Colletotrichum: complex species or species ... - CBS - KNAW

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Damm et al.stimulated by strawberry plant extracts, especially flower extracts(Leandro et al. 2003). According to Buddie et al. (1999) secondaryconidia may be produced directly from germinating primary conidia,and are smaller and more variable in shape, thus obscuringdifferences between taxa. Additionally, C. acutatum forms simplepigmented appressoria, but few or no setae (Simmonds 1965).Guerber & Correll (1997, 2001) described Glomerella acutata,the sexual morph of C. acutatum, as the product of matingexperiments, while some related species are homothallic, includingGa. acutata var. fioriniae (Marcelino et al. 2008), later regarded asa separate species (C. fioriniae, Shivas & Tan 2009) and an isolateof a Glomerella species related to C. acutata from Acer platanoidesin the USA (LoBuglio & Pfister 2008). Talgø et al. (2007) observedthe sexual morph Ga. acutata on naturally infected fruits ofhighbush blueberry in Norway. Numerous studies have shownthat C. acutatum is morphologically and phylogenetically diverse(Sreenivasaprasad et al. 1994, Johnston & Jones 1997, Lardneret al. 1999, Freeman et al. 2001a, Nirenberg et al. 2002, Talhinhaset al. 2002, Guerber et al. 2003, Lubbe et al. 2004, Du et al. 2005,Peres et al. 2005, Sreenivasaprasad & Talhinhas 2005, Talhinhaset al. 2005, Johnston et al. 2008). Sreenivasaprasad et al. (1996)were the first to recognise that C. acutatum was unusually diverse,with strains showing divergence of 5.8 % in ITS-1 sequencecompared with levels of 2–4 % frequently found within otherfungal species, and they suggested splitting C. acutatum into twospecies. Johnston & Jones (1997) recognised four morphologicalgroups, C. acutatum A–C and Glomerella miyabeana. Three ofthese groups were supported by 28S nuclear ribosomal largesubunit rRNA (LSU) sequence data. Lardner et al. (1999), using acombination of RAPDs and morphological/cultural data, identifiedseven subordinate groups within C. acutatum. Sequences of a200-bp intron of the glyceraldehyde-3-phosphate dehydrogenase(GAPDH) and a 900-bp intron of the glutamine synthetase GSdistinguished seven main clades and several subclades withinstrains that originated mainly from North America and New Zealand(Guerber et al. 2003). The recognition of infraspecific groupswas more firmly established by Sreenivasaprasad & Talhinhas(2005), who distinguished clades A1 to A8 based on rDNA ITS andbeta-tubulin DNA (TUB2) sequences. These clades were mostlycorrelated with the groups that had been distinguished previously.Whitelaw-Weckert et al. (2007) recognised an additional group A9.At this point, it was widely presumed that C. acutatum wasa species complex containing a number of constituent taxa, butthere was substantial reluctance to recognise the clades involvedas independent species. This was due to the lack of differentialmorphological and cultural characters. For example, C. lupini wasnot recognised as formally separate from C. acutatum by Talhinhaset al. (2002) or by Sreenivasaprasad & Talhinhas (2005). Therewere some attempts to address these concerns via adoption offormae speciales e.g. C. acutatum f. sp. pineum (Dingley & Gilmour1972), C. acutatum f. sp. hakeae (Lubbe et al. 2004) and C.acutatum f. sp. fioriniae (Marcelino et al. 2008), but this mechanismfor recognition of pathology-related taxa is now rarely used.Gradually, separate species were recognised or accepted aspart of the C. acutatum species complex, e.g. C. lupini (Nirenberget al. 2002) and C. phormii (Farr et al. 2006). In a study using twogenes, ITS and TUB2, combined with morphological data, Shivas& Tan (2009) recognised three distinct groups within C. acutatumstrains from Australia and accepted two new species, C. simmondsiiand C. fioriniae (formerly C. acutatum f. sp. fioriniae) for groups A2and A3. Recently, a new species was described for group A4, C.clavatum (Faedda et al. 2011).Our research aims to present a comprehensive revision ofthe C. acutatum species complex. We thoroughly survey theconstituent taxa and delineate additional species where needed.We have examined a large number of C. acutatum s. lat. strains,isolated from various hosts and in various geographic