Accelerating the development of powdery mildew resistant grapevines through marker assisted selection

AVF Proj. ID: 361 / /Cat.: ,
Primary Investigator(s): Lance Cadle-Davidsonby

The aim of this project is to harness molecular biology in the selection and advancement of improved cultivars having resistance to powdery mildew. Segregating populations from three sources of significant powdery mildew resistance (Vitis davidii, V. rotundifolia, and V. aestivalis), each backcrossed to V. vinifera, were previously generated by Dr. David Ramming. The first objective of this proposal is to characterize the plant-pathogen interactions, in terms of race-specificity and microscopic analysis, for each of the three resistance sources in order to inform the second objective, which is the development of molecular markers that co-segregate with powdery mildew resistance in each of these populations for use by grape breeding programs.

Powdery mildew resistance was assessed in 182 progeny from the three populations using three separate pathogen sources in California and New York. The resulting data suggest the presence of multiple, race-specific resistance genes segregating independently in rotundifolia and aestivalis progeny and suggest that some of the resistance genes would be rapidly overcome if inappropriately deployed. However, some progeny were resistant regardless of the pathogen source, suggesting the presence of all parental resistance alleles as a resistance gene pyramid. The stability of resistance in these individuals and the pathogen-dependent resistance of other individuals were confirmed in 2007. The rotundifolia and aestivalis breeding populations underscore one critical application of marker assisted selection – monitoring and pyramiding all functional resistance genes using a simple molecular assay rather than assaying resistance and durability by complex inoculation studies with multiple pathogen sources.

We also confirmed in 2007 that either of the two putative resistance genes from the davidii resistance source is sufficient for resistance regardless of pathogen source; these genes have the added intrigue of providing resistance against the penetration of the fungus (i.e., the pathogen is unable to access the epidermal cells where it must obtain sustenance to survive). Most powdery mildew penetration resistance genes are effective against all races of powdery mildew, and this appears to hold true with davidii.

To address the second objective, we require molecular markers that are polymorphic (appear different between the two parents) to track regions of the genome that were contributed to progeny by the resistant parent. We have identified 157 Simple Sequence Repeat markers (SSRs) that are polymorphic in these populations. Thus far, we have developed multiplexes for 39 SSRs and used them to screen all progeny in the three populations. In addition, we have identified amplified fragment length polymorphism (AFLP) markers associated with resistance in each of the populations. Our preliminary results support the two-gene models suggested by phenotypic data for the davidii and rotundifolia populations. Marker-trait associations in the aestivalis population will require QTL analysis.

Upon confirmation of which polymorphic markers predict disease resistance, we will focus on providing tightly-linked markers flanking disease resistance genes. From crosses representing each resistance source, we have germinated at least 600 seed and will test the utility of our markers for MAS, while using recombinants to more precisely track resistance genes.

Evaluation of Zinfandel Heritage Selections

AVF Proj. ID: 383 / /Cat.: ,
Primary Investigator(s): Jim Wolpertby

The 2007 growing season was more moderate, without the heat wave of 2006. The result was far less sunburn in 2007. Harvest was compressed with two harvests only two days apart, Sept 11 and Sept 13. Yield varied from a low of 3.7 kg per vine to a high of 6.6 kg. Regarding yield components, berry wt was a very tight range, differing by only 0.2 g, from 1.5 g to 1.7 g per berry. The yield component clusters per vine, as in previous years, was very close among clones due to crop control via pruning. Cluster wt, also following previous the years? pattern differed widely from a low 174 g to almost 300 g. Given the similarity of berry wt, berries per cluster mirrored the difference patterns of cluster wt, with heavy clusters having the most berries and light clusters the least, a range of 108 to 183 berries. Berry samples for comparing selections were taken on a single date just prior to harvest. It shows a range of 24.1 to 26.6 Brix. Values of pH were very low, as is the usual case for Zinfandel at the Oakville Station, and in a relatively tight range, 3.10 to 3.18. Titratable acidity values were also relatively close, ranging from 7.32 to 8.07 (g/L). The patterns of difference in the 2007 data, are similar to those in the 2005 ? 2007 averages. Ripening was a little more uniform in 2007. Cluster numbers are greater in 2007 but this is due to an increase shoot number over year 2005, and a decrease in sunburn loss over year 2006.

