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A molecular analysis of eight described species of seed gall nematode, along with six undescribed isolates from different hosts, has revealed a strong association between nucleotide sequence polymorphism and host status. Each anguinid nematode associated with a unique host produced a unique PCR-RFLP pattern for the ITS1 region. Anguina species that had been synonymized in the past, Anguina agrostis, A. funesta, and A. wevelli (Afrina wevelli), were readily discriminated. Two undescribed species from northern New South Wales and southeastern South Australia, reported to be vectors of Rathyaibacter toxicus in the disease called ‘‘floodplain staggers,’’ were differentiated by a single restriction enzyme, and both could be separated easily from A. funesta, the vector of R. toxicus in annual ryegrass toxicity. Other species differentiated in this study include A. agropyronifloris, A. graminis, A. microlaenae, A. pacificae, and undescribed species from host species Dactylis glomerata, Agrostis avenacea, Polypogon monospeliensis, Stipa sp., Astrebla pectinata, and Holcus lanatus. Phylogenetic analysis of the ITS1 region suggests that considerable anguinid genetic diversification has accompanied specialization on different host species.