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

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Weir et al.grass-inhabiting species of the C. graminicola group and thedevelopment of a more useful taxonomy for this group of fungi(e.g. Hsiang & Goodwin 2001, Du et al. 2005, and Crouch etal. 2006). This group is now recognised as comprising severalhost-specialised, genetically well characterised species, but amodern taxonomy for C. gloeosporioides has yet to be resolved.Von Arx (1970) and Sutton (1980) distinguished the C.gloeosporioides group using conidial shape and size. A few apparentlyhost-specialised, C. gloeosporioides-like taxa were retained by theseauthors, but the basis of their identification was often difficult tounderstand. Prior to the availability of DNA sequence data, taxonomicconcepts within Colletotrichum were based on features such as hostspecies, substrate, conidial size and shape, shape of appressoria,growth rate in culture, colour of cultures, presence or absence ofsetae, whether or not the teleomorph develops, etc. Some studieshave found characters such as these useful for distinguishinggroups within C. gloeosporioides (e.g. Higgins 1926, Gorter 1956,Hindorf 1973, and Johnston & Jones 1997). However, problemsarise because many of these morphological features change underdifferent conditions of growth (dependent upon growth media,temperature, light regime, etc.), or can be lost or change with repeatedsubculturing. Host preference is poorly controlled — even good, welldefinedpathogens causing a specific disease can be isolated bychance from other substrates (e.g. Johnston 2000). Colletotrichumconidia will germinate on most surfaces, form an appressorium,remain attached to that surface as a viable propagule or perhaps asa minor, endophytic or latent infection, and grow out from there intosenescing plant tissue or onto agar plates if given the opportunity.In addition, the same disease can be caused by genetically distinctsets of isolates, the shared pathogenicity presumably independentlyevolved, e.g. the bitter rot disease of apple is caused by membersof both the C. acutatum and C. gloeosporioides species complexes(Johnston et al. 2005).Sutton (1992) commented on C. gloeosporioides that “Noprogress in the systematics and identification of isolates belongingto this complex is likely to be made based on morphology alone”.A start was made towards a modern understanding of this namewith the designation of an epitype specimen with a culture derivedfrom it to stabilise the application of the name (Cannon et al. 2008).Based on ITS sequences, the ex-epitype isolate belongs in astrongly supported clade, distinct from other taxa that have beenconfused with C. gloeosporioides in the past, such as C. acutatumand C. boninense (e.g. Abang et al. 2002, Martinez-Culebras etal. 2003, Johnston et al. 2005, Chung et al. 2006, Farr et al. 2006,Than et al. 2008). However, biological and genetic relationshipswithin the broad C. gloeosporioides clade remain confused and ITSsequences alone are insufficient to resolve them.In this study we define the limits of the C. gloeosporioidesspecies complex on the basis of ITS sequences, the species weaccept within the complex forming a strongly supported cladein the ITS gene tree (fig. 1 in Cannon et al. 2012, this issue). Inall cases the taxa we include in the C. gloeosporioides complexwould fit within the traditional morphological concept of the C.gloeosporioides group (e.g. von Arx 1970, Mordue 1971, andSutton 1980). Commonly used species names within the C.gloeosporioides complex include C. fragariae, C. musae, and C.kahawae. Since the epitype paper (Cannon et al. 2008), severalnew C. gloeosporioides-like species have been described inregional studies, where multi-gene analyses have shown the newspecies to be phylogenetically distinct from the ex-epitype strain ofC. gloeosporioides (e.g. Rojas et al. 2010, Phoulivong et al. 2011,and Wikee et al. 2011).The regional nature of most of these studies, the often restrictedgenetic sampling across the diversity of C. gloeosporioides globally,and the minimal overlap between isolates treated and gene regionstargeted in the various studies, means that the relationship betweenthe newly described species is often poorly understood.While some authors have embraced a genetically highlyrestricted concept for C. gloeosporioides, many applied researcherscontinue to use the name in a broad, group-species concept (e.g.Bogo et al. 2012, Deng et al. 2012, Kenny et al. 2012, Parvin etal. 2012, and Zhang et al. 2012). In this paper we accept bothconcepts as useful and valid. When used in a broad sense, werefer to the taxon as the C. gloeosporioides species complex or C.gloeosporioides s. lat.This paper aims to clarify the genetic and taxonomicrelationships within the C. gloeosporioides species complex usinga set of isolates that widely samples its genetic, biological andgeographic diversity. Type specimens, or cultures derived fromtype specimens, have