A Survey of Xylella fastidiosa in the U.S. State of Virginia Reveals Wide Distribution of Both Subspecies fastidiosa and multiplex in Grapevine.

Global travel and trade in combination with climate change are expanding the geographic distribution of plant pathogens. The bacterium Xylella fastidiosa is a prime example. Native to the Americas, it has spread to Europe, Asia, and the Middle East. To assess the risk that pathogen introductions pose to crops in newly invaded areas, it is key to survey their diversity, host range, and disease incidence in relation to climatic conditions where they are already present. We performed a survey of X. fastidiosa in grapevine in Virginia using a combination of quantitative PCR, multilocus sequencing, and metagenomics. We also analyzed samples from deciduous trees with leaf scorch symptoms. X. fastidiosa subspecies fastidiosa was identified in grapevines in all regions of the state, even in Northern Virginia, where the temperature was below -9°C for 10 days per year on average in the years preceding sampling. Unexpectedly, we also found for the first time grapevine samples infected with X. fastidiosa subspecies multiplex (Xfm). The Xfm lineage found in grapevines had been previously isolated from blueberries in the Southeastern United States and was distinct from that found in deciduous trees in Virginia. The obtained results will be important for risk assessment of X. fastidiosa introductions in other parts of the world.

A NitroPure Nitrocellulose Membrane-Based Grapevine Virus Sampling Kit: Development and Deployment to Survey Japanese Vineyards and Nurseries.

We developed a NitroPure Nitrocellulose (NPN) membrane-based method for sampling and storing grapevine sap for grapevine virus detection. We devised an efficient nucleic acid extraction method for the NPN membrane, resulting in 100% amplification success for grapevine leafroll-associated virus 2 (GLRaV2) and 3 (GLRaV3), grapevine rupestris stem pitting-associated virus (GRSPaV), grapevine virus A, grapevine virus B, and grapevine red blotch virus (GRBV). This method also allowed the storage of recoverable nucleic acid for 18 months at room temperature. We created a sampling kit to survey GLRaV2, GLRaV3, and GRBV in Japanese vineyards. We tested the kits in the field in 2018 and then conducted mail-in surveys in 2020-2021. The results showed a substantial prevalence of GLRaV3, with 48.5% of 132 sampled vines being positive. On the other hand, only 3% of samples tested positive for GLRaV2 and none for GRBV.

Investigating plant disease outbreaks with long-read metagenomics: sensitive detection and highly resolved phylogenetic reconstruction applied to Xylella fastidiosa.

Early disease detection is a prerequisite for enacting effective interventions for disease control. Strains of the bacterial plant pathogen Xylella fastidiosa have recurrently spread to new crops in new countries causing devastating outbreaks. So far, investigation of outbreak strains and highly resolved phylogenetic reconstruction have required whole-genome sequencing of pure bacterial cultures, which are challenging to obtain due to the fastidious nature of X. fastidiosa. Here, we show that culture-independent metagenomic sequencing, using the Oxford Nanopore Technologies MinION long-read sequencer, can sensitively and specifically detect the causative agent of Pierce's disease of grapevine, X. fastidiosa subspecies fastidiosa. Using a DNA sample from a grapevine in Virginia, USA, it was possible to obtain a metagenome-assembled genome (MAG) of sufficient quality for phylogenetic reconstruction with SNP resolution. The analysis placed the MAG in a clade with isolates from Georgia, USA, suggesting introduction of X. fastidiosa subspecies fastidiosa to Virginia from the south-eastern USA. This proof of concept study, thus, revealed that metagenomic sequencing can replace culture-dependent genome sequencing for reconstructing transmission routes of bacterial plant pathogens.

Origin of Pathogens of Grapevine Crown Gall Disease in Hokkaido in Japan as Characterized by Molecular Epidemiology of Allorhizobium vitis Strains.

