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Department of Plant Pathology, University of Kentucky, Lexington, Kentucky 40546-0312
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Colletotrichum species are defined primarily on the basis of host preference and morphology of the organism in planta and in culture. However the genus contains several species complexes that encompass such a broad range of morphological and pathological variation that the species name is of relatively little use either to the taxonomist or plant pathologist. Phylogenetic analyses, primarily based on variable regions of the ribosomal DNA (rDNA) sequences, have indicated that these species complexes comprise a variable number of identifiable monophyletic clades. However rDNA sequences often are insufficiently diverse to fully resolve such closely related lineages. A group of isolates representing three species complexes (C. graminicola, C. gloeosporioides and C. acutatum) were analyzed by using the high mobility group (HMG)-encoding sequence of the MAT1–2 mating type sequence, which has been shown in other fungi to be especially suitable for distinguishing relationships among closely related groups. Results were compared with those obtained from analysis of variable regions of the rDNA as well as from standard morphological classification methods. Results achieved through analysis of MAT1–2 sequences correlated well with those obtained by analysis of rDNA sequences but provided significantly better resolution among the various lineages. Morphological traits, including hyphopodia size, colony appearance, spore size, appresorial shape and size and host preference, frequently were unreliable as indicators of phylogenetic association. Spore shape and hyphopodia shape more often were useful for this purpose.
Key words: Anthracnose, Glomerella, HMG, mating type genes
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Von Arx based his revision of the genus primarily on morphological characters in culture, rather than on host relationships. Since his major treatise the number of recognized species gradually has increased as detailed studies of morphology and pathology have been conducted, so that there are currently approximately 40 generally accepted species of Colletotrichum (reviewed by Sutton 1992).
Because the Glomerella teleomorph is either rare or unknown for most Colletotrichum species, the perfect stage is not commonly considered in classification schemes. Classification of species of Colletotrichum traditionally relies on relatively few characteristics of the anamorph, including the shape and size of conidia and appresoria, appearance of the colony in culture, presence or absence of setae and sclerotia and the host from which it is isolated (Sutton 1980, 1992; Cannon et al 2000). These characters are relatively easy to describe, and so they have some use in species identification. However that use is limited by the small number of traits that typically is considered and by the environmental and genetic plasticity of many of these traits in culture. Furthermore the significance of these characters as indicators of evolutionary relationships among species of Colletotrichum generally is unknown (Cannon et al 2000). Sutton (1980, 1992) has recognized several Colletotrichum species complexes that are so broadly defined, and with such a wide range of morphological and pathological variation, that the species name is of limited use to either the taxonomist or plant health practitioner. Among others these include C. gloeosporioides, C. acutatum and C. graminicola.
Direct analyses of DNA sequences will help to resolve relationships among and within species and species complexes of Colletotrichum. One recent investigation used sequences of introns from two genes (glutamine synthase and glyceraldehyde-3-phosphate dehydrogenase) to evaluate a diverse collection of isolates of C. acutatum (Guerber et al 2003). Several studies have used rDNA sequences for delimiting phylogenetic species of Colletotrichum (e.g. Sherriff et al 1994, 1995; Sreenivasaprasad et al 1994, 1996; Johnston and Jones 1997; Moriwaki et al 2002). These analyses all support the idea that Colletotrichum species complexes each contain several distinct lineages. However in many cases rDNA and intron sequences have not been sufficiently variable to statistically resolve these closely related lineages and considerable confusion remains.
In addition to intron sequences and rDNA sequences, another possible aid for phylogenetic analysis at the subgeneric level appears to be the high mobility group (HMG)-box region of the ascomycete MAT1–2 mating-type idiomorph (reviewed by Turgeon 1998). The degree of nucleotide divergence in the MAT1–2 HMG domain was compared to divergence within the ITS regions of the rDNA, and to divergence within an intron of the glyceraldehyde phosphate dehydrogenase (GPDH) gene, in various Cochliobolus spp. and related taxa (Turgeon 1998, Yun et al 1999). The results suggested that MAT genes have a higher degree of divergence than these other sequences. In addition to a high degree of variation among species, it was observed that there was a correspondingly low degree of variation within species. The high between-species variability of MAT1–2 and the low within-species variability gave strong support for branches, allowing differentiation of closely related Cochliobolus spp. whose relationships were not resolved by ITS or GPDH sequences alone (Turgeon 1998, Yun et al 1999).
