mycological research 113 (2009) 391–400

journal homepage: www.elsevier.com/locate/mycres

Didymella pisi sp. nov., the teleomorph of pisi

Martin I. CHILVERSa,1, Jack D. ROGERSa, Frank M. DUGANb, Jane E. STEWARTa, Weidong CHENb, Tobin L. PEEVERa,* aDepartment of , 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 is one member of a species complex that Received 11 July 2008 causes Ascochyta blight of , 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 Dothidiomycetes Loculoascomycetes Mating type Teleomorph

Introduction 1927). Using morphological criteria, van Warmerlo (1966) placed the perfect state of A. pinodes in .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 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 arietinum AR20 (76501/-) d Iran W.J. Kaiser 1990 Ascochyta sp. d V. hirsuta G11 d Ateni, Georgia W.J. Kaiser 2004 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 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) 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 l1] 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 20 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. (2007). MP analysis was per- WI). Plasmids were sequenced in both directions and formed in PAUP using the heuristic search option with ten 394 M. I. Chilvers et al.

Table 2 – Percentage identity among partial RPB2 nucleotide sequences from teleomorph type species Mycosphaerella and Didymella, and anamorph type species Phoma and Ascochyta Mycosphaerella punctiformis Didymella exigua Phoma herbarum Ascochyta pisi

M. punctiformis 100 ddd D. exigua 66 100 dd P. herbarum 67 88 100 d A. pisi 66 89 89 100

random sequence additions, tree bisection–reconnection Based on results of the mating type PCR assay (Table 1), conidial (TBR) branch swapping, the MULPARS option turned on, and suspensions consisted of either a mix of two isolates of compat- all characters weighted equally. Nodal support was evaluated ible mating types (i.e. AP1 AP2, AP1 AP4, AP2 AP3, and using 1K bootstrapped datasets. For Bayesian analysis, the AP3 AP4) or a single isolate (i.e. AP1, AP2, AP3, and AP4) as con- best fit model of sequence evolution for the dataset was se- trols. Two Whatman no. 1 filter papers were placed on top of lected and model parameter estimates obtained using DT- a large, folded Kimwipe tissue in a 9 cm glass Petri dish, the ModSel (Minin et al. 2003). The TrNþG(Tamura & Nei 1993) papers were moistened with water, excess water was poured model, with equal bases frequencies and three substitution off, and the Petri dishes containing papers were autoclaved. After rates, was selected (Swofford et al. 1996; Felsenstein 2004). inoculation, pea stems were removed from conidial suspensions MrBayes does not allow direct model selection for all possible and placed onto the filter papers in the sterilized glass Petri models so a GTR model with gamma distributed rates was dishes. Petri dishes containing inoculated pea stems were incu- employed as the closest fit (Huelsenbeck & Ronquist 2001). bated on the laboratory bench at ambient temperature for 3 d Two independent MCMC searches were run of four chains prior to being placed in a dark incubator at 10 1 C for eight each (three heated and one cold) for 2M generations. A total weeks. To determine whether a cool temperature incubation of 20,001 trees were generated. The first 5K trees were dis- period was critical for pseudothecia formation as it is for D. rabiei carded as ‘burn in’ for each run. The remaining 15,001 trees (Wilson & Kaiser 1995; Trapero-Casas et al. 1996) Petri dishes of were used to estimate PP for nodes. the cross AP3 AP4 were also placed at 15 1 Cand23 1 C. To compare isolates to published morphologies and mea- Pseudothecia observed under a dissecting microscope surements, isolates were grown on oatmeal agar (Difco, Law- were mounted and crushed in water to assess and measure rence, KS). Conidia of isolates AP3 and AP4 grown on oatmeal asci and . Ascospores were measured with agar for 12 d were mounted in water for microscopy. The a bright-field compound microscope at 400 magnification. length and breadth of 40 conidia were measured, means and The length and breadth of 35 ascospores were measured. standard deviations were calculated without rounding, and Means and standard deviations were calculated without ranges of conidial dimensions were rounded to the nearest rounding, the range of dimensions were rounded full or half micrometre. to the nearest full or half micron. Stems were air-dried and stored at ambient laboratory temperature. Plant inoculations Results One gallon plastic pots containing Sunshine LA 4 potting mix (Sun Gro Horticulture, Bellevue, WA) were sown with four Mating type determination and teleomorph development seeds of pea plants cv. ‘Lifter’ (PI628276) and were grown for three weeks at 20 2 C under natural light conditions in The PCR-based mating type assay revealed that both mating a greenhouse. Forty millilitre conidial suspensions were pre- type 1 (MAT1) and mating type 2 (MAT2) were present in sam- 5 pared from two-week-old V8 agar cultures at 1 10 con- ples from North America, South America, and Eastern Europe 1 idia ml with 1 ml Tween 80. Two replicate pots were (Table 1). Mating type 1 isolates contain the alpha box DNA inoculated by spraying to runoff with isolates Ascochyta pisi binding motif and mating type 2 contain the high mobility (AP4), A. pinodella (PMP3), Didymella pinodes (MP1), and a water group motif (Barve et al. 2003; Turgeon & Yoder 2000). These plus Tween80 control (Table 1). Plants were then placed in results were used to determine the appropriate pairings for a dew chamber for 24 h to promote infection and then trans- the in vitro sexual crosses. Pseudothecia were only formed ferred to a growth chamber with constant 20 1 C and on inoculated pea stems when complementary mating types 80 10 % relative humidity with a 12 h photoperiod. were present. Self-sterility was observed for all four control isolates. Pseudothecia were mature within eight weeks from In vitro sexual crosses and cytological observation the time of inoculation at 10 C. Pseudothecia also initiated and matured at 15 C, although not as profusely as the 10 C Conidia were harvested from 9-d cultures on V8 agar by flooding treatment, but did not develop at 23 C. the surface of the agar with sterile water and gently rubbing with a glass rod. Conidia were enumerated with a haemocytometer Plant inoculations andstoredat4C overnight. Senescent and living pea stems were autoclaved and inoculated by submerging in conidial sus- Pea plants inoculated with Didymella pinodes and Ascochyta pensions of 1 106 conidia ml1 for 1 h (Wilson & Kaiser 1995). pinodella developed lesions within 3 d; in contrast, lesions Didymella pisi sp. nov., the teleomorph of Ascochyta pisi 395

