Detection of Fusarium oxysporum f. sp dianthi in carnation tissue by PCR amplification of transposon insertions

Strains of the carnation wilt pathogen, Fusarium oxysporum f. sp. dianthi, can be distinguished by DNA fingerprint patterns, using the fungal transpos able elements Fot1 and impala as probes for Southern hybridization. The DNA fingerprints correspond to three groups of F. oxysporum f. sp. dianthi str ains: the first group includes isolates of races 1 and 8; the second group includes isolates of races 2, 5 and 6; and the third group includes isolate s of race 4. Genomic DNAs flanking race-associated insertion sites of Foil (from races I, 2, and 8) or impala (from race 4) were amplified by the inve rse polymerase chain reaction (PCR) technique. These regions were cloned an d sequenced, and three sets of primers overlapping the 3' or 5' end of the transposon and its genomic insertion were designed. Using fungal genomic DN A as template in PCR experiments, primer pairs generated amplification prod ucts of 295, 564 and 1,315 bp, corresponding to races 1 and 8; races 2, 5, and 6; and race 4, respectively. When multiplex PCR was performed with geno mic DNA belonging to races I and 8, 2, or 4, single amplimers were generate d, allowing clear race determination of the isolate tested. PCR was success fully performed on DNA extracted from susceptible carnation cv. Indios infe cted with isolates representative of races 1, 2, 4, and 8.