Source: UNIVERSITY OF CALIFORNIA, DAVIS submitted to
BREEDING PIERCE'S DISEASE RESISTANT GRAPES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0194292
Grant No.
(N/A)
Project No.
CA-D-VIT-7086-H
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2012
Project End Date
Sep 30, 2017
Grant Year
(N/A)
Project Director
Walker, M. A.
Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671
Performing Department
Viticulture and Enology
Non Technical Summary
Pierces disease has long limited the growth of V. vinifera grapes in the southern United States. Resistant grape cultivars have been developed, but they lack the fruit and wine quality to compete with V. vinifera cultivars. We have incorporated a novel form of PD resistance into elite V. vinifera cultivars to produce high resistance and high quality fruit.
Animal Health Component
(N/A)
Research Effort Categories
Basic
30%
Applied
70%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011131108080%
2011130108010%
2011132108010%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1131 - Wine grapes; 1130 - Table grapes; 1132 - Raisin grapes;

Field Of Science
1080 - Genetics;
Goals / Objectives
Over the past ten years the Walker lab has developed genetic markers tightly linked to resistance to Pierces disease (PD), a severe bacterial disease of grapevines caused by the bacterium Xylella fastidiosa and spread by a number of sharpshooter insects. This resistance is inherited as a single dominant gene, which we have genetically and physically mapped, and named PdR1. The resistance source is Vitis arizonica b43-17 and we have hybridized this selection with elite cultivars of V. vinifera. We then select the best progeny and backcross them to other elite V. vinifera cultivars increasing the percentage of V. vinifera in each successive generation. This backcross procedure normally utilizes a single elite cultivar, but because the grape genome is heterozygous we have switched V. vinifera cultivars each generation to avoid inbreeding depression. Our goal was to progress through four backcross generations to produce hybrids with about 97 percent V. vinifera parentage while maintaining the PdR1 resistance gene. This process would have been very difficult without the genetic markers linked to PdR1, which allow us to select out all the susceptible individuals as soon as the seeds germinate. It was also greatly expedited by aggressive vine training techniques that reduce the seed-to-seed cycle to two years. Our goal is to evaluate selections from multiple modified backcross generation four populations for the highest resistance to PD, and highest fruit and wine quality prior to commercial release of new varieties. We are also pursuing PD resistance from several other backgrounds in an effort to introgress multiple forms of resistance and thus lessen the chance of resistance breaking down to evolving strains of X. fastidiosa. One of these resistance sources is b42-26 another form of V. arizonica, but with multigenic resistance to PD. We are mapping this resistance source and hope to have effective markers that can optimize the introgression of this resistance into V. vinifera. Additionally, we have an additional form of singe dominant gene resistance, PdR2, from another V. arizonica, which we have mapped and have introgressed with selections carrying PdR1. We are also assisting in the mapping of a multigene resistance source from the Mortensen breeding program in Florida. This resistance is strong but the inheritance is low. The expected output of this research is the release of new PD resistant varieties. We have selections nearing small scale commercial testing at the 94 percent V. vinifera level and will be selecting individuals from thousands of seedlings at the 97 percent V. vinifera level for very small scale wine testing before advancement to small scale commercial testing. In a related project, we hope to characterize the PdR1 resistance gene and verify its function by genetically engineering candidates of this gene into susceptible V. vinifera varieties.
Project Methods
Research in the Walker lab has focused on determining how Xf resistance is inherited, the construction of genetic maps to enable identification of Xf resistance genes, the development of molecular markers that are tightly linked to Xf resistance, and the use of these markers in marker-assisted selection to accelerate the breeding program's efforts to backcross Xf resistance from forms of V. arizonica into elite V. vinifera wine, table and raisin grapes. With the development of tightly linked genetic markers to multiple sources of resistance we are poised to combine several resistances into one background using marker-assisted selection. We have studied the V. arizonica selection, b43-17, and fully described the mode of inheritance of its single dominant resistance gene, PdR1, and discovered that it is homozygous resistant. Genetic mapping has separated the two allelic forms, PdR1a and PdR1b, which provide similar levels of resistance in our greenhouse screen. We are working with both alleles to advance lines, and have recombined them into one line to determine if both are needed for the highest level of resistance; recent results show either allele is sufficient. We are also using resistance from several other sources including a wide range of resistant selections from the southeastern United States. Our screening and evaluation of these sources confirms results from past studies, indicating that multiple genes control resistance in most resistance sources. Earlier breeding efforts were hindered because the ratio of resistant to susceptible plants was very low. We have used multigenic resistance from the V. arizonica selection b42-26, which resulted in relatively high numbers of highly resistant progeny. Genetic mapping efforts to identify key quantitative trait loci in a b42-26 population are underway with the goal of identifying markers to expedite the introgression of this form of resistance. We have well-established screening methodology for Xf resistance under greenhouse conditions based on the widely used needle inoculation technique of Hopkins. A 20 microliter droplet of Xf suspension with about 100 million colony forming units per milliliter is placed on a partially lignified stem just above the petiolar junction. A thin needle is pushed through the droplet five times partly through the width of the shoot. When this is done in a warm sunny greenhouse the suspension is quickly sucked into the stem by vascular pressure. PD symptoms are first seen about 8 weeks after inoculation on susceptible cultivars like Chardonnay. We have used various combinations of Xf strains in the past, and choose those with the most severe symptom expression on V. vinifera cultivars. We are currently using a strain from Napa Valley. We wait between 12 and 16 weeks, depending upon growth rate and weather, before samples are taken for PCR and ELISA evaluation. We score plants for symptom severity and select only those without symptoms and the lowest bacterial levels.

Progress 10/01/12 to 09/30/17

Outputs
Target Audience:This research has developed Pierce's disease resistant grapevines that are currently in the final stages of multiplication and distribution to grapevine nurseries, prior to distribution to grape growers. The target audience will be grape growers in addition to the grape genetics research community to whom publications on genetic mapping and gene characterization are directed. Publications also detail the grape germplasm used in these breeding efforts. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has trained several graduate students - most recently Karla Huerta-Acosta who is pursuing a PhD while examining resistance to Pierce's disease in Mexican and southwestern US grape accessions. How have the results been disseminated to communities of interest?I give many talks each year to industry and academic groups on our progress breeding Pierce's disease resistant grapes. Last year's talks included: The origin of grapes and grape breeding, 3rd International Symposium on Viticulture, Hermosillo, MX, Jan 27 - Invited Speaker; The origin of grape varieties, Daniel Roberts Growers Group, Santa Rosa, CA, Jan 30 - Invited speaker; Vineyard Challenges: Winegrape growing from the ground up, UCD Wine Executive Program, Mar 28 -Invited Speaker; PD issues, Dion Mundy from New Zealand, UCD, Apr 27; Grape variety choices, Constellation - Daniel Bosch and Ollie Davidson, St. Helena, CA, Apr 28 -Grower Assistance; Breeding PD resistant winegrapes, Executive Leadership Board Meeting, Lynmar Winery, Sebastopol, May 5 - Presentation; The Southwestern Vitis: a grape breeding mother lode, ASEV Merit Award, Bellevue, WA, June 29 - Invited Speaker; Vinifera hybrids and resistance to Pierce's disease, Charlottesville, VA, July 12 - Invited Speaker What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objectives Identify unique sources of PD resistance with a focus on accessions collected from the southwestern United States and northern Mexico. Develop F1 and BC1 populations from the most promising new sources of resistance. Evaluate the inheritance of resistance and utilize populations from the most resistant sources to create mapping populations. Provide support to the companion mapping/genetics program by establishing and maintaining mapping populations, and using our optimized greenhouse-based screen to evaluate populations and selections for PD resistance. Develop advanced lines of PD resistant winegrapes from unique resistance sources through four backcross generations to elite V. vinifera cultivars. Evaluate and select on fruit quality traits such as color, tannin content, flavor, and productivity. Complete wine and fruit sensory analysis of advanced selections. Utilize marker-assisted selection (MAS) to stack (combine) different resistance loci from the BC4 generation with advanced selections containing PdR1. Screen for genotypes with combined resistances, to produce new PD resistant grapes with multiple sources of PD resistance and high quality fruit and wine. We continue to make rapid progress breeding PD resistant winegrapes through aggressive vine training, marker-assisted selection, and our rapid greenhouse screen procedures. These practices have allowed us to produce four backcross generations with elite V. vinifera winegrape cultivars in 10 years. We have screened through thousands of seedlings that are 97% V. vinifera with the PdR1b resistance gene from V. arizonica b43-17. Seedlings from these crosses continue to crop and others are advanced to greenhouse testing. We select for fruit and vine quality and then move the best to greenhouse testing, where only those with the highest resistance to X. fastidiosa, after multiple greenhouse tests, are advanced to multi-vine wine testing at Davis and in Pierce's disease hot spots around California. The best of these are being planted in vineyards at 50 to 1,000 vine trials with enough fruit for commercial scale winemaking. We have sent 19 advanced scion selections to FPS over the past four winters to begin the certification and release process. Three Pierce's disease resistant rootstocks were also sent to FPS for certification. Pierce's disease resistance from V. shuttleworthii and BD5-117 is also being pursued, but progress and effort is limited because their resistance is controlled by multiple genes without effective resistance markers. Other forms of V. arizonica are being studied and the resistance of some will be genetically mapped for future efforts to combine multiple resistance sources and ensure durable resistance. Very small-scale wines from 94% and 97% V. vinifera PdR1b selections have been very good, and have been received well at tastings throughout California, Texas and Virginia.