The objective of the work described here was to evaluate the usefulness of phylogenetic analyses of MAT1–2 sequences, compared to morphological analyses and phylogenetic analyses of rDNA sequences, for classification of three Colletotrichum species complexes: C. gloeosporioides, C. acutatum and C. graminicola. The MAT1–2 sequences produced results that were equivalent to or superior to those obtained by analysis of variable regions of the rDNA genes in differentiating among genetically distinct lineages of Colletotrichum within these three species complexes. Morphological traits that are used commonly to define these species complexes, including spore size, appresorial shape and size, colony appearance and host preference, frequently were unreliable in determining phylogenetic association. Spore shape and hyphopodia shape more often were useful for this purpose. The information presented in this paper will provide new tools for researchers as they continue their efforts to clarify relationships among species and species complexes within this important genus.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The isolates originally were assigned to species by the various donors, primarily on the basis of morphological and pathological traits. All isolates were purified genetically in our laboratory by single-sporing and stored as a permanent collection desiccated on silica gel at –80 C. Relevant morphological traits of 56 representative isolates were examined to determine their adherence to published standards for the species and to ascertain the degree of variability within and between the three species complexes. Isolates were grown on potato-dextrose agar (PDA, Difco) at 25 C 7–10 d, except for the C. acutatum isolates, which were grown at 22 C. Photographs were taken of the upper and lower surfaces of the colonies. Conidia were harvested from 14 d old cultures by filtering a conidial suspension through a layer of cheesecloth and resuspending the conidia in water. The lengths and widths of 50 conidia per strain, randomly sampled, were measured at 400x using phase contrast on a Zeiss Axioscop with the measurements module of the Openlab computer program (Improvision, Coventry, England). Appresoria were produced from spores germinated in drops of deionized water on plastic cover slips in a moist chamber overnight (Chaky et al 2000). Hyphopodia were produced from vegetative hyphae grown on blocks of potato-carrot agar (Sutton 1980). Lengths and widths of 50 randomly selected appresoria and up to 50 hyphopodia per strain were measured at 400x with phase contrast on a Zeiss Axioscop with the measurements module of the Openlab computer program. Raw data were analyzed statistically with the Microsoft Excel Spreadsheet and the Duncan’s Multiple Range Test available through the SAS statistical analysis package (SAS Institute Inc. 1997).
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) were used to amplify and sequence the ITS1, 5.8S rDNA and ITS2 regions of the rDNA genes from most of the Colletotrichum strains in this study (TABLE I
). The ITS5 and ITS4 primers were used to amplify DNA from C. gloeosporioides, C. fragariae, C. musae and C. magna. The ITS1 and ITS4 primers were used to amplify DNA from C. graminicola and C. coccodes. Neither of these primer pairs proved to be satisfactory for amplification of DNA from C. acutatum, and so the universal primer ITS4 was combined with a species-specific primer from within the ITS1 sequence of C. acutatum, CaInt2 (Brown et al 1996), to amplify most of the ITS1 region, along with the 5.8S rDNA and ITS2 regions. PCR reactions were incubated on a Perkin Elmer DNA Thermocycler 480 in 50 µL of 10–100 ng of genomic DNA, 1.5 mM MgCl2, 1 x PCR buffer, 0.2 mM each dNTP, 2.5 U of Taq DNA polymerase (Invitrogen, Carlsbad, California) and 0.4 µM of each primer. The amplification cycle consisted of denaturation at 95 C for 5 min followed by 40 cycles consisting of 30 s at 95 C, 30 s at 50 C and 1.5 min at 72 C. PCR products were separated in a 1% agarose gel, stained with ethidium bromide and viewed on a UV transilluminator. PCR products were sequenced, either directly or after band purification by gel electrophoresis, followed by recovery of the band from the gel with a gel extraction system (QIAGEN Inc., Valencia, California), with the BigDye terminator cycle sequencing kit (Applied Biosystems, Foster City, California), and the sequences were analyzed on an ABI 310 automated sequencer (Applied Biosystems).
Amplification and sequencing of the HMG regions of the MAT1–2 mating type genes.— –
Nondegenerate primer pairs were developed to amplify and sequence the HMG box region from isolates of C. graminicola, C. acutatum, C. gloeosporioides and C. fragariae (TABLE II). The primer pairs were formulated on the basis of sequences amplified from at least three representatives of each species complex using the degenerate primers NcHMG1 and NcHMG2, described by Arie et al (1997). PCR products were cloned and sequenced with primers complementary to the cloning vector. Multiple alignments were prepared, and nondegenerate primer pairs were developed for each species (TABLE II). The nondegenerate primer pairs were used to amplify fragments from representative isolates of each species complex (TABLE I). Most of the amplified fragments were sequenced directly with the same primers that were used to amplify them. Amplification of C. acutatum DNA sometimes produced multiple bands, and in these cases the MAT1–2 band was gel-purified before being sequenced directly with the same primers. MAT1–2 sequences from C. musae, C. magna and C. coccodes, each of which was represented by only one isolate in this study, were amplified with NcHMG1 and Nc-HMG2 degenerate primers, cloned and sequenced with primers complementary to the flanking vector sequences. PCR reactions and sequencing were performed with the same conditions and cycling parameters as described above.
