Foundation Plant Services Research Articles

Characterization of Genetic Diversity in the Capsid Protein Gene of Grapevine Fleck Virus and Development of a New Real-Time RT-PCR Assay.

The grapevine fleck virus (GFkV) is a ubiquitous grapevine-infecting virus found worldwide, is associated with the grapevine fleck complex, and is often found in mixed infections with viruses of the grapevine leafroll complex and/or vitiviruses. Although GFkV has been studied for a long time, limited sequence information is available in the public databases. In this study, the GFkV sequence data available in GenBank and data generated at the Foundation Plant Services, University of California, Davis, were used to perform nucleotide sequence comparisons, construct a phylogenetic tree, and develop a new RT-qPCR assay. Sequence comparisons showed high genetic diversity among the GFkV isolates, and the phylogenetic analyses revealed a new group comprised of GFkV isolates identified in the present study. A new assay, referred to as GFkV-CP, was designed and validated using an existing GFkV positive control together with 11 samples known to be infected with combinations of different marafiviruses and maculaviruses but not GFkV. In addition, the newly designed assay was used in a field survey to screen grapevines from diverse geographical locations that are maintained at the United States Department of Agriculture (USDA) National Clonal Germplasm Repository (NCGR) in Winters, CA.

First Report of Watermelon Mosaic Virus Naturally Infecting Coriander (Coriandrum sativum) and causing a leaf mottling disease in California.

Watermelon mosaic virus (WMV, genus Potyvirus, family Potyviridae) is a species of considerable economic importance to cucurbit crops worldwide (Keinath et al. 2017). This virus has a wide host range that includes more than 170 plant species from 27 families (Dong et al. 2017; Lecoq et al. 2011). In 2018, leaves of coriander (Coriandrum sativum) plants in a student garden (C-SG) at UC Davis, and in a home garden in Davis, CA (C-Pet) (~1.1 miles apart) showed symptoms of light green mottling and crumpling. Symptomatic leaves from each location were weakly positive with the general potyvirus immunostrip test (Agdia, Elkhart, IN). In RT-PCR tests with total RNA extracts (RNeasy Plant Mini Kit Qiagen, Germantown, MD) of these leaves and the potyvirus degenerate primer pair CIFor/CIRev (Ha et al. 2008), the expected-size ~0.7 kb fragment was amplified. These fragments were gel-purified and sequenced, and a BLASTn search revealed highest identities of 91.6% (C-SG) and 97.9% (C-Pet) with the sequence of an isolate of WMV from watermelon in the U.S. (TX29, KU246036). Thus, these isolates are designated WMV-C-SG-18 and WMV-C-Pet-18. Mechanical inoculation experiments were next performed with sap prepared with symptomatic coriander leaf tissue in ice-cold 0.01 M phosphate buffer (pH 7.0) in a 1:4 wt/vol ratio. First, to obtain pure isolates, sap was inoculated onto celite-dusted leaves of Chenopodium quinoa plants (3-4 leaf stage). As expected for WMV, leaves inoculated with sap of each isolate developed chlorotic local lesions ~9 d post-inoculation (dpi) (Moreno et al. 2004). One lesion for each isolate was excised, ground in phosphate buffer, and the sap was mechanically inoculated onto leaves of Nicotiana benthamiana plants. By ~14 dpi, newly emerged leaves showed mild mottling and crumpling, and were weakly positive with the potyvirus immunostrip test. To confirm that these plants were only infected with WMV, total RNA was extracted from symptomatic leaves and used for high throughput sequencing (HTS) (Soltani et al. 2021) at the Foundation Plant Services at UC Davis. The HTS analyses revealed infection with only WMV, i.e., no other viral contigs were identified, and allowed for determination of the complete sequences (~10,000 nt) of WMV [US-CA-C-SG-18] and WMV [US-CA-C-Pet-18] with GenBank accession numbers: OM746964 and OM746965, respectively. Whole genome sequence comparisons revealed that the sequences are 99.0% identical, and 97.3% identical to the sequence of WMV TX29. Sap from symptomatic N. benthamiana leaves infected with each isolate was mechanical inoculated onto leaves of coriander plants (30-35 d old). Newly emerged leaves developed epinasty, crumpling and light green mottling by 14 dpi, and WMV infection was confirmed by RT-PCR with the WMV-specific primer pair WMV-UNI-1F and WMV-UNI-1R (Kim et al. 2019). Thus, Koch's postulates were fulfilled for this leaf mottling disease of coriander. Furthermore, the isolates from coriander induced stunting and distortion and mosaic in leaves of melon, pumpkin and squash plants by 7 dpi, whereas watermelon plants developed stunting and small leaves with mild mottling by 20 dpi. Similar results were obtained with sap prepared from infected coriander leaves. Thus, infected coriander plants are a potential inoculum source for cucurbits via several aphid vectors (Keinath et al. 2017). This is the first report of a mottle disease of coriander caused by WMV, and adds to the wide host range of the virus.

