Genetic relationships of pathogenic Typhula species assessed by RAPD, ITS-RFLP and ITS sequencing

Several species of Typhula cause diseases of cereals and grasses at low temperatures. Taxonomic separation of T. phacorrhiza, T. incarnata and T. ishikariensis was investigated using RAPD, ITS-RFLP and nucleotide sequencing of the TTS region. Using 10 RAPD primers on genomic DNA or eight restriction enzymes on PCR-amplified ITS regions, banding patterns were generated for each of 19 isolates from five species or varieties. Polymorphisms were found between species and varieties, with fewer among isolates within species or varieties. RAPD analysis suggested that T. ishikariensis var. idahoensis may be distinct from the other varieties (var. ishikariensis and var. canadensis), although this was not supported by ITS-RFLP. To confirm relationships, ITS nucleotides were obtained from single isolates of each of the five Typhula taxa, and sequence alignments were subjected to distance and parsimony analysis. Although there were differences in branching between dendrograms derived from sequencing vs. RAPD or ITS-RFLP, sequence data supported the distinction of the three main Typhula species, with the T. ishikariensis Varieties grouping together and showing differences at an infraspecific level. ITS sequence data also indicated the closer taxonomic affinity of Typhula species to the agaricoid Tricholoma sejunctum compared to the clavarioid Thelephora terrestris.