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) on the Web (http://prodes.toulouse.inra.fr/multalin/multalin.html). Any nondegenerate primer sequence was removed before alignment. Alignment was verified by eye in MacClade (Maddison 2002), initially by inspecting intronexon junctions and the alignments of coding regions, then by inspecting the intron sequence alignments and adjusting as necessary. Alignments were deposited at TreeBase (No. S1283). Regions where alignment was not obvious due to low complexity were excluded from phylogenetic analysis. Excluded regions included a GC-rich region within rDNA-ITS1, and a region of multiple homopolymer stretches at the 3' end of rDNA-ITS2. The entire sequenced MAT1–2 region was alignable.
Phylogenetic analysis and construction of trees using PAUP.— –
Phylogenetic analyses conducted with PAUP* 4.0b10 (Swofford 1998) included neighbor joining (NJ), maximum parsimony (MP) and maximum likelihood (ML) searches on the aligned sequences. All gave trees with similar topology. For MP analysis, characters were unweighted and character states were unordered. To obtain all exact solutions, branch-and-bound MP searches were conducted on alignments that had sets of identical sequences reduced to one representative each. Parameters for ML analysis were estimated by the protocol of Sullivan et al (1997) as follows. A NJ tree was inferred based on Kimura-2-parameter distances with transition/transversion (ti/tv) ratio assumed to be 2.0, base frequencies assumed to be equal, four classes of mutation rates and the 
-function shape parameter set to 0.5. The search criterion was set to likelihood and these parameters set to estimate: base frequencies, each class of mutation (general time-reversible model), proportion of invariable sites and the -distribution shape parameter. The NJ tree was loaded and described, generating the estimated parameters. All estimated parameters were entered and heuristic likelihood searches were conducted by random taxon addition. After 10 iterations of the search, each with a unique random number seed, the tree with greatest likelihood was reported. Bootstrap analysis employed 1000 replications of MP heuristic searches with random taxon addition, each with a different number seed and no branch swapping.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, 1992). One exception was an isolate of C. gloeosporioides from apple (Pyrus malus), APPY8, which was an uncharacteristic salmon color in culture (FIG. 1). Other exceptions were three C. acutatum isolates from almond (Prunus communis) (ALM-IKS-7Q, ALM-KSH-10 and ALM-NRB-30K) and one isolate of C. acutatum from strawberry (Fragaria chiloensis) (isolate 216). These isolates lacked the pale pink to carmine pigmentation on the colony reverse that is a characteristic of this species in formal descriptions (Sutton 1980, 1992) (FIG. 1). Considerable morphological diversity of colony type was found within each of the species complexes, including in many cases among isolates of the same species from the same host (FIG. 1). Fertile perithecia of the Glomerella cingulata teleomorph were produced in culture by a few of the ex avocado (Persea americana) isolates of C. gloeosporioides but were not observed for any other isolates in our study.
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). Three isolates of C. acutatum collected from almond in Israel (ALM-IKS-7Q, ALM-KSH-10 and ALM-NRB-30K) had spores that were ovoid rather than fusiform and appeared more like the spores of C. gloeosporioides (FIG. 2). Isolates belonging to the C. graminicola complex all produced falcate spores, but isolates from the grass subfamily Poöideae (99396, 99375 and RG1.001) produced more variable spores that ranged in shape from falcate to straight and fusiform (FIG. 2). The two isolates of C. fragariae produced spores that were distinctly tapered toward one end (FIG. 2). The spores of C. magna and C. musae did not differ significantly in shape from the conidia produced by isolates of C. gloeosporioides (FIG. 2).
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) are used to differentiate among species of Colletotrichum. In fact Colletotrichum species produce two very distinct types of appresorial structures, one directly from spores and another type from vegetative hyphae. It is the latter type that is useful taxonomically because those formed directly from spores vary relatively little in shape among species (FIG. 3). We will refer to the type arising from vegetative hyphae as hyphopodia to differentiate them from conidial appresoria. The hyphopodia formed by the isolates in this study matched the descriptions of appresoria provided in the literature (Sutton 1980). Two C. acutatum isolates (ALMIKS7Q and 2.7.15) did not produce hyphopodia under our experimental conditions. It was common among isolates of C. acutatum and C. gloeosporioides to observe chains that apparently had arisen by sequential formation and germination of hyphopodia (FIG. 3). These structures were similar to the "complex appresoria" that Sutton observed occasionally in association with these two species complexes (1980). We never observed these complex hyphopodia among the C. graminicola isolates. Many of the isolates within the C. graminicola species complex formed melanized cells subtending the hyphopodia (FIG. 3). These were not specific to C. graminicola, however, because they also were observed in several isolates of C. gloeosporioides (data not shown).
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). The spores of C. acutatum were on average significantly shorter than spores of C. gloeosporioides. However the size ranges overlapped broadly between the two species complexes (TABLE III). There was significant variation in average spore length among different isolates within the same species complex. The narrowest range was within the C. gloeosporioides species complex (14.7–18.8µ) (TABLE III). Colletotrichum acutatum and C. graminicola had much broader size ranges, with a 1.8-fold difference in each case between the isolates with the largest versus the smallest spores (TABLE III).
