A database to support plant pest diagnostic activities

Methodologies - Colletotrichum

DNA sequences

Genomic DNA of isolates belonging to several species complexes (Damm et al., 2009, 2012a,b, Cannon et al., 2012) was extracted using the method of Damm et al. (2008): Genomic DNA of all isolates was isolated from fungal mycelium grown on PDA plates, placed in a 1.5 mL tube with glass beads and 600 μl hexadecyltrimethyl ammonium bromide (CTAB) extraction buffer (0.2 M Tris, 1.4 M NaCl, 20 mM EDTA, 0.2 g/l CTAB) and crushed 3 min at 30 vibrations per second in a Retsch Mixer Mill MM301 (Retsch, Haan, Germany). Before adding 400 μl chloroform : isoamylalcohol (24 : 1), the tube was placed in a 65 °C water bath for 15 min. The fungal matrix was spun down for 5 min at 15 800 g. The watery supernatant was transferred into a new centrifuge tube and cold ammonium acetate solution (final concentration 2.5 M) and 600 μl cold isopropanol were added. After 15 min incubation at room temperature, the precipitate was spun down for 5 min at 15 800 g and the supernatant discarded. One ml cold 70 % ethanol was added to the pellet, spun down for 5 min at 15 800 g and the supernatant discarded. The DNA pellet was dried and resuspended in 100 μl ddH2O.

Genomic DNA of the isolates belonging to the C. gloeosporioides species complex (Weir et al. 2012) was extracted as follows: Mycelium was collected from isolates grown on PDA agar, and manually comminuted with a micropestle in 420 μL of Quiagen DXT tissue digest buffer; 4.2 μL of proteinase K was added and incubated at 55 °C for 1 h. After a brief centrifugation 220 μL of the supernatant was placed in a Corbett X-tractorGene automated nucleic acid extraction robot. The resulting 100 μL of pure DNA in TE buffer was stored at -30 °C in 1.5 mL tubes until use.

Primer sequences and publication references for the primers can be obtained from the Primer list web page. The following loci and primers were used: