PCR Detection of Fusarium oxysporum f. sp. basilici on Basil

Abstract

Sixty-nine amplified DNA fragments, generated from different isolates of Fusarium oxysporum f. sp. basilici, were tested for F. oxysporum f. sp. basilici-specificity in a dot blot assay. One 1,038-bp fragment hybridized to DNA from all F. oxysporum f. sp. basilici isolates but not to DNA obtained from F. oxysporum isolates nonpathogenic to basil or representatives of other formae speciales of F. oxysporum, or from isolates of F. redolens, F. tabacinum, Rhizoctonia solani, Sclerotinia sclerotiorum, S. minor, and Pythium ultimum obtained from diseased basil. This fragment was cloned and sequenced, and three pairs of F. oxysporum f. sp. basilici-specific primers were designed, giving rise to amplification products of 943, 382, and 330 bp. A nested PCR assay allowed detection of F. oxysporum f. sp. basilici in diseased seedlings and in artificially and naturally contaminated seeds. The theoretical detection limit of this system was 102 fungal propagules per 100 seeds on artificially contaminated samples, while on naturally contaminated commercial seed lots, 32 propagules per 100 seeds were detected.