A Method to Detect and Quantify Eutypa lata and Diplodia seriata-Complex DNA in Grapevine Pruning Wounds.

Trunk diseases are factors that limit sustainability of vineyards worldwide. Botryosphaeria and Eutypa diebacks are caused by several fungi belonging to the Botryosphaeriaceae and Diatrypaceae, respectively, with Diplodia seriata and Eutypa lata being two of the most common species. Previous information indicated that the traditional isolation method used to detect these pathogens from plant samples could underestimate 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 D. seriata-complex (DseCQF/R) and E. lata (ElQF/R) DNA. The design of a species-specific assay was achieved for E. lata. For D. seriata, a species-specific assay could not be designed. The low interspecific diversity across ß-tubulin genes resulted in an assay that could not discriminate D. seriata from some closely related species either not yet reported or presenting a low prevalence on grapevine, such as D. intermedia. We validated our technique on grapevine spur samples naturally and artificially infected with D. seriata and E. 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 E. lata, while low infection frequency (less than 5%) was observed using the reisolation method. For D. seriata-complex, low (5%) to no natural infection frequencies were observed by the qPCR and the reisolation 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-complex 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.

A method to detect and quantify Phaeomoniella chlamydospora and Phaeoacremonium aleophilum DNA in grapevine-wood samples.

Grapevines are sensitive to a wide range of fungal pathogens, including agents such as Phaeomoniella chlamydospora and Phaeoacremonium aleophilum that cause tracheomycosis. In the present study, a procedure for DNA extraction from grapevine woody tissue is first evaluated and shown to be suitable for quantitative analysis. Next, a multiplex real-time PCR method targeting the ß-tubulin gene of the pathogens and the actin gene of plant material is developed and its quantitative capability is verified. This protocol was evaluated in inoculated grapevine-wood samples and in young vines from a nursery and was found to be reliable and highly specific. Results obtained from inoculated cuttings show that the fungal colonization process must be considered regardless of the wood phenotype. An analysis of samples of young vines from the nursery shows that a high rate of contamination occurs at the basis of plants and that this contamination is associated with low quantitative values. This finding provides evidence that in vine nurseries, these fungi may be efficient soil-borne pathogens.