PCR detection of Fusarium oxysporum f.sp gladioli race 1, causal agent of Gladiolus yellows disease, from infected corms

Fusarium oxysporum f.sp. gladioli (FOG) race 1 infects both large- and smal l-flowered Gladiolus cultivars. Race 2 isolates infect only small-flowered cultivars but can be present as epiphytes on large-flowered plants. When 16 0 arbitrary 10-mer oligonucleotide primers were tested on FOG by PCR to fin d RAPD markers specific for race 1, the RAPD primer G12 amplified two discr iminating DNA fragments, AB (609 bp) and EF (1196 bp), in race 1 isolates o nly. Both fragments were cloned and sequenced. Two pairs of race 1-specific primers for multiplex PCR were designed. Tests of 112 F. oxysporum isolate s by PCR showed that, in almost all cases, race 1 isolates of vegetative co mpatibility group 0340 could be distinguished with these primers. Seven put ative race 1 isolates did not react in multiplex PCR; hybridization studies with labelled AB and EF DNA fragments showed that these isolates belong to separate groups. A bioassay was developed to detect corms that were latent ly infected with FOG race 1. Gladiolus corms were homogenized and incubated for 5 days at 28 degrees C in a semiselective medium to induce growth of F usarium. Cultivated mycelium was isolated and subjected to the developed mu ltiplex PCR after standard DNA isolation or disruption by microwave treatme nt.