Vol. 94, No. 2, 2004 171 Biological Control Elicitors of Plant Defense Responses from Biocontrol Strains of Trichoderma virens L. E. Hanson and C. R. Howell First author: U.S. Department of Agriculture-Agricultural Research Service (USDA-ARS), Sugar Beet Research Unit, Fort Collins, CO 80526; and second author: USDA-ARS, SPARC, CPRU, College Station, TX 77845. Accepted for publication 5 September 2003. ABSTRACT Hanson, L. E., and Howell, C. R. 2004. Elicitors of plant defense re- sponses from biocontrol strains of Trichoderma virens. Phytopathology 94:171-176. Effective biocontrol strains of Trichoderma virens can induce the pro- duction of defense-related compounds in the roots of cotton. Ineffective strains do not induce these compounds to significant levels. This elicit- tation was found to be heat stable, insoluble in chloroform, passed through a 5K molecular weight cut-off (MWCO) filter, but not a 3K MWCO filter, and was sensitive to treatment by proteinase K. When the active material was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, several bands were present in the material from bio-control-active strains that were lacking in inactive strains. When eluted and tested for elicitation activity, with or without renaturation, four bands stimulated cotton terpenoid production. One band showed cross-reaction with an antibody to the ethylene-inducing xylanase from T. viride. Another band of approximately 18 kDa, gave significant stimulation of cotton terpenoid production and increased peroxidase activity in cotton radicles in all tests, with or without renaturation. The 18-kDa protein was subjected to amino-terminal sequence analysis, and the first 19 amino acids at the amino terminus were determined to be DTVSYDTGYDN- GSRSLNDV. A database homology search using the BLASTp algorithm showed the highest similarity to a serine proteinase from Fusarium sporotrichioides. Trichoderma spp. have biocontrol activity against a number of different fungi on various hosts. On cotton, biocontrol activity has been reported against Rhizoctonia solani (12), Fusarium oxysporum f. sp. vasinfectum (26), and Verticillium dahliae (10). One proposed mechanism for biocontrol activity in Trichoderma sp. is stimulation of host defense responses (14). Induced resis- tance has been reported with T. harzianum on bean (5), an unidentified Trichoderma sp. on cucumber (18), and T. virens (Gliocladium virens) on cotton (10). Cotton seedlings treated with effective biocontrol strains of T. virens have higher levels of de- fense-related compounds such as terpenoids and higher peroxi- dase activity in the roots than seedlings treated with ineffective isolates (14). Several fungi and oomycetes produce compounds that elicit the production of defense responses in plants (2,4,69,19,21). The majority of these fungi are plant pathogens, but metabolites from Penicillium janczewskii elicit resistance to stem rot in melon and cotton (19) and an ethylene-inducing xylanase (EIX) produced by T. viride (3) elicits the production of the phytoalexin resveratrol in grapevine cells (1). Little is known about the potential production of other elicitors by nonpathogenic plant-associated fungi. Culture filtrates from effective biocontrol strains of T. virens were found to stimulate significantly greater terpenoid levels in cotton than filtrates from noneffective strains or media alone. The purpose of this research was to isolate and identify elicitors from such T. virens culture filtrate. MATERIALS AND METHODS Parent and mutant strains of T. virens were stored as conidia in 25% glycerol at 70C until used. Prior to use, conidia were transferred to potato dextrose agar (PDA, Difco Laboratories, Detroit) plates containing 50 g of rifampicin per ml, and colonies developing from them were used as a source of inoculum. Strains used in this experiment included: T. virens strain G6, an isolate from Texas cotton field soil with good biocontrol activity against R. solani (15) and two UV-induced mutants of G6 (G6-4 and G6-5) that were deficient for both mycoparasitism and gliotoxin production (provided by M. H. Wheeler, College Station, TX). Mutant G6-5 had good biocontrol activity against R. solani, but mutant G6-4 had no detectable biocontrol activity against R. solani (14). To obtain culture filtrates (CFS), the Trichoderma isolates were grown in liquid shake cultures (G-10 gyratory shaker; New Brunswick Scientific Co., New Brunswick, NY) of 100 ml of 5% wheat bran and 1% peat moss (WBPM, pH 4.0) as described by Howell et al. (13). After 7 days incubation at 150 rpm and 27C, the culture contents were filtered through Whatmanns No. 1 filter paper, and the CFS were collected. CFS were either used directly or lyophilized and brought to the desired concentration in sterile water. CFS were filter-sterilized by passage through 0.22-m filters (Sterile Acrodisc; A. Daigger E-mail ad