Returns to public investments in clean plant centers: A case study of leafroll virus-tested grapevines in support of cost-effective grape production systems

AbstractViruses and related graft-transmissible pathogens cause diseases that cost the grape industry billions of dollars annually if left uncontrolled. The National Clean Plant Network (NCPN), a USDA Farm Bill program, is an organization of clean plant centers that produce and maintain virus-tested foundation vine stocks and distribute propagation material derived thereof to nurseries and growers to minimize the introduction of viruses and virus-like diseases into the vineyard. Foundation Plant Services (FPS) is the major NCPN-grapes center. We examined the economic impacts of public investments in FPS from 2006 to 2019. By focusing on grapevine leafroll disease, our analyses revealed a benefit-cost ratio ranging from 22:1 to 96:1, with a 5% and a 20% disease infection rates in commercial vineyards, respectively. A welfare analysis was consistent with grape growers and nurseries capturing most (64–98%) of the benefits from adopting clean planting material compared with winemakers and other actors in the downstream wine supply chain system. This study provided new insights into the returns to public investments in a clean plant center and documented strong financial incentives for higher adoption of clean vines derived from virus-tested stocks, while justifying continued support of NCPN centers from public and private sectors.

First Report of Apricot vein clearing-associated virus Infecting flowering apricot (Prunus mume) in the United States.

Apricot vein clearing-associated virus is the type species of genus Prunevirus, family Betaflexiviridae. The virus was first discovered from an Italian apricot tree (Prunus armeniaca) showing leaf vein clearing and mottling symptoms (Elbeaino et al. 2014). Since then, apricot vein clearing-associated virus (AVCaV) has been reported in symptomatic and asymptomatic plants from other countries (Marais et al. 2015; Kinoti et al. 2017; Kubaa et al. 2014). In 2018, a domestic selection of a flowering apricot (P. mume cv. Peggy Clarke) (PC01) with no discernible foliar virus-like symptoms was received for inclusion in the Foundation Plant Services (UC-Davis) collection. The plant originated from a private Prunus collection located in California. Total nucleic acids (TNA) were isolated from PC01 leaves using MagMax Plant RNA Isolation Kit (Thermo Fisher Scientific). The TNA were analyzed for a panel of 15 Prunus-infecting viruses by reverse-transcription quantitative PCR (RT-qPCR) (Diaz-Lara et al. 2020). In addition, to screen for sap-transmissible viruses, young leaves of PC01 were homogenized in inoculation buffer and were rubbed onto leaves of herbaceous indicator plants, Chenopodium amaranticolor, C. quinoa, Cucumis sativus, and Nicotiana clevelandii (Rowhani et al. 2005). The source PC01 tested negative for the 15 screened viruses. Interestingly, vein clearing symptoms were observed on leaves of C. quinoa and C. amaranticolor plants (Figure S1). These results suggested the presence of a mechanically transmissible virus in PC01. To determine the identity of mechanically transmissible viral agent, symptomatic C. quinoa and PC01 plant were advanced for high throughput sequencing analysis. Aliquots of TNA from PC01 and C. quinoa were rRNA-depleted and used for cDNA library preparation with TruSeq Stranded Total RNA kit (Illumina). The raw reads were trimmed, de novo assembled, and subsequently were annotated using tBLASTx algorithm (Al Rwahnih et al. 2018). A total of 47,261,138 and 8,812,296 single-end reads were obtained from cDNA libraries of PC01 and C. quinoa, respectively. The de novo assembly generated near-complete contigs resembling AVCaV genome ) from both PC01 and C. quinoa, which were 99.8% identical at the nucleotide level. The longest contig (8,342 nucleotides, 73.5x coverage depth) obtained from PC01 was further completed using SMARTer RACE 5'/3' kit (Takara Bio). The complete genome sequence of AVCaV-PC01 is 8,364 nucleotides long (GenBank: MK170158). The full-length virus genome was compared with GenBank database using BLASTn, which the best hit corresponded to KY132099 with 98% identity. Additionally, AVCaV infection was confirmed in both PC01 selection and the symptomatic C. quinoa by RT-PCR as previously described (Marais et al. 2015). Lastly, symptomatic leaves of C. quinoa were used in leaf dip method to visualize virus particles by transmission electron microscope. As a result, flexuous rod-shaped virions were observed from leaf dips of symptomatic C. quinoa plants (Figure S2). Therefore, our results represent the first report of AVCaV in California, USA. Furthermore, mechanical transmission of an AVCaV isolate infecting flowering apricot to herbaceous hosts was confirmed. Field surveys and biological studies are underway to determine the prevalence of AVCaV in commercial orchards and assess its effect on tree performance.

