POLYMERASE CHAIN REACTION-BASED ASSAYS FOR SPECIES-SPECIFIC DETECTIONOF FUSARIUM-CULMORUM, F-GRAMINEARUM, AND F-AVENACEUM

Differential detection assays employing polymerase chain reaction (PCR ) amplification of sequence-characterized amplified regions were devel oped for Fusarium culmorum, F. graminearum, and F. avenaceum. Unique f ragments from randomly amplified polymorphic DNA profiles that differe ntiated F. culmorum and F. graminearum were cloned and sequenced. Base d on the sequences, pairs of 20-mer oligonucleotide primers were desig ned to yield distinguishable amplicons of different molecular weight. Single fragments were amplified with each of the primer pairs that wer e specific for F. culmorum and F. graminearum. Screening a broad range of isolates of Fusarium spp., other cereal pathogens, and potential h ost 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 extract s of infected stem tissue and grains of rye and wheat showing disease symptoms. Additionally, internal transcribed spacer regions (ITS) of n uclear ribosomal DNA were amplified with universal primers and analyze d for sequence variation among the species. The ITS sequences of F. cu lmorum and F. graminearum were not polymorphic enough to design specie s-specific primers. ITS-1 and -2 of both species were compared to thos e of F. avenaceum and revealed sufficient sequence variation, especial ly in ITS-2, to derive primers for specific amplification of F: avenac eum. These specific, sensitive PCR assays represent valuable, versatil e new tools for diagnosis, epidemiology, screening of breeding materia l for Fusarium resistance, and fungal population genetics.