Circumscription of the anthracnose pathogensColletotrichum lindemuthianum and C. nigrum

Fang LiuState Key Laboratory of Mycology, Institute ofMicrobiology, Chinese Academy of Sciences, Beijing,100101, China; Microbiology, Department of Biology,Utrecht University, Padualaan 8, 3584 CH Utrecht,the Netherlands

Lei Cai1

State Key Laboratory of Mycology, Institute ofMicrobiology, Chinese Academy of Sciences, Beijing,100101, China

Pedro W. CrousCBS-KNAW Fungal Biodiversity Centre, Uppsalalaan8, 3584 CT Utrecht, the Netherlands; UtrechtUniversity, Department of Biology, Microbiology,Padualaan 8, 3584 CH Utrecht, the Netherlands;Wageningen University and Research Centre (WUR),Laboratory of Phytopathology, Droevendaalsesteeg 1,6708 PB Wageningen, the Netherlands

Ulrike DammCBS-KNAW Fungal Biodiversity Centre, Uppsalalaan8, 3584 CT Utrecht, the Netherlands

Abstract: The anthracnose pathogen of commonbean (Phaseolus vulgaris) is usually identified asColletotrichum lindemuthianum, while anthracnose ofpotato (Solanum tuberosum), peppers (Capsicumannuum), tomato (S. lycopersicum) and several othercrop plants is often attributed to C. coccodes. In orderto study the phylogenetic relationships of theseimportant pathogens, we conducted a multigeneanalysis (ITS, ACT, TUB2, CHS-1, GAPDH) of strainspreviously identified as C. lindemuthianum, C.coccodes and other related species, as well as repre-sentative species of the major Colletotrichum speciescomplexes. Strains of C. lindemuthianum belonged toa single clade; we selected an authentic specimen aslectotype, and an appropriate specimen and culturefrom the CBS collection to serve as epitype. Twoclades were resolved within C. coccodes s. lat. Oneclade included the ex-neotype strain of C. coccodes onSolanum, while an epitype was selected for C. nigrum,which represents the oldest name of the second clade,which occurs on Capsicum, Solanum, as well as severalother host plants. Furthermore, we recognized C.lycopersici as a synonym of C. nigrum, and C.biologicum as a synonym of C. coccodes.

Key words: Ascomycota, Colletotrichum, epitypifica-tion, morphology, phylogeny, systematics

INTRODUCTION

Anthracnose is one of the most widespread andeconomically important diseases of common bean(Phaseolus vulgaris) (Del Rıo et al. 2002, Goncalves-Vidigal et al. 2004, Kiryakov 2004, Mahuku andRiascos 2004, Bardas et al. 2007, Bardas et al. 2009,Munda et al. 2009), which can lead to significantlosses in bean production (Tu 1992), especially whenthe climatic conditions favor disease development(Gonzalez et al. 1998). Lesions on stems and pods ofcommon bean are gray or brown, slightly sunken withraised dark brown or reddish edges, frequentlybearing conspicuous salmon colored spore masses(Briosi and Cavara 1889).

Bean anthracnose was first reported from Germanyand described as Gloeosporium lindemuthianum Sacc.& Magnus (Saccardo 1878). Subsequently, Briosi andCavara (1889) transferred Gm. lindemuthianum to thegenus Colletotrichum. Von Arx (1957) regarded C.lindemuthianum (Sacc. & Magnus) Briosi & Cavara asa form of C. gloeosporioides, indistinguishable from itbut specialized to Phaseolus vulgaris. Colletotrichumlindemuthianum has been considered closely relatedto C. orbiculare, C. trifolii and C. malvarum (Pain et al.1992, Sherriff et al. 1994, Bailey et al. 1996). Sherriffet al. (1994) considered these species as conspecific,while Liu et al. (2007) found the four species to bedistinct, representing a species complex. This com-plex represents a basal position within the genusColletotrichum (Farr et al. 2006, Cannon et al. 2012).

Farr and Rossman (2012) listed 214 records ofColletotrichum associated with Phaseolus spp., of which142 refer to C. lindemuthianum. On the other hand,C. lindemuthianum was reported on a wide range ofother legumes, such as Dolichos lablab (Zhuang 2001),Lotus corniculatus (Mulenko et al. 2008), Dolichos sp.(Lenne 1990), Vicia faba (Zhuang 2005) and Vignasinensis (Pande and Rao 1998).

While descriptions of C. lindemuthianum in litera-tures agree with each other in colonial characteristicsin having slow growth rate and dark pigmentation,but present considerable variation in conidial mor-phology. According to von Arx (1957), conidiameasure 11–19 3 4–6 mm, form reddish droplets,with abundant setae in culture that are often absent

Submitted 11 Aug 2012; accepted for publication 14 Nov 2012.1 Corresponding author, E-mail: mrcailei@gmail.com

Mycologia, 105(4), 2013, pp. 844–860. DOI: 10.3852/12-315# 2013 by The Mycological Society of America, Lawrence, KS 66044-8897

844

on the host plant. While Sutton (1980) described C.lindemuthianum as having conidia that measure 9.5–11.5 3 3.5–4.5 mm, form in honey- or pale-coloredmasses, with few appressoria and setae.

Very few studies of C. lindemuthianum haveincluded DNA sequence analysis. Prior to our study,there were only 14 ITS sequences and a few sequencesof other gene regions, e.g. Mat1-2 and glutaminesynthetase, lodged in GenBank. A comparison ofthese ITS sequences with other Colletotrichum speciesrevealed that they belong to different species com-plexes (Cannon et al. 2012). Some of these ITSsequences, e.g. EF608059 (Huang et al. 2010) andJN198431 (Wu L and Qin L, unpubl. data), belong tothe C. boninense species complex (Damm et al.2012b).

A recent study by Liu et al. (2011) characterizedand neotypified C. coccodes (Wallr.) S. Hughes., whichis known to represent an important pathogen ofpotato. Farr and Rossman (2012) listed over 30 planthosts for C. coccodes, including many reports onpeppers (Capsicum annuum) and tomato (Solanumlycopersicum syn. Lycopersicon esculentum). How manyof these records are really attributable to C. coccodesremains unknown. For example, Thaung (2008)reported C. coccodes as pathogen of Camellia thea.However, none of our isolates, nor sequences fromthis host on GenBank relate to C. coccodes (unpubl.data); most of them belong to the C. gloeosporioidesspecies complex.

