A database to support plant pest diagnostic activities

Methodologies - Phoma and Phoma-like genera

DNA sequences

In the multilocus sequence identification system, 6 loci can be used for the identification of Phoma: Actin (ACT), β-tubulin (TUB2), Calmodulin (CAL), the Internal transcribed Spacer regions 1 & 2 and the 5.8 S nrRNA gene (ITS), Large Subunit (LSU, 28S nrDNA), and Small Subunit (SSU, 18S nrDNA). It should be noted that it is not always necessary to sequence all loci. Whereas LSU and SSU can give a good indication on the family level of an isolate (De Gruyter et al. 2009), ITS and the house-keeping genes show the highest level of intraspecific variation (Aveskamp et al. 2009a, Woudenberg et al. 2009). Moreover, a relative high level of infraspecific variation is recorded for CAL.

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) + LR7 (Vilgalys & Hester, 1990)
  • Partial actin gene (ACT)
    Primer pair ACT-512F + ACT-783R (Carbone & Kohn, 1999)
  • Partial beta-tubulin gene (TUB2)
    Primer pairs BT2Fd (=TUB2Fd) + BT4R (=TUB4Rd) (Woudenberg et al., 2009)
  • Partial calmoduline gene (CAL)
    Primer pair CAL-228F + CAL-737R (Carbone & Kohn, 1999)
  • Partial chitin synthase 1 gene (CHS-1)
    Primer pair CHS-354R + CHS-79F (Carbone & Kohn, 1999)
  • Partial RNA polymerase II largest subunit gene (RPB1)
    Primer pair RPB1-Af (=RPB1-Ac) (Stiller & Hall, 1997, modification by Giho Sung) + RPB1-Cr (Matheny et al., 2002, modification by Giho Sung)
  • Partial translation elongation factor 1-alpha gene (TEF)
    Primer pair TEF1-983F (AFToL) + TEF1-2218R (AFToL)

A general PCR mixture contains 1x PCR buffer, 2 mM MgCl2, 20 μM (ACT, TUB2, CAL) or 40 μM (ITS, LSU, SSU) dNTP's, 0.2 μM of each primer and 0.5 U Taq polymerase. An annealing temperature of 52 °C was used for ACT, TUB2 and CAL PCR, and 48 °C for the ITS, LSU and SSU PCR. More specific PCR conditions are mentioned in the following articles: Aveskamp et al. 2009a (ACT, TUB2, ITS), Aveskamp et al. 2009b (ACT), De Gruyter et al. 2009 (LSU, SSU) and Woudenberg et al. 2009 (ITS, TUB2). For identification purposes the ITS gene will often provide a good level of solution, but occasionally additional sequencing of the ACT or TUB2 gene is necessary to come to a final identification.