Filtration enrichment method for isolation of auxotrophic mutants of Trichoderma harzianum rifai

ABSTRACTThe isolation of genetic markers, like drug resistance and auxotrophy, is a laborious butimportant step in genetic research. The isolation of auxotrophic mutants of Trichodermaharzianum using the filtration enrichment technique was more effective than using thetotal isolation technique. Most of 12 auxotrophic mutants exhibited similar growth rateand higher sporulation when compared with the wild type, but only two mutants (TWS-410 and TW5-523) could grow in 500µg/L of benomyl.Key-words: Filtration enrichment technique, auxotrophic mutants, Trichodermaharzianum, benomyl Revista de Microbiologia (1999) 30:43-46ISSN 0001-3714* Corresponding author. Mailing address: EMBRAPA Œ Meio Ambiente, Caixa Postal 69, CEP 13820-000, Jaguariœna, SP, Brasil. Fax: ( +5519)867-8740. INTRODUCTIONTrichoderma spp. Rifai are the most promisingproducers of cellulolytic and chitinolytic enzymes, andalso are currently investigated as biological controlagents of plant pathogens. Their sexual state is unknown,but parasexual cycle has been studied with auxotrophicmarkers permitting selection of heterokaryons and ofpossible diploids (5). The classical method used, totalisolation following mutagenic treatment, is laboriousand yields a low frequency of auxotrophics amongtested survivors (6). Several methods have beendescribed for the selection of auxotrophic mutants offungi. These include biotin-starvation methods that havebeen used for Aspergillus nidulans and comparablemethods have been used in other fungi (6). To inducemutants of Aspergillus niger, Bos et al. (4) used lowdoses of the mutagen ultraviolet light (UV) in order toavoid background mutations or chromosomalrearrangements. Usually, this procedure results in highsurvival and low frequency of mutants among survivingprototrophics. Consequently, an efficient enrichmentstep can be a prerequisite. In this paper we describe theisolation of auxotrophic mutants of Trichodermaharzianum by a filtration enrichment technique basedon the technique developed by Silveira and Azevedo(8) for Metarhizium anisopliae.MATERIALS AND METHODSMicroorganism Œ T. harzianum TW5, originallyisolated from a soybean field in Brazil, was obtainedfrom the fungi collection of National Research Centerfor Monitoring and Assessment of EnvironmentalImpact, EMBRAPA, Brazil. This strain has been shownto be antagonist to the plant pathogens Sclerotiniasclerotiorum and Sclerotinia minor.