Oral Presentation Science Protecting Plant Health 2017

Development of quantitative PCR assays to detect and quantify inoculum of grapevine trunk disease pathogens from environmental samples (4010)

Regina Baaijens 1 , Jose Ramon Urbez-Torres 2 , Matthew Ayres 3 , Mark Sosnowski 3 4 , Sandra Savocchia 1
  1. National Wine and Grape Industry Centre, Charles Sturt University, Wagga Wagga, NSW, Australia
  2. Summerland Research and Development Centre, Agriculture and Agri-Food Canada, 4200 Highway 97, Box 5000, Summerland, BC, V0H 1Z0, British Columbia, Canada
  3. South Australian Research and Development Institute, Adelaide, South Australia, Australia
  4. School of Agriculture, Food and Wine, The University of Adelaide, Glen Osmond, SA, Australia

Grapevine trunk diseases (GTDs) are considered a serious threat to viticulture worldwide. Eutypa dieback (ED) and Botryosphaeria dieback (BD) caused by several Diatrypaceae and Botryosphaeriaceae species are the two most important trunk diseases affecting Australian vineyards to date. The ascospores and/or conidia of these pathogens are primarily spread by rain and wind and infect susceptible pruning wounds leading to cankers, dieback and eventually death of vines. The objective of this study was to develop molecular tools to detect and quantify Diatrypaceae and Botryopshaeriaceae inoculum as part of the investigations on spore dispersal patterns of ED and BD pathogens in Australian vineyards. Different DNA extraction protocols were evaluated using artificially-inoculated tapes and Burkard spore trap tapes collected from vineyards and one protocol was found efficient and gave consistent DNA yield. Two sets of multi-species primers for quantitative PCR (qPCR) were further developed to detect and quantify Diatrypaceae and Botryosphaeriaceae spores from a single environmental sample. The multi-species primers for Diatrypaceae and Botryosphaeriaceae species were designed to amplify fragments of the ITS region of the rRNA and β-tubulin genes, respectively. Specificity tests showed the two multi-species primers were highly specific to their corresponding target Diatrypaceae and Botryosphaeriaceae species (nine each) and did not amplify any of the 20 non-target species tested. The two qPCR methods were shown to amplify genomic DNA, synthetic DNA fragments (gBlocks®) and mixed DNA from environmental samples. The limit of detection of the qPCR for the Diatrypaceae and Botryosphaeriaceae species were ~20 fg and ~300 fg of genomic DNA, respectively. In summary, the two qPCR assays were developed for rapid and sensitive detection of ED and BD pathogens from environmental samples. These assays are currently being used to analyse spore trap tape samples collected from different grapevine growing regions in Australia.