Production and immunodetection of ascospores of Mycosphaerella brassicicola: ringspot of vegetable crucifers

Pseudothecia containing abundant ascospores of Mycosphaerella brassicicola were produced in vitro on Brussels sprout decoction agar at 15°C under a 16-hour photoperiod of different light regimes. Spermogonia containing spermatia were also produced on the decoction agar. Ascospores were released when cultures were misted with SDW and placed under continuous light. Germination of ascospores was highest between 20°C and 25°C and spores remained viable at relative humidities above 93.5%. Exposure of ascospores to 55% relative humidity for 24 h reduced their germination to 75%. A polyclonal antiserum raised against whole ascospores was used to detect, by immunofluorescence, the ascospore and mycelial wall of M . brassicicola , following reaction with anti-rabbit IgG FITC conjugate. Autofluorescence of spore and mycelial components of other fungal species could be eliminated using the counterstains Evan's blue and eriochrome black at 0.2% and 0.5%, respectively, in phosphate buffered saline (pH 7.2). A procedure was developed to detect, by immunofluorescence, ascospores of M . brassicicola on artificially inoculated Melinex spore tape. Coating of the spore tape with bovine serum albumin provided a suitable support medium and blocking agent for detection of ascospores in the field. The potential use of the system for selective detection of ascospores of M . brassicicola in infected crops of vegetable brassicas in the presence of other ascosporic fungi is discussed. Keywords : ascospores, immunofluorescence, Mycosphaerella brassicicola , spore production, spore trapping .