EPPO-Q-bank

A database to support plant pest diagnostic activities

Fungi

Methodology

Methodologies - Phytophthora


DNA sequences

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. 


References