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Production of type A trichothecenes has been reported in the closely related species Fusarium langsethiae and F. sporotrichioides. Here, we characterized a collection of Fusarium isolates from Siberia and the Russian Far East (hereafter Asian isolates) that produce high levels of the type A trichothecene T-2 toxin and are similar in morphology to the type A trichothecene-producing F. langsethiae, and to F. poae which often produces the type B trichothecene nivalenol. The Asian isolates possess unique macroscopic and microscopic characters and have a unique TG repeat in the nuclear ribosomal intergenic spacer (IGS rDNA) region. In Asian isolates, the TRI1-TRI16 locus, which determines type A versus type B trichothecene production in other species, is more similar in organization and sequence to the TRI1-TRI16 locus in F. sporotrichioides and F. langsethiae than to that in F. poae. Phylogenetic analysis of the TRI1 and TRI16 gene coding regions indicates that the genes in the Asian isolates are more closely related to those of F. sporotrichioides than F. langsethiae. Phylogenetic analysis of the beta-tubulin, translation elongation factor, RNA polymerase II and phosphate permease gene sequences resolved the Asian isolates into a well-supported sister lineage to F. sporotrichioides, with F. langsethiae forming a sister lineage to F. sporotrichioides and the Asian isolates. The Asian isolates are conspecific with Norwegian isolate IBT 9959 based on morphological and molecular analyses. In addition, the European F. langsethiae isolates from Finland and Russia were resolved into two distinct subgroups based on analyses of translation elongation factor and IGS rDNA sequences. Nucleotide polymorphisms within the IGS rDNA were used to design PCR primers that successfully differentiated the Asian isolates from F. sporotrichioides and F. langsethiae. Based on these data, we formally propose that the Asian isolates together with Norwegian isolate IBT 9959 comprise a novel phylogenetic species, F. sibiricum, while the two subgroups of F. langsethiae only represent intraspecific groups., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The intergenic spacer (IGS) regions of the rDNA of several Fusarium spp. strains obtained from the collaborative researchers (Int. J. Food Microbiol. (2003)) were amplified by polymerase chain reaction (PCR), and an IGS-RFLP analysis was performed. Restriction digestion with AluI, MspI and PstI allowed differentiation between the related Fusarium poae and Fusarium kyushuense species. Fusarium langsethiae was also separated from Fusarium sporotrichioides (including var. minus) on the basis of the banding patterns after MspI digestion, while specific XhoI, AluI and MspI restriction patterns were found in the IGS amplicons of F. sporotrichioides var. minus. According to the phylogenetic analysis of IGS-RFLP patterns, F. langsethiae (except for one strain), F. sporotrichioides, F. poae and F. kyushuense strains formed four well-supported clades with high-bootstrap values. Based on the sequence differences in the IGS region, species-specific primers were designed for the F. langsethiae/F. sporotrichioides group and for F. poae. The specificity and sensitivity of the primers were tested on various Fusarium species and isolates, and on several other important fungal genera associated with cereals. The F. poae-specific primers, designed in this study, showed the same specificity as primers Fp82f/Fp82r developed previously. The two phylogenetic subgroups of F. langsethiae, found by IGS sequencing analysis, were separated on the basis of size differences of the amplification products with primers CNL12/PulvIGSr specific for the F. langsethiae/F. sporotrichioides group. RFLP analysis of the amplified IGS region is a useful molecular assay for characterisation and a phylogenetic study of several related Fusarium species-F. langsethiae, F. sporotrichioides, F. sporotrichioides var. minus, F. poae and F. kyushuense. The primers designed in this study were highly specific and allowed identification of F. poae and the F. langsethiae/F. sporotrichioides group.