Cloning partial endochitinase cDNA of Trichoderma harzianum antagonistic to Colletotrichum falcatum causing red rot of sugarcane

The saprophytic fungus, Trichoderma harzianum strain T5 antagonistic to the red rot pathogen of sugarcane, Colletotrichum falcatum excreted chitinases, viz. N-acetyl-beta-D-glucosaniinidase, 1,4-beta-6-N-N' chitobiosidase and 1,4-beta-D-N-N'-N" chitotriase into the culture medium containing pathogenic cell wall or chitin. The protein profile analysis on SDS-PAGE stained proteins is in the range of 20-124 kDa. The fungus produced a 97 kDa N-acetyl glucosaminidase in medium amended with fungal cell wall or colloidal chitin. Chitobiase isoforms of 66, 56, and 50 kDa and 66 and 50 kDa were detected in fungal cell wall and colloidal chitin amended media respectively. The fungus also excreted chitotriase isoforms of 66 and 50 kDa in cell wall and 50 kDa in colloidal chitin amended media respectively. The chitinase enzymes of 66 and 50 kDa, which degraded both chitobiose and chitotriose, are isoforms of both chitobiase and chitotriase that got separated at the same distance in SDS-PAGE. In an attempt to clone the endochitinase gene from T. harzianum T5, a partial cDNA of 246 bp (Genbank accession number AY762230) was obtained through RTPCR and the deduced sequence showed high level of homology with chitinase sequences of the database.