Fig 2 – Bayesian phylogeny estimated from partial DNA-dependent RNA polymerase II nucleotide sequences (RPB2) from selected Ascochyta, Phoma, and Didymella species. Phylogeny was rooted by Stagonospora nodorum (accession DQ278491). Bayesian PPs (upper numbers) were sampled from 15,001 trees generated from 2M MCMC chains and the lower numbers represent MP BS values based on 1K bootstrapped datasets. Representative isolates of the type species Phoma and Didymella are included as P. herbarum (CBS625.75) and D. exigua (CBS183.55), respectively. A. pisi is the type species for Ascochyta. Isolates of A. pisi used to generate D. pisi teleomorph type material are also included (AP3 [ ATCC 201619 [ CBS 122750 and AP4 [ ATCC 201620 [ CBS 122751). The asterisk represents the PP for P. herbarum placement in Bayesian phylogeny. Placement of this species differed under parsimony analysis where P. herbarum was placed external to the clades containing A. pisi, P. koolunga, D. pinodes, and A. pinodella with 76 % BS support.

were not detected on plants inoculated with A. pisi until 11 d were indistinct (Fig 1A–H). Symptoms caused by D. pinodes and post inoculation (dpi). Only two to three lesions per plant A. pinodella ranged from brown–black flecking on leaves and formed on those inoculated with A. pisi. Symptoms on plants stems to irregular lesions on leaves with an occasional zonate inoculated with D. pinodes and A. pinodella were more severe appearance. All plants recovered from disease, producing with many lesions (Fig 1). Lesions caused by A. pisi were dis- symptomless, apical growth; however, some stems were gir- tinct from those caused by D. pinodes and A. pinodella. Lesions dled by purple to black stem lesions caused by D. pinodes and caused by A. pisi were circular, tan coloured with a dark brown A. pinodella (Fig 1D, H), leading to the collapse of stems and margin (Fig 1I–K). Lesions caused by D. pinodes and A. pinodella the generation of secondary main stems. 396 M. I. Chilvers et al.

