Location: Crops Pathology and Genetics ResearchTitle: A method to detect and quantify Eutypa lata and Diplodia seriata/intermedia complex DNA in grapevine pruning wounds
|Pouzoulet, Jerome - University Of California|
|Rolshausen, Philippe - University Of California|
|Schiavon, Marco - University Of California|
|Bol, Sebastian - University Of California|
|Travadon, Renaud - University Of California|
|Lawrence, Daniel - University Of California|
|Ashworth, Vanessa - University Of California|
|Comont, Gwenaelle - Institut National De La Recherche Agronomique (INRA)|
|Corio-costet, Marie-france - Institut National De La Recherche Agronomique (INRA)|
|Pierron, Romain - University Of Toulouse|
|Besson, Xavier - Lvvd- Loire Vini Viti Distribution|
|Jacques, Alban - University Of Toulouse|
Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/7/2017
Publication Date: 9/7/2017
Citation: Pouzoulet, J., Rolshausen, P.E., Schiavon, M., Bol, S., Travadon, R., Lawrence, D.P., Baumgartner, K., Ashworth, V., Comont, G., Corio-Costet, M., Pierron, R.J., Besson, X., Jacques, A. 2017. A method to detect and quantify Eutypa lata and Diplodia seriata/intermedia complex DNA in grapevine pruning wounds. Plant Disease. 101(8):1470-1480. https://doi.org/10.1094/PDIS-03-17-0362-RE.
Interpretive Summary: Trunk diseases Botryosphaeria dieback and Eutypa dieback limit the sustainability of vineyards worldwide. They are each caused by several fungi belonging to the families Botryosphaeriaceae and Diatrypaceae, respectively, with the species Diplodia seriata and Eutypa lata being two of the most common and widespread species associated with these trunk diseases. We designed two sets of molecular markers for detection of these fungi, using a method known as quantitative real-time PCR (qPCR). We validated our technique on grapevines both naturally and artificially infected with Diplodia seriata and Eutypa lata. Experimental grapevines were located in two Counties of Northern California where the incidence of both pathogens was previously reported. While our assay was specific to E. lata, among other related Diatrypaceae species that cause Eutypa dieback, it could not discriminate Diplodia seriata from Diplodia intermedia, a closely related species in the Botryosphaeriaceae. In comparing our molecular assay to the traditional method of culturing the fungi, we detected more E. lata infections by qPCR (65%) than by culturing (< 5%). For Diplodia seriata, there were low detection rates regardless of the method (5% and 0% by qPCR and culturing, respectively), which is consistent with other studies demonstrating its weak pathogenic capacity. Our assay provides researchers with a more accurate means of evaluating fungicides and other approaches to managing trunk diseases, and will benefit diagnostic labs in the detection of trunk diseases.
Technical Abstract: Trunk diseases are factors that limit sustainability of vineyards worldwide. Bot canker and Eutypa dieback are caused by several fungi belonging to the Botryosphaericeae and Diatrypaceae, respectively with Diplodia seriata and Eutypa lata being two of the most common species. Previous information indicated that traditional isolation method used to detect these pathogens from plant samples could under-estimate their incidence levels. In the present study, we designed two sets of primers that target the ß-tubulin gene and that are amenable for quantitative real-time PCR (qPCR) Sybr-Green assays for the detection and quantification of Diplodia seriata (DseQF/R) and Eutypa lata (ElQF/R) DNA. While the interspecific diversity across ß-tubulin sequences allowed the design of a species-specific assay for E.lata, our assay could not discriminate Diplodia seriata from Diplodia intermedia. We validated our technique on grapevine spur samples naturally and artificially infected with Diplodia seriata and Eutypa lata during the dormant season. Experimental grapevines were located in two Counties of Northern California where the incidence of both pathogens was previously reported. The qPCR assays revealed that a high frequency of pruning wound infections (65%) was achieved naturally by Eutypa lata, while low infection frequency (less than 5%) was observed using the re-isolation method. For Diplodia seriata, low (5%) to no natural infection frequencies were observed by the qPCR and the re-isolation method respectively. These results also provided evidence that our qPCR detection methods were more sensitive to assess the incidence of E. lata and D. seriata in plant samples, than traditional isolation techniques. Benefits of molecular methods for the detection of canker pathogens in the field under natural conditions are discussed.