areas. Multilocusmolecular analysis is the basis of species recognition, butmorphological and cultural characters allowing alternative meansof species recognition are given where possible.MATERIALS AND METHODSIsolatesA total of 331 strains have been studied, mostly previouslyidentified as C. acutatum, as well as other related strains from theCBS, IMI and other culture collections. Type material (holotypesand epitypes) of the species studied are located in the Herbariumof the Centraalbureau voor Schimmelcultures (CBS), Utrecht,The Netherlands, in the IMI Fungarium, which is based in theRoyal Botanic Gardens, Kew (IMI and K(M)), UK, US NationalFungus Collections (BPI), Beltsville, Maryland, USA, the BotanicGarden and Botanical Museum Berlin-Dahlem, Freie UniversitätBerlin (B), Germany and in the dried collection of the BotanischeStaatssammlung München (M), Germany. All descriptions are basedon the ex-type, ex-epitype or ex-neotype culture as appropriate.Features of other strains are added if deviant. Subcultures of thetypes, epitypes and neotypes, respectively, as well as all otherisolates used for morphological and sequence analyses aremaintained in the culture collections of CBS and IMI (Table 1).Morphological analysisTo enhance sporulation, autoclaved filter paper and doubleautoclavedstems of Anthriscus sylvestris were placed onto thesurface of synthetic nutrient-poor agar medium (SNA; Nirenberg1976). SNA and oatmeal agar (OA; Crous et al. 2009) cultures wereincubated at 20 °C under near UV light with 12 h photoperiod for10 d. Measurements and photographs of characteristic structureswere made according to Damm et al. (2007). Appressoria onhyphae were observed on the reverse side of SNA plates.Microscopic preparations were made in clear lactic acid, with 30measurements per structure and observed with a Nikon SMZ1000dissecting microscope (DM) or with a Nikon Eclipse 80i microscopeusing differential interference contrast (DIC) illumination. In the C.acutatum species complex, conidia are usually formed in acervularconidiomata and additionally in the aerial mycelium. Unlessmentioned otherwise, only conidia from conidiomata were used inthis study for morphological examination.Colony characters and pigment production on SNA andOA cultures incubated at 20 °C under near UV light with 12 hphotoperiod were noted after 10 d. Colony colours were ratedaccording to Rayner (1970). Growth rates were measured after 7and 10 d.Phylogenetic analysisGenomic DNA of the isolates was extracted using the method ofDamm et al. (2008). The 5.8S nuclear ribosomal gene with the twoflanking internal transcribed spacers (ITS), a 200-bp intron of theglyceraldehyde-3-phosphate dehydrogenase (GAPDH), and partial38
The Colletotrichum acutatum species complex1 change1001.00881.000.99CBS 109225 Lupinus UkraineCBS 109216 Lupinus BoliviaCBS 513.97 Lupinus Costa RicaCBS 109226 Lupinus CanadaCBS 509.97 Lupinus FranceCBS 466.76 Manihot RwandaIMI 375715 Lupinus AustraliaCBS 122746 Lupinus USACBS 109217 Lupinus GermanyCBS 485.97 Lupinus Germany1001.000.99 CBS 126371 Lupinus NLCBS 507.97 Lupinus FranceCBS 109219 Lupinus GermanyCBS 109220 Lupinus Germany75 CBS 109221 Lupinus Germany1.00 CBS 109222 Lupinus GermanyCBS 109223 Lupinus GermanyCBS 109224 Lupinus AustriaCBS 109227 Lupinus PolandCBS 126525 Lupinus NLCBS 119142 Lupinus ZACBS 119143 Lupinus ZAIMI 351261 Camellia UKCBS 129944 Cinnamomum PortugalCBS 129814 Solanum ColombiaCBS 129811 Solanum Colombia100 CBS 129812 Solanum ColombiaCBS 129813 Solanum Colombia1.00 CBS 129954 Solanum ColombiaCBS 129955 Solanum ColombiaCBS 129956 Solanum ColombiaCBS 129810 Solanum Colombia0.97 CBS 101611 fern Costa Rica74 CBS 129821 Passiflora Colombia1.00 CBS 129820 Passiflora Colombia94 CBS 330.75 Coffea Costa Rica1.00 CBS 211.78 Coffea Costa RicaIMI 304802 Cuscuta DominicaIMI 384185 Caryocar BrazilCBS 129823 Passiflora ColombiaCBS 114.14 Citrus USACBS 159.84 Cucumis BrazilC. lupiniC. tamarilloiC. costaricenseC. cuscutaeC. limetticolaC. melonisA1A8J6J2J3Clade 1Fig. 1. One of 830 most parsimonious trees obtained from a heuristic search of the combined ITS, GAPDH, CHS-1, ACT, HIS3 and TUB2 sequences alignment of theColletotrichum acutatum species complex. Bootstrap support values above 70 % (bold) and Bayesian posterior