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Amrine, K.C.H, B. Blanco-Ulate, S. Riaz, D. Pap, L. Jones, R. Figueroa-Banderas M.A. Walker and D. Cantu. 2015. Comparative transcriptomics of Central Asian Vitis vinifera accessions reveals distinct defense strategies against powdery mildew. Horticultural Research 2: Article number: 15037 (2015)
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Viana, A.P., M.D.V. de Resende, S. Riaz and M.A. Walker. 2016. Genome selection in fruit breeding: application to table grapes. Scientia Agricola 73:142-149
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Pap, D., S. Riaz, I.B. Dry, A. Jermakow, A.C. Tenscher, D. Cantu, R. Olah and M.A. Walker. 2016. Identification of two novel powdery mildew resistance loci, Ren6 and Ren7, from the wild Chinese grape species Vitis piasezkii. BMC Plant Biology Jul 29;16(1):170
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Xie, X., C.B. Ag�ero, Y. Wang and M.A. Walker. 2016. Genetic transformation of grape varieties and rootstocks via organogenesis. Plant, Cell, Tissue and Organ Culture 126:541-552
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: He, Rr; Jiao Wu; Yali Zhang; Shaoli Liu; Chaoxia Wang; Andrew M. Walker; Jiang Lu. 2016 Overexpression of a thaumatin-like protein gene from Vitis amurensis improves downy mildew resistance in Vitis vinifera grapevine. Protoplasma 254(4): 1579-1589


Progress 10/01/15 to 09/30/16

Outputs
Target Audience:This research effort will provide new grape cultivars with resistance to Pierce's disease for use by grape growers in California and across the southern United States. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?The results have been published in Proceedings of conferences and at grower meetings listed below. PD Breeding Progress - report and tour. CDFA Administrators, UC Davis Oct 13 Grape breeding at UC Davis Interview for David Pelletier for International Wine Magazine, UC Davis Oct 13 Breeding PD resistant wine grapes - talk and tasting VEN on the Road, Santa Maria, CA Nov 5 UCD vineyard and winery tour, and PD wine tasting with Darrel Corti. Sacramento Private School support group and auction prize, UC Davis Nov 8 PD resistant winegrapes nearing release. FPS Annual Meeting, UC Davis Nov 10 Breeding PD resistant winegrapes. Napa Vit Tech Meeting, Napa, CA Nov 12 Grape breeding at UC Davis. Guest Lecturer at Chihuahua University, Chihuahua, MX Nov 25 Breeding PD resistant winegrapes. UCD Winegrape Day, UC Davis Dec 2 Walker grape breeding program. UC Cooperative Extension Grape Farm Advisor Meeting, UC Davis Dec 3 PD breeding update and tasting. Oak Knoll Growers Group, Napa, CA Jan 7, 2016 Walker grape breeding program update and tasting. Silverado SIMCO Growers Management Seminar, Napa, CA Jan 13, 2016 PD resistant winegrapes - update and tasting Napa/Sonoma growers meeting, Napa, CA Jan 21, 2016 Rootstock and scion breeding overview. Lodi Grape Day, Lodi, CA Feb. 2 2016 PD resistant winegrape breeding and tasting, Silverado Vineyards meeting, Napa, CA April 4 PD resistant winegrape breeding. Talk and discussion with John Dyson and Williams Salem staff, UC Davis, April 13 PD resistant winegrape breeding and tasting for California Association of Winegrape Growers, Sacramento, CA Apr 18 Breeding PD resistant winegrapes. Temecula Grape Day, Temecula, CA Apr 21 Breeding PD resistant winegrapes. Alan Tenscher presenting to the AVF Board in Livermore, Apr 29 Breeding PD resistant winegrapes. Talk and tasting for Napa winemakers and viticulturists, UC Davis, May 4 Winegrape breeding at UC Davis. Vintage Nursery Open House, Wasco, CA May 18 Winegrape breeding at UC Davis. International Cabernet Sauvignon Conference, Pine Ridge Winery, Napa, CA June 22 2016 Grape breeding Daniel Roberts Growers group, Santa Rosa, CA July 22 Grape breeding at UCD. Chilean table grape growers association, UCD Oct 3 Grape breeding above and below ground. Cal Poly San Luis Obispo, CA Oct 6 What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Over the past ten years the Walker lab has developed genetic markers tightly linked to resistance to Pierces disease (PD), a severe bacterial disease of grapevines caused by the bacterium Xylella fastidiosa and spread by a number of sharpshooter insects. This resistance is inherited as a single dominant gene, which we have genetically and physically mapped, and named PdR1. The resistance source is Vitis arizonica b43-17 and we have hybridized this selection with elite cultivars of V. vinifera. We then select the best progeny and backcross them to other elite V. vinifera cultivars increasing the percentage of V. vinifera in each successive generation. This backcross procedure normally utilizes a single elite cultivar, but because the grape genome is heterozygous we have switched V. vinifera cultivars each generation to avoid inbreeding depression. Our goal was to progress through four backcross generations to produce hybrids with about 97 percent V. vinifera parentage while maintaining the PdR1 resistance gene. This process would have been very difficult without the genetic markers linked to PdR1, which allow us to select out all the susceptible individuals as soon as the seeds germinate. It was also greatly expedited by aggressive vine training techniques that reduce the seed-to-seed cycle to two years. Objectives Identify unique sources of PD resistance with a focus on accessions collected from the southwestern United States and northern Mexico. Develop F1 and BC1 populations from the most promising new sources of resistance. Evaluate the inheritance of resistance and utilize populations from the most resistant sources to create mapping populations. Provide support to the companion mapping/genetics program by establishing and maintaining mapping populations, and using our optimized greenhouse-based screen to evaluate populations and selections for PD resistance. Develop advanced lines of PD resistant winegrapes from unique resistance sources through four backcross generations to elite V. vinifera cultivars. Evaluate and select on fruit quality traits such as color, tannin content, flavor, and productivity. Complete wine and fruit sensory analysis of advanced selections. Utilize marker-assisted selection (MAS) to stack (combine) different resistance loci from the BC4 generation with advanced selections containing PdR1. Screen for genotypes with combined resistances, to produce new PD resistant grapes with multiple sources of PD resistance and high quality fruit and wine. We continue to make rapid progress breeding Pierce's disease (PD) resistant winegrapes. Aggressive vine training and selection for precocious flowering have allowed us to reduce the seed-to-seed cycle to two years. To further expedite breeding progress we are using marker-assisted selection (MAS) for the Pierce's disease resistance gene, PdR1 (see companion report) to select resistant progeny as soon as seeds germinate. These two practices have greatly accelerated the breeding program and allowed us to produce four backcross generations with elite Vitis vinifera winegrape cultivars in 10 years. We have screened through about 2,000 progeny from the 2009, 2010, and 2011 crosses that are 97% V. vinifera with the PdR1b resistance gene from V. arizonica b43-17. Seedlings from these crosses continue to fruit and others are advanced to greenhouse testing. We select for fruit and vine quality and then move the best to greenhouse testing, where only those with the highest resistance to Xylella fastidiosa, after multiple greenhouse tests, are advanced to multi-vine wine testing at Davis and other test sites. The best of these will be advanced to field-testing with commercial-scale wine production, the first of which was planted in Napa in June 2013. We advanced three additional selections to Foundation Plant Services (FPS) last winter to begin the certification and release process. Three PD resistant rootstocks were previously advanced to FPS for certification. Other forms of V. arizonica are being studied and the resistance of some will be genetically mapped for future efforts to combine multiple resistance sources and ensure durable resistance. Stacking of PdR1b with b42-26 Pierce's disease resistance has been advanced to the 92% V. vinifera level using MAS to confirm the presence of PdR1b and greenhouse screening to verify higher than usual levels of Pierce's disease resistance. Pierce's disease resistance from V. shuttleworthii and BD5-117 is also being pursued but progress is limited by their multigenic resistance and the absence of associated genetic markers. Very small scale wines from 94% and 97% V. vinifera PdR1b selections have been very good and have been received well at public tastings in Sacramento (California Association of Winegrape Growers; CAWG) and Santa Rosa (Sonoma Winegrape Commission), Napa Valley (Napa Valley Grape Growers and Winemakers Associations), Temecula (Temecula Valley Winegrape Growers and Vintners), and Healdsburg (Dry Creek Valley and Sonoma Grape Growers and Winemakers).

