was added along the glass wall with gentle agitation to achieve mixing without formation of foam. To each plate, CAS agar was added and the plates were kept in a refrigerator (4C) for 24 hours. 3.4.2.2.3 Inoculation of fluorescent pseudomonads isolates Overnight grown cultures of fluorescent pseudomonads were spotted on CAS agar plates and incubated at 28 2C for 48 hours. Formation of orange coloured zone around the colony was taken as positive for siderophore production. 3.4.3 Production of antimicrobial metabolites The antibiotic activity of the ten selected isolates (selected based on efficacy against different pathogens) was assessed by extracting the metabolites produced by them by following the method of Kraus and Loper (1995) as detailed below. The fluorescent pseudomonads were grown for 48 hours in 5 ml of nutrient broth amended with glucose (2% w/v). The broth culture was spun at 10,000 rpm for 10 minutes and the supernatant extracted with equal volume of chloroform. The metabolites were also extracted from the pellet using chloroform and the extract pooled. The upper aqueous layer was discarded and to the remaining chloroform phase, a pinch of sodium sulphate was added. It was spun at 8000 rpm for eight minutes and the sodium sulphate was pelleted. The clear layer was decanted and the chloroform was removed by flushing in air. The residue was redissolved in 200 l acetone and 70l of extract from this was spotted on to a Thin Layer Chromatography (TLC) plate. The plate was chromatogrammed using chloroform: acetone (9:1) as the solvent system. The plate was observed under UV light at 254 nm. The metabolites were marked and the Rf value calculated. The metabolites were eluted and redissolved in acetone: water. One hundred l of the eluted portion were centrifuged to pellet the silica gel and the clear suspension was further analyzed for the toxicity against the test pathogens. The metabolites were streaked on both sides of the disc of fungal pathogen as explained earlier where as for bacterial pathogens, the individual metabolite was poured in to previously made wells in the plates of Kings B agar which was preseeded with bacterial pathogens. Suitable controls without metabolites for each pathogen were also maintained. The plates were incubated for 48-72 hours at 30C and observations on growth inhibition were made visually.

3.5 Evaluation of biocontrol potential of fluorescent pseudomonads against sclerotial rot of groundnut under greenhouse condition
A pot culture experiment was conducted to assess biocontrol potential of fluorescent pseudomonads against the sclerotial rot of groundnut by dual inoculation of the pathogen (Sclerotium rolfsii) and the biocontrol agent (Ganesan and Gnanamanickam, 1987). The experiment was conducted with nine treatments consisting of 5 isolates and two reference strains of fluorescent pseudomonads, one absolute control (no pathogen and no biocontrol agent) and another control with only pathogen (ordinary control).

3.5.1 Preparation of giant culture of Sclerotium rolfsii Sacc. Sand corn meal medium (Abeyagunawrdena and Wood, 1957) was used to prepare the giant culture of Sclerotium rolfsii Sacc. It was prepared by mixing 95 parts of sand with 5 parts of maize grit. Twelve kg of sand corn meal medium was prepared and transferred to autoclavable polythene bags @ 500g in each bag and sterilized at 15 lbs per sq. inch for 30 minutes. The sterile medium in the bags was inoculated with fresh culture of Sclerotium rolfsii and incubated at 27 1C for 30 days. The giant culture with full cottony growth was used in the pot culture experiment. 3.5.2 Preparation of biocontrol agents The selected strains of fluorescent pseudomonads were inoculated to flasks containing 100 ml of Kings B broth and incubated at 30C for four days. The population of each isolate was estimated by serial dilution plate count method just before use. 3.5.3 Soil, fertilizers and preparation for sowing The medium black soil obtained from plot No.125 in E block of the Main Agriculture Research Station, University of Agricultural Sciences, Dharwad was used for the pot culture experiment. The soil was having a pH of 7.8, 0.37% organic carbon, 212 kg/ha of available nitrogen, 28 kg/ha of available phosphorus and 345 kg/ha of potassium. The soil had a 5 3 3 bacterial population of 4.5 x10 cfu/g, fungi 4.12 x 10 cfu/g, actinomycetes 2.81 x 10 cfu/g and fluorescent pseudomonads 6.42 x 102 cfu/g. The soil 4.5 kg/pot was filled into earthen pots of 5 kg capacity. Calculated quantities of NPK fertilizers and well decomposed FYM were applied to pots at the recommended level (25:50:25, NPK kg/ha and 10 t FYM/ha) just before sowing. The experiment consisted of nine treatments and the treatment details are given below: Treatment details T1 : Reference strain Psedomonads fluorescents NCIM 2099 + Sclerotium rolfsii T2 : Fluorescent pseudomonads (FP) 433(1) + Sclerotium rolfsii T3 : FP 234(1) + Sclerotium rolfsii T4 : FP 173(3) + Sclerotium rolfsii T5 : FP 427 + Sclerotium rolfsii T6 : FP 518(1) + Sclerotium rolfsii T7 : FP 227(2) + Sclerotium rolfsii T8 : Control (only Sclerotium rolfsii) T9 : Absolute control (No pathogen & no biocontrol agent) All the treatments except absolute control were added with 500 g of giant culture of Sclerotium rolfsii per pot. Bold, healthy, and uniform sized seeds of groundnut (var. TMV2) were sown in the pots @ five seed per pot. The seeds were inoculated with fluorescent pseudomonads @ 5 ml per seed and covered with soil. Six replications were maintained for each treatment. The pots were watered regularly to maintain soil moisture at 60 % of its WHC. After germination, thinning was done to retain two plants per pot. The plants were allowed to grow up to 70 days and observation on the per cent disease reduction (% DIR) was calculated by using the formula given below.

No. of plants infected in control No. of plants infected in treatment % DIR= x 100 No. of plants infected in control

The plant growth parameters, viz., number of branches, number of leaves, nodule number and plant dry matter yield were also recorded. The dry matter content of the plants was recorded after drying the plants to constant weight at 60 C.

3.6 Evaluation of biocontrol potential of fluorescent pseudomonads against Ralstonia solanacearum in tomato
A pot culture experiment was conducted using medium block soil obtained from plot no.125 in E block of the Main Research Station, University of Agricultural Sciences Dharwad to evaluate the biocontrol efficacy of the selected antagonistic fluorescent pseudomonads against Ralstonia solanacearum using tomato as test plants. The soil was filled at 2 kg/pot into plastic pots of 3 kg capacity. 3.6.1 Raising of tomato seedlings and treatment imposition Tomato seeds (var. Pusa Ruby) obtained from the Department of Horticulture, University of Agricultural Sciences, Dharwad were sown in sterile soil filled in earthen pots and the seedlings were raised following all the nursery practices. The seedlings were allowed to grow upto 30 days in nursery pots and used for transplanting. Thirty days old seedlings were uprooted gently and two seedlings were transplanted into each pot. The experiment consisted of nine treatments and the treatment details are given below. Treatment details T1 : Reference strain Psedomonads fluorescents NCIM 2099 + Ralstonia solanacearum T2 : Fluorescent pseudomonads (FP) 433(1) + Ralstonia solanacearum T3 : FP 234(1) + Ralstonia solanacearum T4 : FP 173(3) + Ralstonia solanacearum T5 : FP 427 + Ralstonia solanacearum T6 : FP 518(1) + Ralstonia solanacearum T7 : FP 227(2) + Ralstonia solanacearum T8 : Control (only Ralstonia solanacearum ) T9 : Absolute control (No pathogen & no biocontrol agent) As per treatment schedule, the antagonistic strains and bacterial pathogen (Ralstonia solanacearum) were grown separately and inoculated to the seedling roots @ 5 ml/seedling. The seedlings were allowed to grow upto 100 days with regular watering and after care. The seedlings were constantly monitored for the disease expression. The number of diseased plants in each pot were counted and the percent of disease reduction was calculated. The plant growth parameters, viz., stem girth, number of leaves, number of branches and dry matter content were also recorded. The dry matter content of the plants was recorded after drying plants to constant weight at 60C.

3.7 Evaluation of biocontrol potential of fluorescent pseudomonads against root knot nematode (RKN) in tomato
A pot experiment was conducted to evaluate biocontrol potential of fluorescent pseudomonads against the root knot nematode (Meloidogyne incognita) by dual inoculation method (Jothi et al., 2003) using tomato as test plants. Soil Red soil obtained from Horticultural Garden, University of Agricultural Sciences, Dharwad, was used in this experiment. The sandy loam soil had a pH of 5.6, EC-0.20 m mhos/cm, organic carbon 0.30 %, available N: P: K was 43.1:12.5:168.7 kg/ha respectively. The soil was mixed with washed riverbed sand in 1:1 ratio and filled @ 3 kg/pot in earthen pots of 3 kg capacity. The pots were sterilized in an autoclave at 15 lbs for 20 minutes intermittently for 3 times on alternate days. 3.7.1 Raising of tomato seedlings and treatment imposition Tomato (var. Pusa Ruby) seedlings were raised in sterile soil nursery pots for thirty days, and the seedlings were then uprooted and transplanted into pots @ two seedlings/pot.

After one week of transplantation, the treatments were imposed. Totally, there were 11 treatments and the treatment details are given below: Treatment details T1: Fluorescent pseudomonads (FP) isolate 518(1) + Meloidogyne incognita T2: FP isolate 433(1) + Meloidogyne incognita T3: FP isolate 313(1) + Meloidogyne incognita T4: FP isolate 234(1) + Meloidogyne incognita T5: FP isolate 173(3) + Meloidogyne incognita T6: FP isolate 440(1) + Meloidogyne incognita T7: FP isolate 427 + Meloidogyne incognita T8: Reference strain (Pseudomonas fluorescens NCIM 2099) + Meloidogyne incognita T9: Control (only Meloidogyne incognita) T10: Absolute control (no Meloidogyne incognita and no biocontrol agent) For all the treatments except absolute control, Meloidogyne incognita was inoculated @ 500 juveniles/pot. Similarly, the antagonistic strains of fluorescent pseudomonads were inoculated to the seedlings by adding 5ml of 48 hours old culture to each seedling as per treatment schedule. The plants were watered regularly to maintain optimum moisture. The plants were allowed to grow with appropriate care. When the plants started showing yellowing of leaves, which coincided with 50 days after transplantation, the plants were uprooted and observed for gall formation. The number of galls per plant was counted and per cent gall index was calculated. At the same time, plant growth parameters like, stem girth, number of leaves per plant and dry matter content were also recorded. The dry matter content of plants was recorded after drying the plants in an oven to constant weight at 60C.

