Transmission of Grapevine Red Blotch Virus by Spissistilus festinus [Say, 1830] (Hemiptera: Membracidae) between Free-Living Vines and Vitis vinifera 'Cabernet Franc'.

Grapevine red blotch disease emerged within the past decade, disrupting North American vine stock production and vineyard profitability. Our understanding of how grapevine red blotch virus (GRBV), the causal agent of the disease, interacts with its Vitis hosts and insect vector, Spissistilus festinus, is limited. Here, we studied the capabilities of S. festinus to transmit GRBV from and to free-living vines, identified as first-generation hybrids of V. californica and V. vinifera 'Sauvignon blanc' (Vcal hybrids), and to and from V. vinifera 'Cabernet franc' (Vvin Cf) vines. The transmission rate of GRBV was high from infected Vcal hybrid vines to healthy Vcal hybrid vines (77%, 10 of 13) and from infected Vvin Cf vines to healthy Vcal hybrid vines (100%, 3 of 3). In contrast, the transmission rate of GRBV was low from infected Vcal hybrid vines to healthy Vvin Cf vines (15%, 2 of 13), and from infected Vvin Cf vines to healthy Vvin Cf vines (19%, 5 of 27). No association was found between transmission rates and GRBV titer in donor vines used in transmission assays, but the virus titer was higher in the recipient leaves of Vcal hybrid vines compared with recipient leaves of Vvin Cf vines. The transmission of GRBV from infected Vcal hybrid vines was also determined to be trans-stadial. Altogether, our findings revealed that free-living vines can be a source for the GRBV inoculum that is transmissible by S. festinus to other free-living vines and a wine grape cultivar, illustrating the interconnected roles of the two virus hosts in riparian areas and commercial vineyards, respectively, for virus spread. These new insights into red blotch disease epidemiology will inform the implementation of disease management strategies.

Identification of grapevine Pinot gris virus in free-living Vitis spp. located in riparian areas adjacent to commercial vineyards.

Grapevine Pinot gris virus (GPGV) is a recently identified pathogen of grapevines in California. To advance our knowledge about the epidemiology of GPGV, we investigated if free-living Vitis spp. can represent a source of virus infection. In 2019 a field survey of GPGV infection was conducted in Napa County. During the inspection 60 free-living vines in riparian habitats near commercial vineyards with GPGV infection were sampled. Samples were tested by real-time reverse transcription PCR (RT-PCR), identifying 23 free-living Vitis spp. positive for GPGV. Later, GPGV infection was confirmed in these plants via end-point RT-PCR and Sanger sequencing. Based on sequence analysis, detected GPGV isolates are more related to the asymptomatic variant of the virus. Vitis species ancestry was determined by DNA fingerprinting. GPGV-infected material included V. californica, V. californica × V. vinifera hybrids and hybrid rootstock cultivars. Here, GPGV is reported for the first time in free-living Vitis spp. The results of this study will support the development of management strategies for GPGV in California and beyond.

Prevalence and Genetic Diversity of Grabloviruses in Free-Living Vitis spp.

The distribution and diversity of grapevine red blotch virus (GRBV) and wild Vitis virus 1 (WVV1) (genus Grablovirus; family Geminiviridae) were determined in free-living Vitis spp. in northern California and New York from 2013 to 2017. Grabloviruses were detected by polymerase chain reaction in 28% (57 of 203) of samples from California but in none of the 163 samples from New York. The incidence of GRBV in free-living vines was significantly higher in samples from California counties with high compared with low grape production (χ2 = 83.09; P < 0.001), and in samples near (<5 km) to compared with far (>5 km) from vineyards (χ2 = 57.58; P < 0.001). These results suggested a directional spread of GRBV inoculum predominantly from vineyards to free-living Vitis spp. WVV1 incidence was also significantly higher in areas with higher grape production acreage (χ2 = 16.02; P < 0.001). However, in contrast to GRBV, no differential distribution of WVV1 incidence was observed with regard to distance from vineyards (χ2 = 0.88; P = 0.3513). Two distinct phylogenetic clades were identified for both GRBV and WVV1 isolates from free-living Vitis spp., although the nucleotide sequence variability of the genomic diversity fragment was higher for WWV1 (94.3 to 99.8% sequence identity within clade 1 isolates and 90.1 to 100% within clade 2 isolates) than GRBV (98.3% between clade 1 isolates and 96.9 to 100% within clade 2 isolates). Additionally, evidence for intraspecific recombination events was found in WVV1 isolates and confirmed in GRBV isolates. The prevalence of grabloviruses in California free-living vines highlights the need for vigilance regarding potential grablovirus inoculum sources in order to protect new vineyard plantings and foundation stock vineyards in California.