Breeding Rootstocks Resistant to Aggressive Root-knot Nematodes

AVF Proj. ID: 321 / /Cat.: ,
Primary Investigator(s): Peter Cousins, Ph.Dby

The USDA grape rootstock improvement program, based at the Grape Genetics Research Unit, is breeding grape rootstocks resistant to aggressive root-knot nematodes. We define aggressive root-knot nematodes as those which feed on and damage the rootstocks Freedom and Harmony. In 2006 we screened 3622 candidate grape rootstock seedlings for resistance to aggressive root-knot nematodes. We select only those seedlings which completely suppress nematode reproduction and show zero nematode egg masses. These selected seedlings are propagated and then planted into the vineyard. In 2006 we planted 372 nematode resistant rootstock selections in the vineyard. These selections were identified in nematode resistance screening in 2005 and 2004. In 2006 we pollinated 132 clusters of crosses specifically aimed at the breeding of improved rootstocks with resistance to aggressive root-knot nematodes. We tested the propagation ability of 114 nematode resistant selections. We confirmed the resistance of our rootstock selections to aggressive root-knot nematodes and we identified nematode resistant germplasm that may be parents for rootstock breeding.

Development of a National Expert System for Diagnosis of Grape Problems

AVF Proj. ID: 345 / /Cat.: ,
Primary Investigator(s): Edward Hellmanby

Version 1.0 of the diagnosis expert system has been developed with a database containing information on 161 grape problems including 55 arthropods, 48 pathogenic diseases, 25 phytotoxicity problems, 13 nutritional disorders, 9 abiotic stress disorders, 6 wildlife damage, and 5 physiological disorders. Additional problems will be entered into the database in 2007. The system is currently undergoing debugging and refinement prior to evaluation by a test panel. Production of graphics was initiated in 2006 and draft images have been completed for all first-level menu items.

The diagnosis system uses a symptom-based approach that guides the user through a series of directed questions leading to a ranked list of the most probable problems. The user answers questions based on their observations of symptoms and signs of the problem. Questions are organized in hierarchal levels, enabling the software to analyze responses and select the next level of questions on-the-fly based on the previous responses. This approach streamlines the diagnosis process by focusing on discriminating questions and avoiding extraneous ones.

Each vineyard problem is characterized with diagnostic keywords and a Problem Profile. The system uses the keywords to conduct a sorting routine to identify and display a probability-ranked list of possible problems. The Problem Profile contains text and photographs to assist with diagnosis of the problem.

A database was created to hold Diagnostic Keywords and Problem Profiles for known grape problems caused by pathogens, arthropods, vertebrate and other pests, abiotic stresses, nutritional disorders, chemical phytotoxicity, and physiological disorders. Database entries were created based on the personal experience of the principal investigators and from several standard references including the Compendium of Grape Diseases, 1988 APS Press and Grape Pest Management, 1992, University of California, Division of Agriculture and Natural Resources.

The technical accuracy of database entries will be validated by an editorial review process involving experts from around the U.S. Participation in the project by experts will be facilitated by the previous creation of draft versions of Diagnostic Keywords and Problem Profiles, which will minimize the time commitment of reviewers. Experts will be requested to review and edit selected problems. Reviewers will begin testing version 1.0 of the Expert System in 2007 and will be provided password-protected access to the Problem Profile Editor database interface. Reviewers will be credited for their work and will be invited to contribute new problems based on their experience. High-quality photos of symptoms and signs will also be solicited from experts and photo credits acknowledged. The diagnosis expert system will be validated through test use of version 1.0 and 2.0 by a panel comprised of vineyard managers, Extension educators, and students.

Breeding rootstocks resistant to aggressive root-knot nematodes

AVF Proj. ID: 297 / /Cat.: ,
Primary Investigator(s): Peter Cousins, Ph.Dby

The USDA grape rootstock improvement program, based at the Grape Genetics Research Unit, is breeding grape rootstocks resistant to aggressive root-knot nematodes. We define aggressive root-knot nematodes as those which feed on and damage the rootstocks Freedom and Harmony. In 2005 we screened 6201 candidate grape rootstock seedlings for resistance to aggressive root-knot nematodes. We select only those seedlings which completely suppress nematode reproduction and show zero nematode egg masses. These selected seedlings are propagated and then planted into the vineyard. We have 367 nematode resistant selections that will be ready for vineyard planting in spring 2006. In 2005 we planted 38 nematode resistant rootstock selections in the vineyard. These selections were identified in nematode resistance screening in 2004 and 2003. In 2005 we pollinated 857 clusters of crosses specifically aimed at the breeding of improved rootstocks with resistance to aggressive root-knot nematodes.