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). In agreement with a previous report (Browning et al 1999), isolates from Poöideae had smaller spores than ex sorghum and ex maize isolates. In the C. gloeosporioides species complex, isolates from avocado had the largest spores, whereas isolates from apple had the smallest spores (TABLE IV). In the C. acutatum species complex, isolates from anemone (Anemone coronaria) had the largest spores, and spores from ex apple isolates were the smallest (TABLE IV). In each case, though, size ranges overlapped significantly.
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). There was a broader range of hyphopodia sizes: those of C. graminicola were much larger on average than those produced by the other two species complexes (TABLE III). Hyphopodia of ex maize isolates of C. graminicola were significantly larger than those produced by isolates from other grasses (TABLE IV). This difference in size, together with the distinct difference in shape, meant that it was relatively easy to separate ex maize isolates from other C. graminicola isolates on the basis of their hyphopodia. Hyphopodia were not useful for this purpose in the other two species complexes.
Phylogenetic studies.— –
The sequenced region of the MAT1–2 gene included 67 bp of the 3' end of exon 2, all of intron 2 (51–56 bp), and 94 bp of the 5' end of exon 3. The aligned region of the MAT1–2 gene included 218 characters. Of these, 75 were invariant, 129 were parsimony informative and 14 were variable but parsimony uninformative. A branch-and-bound search for all exact solutions gave five MP trees differing only in the branching orders of clades 3, 4, 5 and the sequence from BG1001. The MP trees were similar to the ML tree (FIG. 4). The MP tree length = 321, consistency index = 0.7059 and retention index = 0.9599. Given the amount of variability in this region, the consistency index appeared high.
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distribution of variable site rates (assuming four categories) = 1.03646. The ML tree inferred with these parameters (FIG. 4) had a -Ln likelihood = 1598.55206.
Because of a long G-C rich stretch in the ITS1 region, only the 65 bp at the 3' end of this region were unambiguously aligned for all isolates. The entire ITS2 region was sequenced, and all except the low-complexity 12–13 bp at the 3' end was aligned for all isolates. The aligned region of the rDNA ITS1–5.8S-ITS2 region included 361 characters, excluding regions of ambiguous alignment. Of these characters, 312 were invariant (including all but three sites in the 159 bp 5.8S rRNA coding sequence), 33 were parsimony informative and 16 were variable but parsimony uninformative. A branch-and-bound search for all exact solutions yielded three MP trees similar to the ML tree (FIG. 5). MP trees (differing only in branches within clade 1) were 78 steps long, with consistency index = 0.7692 and retention index = 0.9538.
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distribution of variable site rates (assuming 4 categories) = 0.598118. The ML tree inferred with these parameters (FIG. 5) had a -Ln likelihood = 906.16382.
The inferred ML tree from MAT1–2 sequences (FIG. 4) and the rDNA-ITS ML tree (FIG. 5) are provided. Major clades that appeared in both trees are numbered 1–5, with subclades of clade 2 designated by decimal numbers. Isolates with closely related rDNA sequences invariably had closely related MAT1–2 sequences. However relationships between major clades differed between the trees. For example, although clade 3 of the ITS tree was not a distinct clade in the MAT1–2 ML tree, the MAT1–2 "clade 3" constituted a group of closely related sequences basal to clade 4. In addition the clade 3 MAT1–2 sequences constituted a distinct clade in both MP and Bayesian analysis. Colletotrichum magna (L25) and C. coccodes (155) did not group together with isolates belonging to C. gloeosporiodes, C. graminicola or C. acutatum in either tree. In contrast, C. musae (927) and C. fragariae (CF-63, CF-75) grouped within the C. gloeosporioides clade (clade 1) in both trees. Colletotrichum fragariae was separated from the other isolates in clade 1 with strong support in the MAT1–2 tree. The most dramatic difference between the ITS and MAT1–2 trees was the position of clade 1, which was well separated from all other clades in the MAT1–2 tree but was close to clades 4 and 5 in the rDNA tree. However the branching order of the major clades in the rDNA tree received no significant bootstrap or Bayesian support.