Development of an “ascospore shower” method for inoculating oilseed rape leaves with Leptosphaeria maculans

Two types of inoculum (ascospores and conidia) and four inoculation methods (spore suspension droplet on unwounded leaf, spore suspension droplet on wounded sites on leaf, spore suspension sprayed onto leaves and dry ascospores ‘raining down’ onto leaves) were compared, to develop an efficient method for inoculation with L. maculans when inoculum is limited. Ascospores were more infective than conidia. Leaves on all plants inoculated with ascospores, whether ascospores were produced under natural conditions or from defined crosses between two compatible isolates, developed phoma leaf spots, regardless of the inoculation method. Leaves inoculated with conidial suspensions by droplet inoculation on wounded sites and by spraying developed lesions, while leaves
inoculated by droplet inoculation without wounding produced no lesions. For droplet inoculation, the infection efficiency (% inoculated sites which produced lesions) of ascospores was 100% while the infection efficiency of conidia was 56.1%. With spray inoculation, one lesion developed for every nine ascospores used or for every 2.4 × 106 conidia used. For inoculation with ascospores produced by in vitro crosses, three times as many pseudothecia were needed to inoculate the same number of plants using an ascospore suspension as using the ‘ascospore shower’ method. Since it is difficult to obtain enough ascospores from crosses to make ascospore suspensions, the ‘ascospore shower’ was chosen to study the fitness of near isogenic isolates of L. maculans.