First report of Rose leaf rosette-associated virus infecting rose (Rosa spp.) in California, USA.

Rose leaf rosette-associated virus (RLRaV) is a member of genus Closterovirus, family Closteroviridae. The virus was first discovered in China in 2015 from a mixed infected wild rose (Rosa multiflora Thunb.) showing small leaf rosettes on branches, dieback and severe decline symptoms (He et al. 2015). In 2013, a rose plant (cv. Roses Are Red) was introduced to Foundation Plant Services (FPS, UC-Davis) rose collection. The plant was originated from a private rose breeder collection located in California. In 2019, total nucleic acids (TNA) were isolated from leaf tissues of one asymptomatic plant (Roses Are Red plant) using MagMax Plant RNA Isolation Kit (Thermo Fisher Scientific, USA). Extracted TNA were screened by reverse-transcription quantitative PCR (RT-qPCR) for six common viruses infecting roses, including prunus necrotic ringspot virus (PNRSV), apple mosaic virus (ApMV), rose spring dwarf associated virus (RSDaV), rose yellow vein virus (RYVV), rose rosette virus (RRV), and blackberry chlorotic ringspot virus (BCRV); however, the results were negative. Therefore, the sample was subjected to high throughput sequencing (HTS). Briefly, TNA was depleted of rRNA and advanced for cDNA library preparation using TruSeq Stranded Total RNA kit (Illumina, USA). HTS was performed on Illumina NextSeq 500 platform. The raw reads were trimmed, de novo assembled, and subsequently were annotated using tBLASTx algorithm (Al Rwahnih et al. 2018). HTS generated 23.6 million 75 nucleotide (nt) single-end raw data reads. De novo assembly generated a contig (16,528 nts) resembling RLRaV reference sequence (KJ748003) with 74% identity at the nucleotide level. Putative coat protein and heat shock protein 70-like protein were identified based on >90% identity with RLRaV genes. To confirm HTS results, RT-PCR was performed using two primer sets, 1) Clo-F4916 (5'-GGTGTTCCAACGCTATCGTG-3') and Clo-R5215 (5'- TGTCCTCAAACCGCCTACAT-3') targeting nucleotide sequences of putative polyprotein 1a, and 2) Clo-F10006 (5'-GATTCCGCGGACGAATTAAT-3') and Clo-R10311 (5'-GGTAACCGAAAGGTAAAGTATTC-3') targeting nucleotide sequences of putative protein p25. The RLRaV amplicons with expected size of 300 nt were confirmed using bidirectional Sanger sequencing. The near-complete sequence of the new RLRaV isolate was deposited in GenBank under accession number MW056181. In addition, HTS analysis showed that RLRaV was in mixed infection with two mycoviruses (rose cryptic virus with 8,267 mapped reads and rose partitivirus with 7,283 mapped readss). To our knowledge, this is the first report of RLRaV affecting roses in California. Further research is needed to determine the prevalence of RLRaV in California as well as evaluation of RLRaV effect on rose performance.

Characterization of a New Nepovirus Infecting Grapevine.

