Bioresource Technology 97 (2006) 2330–2334 Self-fusion of protoplasts enhances chitinase production and biocontrol activity in Trichoderma harzianum V.R. Prabavathy, N. Mathivanan ¤ , E. Sagadevan, K. Murugesan, D. Lalithakumari Centre for Advanced Studies in Botany, University of Madras, Guindy Campus, Chennai 600 025, India Abstract Protoplasts were isolated from Trichoderma harzianum strain PTh18 using lysing enzymes and self-fusion of T. harzianum protoplasts was carried out using polyethylene glycol in STC buVer. The fused protoplasts of T. harzianum were regenerated and 15 self-fusants were selected to study the chitinase production and biocontrol activity. High chitinase activity was measured in the culture Wltrates of most of the self-fusants (87%) than the parent. Among the fusants, the strain SFTh8 produced maximum chitinase with a two-fold increase as compared to the parent strain. All the self-fusants exhibited increased antagonistic activity against Rhizoctonia solani than the parent. The crude chitinase preparation of SFTh8 lysed the mycelia of T. harzianum, Trichoderma viride and Trichoderma reesei and released the pro- toplasts in higher number than the crude chitinase preparation of parent strain PTh18.  2005 Elsevier Ltd. All rights reserved. Keywords: Trichoderma harzianum; Protoplast fusion; Chitinase; Antagonistic activity 1. Introduction Trichoderma is one of the most exploited fungal biocon- trol agents in agriculture for the management of plant dis- eases caused by a wide spectrum of fungal pathogens (Elad, 2000; Mathivanan et al., 2000). Production of extracellular hydrolytic enzymes is one of the biocontrol mechanisms exerted by Trichoderma towards fungal pathogens. Several biocontrol agents limit the growth of pathogenic fungi by producing extracellular chitinase, which degrades the chitin polymers of fungal cell wall (Mathivanan et al., 1998). Protoplast fusion is an important tool in strain improve- ment for bringing genetic recombination and developing hybrid strains in Wlamentous fungi (Lalithakumari, 2000). Previously Das et al. (1988) demonstrated the intra-strain protoplast fusion for inducing genetic recombination in Aspergillus niger. Isolation, fusion and regeneration of protoplasts have been carried out in Trichoderma mainly for improving the biocontrol potential (Mrinalini and Lalithakumari, 1998). Ogawa et al. (1989) reported enhanced cellulase production in Trichoderma reesei by inter-speciWc protoplast fusion, but not much work has been focused for increasing the production of other hydro- lytic enzymes such as chitinase in Trichoderma using this technique. Therefore, the present study was aimed to isolate the protoplasts from Trichoderma harzianum and carry out self-fusion of protoplasts with the objective of enhancing the chitinase production. In addition, the antagonistic potential of the fusant strains was also studied and the results are presented in this paper. 2. Methods 2.1. Culture of Trichoderma harzianum A large number of Trichoderma strains were isolated from the soil samples collected from diVerent parts of Southern India by serial dilution on Trichoderma selective medium (Papavizas and Lumsden, 1982). Qualitative and quantitative screening for extracellular chitinase resulted in * Corresponding author. Tel.: +91 44 22350401; fax: +91 44 22352494. E-mail address: prabhamathi@yahoo.com (N. Mathivanan).