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

Abstract

Pseudothecia containing abundant ascospores of Mycosphaerella brassicicola were produced in vitro on Brussels sprout decoction agar at 15ЊC 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 20ЊC and 25ЊC 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.

Methods for Integrated Air Sampling and DNA Analysis for Detection of Airborne Fungal Spores

“…While the potential of these techniques for detection of airborne spores has been recognized for some time (12,14), there have been few reports on progress in this area. The use of immunoassay in the detection of airborne plant pathogenic fungi has been investigated (10,20,21). However, the application of these methods is restricted by the difficulties of developing antibodies showing the required specificity.…”

A New Method To Monitor Airborne Inoculum of the Fungal Plant Pathogens Mycosphaerella brassicicola and Botrytis cinerea

“…Similarly, workers have developed an immunofluorescence test to detect and quantify airborne ascospores of Mycosphaerella brassicicola, the ringspot pathogen of brassicas. Trapped ascospores are labeled directly on B 7-day trap Melinex tape with polyclonal antibodies (PAbs) and anti-rabbit fluorescein isothiocyanate-conjugated antibodies (17). However, when these two methods are used as routine quantification methods, both tests are laborious, require expensive laboratory equipment for analysis, and include processes which require manual operation.…”

“…A second PAb, designated PAb 96/10/4, which was previously raised by using whole ascospores of M. brassicicola, was also used in this study (17).…”

“…B 7-day traps have been used routinely to monitor fungal air spora (12,13,24,26) and in a wide variety of air-sampling studies (3,10). Ascospores on the Melinex spore tape were detected and quantified by immunofluorescence, as described by Kennedy et al (17).…”

“…The plants were visually examined for expression of ringspot lesions. To confirm that ringspot (M. brassicicola) symptoms were present, infected leaf tissue was removed and surface sterilized for 1 min in aqueous sodium hypochlorite (4% [wt/vol] available chlorine), and organisms were isolated by using SLD agar (17).…”