Cloning and Functional Expression of Mycoparasitic Factors in Trichoderma harzianum

Abstract

Species of Trichoderma such as T.harzianum, T.viride and T.atroviride are newlinesome of the most potent antagonistic fungi, and have been used as plant growth newlinepromoters in developed countries. Endochitinase (ech42) gene which is involved in newlinemycoparasitism, was isolated from Trichoderma spp. taken from hot-arid soils of newlineRajasthan, cloned, sequenced and its expression profiling was carried by reverse newlinetranscription-polymerase chain reaction (RT-PCR) technique. The cloned DNA newlinesequence was 1,476 base pairs. Endochitinase gene expression was then observed newlinefor different Trichoderma isolates viz., T.harzianum (T14 and T12) and T.atroviride newline(T5).Among the three, higher expression of endochitinase was observed in T14 and newlineT12, whereas T5 showed lesser expression with respect to T14and T12 strain. The newlineTrichoderma chitinase enzyme activity was monitored for all isolates under study. newlineThe highest chitinase activity was observed in T14and T11 viz., 17.21 (1 enzyme newlineand#1049117;g/ml) and 13.11 enzyme and#1049117;g/ml, respectively. Chitinase gene from Trichoderma newlineharzianum was cloned and heterologously over expressed in M15 Escherichia coli. newlineThe recombinant protein of 42 kDa from E. coli was purified through Ni-NTA newlineaffinity column chromatography. The purified enzyme was active over broad range newlineof pH (2.0 to 8.0) and temperature (10 to 60°C) with the peak activity at pH 5 (0.50 newlineand#1049117;g/ml) and 20°C with enzyme activity value (0.49 and#1049117;g/ml).The purified protein newlinefractions were tested for in vitro antifungal activity against different phytopathogens newlinelike Fusarium oxysporum f.sp.lycopersici, Sclerotioum rolfsii, Alternaria brassicae newlineand Alternaria brassicicola. Purified endochitinase isolated from T. harzianum newlinecaused necrotic lesions, segmentation, branching and hyphal bursting at the newlineconcentration of 200 and#956;g ml-1.Cellulase activity of Trichoderma harzianum was newlineassessed for agro-industrially important cellulase (E.C.3.2.1.4) production. It was newlineused for media optimization studies and effect of pH, temperatures and incubation newlineperiods on cellulase activity. The maximu