Submitted to: Fungal Genetics and Biology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 2/10/2002
Publication Date: 7/1/2003
Citation: BARVE,M.P., ARIE,T., SALIMATH,S.S., MUEHLBAUER,F.J., PEEVER,T.L. CLONING AND CHARACTERIZATION OF THE MATING TYPE (MAT) LOCUS FROM ASCOCHYTA RABIEI (TELE0MORPH: DIDYMELLA RABIEI) AND A MAT PHYLOGENY OF LEGUME-ASSOCIATED ASCOCHYTA SPP. FUNGAL GENETICS AND BIOLOGY. 2003. v. 39. p.151-167. Interpretive Summary: Ascochyta blight of chickpea caused by Ascochyta rabiei is the most damaging disease of the crop throughout the world and in the U.S. Pacific Northwest. The sexual stage of the fungus is considered to be extremely important in long distance spread of the disease and also for development of new genetic combinations that may have an increased ability to cause disease. The fungus has two mating types that are difficult to determine using conventional pathological techniques; however, in this research, a procedure was developed using the Polymerase Chain Reaction (PCR) that can be used to quickly determine mating type of individual Ascochyta isolates. This quick and easily applied procedure has direct use in research on the fungus and the development of the sexual stage and prospects for long distance spread of the disease.
Technical Abstract: The mating type (MAT) locus of Ascochyta rabiei (telemorph: Didymella rabiei) was cloned and sequenced using a combination of TAIL-PCR and inverse PCR. Degenerate primers designed to the high mobility group (HMG) motif of Cochliobolus heterostrophus, C. sativus and Alternaria alternate were used to amplify HMG from A. rabiei. TAIL and inverse PCR reactions extended the MAT1-2 sequence into the conserved flanking DNA and primers designed to the MAT1-2 flanking DNA were used to amplify the entire MAT1-1 idiomoph plus flanking DNA. MAT1-1 and MAT1-2 idiomorphs were 2294 and 2693 bp in length, respectively, and each contained a single putative open reading frame (ORF) and intron. The MAT1-1 ORF was 1152 bp in length and coded for a putative 366 aa protein with an alpha motif. MAT genes were expressed at a high level in rich media and intron splicing was confirmed by sequencing MAT cDNAs. The MAT1-2 ORF was 1111 bp and coded for a putative 351 bp protein with an HMG motif. Putative start and stop codon positions and intron splice sites were highly conserved relative to C. heterostrophus, A. alternate and Mycosphaerella graminicola MAT. MAT-specific PCR primers were designed for use in a multiplex PCR assay and MAT-specific PRC amplicons correlated perfectly to MAT phenotype of 35 ascospore progeny from a genetic cross of MAT1 and MAT 2 isolates and to the MAT phenotype of field-collected isolates from diverse geographic locations. An HMG phylogeny estimated among legume-associated Ascochyta spp. Revealed seven well supported clades which correlated to host of isolation. A. rabiei is phylogenetically distant from other legume associated Ascochyta spp. And the specific status of A. rabiei, A. lentis, A. pisi and A. fabae was confirmed by the HMG data.