Crown gall is a globally distributed and economically important disease of grapevine and other important crop plants. The causal agent of grapevine crown gall is tumorigenic Allorhizobium vitis (Ti) strains that harbor a tumor-inducing plasmid (pTi). The epidemic of grapevine crown gall has not been widely elucidated. In this study, we investigated the genetic diversity of 89 strains of Ti and nonpathogenic A. vitis to clarify their molecular epidemiology. Multi-locus sequence analysis (MLSA) of the partial nucleotide sequences of pyrG, recA, and rpoD was performed for molecular typing of A. vitis strains isolated from grapevines with crown gall symptoms grown in 30 different vineyards, five different countries, mainly in Japan, and seven genomic groups A to F were obtained. The results of MLSA and logistic regression indicated that the population of genetic group A was significantly related to a range of prefectures and that the epidemic of group A strains originated mainly in Hokkaido in Japan through soil infection. Moreover, group E strains could have been transported by infected nursery stocks. In conclusion, this study indicates that both soil infection and transporting of infected nursery stocks are working as infection source in Hokkaido.

Biological control agent Rhizobium (=Agrobacterium) vitis strain ARK-1 suppresses expression of the essential and non-essential vir genes of tumorigenic R. vitis.

OBJECTIVE: To gain insights into the virulence suppressive mechanism of a nonpathogenic strain of Rhizobium vitis ARK-1, we co-inoculated ARK-1 with a tumorigenic (Ti) strain of R. vitis to examine the expression of two essential virulence genes (virA and virG) and one non-essential gene (virD3) of the Ti strain at the wound site of grapevine. RESULTS: Co-inoculation of ARK-1 with a Ti strain VAT03-9 at a 1:1 cell ratio into grapevine shoots resulted in significantly lower expression of the virulence genes virA, virD3, and virG of VAT03-9 at 1 day after inoculation compared with those when shoots were inoculated only with VAT03-9. ARK-1 was not able to catabolize acetosyringone, which is the plant-derived metabolites inducing the entire vir regulon in Ti strains, suggesting the direct effect of ARK-1 on the induction of broad range of vir genes of R. vitis Ti strains.

Evaluation of Quinoxyfen Resistance of Erysiphe necator (Grape Powdery Mildew) in a Single Virginia Vineyard.

The protectant fungicide quinoxyfen has been used against grape powdery mildew (Erysiphe necator) in the United States since 2003. In 2013, isolates of grape powdery mildew with reduced quinoxyfen sensitivity (here designated as quinoxyfen lab resistance or QLR) were detected in a single vineyard in western Virginia, USA. Field trials were conducted in 2014, 2015, and 2016 at the affected vineyard to determine to what extent quinoxyfen might still contribute to disease control. Powdery mildew control by quinoxyfen was similar to, or only slightly less than, that provided by myclobutanil and boscalid in all three years. In 2016, early- versus late-season applications of quinoxyfen were compared to test the hypothesis that early-season applications were more effective, but differences were small. A treatment with two early quinoxyfen applications, at bloom and 2 weeks later, followed by a myclobutanil-boscalid plus a low dose of sulfur rotation provided slightly better control of foliar disease incidence than treatments containing four quinoxyfen applications or two midseason or two late quinoxyfen applications supplemented by myclobutanil and boscalid applications; severity differences were small and nonsignificant. Metrafenone and benzovindiflupyr generally provided excellent powdery mildew control. The frequency of QLR in vines not treated with quinoxyfen slowly declined from 65% in 2014 to 46% in 2016. Further research is needed to explain how, despite this QLR frequency, quinoxyfen applied to grapes in the field was still able to effectively control powdery mildew.

Biological control for grapevine crown gall using nonpathogenic Rhizobium vitis strain ARK-1.

Crown gall of grapevine, which is caused by tumorigenic Rhizobium vitis, is the most important bacterial disease of grapevine throughout the world. Screening tests of biological control agents resulted in the discovery of a nonpathogenic R. vitis strain ARK-1. By soaking grapevine roots with a cell suspension of strain ARK-1 prior to planting in the field, ARK-1 treatment significantly reduced the number of plants with crown gall symptoms. Several field trials result indicated that ARK-1 was very useful in the field, not only for grapevine but also for various other plant species. In experiments where a mixture of ARK-1 and a tumorigenic strain at a 1 : 1 cell ratio was examined in vitro and in planta, expression levels of the virulence genes virD2 and virE2 of the tumorigenic strain were significantly lower. The suppression of virulence genes, which can result in a reduction of gall formation and the pathogen population, seems to be a unique mechanism of ARK-1. These results indicated that ARK-1 is a promising new agent to control grapevine crown gall.

