Methodologies - Phytophthora
The database includes sequences for the following DNA regions: the ribosomal DNA internal transcribed spacer 1 and 2 and intervening 5.8S rDNA, (ITS), mitochondrial cytochrome c oxidase subunit I (COI), ß-tubulin (TUB2), and translation elongation factor 1-alpha (TEF). For some species, data on the Ras-related protein (Ypt1) gene, mitochondrial cytochrome c oxidase subunit II (COII) and the nrRNA Intergenic Spacer 1 (IGS1) are provided.
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 beta-tubulin gene (TUB2)
BTubF1 + BTubR1 (Olson et al., 2011)
Partial cytochrome oxidase I gene (COI)
OomCoxI-Levup + OomCoxI-Levlo (Robideau et al., 2011)
Partialcytochrome oxidase II gene (COX2)
FM75 + FM78 (Martin & Tooley, 2003)
nrRNA Intergenic Spacer 1 (IGS1)
LR12R + Seq5S (Woodhall et al., 2007)
Partial Ras-related protein gene (Ypt1)
Ypt1F + Ypt5R (Schena & Cooke, 2006)
Partial translation elongation factor 1-alpha gene (TEF)
EF1AF + EF1AR (Olson et al., 2011)
Protocol for Bioline Taq:The amplification reactions were performed in a GeneAmp PCR system 9700 (Applied Biosystems, Foster City, California) in a total volume of 12.5 μl. The PCR mixture contained:
0.5 μl diluted genomic DNA (25-40 ng)
1.25 μl of 10× NH4 reaction buffer (Bioline)
0.3 μl of MgCl2 (50mM; Bioline)
0.7 μl DMSO (Sigma)
0.25 μl of each primer (10 pM)
0.25 μl of dNTP (1 mM) mix
0.1 μl Taq (5U/μl; BIOTAQ, Bioline)
8.9 μl sterile water
Conditions for amplification of all loci (ITS, TUB2, COI, TEF, Ypt1) consisted of an initial denaturation step of 5 min at 95 °C, followed by 35 cycles of 30 s at 95 °C, 30 s at 52 °C and 60 s at 72 °C, and a final extension step of 7 min at 72 °C. Amplicons were sequenced with both forward and reverse primers to ensure good quality sequences.
Protocol for Fermentas Taq: The amplification reactions were performed in a GeneAmp PCR system 9700 (Applied Biosystems, Foster City, California) in a total volume of 50 μl. The PCR mixture contained 2 μl diluted genomic DNA (25-40 ng), 300 nM of each primer, 25 μl PCR master mix (Fermentas). Conditions for amplification of the ITS loci consisted of an initial denaturation step of 2 min at 94 °C, followed by 35 cycles of 60 s at 94 °C, 30 s at 58 °C and 90 s at 72 °C, and a final extension step of 10 min at 72 °C. Conditions for COX2, IGS1 and Ras-related gene were identical except annealing temperatures of 52 °C, 50 °C and 55 °C were used respectively. Amplicons were sequenced with both forward and reverse primers to ensure good quality sequences.
- Crous PW, Summerell BA, Carnegie AJ, Wingfield MJ, Hunter GC, Burgess TI, Andjic V, Barber PA, Groenewald JZ (2009). Unravelling Mycosphaerella: do you believe in genera? Persoonia 23: 99-118.
- Martin FN, Tooley PW (2003). Phylogenetic relationships among Phytophthora species inferred from sequence analysis of mitochondrially encoded cytochrome oxidase I and II genes. Mycologia 95: 269-284.
- Olson HA, Carbone I, Bensen DM (2011). Phylogenetic history of Phytophthora cryptogea and P. drechleri isolates from floriculture crops in North Carolina greenhouses. Phytopathology 101: 1373-1378.
- Robideau GP, De Cock AW, Coffey MD, Voglmayr H, Brouwer H, Bala K, Chitty DW, Désaulniers N, Eggertson QA, Gachon CM, Hu CH, Küpper FC, Rintoul TL, Sarhan E, Verstappen EC, Zhang Y, Bonants PJ, Ristaino JB, Levesque CA (2011). DNA barcoding of oomycetes with cytochrome c oxidase subunit I and internal transcribed spacer. Molecular Ecology Resources 11: 1002-1011.
- Schena L, Cooke DE (2006). Assessing the potential of regions of the nuclear and mitochondrial genome to develop a "molecular tool box" for the detection and characterization of Phytophthora species. Journal of Microbiological Methods 67: 70-85.
- White TJ, Bruns T, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR Protocols: A Guide to Methods and Applications (eds. M.A. Innis, D.H. Gelfand, J.J. Sninsky and T.J. White. Academic Press, San Diego: 315-322.
- Woodhall JW, Lees AK, Edwards SG, Jenkinson P (2007). Characterization of Rhizoctonia solani from potato in Great Britain. Plant Pathology 56: 286-95.