Methodologies - Mycosphaerella and its anamorphs

DNA sequences

Genomic DNA of the isolates was extracted using commercial DNA isolation kits.

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., 2009b) + LR5 (Vilgalys & Hester, 1990)
• Partial actin gene (ACT)
  ACT-512F (Carbone & Kohn, 1999) + ACT2Rd (Quaedvlieg et al., 2011)
• Partial beta-tubulin gene (TUB)
  T1(O'Donnell & Cigelnik, 1997) + TUB4Rd (Aveskamp et al., 2009) or TUB2Fd (Aveskamp et al., 2009) + TUB4Rd (Aveskamp et al., 2009)
• Partial calmodulin gene (CAL)
  CAL-228F (Carbone & Kohn, 1999) or CAL-235F (Quaedvlieg et al., 2012) + CAL2Rd (Quaedvlieg et al., 2011)
• Partial RNA polymerase II second largest subunit gene (RPB2)
  fRPB2-5F (Liu et al., 1999) + fRPB2-414R (Quaedvlieg et al., 2011)
• Partial translation elongation factor 1-alpha gene (TEF)
  Primer pair EF1-728F (Carbone & Kohn, 1999) + EF-2 (O'Donnell et al., 1998) or EF1-728F (Carbone & Kohn, 1999) + EF1-986R (Carbone & Kohn, 1999)

The amplification reactions were performed in a 2720 Thermal Cycler (Applied Biosystems, Foster City, California) in a total volume of 25 μl. The PCR mixture contained 2 μl diluted genomic DNA (25-40 ng), 0.1 μM of each primer, 1x PCR buffer (Colorless GoTaq Flexi buffer), 2.0 μl MgCl2 (25 mM stock), 0.15 μl dNTP mix (10 mM stock), 0.7 μl DMSO (optional depending whether other Taq is used) and 0.12 μl Taq DNA polymerase (Promega GoTaq Flexi, 5 U/μl ). Conditions for amplification of both loci consisted of an initial denaturation step of 5 min at 94 °C, followed by 40 cycles of 45 s at 94 °C, 30 s at 52 °C and 90 s at 72 °C, and a final extension step of 6 min at 72 °C. Amplicons were sequenced with both forward and reverse primers to ensure good quality sequences.

References

• 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. and Kohn, L.M. (1999). A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia 91: 553-556.
• Crous PW, Summerell BA, Carnegie AJ, Wingfield MJ, Hunter GC, Burgess TI, Andjic V, Barber PA, Groenewald JZ (2009b). Unravelling Mycosphaerella: do you believe in genera?, Persoonia, 23: 99-118.
• Gardes, M. and Bruns, T.D. (1993). ITS primers with enhanced specificity for basidiomycetes– application to the identification of mycorrhizae and rusts. Molecular Ecology 2: 113-118.
• Hoog, G.S. de, Gerrits, van den Ende, A.H.G. (1998). Molecular diagnostics of clinical strains of filamentous Basidiomycetes. Mycoses 41: 183- 189.
• Liu Y, Whelen S, Hall B (1999). Phylogenetic relationships among ascomycetes: evidence from an RNA polymerse II subunit, Mol Biol Evol, 16: 1799-1808.
• O'Donnell K, Cigelnik E (1997). Two divergent intragenomic rDNA ITS2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous, Molecular Phylogenetics and Evolution, 7: 103-116.
• O'Donnell K, Kistler HC, Cigelnik E, Ploetz RC (1998) Multiple evolutionary origins of the fungus causing Panama disease of banana: concordant evidence from nuclear and mitochondrial gene genealogies. Proceedings of the National Academy of Sciences of the USA (PNAS) 98: 2044-2049.
• Quaedvlieg W, Groenewald JZ, de Jesus Yanez-Morales M, Crous PW (2012). DNA Barcoding of Mycosphaerella species of quarantine importance to Europe. Persoonia 29: 101-115.
• Quaedvlieg W, Kema GHJ, Groenewald JZ, Verkley GJM, Seifbarghi S, Razavi M, Gohari AM, Mehrabi R, Crous PW (2011). Zymoseptoria gen. nov.: a new genus to accommodate Septoria-like species occurring on graminicolous hosts, Persoonia - Molecular Phylogeny and Evolution of Fungi, 26: 57-69.
• 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.