Isolation and Gene Disruption of the Tox5 Gene Encoding Trichodiene Synthase in Gibberella pulicaris. Thomas M. Hohn. Mycotoxin Research Unit, U.S. Department of Agriculture, Agricultural Research Service, National Center for Agricultural Utilization Research, Peoria, IL 61604, U.S.A. Anne E. Desjardins. Mycotoxin Research Unit, U.S. Department of Agriculture, Agricultural Research Service, National Center for Agricultural Utilization Research, Peoria, IL 61604, U.S.A. MPMI 5:249-256. Accepted 24 January 1992. This article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. The American Phytopathological Society, 1992.

The trichodiene synthase gene (Tox5) was isolated from Gibberella pulicaris, and its nucleotide sequence was determined. Tox5 was disrupted through transformation with a plasmid carrying a doubly truncated copy of the coding region and a selectable marker for resistance to hygromycin B (Hyg^r). Analysis of 82 transformants for their ability to produce the trichothecene, 4,15-diacetoxyscirpenol (DAS), resulted in the identification of five DAS‾ strains. Southern hybridization analysis of DAS‾ Hyg^r transformants indicated that the plasmid integrated at the Tox5 locus. The disrupted Tox5 gene was shown to be mitotically stable. Analysis of nine tetrads revealed either the cosegregation of the disrupter plasmid and the DAS‾ phenotype or the loss of the disrupter plasmid. These results demonstrate the feasibility of using gene disruption in G. pulicaris and suggest a general method for obtaining Tox5‾ mutants in other trichothecene-producing fungi.