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Walker, M.A. and A. Tenscher. 2015. Breeding Pierces disease resistant winegrapes. Proceedings of the CDFA Pierces Disease/Glassy-winged Sharpshooter Board, December 2016. Pp. 167-177.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Walker, M.A. and D. Cantu. 2015. Molecular breeding support for the development of Pierce's disease resistant winegrapes. Proceedings of the CDFA Pierces Disease/Glassy-winged Sharpshooter Board, December 2016. Pp. 178-187.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Xie, X., C.B. Ag�ero, Y. Wang and M.A. Walker. 2015. In vitro induction of tetraploids in Vitis X Muscadinia hybrids. Plant Cell, Tissue & Organ Culture 126:541-552.
  • Type: Book Chapters Status: Published Year Published: 2016 Citation: Walker, M.A. 2015. The Genus Vitis, Its Species and Its Rootstocks. IN: Compendium of Grape Diseases, Disorders, and Pests, 2nd Edition, Ed. W.F. Wilcox, W.D Gubler and J.K. Uyemoto et al. APS Press.


Progress 10/01/14 to 09/30/15

Outputs
Target Audience:This research effort will provide new grape cultivars with resistance to Pierce's disease for use by growers in California and across the southern United States. It will also be of benefit to researchers studying resistance to Pierce's disease. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Results have been presented at grower meetings, industry seminars and scientific meetings. Breeding for PD and PM resistance. Napa Valley Grape Growers, Napa, CA March 4 2015 Vineyard challenges. Wine Executive Program lecture, UCD. Mar. 24 Vineyard of the Future Wine Executive Program lecture, UCD. Mar. 27 California viticulture. UC Berkeley DNV Business program Napa, CA. Apr. 18 Breeding for PD and PM resistance. Diageo Winemakers, UC Davis, April 23, 2015 Breeding new winegrape varieties - PD, PM and beyond. Napa Marriot, May 6 A look to the future - what's in store for CA vineyards. Anderson Valley Tech Conference, Philo, CA. May 15 Breeding PD and PM resistant winegrapes - talk and tasting. Daniel Roberts Client Group, Santa Rosa, CA July 10 PD resistant wine grapes. Ventura Farm Press Interview, July 7 Breeding PD and PM resistant winegrapes. Sonoma County Winegrape Commission, Santa Rosa, CA July 31 PD resistant winegrapes - talk and tasting. California Association of Family Farms, Valley Center, CA Aug 7 Grape breeding at UC Davis. Chilean Table Grape Association, UC Davis, Aug 25 Grape rootstock and scion breeding at UC Davis. North American Grape Breeders Association, Geneva, NY Aug 29. What do you plan to do during the next reporting period to accomplish the goals?Continue to pursue the project objectives. The overall objective of this proposal is to develop advanced vinifera-based PD resistant winegrapes that combine resistance from different genetic backgrounds for use in PD "hot-spots" across California and the US. For this purpose, we will continue to screen promising selections at Davis and produce fruit for small-scale fermentations. The best selections from these trials will advance to commercial scale winemaking with grower/winery cooperators. In coming years, we will expand planting of the advanced vinifera-based selections with multiple vines each for small-scale wine evaluation. Our goal is to develop lines representing different PD resistance sources with diverse and elite vinifera backgrounds, stack different lines for durable field resistance, and continue to evaluate new resistant germplasm to optimize the breeding of PD resistant winegrapes. 1. Identify unique sources of PD resistance with a focus on accessions collected from the southwestern United States and northern Mexico. Develop F1 and BC1 populations from the most promising new sources of resistance. Evaluate the inheritance of resistance and utilize populations from the most resistant sources to create mapping populations. 2. Provide support to the companion mapping/genetics program by establishing and maintaining mapping populations, and using the greenhouse screen to evaluate populations and selections for PD resistance. 3. Develop advanced lines of PD resistant winegrapes from unique resistance sources through four backcross generations to elite V. vinifera cultivars. Evaluate and select on fruit quality traits such as color, tannin content, flavor, and productivity. Complete wine and fruit sensory analysis of advanced selections. 4. Utilize marker-assisted selection (MAS) to stack (combine) different resistance loci from the BC4 generation with advanced selections containing PdR1. Screen for genotypes with combined resistances, to produce new PD resistant grapes with multiple sources of PD resistance and high quality fruit and wine.

Impacts
What was accomplished under these goals? The PD resistant winegrape breeding program has made rapid progress. Aggressive vine training and selection for precocious flowering has allowed us to reduce the seed-to-seed cycle to 2 years. The use of molecular markers to facilitate selection of PD resistant progeny as soon as the seeds germinate saves valuable time and resources. These two practices have greatly accelerated the breeding program and allowed us to produce four backcross generations with elite Vitis vinifera winegrape cultivars arriving at the 97% vinifera level in only 10 years. Without molecular breeding support, it could take 20-25 years to get to the same level of resistance and fruit quality. We have fully characterized the resistance locus from V. arizonica/candicans b43-17 and named it PdR1 (Pierce's disease resistance gene 1). We have and generated four backcross generations (BC4 - 97% vinifera). Our companion mapping/genetics project has also identified markers for a second resistant accession V. arizonica b40-14 and we are also working on developing markers for resistance from V. arizonica/girdiana b42-26. We are maintaining backcross 2 (BC2), first filial (F1) cross and backcross 1 (BC1) mapping populations from these two genetic resources to facilitate the research of our mapping/genetics companion project. We have identified 22 new resistant accessions that were collected from the southwestern United States (US) and different regions of Mexico. Many of these have very strong resistance and will be tested to verify that their resistance is different from b43-17. It is critical to exploit the resistance from these diverse genetic backgrounds to broaden the base of PD resistance winegrape breeding and allow stacking (combining) of multiple and different resistant sources into a single line to strengthen resistance. We will begin this process by testing small breeding populations. We will continuing to develop different resistance sources to the 97% vinifera level, advance those with the best fruit and wine quality, expand to small scale wine testing on campus, and advance the best to 100 vine commercial scale field and wine making tests prior to release.

Publications

  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Xie, X., C.B. Ag�ero, Y. Wang and M.A. Walker. 2015. In vitro induction of tetraploids in Vitis X Muscadinia hybrids. Plant Cell, Tissue & Organ Culture DOI 10.1007/s11240-015-0801-8.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Walker, M.A. and A. Tenscher. 2015. Breeding Pierces disease resistant winegrapes. Proceedings of the CDFA Pierces Disease/Glassy-winged Sharpshooter Board, December 2015. Pp. 231-239.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Walker, M.A. and D. Cantu. 2015. Molecular breeding support for the development of Pierces disease resistant winegrapes. Proceedings of the CDFA Pierces Disease/Glassy-winged Sharpshooter Board, December 2015. Pp. 240-247.


Progress 10/01/13 to 09/30/14

Outputs
Target Audience: This research effort will provide new grape cultivars with resistance to Pierce's disease for use by grape growers in California and across the southern United States. It will also be of benefit to researchers studying resistance to Pierce's disease. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Results have been presented at grower meetings, industry seminars and scientific meetings. PD resistant winegrapes. Talk and tasting at Napa Wine and Grape Expo, Napa, CA November 14, 2013 PD resistant wines : tasting. PD discussion and tasting for the Martini Winery and Gallo staff, UC Davis, December 9, 3013 PD resistant winegrapes nearing release. Presented at the CDFA PD/GWSS Research Symposium, Sacramento, CA December 18, 2013 PD resistant winegrapes coming soon. Presented at the Unified Grant Management Seminar, UC Davis February 12, 2014 The history of grape research at the Oakville Station Presented to the Dean's Circle, Oakville, CA March 20, 2014 Walker rootstock breeding program. Presented to the American Vineyard Foundation Oversight Committee, UC Davis, June 17, 2014 New Possibilities: Hope for PD regions. American Society for Enology and Viticulture, Austin, TX, June 26, 2014. Fruit and wine tasting at Walker PD breeding program. UC Davis, August 28, 2014 What do you plan to do during the next reporting period to accomplish the goals? Continue with breeding program goals.

