Enhanced molecular identification of Botrytis spp. from New Zealand onions

Botrytis spp. associated with neck rot disease were isolated from New Zealand onions. The fungi were identified using molecular sequences of the ribosomal internal transcribed spacer (ITS) and intergenic spacer (IGS) regions, and the glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene. Analyses of the sequences showed that the majority of the isolates gathered in 2005–07 were B. aclada. A new high resolution melting analysis (HRMA) assay was developed that allowed fast and simple discrimination between B. aclada and other Botrytis spp. causing onion neck rot in New Zealand. To further verify these results, Botrytis isolates from New Zealand onions, stored in the International Collection of Microorganisms from Plants (ICMP), were also examined. Only a single isolate from the ICMP collection was B. aclada while two isolates were B. byssoidea, one B. squamosa and another closely related to Botryotinia porri. Identification of the remaining Botrytis isolates was more difficult; while IGS and ITS sequences indicated a close relationship to B. allii or B. byssoidea, a previously unreported intron insertion was observed at the 3′ end of the ribosomal small subunit gene in these isolates. No evidence of heterogeneity was observed in the G3PDH gene sequences, as might have been expected of the allodiploid B. allii, but the G3PDH sequence ruled out B. byssoidea as the identity of these isolates.