Assessment of sampling and DNA extraction methods for identification of grapevine trunk microorganisms using metabarcoding
For a deeper understanding of grapevine trunk disease (GTD) in New Zealand, a cheap, rapid, sensitive method for identifying within-vine microbial communities is required. Wood tissue from grapevine trunks was collected and three different DNA extraction methods were compared: a cetyltrimethylammonium bromide (CTAB) method, the Geneaid Plant Genomic DNA Mini Kit and the Qiagen DNeasy Plant Mini Kit. DNA samples from the CTAB and Geneaid methods were used for MiSeq DNA metabarcoding targeting the ribosomal internal transcribed spacer 1 (ITS1) region. DNA produced by the CTAB method was of a greater quantity and quality than for the other two methods, although the majority of the DNA samples provided polymerase chain reaction (PCR) amplification of fungal DNA sequences. Fungal metabarcoding profiles from the CTAB and Geneaid samples indicated the presence of fungi normally associated with GTD in New Zealand. The CTAB method was chosen for subsequent work due to its low-cost, simplicity and effective detection of typical GTD fungi. The complete process of sampling through to metabarcoding is now used annually as part of a wider ecological study, screening more than 600 vines at 12 Marlborough vineyards.
Bertsch C, Ramírezâ€Suero M, Magninâ€Robert M, Larignon P, Chong J, Abouâ€Mansour E, Spagnolo A, Clément C, Fontaine F 2013. Grapevine trunk diseases: complex and still poorly understood. Plant Pathology 62: 243-265. <a href=â€œhttps://doi.org/10.1111/j.1365-3059.2012.02674.x">https://doi.org/10.1111/j.1365-3059.2012.02674.x</a>
Cheng FS, Brown SK, Weeden NF 1997. A DNA extraction protocol from various tissues in woody species. HortScience 32: 921-922.
Hill GN, Evans KJ, Beresford RM, Dambergs RG 2014. Comparison of methods for the quantification of botrytis bunch rot in white wine grapes. Australian Journal of Grape and Wine Research 20: 432–441 <a href=â€œhttps://doi.org/10.1111/ajgw.12101">https://doi.org/10.1111/ajgw.12101</a>
HortNZ 2016. Grape and wine production 2011 & 2016. In: Aitken AG, Hewett EW eds. Freshfacts 2016. New Zealand Institute for Plant & Food Research, Auckland, N.Z. Pp. 8.
Jones PE 2001. The development of diagnostic tools for the grapevine pathogen Eutypa lata. Unpublished MSc thesis, Massey University, Palmerston North, New Zealand. 136 p.
Lawrence DP, Travadon R, Nita M, Baumgartner K 2017. TrunkDiseaseID.org: A molecular database for fast and accurate identification of fungi commonly isolated from grapevine wood. Crop Protection 102: 110-117. <a href=â€œhttps://doi.org/10.1016/j.cropro.2017.08.017">https://doi.org/10.1016/j.cropro.2017.08.017</a>
Lodhi MA, Ye GN, Weeden NF, Reisch BI 1994. A simple and efficient method for DNA extraction from grapevine cultivars and Vitis species. Plant Molecular Biology Reporter 12: 6-13. <a href=â€œhttps://doi.org/10.1007/BF02668658">https://doi.org/10.1007/BF02668658</a>
Martin KJ, Rygiewicz PT 2005. Fungal-specific PCR primers developed for analysis of the ITS region of environmental DNA extracts. BMC Microbiology 5: 28-28. <a href=â€œhttps://doi.org/10.1186/1471-2180-5-28">https://doi.org/10.1186/1471-2180-5-28</a>
Morales-Cruz A, Allenbeck G, Figueroa-Balderas R, Ashworth VE, Lawrence DP, Travadon R, Smith RJ, Baumgartner K, Rolshausen PE, Cantu D 2018. Closed-reference metatranscriptomics enables in planta profiling of putative virulence activities in the grapevine trunk disease complex. Molecular Plant Pathology: 19: 490-503. <a href=â€œhttps://doi.org/10.1111/mpp.12544â€>https://doi.org/10.1111/mpp.12544</a>
Mundy DC, Casonato SG, Manning MA 2009a. Incidence of fungi isolated from grape trunks in New Zealand vineyards. Guest D ed. APPS Plant Health Management: An Integrated Approach. Australasian Plant Pathology Society. Pp. 86.
Mundy DC, Casonato SG, Manning MA 2009b. Sampling techniques for isolating trunk disease fungi from a Nelson vineyard. New Zealand Plant Protection 62: 406.
Mundy DC, McLachlan ARG 2016. Visual symptoms of trunk diseases do not predict vine death. New Zealand Plant Protection 69: 17-24.
Murray MG, Thompson WF 1980. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Research 8: 4321-4325. <a href="https://doi.org/10.1093/nar/8.19.4321">https://doi.org/10.1093/nar/8.19.4321</a>
Porebski LS, Bailey G, Baum BR 1997. Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components. Plant Molecular Biology Reporter 15: 8-15. <a href=â€œhttps://doi.org/10.1007/BF02772108">https://doi.org/10.1007/BF02772108</a>
Siebert JB 2001. Eutypa: the economic toll on vineyards. Wines Vines. April: 50-56.
Sosnowski M, Mundy DC 2016. Sustaining vineyards through practical management of grapevine trunk diseases. NZW 13-100. Adelaide, SA, SARDI. p. 73.
Wicks T, Davies K 1999. The effect of Eutypa on grapevine yield. Australian Grapegrower and Winemaker 426: 15-16.