Capsicum annuum is often severely affected byanthracnose, which causes serious yield losses in manycountries (Oh and Kim 2007). Many Colletotrichumspecies reported from Capsicum annuum belong tothe C. acutatum species complex (C. acutatum, C.brisbanense, C. nymphaeae, C. scovillei) (Than et al.2008a, b; Damm et al. 2012a), C. boninense speciescomplex (C. novae-zelandiae) (Damm et al. 2012b)and C. gloeosporioides species complexes (C. siamense)(Weir et al. 2012) as well as C. truncatum (synonym:C. capsici) (Pakdeevaraporn et al. 2005, Sharma et al.2005, Damm et al. 2009). There are also many reportsof other Colletotrichum species from Capsicumannuum (Farr and Rossman 2012), especially of C.coccodes and C. nigrum. The latter is of uncertainstatus and listed as doubtful species by Hyde et al.(2009). Colletotrichum nigrum was placed in synonymywith C. gloeosporioides by von Arx (1957), who laterresurrected it as a species restricted to Capsicum (vonArx 1981).

Colletotrichum coccodes is also reported as a devas-tating pathogen of tomato (Solanum lycopersicum)causing anthracnose on fruits (Hughes 1958; Chestersand Hornby 1965a, b; Chapin et al. 2006; Alkan et al.2008; Ben-Daniel and Bar-Zvi 2009). There are four

other Colletotrichum species on tomato listed in theUSDA database, C. dematium, C. gloeosporioides, C.lycopersici and C. nigrum (Farr and Rossman 2012).No sequence data of C. lycopersici and C. nigrum areavailable in GenBank. In a preliminary study, the ITSsequences of most of the strains in the CBS collectionoriginally identified as C. lycopersici and C. nigrumcluster with C. coccodes (unpubl. data).

The names C. lindemuthianum, C. lycopersici and C.nigrum are of uncertain application because no ex-type or authentic cultures were retained. The aims ofthis study are therefore, to investigate the phyloge-netic relationships of C. lindemuthianum and C.coccodes and related strains to other species inColletotrichum based on a multilocus phylogeny, tolocate type or authentic material of C. lindemuthia-num, C. lycopersici, C. nigrum, and to select epitypespecimens with living cultures derived from them tofix the application of these names.

MATERIALS AND METHODS

Isolates.—Isolates previously identified as C. lindemuthia-num and C. coccodes as well as other related species wereobtained from the culture collection of the CBS-KNAWFungal Biodiversity Centre, Utrecht, the Netherlands(CBS). Type specimens of the species studied are locatedin the fungaria of the CBS, the Herbarium Hamburgense(HBG), Germany and the US National Fungus Collections(BPI), Beltsville, Maryland, USA. Cultures derived from theepitypes and neotypes, as well as all other isolates used formorphological and phylogenetic analyses are maintained inthe CBS culture collection and presented in TABLE I.

Morphological analysis.—To enhance sporulation, 5 mmdiam plugs from the margin of actively growing cultureswere transferred to the center of 9- cm diam Petri dishescontaining synthetic nutrient-poor agar medium (SNA;Nirenberg 1976) amended with autoclaved filter paperand double-autoclaved stems of Anthriscus sylvestris placedonto the agar surface. The strains were also studied aftergrowth on oatmeal agar (OA) (Crous et al. 2009) and onautoclaved pods of common bean (Phaseolus vulgaris).Cultures were incubated at 20 C under near UV light with a12 h photoperiod for 10 d. Measurements and photographsof characteristic structures were made according to methodsdescribed by Damm et al. (2007). Appressoria on hyphaewere observed on the reverse side of colonies grown on SNAplates. Appressoria were also induced using a slide culturetechnique (Cai et al. 2009). Microscopic preparations weremade in clear lactic acid, with 30 measurements perstructure, and observed with a Nikon SMZ1000 dissectingmicroscope (DM) or with a Nikon Eclipse 80i microscopeusing differential interference contrast (DIC) illumination.Colony characters and pigment production on SNA and OAincubated at 20 C were noted after 10 d. Colony colors wererated according to Rayner (1970). Growth rates weremeasured after 7 and 10 d.

LIU ET AL.: EPITYPIFICATION OF TWO COLLETOTRICHUM SPECIES 845

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LIU ET AL.: EPITYPIFICATION OF TWO COLLETOTRICHUM SPECIES 847

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848 MYCOLOGIA

Sexual compatibility.—Seventeen isolates of C. lindemuthia-num (TABLE I) were crossed with each other in all possiblecombinations on SNA medium amended with sterile stems ofAnthriscus sylvestris, pine needles and autoclaved bean pods.In all tests, two isolates were placed on opposite sides of apiece of A. sylvestris, pine needles or bean pods. Controltests, where isolates were crossed with themselves, wereundertaken to determine whether strains had a heterothallicor homothallic mating system. The plates were incubated at20 C under near UV light with a 12 h photoperiod for 6 wk.Matings were regarded as successful when isolate combina-tions produced perithecia and ascospores.

Phylogenetic analyses.—Genomic DNA of the isolates wasextracted using the method of Damm et al. (2008). Six lociincluding the 5.8S nuclear ribosomal gene with the twoflanking internal transcribed spacers (ITS), a 200-bp intron ofthe glyceraldehyde-3-phosphate dehydrogenase (GAPDH), apartial sequence of the actin (ACT), chitin synthase 1 (CHS-1), beta-tubulin (TUB2) and histon3 (HIS3) gene wereamplified and sequenced using the primer pairs ITS1F(Gardes and Bruns 1993) + ITS4 (White et al. 1990), GDF1+ GDR1 (Guerber et al. 2003), ACT-512F + ACT-783R(Carbone and Kohn 1999), CHS-354R + CHS-79F (Carboneand Kohn 1999), T1 (O’Donnell and Cigelnik 1997) + Bt-2b(Glass and Donaldson 1995) and CYLH3F + CYLH3R (Crouset al. 2004), respectively. The PCR protocols were performedas described by Damm et al. (2009). The DNA sequencesobtained from forward and reverse primers were used toobtain consensus sequences using MEGA5, and subsequentalignments were generated using MAFFT v.6 (Katoh and Toh2010), and manually edited using BioEdit (Hall 1999).

