Health Status of Ready-to-Plant Grapevine Nursery Material in Canada Regarding Crown Gall and Description of the First Allorhizobium vitis Strain OP-G1 Isolated from British Columbia.

Crown gall disease of grapevines caused by Allorhizobium vitis causes significant damage to vineyards in cold-climate viticulture areas such as Canada and the northern United States. Introduction of the disease into vineyards occurs mainly through planting of infected but asymptomatic nursery material. Because A. vitis is not a regulated pest for import into Canada, no information on the health status of nursery material destined for import into Canada has previously been collected. This study evaluated the health status of ready-to-plant nursery material from domestic and international nurseries in regard to crown gall by determining the abundance of A. vitis in different plant sections via Droplet Digital PCR technology. In addition, different rootstocks from one nursery were compared. Results showed that A. vitis was present in planting material from all nurseries tested. The bacteria were nonuniformly distributed in dormant nursery material, and there was no difference in abundance between the rootstocks tested. In addition, the first A. vitis strain OP-G1 isolated from galls in British Columbia is described. Results showed that a minimum of 5,000 bacterial OP-G1 cells were needed for symptom expression, suggesting that the initiation of symptom development is not based on presence of bacteria in nursery material alone; a minimum threshold is needed, and environmental conditions need to be met.

Development of droplet digital PCR assays to quantify genes involved in nitrification and denitrification, comparison with quantitative real-time PCR and validation of assays in vineyard soil.

Quantifying genes in soil is important to relate the abundance of soil bacteria to biogeochemical cycles. Quantitative real-time PCR is widely used for quantification, but its use with environmental samples is limited by poor reaction efficiencies or by PCR inhibition through co-purified soil substances. Droplet digital PCR (ddPCR) is a technology for absolute, sensitive quantification of genes. This study optimized eight ddPCR assays to quantify total bacteria and archaea as well as the nitrification (bacterial and archaeal amoA) and denitrification (nirS, nirK, nosZI, nosZII) genes involved in the generation or reduction of the greenhouse gas nitrous oxide. Detection and quantification thresholds were compared with those of quantitative real-time PCR and were equal to, or improved, in ddPCR. To validate the assays using environmental samples, soil DNA was isolated from two vineyards in the Okanagan valley in British Columbia, Canada, over the 2017 growing season. Soil properties related to the observed gene abundances were determined. Total bacteria, nirK, and nosZII increased with time and the soil C/N ratio and NH4+-N concentration affected total archaea and archaeal amoA negatively. The results, compared with those of other studies, showed that ddPCR is a valid alternative to qPCR to quantify genes involved in nitrification or denitrification.

Quantification of Agrobacterium vitis from Grapevine Nursery Stock and Vineyard Soil using Droplet Digital PCR.

Current detection methodologies for Agrobacterium vitis, causing crown gall of grapevines, are time intensive and lack the ability to quantify pathogen abundance in nursery stock and soil. Information on pathogen abundance is a key component to develop management strategies. The aim of this study was to develop a rapid and sensitive quantification assay for grapevine nursery stock and vineyard soil via droplet digital polymerase chain reaction targeting the virA gene. DNA isolated from roots of dormant grapevines originating from nurseries in Germany, California, and Ontario were tested for virA abundance. Bacterial numbers varied with grapevine origin; plants from California had the highest numbers. In addition, rhizosphere soil from two vineyards in the Okanagan valley in British Columbia was tested over a growing season. Sampling time during the season did not affect virA gene abundance. The older vineyard had higher soil A. vitis populations than the younger vineyard. The assay developed here has potential for use in national clean plant programs to prevent import of infected grapevine nursery stock and to test vineyard soil for abundance of the pathogen before planting.

Identification of a response regulator involved in surface attachment, cell-cell aggregation, exopolysaccharide production and virulence in the plant pathogen Xylella fastidiosa.

Xylella fastidiosa, the causal agent of Pierce's disease of grapevine, possesses several two-component signal transduction systems that allow the bacterium to sense and respond to changes in its environment. Signals are perceived by sensor kinases that autophosphorylate and transfer the phosphate to response regulators (RRs), which direct an output response, usually by acting as transcriptional regulators. In the X. fastidiosa genome, 19 RRs were found. A site-directed knockout mutant in one unusual RR, designated XhpT, composed of a receiver domain and a histidine phosphotransferase output domain, was constructed. The resulting mutant strain was analysed for changes in phenotypic traits related to biofilm formation and gene expression using microarray analysis. We found that the xhpT mutant was altered in surface attachment, cell-cell aggregation, exopolysaccharide (EPS) production and virulence in grapevine. In addition, this mutant had an altered transcriptional profile when compared with wild-type X. fastidiosa in genes for several biofilm-related traits, such as EPS production and haemagglutinin adhesins.

Localization and characterization of Xylella fastidiosa haemagglutinin adhesins.

Xylella fastidiosa is a gram-negative, xylem-inhabiting, plant-pathogenic bacterium responsible for several important diseases including Pierce's disease (PD) of grapevines. The bacteria form biofilms in grapevine xylem that contribute to the occlusion of the xylem vessels. X. fastidiosa haemagglutinin (HA) proteins are large afimbrial adhesins that have been shown to be crucial for biofilm formation. Little is known about the mechanism of X. fastidiosa HA-mediated cell-cell aggregation or the localization of the adhesins on the cell. We generated anti-HA antibodies and show that X. fastidiosa HAs are present in the outer membrane and secreted both as soluble proteins and in membrane vesicles. Furthermore, the HA pre-proteins are processed from the predicted molecular mass of 360 kDa to a mature 220 kDa protein. Based on this information, we are evaluating a novel form of potential resistance against PD by generating HA-expressing transgenic grapevines.