3.8 Green house evaluation of fluorescent pseudomonads for their plant growth promotional activity
A pot culture experiment was conducted to study the influence of the selected isolates of fluorescent pseudomonads on the germination, growth and nutrient uptake of groundnut. Soil Medium black clayey soil obtained from the plot No.125 in E block of the Main Agricultural Research Station, University of Agricultural Sciences, Dharwad and used in previous experiments was also used for this experiment. The sieved soil was filled into earthen pots of 10 kg capacity (30 cm dia.) after mixing it with FYM (@ 10 t/ha on soil weight basis). The recommended dose of fertilizers (25 : 50 : 25 kg NPK/ha) was applied to each pot. N in the form of urea, P in the form of single superphosphate and K in the form of muriate of potash were used. Bold and healthy uniform sized seeds of GPBD-4 variety of groundnut obtained from the Groundnut Breeder, Department of Genetics and Plant Breeding, University of Agricultural Sciences, Dharwad were sown @ 6 seeds/pot. As per the treatment details, 24 h old culture of fluorescent pseudomonads were inoculated to the pots @ 5 ml/pot. The experiment was conducted with 22 treatments consisting of 19 isolates and 2 reference strains of fluorescent pseudomonads and an uninoculated control as detailed below: Treatment details T1: T2: T3: T4: T5: Fluorescent pseudomonad (FP) isolate 47 (2) FP isolate 73 FP isolate 76 FP isolate 139 (2) FP isolate 427

T6: T7: T8: T9: T10: T11: T12: T13: T14: T15: T16: T17: T18: T19: T20: T21: T22:

FP isolate 433 (1) FP isolate 440 (1) FP isolate 494 (2) FP isolate 518 (1) FP isolate 521 (2) FP isolate 327 (2) FP isolate 173 (3) FP isolate 203 (1) FP isolate 227 (2) FP isolate 234 (1) FP isolate 309 (2) FP isolate 313 (1) FP isolate 331 (2) FP isolate 361 (1) Reference strain (Pseudomonas aureofaciens NCIM 2026) Reference strain (Pseudomonas fluorescens NCIM 2099) Control (uninoculated)

After germination, thinning was carried out to retain 2 seedlings in each pot. The pots were watered regularly to maintain optimum moisture and other routine care was taken during the experimentation. Each treatment was replicated 5 times and of which 2 replications were used for recording observations at 50 days after sowing, and the remaining 3 replications were used for observations at harvesting. 3.8.1 Observations The observations on shoot length, root length, shoot and root dry weight, nodule dry weight, number of leaves per plant, number of branches per plant, plant N content and plant P content were recorded at 50 days after sowing and at harvest, whereas the yield parameters like number of pods per plant and pod yield were recorded at harvest. 3.8.2 Plant analysis The plant samples collected at 60 and at the time of harvest, dried and ground in a wiley mill and used for the estimation of nitrogen and phosphorus content by the standard procedures. The total nitrogen content in plant samples was estimated following the micro kjeldahl method (Jackson, 1973) where as phosphorus content in the plant samples was estimated by the Vanadomolybdate phosphoric yellow colour method of Jackson (1973). The N and P uptake was worked out by multiplying the N and P percentage with the corresponding dry matter yield and expressed in mg/plant. 3.8.3 Statistical Analysis The data were subjected to statistical analysis by employing completely randomized design (CRD). The critical differences were calculated at P=0.01 level of significance wherever F test values were significant and interpretation of the data was carried out in accordance with Panse and Sukhatme (1985).

IV. EXPERIMENTAL RESULTS


Investigations were carried out to evaluate the biocontrol potential of fluorescent pseudomonads against the plant pathogens, viz., Sclerotium rolfsii causing collar rot of groundnut, Ralstonia solanacearum causing bacterial/vascular wilt of tomato and root knot nematode Meloidogyne incognita causing root gall disease in solanaceous crops. Attempts were also made to elucidate the mechanisms of biocontrol in the potent antagonistic strains of fluorescent pseudomonads and their plant growth promotional activity was evaluated. The results obtained are presented in this chapter.

4.1 In vitro screening of fluorescent pseudomonads for their biocontrol potential against phytopathogens
A total of 19 fluorescent pseudomonads isolates and two reference strains were tested for their in vitro inhibitory activity against four fungal, three bacterial and one nematode phytopathogens and the results are presented in Tables 4-6. The results in general indicated the potential of the isolates to inhibit multiple pathogens. While seven isolates were inhibitory to all eight pathogens tested (4 fungi, 3 bacteria, and 1 nematode), another nine isolates showed inhibitory activity against seven out of eight pathogens, two isolates against six pathogens and one against five pathogens (Fig 1).

4.1.1 Fungal pathogens


All the isolates showed inhibitory activity against one or the other plant fungal pathogen tested. The results obtained are presented in Table 4. The results clearly indicated that, among the 19 isolates tested, 12 were inhibitory to Rhizoctonia botaticola, 18 to Sclerotium rolfsii and all the 19 isolates were inhibitory to Pythium sp. and Fusarium udum. Out of 19 isolates, eleven isolates recorded biocontrol potential against all the four fungal pathogens and eight isolates showed biocontrol potential against three pathogenic fungi. Thus, many of the fluorescent pseudomonads were inhibitory to more than one pathogen. Among the 12 fluorescent pseudomonad isolates which showed biocontrol potential against Rhizoctonia bataticola, the isolates 427, 518 (1), 327 (2) and 227 (2) showed maximum zone of inhibition (ZOI) of 5.0 cm and were significantly superior over the reference strains (Table 4 and Plate 1). Pseudomonas fluorescens (NCIM 2099) and Pseudomonas aureofaciens (NCIM 2026) both of which recorded inhibition zone of 3.0 cm. The next effective isolates were 47 (2), 494 (2), 521 (2), 173 (3) and 234 (1) with inhibition zone of 4.0 cm whereas three isolates viz., 76, 139(2) and 361(1) recorded inhibition zone of 3.0 cm which was similar to that of the reference strains. Among eighteen isolates inhibitory to Sclerotium rolfsii, the isolate 227 (2) registered maximum ZOI (4.5 cm), which was significantly superior over the rest of the isolates (Table 4 and Plate 2). It was closely followed by the isolates 173 (3) and 427 with ZOI of 4.0 and 3.5 cm respectively. Similarly, twelve isolates showed the ZOI of 2.5 cm, two isolates showed ZOI of 1.5 cm and remaining one isolate recorded ZOI of 1 cm. All the isolates except 313 (1) were found to be significantly superior over the two reference strains. All the 19 isolates tested were able to inhibit the growth of Pythium sp. Among them, isolate 227(2) showed maximum ZOI of 4.5 cm followed by 427, 518 (1) (both with a ZOI of 4.0 cm) and isolate 309 (2) (ZOI of 3.5 cm) (Table 4), all of which were significantly superior to both the reference strains (both recorded ZOI of 3.0 cm). Five isolates viz., 47 (2), 433 (1), 494 (2), 173 (3) and 331 (2) recorded ZOI of 3.0 cm and as such were at par with the reference strains. All the 19 isolates were also inhibitory to Fusarium udum. Three isolates viz., 427, 521 (2), and 227 (2) showed maximum inhibition of growth (4.0 cm) and were significantly superior to all other isolates except the reference strain P. aureofaciens (4.5 cm) with which they were on par (Table 4). The isolates 47 (2) and 234 (1) recorded ZOI of 3.5 cm. Six isolates registered ZOI of 3.0 cm and the remaining isolates recorded less than 2.5 cm ZOI. When compared to the biocontrol potential of the reference strains, eight isolates showed significantly higher potential against Rhizoctonia bataticola, eighteen against Sclerotium rolfsii, four against Pythium sp. and sixteen against Fusarium udum.

Number of pathogens inhibited


FP

47 (2 ) FP 73

FP

FP

Isolates

Fig. 1. Number of plant pathogens inhibited in vitro by fluorescent pseudomonads


Fig. 1. Number of plant pathogens inhibited in vitro by fluorescent pseudomonads
76 13 9( 2) FP 4 FP 27 43 3( 1) FP 44 0( 1) FP 49 4( 2) FP 51 8( 1) FP 52 1( 2) FP 32 7( 2) FP 17 3( 3) FP 20 3( 1) FP 22 7( 2) FP 23 4( 1) FP 30 9( 2) FP 31 3( 1) FP 33 R ef 1( .s 2) FP tr. PA 36 R ef 1( N .s 1 C tr. IM ) PF 20 26 N IC M 20 99 C on tro l

4.1.2 Bacterial pathogens


All the nineteen isolates were tested for their biocontrol potential against three bacterial pathogens. All of them were inhibitory to Xanthmonas campestris, fourteen were inhibitory to Xanthmonas axonopodis and sixteen were inhibitory to Ralstonia solanacearum. Twelve isolates showed biocontrol potential against all the three bacterial pathogens whereas six isolates were inhibitory to two bacterial pathogens (Table 5). Among the isolates of fluorescent pseudomonads which were inhibitory to Xanthmonas campestris, 12 isolates [139 (2), 427, 440 (1), 327 (2), 173 (3), 203 (1), 227 (2), 234 (1), 309 (2), 313 (1), 331 (2) and 361 (1)] recorded maximum ZOI of 4.0 cm and were significantly superior to the other isolates as well as the reference strain P. fluorescens (Table 5). The isolates 47 (2), 73, 433 (1) and 518 (1) recorded ZOI of 3.0 cm and were on par with the reference strain P. fluorescens. Three isolates registered the ZOI of only 2.0 cm. Among the fourteen isolates inhibitory to X. axonopodis, the isolate 227(2) showed maximum ZOI of 4.0 cm and was significantly superior to all other isolates and the reference strains as well. This was followed by the isolate 427, with ZOI of 3.5 cm, which was also significantly superior over the reference strains (Table 5). Five isolates recorded the ZOI of 3.0 cm and seven isolates had the ZOI of 2.0 cm. Out of sixteen isolates inhibitory to R. solanacearum, seven isolates viz., 73, 427, 433 (1), 440 (1), 521 (2), 313 (1) and 331 (2) recorded maximum ZOI of 4.0 cm and were significantly superior to the rest of the isolates but were on par with both the reference strains (Table 5 and Plate 3). The next effective isolate was 309 (2) with the ZOI of 3.5 cm. Six isolates recorded the ZOI of 3.0 cm and two isolates had ZOI of 2.0 cm. When compared with the biocontrol potential of the reference strains viz., P. fluorescens (NCIM 2099) and P. aureofaciens (NCIM 2026), eleven isolates showed greater potential against Xanthmonas campestris, two isolates against Xanthmonas axonopodis and seven isolates showed equal performance as that of reference strain (P. fluorescens NCIM 2099) against Ralstonia solanacearum.

4.1.3 Efficacy of culture filtrate and whole cell culture of fluorescent pseudomonads in killing the plant pathogenic nematode
The efficacy of the culture filtrates and the whole cell cultures of the fluorescent pseudomonads for their ability to immobilize or kill root knot nematode, Meloidogyne incognita was studied under in vitro conditions and the results obtained are presented in Table 6. The results in general indicated that both the whole cell culture and the culture filtrate of all the isolates were able to control the nematode. The whole cell culture and culture filtrate of all the isolates showed almost same killing percentage (87-97%) and were significantly superior over those of the reference strains, which showed killing percentage in the range of 81-85% (Table 6). There was a slight increase in the killing percentage of juveniles due to continuation of incubation up to 72 hrs with culture filtrate of most isolates as well as reference strains. At 72 hours, the culture filtrate of isolate 227(2) showed the higher mortality (99 %) followed by the isolates, 47 (2), 433 (1), 440 (1), 518 (1), 203 (1) and 313 (1) all of which registered 97% juvenile mortality and were significantly superior over other isolates. The remaining twelve isolates recorded 92 to 95 % juvenile mortality. The whole cell cultures of all the fluorescent pseudomonas isolates also recorded significantly higher killing of RKN juveniles over the two reference strains. Among the isolates tested, 433 (1), 518 (1), 227 (2) and 313 (1) recorded significantly higher killing percentage (97.00%) followed by 427 with 96 % juvenile mortality and all were significantly superior over other isolates (Table 6).

4.2

Mechanisms of pseudomonads

biocontrol

of

antagonistic

fluorescent

The mechanism of inhibition of plant pathogens by the antagonistic strains of fluorescent pseudomonads was determined and the results are presented below.