In 2012, dormant canes of a proprietary wine grape (Vitis vinifera L.) accession were included in the collection of the University of California-Davis Foundation Plant Services. No virus-like symptoms were elicited when bud chips from propagated own-rooted canes of the accession were graft-inoculated onto a panel of biological indicators. However, chlorotic ringspot symptoms were observed on sap-inoculated Chenopodium amaranticolor Coste & A. Rein and C. quinoa Willd. plants, indicating the presence of a mechanically transmissible virus. Transmission electron microscopy of virus preparations from symptomatic C. quinoa revealed spherical, nonenveloped virions about 27 nm in diameter. Nepovirus-like haplotypes of sequence contigs were detected in both the source grape accession and symptomatic C. quinoa plants via high-throughput sequencing. A novel bipartite nepovirus-like genome was assembled from these contigs, and the termini of each RNA segment were verified by rapid amplification of complementary DNA ends assays. The RNA1 (7,186-nt) of the virus encodes a large polyprotein 1 of 231.1 kDa, and the RNA2 (4,460-nt) encodes a large polyprotein 2 of 148.9 kDa. Each of the polyadenylated RNA segments is flanked by 5'- (RNA1 = 156-nt; RNA2 = 170-nt) and 3'- (RNA1 = 834-nt; RNA2 = 261-nt) untranslated region sequences with >90% identities. Maximum likelihood phylogenetic analyses of the conserved Pro-Pol amino acid sequences revealed the clustering of the new virus within the genus Nepovirus of the family Secoviridae. Considering its biological and molecular characteristics, and based on current taxonomic criteria, we propose that the novel virus, named grapevine nepovirus A, be assigned to the genus Nepovirus.

Detection and characterization of a second carlavirus in Rosa sp.

A new virus resembling members in the genus Carlavirus was identified in an Out of Yesteryear rose (Rosa sp.) by high-throughput sequencing. The virus was discovered during the screening of a rose virus collection belonging to Foundation Plant Services (UC-Davis). The full genome of the virus is 8825 nt long, excluding a poly(A) tail, and includes six predicted genes coding for replicase, triple gene block, coat protein (CP), and nucleic acid binding protein. The closest relative of the putative virus is rose virus A (RVA; genus Carlavirus), with 75% and 78% aa sequence identity in the replicase and CP, respectively. The relationship with RVA and other carlaviruses was supported by phylogenetic analyses using replicase and CP sequences. Based on genome organization, sequence identity, and phylogenetic analysis, the virus found in the Out of Yesteryear plant represents a new member of the genus Carlavirus and is provisionally named "rose virus B" (RVB). Further testing by reverse transcription PCR confirmed the presence of RVB in the original source and seven additional rose selections from the same collection.

Development of RT-PCR degenerate primers to overcome the high genetic diversity of grapevine virus T

Grapevine virus T (GVT) is a new member of the genus Foveavirus and has been reported to infect grapevines in several European countries. In 2018, GVT was detected for the first time in California in a domestic selection of wine grape, cv. Lambrusca di Alessandria, via high-throughput sequencing (HTS). To further investigate the presence of GVT in other grapevine plants, a two-step reverse transcription (RT)-PCR assay involving degenerate primers was developed. In order to cover the high genetic diversity of GVT, the sequences of available isolates were aligned to identify a conserved region in the coat protein gene that was a suitable target for the assay. The results of the RT-PCR assay showed that GVT was present in three additional grapevine selections among 416 plants integrating the Foundation Plant Services introduction pipeline; all were later confirmed by HTS. A complete and three near-complete genomes of the four GVT isolates were characterized and found to be divergent, sharing an overall 81 % pairwise identity in their nucleotide sequences. This suggested that the new RT-PCR assay was effective in detecting a broad range of GVT variants. The RT-PCR detection method developed in this study would be useful for routine virus testing.

First Report of Grapevine red blotch virus, the Causal Agent of Grapevine Red Blotch Disease, in Vitis vinifera in North Carolina