been examined wherever possible. Althoughwe do not treat all of the names placed in synonymy with C.gloeosporioides or Glomerella cingulata by von Arx & Müller (1954)and von Arx (1957, 1970), we treat all names for which a possibleclose relationship with C. gloeosporioides has been suggested inthe recent literature, along with all subspecific taxa and formaespeciales within C. gloeosporioides and G. cingulata.ITS sequences, the official barcoding gene for fungi (Seifert2009, Schoch et al. 2012), do not reliably resolve relationshipswithin the C. gloeosporioides complex. We define species in thecomplex genetically rather than morphologically, on the basis ofphylogenetic analyses of up to eight genes. Following Cannonet al. (2012, this issue) the generic name Colletotrichum is usedas the preferred generic name for all species wherever possiblethroughout this paper, whether or not a Glomerella state has beenobserved for that fungus, and whether or not the Glomerella statehas a formal name.MATERIALS AND METHODSSpecimen isolation and selectionAn attempt was made to sample the genetic diversity across C.gloeosporioides as widely as possible, with isolates from diversehosts from around the world selected for more intensive study. ABLAST search of GenBank using the ITS sequence of the epitypeculture of C. gloeosporioides (Cannon et al. 2008) provided acoarse estimate for the genetic limit of the C. gloeosporioidescomplex and ITS diversity across the complex was used toselect a genetically diverse set of isolates. Voucher cultures wereobtained from the research groups who deposited the GenBankrecords. To these were added isolates representing the knowngenetic and morphological diversity of C. gloeosporioides fromNew Zealand, isolated from rots of native and introduced fruits,from diseased exotic weeds, and as endophytes from leaves ofnative podocarps. Additional isolates representing ex-type andauthentic cultures of as many named taxa and formae specialeswithin the C. gloeosporioides complex as possible were obtainedfrom international culture collections. Approximately 400 isolatesbelonging to the C. gloeosporioides complex were obtained.GAPDH gene sequences were generated for all isolates as an initialmeasure of genetic diversity. A subset of 156 isolates, selected torepresent the range of genetic, geographic, and host plant diversity,116
The Colletotrichum gloeosporioides species complexwas used in this research (Table 1).Most of the New Zealand isolates had been stored as conidialsuspensions made from single conidium or ascospore cultures andthen stored at -80 °C in a 5 % glycerol/water suspension. Additionalisolates from New Zealand were obtained from the ICMP culturecollection, where isolates are stored as lyophilised (freeze-dried)ampoules or in a metabolically inactive state in liquid nitrogenat -196 °C. The storage history of most of the isolates receivedfrom other research groups is not known. Table 1 lists the isolatesstudied. All those supplying cultures are acknowledged at theend of this manuscript, and additional details on each culture areavailable on the ICMP website (http://www.landcareresearch.co.nz/resources/collections/icmp).Culture collection and fungal herbarium (fungarium)abbreviations used herein are: CBS = Centraalbureau voorSchimmelcultures (Netherlands), ICMP = International Collectionof Microorganisms from Plants, MFLU = Mae Fah Luang UniversityHerbarium (Thailand) MFLUCC = Mae Fah Luang UniversityCulture Collection (Thailand), GCREC = University of Florida,Gulf Coast Research and Education Centre (USA), HKUCC = TheUniversity of Hong Kong Culture Collection (China), IMI = CABIGenetic Resource Collection (UK), MAFF = Ministry of Agriculture,Forestry and Fisheries (Japan), DAR = Plant Pathology Herbarium(Australia), NBRC = Biological Resource Center, National Instituteof Technology and Evaluation (Japan), BCC = BIOTEC CultureCollection (Thailand), GZAAS = Guizhou Academy of AgriculturalSciences herbarium (China), MUCL = Belgian Co-ordinatedCollections of Micro-organisms, (agro)industrial fungi & yeasts(Belgium), BRIP = Queensland Plant Pathology Herbarium(Australia), PDD = New Zealand Fungal and Plant DiseaseCollection (New Zealand), BPI = U.S. National Fungus Collections(USA), STE-U = Culture collection of the Department of PlantPathology, University of Stellenbosch (South Africa), and MCA =M. Catherine Aime’s collection series, Louisiana State University(USA).DNA extraction, amplification, and sequencingMycelium was collected from isolates grown on PDA agar, andmanually comminuted with a micropestle in 420 μL of QuiagenDXT tissue digest buffer; 4.2 μL of proteinase K was added andincubated at 55 °C for 1 h. After a brief centrifugation 220 μL ofthe supernatant was placed in a Corbett X-tractorGene automatednucleic acid extraction robot. The resulting 100 μL of pure DNA inTE buffer was stored at -30 °C in 1.5 mL tubes until use.Gene sequences were obtained from eight nuclear gene regions,actin (ACT) [316 bp], calmodulin (CAL) [756 bp], chitin synthase(CHS-1) [229 bp], glyceraldehyde-3-phosphate dehydrogenase(GAPDH) [308 bp], the ribosomal internal transcribed spacer(ITS) [615 bp], glutamine synthetase (GS) [907 bp], manganesesuperoxidedismutase (SOD2) [376 bp], and β-tubulin 2 (TUB2)[716 bp].PCR Primers used during this study are shown in Table 2.The standard CAL primers (O’Donnell et al. 2000) gave poor ornon-specific amplification for most isolates, thus new primers(CL1C, CL2C) were designed for Colletotrichum based on the C.graminicola M1.001 genome sequence. The standard GS primers(Stephenson et al. 1997) sequenced poorly for some isolates dueto an approx. 