Table 4. In vitro inhibition of different fungal pathogens by fluorescent pseudomonads Treatments (Isolate No.) T1 . FP 47(2) T2 . FP 73 T3 . FP 76 T4 . FP 139(2) T5 . FP 427 T6 . FP 433(1) T7 . FP 440(1) T8 . FP 494(2) T9 . FP 518(1) T10 . FP 521(2) T11 . FP 327(2) T12 . FP 173(3) T13 . FP 203(1) T14 . FP 227(2) T15 . FP 234(1) T16 . FP 309(2) T17 . FP 313(1) T18 . FP 331(2) T19 . FP 361(1) T20 . Ref. str. P. aureofaciens NCIM-2026 T21 . Ref. str. P. fluorescens NCIM2099 T22 . Control S.Em. CD at 1 % R.bataticola 4.0 0.0 3.0 3.0 5.0 0.0 0.0 4.0 5.0 4.0 5.0 4.0 0.0 5.0 4.0 0.0 0.0 0.0 3.0 3.0 Zone of inhibition (cm) S.rolfsii 1.5 2.5 2.5 2.5 3.5 2.5 2.5 0.0 2.5 1.5 2.5 4.0 2.5 4.5 2.5 2.5 1.0 2.5 2.5 1.0 Pythium sp. 3.0 2.5 2.5 2.5 4.0 3.0 2.5 3.0 4.0 2.5 1.5 3.0 2.5 4.5 2.5 3.5 2.0 3.0 2.0 3.0 F. udum 3.5 3.0 2.5 2.5 4.0 2.5 3.0 2.5 3.0 4.0 2.5 3.0 3.0 4.0 3.5 1.5 2.0 2.5 3.0 4.5

3.0

0.5

3.0

2.0

0.0 0.09 0.36

0.0 0.03 0.12

0.0 0.03 0.15

0.0 0.02 0.09

Plate 1. In vitro inhibition of Rhizoctonia bataticola by fluorescent pseudomonads

Plate 2. In vitro inhibition of sclerotium rolfsii by fluorescent pseudomonads

Table 5.

In vitro inhibition of different bacterial pathogens by pseudomonads Diameter of zone of inhibition (cm) X. campestris 3.0 3.0 2.0 4.0 4.0 3.0 4.0 2.0 3.0 2.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 X. axonopodis 2.0 2.0 3.0 3.0 3.5 2.0 0.0 0.0 0.0 0.0 3.0 3.0 0.0 4.0 2.0 2.0 3.0 2.0 2.0 3.0

fluorescent

Treatments (Isolate No) T1 . FP 47(2) T2 . FP 73 T3 . FP 76 T4 . FP 139(2) T5 . FP 427 T6 . FP 433(1) T7 . FP 440(1) T8 . FP 494(2) T9 . FP 518(1) T10 . FP 521(2) T11 . FP 327(2) T12 . FP 173(3) T13 . FP 203(1) T14 . FP 227(2) T15 . FP 234(1) T16 . FP 309(2) T17 . FP 313(1) T18 . FP 331(2) T19 . FP 361(1) T20 . Ref. str. P. aureofaciens NCIM-2026 T21 . Ref. str. P. fluorescens NCIM2099 T22 . Control S.Em. CD at 1 %

R. solanacearum 3.0 4.0 3.0 0.0 4.0 4.0 4.0 0.0 3.0 4.0 3.0 2.0 2.0 3.0 3.0 3.5 4.0 4.0 0.0 4.0

3.0

3.0

4.0

0.0 0.06 0.18

0.0 0.02 0.09

0.0 0.02 0.09

Plate 3. In vitro inhibition of Ralstonia solanacearum by fluorescent pseudomonads

Table 6. Effect of culture filtrate and whole cell culture of fluorescent pseudomonads on nematode juveniles Percent of juveniles killed Treatments (Isolate No) At 24 hrs T1 . FP 47(2) T2 . FP 73 T3 . FP 76 T4 . FP 139(2) T5 . FP 427 T6 . FP 433(1) T7 . FP 440(1) T8 . FP 494(2) T9 . FP 518(1) T10 . FP 521(2) T11 . FP 327(2) T12 . FP 173(3) T13 . FP 203(1) T14 . FP 227(2) T15 . FP 234(1) T16 . FP 309(2) T17 . FP 313(1) T18 . FP 331(2) T19 . FP 361(1) T20 . Ref. str. P. aureofaciens NCIM-2026 T21 . Ref. str. P. fluorescens NCIM2099 T22 . Uninoculated control (distilled water) S.Em. CD at 1 % 93 95 93 95 97 97 95 93 97 93 87 93 95 97 87 93 97 93 91 85 Culture filtrate At 48 hrs 97 95 95 95 95 97 97 95 97 95 91 95 97 99 93 95 97 93 95 91 At 72 hrs 97 95 95 95 95 97 97 95 97 95 92 95 97 99 93 95 97 93 95 95 93 95 93 95 96 97 95 95 97 93 87 93 95 97 87 93 97 93 91 85 Whole cell culture At 24 hrs

81

89

90

81

0 0.20 0.83

0 0.20 0.83

0 0.23 0.94

0 0.20 0.83

Table 7. HCN production and siderophore production by fluorescent pseudomonads Siderphore Production (zone of clearance dia in mm) 13.50 13.00 11.00 15.50 22.00 14.50 13.50 11.50 16.00 13.50 15.50 12.50 14.50 25.50 15.50 13.50 12.00 11.00 12.50 12.00

Isolates FP 47 (2) FP 73 FP 76 FP 139 (2) FP 427 FP 433 (1) FP 440 (1) FP 494 (2) FP 518 (1) FP 521 (2) FP 327 (2) FP 173 (3) FP 203 (1) FP 227 (2) FP 234 (1) FP 309 (2) FP 313 (1) FP 331 (2) FP 361 (1) Ref. str. P. aureofaciens NCIM 2026 Ref. str. P. fluorescence NCIM 2099 Control S.Em. CD at 1 %

HCN production + ++ ++ ++ +++ + + + ++ ++ +++ ++ +++ ++ ++ ++ +++ +++ +++ +++

+++ -

13.50 00.00 0.10 0.42

- No colour change, + Brownish colouration, ++ Brownish to orange, +++ Complete orange

LEGEND
Plate 4 C = Control Fluorescent psuedomonad 427 FP 227(2) PF = Reference strain Pseudomonas fluorescens NCIM 2099 PA = Reference strain Pseudomonas aureofaciens NCIM 2026

Plate 5

47(2)

73

139(2) 433(1) )

427

234(1)

440(1)

494(2) 521(2)

313(1)

227(2) 327(2)

203(1)

173(3) 518(1)

76
NCIM 2099

361(2)

309(2)

331(2) NCIM 2026

Plate 4. HCN production by fluorescent pseudomonads

Plate 5. Siderophore production by fluorescent pseudomonads

4.2.1 HCN production


The In vitro HCN production by the antagonistic strains was tested by the picric acid assay. The results given in Table 7 indicated the ability of all the isolates to produce HCN production. Out of 19 isolates tested, 6 isolates viz., 427, 327 (2), 234 (1), 313 (1), 331(2) and 361(1) were identified as high HCN producers which turned the colour of the filter paper to completely orange (Plate 4). Nine isolates produced moderate quantity of HCN (brownish to orange colour of filter paper). Four isolates were weak HCN producers. Among the isolates examined, six showed greater production of HCN than the reference strain Pseudomonas fluorescens NCIM 2099.

4.2.2 Siderophore production


To examine the ability of isolates to produce siderophores, they were subjected to siderophore assay following the CAS agar plate method. Among the isolates tested, the isolate 227 (2) showed substantial amounts of siderophore production as measured by the zone of clearance surrounding the colonies (25.5 mm), and was followed by the isolate 427 which showed zone of clearance of 22.00 mm. Remaining isolates produced the zones of clearance ranging from 11 to 15.5 mm (Table 7 and Plate 5). Ten isolates produced higher amounts of siderophores than the reference strain Pseudomonas fluorescens NCIM 2099.

4.2.3 Production of antimicrobial compounds


Based on in vitro performance of bacterial strains for biocontrol activity against different plant pathogens (fungal and bacterial), effective strains were selected to ascertain the antibacterial metabolite production by the strains. The metabolic products of each strain were extracted and separated by TLC. Invariably all the strains tested produced more than one metabolite each, which appeared as dark spots under short wave UV light. All the isolates and the two reference strains produced two metabolites each and the Rf values of the metabolites varied from 0.16 to 0.97 (Table 8). The metabolite with an Rf value of 0.97 and 0.83 were produced by the isolates 427 and 521 (2) as in the reference P. fluorescens NCIM 2099. The metabolites from remaining five isolates had Rf values ranging from 0.16 to 0.72. All the unidentified metabolites on TLC plates were scraped, eluted and redissolved in acetone and tested for in vitro inhibition by spotting on a lawn of respective pathogens and all of them inhibited the growth of pathogens (Table 9).

4.3 Biocontrol potentiality of selected fluorescent pseudomonads against Sclerotium rolfsii of groundnut plants
Based on in vitro performance of the fluorescent pseudomonad isolates, seven effective antagonistic isolates were selected and screened under pot culture for their biocontrol potential against S. rolfsii, inciting collar rot of groundnut. The results obtained are presented in Tables 10 and 11.

4.3.1 Percent disease control


The percent disease control by the inoculated fluorescent pseudomonad isolates varied from 66.66 to 100.00% (Table 10 and Fig 2A). The isolates 227 (2), 427 and the reference strain P. fluorescens NCIM 2099 registered the highest (100%) disease control. Among other isolates 433 (1) showed 91.66% control, isolates 234 (1) and 173 (3) recorded 75 % disease control and the isolate 518 (1) recorded disease control of 66.66%. In general, the isolates, which were effective against the test pathogen S. rolfsii under in vitro conditions, have also shown an excellent control of the collar rot disease under green house condition (Plate 6).

Table 8. Rf values of the metabolites produced by the fluorescent pseudomonads with high biocontrol potential.

Sl. No

Isolate no.

Rf value of metabolites

FP 47 (2)

0.16

0.34

FP 427

0.83

0.97

FP 518 (1)

0.20

0.49

FP 521 (2)

0.83

0.97

FP 173 (3)

0.28

0.49

FP 227 (2)

0.61

0.72

FP 234 (1)

0.28

0.69

Ref. str. P. aureofaciens NCIM 2026

0.83

0.93

Ref. str. P. fluorescens NCIM 2099

0.83

0.97

4.3.2 Plant growth parameters


4.3.2.1 Nodule number The numbers of nodules/plant at 70 days of plant growth as influenced by different treatments are presented in Table 11. Seed bacterization with isolate 427 registered the highest number of nodules (103/plant) and was significantly superior to the rest of the treatments. The next superior isolate was 227 (2) with 90 nodules/plant. The remaining five isolates recorded nodule number ranging from 65 to 83 per plant. The plants receiving only pathogen recorded 58 nodules/plant, where as the plants that did not receive the pathogen and antagonists showed 75 nodules/plant. 4.3.2.2 Number of leaves Seed bacterization with isolate 427 registered the highest number of leaves/plant (222) and was significantly superior to the rest of the treatments (Table 11). The next superior treatment in terms of number of leaves/plant was 227 (2) with 219 leaves/plant. The number of leaves in the remaining five isolates ranged from 170 to 198. The uninoculated control, on the other hand, recorded only 186 leaves/plant, where as only pathogen inoculated plants showed 152 leaves/plant. 4.3.2.3 Number of branches Isolate 427 and 227 (2) as well as reference strain P. fluorescens registered the highest number of branches/plant (6) and they were significantly superior over the rest of the treatments. The isolates 433 (1), 234 (1), 173 (3) and 227 (2) recorded 5 branches/plant (Table 11). The remaining treatments recorded 4 branches/plant.

Table 9. Inhibitory activity of eluted metabolites of fluorescent pseudomonads against test pathogens Rf value of inhibitory Metabolites 0.16 0.20 0.28 0.34 0.49 0.61 0.69 0.72 0.83 (FP 427) 0.93 Pathogens inhibited Rhizoctonia bataticola Rhizoctonia bataticola Rhizoctonia bataticola Fusarium udum Pythium sp. Sclerotium rolfsii Rhizoctonia bataticola Fusarium udum Sclerotium rolfsii Rhizoctonia bataticola Fusarium udum Fusarium udum Pythium sp. 0.97 (FP 521 (2)) Rhizoctonia bataticola Sclerotium rolfsii

Table 10. Performance of effective fluorescent pseudomonads in controlling Sclerotium rolfsii causing collar rot of groundnut

Treatments (Isolates)

Total number of plants

Number of diseased plants

Per cent disease controlled

T1 . Ref. str. PF NCIM 2099 + Sr

12

100.00

T2 . FP 433(1) + Sr

12

91.66

T3 . FP 234(1) + Sr

12

75.00

T4 . FP 173(3) + Sr

12

75.00

T5 . FP 427 + Sr

12

100.00

T6 . FP 518(1) + Sr

12

66.66

T7 . FP 227(2) + Sr

12

100.00

T8 . Control (only Sr)

12

12

00.00

T9 . Absolute control (No pathogen & no biocontrol agent) FP Fluorescent pseudomonads Sr Sclerotium rolfsii PF Pseudomonads fluorescens

12

No disease occurrence

LEGEND
Plate 6

A.

