Protoplast fusions are important tools in strain improvement for bringing genetic recombination and developing hybrid strains in filamentous fungi. These are the powerful techniques for engineering of microbial strains with desirable different properties. The molecular characterization of two parent's strains (Trichoderma harzianum NBAII Th 1, Trichoderma viride NBAII Tv 23) and their corresponding fusants, produced by protoplast fusion, was investigated by random amplified polymorphic DNA (RAPD), ISSR (inter-simple sequence repeats) and internal transcribed spacer (ITS) markers. Specific results for fingerprinting were obtained by the 20 primers of each RAPD and ISSR, and four primers of ITS, respectively. These markers produced different fragment patterns with varied number of bands and yielded a total of 419 distinct bands. 13.3% were considered as polymorphic bands and 86.7% were considered as monomorphic bands. For RAPD marker, the OPC-04 primer has showed the highest polymorphism, 58.3%. While in ISSR marker, the UBC-835 primer has showed the highest polymorphism, 57.2%. The dendrogram based on RAPD marker results grouped the two parent's strains and 21 fusants into two different clusters with about 97% genetic similarity. While the dendrogram based on ISSR and ITS markers results grouped the two parent's strains and 21 fusants into two different clusters with about 88% genetic similarity. Moreover, the smallest genetic distance (0.01) was estimated between fusant-10 and fusant-12; moreover, fusant-8 and fusant-20 relatively have the highest genetic distance. Our results are consistent with the conclusion that understanding the genetic variation within parent Trichoderma strains and their corresponding fusants and molecular occurrence of protoplast fusion using RAPD, ISSR and ITS markers.

Six Trichoderma strains (collected from IARI, New Delhi and MTCC, Chandigarh) were tested for their ability to inhibit soil-borne pathogen of groundnut mainly Sclerotium rolfsii (causing stem rot on groundnut). In vitro percent growth inhibition of S. rolfsii by various Trichoderma strains were recorded at 5 th day after inoculation (DAI). Results obtained from the antagonism study indicated that Trichoderma viride (NBAII Tv 23) inhibited 61% growth of phytopathogenic fungi S. rolfsii followed by Trichoderma harzianum (NBAII Th1) (55% growth inhibition of pathogen). The specific activities of cell wall degrading enzymes chitinase, β-1,3 glucanase, protease and cellulase were tested during different incubation period (48, 72 and 96 h) when Trichoderma spp. grew in the presence of pathogen cell wall in synthetic media. The antagonist T. viride (NBAII Tv 23) induced higher chitinase and protease activity. The growth inhibition of pathogen during antagonism were positively correlated with coiling pattern of antagonists at 14 th day after inoculation (DAI) as well as with the induction of chitinase, β-1,3 glucanase and total phenol content. However, the amount of cellulase and polygalacturonase recorded was least in these antagonists treatment. A significant positive correlation (p = 0.01) between percentage growth inhibition of test fungus and lytic enzymes (chitinase, β-1,3-glucanase and protease) in the culture medium of antagonist treatment established a relationship to inhibit growth of fungal pathogen by increasing the levels of these enzymes. Among all the tested Trichoderma strains, T. viride (NBAII Tv 23) was found to be the best strain to be used in biological control of plant pathogen S. rolfsii.

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