Identification ofMagnaporthe poaeby PCR and Examination of Its Relationship to Other Fungi by Analysis of Their Nuclear rDNA ITS-1 Regions

Abstract

Magnaporthe poae is the causal agent of summer patch disease in turfgrasses. Identification of this heterothallic, root-infecting fungus has been difficult because of the lack of diagnostic structures in nature and the time required for the fungus to produce perithecia in culture. Potential probes for the identification of M. poae were obtained by producing a partial genomic library of M. poae isolate 73-15 in the pGEM3Zf + plasmid vector and subsequently screening cloned DNA for strong hybridization to 73-15 isolate genomic DNA. Using Southern blot analysis, a 2.7-kb DNA clone (pMp2-7) that hybridized to six confirmed isolates of M. poae from NJ, PA, NY, and RI was identified. Except for one isolate of Colletotrichum graminicola, the probe did not hybridize to 42 other fungal isolates that commonly inhabit the turfgrass environment. Oligonucleotides that would prime amplification of a 453-nucleotide (nt) fragment from M. poae DNA, but not from C. graminicola DNA, were developed based on sequence from one end of pMp2-7. These oligonucleotides also primed amplification of a similar-sized fragment of one isolate of M. rhizophila (PREM 45952) DNA. DNA from another isolate of M. rhizophila (Mr-2 from PA) did not amplify using these primers. To examine the relationship of M. poae to other fungal species, the internal transcribed spacer region (ITS-1) of nuclear ribosomal DNA for members of the Magnaporthe genus and several other fungi was sequenced. Sequences of three isolates of M. poae, identified through mating, and isolate PREM 45952 were very similar, having only five variable sites of 236. Isolate Mr-2 had seven sites variable to the M. poae isolates, two that were identical to variable sites in PREM 45952. Using maximum parsimony analysis, the M. rhizophila and M. poae isolates grouped in 75% of bootstrap replications. M. rhizophila isolates formed a monophyletic group away from the M. poae isolates in four of five equally parsimonious trees, but this grouping was not supported by more than 50% of bootstrap replications.