OC : Only inoculated with Sclerotium rolfsii AC : No Sclerotium rolfsii no biocontrol agent T1 : Reference strain PF NCIM 2099 + Sclerotium rolfsii T2 : Fluorescent pseudomonads (FP) 433(1) + Sclerotium rolfsii T3 : FP 234(1) + Sclerotium rolfsii

B.

OC : Only inoculated with Sclerotium rolfsii AC : No Sclerotium rolfsii no biocontrol agent T4 : FP 173(3) + Sclerotium rolfsii T5 : FP 427 + Sclerotium rolfsii

C.

OC : Only inoculated with Sclerotium rolfsii AC : No Sclerotium rolfsii no biocontrol agent T6 : FP 518(1) + Sclerotium rolfsii T7 : FP 227(2) + Sclerotium rolfsii

Plate 6. Control of sclerotial rot of groundnut plants simultaneously inoculated with fluorescent pseudomonads and Sclerotium rolfsii

R ef .s tr. PF N C IM
C IM 20 99 N

Dry matter content (g)


ef .s tr. PF

% disease control

100 90 80 70 60 50 40 30 20 10 0

10

12

FP
FP

20 99

Isolates

Isolates

[4 33 (1 FP )] [2 34 (1 FP )] [1 73 (3 )] FP [4 27 FP ] [5 18 (1 FP )] [2 27 (2 )] C Ab on os tro ul l te co nt ro l
[4 33 (1 )] FP [2 34 (1 )] FP [1 73 (3 )] FP [4 27 FP ] [5 18 (1 )] FP [2 27 (2 )] C on tro l

Fig. 2. Control of sclerotial rot (A) and dry matter content (B) in groundnut plants simultaneously inoculated with fluorescent pseudomonads and Sclerotium rolfsii

Table 11. Influence of antagonistic fluorescent pseudomonads on plant growth parameters of groundnut

Treatments (Isolates)

Nodule No

No. of leaves/ plant

No. branches/p lant

Dry matter accumulation (g)

T1 . Ref. str. PF NCIM 2099 + Sr

65

198

9.61

T2 . FP 433(1) + Sr

83

188

8.49

T3 . FP 234(1) + Sr

70

182

6.79

T4 . FP 173(3) + Sr

72

186

7.01

T5 . FP 427 + Sr

103

222

10.97

T6 . FP 518(1) + Sr

60

170

6.06

T7 . FP 227(2) + Sr

90

219

10.14

T8 . Control (only Sr)

58

152

4.30

T9 . Absolute control (No pathogen & no biocontrol agent)

75

186

7.90

S.Em.

0.36

0.57

0.10

0.18

CD at 1% FP Fluorescent pseudomonads Sr Sclerotium rolfsii PF Pseudomonads fluorescens

1.38

2.17

0.38

0.69

4.3.2.4 Dry matter content The dry matter content in groundnut plants was highest (10.97 g/plant) in plants receiving fluorescent pseudomonad isolate 427 which was significantly superior over the rest of the treatments. The isolate 227 (2) recorded dry matter yield of 10.14 g/plant and was significantly superior over all other isolates but was on par with reference strain (9.61 g/plant). The remaining isolates recorded dry matter yield in the range of 6.06 to 8.49 g/plant (Table 11 and Fig 2B). The uninoculated control plants recorded only 7.90 g / plant. Where as plants receiving only pathogen recorded 4.3 g/plant.

4.4 Biocontrol potentiality of selected fluorescent pseudomonads against bacterial wilt of tomato
Based on the in vitro performance, seven effective antagonistic fluorescent pseudomonads isolates were screened under pot culture for their biocontrol potential against R. solanacearum causing bacterial wilt in tomato and the results are presented in Tables 12 and 13.

4.4.1 Percent disease control


The disease control by the inoculated fluorescent pseudomonads varied from 50 % to 91.66% (Table 12 and Fig 3A). Isolate 433 (1) showed maximum disease control (91.66%) followed by the isolates 173 (3) and 427, both of which controlled the disease by 83.33%. The reference strain recorded disease control to the extant of 75.0 % (Plate 7).

4.4.2 Plant growth parameters


4.4.2.1 Number of leaves per plant The numbers of leaves per plant of tomato at 100 days aftr transplantation (DAT) as influenced by different treatments are presented in Table 13. Seed bacterization with isolate 234 (1) registered the highest number of leaves (267 leaves/plant) and was significantly superior over the rest of the treatments. The next superior treatment in terms of number of leaves/plant was 427 with 254 leaves/plant. The remaining five isolates recorded the number of leaves ranging from 123 to 252 per plant. Plants in uninoculated control treatment recorded 198 leaves/plant, where as plants receiving only pathogen inoculation recorded 120 leaves/plant. 4.4.2.2 Stem girth The stem girth of tomato plants at 100 DAT was maximum in treatments with isolate 234 (1) (3.5 cm) and was significantly higher over the rest of the treatments (Table 13). Isolate 427 recorded the stem girth of 3.25 cm which was significantly superior over all other strains including reference strain but was on par with the isolate 433 (1). The stem girth in the remaining five isolates ranged from 3.0 to 3.2 cm. The uninoculated control treatment recorded the least stem girth of 2.5 cm. 4.4.2.3 Dry matter content Among the fluorescent pseudomonads strains tested, the isolate 433 (1) registered the highest dry matter accumulation (25.16 g/plant) and was significantly superior over all other treatments (Table 13 and Fig 3B). Among other isolates, 427 and 173 (3) also recorded significantly higher drymatter content (23 and 22.08 g/plant) over others but they differed significantly among themselves. The reference strain recorded a dry matter content of 19.98 g where as the uninoculated control plants recorded the dry matter content of only 19.57 g/plant, and the plants receiving only pathogen recorded dry matter content of 11.43 g.

4.5 Biocontrol potentiality of selected fluorescent pseudomonads against pathogenic nematode of tomato
Based on in vitro performance of the fluorescent pseudomonad isolates, eight effective antagonistic isolates including one reference strain (P.fluorescens (NCIM 2099) were screened under pot culture for their biocontrol potential against phytopathogenic nematode, Meloidogyne incognita with tomato as test plants and the results are presented in Tables 14 and 15; and Plate 8).

Table 12. Performance of selected isolates of fluorescent pseudomonads in controlling bacterial wilt of tomato

Treatments (Isolates)

Total number of plants

Number of diseased plants

Per cent disease controlled

T1 . Ref. str. PF NCIM 2099 + Rs

12

75.00

T2 . FP 433(1) + Rs

12

91.00

T3 . FP 234(1) + Rs

12

66.66

T4 . FP 173(3) + Rs

12

83.33

T5 . FP 427 + Rs

12

83.33

T6 . FP 518(1) + Rs

12

50.00

T7 . FP 227(2) + Rs

12

75.00

T8 . Control (only Rs)

12

12

0.00

T9 . Absolute control (No pathogen & no biocontrol agent) FP Fluorescent pseudomonads Rs - Ralstonia solanacearum PF Pseudomonads fluorescens

12

No disease occurrence

LEGEND

Plate 7

A.

T8 : Only inoculated with Ralstonia solanacearum T1 : Reference strain PF NCIM 2099 + Ralstonia solanacearum T2 : Fluorescent pseudomonads (FP) 433(1) + Ralstonia solanacearum T9 : No Ralstonia solanacearum and no biocontrol agents

B.

T8 : Only inoculated with Ralstonia solanacearum T3 : FP 234(1) + Ralstonia solanacearum T4 : FP 173(3) + Ralstonia solanacearum T5 : FP 427 + Ralstonia solanacearum T9 : No Ralstonia solanacearum and no biocontrol agents

C.

T8 : Only inoculated with Ralstonia solanacearum T6 : FP 518(1) + Ralstonia solanacearum T7 : FP 227(2) + Ralstonia solanacearum T9 : No Ralstonia solanacearum and no biocontrol agents

Plate 7. Control of bacterial wilt of tomato plants simultaneousaly inoculated with fluorescent pseudomonads and Ralstonia solanacearum

R ef .s tr. PF

Dry matter content (g)


ef .s tr. PF N C IM 20 99

% disease control

10

15

20

25

30

100 90 80 70 60 50 40 30 20 10 0

0
FP

5
Isolates

N C IM 20 FP 99 [4 33 (1 FP )] [2 34 (1 FP )] [1 73 (3 )] FP [4 27 FP ] [5 18 (1 FP )] [2 27 (2 )] C Ab on os tro ul l te co nt ro l

[4 33 (1 )] FP [2 34 (1 )] FP [1 73 (3 )] FP [4 27 FP ] [5 18 (1 )] FP [2 27 (2 )] C on tro l

Fig. 3. Control of bacterial wilt (A) and dry matter content (B) in tomato plants simultaneously inoculated with fluorescent pseudomonads and Ralstonia solanacearum

Table 13. Influence of antagonistic fluorescent pseudomonads on plant growth parameters of tomato

Treatments (Isolates)

Number of leaves/ plant

Stem girth (cm)

Dry matter accumulation (g)

T1 . Ref. str. PF NCIM 2099 + Rs

232

3.0

19.98

T2 . FP 433(1) + Rs

222

3.2

25.16

T3 . FP 234(1) + Rs

267

3.5

21.00

T4 . FP 173(3) + Rs

252

3.0

22.08

T5 . FP 427 + Rs

254

3.25

23.00

T6 . FP 518(1) + Rs

123

3.0

18.92

T7 . FP 227(2) + Rs

198

3.1

19.55

T8 . Control (only Rs)

120

2.5

11.43

T9 . Absolute control (No pathogen & no biocontrol agent)

198

2.2

19.57

S.Em.

0.46

0.02

0.18

CD at 1% FP Fluorescent pseudomonads Rs - Ralstonia solanacearum PF Pseudomonads fluorescens

1.75

0.08

0.69

4.5.1 Reduction in Gall index


All the fluorescent pseudomonads including the reference strain reduced the gall index markedly (Table 14 and Fig 4A). The maximum reduction in gall index was recorded by the fluorescent pseudomonad isolates 518 (1), 173 (3) and 427 (0.66) as against 4.0 in control and they were superior to the reference strain P. fluorescens (NCIM 2099). The isolates 433 (1), 234 (2) and 440 (1) with a gall index of 1.33 were the next effective ones. The isolate 313 (1) recorded gall index of 1.66.

4.5.2 Plant growth parameters


4.5.2.1 Number of leaves Significantly difference existed between the treatments with respect to number of leaves per plant (Table 15). Seed bacterization with isolate 313 (1) registered significantly higher number of leaves (249 leaves/plant) followed by isolates 427 (246 leaves/plant) and 173 (3) (236 leaves per plant) all of which were significantly superior over rest of the treatments and they also differed significantly among themselves. 4.5.2.2 Stem girth Tomato plants receiving isolate 173 (3) registered the highest stem girth (3.5 cm) and were significantly superior to the rest of the treatments (Table 15). The isolate 427 recorded a stem girth of 3.2 cm and was significantly superior over reference strain reference strain P. fluorescens (NCIM 2099) (2.9 cm) and other isolates but was on par with the isolate 518 (1). 4.5.2.3 Dry matter content All the isolates of fluorescent pseudomonads enhanced dry matter content of tomato plants significantly over the reference strain (21.40 g) (Table 15 and fig 4B). Among the isolates, 313 (1) registered the highest dry matter accumulation (24.31 g/plant) but was on par with all other isolates except 433 (1) over which it was significantly superior.

