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

The Colletotrichum gloeosporioides species complexC. kahawae, the biosecurity importance of coffee berry disease,and the ability to distinguish the disease-causing isolates usingcarefully selected genetic markers, we recognise the diseasecausingisolates taxonomically at the subspecific level. Future studyof the comparative pathogenicity of isolates within C. aotearoa, C.clidemiae, and C. theobromicola may reveal genetically distinct,host-specialised pathogenic populations within these species thatfuture workers may also choose to recognise taxonomically.The classification we accept here is deliberately taxonomicallyconservative, minimising nomenclatural changes. This reflectscontinuing uncertainty about sensible species limits within theC. gloeosporioides complex that relate to low levels of geneticdivergence across the complex, gene selection, isolate selection,and a lack of understanding of the mechanisms driving speciesand population divergence amongst these fungi. For example,the two haplotype subgroups of C. fructicola are not distinguishedtaxonomically because collectively they form a monophyletic clade,both subgroups include sets of isolates with similar geographic andhost diversity, and there is no practical need to distinguish themtaxonomically.Molecular tools are increasingly being used for day-to-dayidentification by biosecurity officers and plant pathology researchers,providing a need for both a taxonomy that closely reflects groups thatare resolved genetically, as well as simple and reliable protocols foridentifying those taxa. The internal transcribed spacer region (ITS)has been proposed as the official fungal barcoding gene (Schochet al. 2012). Although ITS is useful at the species complex level, itdoes a poor job of resolving species within the C. gloeosporioidescomplex, resolving only 10 of 22 accepted species. This reflectsthe low number of base changes in the ITS region across the C.gloeosporioides complex; species often distinguished by only oneor two base changes. In some cases, chance variation in the ITSsequence within or between species means that some speciescannot be distinguished (Fig. 6). Examples of taxa with identicalITS sequences include C. clidemiae, C. tropicale, C. ti and someC. siamense isolates; C. fructicola and some C. siamense isolates;and C. alienum, C. aenigma and some C. siamense isolates.Protein-coding genes and their introns often have morevariation than ITS, and the need for secondary barcodes basedon these kinds of genes has been discussed in relation to somegroups of fungi (Fitzpatrick et al. 2006, Aguileta et al. 2008, Weir& Johnston 2011). Ideally, one of the seven protein coding genesthat were used in this study could be proposed as a secondarybarcode to obtain an accurate identification of species within theC. gloeosporioides complex. A preliminary analysis of the genesperformance as barcodes was conducted as part of Cai et al.(2009) with GAPDH, CAL, and ACT performing well, but CHS-1, ITS, and TEF (EF1α) poorly. However, the analysis (Cai et al.2009) included only five species within the C. gloeosporioidescomplex, the Musae and Kahawae clades being treated at the levelof species. With the final classification presented here, none ofthe genes we analysed provides an effective barcode ont its ownacross the entire complex. Of the single genes, TUB2, GS, andGAPDH are amongst the most effective at distinguishing species.However, C. clidemiae is polyphyletic in the TUB2 gene tree andGS sequences are needed to distinguish C. fructicola and C.alienum. With GS, C. aotearoa, C. kahawae subsp. ciggaro, and C.siamense are paraphyletic. GAPDH is the easiest of all the genestested to amplify and sequence, however when using this gene GSsequences are needed to distinguish C. fructicola from C. alienumand C. aeschynomenes from C. siamense, and C. tropicale isparaphyletic. In the species descriptions we provide notes on whichgenes are the best for genetic identifications, and in Table 4 theseare summarised for all species and genes. For species representedby a single or only a few isolates the species boundaries may notbe accurate, we recommend two protein-coding genes in additionto ITS for sequence-based identifications. A meta-analysis ofDNA barcodes across the whole genus will be required to find thecombination of genes that are effective for all species of the genusthat distinguish all Colletotrichum species.Several studies have shown that cultural morphology can beuseful for grouping isolates when they are sampled at a local orregional level (e.g. Johnston & Jones 1997, Prihastuti et al. 2009).However, our experience is that such groups often break downwhen the geographic sample within a clade is extended to a globalscale. Many of the species we accept have few or no diagnosticmorphological or cultural features that can be consistently andreliably used to identify them. Our morphological examinationswere confined to cultures on Difco PDA agar plates, and we willhave missed any features that develop solely in association withplant material. In addition, the cultures we used have been sourcedfrom different labs and collections from around the world, manywith no information on storage history. Storage history and methodhas a major impact on the appearance of Colletotrichum in culture.Cultures can become “stale” during storage, losing the ability toproduce pigments, the aerial mycelium often becoming very denseand felted, and losing the ability to form well-differentiated acervuli,conidia, or perithecia. In some clades, even freshly isolated culturesare highly variable, forming distinct sectors with differences in theproduction of pigment, aerial mycelium, acervuli, and conidia.Some isolates form two very different cultural types from singleconidia or ascospores derived from colonies themselves startedfrom single ascospores. Figure 27F shows single ascosporecultures from an isolate of C. kahawae subsp. ciggaro. One hasthe typical appearance of cultures of this fungus isolated from thefield. The other, with a uniform, dense layer of conidia across thecolony surface without well differentiated acervuli and more or lessno aerial mycelium, is common from single ascospore isolates inculture, but rarely found in cultures isolated directly from the field.This kind of variation, and that revealed from sectoring duringcolony growth, makes morphological variation difficult to interpretfor accurate identification.Many of the species recognised in this work remain poorlyunderstood in terms of their pathogenicity and host preference.This in part reflects a lack of certainty about the biologicalrelationship between the fungi and the plants from which theywere isolated. Species that are pathogenic on one host canalso be isolated from others following opportunistic colonisationof senescing tissue, such as the C. salicis example discussedby Johnston (2000, as Glomerella miyabeana). The multipleColletotrichum spp. associated with a single host are likely tohave a variety of life styles: primary pathogens of healthy tissue,species with the ability to invade and cause minor diseasewhen the host plant is under stress, species that develop latentinfections and fruit only following senescence of the host tissueor ripening of host fruit and endophytic species that sporulateonly following host tissue death. The combination of this range ofdistinct life styles, the fact that several Colletotrichum spp. maybecome established on a single host, and the ability of most ofthese species to also establish on a range of other hosts, hasbeen a large part of the confusion surrounding species limitswithin Colletotrichum.In some cases, apparently clear differences in pathogenicityof isolates in the C. gloeosporioides complex are not reflectedwww.studiesinmycology.org175