9 bp homopolymer T run 71 bp in from the end of theGSF1 primer binding site. A new primer, GSF3, was designed 41 bpdownstream of this region to eliminate the homopolymer slippageerror from sequencing. The reverse primer GSR2 was designed inthe same location as GSR1 with one nucleotide change. Both newGS primers were based on similarity with a C. theobromicola UQ62sequence (GenBank L78067, as C. gloeosporioides).The PCRs were performed in an Applied Biosystems VeritiThermal Cycler in a total volume of 25 μL. The PCR mixturescontained 15.8 μL of UV-sterilised ultra-filtered water, 2.5 μL of 10×PCR buffer (with 20 mM MgCl 2), 2.5 μL of dNTPs (each 20 μM), 1μL of each primer (10 μM), 1 μL of BSA, 1 μL of genomic DNA, and0.2 μL (1 U) of Roche FastStart Taq DNA Polymerase.The PCR conditions for ITS were 4 min at 95 °C, then 35cycles of 95 °C for 30 s, 52 °C for 30 s, 72 °C for 45 s, and then7 min at 72 °C. The annealing temperatures differed for the othergenes, with the optimum for each; ACT: 58 °C, CAL: 59 °C, CHS-1: 58 °C, GAPDH: 60 °C, GS: 54 °C, SOD2: 54 °C, TUB2: 55°C. Some isolates required altered temperatures and occasionallygave multiple bands, which were excised separately from anelectrophoresis gel and purified. PCR Products were purified on aQiagen MinElute 96 UF PCR Purification Plate.DNA sequences were obtained in both directions on an AppliedBiosystems 3130xl Avant Genetic analyzer using BigDye v. 3.1chemistry, electropherograms were analysed and assembled inSequencher v. 4.10.1 (Gene Codes Corp.).Phylogenetic analysesMultiple sequence alignments of each gene were made withClustalX v. 2.1 (Larkin et al. 2007), and manually adjusted wherenecessary with Geneious Pro v. 5.5.6 (Drummond et al. 2011).Bayesian inference (BI) was used to reconstruct most ofthe phylogenies using MrBayes v. 3.2.1 (Ronquist et al. 2012).Bayesian inference has significant advantages over other methodsof analysis such as maximum likelihood and maximum parsimony(Archibald et al. 2003) and provides measures of clade support asposterior probabilities rather than random resampling bootstraps.jModelTest v. 0.1.1 (Posada 2008) was used to carry out statisticalselection of best-fit models of nucleotide substitution using thecorrected Akaike information criteria (AICc) (Table 3). Initialanalyses showed that individual genes were broadly congruent,thus nucleotide alignments of all genes were concatenated usingGeneious, and separate partitions created for each gene withtheir own model of nucleotide substitution. Analyses on the fulldata set were run twice for 5 x 10 7 generations, and twice for 2 x10 7 generations for the clade trees. Samples were taken from theposterior every 1000 generations. Convergence of all parameterswas checked using the internal diagnostics of the standarddeviation of split frequencies and performance scale reductionfactors (PSRF), and then externally with Tracer v. 1.5 (Rambaut& Drummond 2007). On this basis the first 25 % of generationswere discarded as burn-in.An initial BI analysis treated all 158 isolates using a concatenatedalignment for five of the genes, ACT, CAL, CHS-1, GAPDH, and ITS.Colletotrichum boninense and C. hippeastri were used as outgroups.A second BI analysis, restricted to ex-type or authentic isolatesof each of the accepted species, was based on a concatenatedalignment of all eight genes. A third set of BI analyses treatedfocussed on taxa within the Musae clade and the Kahawae clade.For each clade, the ex-type or authentic isolates, together with 2–3additional selected isolates of each accepted taxon where available,were analysed using a concatenated alignment of all eight genes,with C. gloeosporioides used as the outgroup for both analyses.www.studiesinmycology.org117
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Cannon et al.In rare instances, Col
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Cannon et al.Table 1. (Continued).P
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Cannon et al.as did those for Colle
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Cannon et al.110.74 C. cuscutae IMI
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CBS Biodiversity SeriesNo. 11:Taxon
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Studies in Mycology (ISSN 0166-0616
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