4.6 Effect of fluorescent pseudomonads on growth and nutrient uptake in groundnut plants
The effect of 21 fluorescent pseudomonads (19 isolates and 2 reference strains) on the growth and nutrient uptake of groundnut plants was studied in pot culture and the results are presented in Tables 16-19 and Plate 9).

4.6.1 Nodule dry weight per plant at 50 DAS


All the isolates except 203 (1) enhanced the nodule dry weight significantly over uninoculated control (1.50 mg/plant)(Table 16). Among the isolates, 227 (2) showed the highest nodule dry weight (60.50 mg/plant) followed by the isolates 327 (2) (55.50 mg/plant), 427 (33.50 mg/plant) and 518 (1) (21.50 mg/plant) all of which were significantly superior over other strains including the two reference strains and they also differed significantly among themselves. While ten isolates showed significantly higher nodule dry weight over that of the reference strain Pseudomonas aureofaciens (NCIM 2026), five isolates were significantly superior over Pseudomonas fluorescens (NCIM 2099).

4.6.2 Root length at 50 DAS


The highest root length at 50 DAS (22.50 cm) was recorded in groundnut plants inoculated with reference strain Pseudomonas aureofaciens (NCIM 2026) closely followed by the isolate 427 (22.00 cm) which were on par with each other but both were significantly superior over all other strains including the other reference strain Pseudomonas fluorescens (NCIM 2099). Both the reference strains and 17 out of 19 isolates showed significantly higher (13.00 to 22.50 cm) root length over uninoculated control (12.0 cm). However, the isolates 433 (1) (12.5 cm) and 313 (1) (12.0 cm) were on par with uninoculated control (Table 16).

Table 14. Performance of selected fluorescent pseudomonads in controlling root knot nematode in tomato Treatments (Isolates no) T1. FP isolate 518(1) + Mi T2. FP isolate 433(1) + Mi T3. FP isolate 313(1) + Mi T4. FP isolate 234(1) + Mi T5. FP isolate 173(3) + Mi T6. FP isolate 440(1) + Mi T7. FP isolate 427 + Mi T8. Ref. str. (PF NCIM 2099) + Mi T9. Control (only Mi) T10. Absolute control (no Mi and no biocontrol agent) FP Fluorescent pseudomonads Mi - Meloidogyne incognita PF Pseudomonads fluorescens Average No of galls/plant 9.00 15.66 18.66 18.33 4.00 18.66 5.33 10.00 29.00 00.00 Gall index (Avg. of 3 replicates) 0.66 1.33 1.66 1.33 0.66 1.33 0.66 1.00 4.00 0.00

Gall index 1 = 0-25% roots galled Gall index 2 = 26-50% roots galled Gall index 3 = 51-75% roots galled Gall index 4 = 76-100% roots galled

LEGEND

Plate 8

A.

OC : Only inoculated with Meloidogyne incognita AC : No Meloidogyne incognita and no biocontrol agent T1 : Fluorescent pseudomonads (FP) 518(1) + M. incognita T2 : FP 433(1) + Meloidogyne incognita T3 : FP 313(1) + Meloidogyne incognita

B.

OC : Only inoculated with Meloidogyne incognita AC : No Meloidogyne incognita and no biocontrol agent T4 : FP 234(1) + Meloidogyne incognita T5 : FP 173(3) + Meloidogyne incognita T6 : FP 440(1) + Meloidogyne incognita

C.

OC : Only inoculated with Meloidogyne incognita AC : No Meloidogyne incognita and no biocontrol agent T7 : FP 427 + Meloidogyne incognita T8 : Reference strain pseudomonas fluorescens (PF) NCIM 2099 + Meloidogyne incognita

Plate 8. Biocontrol activity of fluorescent pseudomonads against Meloidogyne incognita inoculated to tomato plants

A
4 3.5 3

Gall index

2.5 2 1.5 1 0.5 0

51 8( 1) FP 43 3( 1) FP 31 3( 1) FP 23 4( 1) FP 17 3( 3) FP 44 0( R 1) ef .s FP tr. PF 42 7 N C IM 20 99 C on tro l

FP

Isolates

B
24.5 24 23.5 23 22.5 22 21.5 21 20.5 20 19.5
51 8 FP (1) 43 3 FP (1) 31 3 FP (1) 23 4 FP (1) 17 3 FP (3) 44 R 0( ef 1) .s tr. FP PF 42 N 7 C IM 20 99 Ab C on so tro lu te l co nt ro l FP

Dry matter content (g)

Isolates Fig. 4. Influence of fluorescent pseudomonads on root gall formation (A) and dry matter content (B) of tomato plants inoculated with Meloidogyne incognita

Table 15. Influence of antagonistic fluorescent pseudomonads on plant growth parameters of tomato

Treatments (Isolates no) T1. FP isolate 518(1) + Mi

Number of leaves/plant

Stem girth (cm)

Dry matter accumulation (g)

234

3.00

23.57

T2. FP isolate 433(1) + Mi

228

2.50

22.85

T3. FP isolate 313(1) + Mi

249

2.30

24.31

T4. FP isolate 234(1) + Mi

234

2.50

23.68

T5. FP isolate 173(3) + Mi

236

3.50

23.98

T6. FP isolate 440(1) + Mi

232

2.30

23.19

T7. FP isolate 427 + Mi

246

3.20

24.16

T8. Ref. str. PF NCIM 2099 + Mi T9. Control (only Mi)

222

2.90

21.40

224

2.20

22.44

T10. Absolute control (no Mi and no biocontrol agent) S.Em.

218

1.25

22.06

0.37

0.06

0.32

CD at 1% FP Fluorescent pseudomonads Mi - Meloidogyne incognita PF Pseudomonads fluorescens

1.57

0.24

1.31

Gall index 1 = 0-25% roots galled Gall index 2 = 26-50% roots galled Gall index 3 = 51-75% roots galled Gall index 4 = 76-100% roots galled

4.6.3 Shoot length at 50 DAS


All the fluorescent pseudomonads except the isolates 440 (1), 313 (1) and 331 (2), showed significant increase in the shoot length of groundnut plants at 50 DAS (Table 16). Among the inoculated strains, isolates 139 (2) and 518 (1) recorded the maximum shoot length (43.00 cm in both) and were on par with the reference strain Pseudomonas aureofaciens (NCIM 2026)(42.00 cm) but were significantly superior over all other strains (Table 16). The isolates 173 (3) (41.5 cm), 327 (2) (41.0 cm), 521 (2) (40.5 cm), 227 (2) and 73 (39.5 cm in both) were the other better performing strains. The isolates 440 (1) (31.0 cm), 313 (1) (30.0 cm) and 331 (2) (30.5 cm) were statistically on par with the uninoculated control.

4.6.4 Number of branches per plant


All the isolates enhanced the number of branches/plant significantly over uninoculated control (3.5 branches/plant) (Table 16). Among the isolates, 440 (1) showed the maximum number of branches (8.5 branches/plant) followed by the isolate 433 (1) (7.5 branches/plant) and 203 (1) (7.0 branches/plant), which were significantly superior over other strains including the two reference strains but they differed significantly among themselves. While 16 isolates showed significantly higher number of braches/plant over the reference strain Pseudomonas aureofaciens (NCIM 2026), 5 isolates were significantly superior over Pseudomonas fluorescens (NCIM 2099).

4.6.5 Number of leaves per plant


The two reference strains and all 19 isolates showed significantly higher (130 to 189) leaves/plant over uninoculated control (130 leaves/plant). The maximum number of leaves (189 leaves/plant) was recorded in groundnut plants inoculated with reference strain Pseudomonas fluorescens (NCIM 2099) closely followed by the isolates 139 (2) and 518 (1) (187 leaves/plant in both), which were significantly superior over all other strains including the reference strain Pseudomonas aureofaciens (NCIM 2026) (Table 16).

4.6.6 Root dry weight per plant


All the fluorescent pseudomonads, except 76, 173 (3), 227 (2), 313 (1) showed significant increase in the root dry weight of groundnut plants over uninoculated control at 50 DAS. Among the inoculated strains, 331 (2) recorded the highest root dry weight (11.50 g/plant) (Table 17) followed by the isolates 47 (2) (11.23 g/plant) and the reference strain Pseudomonas aureofaciens (NCIM 2026)(11.00 g/plant) all of which were significantly superior over other strains. The isolates 73 and 203 (1) (10 g/plant in both), the reference strain Pseudomonas fluorescens (NCIM 2099)(9.34 g/plant) and the isolate 518 (1) (8.80 g/plant) were the other better performing strains. The isolates 309 (2)(5.32 g/plant), 76, 173 (3), 227 (2) and 313 (1) (5.0 g in all) were statistically on par with the uninoculated control.

4.6.7 Shoot dry weight per plant at 50 DAS


The highest shoots dry weight (25 g/plant) was recorded in groundnut plants inoculated with the reference strain Pseudomonas aureofaciens (NCIM 2026) and isolate 521 (2) which were significantly superior over all other strains except the isolate 427 (24.5 g/plant) with which they were on par (Table 17). Both the reference strains and 18 out of 19 isolate showed significantly higher (14 to 25 g/plant) shoot dry weight per plant over uninoculated control (13 g/plant).

4.6.8 Total dry weight per plant at 50 DAS


There was significant increase in the total dry matter content of groundnut plants due to inoculation of fluorescent pseudomonads over uninoculated control at 50 DAS (Table 17). The highest total dry matter (36.00 g/plant) was recorded in groundnut plants inoculated with reference strain Pseudomonas aureofaciens (NCIM 2026), which was significantly superior over all other strains. The isolate 331 (2) also showed significantly higher total dry matter (35.00 g/plant) over all other strains including the other reference strain Pseudomonas fluorescens (NCIM 2099)(32.35 g/plant). Both the reference strains and 18 out of 19 isolates showed significantly higher (19.32 to 36.00 g/plant) total dry matter over uninoculated control (18.00 g/plant).

Table 16.

Influence of fluorescent pseudomonads on plant growth parameters of groundnut at 50 DAS Nodule dry weight (mg/plant) 13.50 7.50 5.50 2.50 33.50 5.50 9.50 10.50 21.50 9.50 55.50 6.50 1.50 60.50 11.50 7.50 3.50 2.50 11.50 8.50 Root length (cm) 21.00 18.00 13.00 13.50 22.00 12.50 14.50 16.00 18.50 16.00 15.50 17.00 19.54 16.00 14.00 13.00 12.00 20.50 19.00 22.50 Shoot length (cm) 38.50 39.50 37.00 43.00 37.50 37.00 31.00 37.50 43.00 40.50 41.00 41.50 38.50 39.50 36.50 37.00 30.00 30.50 38.00 42.00 No.of branches/ plant 6.00 6.50 6.00 5.50 4.00 7.50 8.50 6.00 4.00 6.50 5.50 4.50 7.00 6.50 5.50 5.00 5.50 5.50 6.00 4.50 No.of leaves/ plant 167.00 172.00 162.00 187.00 163.50 162.00 134.50 163.00 187.00 176.00 178.50 180.50 167.00 172.00 158.93 161.00 130.00 132.00 165.00 182.50

Treatments (Isolate No) T1 . FP 47(2) T2 . FP 73 T3 . FP 76 T4 . FP 139(2) T5 . FP 427 T6 . FP 433(1) T7 . FP 440(1) T8 . FP 494(2) T9 . FP 518(1) T10 . FP 521(2) T11 . FP 327(2) T12 . FP 173(3) T13 . FP 203(1) T14 . FP 227(2) T15 . FP 234(1) T16 . FP 309(2) T17 . FP 313(1) T18 . FP 331(2) T19 . FP 361(1) T20 .Ref. str. P. aureofaciens NCIM 2026 T21 .Ref. str. P. fluorescens NCIM 2099 T22 .Uninoculated control S.Em. CD at 1 %

11.50

17.00

39.00

5.00

189.00

1.50 0.10 0.42

12.00 0.23 0.90

30.00 0.28 1.09

3.50 0.10 0.38

130.00 0.22 0.85

LEGEND

Plate 9

A.

