Protocols for in Vitro Sporulation, Ascospore Release, Sexual Mating, and Fertility in Crosses of Leptosphaeria maculans. Alemu Mengistu, Department of Plant Pathology, University of Wisconsin, Madison 53706. Roger S. Rimmer, and Paul H. Williams. Department of Plant Science, University of Manitoba, Winnipeg, Canada R3T 2N2; and Department of Plant Pathology, University of Wisconsin, Madison 53706. Plant Dis. 77:538-540. Accepted for publication 13 January 1993. Copyright 1993 The American Phytopathological Society. DOI: 10.1094/PD-77-0538.

Leptosphaeria maculans ascospores were produced in vitro by pairing single ascospore isolates on V8 juice agar. Incubation for 7 days at 24 C with continuous fluorescent lighting provided for intermingling of the developing fungal colonies. Production of pseudothecia and ascospores was stimulated by pouring a 20-ml layer of 2% water agar cooled to 55 C over a culture grown on V8 juice agar, then incubating the dishes for 4 wk at 16 C under black light with a 12-hr photoperiod. Ascospores were recoverable only within 48 hr after they matured. Release of ascospores was facilitated by placing a drop of 5% ?-glucuronidase over the asci, followed in 30–60 sec by two drops of sterile water. The number of pseduothecia varied significantly among the isolates tested. Crosses between isolates WA11 × WA30, WA22 × WA13, WA22 × WA30, WA32 × WA30, WA43 × WA40, and WA74 × WA30 had the highest number of pseudothecia and were the most fertile of the isolates tested.

Keyword(s): black leg, brassica disease, Phoma lingam, spore storage.