A maximum parsimony analysis was performed on themultilocus alignment (ITS, ACT, TUB2, CHS-1, GAPDH)using PAUP v.4.0b10 (Swofford 2002). Ambiguously alignedregions were excluded from all analyses. Unweighted parsi-mony (UP) analysis was performed. Trees were inferred usingthe heuristic search option with TBR branch swapping and1000 random sequence additions. Maxtrees were unlimited,branches of zero length were collapsed and all multipleparsimonious trees were saved. Clade stability was assessed in abootstrap analysis with 1000 replicates, each with 10 replicatesof random stepwise addition of taxa. A second phylogeneticanalysis using a Markov Chain Monte Carlo (MCMC)algorithm was done to generate trees with Bayesian posteriorprobabilities in MrBayes v.3.2.1 (Ronquist and Huelsenbeck2003). Nucleotide substitution models were determined usingMrModeltest v.2.3 (Nylander 2004) for each gene region andincluded in the analyses. Two analyses of four MCMC chainswere run from random trees for ten millions generations andsampled every 1000 generations. The first 25% of trees werediscarded as the burn-in phase of each analysis and posteriorprobabilities determined from the remaining trees. Sequencesderived in this study were deposited in GenBank, and thealignment in TreeBASE (S13363).

RESULTS

The phylogenetic analysis included 83 ingroupstrains, with Monilochaetes infuscans (CBS 869.96) as

outgroup. The dataset of five genes (ACT, CHS-1,GAPDH, ITS, TUB2) comprised 1471 charactersincluding the alignment gaps, of which 683 characterswere parsimony-informative, 103 parsimony-uninfor-mative and 685 constant. Parsimony analysis resultedin nine most parsimonious trees, one of them(Length 5 2978, CI 5 0.547, RI 5 0.911, RC 5

0.498) is shown in FIG. 1. The Bayesian tree agreedwith both the tree topology and bootstrap supports ofthe trees obtained with maximum parsimony, Bayes-ian posterior probability values $ 0.95 are shown asthickened branches on the phylogenetic tree.

The strains studied in this paper formed two mainclades in the multigene phylogeny (FIG. 1). One ofthese clades constitutes the upper part of thephylogenetic tree with isolates previously identifiedas C. coccodes and related strains comprising twosubclades. The first subclade (C. coccodes), with abootstrap support/Bayesian posterior probability val-ue of 94/0.99 respectively, contains 20 strainsincluding the ex-neotype strain of C. coccodes (CBS369.75), while the other 13 strains form a sister clade(98/1.00) to C. coccodes. Both clades have closerelationship with a species complex formed by speciessuch as C. tofieldiae, C. liriopes and C. verruculosum.Seventeen isolates from Phaseolus spp. identified as C.lindemuthianum form a single clade (100/1.00),which is basal to all other clades in the genus.

TAXONOMY

Based on DNA sequence data and morphology, the 17strains of C. lindemuthianum studied belong to asingle species, while a second species C. nigrum wasrevealed closely related to C. coccodes. Both C.lindemuthianum and C. nigrum are characterizedand epitypified below.

Colletotrichum lindemuthianum (Sacc. & Magnus)Briosi & Cavara, Funghi Parass. Piante Colt. od Utili,Fasc. 2:no. 50. 1889. FIG. 2

Basionym: Gloeosporium lindemuthianum Sacc. &Magnus, Michelia 1:129. 1878.On SNA: Vegetative hyphae1–10 mm diam, hyaline to

pale brown, smooth-walled, septate, branched. Chla-mydospores not observed. Conidiomata appearing asaccumulations of conidia on the surface of themedium, conidiophores and setae formed directly onhyphae. Setae not formed in ex-epitype strain, setae ofstrain CBS 146.31, CBS 523.97 and CBS 130841 straightor flexuous, pale brown, 1–4-septate, 53–87.5 mm long,basal cell cylindrical, 3.5–5 mm diam, tip rounded.Conidiophores hyaline, 23–51 mm long, branched, 0–3-septate. Conidiogenous cells hyaline, smooth-walled,

LIU ET AL.: EPITYPIFICATION OF TWO COLLETOTRICHUM SPECIES 849

FIG. 1. One of nine most parsimonious trees obtained from a heuristic search of combined ACT, CHS-1, GAPDH, ITS andTUB2 gene sequences of Colletotrichum species. Bootstrap support values (1000 replicates) above 50% are shown at the nodes.Bayesian posterior probability values $ 0.95 are emphasized by thickened branches. The tree is rooted with Monilochaetesinfuscans. Numbers of ex-holotype, ex-epitype and ex-neotype strains are emphasized in bold. Strain numbers of strainsstudied are followed by host species and country of origin.

850 MYCOLOGIA

cylindrical to ampulliform, 8–14 3 4–5 mm, opening 1–1.5 mm diam, collarette 0.5 mm, periclinal thickeningnot observed. Conidia hyaline, smooth-walled, asep-tate, guttulate, cylindrical, both ends obtuse or with anacute base, (10.5–)11.5–13.5(–14.5) 3 (3–)3.5–4(–4.5)mm, mean 6 SD 5 12.6 6 1.1 3 3.8 6 0.4 mm, L/Wratio 5 3.3. Appressoria not formed in ex-epitypestrain, appressoria of strain CBS 146.31 single or inloose groups, (pale to) medium brown, smooth-walled,outline subglobose, ovoid to ellipsoidal, with entire or

undulate edge, (5–)5.5–8.5(–12) 3 (4.5–)5–6.5(–7)mm, mean 6 SD 5 7 6 1.7 3 5.6 6 0.7 mm, L/W ratio 5

1.3.On Anthriscus stem: Conidiomata acervular, conid-

iophores and setae formed from a cushion of palebrown, angular cells, 2.5–6 mm diam. Setae pale tomedium brown, 1–4-septate, 30–93 mm long, basal cellcylindrical, inflated or constricted, 3.5–5 mm diam, tipround. Conidiophores hyaline to pale brown, smooth-walled, branched, 1-septate, up to 20 mm long.