Control: No Plant Growth Promoting Rhizobiacteria (PGPR) T14 : Fluorescent pseudomonads (FP) 227(2) T1 : FP 47(2)

B.

Control: No PGPR T3 : FP 76 T5 : FP 427 T2 : FP 73

C.

Control: No PGPR T11 : FP 327(2) T10 : FP 521(2) T9 : FP 518(1)

D.

Control: No PGPR T12 : FP 173(3) T13 : FP 203(1) T19 : FP 361(1)

Plate 9. Plant growth promotional activity of fluorescent pseudomonads in groundnut

4.6.9 Nitrogen uptake at 50 DAS


All the fluorescent pseudomonads showed significant increase in N-uptake in groundnut plants over uninoculated control at 50 DAS (Table 17). Among the inoculated strains, 518 (1) recorded the maximum nitrogen uptake (352.10 mg/plant) followed by the isolate 427 (345.85 mg/plant) which were on par with each other but both were significantly superior over rest of the isolates including the reference strains. Among others, 331 (2) (303.05 mg/plant) also recorded significantly higher N-uptake over all other strains including the two reference strains. The reference strain Pseudomonas aureofaciens (NCIM 2026) (275.25 mg/plant), isolates 521 (2) (254.55 mg/plant), 494 (2) (244.60 mg/plant) and 203 (1) (239.70 mg/plant) were the other better performing strains. While 3 isolates were significantly superior over reference strain Pseudomonas aureofacience, 11 isolates were significantly superior over Pseudomonas fluorescens (NCIM 2099).

4.6.10 Phosphorus uptake at 50 DAS


The highest phosphorus uptake (114.44 mg/plant) was recorded in groundnut plants inoculated with strain 227 (2) followed by the isolate 427 (103.33 mg/plant) both of which were significantly superior over all other strains including two reference strains and they also differed significantly among themselves (Table 17). While 6 isolates were significantly superior over the reference strain Pseudomonas aureofaciens (NCIM 2026) (88.32 mg/plant) 17 isolates were significantly superior over the other reference strain Pseudomonas fluorescens (NCIM 2099) (69.84 mg/plant). All isolates including two reference strains showed significantly higher P-uptake (69.84 to 114.44 mg/plant) in groundnut plants over the uninoculated control (67.42 mg/plant) at 50 DAS.

4.7 Root length at harvest


All the inoculated strains, except 139 (2), 433 (1) and 234 (1), enhanced the root length significantly over uninoculated control (16.00 cm) (Table 18). Among the isolates, 73 showed the maximum root length (27.00 cm) and was significantly superior over all other strains including the two reference strains. The isolates 47 (2) and 227 (2) (23.33 cm in both) were also significantly superior over all other strains except 331 (2) (23.00 cm) with which they were on par. While 14 isolates showed significant increase in root length over the reference strain Pseudomonas fluorescens (NCIM 2099) (17.33 cm), three isolates were significantly superior over Pseudomonas aureofaciens (NCIM 2026) (22.33 cm).

4.7.1 Shoot length at harvest


The highest shoot length (43.66 cm) at harvest was recorded in groundnut plants inoculated with isolate 139 (2) closely followed by the isolate 518 (1) (43.00 cm) which were on par with each other but both were significantly superior over all other strains including the two reference strains (Table 18). Both the reference strains and 17 out of 19 isolates showed significant increase in shoot length (32 to 43.66 cm) over uninoculated control (31.00 cm). However, the isolates 313 (1) and 331 (2) (31.00 and 31.33 cm respectively) were on par with uninoculated control.

4.7.2 Root dry weight per plant at harvest


All the inoculated strains except isolate 433 (1) enhanced the root dry weight significantly over uninoculated control (8.50 g/plant) at harvet (Table 18). Among the isolates, 47 (2) (13.44 g/plant) showed the highest root dry weight and was significantly superior over all other strains. The next best strain in terms of increasing root dry weight was isolate 73 (12.55 g/plant) which was significantly superior over all other strains except isolate 331 (2) and 227 (2) (12.39 and 12.24 cm respectively) with which it was on par. While 4 isolates showed significantly higher root dry weight over that of the reference strain Pseudomonas aureofaciens (NCIM 2026) (11.59 g/plant), 13 isolates showed significantly higher root dry weight over the other reference strain Pseudomonas fluorescens (NCIM 2099) (9.48 g/plant).

Table 17.

Influence of fluorescent pseudomonads on plant growth parameters and nutrient uptake by groundnut at 50 DAS Shoot dry weight (g/plant) 18.50 18.40 23.00 23.00 24.50 17.00 15.00 19.50 24.00 25.00 19.80 18.50 19.50 23.50 14.50 14.00 13.00 23.50 19.00 25.00

Treatments (Isolate No) T1 . FP 47(2) T2 . FP 73 T3 . FP 76 T4 . FP 139(2) T5 . FP 427 T6 . FP 433(1) T7 . FP 440(1) T8 . FP 494(2) T9 . FP 518(1) T10 . FP 521(2) T11 . FP 327(2) T12 . FP 173(3) T13 . FP 203(1) T14 . FP 227(2) T15 . FP 234(1) T16 . FP 309(2) T17 . FP 313(1) T18 . FP 331(2) T19 . FP 361(1) T20 .Ref. str. P. aureofaciens NCIM 2026 T21 .Ref. str. P. fluorescens NCIM 2099 T22 .Uninoculated control S.Em. CD at 1 %

Root dry weight (g/plant) 11.23 10.00 5.00 5.50 7.00 5.50 5.50 8.00 8.80 7.00 8.50 5.00 10.00 5.00 6.00 5.32 5.00 11.50 8.73 11.00

Total dry weight (g/plant) 29.74 28.40 28.00 28.50 31.53 22.50 20.50 27.51 32.52 32.00 28.35 25.50 29.50 28.56 20.51 19.32 18.00 35.00 27.74 36.00

N-uptake (mg /plant) 211.75 231.60 159.40 162.00 345.85 164.00 163.55 244.60 352.10 254.55 215.30 169.85 239.70 225.05 126.85 154.65 131.45 303.05 213.45 275.25

P-uptake (mg /plant) 70.45 72.30 84.23 72.57 103.33 68.32 99.30 83.53 75.17 98.16 78.49 76.13 90.46 114.44 73.15 77.14 79.29 96.30 80.33 88.32

9.34

23.00

32.35

206.00

69.84

5.00 0.09 0.36

13.00 0.15 0.59

18.00 0.19 0.77

115.25 3.41 13.61

67.42 0.22 0.88

Table 18.

Influence of fluorescent pseudomonads on plant growth parameters of groundnut at harvest Root length (cm) 23.33 27.00 19.00 16.33 22.00 16.00 18.33 18.66 20.83 19.00 17.66 21.66 19.66 23.33 16.00 17.00 18.33 23.00 21.00 22.33 Shoot length (cm) 39.66 40.66 38.66 43.66 38.33 39.00 32.00 39.33 43.00 40.33 41.00 41.66 39.33 40.33 37.33 38.00 31.00 31.33 38.00 42.00 Root dry weight (g/plant) 13.44 12.55 9.46 8.60 11.32 8.27 9.53 10.14 9.92 10.32 10.48 11.52 10.04 12.24 8.77 8.99 9.94 12.39 10.80 11.59 Shoot dry weight (g/plant) 19.84 24.13 24.32 23.54 25.51 20.62 19.18 21.09 24.63 25.83 20.53 18.97 20.23 24.19 20.26 22.23 22.47 24.21 19.64 25.95 Total dry weight (g/plant) 33.29 36.68 33.79 32.14 36.84 28.89 28.71 31.24 34.55 36.15 31.02 30.49 30.27 36.44 29.23 31.22 32.41 36.60 30.45 37.54

Treatments (Isolate No) T1 . FP 47(2) T2 . FP 73 T3 . FP 76 T4 . FP 139(2) T5 . FP 427 T6 . FP 433(1) T7 . FP 440(1) T8 . FP 494(2) T9 . FP 518(1) T10 . FP 521(2) T11 . FP 327(2) T12 . FP 173(3) T13 . FP 203(1) T14 . FP 227(2) T15 . FP 234(1) T16 . FP 309(2) T17 . FP 313(1) T18 . FP 331(2) T19 . FP 361(1) T20 .Ref. str. P. aureofaciens NCIM 2026 T21 .Ref. str. P. fluorescens NCIM 2099 T22 .Uninoculated control S.Em. CD at 1 %

17.33

40.00

9.48

24.09

33.57

16.00 0.23 0.86

31.00 0.13 0.47

8.50 0.09 0.33

15.58 0.25 0.96

27.54 0.26 1.01

4.7.3 Shoot dry weight per plant at harvest


The highest shoot dry weight (25.95 g/plant) at harvest was recorded in groundnut plants inoculated with the reference strain Pseudomonas aureofaciens (NCIM 2026) closely followed by the isolates 521 (2)(25.83 g/plant) and 427 (25.51 g/plant) which were on par with each other but were significantly superior over all other strains including the reference strain Pseudomonas fluorescens (NCIM 2099) (Table 18). Both the reference strains and all 19 isolates showed significantly higher (18.97 to 25.95 g/plant) shoot dry weight over uninoculated control (15.58 g/plant).

4.7.4 Total dry weight at harvest


All the strains enhanced the total dry weight of groundnut plants at significantly over uninoculated control (27.54 g/plant) (Table 18 and Fig. 5A). Among the strains, the reference strain Pseudomonas aureofaciens (NCIM 2026) showed the highest total dry weight (37.54 g/plant) followed by the isolates 427 (36.84 g/plant), 73 (36.68 g/plant) and 331 (2) (36.60 g/plant), which were on par among themselves but were significantly superior over all other strains. The other better performing strains were 227 (2) (36.44 g/plant) and 521 (2) (36.15 g/plant).

4.7.5 Number of pods per plant


The highest number of pods per plant (30 pods/plant) was recorded in groundnut plants inoculated with isolate 227 (2) followed by the isolates 440 (1) (22 pods/plant), 427 and 521 (2) (21 pods/plant in both) (Table 19) which were on par with each other but were significantly superior over all other strains including both the reference strains. Reference strain Pseudomonas aureofaciens (NCIM 2026) and 12 out of 19 isolates showed significantly higher number of pods/plant (16 to 30 pods/plant) over uninoculated control (15.0 pods/plant). However, the reference strain Pseudomonas fluorescens (NCIM 2099), 309 (2), 173 (3) (15.66 pods/plant in all three), 47 (2), 433 (1), 518 (1) (15.33 pods/plant in all three) were on par with uninoculated control.

4.7.6 Pod yield per plant


All the inoculated strains, except 433 (1) and the reference strain Pseudomonas fluorescens (NCIM 2099) enhanced the pod yield significantly over uninoculated control (14.42 g/plant) (Table 19 and Fig. 5B). Among the isolates, 227 (2) showed the highest pod yield (27.65 g/plant) followed by the isolates 427 (24.54 g/plant) and 440 (1) (23.52 g/plant), which were significantly superior over all other strains including the two reference strains, and they also differed significantly among themselves. While five isolate showed significantly higher pod yield/plant over the reference strain Pseudomonas aureofaciens (NCIM 2026) (20.21 g/plant), 17 isolates recorded significantly higher pod yield over Pseudomonas fluorescens (NCIM 2099) (14.94 g/plant).

4.7.7 Nitrogen uptake at harvest


The highest nitrogen uptake at harvest (762.87 mg/plant) was recorded in groundnut plants inoculated with isolate 518 (1) followed by the isolate 427 (586.93 mg/plant), reference strain Pseudomonas aureofaciens (NCIM 2026)(533.77 mg/plant) and isolate 521 (2) (504.23 mg/plant) all of which were significantly superior over other strains and they also differed significantly among themselves (Table 19 and Fig. 6). Both the reference strains and all 19 isolates showed significantly higher (278.90 to 762.87 mg/plant) nitrogen uptake over uninoculated control (208.87 mg/plant). While two isolates showed significantly higher nitrogen uptake over the reference strain Pseudomonas aureofaciens (NCIM 2026), eight were significantly superior over the other reference strain Pseudomonas fluorescens (NCIM 2099).