Fig 3 – Didymella pisi (holotype WSP 71448). Large black bodies on Pisum sativum stem are pseudothecia, smaller structures are pycnidia, (A) before and (B) after drying. (C–E) Asci stained with iodine. (F-G) Asci mounted in water. Bars [ (A–B) 1 mm; (C–G) 10 mm.

RPB2 phylogeny of pea in Australia (Davidson et al. in press). Within clade 1 (Fig 2), Ascochyta pisi clustered into a well-supported subclade with The degenerate primers RPB2 R2-4Fa and RPB2-7R amplified A. fabae. Clade 2 (Fig 2) contained the other two Ascochyta ca 995 bp amplicons from all isolates, except Mycosphaerella blight pathogens of pea, D. pinodes and A. pinodella, as well as punctiformis for which the amplicon was 1039 bp. RPB2 the type species of Didymella, D. exigua. P. herbarum, the type sequences of M. punctiformis were highly divergent (66–67 % species for Phoma, and D. bryoniae were not found in either identity) from those of the type species of Didymella, Phoma, of these clades. Phylogenetic placement of P. herbarum was in- and Ascochyta (Table 2), and were excluded from phyloge- consistent between the Bayesian and MP analyses. Bayesian netic analyses. MP and Bayesian analyses yielded phyloge- analysis placed P. herbarum sister to the well-supported A. nies with similar topologies (Fig 2). For parsimony analyses, pisi clade, which included the Phoma sp. from pea. In parsi- sequences were trimmed to 1002 characters, of these 375 mony analysis, P. herbarum was placed outside the clades con- characters were variable and 225 were parsimony- taining A. pisi, P. koolunga, D. pinodes, and A. pinodella. informative. RPB2 revealed similar phylogenetic signal compared to a glyceraldehyde-3-phosphate-dehydrogenase gene (G3PD) and more resolution than the ITS of rDNA (Peever et al. 2007). The majority of taxa clustered into two well-supported Didymella pisi M.I. Chilvers, J.D. Rogers, & T.L. Peever sp. nov. clades. The first clade contained the type species for Ascochyta, (Fig 3) A. pisi, as well as D. fabae, A. viciae-villosae, D. lentis, D. rabiei,an MycoBank no.: MB 508297 Ascochyta sp. isolated from Astragalus sp., P. medicaginis, and Etym: derived from the anamorph. the recently described Phoma koolunga causing Ascochyta blight Anam: Ascochyta pisi Lib., Pl. Crypt. Ard. (Fasc. 1) No. 59, 1830. Didymella pisi sp. nov., the teleomorph of Ascochyta pisi 397

Pseudothecia rotundata vel irregularia, 200–400 mm diam, cum formed when opposite mating types were present. Sterility ostiolo inconspicuo, brunnea vel nigricantia, mollia. Asci bitunicati, was observed for all four isolates when inoculated onto stems cylindrici vel saccaformia, octospori cum ascosporis uniseriatis, alone. 46–168 mm longitudine tota stipite diminuto vel nullo, 10–15 mm Colonies on oatmeal agar (Difco) produced abundant pyc- lati. Ascosporae hyalinae, bicellulares partibus plus minusve aequis, septo constricto, utrinque rotundatae vel unca plus acuta, leves, nidia, being pale luteous (12) in colour around the edges to 12–17.5 6.5–8.5 mm. Hamathecium sparsum vel nullum. Status ochreous (44) in the centre, with the reverse side of the plate anamorphosis Ascochyta pisa Lib. being similar (Rayner 1970). Aerial hyphae ranged from sparse Typus: USA: Washington:Pullman,in vitro on sterilized stems around the central fruiting region to densely lanose (woolly). of Pisum sativum inoculated with Ascochyta pisi isolates AP3 Punithalingam & Holliday (1972a) noted on oatmeal agar the (ATCC 201619, CBS 122750) and AP4 (ATCC 201620; CBS production of abundant pycnidia with carrot red cirrhi. Coni- 122751) 2008, M. I. Chilvers (WSP 71448dholotypus; BPI dia were hyaline, straight, or slightly curved, the majority 878440disotypus; K 157110disotypus). were 1-septate, cylindrical and rounded at both ends and Pseudothecia rotund to irregular, 200–400 mm diam, with in- slightly constricted at the septum matching the description conspicuous ostiole, brown to blackish, soft. Asci bitunicate, of Punithalingam & Holliday (1972a). Conidia were 8.5–17 mm cylindrical to saccate, 8-spored, the ascospores usually uni- in length (mean 13 mm, S.D.1.7 mm) 3.5–6 mm in breadth seriately arranged, 46–168 mm total length with stipe short or (mean 4.6 mm, S.D.0.7 mm; n ¼ 40, AP3; n ¼ 40, AP4). Conidia obsolete, 10–15 mm broad. Ascospores hyaline, more or less of Ascochyta pisi produced on V8 agar were compared with equally bicellular, constricted at the septum, rounded at other Ascochyta spp. of pea and the closely related Didymella both ends or with one end more acute, smooth, 12– fabae. A. pinodella is distinguished from the other Ascochyta 17.5 6.5–8.5 mm [mean length 15 mm, S.D. 1.4 mm; mean spp. by its smaller and predominantly aseptate conidia breadth 7.2 mm, S.D. 0.6 mm(n ¼ 35)]. Hamathecial elements (Fig 4). The other three species D. pinodes, A. pisi, and D. fabae sparse or absent. Pseudothecia were formed on pea stems after produce larger conidia of overlapping size ranges, which are eight weeks of incubation at 10 C. Pseudothecia were only predominantly 1-septate (Table 3).

