Two–dimensional and chitinase activity analysis of a novel mutant of Trichoderma koningii for biodegradation of Macrophomina phaseolina cell walls

The role of extracellular enzymes in mycoparasitism of Trichoderma spp. have been demonstrated in several cases. Trichoderma spp . produces several chitinolytic enzymes and this fungus is known as a powerful antagonist against Macrophomiona phaseolina, the causal agent of charcoal rot disease of soybean. In this study, two–dimensional protein pattern analysis and chitinaseactivity evaluation were performed for an Iranian Trichoderma koningii strain (NAS–K1) and its generated mutants were measured to indicate the potential role of endochitinases (N–acetylglucosam–inidase (NAG–I and NAG–II)) in its mycoparasitism. The results of chitinase activity assay using chitin and cell walls of M. phaseolina as substrates showed that the mutant isolates have significantly more enzyme activity compared to the wild type strain. The specific endochitinase enzyme activity in the mutant NAS–K1M25 was increased to approximately 2.5 times more and secreted three times more endochitinase than that of the wild type strain. This superior mutant showed up to 65% growth inhabitation against M. phaseolina in dual culture test (five times more than the wild type strain). Moreover, this strain showed sharper spots belong to endochitinase, and N–acetylglucosaminidase (I & II) presented in SDS–PAGE and 2D electrophoresis. Overall, induced mutation by gamma irradiation could be a useful method to develop such superior mutants, and the mutant NAS–K1M25 could be used as a potential biological control agent candidate for plant disease management programs of M. phaseolina. However, more detailed fermentation, formulation and field trial studies should be performed to finalize its biocontrol potentials.