A database to support plant pest diagnostic activities

Methodologies - Melampsora

DNA sequences

Genomic DNA of the fungal material was extracted using the Wizard Food kit (Promega) in conjunction with a Kingfisher ML magnetic particle processor (Thermo Electron Corporation) with DNA eluted into 200 µl of TE buffer. Due to the biotrophic nature of Melampsora, the presence of host plant DNA in the sample was unavoidable.

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 28S nrRNA gene (LSU) LSU1Fd (Crous et al., 2009) + LR5 (Vilgalys & Hester, 1990)
  • Partial cytochrome oxidase 1 gene (COI) Cox1MlpAF + Cox1MlpAR (Feau et al., 2009)

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 the LSU and Cox were identical except annealing temperatures of 50°C and 47 °C were used respectively. Amplicons were sequenced with both forward and reverse primers to ensure good quality sequences.