Molecular characterization and inter- and intra-species phylogenetic relationships in the fungal plant pathogen Colletotrichum

Colletotrichum is a large morphologically diverse fungal ascomycete genus which causes economically important "anthracnose" diseases on cereals, legumes, vegetables, fruits, perennial crops, ornamentals, and native plants worldwide. The species concept within Colletotrichum is poorly defined as traditional criteria, namely morphology, have not adequately delineated biological or genetically distinct subgroups. Furthermore, few studies have adequately examined the phylogenetic relationships within the genus in any statistically robust manner. The goal of this research was to examine inter- and intra species phylogenetic relationships using sequence data from two introns of two unlinked nuclear genes [glutatmine synthetase (GS) and glyceraldehyde phosphate dehydrogenase (GPDH)]. The sequence analysis of the GS and GPDH introns indicated that they were exceptionably statistically robust for making phylogenetic inferences, had a high level of resolution for characterizing inter as well as intraspecific diversity, and could readily be used to develop a phylogenetic species concept for Colletotrichum . In addition, the two introns were genealogically concordant further supporting their utility for making phylogenetic inferences.;RFLP analysis of a 1-kb GS intron could effectively qualitatively differentiate each of the Colletotrichum species examined; sequence analysis of both the 1-kb GS and the 200-bp GPDH introns were congruent and clearly delineated the various species as well as showed the phylogenetic relationships among species. The RFLP and sequence analysis was equally effective in characterizing subgroups within several species "complexes", namely, C. orbiculare and C. acutatum. The C. orbiculare complex consisted of C. orbiculare, C. trifolii, C. lindemuthianum and C. malvarum. The intron sequence data indicated that all of these taxa represent a monophyletic group; however, fixed differences within each of the introns could be used to consistently delineate the various taxa. Furthermore, distinct differences in other phenotypic characteristics, namely host specificity, also could be used to discriminate these taxa and would support keeping them as distinct species.;The C. acutatum complex also was examined and the intron sequence data indicated that C. acutatum was composed of a number of phylogenetically distinct species. Interestingly, two phylogenetically distinct subgroups, namely C1 and J4, included isolates that were capable of mating and producing the sexual stage Glomerella acutata under laboratory conditions. Genetic recombination also was evident in these matings. Thus genetic isolation may have preceded reproductive isolation.;Overall, the molecular analysis demonstrated that the 1-kb GS and the 200-bp GPDH intron sequence data was genealogically concordant and was a powerful approach to examine molecular evolution and phylogenetic relationships in Colletotrichum. In addition, RFLP analysis of the 1-kb GS intron may provide an easy, rapid, and inexpensive method to objectively delineate species within this genus.