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, O’Donnell et al 2004, Pöggeler 1999, Steenkamp et al 2000, Turgeon 1998). This is in spite of the suggestion that the fungal mating-type genes, as the primary determinants of sexual compatibility, may be particularly well suited for identification of species boundaries. The hypothesis is that MAT gene evolution would be highly constrained by purifying selection because of the need to maintain the function of the MAT proteins, which interact in heteroallelic pairs to activate sexual development (Turgeon 1998, Yun et al 2000). There has been some question of whether the MAT genes would be as useful for defining species boundaries in genera like Colletotrichum, in which many of the species appear to have lost the ability to reproduce sexually. It has been suggested that purifying selective pressure on the genes would be removed in asexual lineages. This prediction was addressed when the mating-type genes of the Fusarium graminearum clades recently were subjected to a rigorous phylogenetic analysis (O’Donnell et al 2004). The conclusion of that study was that the mating-type genes were under purifying selective pressure, even within apparently asexual clades, and that they provided a valuable indication of phylogenetic relatedness in both sexual and asexual lineages. In fact, when intergenic regions also were considered, the MAT genes were more informative than other regions of the genome that also were tested. Our phylogenetic analysis of the alignable rDNA ITS1–5.8S-ITS2 region and of the MAT1–2 HMG region in three Colletotrichum species complexes indicated similar relationships overall among the various isolates. This result suggests that in the recent past there has been little or no recombination between these loci among members of different clades. It is possible that the discordant placement of clade 1 in the two gene trees reflects an ancient recombination event, but the support for the clade 1 position in the rDNA tree was weak. Thus any discordance between the gene trees appears to be due mainly to the difference in resolving power of the two datasets. The MAT1–2 HMG sequences provided a better indication of the interrelationships of major groups within the genus. This is probably due to the fact that the MAT1–2 sequences provided many more alignable variable sites than the rDNA region, allowing for greater resolution and stronger support of most branches than the rDNA. Much of the ITS1 sequence and part of the ITS2 sequence has low complexity and cannot be unambiguously aligned. The extremely high GC content of the low-complexity ITS1 region made sequencing the 5' end of ITS 1 difficult, further reducing the number of useful characters. In contrast the HMG-box region and its intervening sequence in MAT1–2 were relatively easy to amplify, sequence and align, and the coding sequence as well as the intron displayed considerable phylogenetically informative variation. Thus the MAT1–2 locus is more useful that the rDNA ITS sequences for discriminating among closely related lineages within species complexes in the genus Colletotrichum.
Sutton suggested nearly 40 y ago that C. graminicola, as revised by von Arx (1957), is a species complex that actually contains at least five different host-specialized species (Sutton 1965, 1968; reviewed by Sutton 1992). Several more studies have provided further support for this separation. ( Jamil and Nicholson 1987, Huguenin et al 1982, Vaillancourt and Hanau 1992, Randhir and Hanau 1997, Browning et al 1999, Hsiang and Goodwin 2001). On the basis of sequence data and other traits, most authorities now agree that the strains of Colletotrichum that infect sorghum comprise a separate species, referred to as C. sublineolum. However this distinction has not been accepted by all workers, with some continuing to refer in the literature to isolates infecting sorghum as C. graminicola.
In our study the C. graminicola complex exhibited considerable correspondence of phylogenetic and host relationships. Isolates from maize grouped together (clade 4) as did isolates from Sorghum spp. (clade 5, with one exception discussed below). Most isolates from poöid grasses (Agrostis palustris, Dactylis glomerata, Lolium perenne and Poa annua) grouped into another distinct clade (clade 3) and isolate BG1.001, from the panicoid grass Echinochloa crusgalli, was separate from the other three clades in both trees. The one exception was isolate W1.001 from wheat (Triticum aestivum), a poöid grass. This isolate however was from a volunteer plant in an African field of sorghum under high infection pressure and might not be indicative of wheat-adapted strains (El Hilu Omer pers comm). Because wheat is a known host of C. graminicola, it will be of interest in the future to investigate more isolates from this species and to see if they also support the host-based grouping that typifies most of the C. graminicola-complex isolates in our study.
Colletotrichum acutatum is a broadly defined species that has been reported to cause anthracnose diseases on 34 host genera in 22 families (Sutton 1992). Previous phylogenetic studies based on rDNA, and on glutamine synthase and glyceraldehyde-3-phosphate dehydrogenase intron sequences, have indicated that this species complex comprises several distinct clades that probably represent different phylogenetic species (Sreenivasaprasad et al 1996, Johnston and Jones 1997, Guerber et al 2003). Some clades were reported to be fertile in culture, and with a few rare exceptions phylogenetic species corresponded with biological species. All these studies significantly found that host range is not associated with phylogenetic species in C. acutatum. There are other reports that also demonstrate this (Förster and Adaskaveg 1999; Freeman et al 1998, 2000a, b). In contrast morphological features including growth rate, colony color and texture, and conidial shape, often were reported to be reliable predictors of phylogenetic association in the C. acutatum species complex.