FIG. 2. Colletotrichum lindemuthianum (a–c, f–h, n–o from ex-epitype strain CBS 144.31, d–e from CBS 130841, i–m fromstrain CBS 146.31). a–b. Acervuli; c. Setae; d. Tip of seta; e. Basal part of seta; f–h. Conidiophores; i–m. Appressoria; n–o.conidia; a, c, g, n: from Anthriscus stem; b, d–e, f, h, i–m, o: from SNA. a–b: DM; c–o: DIC. — Scale bars: a 5 100 mm; c 5 10 mm;a applies to a–b; c applies to c–o.

LIU ET AL.: EPITYPIFICATION OF TWO COLLETOTRICHUM SPECIES 851

Conidiogenous cells hyaline to pale brown, smooth,8–13.5 3 3–5 mm, opening 1–1.5 mm diam, collarette0.5 mm, periclinal thickening sometimes visible.Conidia hyaline, smooth-walled, aseptate, guttulate,cylindrical, both ends obtuse or with base acute,(10.5–)11.5–14(–15.5) 3 (3–)3.5–4(–4.5) mm, mean6 SD 5 12.9 6 1.2 3 3.8 6 0.4 mm, L/W ratio 5 3.4.

Culture characteristics: Colonies on SNA flat withentire margin, medium hyaline, filter paper grayolivaceous, acervuli aggregated close to the Anthriscusstem, aerial mycelium lacking, 21–24 mm in 7 d (29–31 mm in 10 d). Colonies on OA flat with entiremargin, surface gray olivaceous to olivaceous black,covered by long hyaline and sparse aerial mycelium,conidial masses whitish, abundant, reverse olivaceousgray to iron gray, 26–27 mm in 7 d (41–43 mm after10 d). Conidia in masses whitish to pale salmon.

Materials examined. BRAZIL. On Phaseolus vulgaris, Mar1997, J.A. Bailey, culture CBS 571.97 5 Lars 83. COSTARICA. On P. coccineus, Feb 1997, J.A. Bailey, culture CBS523.97 5 LARS 798; On P. coccineus, Feb 1997, J.A. Bailey,culture CBS 524.97 5 LARS 800. EUROPE. Unknowncountry. On P. vulgaris, Mar 1997, J.A. Bailey, culture CBS569.97 5 LARS 9 5 ATCC 56897. FRANCE. Paris, RousselLab. Ltd. On pod of P. vulgaris, Jul 1956, Uclaf, culture CBS151.56 5 IMI 063364 5 ATCC 12611 5 UCLAF 230.GERMANY. Bonn, Poppelsdorf, fruit and vegetable gardenof the Agricultural Institute Poppelsdorf. On pods ofPhaseolus vulgaris, 23 Aug 1875, H. Lindemuth (HBG26/2270, ex herb. P. Magnus–syntype of Gloeosporium linde-muthianum, lectotype here designated); Bonn. On P.vulgaris, Aug 1931, E. Schaffnit (CBS H-20954, epitypehere designated, culture ex-epitype CBS 144.31); Schonin-gen. On P. vulgaris, Feb 1928, E. Schaffnit, culture CBS150.28; Schoningen. On P. vulgaris, Feb 1928, E. Schaffnit,culture CBS 151.28; Schoningen. On P. vulgaris, Feb 1928,E. Schaffnit, culture CBS 153.28; Berlin, Malchow. On P.vulgaris, Aug 1931, E. Schaffnit, culture CBS 143.31;Dresden. On P. vulgaris, Aug 1931, E. Schaffnit, cultureCBS 146.31; Kirchwarder near Hamburg. On P. vulgaris,Aug 1931, E. Schaffnit, culture CBS 147.31. THE NETHER-LANDS. Amsterdam. On P. vulgaris, Feb 1928, E. Schaffnit,culture CBS 152.28. USA. MARYLAND: Takoma park. On P.vulgaris, Jul 1906, C.L. Shear (BPI 597216); WASHINGTON:District of Columbia. On P. vulgaris, Dec 1908, C.L. Shear(BPI 597217); VIRGINIA: Arlington. On Phaseolus sp., Jul1942, C.L. Shear (BPI 597215); KENTUCKY. On P. vulgaris,2007, unknown collector, culture CBS 130841 5 C1KY1;NEW YORK: Ithaca, Cornell University. On P. vulgaris, Jan1957, unknown collector (isolated by W.H. Burkholder),culture CBS 131.57; NEW YORK: Ithaca, Cornell University.On P. vulgaris, Jan 1957, unknown collector (isolated byW.H. Burkholder), culture CBS 132.57; NEW YORK: Ithaca,Cornell University. On P. vulgaris, Jan 1957, unknowncollector (isolated by W.H. Burkholder), culture CBS 133.57.

Notes: Gloeosporium lindemuthianum was describedfrom common beans collected in 1875 from Poppels-dorf, near Bonn, Germany and in the same year in

Padova, Italy (Saccardo 1878). We could not locate thespecimen from Italy, but we located the authenticherbarium specimens of Gm. lindemuthianum in theHerbarium Hamburgense (HBG). Two of these spec-imens were collected by H. Lindemuth prior to thedescription of Gm. lindemuthianum. The label of one ofthem contains the same collection data as the originaldescription, and was therefore selected as lectotype.

Conidia on the lectotype specimen are hyaline andcylindrical, 11.5–21 3 3.5–5 mm, mean 6 SD 5 15.5 6

1.8 3 4.5 6 0.4 mm, and formed light salmon to pinkmasses. The conidial size is concordance with theoriginal description (16–19 3 4.5–5.5 mm) (Saccardo1878) and von Arx (1957) (11–19 3 4–6 mm), butlarger than that from Sutton (1980, 1992) (9.5–11.5 3

3.5–4.5 mm). Conidial dimensions of the ex-epitypestrain CBS 144.31 formed on SNA, Anthriscus stemsand autoclaved bean pods (9.5–22 3 3.5–5.5 mm,mean 6 SD 5 13 6 2.1 3 4.6 6 0.4 mm) are alsoconcordance with the conidial dimensions of thelectotype specimen and original description.

