mycological research 113 (2009) 391–400 journal homepage: www.elsevier.com/locate/mycres Didymella pisi sp. nov., the teleomorph of Ascochyta pisi Martin I. CHILVERSa,1, Jack D. ROGERSa, Frank M. DUGANb, Jane E. STEWARTa, Weidong CHENb, Tobin L. PEEVERa,* aDepartment of Plant Pathology, Washington State University, Pullman, WA 99164-6430, USA bUSDA-ARS, Washington State University, Pullman, WA 99164, USA article info abstract Article history: The anamorphic pycnidial fungus Ascochyta pisi is one member of a species complex that Received 11 July 2008 causes Ascochyta blight of pea, a potentially devastating disease. The teleomorphic state Received in revised form of this fungus was induced under laboratory conditions. Using morphological and molec- 14 November 2008 ular characters, we placed the teleomorph within the genus Didymella as D. pisi and de- Accepted 26 November 2008 scribe a heterothallic mating system using a PCR-based mating type assay and in vitro Published online 24 December 2008 crosses. We compare D. pisi with other Didymella spp. with which it might be confused. Corresponding Editor: ª 2008 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. D. L. Hawksworth Keywords: Ascochyta blight Ascomycete Cool season legume Dothidiomycetes Loculoascomycetes Mating type Teleomorph Introduction 1927). Using morphological criteria, van Warmerlo (1966) placed the perfect state of A. pinodes in Mycosphaerella.However,based A number of morphologically and/or phylogenetically distinct on molecular data, Peever et al. (2007) showed that M. pinodes taxa cause foliar, stem, and pod diseases of pea (Pisum sativum). clustered with Didymella taxa rather than with Mycosphaerella A taxon of special interest to us is the pycnidial anamorph, Asco- taxa, including a representative strain of M. punctiformis,the chyta pisi, which is the type species for Ascochyta and one of the type species for Mycosphaerella. They proposed that D. pinodes, causal agents of Ascochyta blight of pea. Based on inoculation asynonymofM. pinodes, be the accepted name. Peever et al. and cultural studies, Stone (1912) erroneously considered A. (2007) also showed that representative isolates of Ascochyta pisi pisi to be the anamorph of Sphaerella pinodes (syn. Mycosphaerella (the type species for Ascochyta) clustered with Didymella taxa pinodes). Subsequently, it was shown that the anamorph of M. and several other Ascochyta species. Peever et al. (2007) hypothe- pinodes was Ascochyta pinodes (Jones 1927; Linford & Sprague sized that when and if a teleomorph of Ascochyta pisi was found, * Corresponding author. Tel.: þ1 509 335 3754. E-mail address: [email protected] 1 Present address: Department of Plant Pathology, Michigan State University, East Lansing, MI 48824, USA. 0953-7562/$ – see front matter ª 2008 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2008.11.017 392 M. I. Chilvers et al. Table 1 – Ascochyta and other fungal isolates used in this study Anamorpha Teleomorphb Host Isolate code Mating Origin Collector Year (ATCC/CBS) typec Ascochyta pisi Didymella pisi Pisum sativum AP1 (201617/122748) 1 Bulgaria W.J. Kaiser 1992 A. pisi D. pisi P. sativum AP2 (201618/122749) 2 Poland E. Swiecicki Unknown A. pisi D. pisi P. sativum AP3 (201619/122750) 1 ID, USA D. Webster 1995 A. pisi D. pisi P. sativum AP4 (201620/122751) 2 Canada B. Gossen 1996 A. pisi D. pisi P. sativum AP5 1 Canada B. Gossen 1996 A. pisi D. pisi P. sativum AP6 1 Canada B. Gossen 1996 A. pisi D. pisi P. sativum AP7 1 Canada B. Gossen 1996 A. pisi D. pisi P. sativum AP8 1 Bolivia W.J. Kaiser 1999 A. pisi D. pisi P. sativum AP9 2 Bolivia W.J. Kaiser 1999 A. pisi D. pisi P. sativum 07KRP4 2 ND, USA R. Goswami 2007 A. pisi D. pisi P. elatius G-6 2 Georgia W.J. Kaiser 2004 A. pisi D. pisi P. elatius G-7 2 Georgia W.J. Kaiser 2004 A. pisi D. pisi P. elatius G-12 2 Georgia W.J. Kaiser 2004 A. fabae D. fabae Vicia faba AF1 (96418/-) 2 Saskatoon, B. Vandenberg 1992 Canada Ascochyta sp. d V. lathroides AV8 1 WA, USA W.J. Kaiser 1994 A. lentis D. lentis Lens culinaris AL1 (96419/-) 2 Australia W.J. Kaiser Unknown A. rabiei D. rabiei Cicer arietinum AR20 (76501/-) d Iran W.J. Kaiser 1990 Ascochyta sp. d V. hirsuta G11 d Ateni, Georgia W.J. Kaiser 2004 Phoma medicaginis d Medicago sativa AS1 d WA, USA T.L. Peever 2001 A. pinodes D. pinodes P. sativum MP1 (201628/-) d OR, USA J. Baggett Unknown A. pinodes D. pinodes P. sativum MP2 (201629/-) d Ireland J. Kraft Unknown A. pinodes D. pinodes P. sativum MP19 (201632/-) d Argentina D. Webster 1996 A. pinodes D. pinodes P. sativum AWPP7B1I0 d ID, USA M.I. Chilvers 2007 A. pinodella d L. culinaris PMP1 dd W.J. Kaiser Unknown A. pinodella d L. culinaris PMP3 (58660/-) d WA, USA W.J. Kaiser 1996 A. pinodella d P. sativum AWPP4B1I0 d ID, USA