Methodologies - Phoma and Phoma-like genera
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.
- Aveskamp MM, Verkley GJM, de Gruyter J, Murace MA, Perello A, Woudenberg JHC, Groenewald JZ, Crous PW. 2009. DNA phylogeny reveals polyphyly of Phoma section Peyronellaea and multiple taxonomic novelties, Mycologia, 101: 363-382.
- Carbone I, Kohn LM. 1999. A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia 91: 553-556.
- De Hoog GS, Gerrits van den Ende AHG. 1998. Molecular diagnostics of clinical strains of filamentous Basidiomycetes. Mycoses 41: 183-189.
- Gardes M, Bruns TD. 1993. ITS primers with enhanced specificity for basidiomycetes – application to the identification of mycorrhizae and rusts. Molecular Ecology 2: 113-118.
- Matheny PB, Liu YJ, Ammirati JF, Hall BD 2002. Using RPB1 sequences to improve phylogenetic inference among mushrooms (Inocybe, Agaricales). American Journal of Botany 89: 688-698.
- Rehner SA, Samuels GJ. 1994. Taxonomy and phylogeny of Gliocladium analysed from nuclear large subunit ribosomal DNA sequences. Mycological Research 98: 625-634.
- Stiller JW, Hall BD. 1997. The origin of red algae: Implications for plastid evolution. Proceedings of the National Academy of Sciences (USA) 94: 4520-4525.
- Vilgalys R, Hester M (1990). Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species, J Bacteriol, 172: 4238-4246.
- 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.
- 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.