Impacts
What was accomplished under these goals? We continue to make rapid progress breeding Pierce's disease (PD) resistant winegrapes. Aggressive vine training and selection for precocious flowering has allowed us to reduce the seed-to-seed cycle to 2 years. We are also using marker-assisted selection (MAS) for the PD resistance gene, PdR1, to select resistant progeny as soon as seeds germinate. These two practices have greatly accelerated the breeding program and allowed us to produce four backcross generations with elite Vitis vinifera wine grape cultivars in 10 years. In 2009, 2010 and 2011 we created thousands of progeny with PdR1 and about 97% V. vinifera parentage. From these 10 to 20 will be selected for release. As part of the release process the selections are certified at our Foundation Plant Services (FPS). They are encouraging us to send selections for certification before final testing is completed so that release can be accelerated. FPS now has seven 94% V. vinifera selections. Four are red wine selections with the following V. vinifera cultivars as the last parent: Chardonnay 1; Lacrima Cristi 1; and Petite Sirah 2, and three are white wine selections with a Cabernet Sauvignon x Carignane hybrid as the last parent. We also sent eight 97% V. vinifera selections of which are red wine grapes with the following cultivars as the last parent: Cabernet Sauvignon 1; Zinfandel 3; and Chardonnay 4. Many more selections are in the final stages of testing. This testing includes wine production at the 10 to 15 liter level and three repeats of the greenhouse screen during which we select for the lowest levels of Xylella fastidiosa in the stem tissue. We have also sent three rootstock selections to FPS with very strong PD resistance. The first is a cross of 101-14Mgt X F8909-08 (the source of PdR1 resistance). The other two are crosses of this selection crossed to GRN-4 (a recently patented rootstocks with broad nematode resistance). Given the single gene resistance of PdR1 and its risk of being overcome after long-term use, we have been searching for additional resistance sources. Over 200 accessions, acquired across the southern US, have been tested for resistance. These accessions were also evaluated with SSR and chloroplast markers to evaluate their relatedness. Optimally we would select highly resistant and genetically different selections to increase of chances of finding novel and useful resistance sources that can be crossed with advanced 97% V. vinifera PdR1 selections in an effort to stack resistance genes and broaden PD resistance. Thus far we have identified 20 new and highly resistant accessions and made crosses to create 8 new resistant lines. To date we have created genetic maps for PD resistance in three backgrounds, b43-17, b40-14 and b42-26, all of which are forms of V. arizonica. PD resistance is inherited as a single gene in the first two, but is a quantitative trait in b42-26. We have created SSR-based markers for b43-17 and b40-14 (Pdr1a, Pdr1b and PdR1c) and are getting closer to useful quantitative trait loci for resistance from b42-26. We have also physically mapped PdR1 and found that it consists of six open reading frames, and have sequenced and analyzed them. In a first round of testing to confirm gene function constructs were made with each of these genes with CaMV 35S and they are engineered into tobacco, which is susceptible to PD. There was evidence that some of the genes suppressed symptom expression, but none impacted X. fastidiosa replication and movement. We then engineered the same constructs into Chardonnay and again none worked to suppress PD. Since then we have more carefully evaluated the sequence and believe we have a more likely gene candidate. We are also designing native grape promoters and will soon be engineering and testing this newly developed gene construct in Chardonnay.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Lin, H., M. S. Islam, L. Morano, R. Groves, B. Bextine, E. Civerolo, M. A. Walker. 2013. Genetic variation of Xylella fastidiosa associated with grapevines in two major viticultural regions in the United States  California and Texas. Journal of Plant Pathology. 95:329-337.
  • Type: Journal Articles Status: Published Year Published: 2014 Citation: Walker, M. A., S. Riaz and A. Tenscher. 2014. Optimizing the breeding of Pierces disease resistant winegrapes with marker-assisted selection. Acta Horticulturae 1046:139-143.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Ag�ero, C., S. Riaz and A. Walker. 2013. Molecular characterization of the putative Xylella fastidiosa resistance gene(s) from b43-17 (V. arizonica/candicans). Proceedings of the CDFA Pierces Disease/Glassy-winged Sharpshooter Board, Sacramento, CA, Dec 16-18, p.208-213.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Walker, A. and S. Riaz. 2013. Genetic mapping of Xylella fastidiosa resistance genes (s) in grape germplasm from the southern United States. Proceedings of the CDFA Pierces Disease/Glassy-winged Sharpshooter Board, Sacramento, CA, Dec 16-18, p200-207.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Walker, A. and A. Tenscher. 2013. Breeding Pierces disease resistant winegrapes. Proceedings of the CDFA Pierces Disease/Glassy-winged Sharpshooter Board, Sacramento, CA, Dec 16-18, p192-199.


Progress 01/01/13 to 09/30/13

Outputs
Target Audience: The goal of this project is the release of novel Pierce’s disease (PD) resistant wine grape varieties. These varieties will permit viticulture where PD is now endemic and provide PD resistant wine grape varieties for use if the glassy-winged sharpshooter spreads PD more widely. We are also expecting that PD resistant releases will have potential use across the southern US where PD is common. This project also tests and selects populations and genotypes for use in the development of genetic maps, from which genetic markers associated with resistance, are derived and used in marker-assisted selection. The target audience of this research are grape growers and winemakers in California, particularly those impacted by Pierce’s disease. This research will also be utilized by geneticists, pathologists and breeders working on the genetics and mechanisms of resistance to this disease. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Presentations to the grape and wine industryGrape breeding. Napa Valley Vintners. UC Davis, Feb. 27, 2013. Grape diseases and pests. Wine Executive Program, UC Davis, March 26, 2013 PD resistant wine tasting. Napa County Grape Growers and Winemakers, Oakville, CA April 5, 2013 Sustainable Viticulture. Haas Business School, DNV Top Tech Program, Mondavi Winery, Oakville, CA, April 20, 2013 Pest and disease threats: Decisions and the future of farming. Napa Valley 2030 – Ahead of the Curve, Napa Valley Grape Growers, Napa, May 7, 2013 Marker-assisted selection to optimize grape breeding. Grape Genetics Research Coordination Network. UC Davis, July 11, 2013. PD resistant wine tasting. Temecula Wine Association, Temecula, CA, July 17, 2013. PD resistant wine tasting. Healdsburg/Dry Creek Growers and Wineries, Clos du Bois, Healdsburg, CA, July 31, 2013. What do you plan to do during the next reporting period to accomplish the goals? We will continue to address the long-term goals of this project: 1. Breed PD resistant winegrapes through backcross techniques using high quality V. vinifera winegrape cultivars and X. fastidiosa resistant selections and sources characterized from our previous efforts. 2. Continue the characterization of X. fastidiosa resistance and winegrape quality traits (color, tannin, ripening dates, flavor, productivity, etc.) in novel germplasm sources, in our breeding populations, and in our genetic mapping populations. 3. Understand the genetic control of PD resistance and characterize resistance in other grape species backgrounds to allow the stacking of different resistance genes to enable broad and durable resistance

Impacts
What was accomplished under these goals? We continue to make rapid progress breeding Pierce’s disease (PD) resistant winegrapes. Aggressive vine training and selection for precocious flowering have allowed us to reduce the seed-to-seed cycle to 2 years. We are also using marker-assisted selection (MAS) for the PD resistance gene, PdR1 to select resistant progeny as soon as seeds germinate. These two practices have greatly accelerated the breeding program and allowed us to produce four backcross generations with elite V. vinifera wine grape cultivars in 10 years. We have screened through about 2,000 progeny from the 2009, 2010 and 2011 crosses that are 97% vinifera with the PdR1b resistance gene from V. arizonica b43-17. Seedlings from these crosses continue to crop and others are advanced to greenhouse testing. We select for fruit and vine quality and then move the best to greenhouse testing, where only those with the highest resistance to X. fastidiosa, after multiple greenhouse tests, are advanced to multi-vine wine testing at Davis and at a PD hot spot in Napa. The best of these will be advanced to 100 vine commercial wine testing the first of which was planted in Napa this past June. We advanced 10 selections to Foundation Plant Services this winter to begin the certification and release process. Three PD resistant rootstocks were also advanced to FPS for certification. PD resistance from V. shuttleworthii and BD5-117 are also being pursued but progress is limited by their multigenic resistance and the absence of corresponding genetic markers. Other forms of V. arizonica are being studied and the resistance of some will be genetically mapped for future efforts to combine multiple resistance sources and ensure durable resistance. Very small scale wines from 94% and 97% vinifera PdR1b selections have been very good and have been received well at pubic tastings across the State. This program is also studying the genetics of resistance to this disease and is attempting to broaden the genetic base of resistance by searching for and characterizing new forms of PD resistance. Previously, we reported on the screening of 52 accessions of grape species that were collected from across the southern US and northern Mexico. Greenhouse screening of these plants identified 20 new resistant accessions. We expanded this work to over 200 accessions that were acquired from States along the Gulf of Mexico, utilized 22 SSR markers and 14 chloroplast markers to develop fingerprint profiles for them. Analysis with two different programs revealed three major groups. The V. arizonica like group was composed of several species with distinct maternal and paternal inheritance. The species within this group are also very distinct from southeastern PD resistant species, once thought to be the only source of PD resistance. Greenhouse screening was completed on a subset of genotypes, and crosses with 8 new resistant lines were made in 2012 and 2013; the remaining germplasm is in the process of being screened. This germplasm screening provides opportunities to explore and identify resistance loci that may provide different resistance mechanisms allowing us to expand the genetic base of the PD resistance-breeding program. To date, we have utilized 3 different genetic resources to identify PD resistance. Progress was made with b43-17 and b40-14 both of which carry a major locus on chromosome 14, as well as minor QTLs on different chromosomes. For V. arizonica/candicans b43-17, a minor QTL has identified on chromosome 19 (PdR2) and for V. arizonica b40-14, a minor QTL was identified on chromosome 5. Mapping of a multigenic source of PD resistance from V. arizonica/girdiana b42-26 continues. A total of 916 markers have been tested, and 185 polymorphic markers (60 more since the previous report) have been added to the entire population of 239 seedlings. A framework genetic map was developed for 198 seedlings, which had repeated greenhouse screening. Preliminary QTL analysis identified QTLs on chromosome 8, 12 and 14 that explained over 25% phenotypic variation. Currently, we are saturating maps of these three chromosomes and associating SSR markers that are in linkage with resistance for marker-aided breeding. We plan to combine these multiple resistance sources in our breeding program to ensure broad and durable PD resistance. This project provides the genetic markers critical to the successful classical breeding of PD resistant wine, table and raisin grapes. Identification of markers for PdR1 has allowed us to reduce the seed-to-seed cycle to 2 years and produce selections that are PD resistant and 97% vinifera. These markers have also led to the identification of 6 genetic sequences that may house the PD resistance gene, and which are being tested to verify their function. These efforts will help us better understand how these genes function and could also lead to PD resistance genes from grape that would be available to genetically engineer PD resistance in V. vinifera cultivars.