Grapevine red blotch virus (GRBV), genus Grablovirus, family Geminiviridae, is the causal agent for grapevine red blotch disease (GRBD) (Al Rwahnih et al. 2013; Yepes et al. 2018). GRBV has been found in several wine-grape growing regions in the United States (Krenz et al. 2014), as well as in germplasm repositories (Al Rwahnih et al. 2015). However, the incidence of GRBV in parts of the southeastern United States is unknown. Initial observations between August and October of 2017 in North Carolina (NC) vineyards revealed that the commonly grown red cultivars Vitis vinifera ‘Merlot’, ‘Malbec’, and ‘Cabernet franc’ frequently exhibited leaf symptoms similar to those associated with GRBD. The symptoms were seemingly unrelated to location, management, or age of the plants. A total of 80 grapevine samples were collected from eight blocks in six vineyards (10 vines per block) in three different wine-grape growing regions in NC (Upper Hiwassee Highlands American Viticultural Area [AVA], Crest of the Blue Ridge Henderson County AVA, and Yadkin Valley AVA), following the recommended sampling strategy from Foundation Plant Services (FPS, University of California–Davis) (http://fps.ucdavis.edu/samplecollection.cfm). All samples were collected in October 2018. Blocks were selected based on the cultivar and the visibility of symptomatic leaves. Ten vines with leaf symptoms were selected randomly within each block. Three samples per grapevine were collected, sampling both cordons. Samples were directly stored at 4°C and shipped overnight to the NC State University Micropropagation and Repository Unit for further processing. Following FPS protocols, total nucleic acid (TNA) was extracted from leaf petiole tissue using the RNeasy Mini Kit (Qiagen), and samples were analyzed by quantitative reverse transcription PCR (RT-qPCR) for a panel of viruses known to infect grapevine, including GRBV. GRBV was identified in 21 samples of cultivars Merlot and Malbec in Yadkin and Surry Counties, NC (both Yadkin Valley AVA). All positive vines showed GRBD symptoms. To confirm the RT-qPCR results, aliquots of TNA from GRBV-positive samples were run by end-point PCR for further sequencing. GRBV-specific primers as described by Al Rwahnih et al. (2013) were used. RT-PCR yielded products of the expected size (557 bp) for all positive samples, and they were directly sequenced. All the positive samples shared 100% identity with 22 GRBV GenBank accessions, with the top hit being an isolate from Washington state (MF795177), based on BLASTN analysis. Complete genomes of three representative GRBV samples, one from each positive vineyard, were amplified using Phusion Hot Start Flex 2× Master Mix (NEB) and overlapping primers, GRBV-OL-F (5′-ATTCCTGCAGTTCTAGTGAAAG-3′) and GRBV-OL-R (5′-TAGAACTGCAGGAATCGC-3′). Sequencing of the 3.2-kb amplicons was done and the sequences deposited in GenBank (accession nos. MN186403 to MN186405). The GRBV sequences from NC shared 97 to 99% complete genome identities with isolates from Washington State (MF795161 and MF795177). Phylogenetic analysis revealed the clustering of the three NC isolates into GRBV clade 2 (Krenz et al. 2014). This report is part of a comprehensive survey of NC vineyards to study grapevine virus incidence in the state. The source of the virus is unclear; however, studies are underway to determine source and potential vector capacity in NC. To our understanding, this is the first time GRBV is being reported in NC.

Evaluation of Rosa Species Accessions for Resistance to Eriophyid Mites1

Abstract Rose rosette disease, caused by rose rosette virus (RRV), is an epidemic affecting nearly every rose cultivar in the United States. The only hosts for Phyllocoptes fructiphilus, the eriophyid mite that vectors RRV, are Rosa species. Eighteen Rosa species were evaluated for mite resistance by collecting foliage samples from July to November in 2016 and 2017, from which mites were extracted. Mites were isolated through a series of sieves and counted using a stereomicroscope. The response variable was expressed as the number of mites per gram of optimal rose tissue. Mite data were evaluated to determine the peak week for mite populations for each year. The mite populations varied by rose species (α = 0.05) in 2016 but not 2017. Due to high variability in mite counts, the species were not as clearly distinguishable as expected. This high variability is likely due to factors such as differential growth rates of the roses, weather, presence of RRV in the rose, and the quality of the tissue collected throughout the season. Experimental design revisions are proposed for future studies looking at Rosa species resistance to eriophyid mite populations. Index words: rose rosette virus, rose rosette disease, Phyllocoptes fructiphilus Keifer, virus, vector. Species used in this study: Phyllocoptes fructiphilus (Keifer), Prairie Rose [Rosa arkansana (Porter), Forest Farm]; Carolina Rose [Rosa carolina (L.), Forest Farm]; Rosa clinophylla (Thory), Rogue Valley Rose; White Prairie Rose [Rosa foliolosa (Nutt.), Rogue Valley Rose]; White Prairie Rose [Rosa foliolosa (Nutt.) Antique Rose Emporium]; Father Hugo Rose [Rosa hugonis, Rogue Valley Rose]; Musk Rose [Rosa moschata (J. Herrm.), Antique Rose Emporium]; Multiflora Rose [Rosa multiflora (Thunb.)]; Shining Rose [Rosa nitida (Willd.), Rogue Valley Rose]; Shining Rose [Rosa nitida (Willd.), Antique Rose Emporium]; Nootka Rose [Rosa nutkana (C. Presl.), Rogue Valley Rose]; Tea Rose [Rosa odorata (Andrews), Foundation Plant Services, Davis, CA]; Swamp Rose [Rosa palustris (Marshall), Antique Rose Emporium]; Swamp Rose [Rosa palustris (Marshall), Ever Blooming Antique Rose Emporium]; Chestnut Rose [Rosa roxburghii (Tratt.), Antique Rose Emporium]; ‘Plena' Chestnut Rose [Rosa roxburghii (Tratt.), Rogue Valley Rose]; Rugosa Rose [Rosa rugosa (Thunb.), Bailey's Nursery]; ‘Alba' Rugosa Rose [Rosa rugosa (Thunb.), Bailey's Nursery]; Climbing Prairie Rose [Rosa setigera (Michx.), Antique Rose Emporium]; Rosa soulieana (Crép.), Ralph Moore; Virginia Rose [Rosa virginiana (Mill.), Forest Farm]; Porterfolia Memorial Rose [Rosa wichuraiana (Crép.), Antique Rose Emporium]; Mountain Woods' Rose [Rosa woodsii (Lindl.), Rogue Valley Rose].