Pod yield/ plant (g)


FP

Dry matter (g/plant)


30
FP

10

15

20

25

10

15

20

25

30

35

40

0 0 5
47 (2 ) 76 FP FP FP FP FP FP FP FP R FP ef .s tr. P F N 42 7 44 0( 1) 51 8( 1) 32 7( 2) 20 3( 1) 23 4( 1) 31 3( 1) 36 1( 1) IC M 20 99

47 (2 ) 76

FP FP 42 7 FP FP FP FP FP FP R ef .s tr. P F N IC M 20 99 36 1( 1) FP 31 3( 1) 23 4( 1) 20 3( 1) 32 7( 2) 51 8( 1) 44 0( 1)

Isolates

Isolates

Fig. 5. Dry matter yield (A) and pod yield (g) of groundnut plants as influenced by fluorescent pseudomonads

Table 19.

Influence of fluorescent pseudomonads on yield and nutrient uptake by groundnut No.of Pods/ plant 15.33 15.00 18.00 16.66 21.00 15.33 22.00 17.33 15.33 21.00 16.00 15.66 20.00 30.00 15.00 15.66 15.33 18.00 18.66 16.66 Pod yield/ plant (g) 15.47 15.28 19.19 20.36 24.54 14.46 23.52 19.93 16.60 22.06 18.55 16.89 20.48 27.65 16.51 18.02 18.57 21.58 19.98 20.21 N uptake (mg/plant) 378.67 396.70 341.80 407.13 586.93 291.53 388.63 444.10 762.87 504.23 356.93 403.90 440.80 500.43 278.90 369.57 392.47 480.67 461.13 533.77 P uptake (mg/plant) 118.33 119.23 168.34 178.32 195.28 115.34 192.38 168.31 173.06 191.56 145.41 128.47 175.07 198.50 121.58 132.48 157.20 183.49 161.77 171.24

Treatments (Isolate No) T1 . FP 47(2) T2 . FP 73 T3 . FP 76 T4 . FP 139(2) T5 . FP 427 T6 . FP 433(1) T7 . FP 440(1) T8 . FP 494(2) T9 . FP 518(1) T10 . FP 521(2) T11 . FP 327(2) T12 . FP 173(3) T13 . FP 203(1) T14 . FP 227(2) T15 . FP 234(1) T16 . FP 309(2) T17 . FP 313(1) T18 . FP 331(2) T19 . FP 361(1) T20 .Ref. str. P. aureofaciens NCIM 2026 T21 .Ref. str. P. fluorescens NCIM 2099 T22 .Uninoculated control S.Em. CD at 1 %

15.66

14.94

373.37

117.09

15.00 0.19 0.73

14.42 0.20 0.79

208.87 5.04 19.20

115.28 0.13 0.50

800

N uptake (mg/plant) P uptake (mg/plant)

700

600

500

mg/plant

400

300

200

100

0
76 13 9( 2) FP 4 FP 27 43 3( 1) FP 44 0( 1) FP 49 4( 2) FP 51 8( 1) FP 52 1( 2) FP 32 7( 2) FP 17 3( 3) FP 20 3( 1) FP 22 7( 2) FP 23 4( 1) FP 30 9( 2) FP 31 3( 1) FP R 33 ef 1( .s 2) FP tr. PA 36 R ef 1( N .s 1) C tr. I PF M 2 02 N 6 IC M 20 99 C on tro l FP 47 (2 ) FP 73 FP

FP

Isolates

Fig. 6. Nutrient uptake in groundnut plants as influenced by fluorescent pseudomonads

Fig. 6. Nutrient uptake in groundnut plants as influenced by fluorescent pseudomonads

4.7.8 Phosphorus uptake at harvest


All the fluorescent pseudomonads except isolate 433 (1) showed significant increase in the phosphorus uptake of groundnut plants over uninoculated control (115.28 mg/plant) at harvest (Table 19 and Fig. 6). Among the inoculated strains, 227 (2) recorded the maximum phosphorus uptake (198.50 mg/plant) followed by the isolates 427 (195.28 mg/plant), both of which were significantly superior over all other strains including two reference strains and they also differed significantly among themselves.

V. DISCUSSION
Soil is a dynamic living matrix and is a critical resource not only for agricultural production and food security but also for maintaining the life processes on earth. Soil is truly living because of the presence of microorganisms, which are central to nutrient recycling. Although the space occupied by microorganisms in soil is said to be less than 5% of the total space, soil is considered as a store house of microorganisms and their activities. In soil, the microbial activity is higher in localities where there is accumulation of organic matter and in the rhizosphere. Rhizosphere is the zone, which is under continuous influence of living roots and is a unique habitat for soil microorganisms. The rich nutrient supply and the close physical contact with living roots place the rhizosphere-inhabiting microorganisms in a favourable position to directly influence the plant growth. Bacteria that colonize the roots effectively are termed rhizobacteria (Schroth and Hancock, 1982). Rhizobacteria that establish positive interaction with plant roots are called plant growth promoting rhizobacteria (PGPR). PGPR play important role in phytostimulation, phytoremediation and biofertilization. The important traits of PGPR that are involved in plant growth stimulation include production of plant hormones, siderophores, bacteriocins, exopolysaccharides, P-solubilization, nitrogen fixation, etc. A number of bacterial species associated with the plant rhizosphere belonging to genera Azospirillum, Azotobacter, Rhizobium, Alkaligenes, Arthrobacter, Acenitobacter, Bacillus, Burkholderia, Serratia, Pseudomonas are able to exert a beneficial effect on plant growth and can be considered as PGPRs. Among the PGPRs, fluorescent pseudomonads stand out because of their high level of genetic variability and competitiveness in the rhizosphere, their ability to release plant growth promoting substances, alter the root physiology, increase the nutrient availability to plants and to control plant pathogens (Kloepper, 1993). Pseudomonas sp including fluorescent pseudomonads are a more versatile group with their ability to utilize a wide range of substances as energy and carbon sources. They are known to inhibit the growth of plant pathogens by diverse mechanisms such as antibiotic production (Hill et al., 1994), siderophore production (Loper, 1988), HCN release (Voisard et al., 1989), induction of systemic resistance and competitive colonization of plant roots (Weller, 1985). Hence, they have been advocated as ideal biocontrol agents and plant growth promoting rhizobacteria. The functional and molecular diversity of fluorescent pseudomonads from the Western Ghat regions have been studied (Suneesh, 2005; Megha, 2006) and several of them are known to possess multiple beneficial functions relevant to agriculture. It was in this background that the investigations were carried out on the biocontrol potential and plant growth promotional activities of 19 selected fluorescent pseudomonads of Western Ghats that were maintained in the culture bank of the Department of Agricultural Microbiology, University of Agricultural Sciences, Dharwad. These 19 selected isolates of fluorescent pseudomonads and two reference strains (Pseudomonas aureofaciens NCIM 2026 and Pseudomonas fluoresces NCIM 2099) were tested for their in vitro biocontrol potential against four fungal pathogens (Rhizoctonia bataticola, Sclerotium rolfsii, Pythium sp. and Fusarium udum), three bacterial pathogens (Xanthomonas campestris, X. axonopodis and Ralstonia solanacearum) and a plant pathogenic nematode, Meloidogyne incognita. The results clearly indicated the potential of seven isolates to inhibit all the eight pathogens tested while nine isolates were able to control seven plant pathogens. Two of the isolates showed inhibitory activity against six pathogens where as one isolate inhibited five of the pathogens tested. Two reference strains used in the present study were also inhibitory to all the pathogens tested. Among the two reference strains, P. aureofaciens (NCIM 2026) was found to perform better over P. fluorescens (NCIM 2099) with respect to inhibition of plant pathogenic organisms. As compared to the better performing reference strain (P. aureofaciens, NCIM 2026) with respect to in vitro biocontrol potential against individual plant pathogens, nine isolates performed better over the reference strain in inhibiting R. bataticola. Similarly, 17 isolates showed better inhibition of S. rolfsii, four isolates against Pythium sp. and two isolates against X. axonopodis. While none of the isolates were superior to P. aureofaciens in inhibiting F. udum, 12 isolates showed the same activity against X. campestris and seven isolates showed similar inhibitory activity against R. solanacearum as that of P. aureofaciens.

These results clearly indicate the higher potential of several of the isolates over the two reference strains in inhibiting a number of plant pathogenic organisms. Similar observations on inhibition of different plant pathogenic organisms by fluorescent pseudomonads (Dwivedi and Johri, 2003), P. fluorescens (Bhowmik et al., 2002) and P. aeruginosa (Saikia et al., 2004) have been made earlier. The inhibitory activity of fluorescent pseudomonads against plant pathogenic organisms is said to be due to production of secondary metabolites such as phenazines, acetyl phloroglucinols and cyanides (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 (Kloepper et al., 1980; Davison, 1986). Pseudomonads produce pyoverdine type siderophores, which are high affinity iron chelaters (Voisard et al., 1989; Neilands and Leong, 1986). Besides, the aggressive root colonization character of fluorescent pseudomonads is also reported to play an important role in rhizosphere competence and associated biocontrol activity (Neilands and Leong, 1986). It was on these lines, all the isolates of present investigations were tested for production of HCN and siderophores. The results indicated the ability of all the isolates to produce both HCN and siderophores. Among the isolates, 427, 327 (2), 203 (1), 313 (1), 331 (2), 361 (1) and the two reference strains were strong HCN producers and were able to inhibit almost all the plant pathogens studied. A few isolates like 227 (2), 427, 139 (2), 327 (2), 234 (1), 203 (1) and 433 (1) showed significantly higher production of siderophores over other isolates as well as the two reference strains. Production of HCN and siderophores by fluorescent pseudomonads has been reported earlier (Bhatia et al., 2005; Gupta et al., 2002; Schwyn and Neilands, 1987; Bakker and Schippers, 1987) and with the results of present investigation are in line with the findings of these reports. Seven selected isolates along with the two reference strains were also tested for production of antimicrobial metabolites and all of them were found to produce two metabolites each. The eluted metabolites of each organism were also tested for inhibitory activity against the fungal pathogens. The metabolites with Rf values of 0.16, 0.20, 0.28, 0.61, 0.83 and 0.97 were inhibitory to Rhizoctonia bataticola whereas the metabolites with Rf values 0.34, 0.69, 0.83 and 0.97 were inhibitory to Fusarium udum. The metabolites with Rf value of 0.49 and 0.97 inhibited the growth of Pythium sp. and the metabolites with Rf values of 0.49, 0.72 and 0.97 inhibited the growth of Sclerotium rolfsii. These results indicate the ability of the isolates to produce different metabolites, which are able to inhibit the growth of fungal pathogens. Saikia et al. (2004) observed production of an antifungal compound by P. aeroginosa RsB29 and the compound with an Rf value of 0.72 was inhibitory to different plant pathogenic fungi. Production of antibiotics by the several strains of florescent pseudomonads has been recognized as a major factor in suppression of pathogens. A number of disease suppressive compounds have been characterized including N- containing compounds such as phenazines, pyrrol-type antibiotics, pyo-compounds and indole derivatives (Dwivedi and Johri, 2003). Hence, the ability of the fluorescent pseudomonads of present investigation to inhibit the growth of plant pathogenic organisms could be due to their multifunctional attributes such as production of HCN, siderophores and antimicrobial metabolites. The better performing isolates of fluorescent pseudomonads were further examined for their biocontrol potential against one each of fungal, bacterial and nematode pathogens in soil under green house conditions following dual inoculation. Out of seven strains tested (six isolates and one reference strain) for their biocontrol potential against S. rolfsii in groundnut, two isolates (427 and 227 [2]) and the reference strain P. fluorescens (NCIM 2099) exhibited one hundred per cent reduction in disease incidence whereas others showed disease incidence reduction in the range of 67 - 92 per cent. These results are in line with those of Ganesan and Gnanamanickam (1987) who recorded 99 per cent protection of groundnut plants from Sclerotium rolfsii infection when they were inoculated with the native strain of P. fluorescence under green house conditions. Bhatia et al. (2005) also noticed significant reduction in the incidence of collar rot of sunflower in Sclerotium rolfsii infested soil due to seed bacteriazation with florescent pseudomonads. Besides, the two isolates, viz., 427 and 227 (2) also increased the growth parameters of groundnut plants significantly over other strains and uninoculated control. In addition to their biocontrol potential, these antagonistic fluorescent pseudomonads are known to produce plant growth promoting substances such as