Publications

  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Walker, A. 2013. Breeding Pierces disease resistant winegrapes. Proceedings of the Pierces Disease Research Symposium. On line at: http://www.cdfa.ca.gov/pdcp/Research.html
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Walker, A. 2013. Genetic mapping of Xylella fastidiosa resistance gene(s) in grape germplasm from the southern United States. Proceedings of the Pierces Disease Research Symposium. On line at: http://www.cdfa.ca.gov/pdcp/Research.html
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Walker, A. 2013. Molecular and functional characterization of the putative Xylella fastidiosa resistance gene(s) from b43-17 (Vitis arizonica). Proceedings of the Pierces Disease Research Symposium. On line at: http://www.cdfa.ca.gov/pdcp/Research.html
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Lin, H., Isalam, M.S., Morano, L., Groves, R., Bextine, B., Civerolo, E. and M.A. Walker. 2013. Genetic variation of Xylella fastidiosa associated with grapevines in two major viticultural areas in n the United States  California and Texas. Journal of Plant Pathology. 95:329-337.


Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: We continue to make rapid progress breeding Pierce's disease (PD) resistant winegrapes. Aggressive vine training and selection for precocious flowering has allowed us to reduce the seed-to-seed cycle to 2 years. We are also using marker-assisted selection (MAS) for the PD resistance gene, PdR1, to select resistant progeny as soon as seeds germinate. These two practices have greatly accelerated the breeding program and allowed us to produce four backcross generations with elite Vitis vinifera wine grape cultivars in 10 years. We have screened through about 1,000 progeny from the 2009 and 2010 crosses that are 97 percent (modified backcross generation 4) V. vinifera with the PdR1b resistance gene from V. arizonica b43-17. Seedlings from these crosses continue to crop and others are advanced to greenhouse testing. We select for fruit and vine quality first and then move the best to greenhouse testing, where only those with the highest resistance to the PD bacteria, Xylella fastidiosa, after multiple greenhouse tests, will be advanced to multi-vine wine testing at Davis and at a PD hot spot in Napa Valley, California. The best of these will be advanced to 100 vine commercial wine testing. We plan to move 9 advanced selections to UC Davis' Foundation Plant Services (FPS) this winter to begin the certification and release process. Two PD resistant rootstocks will also be advanced to FPS certification. PD resistance from V. shuttleworthii and BD5-117 are also being pursued but progress is limited by their multigenic resistance, which results in few resistant seedlings per generation. Other forms of V. arizonica are being studied and the resistance of some will be genetically mapped for future efforts to combine multiple resistance sources and ensure durable resistance. Small scale wines from 94% and 97% V. vinifera PdR1b selections have been very good and have been received well at pubic tastings. Mapping of a multigenic source of PD resistance from V. arizonica/girdiana b42-26 continues - a total of 916 markers were tested, and 170 polymorphic markers (45 more since the previous report) were added to the entire population of 239 seedlings. An initial genetic map was developed to assess the level of coverage on all 19 chromosomes. All markers were grouped and 15 linkage groups were established. The whole population is currently being rescreened for PD resistance due to the relatively high variation in ELISA values encountered in previous tests. Fifty-two accessions were selected from Vitis species collections we have made from across the southern US and northern Mexico. Greenhouse screening identified 20 new resistant accessions. Next we used 18 SSR markers to enable an assessment of the genetic diversity and similarity among these accessions. Four major groups of resistant germplasm were identified. The breeding program has used resistance from only two of these groups. This germplasm screening provides opportunities to explore and identify resistance loci that may provide different resistance mechanisms to expand the genetic base of the PD resistance breeding program. PARTICIPANTS: Dr. David Ramming, Grape Breeder for the USDA/ARS - Parlier, California and I are collaborating on this project, particularly with respect to table and raisin grape breeding. My lab supplies resistant plant material and marker-assisted selection for the PdR1 locus and his lab provides elite table and raisin grape selections. TARGET AUDIENCES: This research effort will provide new grape cultivars with resistance to Pierce's disease for use by grape growers in California and across the southern United States PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The goal of this project is the release of novel Pierce's disease resistant wine grape varieties. These varieties will permit viticulture where PD is now endemic and provide PD resistant wine grape varieties for use if the glassy-winged sharpshooter spreads PD more widely. We are also expecting that PD resistant releases will have potential use across the southern US where PD is common. This project also tests and selects populations and genotypes for use in the development of genetic maps, from which genetic markers associated with resistance, are derived and used in marker-assisted selection.

Publications

  • Walker, A. 2012. Molecular characterization of putative Xylella fastidiosa gene(s) from b43-17 (V. arizonica/candicans). CDFA PD Reports www.cdfa.ca.gov/pdcp/Documents/.../2012_Progress_Rpts.
  • Riaz, S., R. Hu and M. A. Walker. 2012. A framework genetic map of Muscadinia rotundifolia. Theoretical and Applied Genetics 125:1195-1210.
  • Sun, Q., Y. Sun, M. A. Walker and J. M. Labavitch. 2013. Vascular occlusions in grapevines with Pierce's disease make disease symptom development worse. Plant Physiology, January 2013 pp.112.208157


Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: We continue to make rapid progress breeding Pierce's disease (PD) resistant winegrapes. Aggressive vine training and selection for precocious flowering has allowed us to reduce the seed-to-seed cycle to 2 years. Breeding efforts have been further facilitated by the use of marker-assisted selection (MAS) for the PD resistance gene, PdR1, to select resistant progeny as soon as seeds germinate. These practices have greatly accelerated the breeding program and allowed us to produce four backcross generations with elite V. vinifera wine grape cultivars in 10 years. In Spring 2010, we planted about 2,000 97% vinifera seedlings with PdR1. We finished evaluating the fruit quality of over 1,200 of these in September 2011, and produced a small-scale wine of one, 09333-178. We are preparing to greenhouse test the best of these to verify which have the highest level of resistance to PD prior to multiplication and grafting for larger scale field trials. We plan to release commercially useful varieties from populations at this 97% vinifera level. The resistance above is based on 8909-08, which has one of the two alleles, PdR1b, from the Vitis arizonica/candicans b43-17 resistance source. The other resistance allele, PdR1a, is in 8909-17 and we have advanced this resistance to the 94% vinifera level and have combined it with the PdR1b allele to determine whether resistance with both alleles is stronger. There is also strong resistance in b42-26 a form of V. arizonica/girdiana form Baja California. b42-26's resistance is controlled by multiple genes, as opposed to the single gene resistance found in b43-17. We made crosses this year to advance the b42-26 resistance to the 87% vinifera level and have been surprised no only by the strength of resistance but also by the relatively large number of resistant progeny each generation. We are now re-evaluating its resistance markers to verify that it is not another form of PdR1. Finally, we evaluated the first set of about 50 accessions collected across the southwestern US for PD resistance. There were many with very strong resistance and these will be tested to verify that their resistance is different from b43-17's. This year's wine making also included wines made as blends with elite vinifera winegrapes, a likely use of our eventual releases. These selections could be used in severe PD hot spots and the fruit could be blended into the rest of the vineyard in a 25/75% ratio. We used Napa Valley (Oakville Station) Sauvignon blanc and Merlot with 07713-051 and 07355-075, respectively. We also made wine at the same scale with the Napa Sauvignon blanc and Merlot, and Davis Sauvignon blanc and Merlot (as growing region controls). PARTICIPANTS: Dr. David Ramming, Grape Breeder for the USDA/ARS - Parlier, California and I are collaborating on this project, particularly with respect to table and raisin grape breeding. My lab supplies resistant plant material and marker-assisted selection for the PdR1 locus and his lab provides elite table and raisin grape selections. TARGET AUDIENCES: This research effort will provide new grape cultivars with resistance to Pierce's disease for use by grape growers in California and across the southern United States. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The goal of this project is the release of novel Pierce's disease resistant wine grape varieties. These varieties will permit viticulture where PD is now endemic and provide PD resistant wine grape varieties for use if the glassy-winged sharpshooter spreads PD more widely. We are also expecting that PD resistant releases will have potential use across the southern US where PD is common. This project also tests and selects populations and genotypes for use in the development of genetic maps, from which genetic markers associated with resistance, are derived and used in marker-assisted selection.

