Methodologies - Colletotrichum
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:
- 5.8S nrRNA gene with the two flanking internal transcribed spacers (ITS) Primer pairs ITS1F or ITS-5 or V9G + ITS-4 or LR5 For a detailed protocol for amplification of the ITS region, please see the Molecular Decision Scheme. The ITS1F primer is fungal-specific and can be used for selective amplification of pure fungal material present on host material.
- Partial 18S nrRNA gene (SSU) NS1 (White et al., 1990) + NS4 (White et al., 1990)
- Partial 28S nrRNA gene (LSU) LR0R (Rehner & Samuels, 1994) + LR5 (Vilgalys & Hester, 1990)
- Partial actin gene (ACT) Primer pair ACT-512F + ACT-783R (Carbone & Kohn, 1999)
- Partial Apn2, the intergenic region between Apn2 and Mat1, and partial Mat1 gene (Mat1/APN2) Primer pairs Apn1W1F + Apn1W1R and Mat1M72F + Mat1M72R (Crouch et al., 2009)
- Partial apurinic DNA lyase gene (APN2) Primer pair Apn1W1F + Apn1W1R (Crouch et al., 2009)
- Partial beta-tubulin gene (TUB2) Primer pairs BT2Fd (=TUB2Fd) + BT4R (=TUB4Rd) (Woudenberg et al., 2009) or T1 + T2 (O'Donnell and Cigelnik, 1997) or T1 + Bt-2b (Glass & Donaldson 1995)
- Partial calmoduline gene (CAL) Primer pair CAL-228F + CAL-737R (Carbone & Kohn, 1999) or CL1 + CL2A (O’Donnell et al. 2000) or CL1C + CL2C (Weir et al., 2012)
- Partial chitin synthase 1 gene (CHS-1) Primer pair CHS-354R + CHS- 79F (Carbone & Kohn, 1999)
- Partial glutamine synthetase gene (GS) Primer pair GSF1+ GSR1 (Stephenson et al. 1997) or GSF3 + GSR2 (Weir et al., 2012)
- Partial glyceraldehyde-3- phosphate dehydrogenase gene (GAPDH) Primer pair GDF1 + GDR1 (Guerber et al., 2003) or GDF + GDR (Templeton et al., 1992)
- Partial histone H3 gene (HIS3) Primer pair CYLH3F + CYLH3R (Crous et al., 2004)
- Partial manganese superoxide dismutase gene (SOD2) Primer pair SODglo2-F + SODglo2-R (Moriwaki and Tsukiboshi, 2009) or SOD625F + SOD625R or SOD507F + SOD507R (White et al., 1990, Crouch et al., 2006)
- Partial mating type locus gene (MAT1-2) Primer pair Mat1M72F + Mat1M72R (Crouch et al., 2009)
- Partial RNA polymerase II second largest subunit gene (RPB2) Primer pair fRPB2-5F + fRPB2-7R (Liu et al., 1999)
The amplification reactions of isolates belonging to several species complexes (Damm et al., 2009, 2012a,b, Cannon et al., 2012) were performed in a 2720 Thermal Cycler (Applied Biosystems, Foster City, California) in a total volume of 12.5 μl. The PCR mixture contained 1 μl 20x diluted genomic DNA, 0.2 μM of each primer, 1x PCR buffer (Bioline, Luckenwalde, Germany), 2 mM MgCl2, 20 μM of each dNTP, 0.7 μl DMSO and 0.25 U Taq DNA polymerase (Bioline). Conditions for amplification of all loci except ITS consisted of an initial denaturation step of 5 min at 94 °C, followed by 40 cycles of 30 s at 94 °C, 30 s at 52 °C and 30 s at 72 °C, and a final denaturation step of 7 min at 72 °C. Conditions for PCR amplification of ITS consisted of an initial denaturation step of 5 min at 94 °C, followed by 40 cycles of 30 s at 94 °C, 30 s at 48 °C and 60 s at 72 °C and a final extension step of 7 min at 72 °C.
The PCRs of strains belonging to the C. gloeosporioides species complex (Weir et al. 2012) were performed in an Applied Biosystems Veriti Thermal Cycler in a total volume of 25 μL. The PCR mixtures contained 15.8 μL of UV-sterilised ultra-filtered water, 2.5 μL of 10× PCR buffer (with 20 mM MgCl2), 2.5 μL of dNTPs (each 20 μM), 1 μL of each primer (10 μM), 1 μL of BSA, 1 μL of genomic DNA, and 0.2 μL (1 U) of Roche FastStart Taq DNA Polymerase. The PCR conditions for ITS were 4 min at 95 °C, then 35 cycles of 95 °C for 30 s, 52 °C for 30 s, 72 °C for 45 s, and then 7 min at 72 °C. The annealing temperatures differed for the other genes, with the optimum for each; ACT: 58 °C, CAL: 59 °C, CHS-1: 58 °C, GAPDH: 60 °C, GS: 54 °C, SOD2: 54 °C, TUB2: 55 °C.
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- Crouch JA, Clarke BB, Hillman BI. 2006. Unraveling evolutionary relationships among the divergent lineages of Colletotrichum causing anthracnose disease in turfgrass and maize. Phytopathology 96: 46-60.
- Crouch JA, Tredway LP, Clarke BB, Hillman BI. 2009. Phylogenetic and population genetic divergence correspond with habitat for the pathogen Colletotrichum cereale and allied taxa across diverse grass communities. Molecular Ecology 18: 123-135.
- Crous PW, Groenewald JZ, Risede JM, Hywel-Jones NL. 2004. Calonectria species and their Cylindrocladium anamorphs: species with sphaeropedunculate vesicles. Studies in Mycology 50: 415-430.
- Crous PW, Verkley GJM, Groenewald JZ, Samson RA. 2009. Fungal Biodiversity. CBS Laboratory Manual Series 1. Centraalbureau voor Schimmelcultures, Utrecht, Netherlands.
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- Glass NL, Donaldson G. 1995. Development of primer sets designed for use with PCR to amplify conserved genes from filamentous ascomycetes. Applied and Environmental Microbiology 61: 1323-1330.
- 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.
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- O'Donnell K, Cigelnik E. 1997. Two divergent intragenomic rDNA ITS2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous. Molecular Phylogenetics and Evolution 7: 103-116.
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- Weir B, Johnston PR, Damm U. 2012. The Colletotrichum gloeosporioides species complex. Studies in Mycology 73: 115-180.
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- Woudenberg JHC, Aveskamp MM, Gruyter J de, Spiers AG, Crous PW. 2009. Multiple Didymella teleomorphs are linked to the Phoma clematidina morphotype. Persoonia 22: 56-62.