ORIGINAL ARTICLE Biosurfactants are involved in the biological control of Verticillium microsclerotia by Pseudomonas spp. J. Debode, K. De Maeyer, M. Perneel, J. Pannecoucque, G. De Backer and M. Ho ¨ fte Department of Crop Protection, Laboratory of Phytopathology, Ghent University, Gent, Belgium Introduction Verticillium wilt is an important disease responsible for dramatic yield losses in many crops all over the world (Pegg and Brady 2002). Verticillium dahliae and Verticillium longisporum produce melanized resting structures, microsclerotia, which can survive for more than a decade in soil. Microsclerotia are stimulated to germinate by root exudates. The infectious hyphae that emerge from the microsclerotia penetrate the roots of the host plant (Schnathorst 1981). Because the microsclerotia are the most important structures of the pathogen for survival and in causing initial infections, the microsclerotia are regarded as one of the direct targets of biological control of Verticillium (Tjamos 2000). In the past, studies on biological control of Verticillium microsclerotia have typically focused on the use of the fungus Talaromyces flavus. This fungal antagonist kills individual Verticillium microsclerotia in vitro, because of the production of glucose oxidase that converts glucose to hydrogen peroxide (Fravel et al. 1987; Kim et al. 1988; Fahima et al. 1992). In recent years, bacterial antagonists, e.g. Pseudomonas spp., have also been identified as poten- tial biological control agents for Verticillium wilt (e.g. Leben et al. 1987; Berg et al. 2001; Mercado-Blanco et al. 2004). These previous studies have specifically targeted the suppression of the hyphal growth of Verticillium. However, to our knowledge, no research has ever looked at the direct effect of these bacterial antagonists on the viability of individual Verticillium microsclerotia in vitro. As mentioned, a better control of microsclerotia is Keywords biological control, biosurfactants, competition, phenazines, Pseudomonas, Verticillium dahliae, Verticillium longisporum. Correspondence Monica Ho ¨ fte, Department of Crop Protection, Laboratory of Phytopathology, Ghent University, Coupure Links 653 B-9000, Gent, Belgium. E-mail: monica.hofte@UGent.be 2006 ⁄ 1474: received 23 October 2006, revised 15 January 2007 and accepted 30 January 2007 doi:10.1111/j.1365-2672.2007.03348.x Abstract Aims: To examine the effect of previously described bacterial antagonists on the viability of Verticillium microsclerotia in vitro and to elucidate the possible modes of action of bacterial strains in the suppression of Verticillium micro- sclerotia viability. Methods and Results: A microplate assay was developed to test the suppressive effect of well-defined Pseudomonas spp. on the viability of Verticillium micro- sclerotia in vitro. Experiments using phenazine- and biosurfactant-deficient mutants indicated that biosurfactants and phenazine-1-carboxylic acid play a role in the suppression of microsclerotia viability by Pseudomonas spp. In addi- tion, microsclerotia colonization tests revealed that Pseudomonas spp. are able to colonize the surface of the microsclerotia, but not the inner matrix. Growth response curves showed that the population levels of Pseudomonas spp. increased when they were in the vicinity of Verticillium microsclerotia, indica- ting that Pseudomonas spp. may utilize nutrients from the microsclerotia for their growth. Conclusions: Pseudomonas spp. seem to be good candidates for Verticilllium microsclerotia biocontrol. Biosurfactant production is one of the main mecha- nisms involved in their mode of action. Significance and Impact of the Study: This line of work may contribute to a better understanding of biological control agents and their working mecha- nisms. Journal of Applied Microbiology ISSN 1364-5072 ª 2007 The Authors Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 1