Phylogeny, Molecular Detection, and Genetic Variation of Fusarium oxysporum, Vascular Wilt Pathogen of Lettuce

Abstract

This work encompasses studies on the phylogeny of F. oxysporum f. sp. lactucae, the development of a PCR-based seed assay for the detection of this fungus in seed, the potential of seed transmission of the fungus that may result in seed dissemination, and the genetic variation existing within pathogen populations. In phylogenetic analysis, the mtSSU and EF-1α sequences provided limited phylogenetic resolution and did not differentiate the lactucae isolates from other F. oxysporum isolates, while the IGS region resolved lactucae race 1 isolates as a monophyletic group with three other f. spp. of F. oxysporum. In all analyses, lactucae race 2 isolates comprised a separate lineage that was phylogenetically distinct. Based the IGS, PCR primers were designed for detection of the fungus, and a PCR-based seed assay was developed for detection of the fungus in seed. This assay allowed for detection of the pathogen from artificially infested seed lots with infestation rates as low as 0.5%. To investigate seedborne transmission, the moderately resistant cultivars Sharpshooter, Vulcan, and King Henry were inoculated and grown to maturity in the greenhouse. The pathogen was recovered from sections of surface disinfested inflorescence stalks at rates of 14.3 - 62.7% but not from the floral parts. The incidence of recovery from nondisinfested seeds was between 0.02% and 0.08%. The pathogen was not isolated from surface disinfested seeds suggesting that it was externally seedborne. The pathogen was recovered from pathogen-free seeds mixed with infested debris suggesting infested seed may contribute to recently documented dissemination of this pathogen worldwide. Isolates of Fusarium oxsyporum f. sp. lactucae were analyzed for genetic diversity using inter-simple sequence repeat molecular markers. Results revealed 2 main groups within the Arizona isolates corresponding to eight haplotypes in 2005, which evolved from 2 haplotypes in 2001. Haplotype 1-05 was widespread, occurring in two of the four countries where F. o. f. sp. lactucae has been reported. 23 haplotypes were identified among the California isolates that clustered into two subgroups. The clustering of isolates from Arizona suggests that there has been more than one introduction of the pathogen into Arizona.