The two C. lindemuthianum strains from P.coccineus (CBS 523.97 and CBS 524.97) differed fromthe strains from P. vulgaris in the morphology oftheir conidiophores. In addition to the conidiophorestypical for C. lindemuthianum described above,conidia were also formed from up to 89 mm longhyphae that resemble a transitional stage betweensetae and conidiophores, with a conidiogenous cellthat is usually cylindrical and 20–28.5 mm long. Bothtypes of conidiophores are branched and septate.Sometimes setae and conidiophores extend from thesame node. The phylogeny of the multigene datasetgenerated in this study only supports one species(FIG. 1), suggesting that these morphological differ-ences fall within the variability of the species.

Shear and Wood (1913) first induced the sexualstage of C. lindemuthianum, Glomerella lindemuthiana,by incubating a strain from Takoma Park, Maryland,USA on living bean pod tissue with acervuli on cornmeal agar. However, Shear and Wood (1913) failed todesignate a type specimen for Glomerella lindemuthi-ana and only provided a morphological description.Three Glomerella lindemuthiana specimens (plantmaterials) collected by Shear in the United States inthe years 1906 (BPI 597216), 1908 (BPI 597217) and1942 (BPI 597215) are available in the BPI herbari-um, one of them (BPI 597217) was collected inWashington. This specimen contained cylindricalconidia, measuring 10–14.5 3 4–5.5 mm, mean 6 SD5 12.2 6 1.2 3 4.7 6 0.3 mm, L/W ratio 5 2.6 andseptate setae measuring 40–120 mm long, whichagrees with those of the ex-epitype culture (CBS144.31). However, ascomata were not observed fromBPI597217.

852 MYCOLOGIA

In this study, several methods were unsuccessfullytested to induce the teleomorph stage of C.lindemuthianum. Previous morphological descrip-tions for teleomorph are doubtful and conflict witheach other. Kimati and Galli (1970) induced anascogenous stage of C. lindemuthianum by pairingisolates from Phaseolus sp. from Brazil in semi-sytheticmedium, and was referred as Glomerella cingulata f.sp. phaseoli. They observed two types of asci andascospores: (1–)4(–8)-spored asci, with mean asco-spores dimension 20 3 6.5 mm, and 8-spored asci, withellipsoidal ascospores, measuring on average 10 3

4 mm, which indicates that another fungus may beinvolved. In addition, fig.1 of Rodrıguez-Guerra et al.(2005) shows a sexual stage with 8-spored asci andascospores that are longer and wider (15.5–29 3 4.5–7 mm) than those formed in 8-spored asci described byKimati and Galli (1970) (av. 10 3 4 mm). In spite ofthis, the occurrence of sexual reproduction in Ga.lindemuthiana under field conditions has not yetbeen observed. Furthermore, most of the previousstudies reporting sexual stages lack molecular data,and the few studies that include them show diver-gence from C. lindemuthianum. For example, one ofthe six RAPD groups found by Talamini et al. (2006)formed a sexual stage (referred to as Glomerellacingulata f. sp. phaseoli) and was divergent from theother groups. Phylogenies based on ITS and HMGsequences by Barcelos et al. (2011) show that strainsfrom common beans with and without sexual stagesbelong to different clades within Colletotrichum. Weare not aware of any data that shows C. lindemuthia-num s. str. forms a sexual stage. We assume that sexualstages linked to C. lindemuthianum in the past belongto other species. This would explain why attempts torepeat inducing sexual stage of C. lindemuthianumfailed (Edgerton 1915, Muller 1927, Batista andChaves 1982, Bryson 1990).

Another species described as causing anthracnoseof Phaseolus vulgaris is C. caulicolum. However theconidia of C. caulicolum are falcate and measure 18–30 3 3.5–4 mm (Heald and Wolf 1911). This speciesmay be a synonym of C. truncatum, which has alsobeen reported from beans (Damm et al. 2009).

Colletotrichum coccodes (Wallr.) S. Hughes, Can. J.Bot. 36:754. 1958.

Basionym: Chaetomium coccodes Wallr., Fl. Crypt.Germ. (Nurnberg) 2:265. 1833.5 Colletotrichum biologicum Chaudhuri, Ann. Bot. 38:735.

1924.

For neotype and description of C. coccodes refer toLiu et al. (2011).

Materials examined. BULGARIA. Shumen. On Solanumtuberosum, 1976, E. llieva, CBS H-10565, culture CBS

527.77. CZECH REPUBLIC. Prague. On Cucurbita pepo,unknown collector and collection date (isolated by A.Kubatova), culture CBS 125352 5 CCF 3825. INDIA? Ondying stem of Solanum tuberosum, unknown collection dateand collector (deposited in CBS collection Feb. 1925 by H.Chaudhuri), culture CBS 122.25; On Solanum tuberosum,unknown collector and collection date (isolated by H.Nirenberg), culture CBS 109213 5 BBA 62126; Munster.On leaf of Anthurium sp., unknown collection date, P.Kotthoff, CBS H-10654, culture CBS 150.33. IRELAND. OnHeterodera pallida, unknown collector and collection date,culture IMI 345429 5 CPC 20243. THE NETHERLANDS.On Solanum tuberosum, unknown collector and collectiondate (isolated by H.M. Quanjer), culture CBS 103.16; Onstem of Solanum tuberosum, unknown collector andcollection date (isolated by Naktuinbouw), culture CBS125963 5 NB 596; Groningen. On tuber of Solanumtuberosum, 1975, G. Jager, CBS H-10573, culture CBS369.75; Wageningen. On Solanum tuberosum, unknowncollector and collection date, culture CBS 164.49 ; Fromsoil, Oct 1977, Q. Jager, CBS H-10575, CBS H-10571,culture CBS 528.77. SOUTH AFRICA. University ofStellenbosch. On Solanum tuberosum, unknown collectorand collection date, culture CBS 126378 5 C 101.SWITZERLAND. On Globodera rostochiensis, unknowncollector and collection date (isolated by A. Papert), cultureCBS 641.97. UK. On Solanum tuberosum, unknown collec-tor and collection date, culture IMI 78352 5 CPC 16810.UNKNOWN LOCATION. On Solanum tuberosum, unknowncollector and collection date (isolated by G. Van den Ende),culture CBS 125.57; On Beta vulgaris, unknown collectorand collection date (isolated by G. Van den Ende), cultureCBS 126.57; Unknown substrate, unknown collector andcollection date (isolated by F. Labrousse), culture CBS134.30. USA. Unknown substrate, unknown collector andcollection date (isolated by C.L. Shear), culture CBS121.24. YUGOSLAVIA. Novisad. On Capsicum sp., 1965,M. Acimovic, culture CBS 125342 5 MUCL 8255.ZIMBABWE. On Solanum tuberosum, unknown collectiondate, P. Cannon, culture CBS 112897 5 IMI 61249.

