PCR PRIMERS THAT ALLOW INTERGENERIC DIFFERENTIATION OF ASCOMYCETES AND THEIR APPLICATION TO VERTICILLIUM SPP

A pair of conserved PCR primers, designated NMS1 and NMS2, that amplif y a region in the mitochondrial small rRNA gene region were designed f or fungi belonging to the class Ascomycetes. These primers were tested with members of eight fungal genera (Aspergillus, Fusarium, Magnaport he, Mycospharella, Neurospora, Saccharomyces, Sclerotinia, Verticilliu m) and 10 Verticillium species (Verticillium albo-atrum, Verticillium chlamydosporium, Verticillium cinnebarium, Verticillium dahliae, Verti cillium fungicola, Verticillium lecanii, Verticillium lateritium, Vert icillium nigrescens, Verticillium psaliotae, and Verticillium tricorpu s). The primers were also tested with 35 isolates of V. dahliae obtain ed from diverse geographic areas and diverse hosts. The! results of a restriction fragment length polymorphism analysis of the region amplif ied by the primers differentiated the genera examined and the results of a DNA sequence analysis of the amplified region differentiated the Verticillium species. Two Fusarium species were also differentiated by the results of the restriction fragment length polymorphism analysis. On the basis of the nucleotide sequences of the amplified regions, we obtained a pair of PCR primers that could be used to differentiate V. dahliae from the other fungal isolates tested, including V. albo-atru m, a closely related plant-pathogenic species. The V. dahliae-specific PCR primer may aid in more rapid and specific detection of the pathog en directly in plant and/or soil samples. PCR primers NMS1 and NMS2 ma y be used as potential mitochondrial markers for studying fungal cytop lasmic inheritance of ascomycetes and for identifying DNA probes that are informative at or below the genus level.