Grapevine red blotch-associated virus is Present in Free-Living Vitis spp. Proximal to Cultivated Grapevines.

Red blotch is an emerging disease of grapevine associated with grapevine red blotch-associated virus (GRBaV). The virus spreads with infected planting stocks but no vector of epidemiological significance has been conclusively identified. A vineyard block of red-blotch-affected Vitis vinifera 'Cabernet franc' clone 214 was observed in California, with a clustering of infected, symptomatic vines focused along one edge of the field proximal to a riparian habitat with free-living Vitis spp. No genetic heterogeneity was observed in a 587-nucleotide region of the GRBaV genome in a population of 44 Cabernet franc clone 214 isolates. By contrast, genetic differences were observed in isolates from other cultivars and clones growing in adjacent blocks. GRBaV was confirmed infecting four free-living vines, two of which were shown to be V. californica × V. vinifera hybrids. The genomes of three free-living GRBaV vine isolates and seven from V. vinifera cultivars were compared; free-living vine isolates were shown to be more similar to each other and a 'Merlot' isolate than to the other cultivated vine isolates. The finding that GRBaV is present in free-living Vitis spp. indicates the virus can be spread by natural (nonhuman-mediated) means, and we hypothesize that in-field spread of GRBaV is occurring.

Discovery of non-climacteric and suppressed climacteric bud sport mutations originating from a climacteric Japanese plum cultivar (Prunus salicina Lindl.).

Japanese plums are classified as climacteric; however, some economically important cultivars selected in California produce very little ethylene and require long ripening both "on" and "off" the tree to reach eating-ripe firmness. To unravel the ripening behavior of different Japanese plum cultivars, ripening was examined in the absence (air) or in the presence of ethylene or propylene (an ethylene analog) following a treatment or not with 1-methylcyclopropene (1-MCP, an ethylene action inhibitor). Detailed physiological studies revealed for the first time three distinct ripening types in plum fruit: climacteric, suppressed-climacteric, and non-climacteric. Responding to exogenous ethylene or propylene, the slow-softening supressed-climacteric cultivars produced detectable amounts of ethylene, in contrast to the novel non-climacteric cultivar that produced no ethylene and softened extremely slowly. Genetic analysis using microsatellite markers produced identical DNA profiles for the climacteric cultivars "Santa Rosa" and "July Santa Rosa," the suppressed-climacteric cultivars "Late Santa Rosa," "Casselman," and "Roysum" and the novel non-climacteric "Sweet Miriam," as expected since historic records present most of these cultivars as bud-sport mutations derived initially from "Santa Rosa." This present study provides a novel fruit system to address the molecular basis of ripening and to develop markers that assist breeders in providing high-quality stone fruit cultivars that can remain "on-tree," increasing fruit flavor, saving harvesting costs, and potentially reducing the need for low-temperature storage during postharvest handling.

Hybridization of cultivated Vitis vinifera with wild V. californica and V. girdiana in California.

Hybridization of introduced domesticates and closely related natives is well documented in annual crops. The widespread introduction of the domesticated grapevine, Vitis vinifera, into California where it overlaps with two native congenerics, with which it is interfertile, provides opportunity to investigate hybridization between woody perennials. Although geographically widespread, the introduction over the past two centuries has been limited to a few elite clonal cultivars, providing a unique opportunity to study the effects of hybridization on the native species. The amount of hybridization with V. vinifera and the genetic diversity of wild-growing Vitis californica and Vitis girdiana were examined using nineteen microsatellite markers. STRUCTURE analysis was used to define hybrid and introgressed individuals and to analyze genetic structure of the native species. FAMOZ software was used to identify which V. vinifera cultivars served as parents of F 1 hybrids. The three species were clearly distinguished by STRUCTURE analysis. Thirty percent of 119 V. californica vines were hybrids. The domesticated parent was identified for 16 F 1 hybrid vines; the original California cultivar, 'Mission', was the parent of eight. Backcrosses were also found, showing introgression into subsequent generations. Similar results were obtained for a small sample of V. girdiana. Removing hybrids greatly reduced the genetic variation of the presumed pure species, among which there was essentially no genetic structure. Limited genetic variability indicates the California natives may be threatened by genetic erosion. The discovery of F 1 hybrids of 'Mission', a cultivar not grown in the areas for ~100 years, suggests long generation times for wild vines that, often, grow into expansive liana and propagate by layering, all factors that limit recruitment in populations already disjunct by habitat lose. Hermaphroditic flowers and fruit that is more attractive to birds may favor the production of backcross seed and establishment of introgressed individuals.