Notes: Colletotrichum biologicum was first reportedfrom the dying stalks of potatoes (Solanum tuberosum)in the garden of the ‘‘Biologische Reichsanstalt furForst- und Landwirtschaft’’ at Berlin-Dahlem, Ger-many (Chaudhuri 1924). In February 1925, Chaud-huri deposited one culture in the CBS collection(CBS 122.25). According to the CBS database thisstrain is from India, which is doubtful. It is more likelythat this is a strain from the original collection fromGermany that H. Chaudhuri sent to CBS afterreturning to India. The strain is included in thecurrent study and clusters with the ex-neotype strainof C. coccodes (CBS 369.75) in FIG. 1, which indicatesthat C. biologicum is the synonym of C. coccodes.

Colletotrichum coccodes, originally described frompotato black dot as Chaetomium coccodes Wallr. (Wall-roth 1833), is not host-specific. In our study, severalstrains from other hosts cluster in the C. coccodes

LIU ET AL.: EPITYPIFICATION OF TWO COLLETOTRICHUM SPECIES 853

FIG. 3. Colletotrichum nigrum (a–f, h–q from strain CBS 128507, g from strain CBS 132451). a–c. Acervuli; d. Tip of seta; e.Basal part of seta; f–h. Conidiophores; i–o. Appressoria; p–q. Conidia; a, d–f, i–k, q: from Anthriscus stem; b–c, g–h, l–p: fromSNA. a–c: DM; d–p: DIC. — Scale bars: a 5 100 mm; h 5 10 mm; a applies to a–c; c applies to d–q.

854 MYCOLOGIA

clade, e.g. strains from Beta vulgaris, Curcubita pepoand Lycopersicon esculentum. These strains form largerconidia (average 17.5–19.5 3 3.5 mm on SNA, average19–20 3 3.5 mm on Anthriscus stem) than those frompotato (average 12 3 3.5 mm on SNA, average 14–163 4 mm on Anthriscus stem).

Colletotrichum nigrum Ellis & Halst., Bull. TorreyBot. Club 18:15. 1891. FIG. 35 Colletotrichum lycopersici Chester, Bull. Torrey Bot.

Club 18:372. 1891.

On SNA: Vegetative hyphae hyaline to mediumbrown, smooth-walled, septate, branched, 1–7 mmdiam. Chlamydospores not observed. Conidiomataacervular, or appearing as accumulation of conidia onsurface of medium, conidiophores formed from acushion of pale brown, angular cells or directly onhyphae. Setae not observed. Conidiophores hyaline,branched, septate. Conidiogenous cells hyaline,smooth-walled, cylindrical, 15.5–31.5 3 1.5–3.5 mm,opening 1–1.5 mm diam. Conidia hyaline, smooth-walled, aseptate, guttulate, cylindrical, apex subacuteor obtuse, (9–)15–20(–24) 3 (3.0–)3.5–4(–4.5) mm,mean 6 SD 5 17.5 6 2.5 3 4 6 0.4 mm, L/W ratio 5

4.4. Appressoria single, brown, smooth-walled, glo-bose, obvoid, clavate, or irregular outline, with anentire or undulate edge, (4.5–)7–13(–21) 3 (4.5–)5.5–7.5(–9) mm, mean 6 SD 5 10 6 2.9 3 6.5 6

1.0 mm, L/W ratio 5 1.5.On Anthriscus stem: Conidiomata acervular, com-

pact fruiting structures composed of cushions ofbrown to black angular cells, or appearing asaccumulation of conidia on stem. Setae abundant,straight or flexuous, gradually tapering to the tip, 1–5-septate, medium brown, 38–116 mm long, basal cellcylindrical, inflated or constricted, 3–7 mm diam, tipusually acute. Conidiophores hyaline to pale brown,branched, septate. Conidiogenous cells hyaline topale brown, cylindrical to ampulliform, 7–12.5 3 2.5–4.5 mm, opening 1–2 mm diam. Conidia hyaline,smooth-walled, aseptate, guttulate, cylindrical, apexsubacute or obtuse, (13.5–)17.5–21.5 (–32) 3 (3–)3.5–4(–4.5) mm, mean 6 SD 5 19.5 6 1.9 3 3.5 6

0.3 mm, L/W ratio 5 5.6.Culture characteristics: Colonies on SNA flat with

entire margin, hyaline, aerial mycelium lacking,abundant salmon or black acervuli aggregated onAnthriscus stem and filter paper, few acervuli onmedium, 40–48 mm in 7 d (58–66 mm in 10 d).Colonies on OA flat with entire margin, entirelycovert with dense, smoke gray aerial mycelium,reverse mouse gray to dark mouse gray, 42–43 mmin 7 d (59–61 mm in 10 d). Conidia in masses salmon.