Publications

  • 1. Walker, A. and A. Tenscher. 2011. Breeding Pierce's disease resistant winegrapes. Proceedings of the CDFA Pierces Disease/Glassy-winged Sharpshooter Board, San Diego, CA, Dec 13-15, p204-209.
  • 2. Walker, A. and Riaz, S. 2011. Genetic mapping of Xylella fastidiosa resistance genes in grape germplasm form the southern United States. Proceedings for the CDFA Pierce's Disease/Glassy-winged Sharpshooter Board, San Diego, CA Dec 13-15, p261-266.
  • 3. Walker, A. and C. Aguero. 2011. Molecular characterization of putative Xylella fastidiosa gene(s)from b43-17 (V. arizonica/candicans). Proceedings of the CDFA Pierce's Disease/Glassy-winged Sharpshooter Board, San Diego, CA Dec 13-15, p193-197.
  • 4. Ramming, D. W. and A. Walker. 2001. Breeding Pierce's Disease resistant table and raisin grapes and the development of markers for additional sources of resistance. Proceedings of the CDFA Pierce's Disease/Glassy-winged Sharpshooter Board, San Diego, CA, Dec 13-15, p186-192.


Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: This project is aimed at creating wine, table and raisin grape cultivars with resistance to Pierce's disease (PD), a bacterial disease caused by the xylem inhabiting Xylella fastidiosa. This disease exists from southern North America to central South America and widely grown Vitis vinifera wine, table and raisin grape cultivars are highly susceptible. Many grape species resist PD but in most cases resistance is controlled by multiple genes. We discovered several sources of single gene resistance in accessions of Vitis arizonica from northern Mexico and have used these to rapidly introgress PD resistance into elite cultivars of wine, table and raisin grape. One accession, b43-17 was used to create a mapping population, and genetic mapping efforts with simple sequence repeat markers (SSR) identified the primary locus responsible for PD resistance, which is located on chromosome 14 and which we named PdR1. We have now back crossed PdR1 to elite wine grapes for four generations to produce selections that are about 97% Vitis vinifera. We produced about 10,000 seed of this fourth backcross generation (modified BC4) in 2009. These seeds were germinated and about 4,500 seedlings were tested for the presence of PdR1 using SSR markers that are tightly linked to this locus. About 2,000 seedlings with PdR1 were planted in the vineyard in May 2010 and many of them are expected to fruit this year and allow fruit quality evaluations. Based on these evaluations we will propagate selections and plant up to 10 vine sets for small-scale wine evaluation, which will begin in 2013. We have also reached the BC4 generation while introgressing PdR1 into table and raisin grapes. This effort is in collaboration with David Ramming at the USDA-ARS Parlier. While the resistant table grape types need some further effort to reach current commercial standards, there are raisin selections ready for field testing. This year we continued to make crosses with elite wine grape cultivars to further the number of 97% vinifera populations we can select from, emphasizing red wine quality (using Cabernet Sauvignon as a parent), good acidity (using Barbera as a parent), and aromatics for white wines (using Muscat blanc, Riesling and Viognier as parents). These crosses produced about 3,300 seeds that are now germinating and will be tested for the PdR1 markers in February. We will be broadening the range of high quality wine grapes used for parents in this Spring's crosses. We also made crosses utilizing another accession of Vitis arizonica, b42-26. The PD resistance in this accession is controlled by multiple genes, which results in fewer resistant progeny in each generation. We are developing genetic markers to this resistance to expedite the incorporation of this resistance with PdR1, with the goal of broaden PD resistance in an effort to make it more durable. Finally, we are also beginning to introgress resistance to powdery mildew into the 97% vinifera lines. The powdery mildew resistance breeding efforts are also progressing using marker-assisted selection to introgress four different resistance loci. PARTICIPANTS: Dr. David Ramming, Grape Breeder for the USDA/ARS - Parlier, California and I are collaborating on this project, particularly with respect to table and raisin grape breeding. My lab supplies resistant plant material and marker-assisted selection for the PdR1 locus and his lab provides elite table and raisin grape selections. TARGET AUDIENCES: This research effort will provide new grape cultivars with resistance to Pierce's disease for use in California and across the southern United States. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
New Pierce's disease resistant wine grape varieties are being produced. These resistant varieties will permit viticulture where PD is now endemic and provide PD resistant wine grape varieties for use if the glassy-winged sharpshooter spreads PD more widely. We are also expecting that PD resistant releases will have potential use across the southern US where PD is common. We are also researching the genetics of resistance to optimize breeding through marker-assisted selection and eventually enable effective genetic engineering of grape resistance genes into high quality susceptible varieties.

Publications

  • Riaz, S., A.C. Tenscher, D.W. Ramming and M.A. Walker. 2011. Using a limited mapping strategy to identify major QTLs for resistance to grapevine powdery mildew (Erysiphe necator) and their use in marker-assisted breeding. Theoretical and Applied Genetics 122: 1059-1073.


Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: This project is aimed at creating wine, table and raisin grape cultivars with resistance to Pierce's disease(PD), a bacterial disease caused by the xylem inhabiting Xylella fastidiosa. This disease exists from southern North America to central South America and widely grown Vitis vinifera wine, table and raisin grape cultivars are highly susceptible. There are many grape species that resist this disease, but the genetic control of resistance is very complex in most of species, which has limited breeding progress. We are researching PD resistance and creating new disease resistant wine, table and raisin grapes. Resistance from several sources (including Vitis arizonica, Vitis aestivalis, Vitis champinii, Vitis shuttleworthii, and Muscadinia rotundifolia) is being utilized, but the most rapid progress is being made using resistance from a selection of Vitis arizonica. Olmo collected seed of this plant near Monterrey, Mexico and one of the progeny, b43-17, was later used in crosses with Vitis rupestris. We have determined that b43-17 is homozygous resistant to Xylella fastidiosa and that this resistance is controlled by a single dominant locus, named PdR1 that has been genetically mapped to chromosome 14. A multigenerational modified backcross program has been underway to introgress PdR1 into elite Vitis vinifera wine, table and raisin grape backgrounds. We have been collaborating with David Ramming of the USDA-ARS, Parlier to backcross PdR1 into his advanced large berried seedless table grapes and early maturity raisin grapes. We are breeding Pierce's disease resistant winegrapes using a modified backcross (BC) technique by utilizing a different elite Vitis vinifera winegrape in each generation while creating numerous lines that originate from cultivars within the major winegrape regions of Europe. We are avoiding inbreeding depression by not crossing back to the same recurrent parent. In 2009 we produced over 10,000 seeds with an assortment of winegrape types with our PdR1 resistance source that are 97 percent vinifera, the modified (BC4) generation. We hope to release PD resistant selections from this generation. Last year we made wines from the BC2 87.5 percent vinifera generation and the quality was good although not equal to vinifera (the hybrids possessed purple/blue pigments and off-aromas). In Fall 2009 we made wines from BC3 93.75 percent vinifera selections both wine and red types. The quality was much better, and the whites scored very well. We expect to have the first fruit on the BC4 97 percent vinifera in Summer 2011 and selections will be made for expanded planting and small-scale winemaking in 2011 and 2012. PARTICIPANTS: Dr. David Ramming, Grape Breeder for the USDA/ARS - Parlier, California and I are collaborating on this project, particularly with respect to table and raisin grape breeding. My lab supplies resistant plant material and marker-assisted selection for the PdR1 locus and his lab provides elite table and raisin grape selections. TARGET AUDIENCES: This research effort will provide new grape cultivars with resistance to Pierce's disease for use in California and across the southern United States. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
New Pierce's disease resistant wine grape varieties are being produced. These resistant varieties will permit viticulture where PD is now endemic and provide PD resistant wine grape varieties for use if the glassy-winged sharpshooter spreads PD more widely. We are also expecting that PD resistant releases will have potential use across the southern US where PD is common. We are also researching the genetics of resistance to optimize breeding through marker-assisted selection and eventually enable effective genetic engineering of grape resistance genes into high quality susceptible varieties.

Publications

  • Cheng, D.W., H. Lin, M.A. Walker, D.C. Stenger, and E.L. Civerolo. 2009. Effects of grape xylem sap and cell wall constituents on in vitro growth, biofilm formation, and cellular aggregation of Xylella fastidiosa. European Journal of Plant Pathology 125:213-222.
  • Riaz, S., A.C. Tenscher, R. Graziani, A.F. Krivanek, D.W. Ramming and M.A. Walker. 2009. Using marker-assisted selection to breed Pierce's disease resistant grapes. American Journal of Enology and Viticulture 60:199-207


Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: This project is aimed at creating wine, table and raisin grape cultivars with resistance to Pierce's disease (PD), a bacterial disease caused by the xylem inhabiting Xylella fastidiosa. This disease exists from southern North America to central South America and widely grown Vitis vinifera wine, table and raisin grape cultivars are highly susceptible. There are many grape species that resist this disease, but the genetic control of resistance is very complex in most of species, which has limited breeding progress. We are researching PD resistance and creating new disease resistant wine, table and raisin grapes. Resistance from several sources (including Vitis arizonica, Vitis aestivalis, Vitis champinii, Vitis shuttleworthii, and Muscadinia rotundifolia) is being utilized, but the most rapid progress is being made using resistance from a selection of Vitis arizonica. Olmo collected seed of this plant near Monterrey, Mexico and one of the progeny, b43-17, was later used in crosses with Vitis rupestris. We have determined that b43-17 is homozygous resistant to Xylella fastidiosa and that this resistance is controlled by a single dominant locus, named PdR1 that has been genetically mapped to chromosome 14. A multigenerational modified backcross program has been underway to introgress PdR1 into elite Vitis vinifera wine, table and raisin grape backgrounds. We have been collaborating with David Ramming of the USDA-ARS, Parlier to backcross PdR1 into his advanced large berried seedless table grapes and early maturity raisin grapes. We are breeding Pierce's disease resistant winegrapes using a modified backcross technique by utilizing a different elite Vitis vinifera winegrape in each generation while creating numerous lines that originate from cultivars within the major winegrape regions of Europe. We are avoiding inbreeding depression by not crossing back to the same recurrent parent. Over the last two years we have made wine from selections containing PdR1 in a 87.5 percent Vitis vinifera background (modified backcross generation 2); one line with 50 percent Chardonnay and the other with 50 percent Syrah. These selections were fermented along with similar quantities of UC Davis grown Cabernet Sauvignon, Pinot noir and Lenoir (a Pierce's disease resistant cultivar from the southeastern United States). The selections 0502-01, 0502-10 and 0502-12 were judged by a trained tasting panel to be comparable to the Vitis vinifera winegrapes, Cabernet Sauvignon and Pinot noir, and judged to be better than Lenoir. These selections are now planted in two highly infected vineyards in Texas. In 2008 we chose six PdR1 containing winegrape selections with 93.75 percent Vitis vinifera and replicated them eight times to enable small scale wine making in Fall 2009. These and other selections from the modified backcross generation 3 will be crossed with elite Vitis vinifera to create 96.88 percent Vitis vinifera seed in 2009. Small scale wines will be made from these lines in 2011 with decisions on selections for the first larger scale commercial trials to be made in 2012. PARTICIPANTS: Dr. David Ramming, Grape Breeder for the USDA/ARS - Parlier, California and I are collaborating on this project, particularly with respect to table and raisin grape breeding. My lab supplies resistant plant material and marker-assisted selection for the PdR1 locus and his lab provides elite table and raisin grape selections. TARGET AUDIENCES: This research effort will provide new grape cultivars with resistance to Pierce's disease for use in California and across the southern United States. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
New Pierce's disease resistant wine grape varieties are being produced. These resistant varieties will permit viticulture where PD is now endemic and counter the possible wide scale incidence of this disease if the introduced glassy winged sharpshooter vector spreads. We are researching the genetics of resistance to optimize breeding through marker-assisted selection and eventually enable effective genetic engineering of grape resistance genes into high quality susceptible varieties.

Publications

  • Fritschi, F.B., H. Lin and M.A. Walker. 2008. Scanning electron microscopy reveals different plant-pathogen interaction pattern in four Vitis genotypes infected with Xylella fastidiosa. Plant Disease 92:276-286.
  • Riaz, S, A.C. Tenscher, B.P. Smith, D.A. Ng and M.A. Walker. 2008. Use of SSR markers to assess identity, pedigree, and diversity of cultivated muscadine grapes. Journal of the American Society for Horticultural Science 133: 559-568.
  • Doddapaneni, H., H. Lin, M.A. Walker, J. Yao and E.L. Civerolo. 2008. VitisExpDB: a database resource for grape functional genomics. BMC Plant Biology 8:23 (online http://www.biomedcentral.com/1471-2229/8/23)
  • Riaz, S., A.C. Tenscher, J. Rubin, R. Graziani, S.S. Pao and M.A, Walker. 2008. Fine-scale genetic mapping of two Pierce's disease resistance loci and a major segregation distortion region on chromosome 14 of grape. Theoretical and Applied Genetics 117:671-681.
  • Lowe, K.M., S. Riaz and M.A. Walker. 2008. Variation in recombination rates across Vitis species. Tree Genetics and Genomes 5:71-80.
  • Stover, E., S. Riaz and M.A. Walker. 2008. PCR screening for Xylella fastidiosa in grape genebank accessions collected in the southeastern United States. American Journal of Enology and Viticulture 59:437-439.


Progress 01/01/07 to 12/31/07

Outputs
This project is aimed at creating wine, table and raisin grape cultivars with resistance to Pierce's disease (PD), a bacterial disease caused by the xylem inhabiting Xylella fastidiosa. This disease exists from southern North America to central South America and widely grown Vitis vinifera wine, table and raisin grape cultivars are highly susceptible. There are many grape species that resist this disease, but the genetic control of resistance is very complex in most of species, which has limited breeding progress. We are researching PD resistance and creating new disease resistant wine, table and raisin grapes. Resistance from several sources (including Vitis arizonica, Vitis aestivalis, Vitis champinii, Vitis shuttleworthii, and Muscadinia rotundifolia) are being utilized, but the most rapid progress is being made using resistance from a selection of Vitis arizonica. A selection of this species, which appears to be introgressd with Vitis candicans, b43-17, is the source of the Xylella fastidiosa resistance locus, PdR1, which has now been mapped to chromosome 14. SSR markers tightly linked to PdR1 are being used to efficiently introgress PD resistance into elite wine grape cultivars. This resistance is from a single dominant locus (gene) and yields 50% resistant progeny in each generation. We are planting selections that are 93.75% Vitis vinifera from elite winegrape cultivars. We also made wine from advanced selections with 87.5% Vitis vinifera winegrapes to prove that high quality wines could be made at this percentage of Vitis vinifera. These wines were rated by a tasting panel in comparison to similarly sized fruit and wine lots of Cabernet Sauvignon, Pinot noir, Lenoir and Midsouth. The goal of the breeding program is to begin release of PD resistant cultivars once we complete another round of modified backcrossing to 10 or more elite Vitis vinifera winegrape cultivars. The speed at which this breeding program is progressing is only possible through marker-assisted selection (MAS) for the PdR1 locus. We are now mapping alternate forms of PD resistance in a form of Vitis arizonica which is introgressed with Vitis girdiana and whose resistance is quantitatively inherited, and a pure Vitis arizonica form whose resistance is a single dominant gene, but different from PdR1. The goal of these efforts is to use MAS with markers linked to these alternate forms of PD resistance so that resistance genes can be multiplexed to add breadth and durability to PD resistance.

Impacts
New Pierce's disease resistant wine grape grape varieties are being produced. These resistant varieties will permit viticulture where PD is now endemic and counter the possible wide scale incidence of this disease if the introduced glassy winged sharpshooter vector spreads. We are researching the genetics of resistance to optimize breeding through marker-assisted selection and eventually enable effective genetic engineering of grape resistance genes into high quality susceptible varieties.

Publications

  • Doddapaneni, H., J. Yao, H. Lin, M. A. Walker and E.L. Civerolo. 2006. Analysis of the genome-wide variations among multiple strains of the plant pathogenic bacterium Xylella fastidiosa. BMC Genomics. Available: http://www.biomedcentral.com/1471-2164/7/225
  • Riaz, S., A. Doligez, R. J. Henry and M. A. Walker. 2007. Grape. Pp. 63-101. In, Genome Mapping and Molecular Breeding in Plants, Volume 4, Fruits and Nuts, C. Kole (Ed.), Springer, Heidelberg.
  • Lin, H., H. Doddapanneni, Y. Takahashi and A. Walker. 2007. Comparative analysis of ESTs involved in grape responses to Xylella fastidiosa infection. BMC Plant Biol. 7:8, doi:10.1186/1471-2229-7-8.
  • Fritschi, F.B., H. Lin and M.A. Walker. 2007. Xylella fastidiosa population dynamics in grapevine genotypes differing in susceptibility to Pierces disease. American Journal of Enology and Viticulture 58:326-332.
  • Riaz, S., S. Vezzulli, E.S. Harbertson, and M.A. Walker. 2007. Use of molecular markers to correct grape breeding errors and determine the identity of novel sources of resistance to Xiphinema index and Pierces disease. American Journal of Enology and Viticulture 58:494-49


Progress 01/01/06 to 12/31/06

Outputs
This project is aimed at creating wine, table and raisin grape cultivars with resistance to Pierce's disease (PD), a bacterial disease caused by the xylem inhabiting Xylella fastidiosa. This disease exists from southern North America to central South America and widely grown Vitis vinifera wine, table and raisin grape cultivars are highly susceptible. There are many grape species that resist this disease, but the genetic control of resistance is very complex in most of species, which has limited breeding progress. We are researching PD resistance and creating new disease resistant wine, table and raisin grapes. Resistance from several sources (including Vitis shuttleworthii, Vitis aestivalis, and Muscadinia rotundifolia) is being utilized, but the most rapid progress is being made using resistance from a selection of Vitis arizonica. This selection, b43-17, is the source of the Xylella fastidiosa resistance locus, PdR1, which has now been mapped to chromosome 14. SSR markers tightly linked to PdR1 are being used to efficiently introgress PD resistance into elite wine grape cultivars. This resistance is from a single dominant locus (gene) and yields 50% resistant progeny in each generation. We have planted selections that are 87.5% V. vinifera, contain PdR1, and that will fruit in 2007. Many seedling populations have been created from wide range of crosses to PdR1 carrying wine grape parents that are also 87.5% V. vinifera, and crosses to produce 94% V. vinifera with PdR1 progeny will be made in 2007. Pierce's disease resistant populations and selections with resistance from a wide range of other sources (V. shuttleworthii, M. rotundifolia, V. aestivalis, and others) are also being advanced and will be used to integrate resistance sources in the future. The mapping resolution we now possess allows us to characterize the resistance gene(s) at the PdR1 locus, and efforts to physically map and characterize PdR1 have commenced. The 9621 population was used to map PdR1 and is a cross of D8909-15 x F8909-17, both resistant to Xylella fastidiosa but only F8909-17 has PdR1. Analysis of breeding populations found that b43-17 is homozygous resistant at PdR1, but each of the chromatids has a different resistance allele; the siblings F8909-08 and F8909-17 have different forms of PdR1 from these chromatids. This information led to the development of three new mapping populations by crossing b43-17, F8909-08 and F8909-17 to highly susceptible Vitis vinifera. The screening and analysis of these populations will be critical to the success of physical mapping efforts.

