Ribosomal DNA Sequence Analysis Reveals New Species Groupings in the Genus Colletotrichum

Colletotrichum isolates related to Anthracnose of cashew trees in Brazil: morphological and molecular description using LSU rDNA sequences

“…Comparing the aligned nucleotide sequences of the D2 region (180 nucleotides) of the studied isolates, along with data of other previously-identified Colletotrichum species from LARS collection Moses et al, 1996;Sherriff et al, 1994), the maximum number of differences among the aligned bases was 16, i.e. 8.9%.…”

“…Conidial suspensions from CM flask cultures were used to inoculate 100 ml of Czapek Dox-V8 liquid medium in 250 ml flasks (Sherriff et al, 1994). The flasks were incubated at 25 o C on an orbital shaker (126 rpm) for 48 h. To test for yeast or bacterial contamination, LB medium (Gibco BRL) was inoculated with a small aliquot of the culture fluid, just before harvest of the mycelium, and incubated for 48 h (25 o C dark).…”

“…The 10X PCR buffer contained 100 mM Tris-HCl pH 8.3, 500 mM KCl, 15 mM MgCl 2, 0.01% (w/v) and gelatin, and the dNTPs were dATP, dCTP, dGTP and dTTP. Domain 2 within the 28S gene was amplified using the primers Pn 2 (5'-GTT CAC CAT CTT TCG GGT CC-3') and Pn 9 (5'-CTT AAG CAT ATC AAT AAG CGG AGG-3') (Guadet et al, 1989;Sherriff et al, 1994). The reaction mixture per PCR tube consisted of 10 µl of water-diluted DNA sample (0.1 µg/µl), 2 µl of each primer (50 pmol/µl, 2 µl = 1 µM of each), 2 µl of each dNTP (0.2 mM of each dATP, dCTP, dGTP and dTTP), 10 µl of PCR buffer, 0.5 µl of AmpliTaq DNA polymerase (2.5 Units) and 67.5 µl of SDW.…”

“…The developed gel was read manually, and each sequence was aligned using GCG PileUp. Data obtained from the sequencing of the D2 region of all isolates were compared with those of the D2 regions of some Colletotrichum species previously studied Moses et al, 1996;Sherriff et al, 1994), using the neighbour-joining method of Kumar et al (1993), and a dendrogram depicting similarities was constructed using the MEGA (Molecular Evolutionary Genetic Analysis) software (version 1.01).…”

“…Species concepts based on morphological criteria are generally broad for the genus Colletotrichum, and wide variations for cultural and morphological characters, pathogenicity and host range have been reported among isolates of C. gloeosporioides (Alahakoon and Brown, 1994;Gonçalves-Vidigal et al 2008;Jeffries et al, 1990;Skipp et al, 1995;Menezes and Hanlin, 1996a, b;Sera et al 2007). Sequence analyses of the intergenic transcribed spacers (ITS 1, ITS 2) and Domains (D1,D2) of large subunit (LSU) ribosomal DNA (rDNA) have been valuable for delineating isolates and species from this genus (Adaskaveg and Hartin, 1997;Forseille, 2007;Freeman et al, 2001;Johnston, 2000;Johnston and Jones, 1997;Moriwaki et al, 2002;Sherriff et al, 1994;Vinnere et al, 2002). In a study of C. gloeosporioides isolates from Stylosanthes spp.…”