Research Article TRICODERMA SP AS A MICROBIAL ANTAGONIST AGAINST RIZOCTONIA SOLANI M. PADMAJA, J. SWATHI, K. NARENDRA, K. M. SOWJANYA, P. JAWAHAR BABU, A. KRISHNA SATYA* Department of Biotechnology, Acharya Nagarjuna University, Guntur-522510 India. Email: akrishnasatya78@gmail.com Received: 02 Sep 2013, Revised and Accepted: 13 Oct 2013 ABSTRACT Objective: The antagonistic potential of native Tricoderma isolates was investigated in vitro with an objective of selecting an efficient native bio- control agent against the most prevalent soil borne pathogen against R. solani the causal agent of sheath blight in rice. Methods: Ten native Tricoderma isolates and a commercial formulation of Tricoderma were screened by using Dual culture method. Eight isolates have shown effective inhibition of pathogen growth. Four isolates showed maximum growth inhibition of 70 to 76%. Microscopic observation of cultures taken from interaction zone of dual culture plates was done in order to find the mycoparasitic interaction between pathogen and antagonist which showed that the hyphae of Trichoderma isolates could grow parallel to the hyphae of Rhizoctonia solani, coiled around the hyphae and formed appresoria and hook-like structures. Four Trichoderma isolates caused lysis and over growth on R. solani. Results: These native antagonists have the properties of potential bio control agent like in vitro antagonism towards the test pathogen, producing fungal cell wall degrading enzymes and mycoparasitic behaviour. Conclusion: The native isolates proved effective in controlling the pathogen in-vitro than the commercial formulation, indicating their superiority in the bio control of phyto pathogens. Keywords: Bio control agent, Mycoparasitism, Rhizoctonia solani, Trichoderma. INTRODUCTION Soil born, plant pathogenic fungi such as Sclerotium, Rizoctonia, Pithium, Fusarium cause diseases in most of the economically important crops. Sheath blight caused by Rhizoctonia solani is one of the most important destructive diseases of rice next to rice blast [1]. R. solani is a soil borne necrotrophic fungus with wide host range and survives in the soil as hard, resistant sclerotial bodies.[2] Besides rice, it can infect crops of nearly 50 species, including barley, lettuce, tomato, sorghum, and maize which is responsible for damping-off, blackspot and root rot diseases. No effective fungicides are available against Rizoctonia diseases that damages vegetables and more over chemical control has undesirable effects like phyto toxicity and environmental pollution. [3] In this regard biological control offers an alternative solution for long term sustainability and effective management of soil borne diseases. Tricoderma sp which is a common saprophytic filamentous fungi in almost any soil and rhizosphere microflora, is well recognized as bio control agent against various plant pathogenic fungi. Different mechanisms have been suggested for its bio-control activity, which include competition for space and nutrients, secretion of lytic enzymes, mycoparasitism and production of inhibitory compounds.[4] The objective of this study was to evaluate the use of Trichoderma spp. in the biocontrol against R. solani in vitro. MATERIALS AND METHODS Isolation & Identification of pathogen and antagonistic mycoflora Test pathogen Rhizoctonia Solani was isolated from diseased plants. Diseased portions along with visible mycelia transferred aseptically on to petriplates that contained PDA medium and incubated at 28 ± 1 0 C. After two days of incubation, hyphal growth was observed, which was further purified by hyphal tip method. The identification of the fungus isolated during the study was done based on morphological characters such as hyphal branching, septal pore type and sclerotial formation. Pathogenicity of the fungus was tested on plants by seed treatment method. Pathogen was reisolated from infected plants and compared with original isolate. R. solani  Mycelium was white initially, turned to brown  Hyphae pale brown, branched with nearly right angled side branches constricted basally, septate.  Sclerotia brown to dark brown Isolation of Trichoderma spp. from soil samples was done by serial dilution method. By using these pure cultures, identification of species of Trichoderma was done based on their cultural and morphological characteristics described by Gams and Bisset [5] For eliminating the bacteria antibiotic was added to the media. One sample was isolated from commercial formulation available in the market which is used by the farmers. Colony radial growth was measured and rated as fast, medium and slow growth. Finally Ten Tricoderma isolates are selected based on their radial growth from many fungal isolates obtained from different soil samples collected from organic fields, and farmer’s fields of natural cropping system of the area (paddy, groundnut and vegetable). These isolates are different from one another by their source, colony colour and radial growth. Screening of antagonists Ten isolates of native Tricoderma, and Commercial formulation were tested by dual culture technique. [6] Twenty ml of autoclaved PDA was poured aseptically in to sterile Petri dishes of 9cm diameter A 2 mm mycelial disc of Rhizoctonia solani and Trichoderma were placed opposite to each other near the periphery of the petri plate and incubated at 29± 1 0 C. Rhizoctonia solani alone inoculated plate served as control. Mycelial growth of the pathogen was measured and observations were recorded on formation of inhibition zone, over growth and lysis of pathogen mycelium. Mycoparasitism Hyphae from the interaction zone of the dual culture plates was observed under Labomed LX400) compound microscope. Detection of the in vitro activities of protease and cellulase by Tricoderma isolates The antagonistic isolates were evaluated for the in vitro activities of protease and cellulase. To determine protease activity indicated by casein degradation, fungal isolates were plated on skimmed milk agar (50 ml of sterilized skim-milk mixed with 50 ml of 4% water agar at 55 0 C), and the width of each resulting clearing halo was recorded as an indicator of the level of protease activity.[7] The International Journal of Pharmacy and Pharmaceutical Sciences ISSN- 0975-1491 Vol 5, Suppl 4, 2013 A A c c a a d d e e m mi i c c S S c c i i e e n n c c e e s s