Methodologies - Phyllosticta and Guignardia

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)
  LR0R (Rehner & Samuels, 1994) + LR5 (Vilgalys & Hester, 1990) or LSU1Fd (Crous et al., 2009b) + LR5 (Vilgalys & Hester, 1990)
• Partial actin gene (ACT)
  ACT-512F (Carbone & Kohn, 1999) + ACT-783R (Carbone & Kohn, 1999)
• Partial glyceraldehyde-3- phosphate dehydrogenase gene (GAPDH)
  Primer pair Gpd1-LM + Gpd2-LM (Myllys et al., 2002); and for Phyllosticta citricarpa isolates GDF1 (Guerber et al., 2003) + GPDHR2 (Glienke 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 standard 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 mM MgCl2, 20 μM dNTP mix, 5.6% v/v 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 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 7 min at 72 °C. Amplicons were sequenced with both forward and reverse primers to ensure good quality sequences.

For the approximately 1 kb fragment of GAPDH of the Phyllosticta citricarpa isolates, 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), 0.12 μM of each primer, 1x PCR buffer (Colorless GoTaq Flexi buffer), 2.5 mM MgCl2, 40 μM dNTP mix, and 0.12 μl Taq DNA polymerase (Promega GoTaq Flexi, 5 U/μl ). Conditions for amplification of loci consisted of an initial denaturation step of 5 min at 95 °C, followed by 35 cycles of 30 s at 95 °C, 45 s at 50 °C and 90 s at 72 °C, and a final extension step of 7 min at 72 °C.

References

• Carbone I, Kohn LM. (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, Bruns TD. (1993). ITS primers with enhanced specificity for basidiomycetes– application to the identification of mycorrhizae and rusts. Molecular Ecology 2: 113-118.
• Glienke C, Pereira O, Stringari D, Fabris J, Kava-Cordeiro V, et al. (2011). Endophytic and pathogenic Phyllosticta species, with reference to those associated with Citrus Black Spot. Persoonia 26: 47–56.
• Guerber JC, Liu B, Correll JC, Johnston PR. (2003). Characterization of diversity in Colletotrichum acutatum sensu lato by sequence analysis of two gene introns, mtDNA and intron RFLPs, and mating compatibility. Mycologia 95: 872-895.
• Hoog, GS de, Gerrits AHG van den Ende. (1998). Molecular diagnostics of clinical strains of filamentous Basidiomycetes. Mycoses 41: 183- 189.
• Myllys L, Stenroos S, Thell A. (2002). New genes for phylogenetic studies of lichenized fungi: glyceraldehyde-3-phosphate dehydrogenase and beta-tubulin genes. Lichenologist 34: 237–246.
• 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, 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.
• Rehner SA, Samuels GJ. 1994. Taxonomy and phylogeny of Gliocladium analysed from nuclear large subunit ribosomal DNA sequences. Mycological Research 98: 625-634
• Templeton MD, Rikkerink EHA, Solon SL, Crowhurst RN. (1992). Cloning and molecular characterization of the glyceraldehyde-3-phosphate dehydrogenaseencoding gene and cDNA from the plant pathogenic fungus Glomerella cingulata. Gene 122: 225-230.
• 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.