Elimination of rose viruses using microshoot tip tissue culture

Virus elimination therapy for roses is very challenging. Foundation Plant Services at the University of California, Davis provides virus-tested rose propagation material to the rose nursery industry in the United States. Before 1999, heat treatment of potted plants was used to eliminate viruses. Success was varied and often the plant died during the treatment or was still virus-infected at the end of treatment period. In 2007 we reported on successful elimination of Apple mosaic virus (ApMV) and Prunus necrotic ringspot virus (PNRSV) using microshoot tip culture for six cultivars. However, survival was low and was cultivar dependent. In the period 2013 to 2016, 35 rose cultivars were subjected to microshoot tip tissue culture therapy. Microshoot tips measuring <0.5 to 0.7 mm, consisting of the meristem dome plus several pairs of leaf primordia, were excised from actively growing vegetative shoots and cultured in aseptic conditions. Different media, growth factors and light intensity were trialed. To date the most promising medium is: Murashige and Skoog (MS) salts and vitamins with 3% sucrose, 2.5 µmol 6-benzyladeninepurine (BA) and 0.4 µmol indole-3-acetic acid (IAA). Rooting was ex vitro by transplanting shoots that were approximately 5 cm long into a sealed container of saturated perlite to keep humidity high. One or more plants survived from 20 cultivars of the 35 that were treated (56%). Overall percent survival averaged 3% and ranged from 0 to 22%, depending on cultivar. Preliminary testing by ELISA shows that ApMV was eliminated; plants were still infected with PNRSV. Further testing using qPCR and other methods and trials are ongoing.

Characterization of grapevine leafroll-associated virus 3 genetic variants and application towards RT-qPCR assay design.

Grapevine leafroll-associated virus 3 (GLRaV-3) is the most widely prevalent and economically important of the complex of RNA viruses associated with grapevine leafroll disease (GLD). Phylogenetic studies have grouped GLRaV-3 isolates into nine different monophyletic groups and four supergroups, making GLRaV-3 a genetically highly diverse virus species. In addition, new divergent variants have been discovered recently around the world. Accurate identification of the virus is an essential component in the management and control of GLRaV-3; however, the diversity of GLRaV-3, coupled with the limited sequence information, have complicated the development of a reliable detection assay. In this study, GLRaV-3 sequence data available in GenBank and those generated at Foundation Plant Services, University of California-Davis, was used to develop a new RT-qPCR assay with the capacity to detect all known GLRaV-3 variants. The new assay, referred to as FPST, was challenged against samples that included plants infected with different GLRaV-3 variants and originating from 46 countries. The FPST assay detected all known GLRaV-3 variants, including the highly divergent variants, by amplifying a small highly conserved region in the 3’ untranslated terminal region (UTR) of the virus genome. The reliability of the new RT-qPCR assay was confirmed by an enzyme linked immunosorbent assay (ELISA) that can detect all known GLRaV-3 variants characterized to date. Additionally, three new GLRaV-3 divergent variants, represented by four isolates, were identified using a hierarchical testing process involving the FPST assay, GLRaV-3 variant-specific assays and high-throughput sequencing analysis. These variants were distantly related to groups I, II, III, V, VI, VII and IX, but much similar to GLRaV-3 variants with no assigned group; thus, they may represent new clades. Finally, based on the phylogenetic analysis, a new GLRaV-3 subclade is proposed and named as group X.

