Polymerase chain reaction-based assays for species-specific detection of Fusarium culmorum, F-graminearum, and F-avenaceum

Differential detection assays employing polymerase chain reaction (PCR) amplification of sequence-characterized amplified regions were developed for Fusarium culmorum, F. graminearum, and F. avenaceum. Unique fragments from randomly amplified polymorphic DNA profiles that differentiated F. culmorum and F. graminearum were cloned and sequenced. Based on the sequences, pairs of 20-mer oligonucleotide primers were designed to yield distinguishable amplicons of different molecular weight. Single fragments were amplified with each of the primer pairs that were specific for F. culmorum and F. graminearum. Screening a broad range of isolates of Fusarium spp., other cereal pathogens, and potential host plants revealed no significant cross-reactions for any assay. The assays were capable of detecting less than 10(-12) g of fungal DNA and enabled the detection of individual Fusarium spp. directly in extracts of infected stem tissue and grains of rye and wheat showing disease symptoms. Additionally, internal transcribed spacer regions (ITS) of nuclear ribosomal DNA were amplified with universal primers and analyzed for sequence variation among the species. The ITS sequences of F. culmorum and F. graminearum were not polymorphic enough to design species-specific primers. ITS-1 and -2 of both species were compared to those of F. avenaceum and revealed sufficient sequence variation, especially in ITS-2, to derive primers for specific amplification of F: avenaceum. These specific, sensitive PCR assays represent valuable, versatile new tools for diagnosis, epidemiology, screening of breeding material for Fusarium resistance, and fungal population genetics.