Identification of mildew resistance in wild and cultivated Central Asian grape germplasm.

BACKGROUND: Cultivated grapevines, Vitis vinifera subsp. sativa, evolved from their wild relative, V. vinifera subsp. sylvestris. They were domesticated in Central Asia in the absence of the powdery mildew fungus, Erysiphe necator, which is thought to have originated in North America. However, powdery mildew resistance has previously been discovered in two Central Asian cultivars and in Chinese Vitis species. RESULTS: A set of 380 unique genotypes were evaluated with data generated from 34 simple sequence repeat (SSR) markers. The set included 306 V. vinifera cultivars, 40 accessions of V. vinifera subsp. sylvestris, and 34 accessions of Vitis species from northern Pakistan, Afghanistan and China. Based on the presence of four SSR alleles previously identified as linked to the powdery mildew resistance locus, Ren1, 10 new mildew resistant genotypes were identified in the test set: eight were V. vinifera cultivars and two were V. vinifera subsp. sylvestris based on flower and seed morphology. Sequence comparison of a 620 bp region that includes the Ren1-linked allele (143 bp) of the co-segregating SSR marker SC8-0071-014, revealed that the ten newly identified genotypes have sequences that are essentially identical to the previously identified mildew resistant V. vinifera cultivars: 'Kishmish vatkana' and 'Karadzhandal'. Kinship analysis determined that three of the newly identified powdery mildew resistant accessions had a relationship with 'Kishmish vatkana' and 'Karadzhandal', and that six were not related to any other accession in this study set. Clustering procedures assigned accessions into three groups: 1) Chinese species; 2) a mixed group of cultivated and wild V. vinifera; and 3) table grape cultivars, including nine of the powdery mildew resistant accessions. Gene flow was detected among the groups. CONCLUSIONS: This study provides evidence that powdery mildew resistance is present in V. vinifera subsp. sylvestris, the dioecious wild progenitor of the cultivated grape. Four first-degree parent progeny relationships were discovered among the hermaphroditic powdery mildew resistant cultivars, supporting the existence of intentional grape breeding efforts. Although several Chinese grape species are resistant to powdery mildew, no direct genetic link to the resistance found in V. vinifera could be established.

Genetic structure and differentiation in cultivated grape, Vitis vinifera L.

222 cultivated (Vitis vinifera) and 22 wild (V. vinifera ssp. sylvestris) grape accessions were analysed for genetic diversity and differentiation at eight microsatellite loci. A total of 94 alleles were detected, with extensive polymorphism among the accessions. Multivariate relationships among accessions revealed 16 genetic groups structured into three clusters, supporting the classical eco-geographic grouping of grape cultivars: occidentalis, pontica and orientalis. French cultivars appeared to be distinct and showed close affinity to the wild progenitor, ssp. sylvestris from south-western France (Pyrenees) and Tunisia, probably reflecting the origin and domestication history of many of the old wine cultivars from France. There was appreciable level of differentiation between table and wine grape cultivars, and the Muscat types were somewhat distinct within the wine grapes. Contingency chi2 analysis indicated significant heterogeneity in allele frequencies among groups at all loci. The observed heterozygosities for different groups ranged from 0.625 to 0.9 with an overall average of 0.771. Genetic relationships among groups suggested hierarchical differentiation within cultivated grape. The gene diversity analysis indicated narrow divergence among groups and that most variation was found within groups (approximately 85%). Partitioning of diversity suggested that the remaining variation is somewhat structured hierarchically at different levels of differentiation. The overall organization of genetic diversity suggests that the germplasm of cultivated grape represents a single complex gene pool and that its structure is determined by strong artificial selection and a vegetative mode of reproduction.