Pest Management in Horticultural Ecosystems, Vol. 20, No. 2 pp (2014)

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1 Documentation of bio-efficacy of DAPG producing Pseudomonas fluorescens- 1% A.S. on Meloidogyene incognita and Ralstonia solanacearum infecting tomato in different agro-climatic regions M. S. RAO, M. KUSUM DWIVEDI*, S. P. TIWARI, R. MANOJ KUMAR, R. RAJINIKANTH, M. K. CHAYA, G. N. GRACE, K. PRITI, K. RATNAMMA, M. KAMALNATH, P. PRABU, N. VIDYA SHREE, C. GOPALA KRISHNA and T. N. SHIVANANDA ICAR-Indian Institute of Horticultural Research, Hessaraghatta Lake P. O., Bengaluru , India *Department of Entomology, C. S. Azad University of Agriculture and Technology, Kanpur, Uttar Pradesh, India Department of Plant Patholgy, Jawaharlal Nehru KrishiVishwa Vidyalaya (JNKVV), Jabalpur, Madhya Pradesh msraobio45@gmail.com ABSTRACT : Bio-efficacy of Pseudomonas fluorescens- 1% A.S. was evoluated at three agro-climatic regions of India against Meloidogyne incognita (Kofoid and White) Chitw and Ralstonia solanacearum infecting tomato (Solanum lycopersicum). Through these field trials we intend to document bio-efficacy data of this potential bio-pesticide in the management of disease complex of Tomato in Indian Institute of Horticultural Research, Bengaluru, C. S. Azad University of Agriculture & Technology (CSAUA&T), Kanpur, Uttar Pradesh and Jawaharlal Nehru KrishiVishwavidyalaya (JNKVV), Jabalpur, Madhya Pradesh. Data indicated that the seed 20 ml of P. fluorescens/kg and application of 5 tons of FYM enriched with 5 lit P. fluorescens/ha proved significantly effective in management of disease complex caused by Meloidogyne incognita and R. solanacearum infecting Tomato. This bio-pesticide can lead to the sustainable production of tomato with the significant increase in the yield of tomato. Keywords: Pseudomonas fluorescens-1% A.S., liquid formulation, Ralstonia solanacearum, Meloidogyne incognita, tomato INTRODUCTION Technical, economical and environmental factors are forcing to adopt new sustainable methods, such as the use of microbial antagonists, for the control of soilborne pathogens. Meeting the goal of improving soilborne disease control for efficient and sustainable production systems requires reduction of the chemical inputs in agriculture. Plants are constantly exposed to numerous pathogenic organisms many of which are common components of the soil biosphere. The soil-borne pathogens like nematode, fungi and bacteria infect plant roots and hence, the plant is not able to absorb nutrients from soil and often these results in death of the plant. Nematodes are of tremendous importance as components of disease complex along with other disease causing agents. The yield of tomato suffered 46.2 percent loss due to Meloidogyene incognita 3-4 larvae per gram soil under field conditions in India (Bhatti, 1994). The bacterial wilt caused by R. solanacearum (Smith, 1896) is one of the most devastating bacterial plant diseases in the tropical and subtropical regions of the world. It affects a wide range of economically important crops such as tomato, potato, eggplant, chilli and nonsolanaceous crops such as banana and groundnut in India. M. incognita and R. solanacearum together cause disease complex of tomato or it is also referred as nematode induced disease complex (Hayward, 1991). IIHR P. fluorescens strain IIHR (Pf-2) accession number ITCC B0034 was found to be having both nematicidal and bactericidal properties. This strain was found to produce an antibiotic called diacetylpholoroglucinol (DAPG) (confirmed by molecular characterization) and hence was evaluated in various centers of All India Coordinated Research Project (Nematology). This paper documents the results of bio-efficacy of DAPG producing P. fluorescens on the disease complex of tomato caused by M. incognita and R. solanacearum in these agro-climatic regions mentioned above. 222

