Methodologies - Verticillium

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.

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 actin gene (ACT)
ACT-512F + ACT-783R (Carbone & Kohn, 1999)

Partial calmodulin gene (CAL)
CAL-228F + CAL-737R (Carbone & Kohn, 1999)

nrRNA Intergenic Spacer 1 (IGS) 
LR12R (Vilgalys lab) & Seq5S (Woodhall et al., 2007)

Partial cytochrome oxidase I gene (COI)
PezizF + PezizR (Nguyen & Seifert, 2008)

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 other loci were identical except annealing temperatures differed as follows: 50 °C (LSU), 48°C (ACT), 50 °C (CAL), 50 °C (COI) and 50 °C (IGS). Amplicons were sequenced with both forward and reverse primers to ensure good quality sequences.

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 (2009). Unravelling Mycosphaerella: do you believe in genera? Persoonia, 23: 99-118.
• Nguyen HDT, Seifert KA (2008). Description and DNA barcoding of three new species of Leohumicola from South Africa and the United States. Persoonia 21, 2008: 57-69.
• Vilgalys R, Hester M (1990). Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. Journal of Bacteriology, 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.
• Woodhall JW, Lees AK, Edwards SG, Jenkinson P (2007). Characterization of Rhizoctonia solani from potato in Great Britain. Plant Pathology 56: 286-95.