Molecular identification of Fusarium species occurring on white pine seedlings and methods to differentiate pathogenic and nonpathogenic isolates of Fusarium oxysporum f.sp. pini.

摘要

Eighteen isolates representing six Fusarium species from diverse hosts and geographical origins were evaluated to determine ribosomal DNA variation using polymerase chain reaction and restriction fragment length polymorphisms. No length variation was observed for amplified 18S and 28S regions. However, amplification of the ITS region showed one isolate, a F. oxysporum to be about 120bp larger than the remaining seventeen. Restriction digestions in the 18S region revealed polymorphisms within species of F. oxysporum and F. solani. An amplified variable stretch of the 28S gene showed restriction site differences between F. avenecum, F. sambucinum and F. sporotrichioides . A large degree of polymorphism. was observed both between and within species in the ITS region. Therefore, entire sequences of the ITS and the 5.8S subunit were obtained for seventeen of the eighteen isolates. These sequences, along with those from eight additional isolates, were analysed using PAUP to assess the occurrence of DNA sequence divergence within the ITS region. The lack of correlation between molecular based relationships and species affinities inferred from morphology for some isolates indicates that species designation can be unreliable using morphological data alone.The rDNA of 50 collected field isolates of F. oxysporum f.sp. pini was examined. The ITS region and the variable region in the 28S gene encompassing the D1 and D2 domains were used as a baseline for confirming the identity of all F. Oxysporum f. sp. pini isolates subsequently studied. Various approaches were utilized to determine pathogenicity within the population. of the RAPD primers used, none, either singly or in combination could differentiate between pathogenic or nonpathogenic isolates. However, a simple, quick lab procedure was designed and successfully used to determine the pathogenic nature of the isolates. The mtDNA of 33 isolates of F. oxysporum f.sp. pini , all divided based on pathogenicity, was studied. Mt RFLP data was used to assess interstrain relationships. Twelve mt RFLP haplotypes were detected. The diversity of mtDNA particularly in relation to virulence appear to be unrelated.The IGS region of F. oxysporum f.sp. pini was examined due to its potential for intraspecific variation. The entire IGS sequence of a pathogenic isolate, 2691bp in length, was derived. The region was selected from the lamda genomic library and cloned into the appropriate vectors prior to sequencing. Using a RFLP map constructed from the sequence, intraspecific variation with the population of F. oxysporum f.sp. pini was determined. A total of 6 IGS haplotypes were identified. Interisolate affinities among pathogenic and nonpathogenic isolates based on polymorphisms in the IGS region were not always consistent with polymorphisms in the mtDNA.