2 Bio-efficacy of DAPG producing Pseudomonas fluorescens MATERIALS AND METHODS Field trials were conducted in two seasons in three locations mentioned above with the effective isolate of P. fluorescens IIHR (Pf-2) accession number ITCC B0034. The effect of seed ml/kg of seed with liquid formulation of P. fluorescens 1% A.S. produced at IIHR, Bengaluru was studied. The seeds were sown in the raised beds of 1 sq. m each drenched with 50 ml of this formulation. P. fluorescens 1% A. S. (CFU 2 x 10 8 /g) was added to FYM and left under shade for 15 days with moisture content of 25-28% for its enrichment with the bio-pesticide. The experiments were conducted at the different agro climatic regions such as IIHR, Bengaluru, Karnataka (13º58 N, 78 E, and at 890 m above mean sea level), C.S. Azad University of Agriculture & Technology Kanpur (25 o 26 to 26º58 N, 79 o 31 to 80 o 34 E and m above mean sea level) and Jawaharlal Nehru Krishi Vishwavidyalaya (JNKVV), Jabalpur, Madhya Pradesh (23 o 10' N latitude and 79 o 57' E longitude with altitude of 393 meters mean sea level) in sick plots. The experiment was carried out in 4 x 2.5 m plots in all these centers. Initial population densities of nematode in soil at the centers were in the range of J 2 per 100 g of soil. The soil in the experimental fields was incorporated with bio-pesticide enriched FYM. Control was maintainedwithout any treatment. All the treatments were replicated ten times in a randomized block design. After three months of transplanting of tomato, data were recorded on root-knot index on a 1-5 scale of Bridge and Page (1980), densities of M. incognita / 10 g root, disease incidence and yield/plot. Root populations of the M. incognita were estimated from 10 g samples of roots from each plant. The root samples were stained using acid fuchsin following the method of Bridge et al. (1982), homogenised, and the numbers of nematodes in the roots were recorded. The experiments were conducted in two seasons during in these agro-climatic regions.the date of sowing, transplanting and termination of experiment using Arka Vikas variety of tomato were March 11 th, 2012, April 12 th, 2012 & July 20 th, 2012in the year 2012 and July 18 th, 2013, Aug 19 th, 2013 and Nov 28 th 2013 respectively at IIHR centre in the year The date of sowing, transplanting and termination of experiment using PUSA RUBY variety of Tomato were Jan 10 th, 2012, Feb 2 nd 2012 and June 11 th, 2012 during the year, And March 12 th, April 10 th and Aug 22 nd in the year, 2013 (CSAUA&T) centre.the date of sowing, transplanting and termination of experiment using tomato were July 20 th, Aug 20 th, Oct 28 th 2012 in the year, 2012 and July 14 th, 2013, Aug 17 th, 2013 and Nov 28 th 2013 respectivelyat (JNKVV) centre in the year The data were analyzed by using standard statistical methods. The treatments were as follows: T 1 T 3 T 4 T 5 Seed treatment with P. fluorescens 1% A.S. 20ml/ kg of seed T 1 + nursery bed treatment with 50ml P. fluorescens 1% A.S. T2 + 5 tons FYM 2 lit P. fluorescens 1% A.S./ha + 5 ton FYM enriched with 3.5 lit P. fluorescens 1% A.S./ha + 5 ton FYM enriched with 5 lit P. fluorescens 1% A.S./ha T 6 Chemical treatment (Carbofuran 1.0 kg a.i./ha + streptocycline 1kg/ha) T 7 Control RESULTS AND DISCUSSION Management of bacterial wilt in tomato and in other crops has been difficult. The disease still threatens commercial production of tomato, as management including cultural practices and crop rotation had limited success. Chemicals cannot be an alternative method in controlling many bacterial diseases, as they potentially cause negative impact on ecosystem. These investigations revealed significant reduction in the disease complex on tomato roots caused by M. incognita and R. solanacearum in the treatment where seeds were treated with 20 ml/kg and incorporated 5 tons of FYM enriched with 5 lit of P. fluorescens 1% A.S. at all the centers mentioned above (Tables 1-3). This treatment had significantly increased the yield of the crop and is at par to chemical treatment (chemical check) in all three agro-climatic regions (Table 1-3). Fluorescent pseudomonads are among the most effective rhizospheric bacteria used to suppress diseases caused by soil-borne plant pathogens. P. fluorescens has been reported to be effective against Fusarium oxysporum infecting tomato (Benhamou et al., 1996). Various researchers started working on the use of P. fluorescens for the management of nematodes also in various crops. C. M. Weller and L.S. Thomashow in Pullman (WA) have shown that 2,4-diacetylpholoroglucinol (DAPG) - producing fluorescent pseudomonas play a key role in 223