Impacts
New Pierce's disease resistant grape varieties are being produced. These resistant varieties will permit viticulture where PD is now endemic and counter the possible wide scale incidence of this disease if the introduced glassy winged sharpshooter vector spreads. We are researching the genetics of resistance to optimize breeding and eventually enable effective genetic engineering of grape resistance genes into high quality susceptible varieties.

Publications

  • Krivanek, A.F., S. Riaz and M.A. Walker. 2006. The identification of PdR1, a primary resistance gene to Pierces disease in Vitis. Theoretical and Applied Genetics 112:1125-1131.
  • Ruel, J.J. and M.A. Walker. 2006. Resistance to Pierces Disease in Muscadinia rotundifolia and other native grape species. American Journal of Enology and Viticulture 57:158-165.
  • Riaz, S., A.F. Krivanek, K. Xu and M.A. Walker. 2006. Refined mapping of the Pierces disease resistance locus, PdR1, and Sex on an extended genetic linkage map of Vitis rupestris x V. arizonica. Theoretical and Applied Genetics 113:1317-1329


Progress 01/01/05 to 12/31/05

Outputs
We determined the inheritance of resistance to the bacterial causal agent of Pierces disease (PD), Xylella fastidiosa, in Vitis rupestris x Vitis arizonica/candicans, a key source of resistance in our grape breeding program. Measurements of disease progression under greenhouse conditions were based on quantitative assessment of bacteria populations in stem tissues and on three phenotypic scores: leaf scorch, a cane maturation index, and an index that incorporated shoot stunting into the cane maturation index. Measurement of bacterial populations yielded the highest broad-sense heritability for PD resistance on a genotype mean basis (0.97), indicating that this measure of resistance was the least effected by environmental variation. Narrow-sense heritability of PD resistance based on bacterial numbers in stems was moderately high at 0.52. Complex segregation analysis strongly indicated the existence of a major gene for PD resistance, which accounted for 91% of the total genetic variance. Conversion of the quantitative data into qualitative resistance levels and evaluation via a chi-square analysis showed that 15 of the 16 families segregated in accordance with a single gene hypothesis with a dominant allele controlling PD resistance. Our genetic mapping efforts locate this resistance locus, PdR1, on linkage group 14, and the tightly linked microsatellite markers are being used in marker-assisted selection. Other resistance sources appear to be quantitatively controlled.

Impacts
We are producing new Pierces disease resistant grape varieties. These resistant varieties will permit viticulture where PD is now endemic and counter the possible wide scale incidence of this disease if the introduced glassy winged sharpshooter vector spreads. We are researching the genetics of resistance to optimize breeding and enable effective genetic engineering of grape resistance genes into high quality susceptible varieties.

Publications

  • Buzkan, N., L. Kocsis, and M.A. Walker. 2005. Detection of Xylella fastidiosa from resistant and susceptible grapevine by tissue sectioning and membrane entrapment immunofluorescence. Microbiological Research 160:225-231.
  • Krivanek, A.F., T.R. Famula, A. Tenscher, and M.A. Walker. 2005. Inheritance of resistance to Xylella fastidiosa within a Vitis rupestris x Vitis arizonica hybrid population. Theoretical and Applied Genetics 111:110-119.
  • Lin, H, E.L. Civerolo, R. Hu, S. Barros, M. Francis and M.A. Walker. 2005. Multi-locus simple sequence repeat (SSR) markers for differentiating strains and evaluating genetic diversity of Xylella fastidiosa. Applied Environmental Microbiology 71:4888-4892.


Progress 01/01/04 to 12/31/04

Outputs
We are breeding grapes with resistance to Pierces disease (PD) by incorporating resistance from southern US grape species and cultivars. The table grape breeding efforts are in collaboration with David Ramming, USDA/ARS-Parlier. Available sources of resistance have very poor fruit quality and must be hybridized with highly susceptible, but high fruit quality Vitis vinifera cultivars. We are now in the third generation of backcrosses and fruit quality of table, raisin and wine grapes has markedly improved. This project has generated and is maintaining mapping populations being used to develop markers for resistance to Xylella fastidiosa, the bacterial causal agent of PD. We have optimized the resistance screen and have classified resistance as suppressed Xylella fastidiosa population development. Artificially inoculated plants with bacterial levels below 100,000 cfu/ml, based on ELISA testing, are highly resistant to the bacteria and disease as demonstrated through greenhouse testing and comparisons to past field testing. Inheritance testing was completed this year and determined that although resistance is controlled by multiple genes in most resistance sources, it is controlled by a single dominant gene from a Vitis arizonica-Vitis candicans hybrid. This resistance locus, PdR1, has been mapped to chromosome 14 and linked SSR markers are now being used in the breeding program.

Impacts
We are producing new Pierce's disease resistant grape varieties. These resistant varieties will permit viticulture where PD is now endemic and counter the possible wide scale incidence of this disease if the introduced glassy winged sharpshooter vector spreads. We are researching the genetics of resistance to optimize breeding and enable effective genetic engineering of grape resistance genes into high quality susceptible varieties.

Publications

  • Doucleff, M, Y. Jin, F. Gao, S. Riaz, A.F. Krivanek and M.A. Walker. 2004. A genetic linkage map of grape utilizing Vitis rupestris x Vitis arizonica. Theoretical and Applied Genetics 109:1178-1187.
  • Buzkan, N. L. Kocsis and M.A. Walker. 2005. Detection of Xylella fastidiosa from resistant and susceptible grapevine by tissue sectioning and membrane entrapment immunofluorescence. Microbiological Research 159 (In Press).
  • Krivanek, A.F., J.F. Stevenson and M.A. Walker. 2005. Development and comparison of symptom indices for quantifying grapevine resistance to Pierces disease. Phytopathology 95:36-43.
  • Krivanek, A.F. and M.A. Walker. 2005. Vitis resistance to Pierces disease is characterized by differential Xylella fastidiosa populations in stems and leaves. Phytopathology 95:44-52.


Progress 01/01/03 to 12/31/03

Outputs
In collaboration with David Ramming, USDA ARS Parlier, we are breeding grapes with resistance to Pierce's disease (PD) by incorporating resistance from southeastern US grape species and cultivars. These sources of resistance have had limited application because their fruit quality does not compare well with fruit from highly susceptible and widely grown Vitis vinifera cultivars. This project has not yet released resistant cultivars, but we are in the third generation of backcrossing to high quality Vitis vinifera parents. This project is closely tied to the development of genetic markers to optimize screening, and efforts to genetically map and characterize PD resistance. We have optimized screening and base resistance on incomplete wood development of inoculated canes, and titer and movement of the bacterial causal agent, Xylella fastidiosa, from the point of inoculation based on ELISA. We have discovered a novel source of resistance in Vitis arizonica and Vitis arizonica / Vitis candicans type hybrids. These selections were gathered in northern and central Mexico, have very high resistance and have been used in breeding. We continue to make many crosses, produce thousands of seeds and embryos, and about four thousand plants in the field each year. We are using embryo rescue to produce a large number of progeny from crosses of resistant males to seedless females including 265 plants from second generation backcrosses. This process generates high numbers of seedless progeny and improving chances of selecting new table and raisin grapes. This year (2,702 ovules) produced 484 embryos that are now germinating.

Impacts
We are producing new Pierce's disease resistant grape varieties. These resistant varieties will permit viticulture where PD is now endemic and counter the possible wide scale incidence of this disease if the introduced glassy winged sharpshooter vector spreads. We are researching the genetics of resistance to optimize breeding and enable effective genetic engineering of grape resistance genes into high quality susceptible varieties.

Publications

  • Buzkan, N., L. Kocsis, A.F. Krivanek, A.F. and M.A. Walker. 2003. Developing rapid evaluations for resistance to Xylella fastidiosa, the causal agent of Pierce's disease. Acta Horticulturae 603:433-440.
  • Buzkan, N. A.F. Krivanek, A. Eskalen and M.A. Walker. 2003. Improvements in sample preparation and polymerase chain reaction detection techniques for Xylella fastidiosa in grapevine tissue. American Journal of Enology and Viticulture 54:307-312.
  • Krivanek, A.F. and M.A. Walker. 2003. Progress in understanding the genetics of resistance from the grape Muscadinia rotundifolia to the bacterial pathogen Xylella fastidiosa. Acta Horticulturae 603:429-432.