probability values above 0.95 are shown at the nodes.Colletotrichum orchidophilum CBS 632.80, CBS 631.80, IMI 309357 and CBS 119291 are used as outgroup. Numbers of ex-holotype, ex-neotype and ex-epitype strains areemphasised in bold. Strain numbers are followed by substrate (host genus) and country of origin, NL = Netherlands, NZ = New Zealand, ZA = South Africa. Branches that arecrossed by diagonal lines are shortened by 50 %. Corresponding groups of Sreenivasaprasad & Talhinhas 2005 are emphasised in red, mtDNA RFLP haplotypes of Guerberet al. (2003) are emphasised in blue.sequences of the chitin synthase 1 (CHS-1), histone3 (HIS3), actin(ACT) and beta-tubulin (TUB2) genes were amplified and sequencedusing the primer pairs ITS-1F (Gardes & Bruns 1993) + ITS-4 (Whiteet al. 1990) or V9G (de Hoog & Gerrits van den Ende 1998) + ITS-4, GDF1 + GDR1 (Guerber et al. 2003), CHS-354R + CHS-79F(Carbone & Kohn 1999), CYLH3F + CYLH3R (Crous et al. 2004b),ACT-512F + ACT-783R (Carbone & Kohn 1999) and BT2Fd + BT4R(Woudenberg et al. 2009) or T1 (O’Donnell & Cigelnik 1997) + Bt-2b(Glass & Donaldson 1995), respectively. The PCRs were performedin a 2720 Thermal Cycler (Applied Biosystems, Foster City,California) in a total volume of 12.5 μL. The GAPDH, CHS-1, HIS3,ACT and TUB2 PCR mixture contained 1 μL 20x diluted genomicDNA, 0.2 μM of each primer, 1x PCR buffer (Bioline, Luckenwalde,Germany), 2 mM MgCl 2, 20 μM of each dNTP, 0.7 μL DMSO and0.25 U Taq DNA polymerase (Bioline). Conditions for PCR of thesegenes constituted an initial denaturation step of 5 min at 94 o C,followed by 40 cycles of 30 s at 94 o C, 30 s at 52 o C and 30 s at72 o C, and a final denaturation step of 7 min at 72 o C, while the ITSPCR was performed as described by Woudenberg et al. (2009). TheDNA sequences generated with forward and reverse primers wereused to obtain consensus sequences using Bionumerics v. 4.60(Applied Maths, St-Marthens-Lathem, Belgium), and the alignmentassembled and manually adjusted using Sequence Alignment Editorv. 2.0a11 (Rambaut 2002).To determine whether the six sequence datasets werecongruent and combinable, tree topologies of 70 % reciprocalNeighbour-Joining bootstrap with Maximum Likelihood distances(10 000 replicates) with substitution models determined separatelyfor each partition using MrModeltest v. 2.3 (Nylander 2004) werecompared visually (Mason-Gamer & Kellogg 1996). A maximumparsimony analysis was performed on the multilocus alignment(ITS, GAPDH, CHS-1, HIS3, ACT, TUB2) as well as for each geneseparately with PAUP (Phylogenetic Analysis Using Parsimony) v.4.0b10 (Swofford 2000) using the heuristic search option with 100random sequence additions and tree bisection and reconstruction(TBR) as the branch-swapping algorithm. Alignment gaps weretreated as missing and all characters were unordered and of equalweight. No more than 10 trees of score (length) greater than or equalto 10 were saved in each replicate. The robustness of the treesobtained was evaluated by 10 000 bootstrap replications using theFast-stepwise addition algorithm (Hillis & Bull 1993). Tree length,consistency index (CI), retention index (RI), rescaled consistencyindex (RC) and homoplasy index (HI) were calculated for theresulting tree. A Markov Chain Monte Carlo (MCMC) algorithm wasused to generate phylogenetic trees with Bayesian probabilitiesusing MrBayes v. 3.1.1 (Ronquist & Huelsenbeck 2003) for thecombined sequence datasets. Models of nucleotide substitutionfor each gene determined by MrModeltest v. 2.3 were included foreach gene partition. The analyses of two MCMC chains were runfrom random trees for 1000 000 generations and sampled every100 generations. The likelihood score of the two runs were 2 500and 2 200 and therefore, the first 2 350 (the average of both) treeswere discarded as the burn-in phase of the analysis and posteriorprobabilities determined from the remaining trees. For additionalcomparison, a Neighbour-Joining analysis was performed on themultigene alignment using PAUP and 1000 bootstrap replications.Sequences derived in this study have been lodged at GenBank, thealignment in TreeBASE (www.treebase.org/treebase-web/home.html), and taxonomic novelties in MycoBank (Crous et al. 2004a).RESULTSPhylogenyThe six sequence data sets did not show any conflicts in treetopology for the 70 % reciprocal bootstrap trees, which allowed uswww.studiesinmycology.org39