Detection of Grapevine leafroll-associated virus 7 using real time qRT-PCR and conventional RT-PCR

Nine isolates of Grapevine leafroll-associated virus 7 (GLRaV-7) from diverse geographical regions were sequenced to design more sensitive molecular diagnostic tools. The coat protein (CP) and heat shock protein 70 homologue (HSP70h) genes of these nine isolates were sequenced. Sequences were then used to design more sensitive molecular diagnostic tools. Sequence identity among these isolates ranged between 90 to 100% at the nucleotide and amino acid levels. One RT-PCR and two qRT-PCR assays were used to survey 86 different grapevines from the University of California, Davis Grapevine Virus Collection, the Foundation Plant Services collection and the USDA National Clonal Germplasm Repository, Davis, CA with primers designed in conserved regions of the CP and HSP70h genes. Results revealed that qRT-PCR assays designed in the HSP70h gene was more sensitive (29.07% positives) than that designed in the CP gene (22.09% positives) and both qRT-PCR assays proved to be more sensitive than RT-PCR.

Yield Components and Fruit Composition of Five Barbera Grapevine Selections in the San Joaquin Valley, California

Barbera grapevine (<i>Vitis vinifera</i>) selections from Foundation Plant Services (FPS), University of California, Davis, were evaluated in Parlier, Fresno County, California. Green potted plants of Barbera, FPS selections 02, 03, 04, 05, and 06, were planted in the summer of 2000, and fruit yield and quality were assessed annually between 2003 and 2006. Selection 02 performed similarly to several other selections with respect to fruit composition and yield, but its berries were 10 to 25% heavier than the others. Large berries made the clusters of FPS 02 excessively compact; the incidence of sour rot was sometimes two to three times higher for FPS 02 than for other selections. Selections 03 and 05 originated from the same mother vine and performed identically. Their fruit composition was similar to several other selections, but they had the highest, or among the highest, yield of the selections tested and were moderately susceptible to sour rot. Selection 04 was generally similar to selections 03 and 05 except that 04 had lower yields in two of the four years tested. Selection 06 was distinctive in that it was generally the earliest ripening, had the smallest berries, the lowest yields, the lowest yield:pruning weight ratios, and was the least susceptible to sour rot.

Viticultural Evaluation of French and California Pinot noir Clones Grown for Production of Sparkling Wine

Twenty Pinot noir clonal selections were evaluated for viticultural characteristics in a vineyard managed for production of sparkling wine in the Los Carneros American Viticultural Area, Sonoma County, CA. Eight clones were of California origin from Foundation Plant Services (FPS), University of California, Davis: 2A, 4, 13, 17, 22, 31, 32, and 33. Twelve clones were of French (Champagne) origin: 389, 521, 665, 666, 668, 743, 779, 780, 870, 871, 872, and 927. Vine yield, yield components, fruit composition (soluble solids, pH, and titratable acidity), and vegetative growth were measured over three growing seasons, 1999 through 2001. Clones were harvested at similar soluble solids levels. Yield and all yield components differed significantly among clones. FPS 33 and clones 389 and 666 had significantly highest yields while FPS 13 and 22 and clone 870 had the lowest yields. Yield differences were primarily due to number of berries per cluster and number of clusters per shoot. Juice pH and titratable acidity differed significantly among clones, although the range for both measurements was small. Clones 521 and 870 had the highest pruning weights while clones 780, 668, and 665 and FPS 31 had the lowest pruning weights. Ravaz index values were high (&gt;12) for 780, 668, and FPS 31, while values were lowest (~5) for clones 870, 521, and FPS 17.

Viticultural Evaluation of French and California Chardonnay Clones Grown for Production of Sparkling Wine

Thirteen Chardonnay (<i>Vitis vinifera</i> L.) clones were evaluated for viticulture performance in a vineyard managed for sparkling wine production in the Los Carneros American Viticultural Area, Sonoma County, California. Two clones were of California origin (Foundation Plant Services [FPS] 4 and 72) and 11 were of French origin, reported to be from either Dijon (clones 75, 76, 78, 96, and 352) or Champagne (clones 118, 121, 124, 130, 131, and 132). Vine yield and yield components, fruit composition (soluble solids, pH, and titratable acidity), and vegetative growth were measured over four growing seasons, from 1998 through 2001. Clones were harvested on a soluble solids basis. Yield, berries per cluster, berry weight, cluster weight, and clusters per shoot differed significantly among clones. FPS 4 and French clones 96 and 352 had highest yields, and FPS 72 the lowest, with no great differences among other clones. Low yield of FPS 72 was due to very low berry weight and fewer berries per cluster. Yield components of other clones differed significantly over the four years of study. FPS 4 and FPS 72 retained the highest levels of titratable acidity at harvest. Vegetative growth was greatly influenced by both clone and year. FPS 72 had the highest pruning weights and clone 118 the lowest. Yield-to-pruning weight ratios were lowest for FPS 72 and highest for FPS 4.

