Abstract
The Colletotrichum gloeosporioides species complex is among the most destructive fungal plant pathogens in the world, however, identification of member species which are of quarantine importance is impacted by a number of factors that negatively affect species identification. Structural information of the rRNA marker may be considered to be a conserved marker which can be used as supplementary information for possible species identification. The difficulty in using ITS rDNA sequences for identification lies in the low level of sequence variation at the intra-specific level and the generation of artificially-induced sequence variation due to errors in polymerization of the ITS array during DNA replication. Type and query ITS sequences were subjected to sequence analyses prior to generation of predicted consensus secondary structures, including the pattern of nucleotide polymorphisms and number of indel haplotypes, GC content, and detection of artificially-induced sequence variation. Data pertaining to structure stability, the presence of conserved motifs in secondary structures and mapping of all sequences onto the consensus C. gloeosporioides sensu stricto secondary structure for ITS1, 5.8S and ITS2 markers was then carried out. Motifs that are evolutionarily conserved among eukaryotes were found for all ITS1, 5.8S and ITS2 sequences. The sequences exhibited conserved features typical of functional rRNAs. Generally, polymorphisms occurred within less conserved regions and were seen as bulges, internal and terminal loops or non-canonical G–U base-pairs within regions of the double stranded helices. Importantly, there were also taxonomic motifs and base changes that were unique to specific taxa and which may be used to support intra-specific identification of members of the C. gloeosporioides sensu lato species complex.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-3-684) contains supplementary material, which is available to authorized users.
Highlights
Colletotrichum gloeosporioides is one of the most ubiquitous fungal plant pathogens in the world (Sutton 1992; Cannon et al, 2008) and has been associated with at least 1,972 different fungus-host combinations in the fungal databases including many tropical fruit crops (See Phoulivong et al 2010 for a review of Colletotrichum species infecting tropical fruits)
Some of the problems encountered with this approach include (i) some isolates still had ambiguous phylogenetic placement based on separate gene tree assessment or when a concatenated data set was used (Weir et al 2012, (ii) other isolates are recalcitrant to amplification (e.g. CAL primers) and/or multiple bands are produced after PCR amplification which requires gel extraction and purification prior to sequencing (Weir et al 2012), and (iii) there is information bias among the different genes used to identify member species of this complex (Rampersad et al 2014)
Four ITS1 ribotypes were proposed based on variations in type sequences: Ribotype 1 – C. gloeosporioides sensu stricto, Ribotype 2: C. asianum, Ribotype 3: C. fructicola, Ribotype 4: C. siamense and C. tropicale (Figure 2)
Summary
Colletotrichum gloeosporioides is one of the most ubiquitous fungal plant pathogens in the world (Sutton 1992; Cannon et al, 2008) and has been associated with at least 1,972 different fungus-host combinations in the fungal databases (http://nt.ars-grin.gov/fungaldatabases/) including many tropical fruit crops (See Phoulivong et al 2010 for a review of Colletotrichum species infecting tropical fruits). Non-molecular traits commonly used to assign intra-specific ranking to these segregate taxa do not demonstrate adequate variability (e.g. using morphological characters), and/or are homoplasious (e.g. morphology and host range criteria). In view of this difficulty, there is a preference among many practitioners to refer to the broad, group-species concept rather than referring to names at the intra-specific level. Multi-locus phylogeny must be used for correct identification of Colletotrichum species (Weir et al 2012). Some of the problems encountered with this approach include (i) some isolates still had ambiguous phylogenetic placement based on separate gene tree assessment or when a concatenated data set was used (Weir et al 2012, (ii) other isolates are recalcitrant to amplification (e.g. CAL primers) and/or multiple bands are produced after PCR amplification which requires gel extraction and purification prior to sequencing (Weir et al 2012), and (iii) there is information bias among the different genes used to identify member species of this complex (Rampersad et al 2014)
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