Materials examined. ARGENTINA. On Capsicum sp.,unknown collection date and collector (CBS H-21032,

epitype here designated, ex-epitype culture CBS 169.49).Quarantine interception. On Fragaria sp., petiole, unknowncollection date and collector (deposited in IMI collection1994 with no. B11/12), culture IMI 363582 5 CPC 18083;On Fragaria sp., petiole, unknown collection date andcollector (deposited in IMI collection 1994 with no. A10/11), culture IMI 363581 5 CPC 18133. CANADA. On Lensculinare, 1992, unknown collector, culture IMI 352646 5

CPC 19381. CHILE. San Ignacio. On Cichorium intybus,unknown collection date, A. Schilder, culture CBS 1275625 CPC 16433. INDONESIA. Java. On Capsicum annuum,Feb 24 1939, J.T. Beauchamp (BPI 395383). NEW ZEA-LAND. Bay of Plenty. On Capsicum annuum (sweetpepper), fruit rot, Mar 1990, P.R. Johnston, CBS H-21033,culture CBS 128507 5 ICMP 12927 5 C1147-2; Bay ofPlenty. On Solanum lycopersicum, fruit rot, Mar 1990, P.R.Johnston, culture CBS 128553 5 ICMP 12929 5 C1167-1.SERBIA AND MONTENEGRO. Novi Sad, Institute of Fieldand Vegetable Crops. On Helianthus tuberosus, Feb 1981, T.Verbalov, culture CBS 288.81. UNKNOWN LOCATION. OnCapsicum annuum fruit, Autumn 1903, unknown collector(BPI 395380); Unknown substrate, Autumn 1903, unknowncollector (BPI 395374); Probably on Solanum lycopersicum,unknown collection date and collector, culture CBS 175.59;Probably on Solanum lycopersicum, unknown collection dateand collector, culture CBS 174.59 5 ATCC 12521 5 SC2145. USA. NEW JERSEY: probably Swedesboro, GloucesterCo. On fruit of Capsicum annuum, fruit anthracnose,probably Aug 1890, probably B.D. Halsted (NY, Elliscollection, no. 158 [not seen] lectotype designated here,BPI 797145 isolectotype); NEW JERSEY: New Brunswick.On Capsicum annuum fruits, Aug 1896, B.D. Halsted (BPI399228); NEW JERSEY: Vineland Market. On Capsicumannuum, Oct 1889, J.B. Ellis (BPI 395379, possible type ofGloeosporium piperatum); NEW JERSEY: Vineland Market.On Capsicum annuum, Oct 1889, J.B. Ellis (BPI 797997);NEW JERSEY: New Brunswick. On cultivated Capsicumannuum, May 1896, B.D. Halsted (BPI 395373); CONNEC-TICUT: Hamden. On Capsicum annuum, Oct 1903, G.P.Clinton (BPI 395382); DELAWARE: Newark. On Capsicumannuum, Nov 1903, C.O. Smith (BPI 395381); DELAWARE:Newark. On Lycopersicon esculentum, Oct 1891, F.D. Chester(BPI 399511, possible type of Colletotrichum lycopersici);DELAWARE: Newark. On Lycopersicon esculentum fruit, Oct1891, F.D. Chester (BPI 399513); DELAWARE: Newark. OnLycopersicon esculentum fruit, Oct 1891, F.D. Chester (BPI398647); DELAWARE: Newark. On Lycopersicon esculentumfruit, Oct 1891, F.D. Chester (BPI 398648); OHIO. OnSolanum lycopersicum, unknown collection date and collec-tor, culture CBS 132451 5 Sa3; OHIO. On Solanumlycopersicum, unknown collection date and collector, cultureCBS 132450 5 Hu20; WASHINGTON. On Solanumlycopersicum, 1921, F.T. Brooks, culture IMI 17310 5 CPC20244 5 NCTC 1130.

Note: Anthracnose of peppers was reported fromNew Jersey, USA by Halsted (1891a, b), who describedthe causal agents as C. nigrum and Gm. piperatum.Halsted (1891b) observed that the two species causeddifferent symptoms on peppers. Colletotrichum nigrum

LIU ET AL.: EPITYPIFICATION OF TWO COLLETOTRICHUM SPECIES 855

formed decayed patches that turned black due to thesetae of the fungus, while the color of decayedpatches caused by Gm. piperatum remained un-changed. The conidia of C. nigrum are cylindrical,nearly straight, hyaline, and measure 20–22 3 4 mm(Halsted 1891a). Later, Halsted (1896) reported C.nigrum as the main cause of anthracnose of peppersin the USA.

We located authentic specimens of C. nigrum andGm. piperatum from the herbarium BPI. The label ofone of the C. nigrum specimens (BPI 797145) states,‘‘158, New York Botanical Garden, Colletotrichumnigrum E & H, on pepper fruit’’ with a stamp ‘‘CO-TYPE’’. The morphology of this fungus agrees with theoriginal description of C. nigrum by Halsted (Halsted1891a). The small size and shape of this specimen aswell as the number ‘‘158’’ identifies it as part of aspecimen located in the New York Botanical Gardenherbarium. There was no type designated either in theoriginal description of C. nigrum (Halsted 1891a) or inthe following reports of this species by Halsted (1891b,1896). The label of this NY specimen does not statethat it is the holotype, but we are confident that it isauthentic material of C. nigrum, and thereforedesignate it here as the lectotype.

We also examined a possible type specimen of Gm.piperatum (BPI 395379), which differed from C.nigrum in forming fusiform conidia, indicatingaffinity to the C. acutatum species complex.

The ex-epitype culture (CBS 169.49) was collectedin Argentina (South America), which is nearest to thelocation where the type specimen was collected(USA). Unfortunately, strain CBS 169.49 is sterile,thus we chose a genetically identical strain, CBS128507 from Capsicum in New Zealand, as basis forthe morphological description. Several strains fromother hosts collected in the USA confirm theoccurrence of C. nigrum in this country.

Colletotrichum nigrum forms significantly longerconidia than C. coccodes with a larger L/W ratio bothon SNA medium (average size of conidia of C.nigrum, CBS 128507, 17.5 3 4 mm, L/W 5 4.4 vs. C.coccodes, CBS 369.75, 12 3 3.5 mm, L/W 5 3.4) and onAnthriscus stem (average size of conidia of C. nigrum,CBS 128507, 19.5 3 3.5 mm, L/W 5 5.5 vs. C. coccodes,CBS 369.75, 14 3 4 mm, L/W 5 3.5). According to ourresults, C. nigrum is not host-specific, and can alsoinfect Cichorium intybus, Fragaria sp., Helianthustuberosus, Lens culinare and Solanum lycopersicum.