Fig 4 – Conidia of three Ascochyta spp. responsible for Ascochyta blight of pea, (A) Didymella pinodes isolate MP1, (B) A. pinodella isolate PMP3, and (C) A. pisi isolate AP4, and the causal agent of Ascochyta blight of faba bean (D) D. fabae isolate AF1. Conidia were collected from colonies grown on V8 agar for 16 d and stained with aniline blue. Bar [ 20 mm. 398 M. I. Chilvers et al.

Table 3 – Some common and recognized Ascochyta spp. that cause disease on Anamorph Teleomorph Host Conidia Ascospore Mating system

Ascochyta pisi Didymella pisi Pisum sativum a10–16 3–4.5 12–17.5 6.5–8.5 Heterothallic 1-Septate, some unicellular A. pinodella bNot named P. sativum a5–8(12) 2.5(4) b25–35 12.5–19 Heterothallic Unicellular rarely 1-septate A. pinodes D. pinodes P. sativum a8–16(18) 3–4.5(5) a12–18 4–8 Homothallic 1-Septate (sometimes 2-3) A. fabae D. fabae Vicia faba a16–24 3.5–6 c15–18 5.5–6.5 Heterothallic 1-Septate (sometimes 2–3) A. lentis D. lentis Lens culinaris d10–23 4–8 e14.7–17.6 6.6–8 Heterothallic 1-Septate (sometimes multicellular) A. viciae-villosae Not described V. villosa f15–25 3.5–4.5 d Heterothallic A. rabiei D. rabiei Cicer arietinum a10–16 3.5 g9.5–16 4.5–7 Heterothallic 1-Septate

a CMI descriptions (Punithalingam & Holliday 1972c; Punithalingam & Holliday 1972a; Punithalingam & Holliday 1972b; Punithalingam & Holli- day 1975; Kinsey 2002). b The teleomorph for Ascochyta pinodella has been reported in culture, however, it has not been named (Bowen et al. 1997). c Jellis & Punithalingam 1991. d Morrall & Sheppard 1981. e Kaiser et al. 1997. f Mel’nik et al. 2000. g Trapero-Casas & Kaiser 1992.