3 Rao et al. Table 1. Bio-efficacy of P. fluorescens 1% A.S. against disease complex of tomato at CSAUAT, Kanpur T T T T T T T % Table 2. Bio-efficacy of P. fluorescens 1% A.S. against disease complex of tomato at IIHR, Bengaluru T T T T T T T % Table 3. Bio-efficacy of P. fluorescens 1% A.S. against disease complex of tomato at JNKVV, Jabalpur T T T T T T T %

4 Bio-efficacy of DAPG producing Pseudomonas fluorescens declining the population of the pathogens (Raajimakers et al, 1998 and 2001). Siddiqui et al. (2003) reported that HCN, a secondary metabolite produced by a strain of P. fluorescens was responsible for its bio-control activity on M. javanica on tomato and they found exogenous cyanide inhibiting egg hatch and causing mortality of juveniles of M. javanica in vitro. Production of the antibiotic 2, 4-DAPG contributes to biological control activity of many beneficial strains of the bacterium P. fluorescens (McSpadden Gardener, 2007; Weller, 2007). DAPG is active against numerous organisms, including plants, fungi, viruses, bacteria and nematodes. Increased production of DAPG has been associated with enhanced activity against plant pathogens (Keel et al. 1992; Maurhofer et al. 1992; Mazzola et al. 1995; Cronin et al. 1997; Delany et al. 2001; Dwivedi and Johri, 2003; Siddiqui and Shaukat, 2003a; 2003b; 2004a). Consequently application of DAPGproducing pseudomonads can result in increased crop yields (Mcspadden Gardener et al. 2006a; 2006b) Application of huge quantity of carbofuran and streptocycline to the soil ecosystem will have tremendous adverse effect in the long run. As such kg of nematicide and 2-3 kg of bactericide chemicals cannot be applied to treat the soil uniformly as these target pathogens are spread in top cm of soil in the fields. Hence, these investigations were carried out to develop a delivery system for the application of this potential bio-pesticide in different agro-climatic regions in India. Any suitable organic material which could be enriched with this bio-pesticide for the application in the main field conditions can serve the purpose. Among organic manures such as vermincompost; city compost and farm yard manure (FYM) we found FYM is comparatively better. City compost was found to contain lot of heavy metals. Vermicompost if available at cheaper cost, it could be also a better option. As FYM is available every where, we used FYM to enrich with this P. fluorescens-1% A.S. Bio-pesticides enriched FYM could be easily applied to the soil. In earlier experiments we observed seedlings produced by seed and substrate treatment of P. fluorescens resulted in very good colonization of bio-agent (unpublished). So with these treatments, we transplanted P. fluorescens colonized seedlings to the main infield. In this way seedlings get initial protection from target pathogens attack in mainfield soon after transplanting. Through the use of P. fluorescens for seed, substrate and mainfield treatment, this bio-agent is able to colonize and establish in the rhizosphere which we computed and expressed as root colonization of bio-agent (Table-1-3). Successful root colonization of any bio-agent is an important criteria to judge the rhizospheric competency of any bio-agent. Further P. fluorescens is an endophyte and gets inside the tomato plant system and trigger induced systemic resistance which would offer greater scope for the management of virulent strains of R. solanacearum. These studies are being carried out and the results will be communicated in subsequent publications. The dosage of 5 tons of FYM enriched with 5 l of P. fluorescens 1% A.S. was significantly effective in reducing the root-knot index, nematode population and disease incidence in tomato. Root colonization of P. fluorescens was significantly higher at this dosage when compared to other dosages. The experiments were repeated in two seasons during the year 2012 to 2013 to confirm the results on the efficacy of application of this bio-pesticide enriched FYM on the management of root-knot nematodes on tomato under the field conditions at different agro climatic regions given in the text. ACKNOWLEDGEMENT The authors thank the Director, IIHR, Bangalore, Directors of Research of C. S. 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