Our results agree with these studies. Host preference was not a good predictor of phylogenetic association among our isolates of C. acutatum, whereas there was a link between certain morphological features and phylogenetic relationships. For instance, one morphotype was recognized (the "red" morphotype) that produced a deep red pigment in culture (especially noticeable on the colony reverse) and with conidia formed in concentric rings (FIG. 1). The red morphotype included pathogens of apple (1.4.57, 5.7.52, APPY3, 2.7.3 and ATCC28992), anemone (ANE-4 and ANE-NL12) and strawberry (2.7.15). These isolates formed a group with identical rDNA sequences and nearly identical MAT1–2 sequences (clade 2.2). Isolate 1.4.51 from apple, originally labeled as C. gloeosporioides, probably was misidentified because it also grouped with these C. acutatum isolates and was morphologically similar to them. A second morphotype could be recognized that had gray, felty mycelium, and that lacked any red pigment. This group had ovoid rather than fusiform conidia that were produced over the entire surface of the colony, rather than in concentric rings. Members of this morphotype all were isolated from almond in Israel, and they shared identical MAT1–2 and rDNA haplotypes (clade 2.3). Thus, in contrast to C. graminicola, host range is not a useful criterion for classification of C. acutatum, whereas certain morphological features, including colony appearance and spore shape, are useful. It is possible that significant cross-infection occurs among different host families in C. acutatum.
C. gloeosporioides has the broadest host range of all the Colletotrichum species, and it displays the greatest degree of morphological and biological diversity. This makes it by far the most challenging of the species complexes to resolve. Complicating the matter even more, there are several generally recognized Colletotrichum species whose separation from C. gloeosporioides is questionable. These species were lumped together with C. gloeosporioides by von Arx (1957) based on morphological similarity, but many workers prefer to keep them separate because of distinctions in host preference and cultural characters. For example C. musae is widely recognized as a separate species because its host range appears to be limited to banana (Musa spp.) and C. magna is morphologically similar to C. gloeosporioides but pathogenically distinct with a host range limited to cucurbits. Colletotrichum fragariae and C. gloeosporioides both cause root and crown rot, fruit rot and stolon lesions on strawberry (Gunnell et al 1992, Howard and Albregts 1983), but C. gloeosporioides attacks various hosts (Mordue 1971, Dyko and Mordue 1979), whereas C. fragariae seems to be confined to strawberry. There is also a morphological distinction between C. fragariae and C. gloeosporioides. The shape of conidia of C. fragariae is described as narrowly obovate tapering to the base, while C. gloeosporioides has straight, untapered conidia. Phylogenetic analyses of C. musae and C. fragariae based on variable regions of the rDNA sequences have provided no basis for separating them from C. gloeosporioides (Sreenivasaprasad 1992, 1994; Johnston and Jones 1997). Thus we are left with the unsatisfying choice of separating isolates that appear to belong to the same monophyletic clade, versus lumping isolates together despite distinct and pathologically meaningful differences. Identification of sequences that provide greater discrimination than the variable regions of the rDNA will be especially helpful in resolving this problem.
In our study no obvious relationship among morphology, host range and phylogenetic groupings was revealed among our relatively limited sample of C. gloeosporioides isolates. Some minor morphological differences were noted among isolates from different hosts. For example conidia of ex apple isolates were smaller than those of isolates from other hosts; they were comparable in size to spores of ex apple isolates of C. acutatum. Our phylogenetic analysis based on the ITS1 and ITS2 sequences of the rDNA did not let us distinguish separate lineages among our group of isolates, and C. musae and C. fragariae could not be separated although C. magna was clearly unique. The MAT1–2 sequences provided significantly greater resolution, allowing C. fragariae (but not C. musae) to be statistically separated from isolates of C. gloeosporioides. It was possible to separate the C. gloeosporioides isolates with a high degree of confidence into two groups, one containing a range of isolates from avocado, apple and strawberry collected from Israel and various parts of the United States, and the other containing a geographically more limited set of three isolates from apple (APPR1, APPY8 and NC329) and one from strawberry (2489) from Kentucky and North Carolina. One isolate from avocado from Florida (1.3.62) grouped together with the three other isolates from that host in the rDNA tree but was divergent from them in the MAT1–2 tree. This might be evidence of a recombination event between the MAT1–2 and rDNA loci in the past. This indication of recombination is consistent with the fact that the Glomerella cingulata teleomorph of C. gloeosporioides occurs frequently in nature. In contrast the teleomorphs of C. acutatum (G. acutata) and of C. graminicola (G. graminicola) have been observed only in culture.
Our analysis has demonstrated that the MAT1–2 HMG sequence is a powerful tool for dissection of Colletotrichum phylogenetic relationships. Our primers and methods should be useful in the continuing effort to clarify relationships among species and species complexes in this important genus. Our results confirm and expand the findings of previous studies, demonstrating that Colletotrichum species complexes contain multiple clades, each of which may represent a genetically isolated species. Results achieved through analysis of MAT1–2 sequences correlated well with those obtained by analysis of rDNA sequences but typically provided greater resolution among the various lineages, especially within the particularly recalcitrant C. gloeosporioides species complex. Morphological features and host preference sometimes were unreliable as indicators of phylogenetic association. For example, although host preference appears to correlate well with phylogenetic relationships in the C. graminicola species complex, the association is comparatively poor in the C. acutatum and C. gloeosporioides complexes. Colony morphology appears to be a useful trait for classification in C. acutatum but is far less useful for C. graminicola and C. gloeosporioides. Hyphopodial shape and size and spore shape and size appear to be informative in the C. graminicola complex but not in C. acutatum and C. gloeosporioides. Expanded use of the MAT1–2 and other sequences for phylogenetic analysis of Colletotrichum is essential and will do much to improve our understanding of relationships among species and species complexes within this important genus. This in turn should improve our ability to manage the diseases caused by these fungi.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This is contribution number 05-12-055 of the Kentucky Agricultural Experiment Station, published with the approval of the director. 