Temporal Effects on the Incidence and Severity of Brown Marmorated Stink Bug (Hemiptera: Pentatomidae) Feeding Injury to Peaches and Apples during the Fruiting Period in Virginia.

Exclusion cages were used to compare the incidence and severity of feeding injury from brown marmorated stink bug, Halyomorpha halys (Stål) (Hemiptera: Pentatomidae), on 'Redhaven' peaches, 'Golden Delicious' apples, and 'Smoothee Golden' apples at harvest, following sequential periods of exposure to natural H. halys populations during the 2011 and 2012 growing seasons in Virginia. The fruit used in these experiments were in orchards or on trees that were not managed for H. halys. Treatments were sets of 50 fruit that were always caged, never caged, or exposed during one interval during the fruiting period of peaches and apples in the Mid-Atlantic region of the United States. The cages effectively prevented feeding injury from H. halys. Peaches and apples that were never caged showed the highest percentages of injured fruit at harvest. Exposure treatment had a significant effect on the percentage of fruit showing external injury at harvest in both years for apples and in 2012 for peaches, and a significant effect on the percentage of apples and peaches showing internal injury at harvest in both years. There was no consistent effect of each exposure period on peach injury, but apples exposed during the mid- to latter portion of the season tended to show most injury. Across all exposure periods, more internal than external injuries were recorded at harvest from peaches, while apples tended to have equal or very similar numbers of both kinds of injury. The implications of these results to H. halys management in eastern apple orchards are discussed.

Geographic distribution of cryptic species of Plasmopara viticola causing downy mildew on wild and cultivated grape in eastern North America.

The putative center of origin of Plasmopara viticola, the causal agent of grape downy mildew, is eastern North America, where it has been described on several members of the family Vitaceae (e.g., Vitis spp., Parthenocissus spp., and Ampelopsis spp.). We have completed the first large-scale sampling of P. viticola isolates across a range of wild and cultivated host species distributed throughout the above region. Sequencing results of four partial genes indicated the presence of a new P. viticola species on Vitis vulpina in Virginia, adding to the four cryptic species of P. viticola recently recorded. The phylogenetic analysis also indicated that the P. viticola species found on Parthenocissus quinquefolia in North America is identical to Plasmopara muralis in Europe. The geographic distribution and host range of five pathogen species was determined through analysis of the internal transcribed spacer polymorphism of 896 isolates of P. viticola. Among three P. viticola species found on cultivated grape, one was restricted to Vitis interspecific hybrids within the northern part of eastern North America. A second species was recovered from V. vinifera and V. labrusca, and was distributed across most of the sampled region. A third species, although less abundant, was distributed across a larger geographical range, including the southern part of eastern North America. P. viticola clade aestivalis predominated (83% of isolates) in vineyards of the European winegrape V. vinifera within the sampled area, indicating that a single pathogen species may represent the primary threat to the European host species within eastern North America.

Characterization of Components of Partial Resistance, Rps2, and Root Resistance to Phytophthora sojae in Soybean.

ABSTRACT Phytophthora root and stem rot of soybeans caused by Phytophthora sojae is a serious limitation to soybean production in the United States. Partial resistance to P. sojae in soybeans is effective against all the races of the pathogen and is a form of incomplete resistance in which the level of colonization of the root is reduced following inoculation. Other forms of incomplete resistance include the single dominant gene Rps2 and Ripley's root resistance, which are both race-specific. To differentiate partial resistance from the other types of incomplete resistance, the components lesion length, numbers of oospores, and infection frequency were measured in eight soybean genotypes inoculated with two P. sojae isolates. The Rps2 and root-resistant genotypes had significantly lower oospore production and infection frequency compared with the partially resistant genotype Conrad, while the root-resistant genotype also had significantly smaller lesion lengths. However, the high levels of partial resistance in Jack were indistinguishable from Rps2 in L76-1988, based on the evaluation of these components. Root resistance in Ripley and Rps2 in L76-1988 had similar responses for all components measured in this study. Partial resistance expressed in Conrad, Williams, Jack, and General was comprised of various components that interact for defense against P. sojae in the roots, and different levels of each component were found in each of the genotypes. However, forms of incomplete resistance expressed via single genes in Ripley and Rps2 in L76-1988, could not be distinguished from high levels of partial resistance based on lesion length, oospore production, and infection frequency.