Poster Presentation Science Protecting Plant Health 2017

Development of a multiplex PCR for the detection of Eutypa lata and Cryptovalsa ampelina associated with Eutypa dieback of grapevines (#209)

Regina Billones-Baaijens 1 , Meifang Liu 1 , Matthew Ayres 2 , Mark Sosnowski 2 3 , Sandra Savocchia 1
  1. National Wine and Grape Industry Centre, Charles Sturt University, Wagga Wagga, NSW, Australia
  2. South Australian Research and Development Institute, Adelaide, South Australia, Australia
  3. School of Agriculture, Food and Wine, The University of Adelaide, Glen Osmond, SA, Australia

Eutypa dieback (ED) caused by the fungus Eutypa lata is one of the important trunk diseases affecting grapevines in Australian vineyards. The pathogen primarily infects pruning wounds and slowly colonises the vascular tissues resulting in cankers, dieback and eventually death of vines. Grapevines affected by ED also display foliar symptoms including chlorosis, necrosis, cupping of leaves and shortening of internodes which is due to phytotoxins produced by E. lata. Recent studies, however, showed that in addition to E. lata, other fungi belonging to the Diatrypaceae family are also associated with ED including Cryptovalsa ampelina, another prevalent species found in Australian vineyards. The objective of this study was to develop a multiplex PCR for rapid detection of E. lata and C. ampelina in a single sample. Two sets of primers specific for E. lata (BtEuF1 and BtEuR1) and C. ampelina (CampBtF1 and CampBtr1) were designed to amplify 327 bp and 215 bp fragments of the β-tubulin gene, respectively. The two sets of primers were optimised using single and mixed DNA of E. lata and C. ampelina in a single reaction. The assay was able to amplify two distinct PCR products specific to E. lata and C. ampelina in a mixed DNA sample demonstrating it is suitable for multiplex-PCR. Specificity tests also showed the two primer pairs can amplify all E. lata and C. ampelina DNA while no PCR products were observed with the DNA from seven other Diatrypaceae species and 20 other non-target fungal species associated with grapevines. The multiplex-PCR developed in this study is suitable for rapid detection of two ED pathogens in a single sample. This assay can be used for rapid diagnosis of ED from spore trap tapes collected in different grapevine growing regions in Australia and from grapevine tissue samples.