2 Current address: Department of Religion, University of Georgia, Athens, Georgia 30602-7271.
3 Corresponding author. E-mail: vaillan{at}uky.edu
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Barve MP, Arie T, Salimath SS, Muehlbauer FJ, Peever TL. 2003. Cloning and characterization of the mating type (MAT) locus from Ascochyta rabiei (teleomorph: Didymella rabiei) and a MAT phylogeny of legume-associated Ascochyta spp. Fungal Genet Biol 39:151–167.[CrossRef][Medline]
Brown AE, Sreenivasaprasad S, Timmer LW. 1996. Molecular characterization of slow-growing orange and key lime anthracnose strains of Colletotrichum from citrus as C. acutatum. Phytopathology 86:523–527.[CrossRef]
Browning M, Rowley LV, Zeng P, Chandlee JM, Jackson N. 1999. Morphological, pathogenic, and genetic comparisons of Colletotrichum graminicola isolates from Poaceae. Plant Dis 83:286–292.[CrossRef]
Cannon PF, Bridge PD, Monte E. 2000. Linking the past, present, and future of Colletotrichum systematics. In: Prusky D, Freeman S, Dickman M, eds. Colletotrichum: host specificity, pathology, and host-pathogen interaction. St Paul, Minnesota: APS Press. p 1–20.
Chaky J, Anderson K, Moss M, Vaillancourt L. 2001. Surface hydrophobicity and surface rigidity induce spore germination in Colletotrichum graminicola. Phytopathology 91:558–564.[Medline]
Corpet F. 1988. Multiple sequence alignment with hierarchical clustering. Nucleic Acid Res 16:10881–10890.
Dyko BJ, Mordue JEM. 1979. Colletotrichum acutatum. CMI descriptions of pathogenic fungi and bacteria. No. 630. Commonwealth mycological institute, Kew, United Kingdom. 2 p.
Förster H, Adaskaveg JE. 1999. Identification of subpopulations of Colletotrichum acutatum and epidemiology of almond anthracnose in California. Phytopathology 89: 1056–1065.[Medline]
Freeman S, Katan T. 1997. Identification of Colletotrichum species responsible for anthracnose and root necrosis of strawberry in Israel. Phytopathology 87:516–521.[CrossRef][Medline]
———, ———, Shabi E. 1998. Characterization of Colletotrichum species responsible for anthracnose diseases of various fruits. Plant Dis 82:596–605.[CrossRef]
———, Minz D, Jurkevitch E, Maymon M, Shabi E. 2000a. Molecular analyses of Colletotrichum species from almond and other fruits. Phytopathology 90:608–614.[Medline]
———, Shabi E, Katan T. 2000b. Characterization of Colletotrichum acutatum causing anthracnose of anemone (Anemone coronaria L.). Appl Env Microbiol 66:5267–5272.
Gardes M, White TJ, Fortin JA, Bruns TD, Taylor JW. 1991. Identification of indigenous and introduced symbiotic fungi in ectomycorrhizae by amplification of nuclear and mitochondrial ribosomal DNA. Can J Bot 69:180–190.
Guerber JC, Liu B, Correll JC, Johnston PR. 2003. Characterization of diversity in Colletotrichum acutatum sensu lato by sequence analysis of two gene introns, mtDNA and intron RFLPs, and mating compatibility. Mycologia 95:872–895.
Gunnell PS, Gubler WD. 1992. Taxonomy and morphology of Colletotrichum species pathogenic to strawberry. Mycologia 84:157–165.[CrossRef]
Howard CM, Albregts EE. 1983. Black leaf spot phase of strawberry anthracnose caused by Colletotrichum gloeosporioides (C. fragariae). Plant Dis 67:1144–1146.[CrossRef]
Hsiang T, Goodwin PH. 2001. Ribosomal DNA sequence comparisons of Colletotrichum graminicola from turf-grasses and other hosts. Eur J Plant Pathol 107:593–599.[CrossRef]
Huguenin B, Lourd M, Geiger JP. 1982. Comparison entre isolates de Colletotrichum graminicola sur la base de leurs caracteristique morphologiques, physiologiques, et pathologiques. Phytopath Z 105:293–304.[CrossRef]
Jamil F, Nicholson RL. 1987. Susceptibility of corn to isolates of Colletotrichum graminicola pathogenic to other grasses. Plant Dis 71:809–810.[CrossRef]
Johnston PR, Jones D. 1997. Relationships among Colletotrichum isolates from fruit-rots assessed using rDNA sequences. Mycologia 89:420–430.[CrossRef]
Lardner R, Johnston PR, Plummer KM, Pearson MN. 1999. Morphological and molecular analysis of Colletotrichum acutatum sensu lato. Mycol Res 103:275–285.[CrossRef]
Lee SB, Taylor JW. 1992. Phylogeny of five fungus-like protoctistan Phytophthora species, inferred from the internal transcribed spacers of ribosomal DNA. Mol Biol Evol 9:636–653.[Abstract]
Maddison DR, Maddison WP. 2000. MacClade 4.0. Sunderland, Massachusetts.
