Cloning of the mating type locus from Ascochyta lentis (teleomorph: Didymella lentis) and development of a multiplex PCR mating assay for Ascochyta species
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The mating type (MAT) locus of the lentil pathogen, Ascochyta lentis, was cloned and characterized using thermal asymmetric interlaced and inverse PCR with primers designed to the HMG-box of Ascochyta rabiei. A multiplex PCR assay for mating type was developed based on MAT idiomorph and flanking sequences. Primers were designed to specifically amplify MAT from several Ascochyta spp. including A. pisi, A. fabae and A. viciae-villosae in addition to A. lentis. Four hundred and fifty and 700 bp fragments were amplified from MAT1-1 and MAT1-2 isolates, respectively, and fragment size correlated perfectly with laboratory crosses using mating type tester strains. MAT-specific PCR allowed rapid scoring of mating type in crude DNA extracts from geographically diverse population samples of A. viciae-villosae from California and Washington State, USA. This co-dominant MAT-specific PCR assay will be a valuable tool for studying the population structure, biology and epidemiology of these fungi.
KeywordsAscochyta lentis High mobility group HMG box Inverse PCR Thermal asymmetric interlaced (TAIL)-PCR Multiplex PCR
A portion of this research was conducted during the visit of M. Chérif to the laboratory of T.L. Peever supported by a Fulbright Fellowship and USDA-STEEP for partial funding. The authors would like to thank Tamara Horton for technical assistance. Nucleotide sequence data reported are available in the GenBank database under the accession numbers DQ341314 and DQ341315.
- Goodwin SB, Waalwijk C, Kema GHJ, Cavaletto JR, Zhang G (2003) Cloning and analysis of the mating-type idiomorphs from the barley pathogen Septoria passerinii. Mol Gen Genomics 269:1–12Google Scholar
- Hernandez-Bello MA, Chilvers MI, Akamatsu H, Peever TL (2006) Host specificity of Ascochyta species infecting legumes of the Viciae and Cicerae tribes and pathogenicity of an interspecific hybrid. Phytopathology (in press)Google Scholar
- Kovachevski IC (1936) The blight of chickpea (Cicer arietinum), Mycosphaerella rabiei n. sp. In: Ministry of Agriculture and Natural Domains, Plant Protection Institute, Sofia, BulgariaGoogle Scholar
- Lee SB, Taylor JW (1990) Isolation of DNA from fungal mycelia and single spores. In: Innis MA, Gelfand DH, Snisky JJ, White TJ (eds) PCR protocols: a guide to methods and applications. Academic, San DiegoGoogle Scholar
- Mel’nik VA, Braun U, Hagedorn G (2000) Key to the fungi of the genus Ascochyta Lib. (Coelomycetes)/Opredelitel gribov roda Ascochyta Lib. Parey, BerlinGoogle Scholar
- Navas-Cortés JA, Pérez-Artés E, Jiménez-Díaz RM, Llobell A, Bainbridge BW, Heale JB (1998a) Mating type, pathotype and RAPDs analysis in Didymella rabiei, the agent of Ascochyta blight of chickpea. Phytoparasitica 26:199–212Google Scholar
- Nene YL, Reddy MV (1987) Chickpea diseases and their control. In: Saxena MC, Singh KB (eds) The chickpea. CAB International, OxonGoogle Scholar
- Nene YL, Hanounik SB, Qureshi SH, Sen B (1988) Fungal and bacterial foliar diseases of pea, lentil, faba bean and chickpea. In: Summerfield RJ (ed) World crops: cool season food legumes. Kluwer Academic Publishers, DordrechtGoogle Scholar
- Peever TL, Hernandez-Bello MA, Barve MP, Kaiser WJ (2005) Evolution of Ascochyta species on wild and cultivated legumes. In: 23rd fungal genetics conference, AsilomarGoogle Scholar
- Rozen S, Skaletsky HJ (2000) Primer3 on the WWW for general users and for biologist programmers (http://www.frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). In: Krawetz S, Misener S (eds) Bioinformatics methods and protocols: methods in molecular biology. Humana, Totowa, pp. 365–386
- Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Press, PlainviewGoogle Scholar