Inter-specific protoplast fusion in Trichoderma spp. For enhancing enzyme production and biocontrol activity

Abstract

The enzymes, particularly cellulase, have got tremendous industrial application. These enzymes are employed for the conversion of wood and its products, cellulosic agricultural by-products to fermentable substances and in the conversion of lignocellulosic materials into biofuels like ethanol and single cell proteins. In Plant disease management, biological control plays a vital role. The cell-wall degrading enzymes chitinase have been suggested to be essential for the mycoparasitic action of Trichoderma species against plant fungal pathogens. In order to develop an unique effective strain as a single source of both these vital enzymes, it was intended to integrate the characterstics of high cellulase producing Trichoderma reesei and high chitinase producing Trichoderma harzianum by fusing their protoplasts. Protoplasts were isolated from 16 h old cultures of T. harzianum and T. reesei using Lysing Enzyme. The fusion of protoplasts was achieved by using Polyethylene glycol (PEG) as fusing agent. Among the 20 fast growing fusants, six were selected based on their enhanced growth on selective media and antagonistic activity. All the six selected fusants exhibited morphological variations such as mycelial growth, pigmentation, sporulation and spore coloration among themselves further; they also possessed some common morphological characteristics of the parent strains. The extent of fusion in the fusant strains was confirmed by PCR-RFLP method, which exhibited the presence of DNA from both the fusing parents. The fusant strains were growing 60%-70% faster than the parents, upto 3^rd generation. The fusant strains displayed 40-50% increased cellulase production and 10-20% increase in chitinase production than the parent strains, and hence 7%-8% higher antagonistic activity than the parents.