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Damm et al.Table 2. Conidia measure
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Damm et al.Table 2. (Continued).Spe
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Damm et al.Table 3. Appressoria mea
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Damm et al.Fig. 25. Colletotrichum
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Damm et al.to olivaceous grey, filt
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Damm et al.are formed directly from
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Damm et al.Arx JA von (1957). Die A
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Damm et al.Masaba DM, Waller JM (19
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114
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Weir et al.grass-inhabiting species
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Weir et al.Table 1. A list of strai
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Weir et al.Table 1. (Continued).Spe
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Weir et al.Table 1. (Continued).Spe
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Weir et al.Table 2. Primers used in
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Weir et al.11ICMP 17789 Malus USA1I
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Weir et al.Kahawae cladeC. alataeC.
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Weir et al.A0.991ICMP 17905 Coffea
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Weir et al.Conidial width variation
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Weir et al.G. c.“f.sp. camelliae
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Weir et al.Fig. 10. Colletotrichum
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Weir et al.Fig. 18. Glomerella cing
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Weir et al.images under C. fructico
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Weir et al.Specimen examined: Thail
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Weir et al.Notes: First described f
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Weir et al.gossypii var. cephalospo
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Weir et al.to utilise either citrat
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Weir et al.(ex-epitype culture CBS
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Weir et al.of this species to C. ps
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Weir et al.Table 4. Performance of
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Weir et al.Crous PW, Gams W, Stalpe
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Weir et al.Simmonds JH (1968). Type
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Cannon et al.182
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Cannon et al.In rare instances, Col
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Cannon et al.Table 1. Summary of pr
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Cannon et al.Table 1. (Continued).P
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Cannon et al.Table 2. Colletotrichu
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Cannon et al.as did those for Colle
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Cannon et al.Table 3. Authentic seq
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Cannon et al.Table 3. (Continued).S
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Cannon et al.110.74 C. cuscutae IMI
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Cannon et al.C. lindemuthianum AB08
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Cannon et al.A further important ac
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Cannon et al.Hawksworth DL (2011).
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Cannon et al.Rodríguez-Guerra R, R
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Studies in MycologyStudies in Mycol
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CBS Biodiversity SeriesNo. 11:Taxon
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Studies in Mycology (ISSN 0166-0616
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