Degenerate primers designed to correspond to conserved regions of the: high mobility group (HMG), protein encoded by the MATI-2 gene of Cochliobohis heterostrophus,, Cochliobolus sativus, and Alternaria alternata were used to amplify the portion of the sequence corresponding to the HMG box motif from Ascochyta, rabiei (teleomorph: Didymella rabiei). A combination of TAIL and inverse PCR extended the MATI-2 sequence in both directions, then primers designed to, MATI-2 flanking DNA were used to amplify the entire MATI-1 idiomorph. MATI-1 and: MATI-1 and diomorphs. were 2294 and 2693 bp in length, respectively, and each contained a single putative open reading frame (ORF) and intron similar to,MAT loci of other loculoascomycete fungi, MAT genes; were expressed at high levels in rich medium. MAT-specific PCR primers were designed for use in a multiplex PCR assay and MAT-specific PCR amplicons correlated perfectly to mating phenotype of 35 ascospore, progeny from a cross of MATI-1 by MATI-2 isolates and to the mating phenotype of field collected: isolates from diverse geographic locations. MAT-specific PCR was used to rapidly determine the mating type of isolates of A. rabiei sampled from chickpea fields in the US Pacific Northwest. Mating type ratios were not significantly different from: 1: 1 among isolates sampled from two commercial chickpea fields consistent with. the hypothesis that these A. rabiei populations, were randomly mating. The mating type ratio among isolates sampled, from an experimental. chickpea field where asexual reproduction was enforced, differed significantly from 1:1. A phylogeny estimated among legume-associated Ascochyta spp. and related loculoascocmycete fungi using sequence data from the nuclear ribosomal, internal. transcribed spacer (ITS) demonstrated the monophyly of Ascochytal Didymella spp. associated with legumes but was insufficiently variable to differentiate isolates associated with different legume hosts. In contrast, sequences of the HMG region of MATI-2 were substantially more variable, revealing seven well-supported clades that correlated to host of isolation. A rabiei, on! chickpeas is phylogenetically distant from: other legume-associated Ascochyta: spp and the specific status of A. rabiei, A. lentis, A. pisi, ands A. fabae was confirmed by the HMG phylogeny (C) 2003 Elsevier Science (USA). All rights reserved.
