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Chytridiomycosis caused by Batrachochytrium dendrobatidis (Chytridiomycota) has been implicated in declines of amphibian populations on four continents. We have developed a sensitive and specific polymerase chain reaction-based assay to detect this pathogen. We isolated B. dendrobatidis from captive and wild amphibians collected across North America and sequenced the internal transcribed spacer regions of the rDNA cassette of multiple isolates. We identified two primers (Bd1a and Bd2a) that are specific to B. dendrobatidis under amplification conditions described in this study. DNA amplification with Bd1a/Bd2a primers produced a fragment of approximately 300 bp from B. dendrobatidis DNA but not from DNA of other species of chytrids or common soil fungi. The assay detected 10 zoospores or 10 pg of DNA from B. dendrobatidis and detected infections in skin samples from a tiger salamander (Ambystoma tigrinum), boreal toads (Bufo boreas), Wyoming toads (Bufo baxteri), and smooth-sided toads (Bufo guttatus). This assay required only small samples of skin and can be used to process a large number of samples.

Canadian isolates of Leptosphaeria maculans, the causal agent of blackleg of crucifers, were examined for genetic relatedness by the random amplified polymorphic DNA assay. DNA polymorphisms amplified with random decamer primers were used to distinguish three groups of isolates. Group 1 contained all isolates of the virulent pathotype, group 2 contained isolates of the avirulent pathotype from western Canada, and group 3 contained avirulent pathotype isolates from Ontario. These results agreed with other reports which showed many genetic differences between pathotypes and were consistent with the hypothesis that the virulent pathotype was recently introduced into Canada and has diverged relatively little. In contrast, the avirulent pathotype has probably been present in Canada for a longer time and has diverged with geographic isolation. In addition to establishing genetic relationships, DNA fingerprints generated by the random amplified polymorphic DNA assay have potential applications in pathotype identification and blackleg disease management. Recent publications on the genome size and RFLP patterns of isolates of L. maculans demonstrate that the A and V pathotypes are genetically very different and suggest that they are distinct species (10a, 21a).

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