ABSTRACTA nested polymerase chain reaction-based (nPCR) assay was developed and evaluated for the rapid detection of Fusarium oxysporum f. sp. lactucae in seed of lettuce. Three primers were designed from sequences of the intergenic spacer region of the rDNA and were used in the PCR amplifications. The first amplification employed the primer pair GYCF1 and GYCR4C and produced a product of 2,270 bp. The second amplification employed the forward primer GYCF1 and the nested primer R943 and produced a single 936-bp PCR product. The nPCR protocol developed successfully detected the target sequence in genomic DNA at 1 fg/μl. A seed assay was tested that included a 4-day incubation step in which seed were maintained under high humidity conditions to increase fungal biomass for DNA extraction. In seed lots prepared by mixing known amounts of F. oxysporum f. sp. lactucae--infested seed with noninfested seed, this assay permitted the detection of the pathogen from lots with infestation rates as low as 0.1%. Samples of lettuce seed obtained from 88 commercial lettuce seed lots were assayed for the pathogen by direct plating and by using the nPCR assay. The pathogen was not detected by either diagnostic method, suggesting the seed lots were pathogen free or the level was below detection limits.