Mordue JEM. 1971. Glomerella cingulata. CMI descriptions of pathogenic fungi and bacteria. No. 315. Commonwealth Mycological Institute, Kew, United Kingdom. 2 p.
Moriwaki J, Tsukiboshi T, Sato T. 2002. Grouping of Colletotrichum species in Japan based on rDNA sequences. J Gen Plant Pathol 68:307–320.[CrossRef]
Pöggeler S. 1999. Phylogenetic relationships between mating-type sequences from homothallic and heterothallic ascomycetes. Curr Genet 36:222–231.[CrossRef][Medline]
Randhir RJ, Hanau RM. 1997. Size and complexity of the nuclear genome of Colletotrichum graminicola. Appl Environ Microbiol 63:4001–4004.[Abstract]
Sherriff C, Whelan MJ, Arnold GM, Lafay J, Brygoo Y, Bailey JA. 1994. Ribosomal DNA sequence analysis reveals new species groupings in the genus Colletotrichum. Experim Mycol 18:121–138.[CrossRef]
———, ———, ———, Bailey JA. 1995. rDNA sequence analysis confirms the distinction between Colletotrichum graminicola and Colletotrichum sublineolum. Mycol Res 99:475–478.
Sreenivasaprasad S, Brown AE, Mills PR. 1992. DNA sequence variation and interrelationships among Colletotrichum species causing strawberry anthracnose. Physiol Molec Plant Pathol 41:265–281.[CrossRef]
———, Mills PR, Brown AE. 1994. Nucleotide sequence of the rDNA spacer 1 enables identification of isolates of Colletotrichum as C. acutatum. Mycol Res 98:186–188.
———, ———, Meehan BM, Brown AE. 1996. Phylogeny and systematics of 18 Colletotrichum species based on ribosomal DNA spacer sequences. Genome 39:499–512.[Medline]
Steenkamp ET, Wingfield BD, Coutinho TA, Zeller KA, Wingfield MJ, Marasas WFO, Leslie JF. 2000. PCR-based identification of MAT-1 and MAT-2 in the Gibberella fujikuroi species complex. Appl Environ Microbiol 66: 4378–4382.
Sullivan J, Markert JA, Kilpatrick CW. 1997. Phylogeography and molecular systematics of the Peromyscus aztecus species group (Rodentia: Muridae) inferred using parsimony and likelihood. Systematic Biol 46:426–440.[CrossRef]
Sutton BC. 1965. Studies on the taxonomy of Colletotrichum Cda with especial reference to C. graminicola. (Ces.) Wilson [Doctoral thesis]. University of London.
———. 1968. The appresoria of Colletotrichum graminicola and C. falcatum. Can J Bot 46:873–876.
———. 1980. The Coelomycetes. Commonwealth Mycological Institute, Kew, Surrey, England. p 522–537.
———. 1992. The genus Glomerella and its anamorph Colletotrichum. In: Bailey JA, Jeger MJ, eds. Colletotrichum: biology, pathology, and control. Wallingford, UK: CAB International. p 1–26.
Swofford DL. 1998. PAUP*. Phylogenetic Analysis Using Parsimony (*and other methods). Sunderland, Massachusetts: Sinauer Associates.
Turgeon BG. 1998. Application of mating type gene technology to problems in fungal biology. Annu Rev Phytopath 36:115–137.[CrossRef][Medline]
Vaillancourt LJ, Hanau RM. 1992. Genetic and morphological comparison of Glomerella (Colletotrichum) isolates from maize and from sorghum. Experim Mycol 16:219–229.
———, Du M, Wang J, Rollins J, Hanau R. 2000a. Genetic analysis of cross fertility between two self-sterile strains of Glomerella graminicola. Mycologia 92:430–435.[CrossRef]
von Arx J. 1957. Die arten der gattung Colletotrichum Cda. Phytopath Z 29:413–468.
White TJ, Bruns T, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR Protocols: a guide to methods and applications. San Diego: Academic Press. p 482.
Wiese MV. 1977. Compendium of wheat diseases. APS Press. p 27–28.
Yun S-H, Berbee ML, Yoder OC, Turgeon BG. 1999. Evolution of the fungal self-fertile reproductive life style from self-sterile ancestors. Proc Nat Acad Sci USA 96: 5592–5597.
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