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Didymella bryoniae (anamorph Phoma cucurbitacearum) is the causal agent of gummy stem blight, although other Phoma species are often isolated from cucurbit plants exhibiting symptoms of the disease. The molecular and phylogenetic relationships between D. bryoniae and these Phoma species are unknown. Isolates of D. bryoniae and Phoma obtained from cucurbits grown at various geographical locations in the United States were subjected to random amplified polymorphic DNA (RAPD) analysis and internal transcribed spacer (ITS) sequence analysis (ITS-1 and ITS-2) to determine the molecular and phylogenetic relationships within and between these fungi. Using RAPD fingerprinting, 59 isolates were placed into four phylogenetic groups, designated RAPD group (RG) I, RG II, RG III, and RG IV. D. bryoniae isolates clustered in either RG I (33 isolates), RG II (12 isolates), or RG IV (one isolate), whereas all 13 Phoma isolates clustered to RG III. There was greater than 99% sequence identity in the ITS-1 and ITS-2 regions between isolates in RG I and RG II, whereas isolates in RG III, P. medicaginis ATCC 64481, and P. exigua ATCC 14728 clustered separately. On muskmelon seedlings, a subset of RG I isolates were highly virulent (mean disease severity was 71%), RG II and RG IV isolates were slightly virulent (mean disease severity was 4%), and RG III isolates were nonpathogenic (disease severity was 0% for all isolates). The ITS sequences indicate that RG I and RG II are both D. bryoniae, but RAPD fingerprints and pathogenicity indicate that they represent two different molecular and virulence subgroups.

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The causal agent of gummy stem blight, Didymella bryoniae, often is isolated from infected cucurbits together with other Phoma spp. Polymerase chain reaction (PCR) primers specific to D. bryoniae and Phoma were used to develop and evaluate a microtiter-based PCR-enzyme-linked immunosorbent assay (ELISA) technique. Primers were modified by addition of a fluorescein and a biotin label to the 5′ ends of the forward and reverse primers, respectively. After amplification, PCR products were detected in an ELISA using horseradish peroxidase-conjugated antifluorescein antibody and three substrates that yielded three colored products, one for each fungal group. The most sensitive substrate (highest signal:noise ratio) was 2,2′ -azino-bis[3-ethylbenz-thiazoline-6-sulfonic acid]. PCR-ELISA successfully detected 45 of 46 D. bryoniae and all 13 Phoma isolates that were used. Results were comparable to those obtained with gel electrophoresis. Only one D. bryoniae isolate could not be detected with PCR-ELISA; this isolate also produced a fragment larger than other D. bryoniae isolates on agarose gels. PCR-ELISA was used successfully on crude extracts of “blind” fungal samples and identified seven of seven isolates as D. bryoniae or Phoma. Although less sensitive than gel electrophoresis, PCR-ELISA was a highly specific, yet simple, rapid and convenient assay for detection of D. bryoniae and Phoma sp.