Viticultural Performance of Three Malbec Clones on Two Rootstocks in Oakville, Napa Valley, California

Three clones of Malbec (<i>Vitis vinifera</i> L., syn. Cot) from Foundation Plant Services (FPS) were evaluated in Oakville, Napa Valley, California. Clones FPS 4, FPS 6, and FPS 8 were grown on two rootstocks, 110 Richter (110R) and Teleki 5C (5C). Vine yield components, fruit composition, and vegetative growth were measured over four growing seasons, 1997 through 2000. Shoot number per vine was held constant for all clones in a given year. Yield per vine, berries per cluster, cluster weight, clusters per shoot, and cluster number differed significantly among clones and between rootstocks. Berry weight differed significantly among clones, but not between rootstocks. All yield components varied significantly over the four vintages. FPS 8 had the highest yield and yield components, while FPS 6 had the lowest. Rootstock 110R had significantly higher yield than 5C by having more clusters and more berries per cluster. Yield component berries per cluster was most strongly correlated with yield differences. Pruning weight was inversely related to yield, where FPS 8 had the lowest pruning weight and FPS 6 the highest. The Ravaz index (yield to pruning weight ratio) was highest for FPS 8 and lowest for FPS 6. In years of greater yield, FPS 8 had greater soluble solids than FPS 4 or 6.

Fruit Characteristics of Six Merlot Grapevine Selections in the Central San Joaquin Valley, California

Merlot grapevine (<i>Vitis vinifera</i>) selections from Foundation Plant Services were evaluated near Fresno, California. Merlot selections FPS 1, 3, 9, 10, 11, and 14 were planted in April 1997 as own-rooted cuttings, and fruit yield and composition were assessed annually between 2000 and 2003. Vines of selection 10 performed best, producing consistently higher yields of fruit than vines of other selections, and the fruits of selection 10 had among the lowest pH and highest titratable acidity of the selections tested. In contrast, selection 11 was undesirable as it produced the largest berries, was the most susceptible to sour rot, and the juice of its fruit had the highest pH. Selection 14 produced fruit of good composition, but its yield was lower than the other selections. Selection 3, the current industry standard, performed similarly to selections 1 and 9, and was not superior to selection 10.

Yield Components and Fruit Composition of Six Chardonnay Grapevine Clones in the Central San Joaquin Valley, California

Chardonnay grapevine (<i>Vitis vinifera</i>) clones from Foundation Plant Services (FPS), University of California, Davis, were evaluated near Fresno, California. Chardonnay selections FPS 4, 6, 15, 18, 20, and 37 were planted in April 1997 as own-rooted cuttings, and fruit yield and quality were assessed annually between 2000 and 2003. Yield differences among clones were inconsistent, but vines of clone 4 generally had fewer and heavier clusters than vines of other clones, whereas vines of clones 6 and 15 generally had more and lighter clusters. Differences in cluster weight were mainly due to the number of berries per cluster. The fruits of clone 4 had higher soluble solids, lower pH, and similar or higher titratable acidity than the fruits of other clones. However, clone 4 had similar or worse sour rot incidence than the other clones, in contrast with clone 15, which usually had much less sour rot than the others. Results suggest that growers in the San Joaquin Valley, or in other warm-climate areas, might consider planting clone 15 instead of clone 4.

Viticultural Evaluation of Eleven Clones and Two Field Selections of Pinot noir Grown for Production of Sparkling Wine in Los Carneros, California

Eleven clones and two field selections of Pinot noir (<i>Vitis vinifera</i> L.) were evaluated for three years, 1992 through 1994, for viticulture characteristics in a vineyard managed for sparkling wine production. Clones from Foundation Plant Services (FPS), University of California, Davis, included Pinot noir FPS 1, 2A, 13, 23, 29, 31, 32, and 33. Also evaluated were three clones sourced from a plant importation program at Oregon State University, Espiguette 236, Espiguette 374, and Colmar 538, as were two field selections referred to locally in California as Wente and Gamay Beaujolais. The trial was established in 1988 in the Los Carneros American Viticultural Area in Sonoma County, California. Great variation among the clones was seen in vegetative growth and yield parameters. Pruning weight varied by a factor of 2 and yield by a factor of 1.5. Yield was influenced by cluster number, berries per cluster, and berry weight. There was a range of 4 Brix at harvest across all treatments in all three years and Brix was closely correlated with vine yield.