There are two other names related to this species,Gm. phomoides and C. lycopersici, both described fromS. lycopersicum. Gloeosporium phomoides, describedfrom tomato in Italy by Saccardo (Saccardo 1884),was prior to C. lycopersici (Chester 1891) and C.nigrum (Halsted 1891a). Gloeosporium phomoides was

described as having conidia with an abruptly attenu-ate base and a rounded apex, oblong to clavate(10–12 3 2.5–3 mm), characters typical of C. coccodes.We were unable to locate the type specimen of Gm.phomoides. In 1884, Arthur reported the occurrenceof the fungus in New York, and in 1891, Chesterreported a fungus in the USA similar to thatdescribed by Saccardo, but lacking setae. He de-scribed it as a new species, C. lycopersici (Chester1891). In a later publication, Chester (1893) recog-nized that setae may be present or absent on artificialmedium and regarded C. lycopersici as a synonym ofGm. phomoides and therefore combined it in Colleto-trichum as C. phomoides. Chester (1891) described C.lycopersici as forming oblong conidia, with subacuteends, measuring 16–22 3 4 mm, similar to the size ofC. nigrum. Our examination of the possible typespecimen of C. lycopersici (BPI 399513) confirmedthis. Two strains from tomato in the USA (CBS132450 and CBS 132451) are included in this study,which cluster together with C. nigrum (FIG. 1).Therefore we regard C. lycopersici as a synonym ofC. nigrum. While Glomerella lycopersici W. Kruger(anamorph Gloeosporium lycopersici) (Kruger 1913) isa synonym of C. salicis that belongs to the C.acutatum species complex (Damm et al. 2012a), andis not closely related to C. lycopersici.

There are two species described from Lensculinaris: C. savulescui Sandu-Ville from Romania(Sandu-Ville 1959) and Glomerella truncata fromCanada (Armstrong-Cho and Banniza 2006). Glomer-ella truncata is more closely related to C. destructivum(Gossen et al. 2009, O’Connell et al. 2012). Conidiaof C. savulescui are hyaline, cylindrical with both sidesrounded, straight or slightly curved, measuring 7.5–183 3–4.5 mm. The shape of the conidia is often slightlycurved which indicates this species might be asynonym of Glomerella truncata, which however hasconidia that are usually larger (18–24 3 4–6 mm)(Armstrong-Cho and Banniza 2006).

DISCUSSION

Colletotrichum isolates from bean (Phaseolus sp.) areusually identified as C. lindemuthianum based onmorphological characteristics (Bigirimana et al. 2000,Ansari et al. 2004, Goncalves-Vidigal et al. 2008).However, morphology does not always reflect phylo-genetic relationships between species in this genus.For example, C. petchii with conidia resembling C.gloeosporioides does not belong to the C. gloeosporioidesspecies complex, but to the C. boninense speciescomplex (Damm et al. 2012b). Our results show thatalthough C. lindemuthianum produces conidia andappressoria with shapes similar to C. gloeosporioides s.

856 MYCOLOGIA

str. (Cannon et al. 2008), it does not belong to the C.gloeosporioides species complex. Our analysis revealsthat C. lindemuthianum is basal to the other speciesincluded (FIG. 1). The epitypification of other speciesin this complex, e.g. C. orbiculare, C. malvarum and C.trifolii are still needed.

Based on this study, C. lindemuthianum seems to berestricted to Phaseolus vulgaris and P. coccineus.Other host plants from which this species has beenreported need to be reconfirmed based on acomparison to the designated ex-epitype strain.

The infraspecific morphological variation observedamong the 17 C. lindemuthianum strains studied maybe due to the different age and repeated subculturingthat can result in degeneration. The strains includedin this study were up to 84 years old. Many of the oldstrains in this study and even a more recentlycollected strain (CBS 569.97, collected in 1997)produced a yellow pigment and did not sporulate,while well-sporulating cultures of C. lindemuthianumare gray olivaceous to olivaceous black.

In the present study, 33 strains previously identifiedas C. coccodes, segregated into two clades in themultigene phylogeny obtained. There were isolatesfrom peppers and tomato in both clades, but allisolates from potato (and potato cyst nematodes)clustered with the ex-neotype strain of C. coccodes.The second clade includes the ex-epitype strain of C.nigrum. Both C. coccodes and C. nigrum can causeanthracnose of peppers and tomato, while potato isonly infected by C. coccodes.

ACKNOWLEDGMENTS

We thank the curators of the CBS and CABI culturecollections as well as Prof. dr Lisa Vaillancourt (Departmentof Plant Pathology, University of Kentucky, USA), Dr PeterJohnston and Dr Bevan Weir (Landcare Research, Auck-land, New Zealand), Prof. dr Annemiek C. Schilder(Department of Plant Pathology, Michigan State University,USA), Dr Alena Kubatova (Culture Collection of Fungi(CCF), Charles University in Prague, Prague, CzechRepublic), Dr Ellis T.M. Meekes (Naktuinbouw, Research& Development, Roelofarendsveen, the Netherlands), DrLizel Mostert (Department of Plant Pathology, University ofStellenbosch, South Africa) and Dr Francoise Munot(Mycotheque de l’Universite catholique de Louvain, Unitede Microbiologie, Louvain-la-Neuve, Belgique) for kindlysupplying isolates for this study. We kindly thank thecurators of the fungaria at the Herbarium Hamburgense,Hamburg, Germany and the US National Fungus Collec-tions, Beltsville, Maryland, USA for providing us withimportant herbarium specimens. This study was financiallysupported by CAS KSCX2-YW-Z-1026 and the NationalNatural Science Foundation of China (NSFC 31070020).This research was also supported by the Dutch Ministry ofAgriculture, Nature and Food Quality through an endow-

ment of the FES programme ‘‘Versterking infrastructuurPlantgezondheid’’.

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