indole acetic acid and gibberellic acid and solubilize insoluble phosphates (Suneesh, 2004; Megha, 2006) which might have resulted in increased plant growth. The same six isolates along with the reference strain (Pseudomonas fluorescens NCIM 2099) were also tested for their biocontrol potential against bacterial wilt of tomato caused by Ralstonia solanacearum. Among the six isolates, 433 (1) recorded maximum reduction in disease incidence (91.00%), followed by 173 (3) and 427 (both with 83.33% DR) all of which were much superior to the reference strain. Others including the reference strain showed disease reduction in the range of 50-75 per cent. Pradeep Kumar and Sood (2001) also observed significant reduction in bacterial wilt incidence in tomato plants inoculated with Pseudomonas fluorescens. Meenakumari et al. (2003) reported the significance of native isolates of fluorescent pseudomonads in suppressing bacterial wilt in chilli and tomato. Seven selected isolates along with the reference strain P. fluorescens were also tested for their biocontrol potential against root knot nematode, Meloidogyne incognita in tomato. Among the seven isolates, 518 (1), 173 (3) and 427 recorded minimum number of galls per plant as well as gall index (0.66) and were much superior to the reference strain, Pseudomonas fluorescens (gall index of 1.00) and other isolates. These results are in line with those of Devi and Dutta (2002) who observed significantly increased plant growth and reduced root knot galling in okra plants treated with P. fluorescens. Jothi et al. (2003) also observed maximum increase in yield (64.3%) and minimum soil population of root knot nematode (56%) in tomato plants treated with P. fluorescens. Similar effects of P. fluorescence in tomato and brinjal nurseries against Meloidogyne incognita have also been recorded by Santhi and Sivakumar (1995). In addition to their potential to control plant diseases in above three experiments, most of the inoculated fluorescent pseudomonads enhanced the growth parameters and dry matter content of groundnut and tomato plants significantly over uninoculated control indicating their plant growth promotional potential. Since all these strains are known producers of IAA and GA and also possess P-solubilizing ability (Suneesh 2004; Megha, 2006), they might have contributed to better growth of plants. The plant growth promotional activity of all nineteen fluorescent pseudomonads along with two reference strains was also tested using groundnut as test plants under green house condition. Most of the inoculated strains showed significant increase in nodulation, all the plant growth parameters, nutrient uptake and yield of groundnut plants over uninoculated control. Such beneficial microorganisms are reported to have potential to increase the plant growth parameters, nutrient uptake and yield (Burr and Caelor, 1984; Clarkson, 1985; Gaskins et al., 1985; Fredrickson and Elliott, 1985). Although a large variation existed between the strains of present investigation in promoting plant growth, a few strains like, 227 (2), 427, 440 (1), 521 (2), 331 (2), performed significantly better over the reference strains in improving the pod yield of groundnut plants. The observed improvement in growth stimulation in plants inoculated with PGPR strain could be attributed to production of plant growth promoting substances like IAA and GA by these strains (Suneesh, 2004; Megha, 2006). Production of phytohormones by fluorescent pseudomonads has been reported by earlier workers (Neito and Frankenberger, 1989; Hussain and Vencura, 1970). Phytohormones such as auxins and cytokinins are known to cause enhanced cell division and root development (Arshad and Frankenberger, 1993). The increased growth and biomass of crop plants as a result of inoculatation with PGPR strains has been previously reported (Alstrom, 1987; Dileepkumar and Dubey, 1991; Burr et al., 1978) and have been implicated to be due to production of plant growth promoting substances (Gaskins et al., 1985) and increased availability and uptake of nutrients. Since all the strains used in present study are known to solubilise phosphorus (Suneesh, 2004; Megha, 2006), they might have improved phosphorus uptake of plants. Phosphorus is known to improve root growth and nodulation of legumes thereby improve the N content of plants through nitrogen fixation (Pal et al., 2003). Increased N and P uptake probably resulted in improved growth and higher yield of plants due to inoculation with fluorescent pseudomonads. Similar results of increased growth and yield of groundnut (Pal et al., 2003), sugarbeet (Suslow and Schroth, 1982), ornamental plants (Yuen and Schroth, 1986), potato (Vrany and Fiker, 1984) and fur and spruce (Zargar et al., 2005) have been obtained earlier due to inoculation with fluorescent pseudomonads having multifunctional traits.

The findings of present investigations have clearly brought out the potential of fluorescent pseudomonads of Western Ghats of Uttara Kannada district of Karnataka to reduce the incidence of different diseases of crop plants as well as to enhance the nutrient uptake and yield of groundnut and tomato plants under green house conditions. These strains could be of potential to develop as biofertilizers with biocontrol potential after testing their performance under field conditions either alone or as components of integrated disease/nutrient management systems.

VI. SUMMARY
Nineteen selected isolates of fluorescent pseudomonads of Western Ghats were assessed for their biocontrol potential against fungal, bacterial and nematode pathogens under in vitro and green house conditions. An attempt was also made to study their plant growth promotional activity. The salient features of the findings are outlined below. All 19 selected isolates of fluorescent pseudomonads and two reference strains (Pseudomonas aureofaciens NCIM 2026 and Pseudomonas fluorescens NCIM 2099) were tested for their in vitro biocontrol potential against four fungal pathogens (Rhizoctonia bataticola, Sclerotium rolfsii, Pythium sp. and Fusarium udum), three bacterial pathogens (Xanthomonas campestris, X. axonopodis and Ralstonia solanacearum) and a plant pathogenic nematode, Meloidogyne incognita. The results clearly indicated the potential of seven isolates to inhibit all the eight pathogens tested while nine of them were able to control seven plant pathogens. Two of the isolates showed inhibitory activity against six pathogens where as one isolate inhibited five of the pathogens tested. Two reference strains used in the present study were also inhibitory to all the pathogens tested. These results also clearly indicated the higher potential of several of the isolates over the two reference strains in inhibiting the number of plant pathogenic organisms. All the isolates of present investigations were also tested for production of HCN and siderophores. The results indicated the ability of all the isolates to produce both HCN and siderophores. Among the isolates, 6 isolates were identified as high HCN producers, nine as moderate HCN producers and four were weak HCN producers. All the isolates produced siderophores and a few isolates showed significantly higher production of siderophores over other isolates as well as the two reference strains. Seven selected isolates along with the two reference strains were tested for production of antimicrobial metabolites and invariably all the strains tested produced two metabolites each. The Rf values of the metabolites varied from 0.16 to 0.97. A few better performing isolates of fluorescent pseudomonads were further examined for their biocontrol potential against one each of fungal, bacterial and nematode pathogens in soil under green house conditions following dual inoculation methods. Out of seven strains examined (six isolates and one reference strain) for their biocontrol potential against S. rolfsii in groundnut under green house condition, two isolates (427 and 227 [2]) and the reference strain P. fluorescence (NCIM 2099) exhibited cent percent reduction of disease incidence whereas others showed disease incidence reduction in the range of 66.66 - 91.66 per cent. Among the six isolates tested for their biocontrol potential against Ralstonia solanacearum inciting the bacterial wilt of tomato in green house, the isolate 433 (1) recorded maximum reduction in disease incidence (91.00%), followed by 173 (3) and 427 (both with 83.33% DIR) all of which were much superior over the reference strain. Others including the reference stain showed disease incidence reduction in the range of 50-75 per cent. Among the seven isolates tested for their biocontrol potential against root knot nematode under green house condition, the isolates 518 (1), 173 (3) and 427 recorded minimum number of galls per plant as well as gall index (0.66) and were much superior to the reference strain, Pseudomonas fluorescens (gall index of 1.00) and other isolates. In addition to their potential to control plant diseases in above three experiments, most of the inoculated fluorescent pseudomonads enhanced the growth parameters and dry matter content of groundnut and tomato plants significantly over uninoculated control indicating their plant growth promotional potential. The plant growth promotional activity of all nineteen fluorescent pseudomonads along with two reference strains was also tested using groundnut as test plants under green house condition. Most of the inoculated strains showed significant increase in nodulation, all the plant growth parameters, nutrient uptake and yield of groundnut plants over uninoculated control. Although large variation existed between the strains of present investigation in promoting plant growth, a few strains like, 227 (2), 427, 440 (1), 521 (2), 331 (2), performed significantly better over the reference strains in improving the pod yield of groundnut plants.

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compete with Agrobacterium Microbiology, 56: 2834-2839.

tumefaciens.

Applied

and

Environmental

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APPENDIX
Media Composition
1. Nutrient agar (Anon, 1957) Peptone Beef extract Sodium chloride Distilled water pH Agar 2. Kings B agar medium (King et al., 1954) Peptone K2HPO4 MgSO4 Glycerol Agar Distilled water 3. Trypticase soy agar: (Frazier et al., 1987) Tryptone Soyatone Glucose Agar Distilled water 4. Potato dextrose agar (Okon et al., 1977) Potato Sucrose Yeast extract Distilled water Agar 5. Reagents used in HCN assay Picric acid solution (g/l) Picric acid NA2CO3

: 2.5 : 12.5

5.0 g 3.0 g 5.0 g 1000 ml 6.8 7.2 18.00 g 16.0 g 1.6 g 1.6 g 10.0 g 18.00 g 1000 ml 17 g 3g 2.5 g 18 g 1000 ml 200 g 20 g 0.5 g 1000 ml 18.00 g

BIOCONTROL POTENTIAL AND PLANT GROWTH PROMOTIONAL ACTIVITY OF FLUORESCENT PSEUDOMONADS OF WESTERN GHATS
SHIVAKUMAR B. 2007 Dr. A. R. ALAGAWADI MAJOR ADVISOR

ABSTRACT
Nineteen fluorescent pseudomonads of Western Ghats of Uttar Kannada district and two reference strains were studied for their in vitro biocontrol potential against four fungal pathogens, three bacterial pathogens and a plant pathogenic nematode. Seven isolates and two reference strains showed inhibitory activity against all the eight pathogens. While nine isolates were able to control seven plant pathogens, two isolates showed inhibitory activity against six pathogens and one isolate inhibited five of the pathogens tested. All isolates produced HCN and siderophores. Among the isolates, 6 isolates were identified as high HCN producers and a few isolates showed significantly higher production of siderophores over other isolates as well as the two reference strains. Seven selected isolates along with the two reference strains also produced antimicrobial metabolites having Rf values ranging from 0.16 to 0.97. The better performing isolates of fluorescent psuedomonads were further studied for their biocontrol potential against fungal, bacterial and nematode pathogens inoculated to soil under green house conditions. Two isolates (427 and 227(2)) exhibited one hundred per cent control of collar rot of groundnut caused by Sclerotium rolfsii. Similarly, in another experiment isolate 433(1) showed 91.00 per cent control of bacterial wilt of tomato caused by Ralstonia solanacerum. In the third experiment the isolates 518(1), 173(3) and 427 performed much better against root knot nematode, Meloidogyne incognita, in tomato by recording minimum number of galls per plant as well as gall index (0.66) as compared to reference strain, other isolates and the control. Out of 19 strains tested for growth promotional activity of groundnut plants under green house condition, most of the strains showed significant increase in the plant growth parameters, dry matter content, nutrient uptake and pod yield of groundnut plants over uninoculated control.