Phoma pinodella)(Bowen et al. 1997). Although the teleomorph Discussion for A. pinodella has been reported, no formal description exists (Bowen et al. 1997). In agreement with previous studies, le- Ascochyta pisi is one member of a species complex that causes sions produced by A. pisi were distinct from, and caused less Ascochyta blight of pea (Pisum sativum), a potentially devastat- plant damage than, those caused by D. pinodes and A. pinodella ing disease. Although A. pisi appears to be the least damaging (Fig 1)(Jones 1927; Kraft & Pfleger 2001; Bailey et al. 2003). of the Ascochyta blight species under most environmental con- Plants inoculated with D. pinodes and A. pinodella developed ditions (Wallen 1965), a recent epidemic in Spain was primar- disease symptoms that were virtually indistinguishable. How- ily attributed to A. pisi (Kaiser et al. 2008). The teleomorph of ever, conidia of D. pinodes and A. pinodella produced on V8 agar A. pisi has not previously been reported, but may play a signif- could be differentiated by size and septa (Fig 4), as has been icant role in the epidemiology of Ascochyta blight of pea as it previously observed (Onfroy et al. 1999). Conidia of D. pinodes, does in the closely related Didymella rabiei, the causal agent A. pisi, and D. fabae were virtually indistinguishable, with over- of Ascochyta blight of (Trapero-Casas et al. 1996; lapping size ranges. Recently, a fourth Ascochyta blight patho- Peever et al. 2004; Gamliel-Atinsky et al. 2005). An extended gen of pea P. koolunga was reported to occur in the state of low temperature incubation period is required for D. pisi to South Australia (Davidson et al. 2009). Although we were not develop, similar to that required for D. rabiei (Wilson & Kaiser able to obtain a live specimen of this species for morphological 1995). This study also confirmed the bipolar, heterothallic na- and pathogenic comparison, we were able to obtain DNA from ture of the A. pisi mating system using a molecular assay and this species. Analysis of partial RPB2 and partial glyceralde- in vitro crosses. In a previous study, Mendelian segregation hyde-3-phosphate-dehydrogenase (G3PD; data not shown) was observed for 13 out of 15 molecular markers and mating demonstrate that P. koolunga is closely related to the known type among progeny from a cross between A. pisi isolates Ascochyta blight pathogens of pea, A. pisi, D. pinodes, and A. AP1 and AP2 (Hernandez-Bello et al. 2006). The PCR-based mat- pinodella (Fig 2). Partial RPB2 sequence data demonstrated ing type assay demonstrated that both mating types are pres- that the type species for Didymella, D. exigua, is closely related ent in North America, South America, and Eastern Europe. If to the Ascochyta pathogens of pea, including the newly discov- both mating types are present locally within infected crops ered P. koolunga. Thus, Didymella is considered to be a more ap- or wild pea species, the teleomorph may be capable of forming propriate genus for D. pisi than Mycosphaerella. under the appropriate environmental conditions and contrib- The type species for Didymella is D. exigua and the type for ute to the epidemiology of Ascochyta blight of pea. Further sur- Mycosphaerella is M. punctiformis (Verkley et al. 2004). RPB2 veys of mating type distributions on a field-scale and sequences of M. punctiformis were highly divergent from epidemiological studies are needed to test this hypothesis RPB2 sequences of the type species of Ascochyta, Phoma, and and determine the prevalence and significance of the sexual Didymella. The present results are supported by the ITS state in the biology of A. pisi. sequence analysis of Peever et al. (2007) who demonstrated The Ascochyta blight complex also includes two other spe- that M. punctiformis (isolate CBS724.79), M. graminicola (Gen- cies for which teleomorphs have been reported, D. pinodes Bank accession no. AF181694), and M. fijiensis (GenBank acces- and A. pinodella (syn. Phoma medicaginis var. pinodella and sion no. AF297225) were distantly related to a well-supported Didymella pisi sp. nov., the teleomorph of Ascochyta pisi 399

‘Didymella’ clade, which included A. pisi, D. pinodes, and A. pino- references della. The M. punctiformis isolate CBS942.97 that we sequenced is considered to be M. punctiformis s. str. (Verkley et al. 2004). The RPB2 phylogeny presented here also demonstrates that Bailey KL, Gossen BD, Gugel RK, Morrall RAA, 2003. Diseases of Field A. pisi is distinct from the other two Ascochyta blight patho- Crops in Canada, 3rd edn. The Canadian Phytopathological gens of pea, D. pinodes and A. pinodella. Several previous stud- Society, University Extension Press, Saskatchewan. ies have also examined genetic relatedness among members Barve MP, Arie T, Salimath SS, Muehlbauer FJ, Peever TL, 2003. 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