M.I. Chilvers 2007 Ascochyta sp. d Astragalus sp. 3A d ID, USA W.J. Kaiser Unknown P. koolunga d P. sativum DAR 78535 d SA, Australia J.A. Davidson Unknown d D. exigua Rumex arifolius CBS183.55 d France E. Mu¨ller 1953 P. herbarum d Rosa multiflora CBS615.75 d Netherlands G.H. Boerema 1973 A. cucumis D. bryoniae Cucumis melo ATCC16241d d Florida Unknown Unknown Stagonospora nodorum Phaeosphaeria nodorum Triticum aestivum Sn37-1e d Poland Unknown Unknown Ramularia sp. Mycosphaerella punctiformis Quercus sp. CBS942.97 d Belgium A. Aptroot 1997 a d, no described anamorph. b d, no teleomorph described. c Mating type as determined with PCR assay; mating type 2 corresponds to the allele coding for the high mobility group protein (Turgeon & Yoder 2000). d, mating type unknown or not tested. d RPB2 sequence data obtained from GenBank. Culture was listed as Mycosphaerella citrullina by ATCC, but is considered to be Didymella bryoniae (Corlett 1991; Goodwin et al. 2001). e RPB2 sequence data obtained from GenBank (Malkus et al. 2006). it would be assigned to Didymella. Herein, we report on the dis- characterized (Barve et al. 2003; Che´rif et al. 2006). Che´rif et al. covery of the Didymella teleomorph of Ascochyta pisi, and show (2006) developed a PCR-based mating type assay that could be the phylogenetic placement of that fungus with respect to a rep- used to determine the mating type of A. lentis and A. pisi, as resentative isolate of D. exigua,thetypespeciesofDidymella. well as the faba bean and vetch pathogens, A. fabae, and A. Ascochyta and Phoma species are morphologically similar and viciae-villosae. The objectives of this research were to: (1) describe for the purposes of identification have been primarily defined the teleomorph of A. pisi; (2) to determine the mating system of by the production of two-celled conidia in the former and one- A. pisi through the application of the PCR-based mating type celled conidia in the latter (Mel’nik et al. 2000; Boerema et al. assay and in vitro sexual crosses; (3) use morphological, patho- 2004). To investigate the phylogenetic relationship between genic, and phylogenetic methods to compare D. pisi with other the two genera we included a representative isolate of P. herba- Didymella spp. colonizing pea with which it might be confused. rum, the type species of Phoma. Sexual reproduction in heterothallic ascomycetes is con- trolled by two highly divergent alternate alleles (idiomorphs) Materials and methods at a single mating type (MAT ) locus, each encoding a single regulatory gene (Metzenberg & Glass 1990; Turgeon & Yoder Fungal isolates, culturing and molecular methods 2000). Although the mating type locus of A. pisi has not been pre- viously characterized, the mating type loci of A. rabiei and the Single-conidia isolates of Ascochyta pisi were obtained from closely related lentil pathogen, A. lentis, have been cloned and the USDA Western Regional Plant Introduction Station, Didymella pisi sp. nov., the teleomorph of Ascochyta pisi 393 Fig 1 – Lesions caused by three Ascochyta blight pathogens of pea on Pisum sativum cv. ‘Lifter’. (A–D) Didymella pinodes isolate MP1. (A–B) Eight dpi, (C) 18 dpi, (D) 38 dpi. (E–G) Ascochyta pinodella isolate PMP3. (E–F) Eight dpi, (G) 18 dpi. (H–J) Ascochyta pisi isolate AP4 18 dpi. Bars [ 5 mm. Pullman, WA (Table 1). Isolates were grown on V8 juice agar contiguous sequences were aligned and manipulated with [200 ml V8 juice (Campbell Soup Company, NJ), 3 g CaCO3, Vector NTI 10.1.1 (Invitrogen, Carlsbad, CA). All RPB2 se- 20 g agar lÀ1] on lighted growth shelves at 22 C with a 12 h quences generated in this study were submitted to GenBank photoperiod and stored dry on sterile filter paper at À20 C. under accession numbers (EU874849–EU874867). Growth of mycelium, DNA extraction, and application of MP and Bayesian phylogenetic analyses of the RPB2 region PCR-based mating type assay were performed as previously were conducted using PAUP (Swofford 1999) and Mr Bayes described (Che´rif et al. 2006). Partial RPB2 sequences encoding v.3.0b4 (Huelsenbeck & Ronquist 2001). Additional taxa in- DNA-dependent RNA polymerase II were amplified with RPB2 cluding Didymella bryoniae (ATCC16241) and Stagonospora nodo- R2-4Fa (GCNACNGGNAAYTGGGG) and RPB2-7R (CCCATWG- rum were obtained from GenBank (accession numbers CYTGCTTMCCCAT) (Liu et al. 1999; AFTOL website) from rep- AF107801 and DQ278491) (Malkus et al. 2006). Sequences resentative isolates (Table 1) ligated into the pGEM-T Easy were aligned using Clustal X 1.83 (Thompson et al. 1997). S. Vector and transformed into competent Escherichia coli cells nodorum (accession number DQ278491) served as the outgroup following the manufacturers’ instructions (Promega, Madison, for analyses after Peever et al.