298 Indian Phytopathology 69 (3) : 298-303 (2016) Indian Phytopath. 69 (3) : 298-303 (2016) Morphological and molecular characterization of Trichoderma asperellum strain Ta13 R. THAVA PRAKASA PANDIAN, M. RAJA, A. KUMAR and PRATIBHA SHARMA* Division of Plant Pathology, ICAR-Indian Agricultural Research Institute, New Delhi 110 012, India ABSTRACT: Morphological characters are not sufficient to properly identify different species of Trichoderma. Recently, multi-gene phylogeny in combination with morphological characters is used to identify Trichoderma at species level. The current study was focused to characterize Trichoderma asperellum strain Ta13 based on morphology and molecular analysis using genes such as ITS, tef1, rpb2, act and cal. Light and scanning electron microscopy (SEM) results showed that Ta13 has regularly branched, typically paired conidiophores with straight phialides and globose to sub-globose shaped conidia having size of 2.91μm x 2.37μm with inconspicuous ornamentation. Sequence similarity analysis with reference T. asperellum isolates available in ISTH database showed 99.30, 90.60, 99.20, 98.70, 100 and 99.80 per cent nucleotide similarity for ITS1 and ITS2, tef1 intron4 (large), tef1 intron5 (short), rpb2, cal and act respectively. Confrontation assay clearly showed that Ta13 inhibited several fungal plant pathogens viz. Rhizoctonia solani (88.69 per cent), Fusarium oxysporum f. sp. lycopersici (85.27 per cent), F. moniliforme (84.55 per cent), S. sclerotiorum (80.17 per cent), S. rolfsii (78.26 per cent), and A. brassicicola (77.5 per cent). Key words: Biocontrol agents, confrontation assay, multi-gene analysis, Trichoderma asperellum RESEARCH ARTICLE *Corresponding author: psharma032003@yahoo.co.in Trichoderma species (Perfect stage: Hypocrea) is a filamentous ascomycetous fungus widely used as bio- control agent (BCA) against plant pathogens. It comprises different economically important species viz., T. harzianum, T. asperellum, T. viride, T. atroviride and T. virens and T. reesei (Sharma et al., 2014). The above species are well known for their biocontrol activity, rhizosphere competence and production of hydrolytic enzymes such as cellulases, chitinases, glucanases and proteases as well as many secondary metabolites (Sharma et al., 2011; Nicolus et al., 2014). Trichoderma asperellum Samuels Lieckf. & Nirenberg is morphologically indistinguishable from its cryptic species T. asperelloides (Samuels et al., 2010). Molecular tools such as multi-gene phylogeny were commonly used to define Trichoderma species. Very recently, Trichoderma viride is re-classified (Sriram et al., 2013; Mukherjee, 2015). The present work was mainly focused on characterization of Trichoderma asperellum strain Ta13 based on morphological and molecular analysis. MATERIALS AND METHODS Fungal cultures Fungal biocontrol agent, T. asperellum (Ta13) (ITCC number-7766) and fungal plant pathogens were obtained from biological control laboratory, Division of Plant Pathology, ICAR-Indian Agricultural Research Institute, New Delhi. Cultures were routinely grown in potato dextrose agar (PDA) (HIMEDIA Laboratories Pvt. Ltd., India) plates and incubated at 28±2°C for 5-7 days. Confrontation assay Antagonistic potential of Ta13 was studied by dual culture assay against various fungal plant pathogens by keeping the pathogen inoculated PDA plates as control (Dennis and Webster, 1971). Six replications of the each culture plates were incubated at 28±2°C for measuring colony growth both in control and dual culture plates. The plates were evaluated based on the grading method (Bell et al., 1982). The radial growth of the pathogen was measured after full growth and the percent inhibition was calculated as follows: PI = (C - T) × 100 / C, where PI is the percent inhibition of mycelial growth; C is the radial growth of pathogen in control plates (cm) and T is the radial growth of pathogen in dual culture (cm). The antagonistic potential was further evaluated by cell-free-culture filtrate assays by growing in liquid cultures. Discs of actively growing fungal mycelium (5mm) was inoculated and then incubated at 28±2°C for 10 days in shaker cultures and filtered through Millipore filters (Pore size 450 nm). Poisoned food technique was followed to analyze the inhibitory properties of Ta13 against the pathogen and expressed as per cent of inhibition over the control. Microscopy Light microscopy Single spore culture of Ta 13 was prepared and recorded for its cultural characters viz. growing pattern, colour and texture as well as morphological features viz. conidiophores branching pattern, phialides structures, conidial shape and size (Olympus CH20i, Olympus opto system India Pvt. Ltd., India).