IMMUNODETECTION OF ASCOSPORES OF THE RINGSPOT PATHOGEN MYCOSPHAERELLA BRASSICICOLA R KENNEDY and AJ WAKEHAM Horticulture Research International, Wellesbourne, Warwick CV35 9EF, UK. Background and Objectives The ringspot pathogen Mycosphaerelia brassicicola, is a major air-borne disease of vegetable brassicas in the U.K. The characteristic light brown to black necrotic lesions contain black perithecia (fruiting bodies) which are dependent on periods of leaf surface wetness for their formation. Perithecia are often formed concentric rings and at maturity release ascospores which are wind-borne over considerable distances. The pathogen has a relatively long latent/incubation period. In areas of intensive vegetable brassica cultivation control of the disease is problematical due to only very short periods of favourable environmental conditions required for infection and the wind-borne nature of the inoculum. Models [1] have been developed for this disease which can accurately predict the production of inoculum within the crop.By measuring environmental variables predictions on the likely inoculum availability from lesions within the crop can be ascertained. However infection resulting from long distance dispersal of inoculum cannot be predicted. This source of disease is potentially important if improved timing of fungicide application to the crop is to be used to improve fungicide efficiency. Improved control of ringspot could be achieved by using accurate detection systems for ascosporic inoculum in conjunction with disease forecasting models. Conventional methods of detecting ascospore presence has proved laborious and unreliable. Employing immunofluorescence (IF), the use of a polyclonal antiserum (PAb) to facilitate rapid detection of M. brassicicoia ascospores is described in this paper. Materials and Methods A Pab was produced in a New Zealand White rabbit to whole ascospores of M. brassicicoia
  • Employing IF [21 the specificity of the PAb was assessed against a range of air and soil-borne fungi. To block spore and mycelial autofluorescence the counterstains Evans blue and Eriochrome black were used [3]. A Melinex tape was incubated for 3 hours at room temperature in 5 % Bovine serum albumin. Following air-drying the tape was exposed, under white fluorescent lighting, to a lightly misted sporulating culture of M. brassicicola over a 24 hr period at 12 'C. The tape was removed and attached to a glass microscope slide using double sided adhesive tape. Following IF the tape was detached from the holding adhesive tape and using Gelvatol (Burkard Scientific, Uxbridge, U.K.) affixed directly onto a glass slide, mounted with PPDG (90 mi glycerol, 10 m] PBS, 1 mg mi -' p-phenylendiamine) and viewed under Episcopic-fluorescence. Results and Conclusions Of the fungal species tested, only low levels of fluorescence of the conidial wall of Eurotium amstelodami was observed. Using the counterstains Evans blue and Eriochrome black autofluorescence of spore and mycelial components tested was eliminated. Spore tape, with both non germinated and germinating ascospores were identified by the intense fluorescence of cell wall components. Fluorescence was not reduced with the addition of either counterstain. Further investigation of ascospore attachment to the pre-coated Melinex tape in comparison to conventional melinex tape coatings and the specificity of the PAb will enable the development of an assay system which could be used to detect ascospores within crop canopies. Future studies will investigate the use of more automated test formats. Such rapid and direct detection of the presence or absence of ascospores used in conjunction with disease forecasting models would enable optimisation of fungicide applications within areas of intensive brassica production. References 1. Cullington JE, 1995. Ph.D. Thesis. University of Birmingham 2. De Boer S, 1990. Serological Methods for Detection and Identification of Viral and Bacterial Antigens IV.,295-299. 3. Montenegro-James, S. and James, M A. 1985. American Journal of Vetinary Research 1 1, 2401-2403.