Development, Testing and Introduction of Grape Rootstocks with Broad and Durable Nematode Resistance

AVF Proj. ID: 304 / /Cat.: ,
Primary Investigator(s): Howard Ferris, Andrew Walkerby

We continue toward our goal of developing and releasing grape rootstocks with broad and durable resistance to nematode species that are important in California vineyards. In previous years, we have screened rootstock candidates against the root-knot nematode (Meloidogyne incognita race 3), two strains of root-knot nematode that overcome the resistance of Harmony rootstock (Meloidogyne arenaria strain A and Meloidogyne incognitastrain C), and the dagger nematode (Xiphinema index). Fourteen rootstock candidates exhibit broad resistance to those nematodes. This year, we continued to test the breadth of that resistance beyond the range of the primary screen species by evaluating the resistance of the 14 candidates to the ring nematode, Mesocriconema xenoplax, in the presence of other nematode species.

We also evaluated ring nematode resistance in the parents of the current rootstock candidates and in some other Vitis sources. Only two of the rootstock candidates exhibit any resistance to the ring nematode and that may not be durable when other nematodes are present. We continue to seek new sources of resistance. We also continued to test the durability of nematode resistance of the rootstock candidates when they are exposed to combinations of nematode species by determining the durability of resistance at different temperatures. Resistance of the parents of the rootstock candidates to several root-knot nematode variants was compromised at soil temperatures of 30°C and above but not below 27°C. However, some of the rootstock parents maintained resistance to even the virulent Meloidogyne arenaria strain A at high temperatures, indicating that there is durability to temperature among the parentage.

Field testing of the rootstock candidates continues in fields that were heavily infested with root-knot nematodes. Nematode population levels are declining in the root-zones of all rootstock candidates, indicating that reproduction of root-knot nematodes is not occurring. However, population levels of ring nematodes at the field site are high on most of the selections, underscoring the need for obtaining new sources of resistance to that nematode.

Expression of Anti-microbial Genes in Transgenic Grapevines for Enhanced Disease Resistance

AVF Proj. ID: 311 / /Cat.: ,
Primary Investigator(s): Bruce Reischby

Grapevines are susceptible to numerous diseases harming both plants and profits. Transgenic grapevines that resist disease would provide better disease control as well as economic benefits from the reduction in spray applications. Our overall goal has been to research and develop methods to create transgenic selections of elite cultivars with improved resistance to diseases. The transgenic strategy is especially appropriate for clonally-propagated crops, such as grapevines, where the wine industry is rooted in traditional European grapes with strong name recognition and very high disease susceptibility. During the past year, we screened six different antimicrobial peptides, which are small proteins known to be inhibitory to a range of bacteria and fungi, to determine which might best provide resistance to bunch rot (Botrytis) and crown gall (Agrobacterium vitis). These same peptides are also being tested for their effects on germinationof powdery mildew conidia. Based on the incoming results from peptide screening, development of new gene constructs is underway. These constructs will be inserted into Chardonnay and the resulting vines will be tested for improvements in disease resistance.

Application of molecular advances from tomato root-knot nematode Resistance to Use with Grape Rootstalks

AVF Proj. ID: 314 / /Cat.: ,
Primary Investigator(s): Andrew Walker, Brady Smithby

The overall goal of this research is to characterize resistance to root-knot nematodes (RKN) in grapevine rootstocks. These nematodes are an important grape pest, and strategies to minimize nematode damage are an important component of grape production in many grapegrowing regions worldwide. Numerous genes that confer resistance against plant parasitic nematodes have been described, and several of these have now been cloned. The best studied of these genes is the tomato gene Mi-1, which confers resistance against several species of RKN including Meloidogyne incognita, M. javanica, and M. arenaria. Response to RKN in resistant grape rootstocks resembles the Mi-1-mediated resistance in tomato. For example, root tips of the resistant grape rootstock Harmony develop a hypersensitive response when penetrated by juveniles from M. incognita Race 3; in addition, the resistance phenotype in both tomato and grape are effective only at temperatures below 32 °C. Since no nematode resistance gene from grape has yet been cloned, we are interested in applying molecular tools and knowledge developed from studying the tomato Mi-1 gene to grapevine-nematode interactions.

From previous studies, we have identified 77 unique cDNA candidate clones whose mRNAs may be expressed more abundantly preceding Mi-1 associated cell death by suppression subtractive hybridization. Here we propose to use an established set of assays to test those cDNA clones to identify signaling pathway components leading to nematode resistance and their relationship to cell death. These assays include determining the effect of altered expression levels of a candidate cDNA on cell death in a leaf infiltration assay and determination of the nematode resistance phenotype in transformed roots. To examine the biological function of these candidate genes relative to cell death, we employed virus-induced gene silencing (VIGS) using the potato virus X (PVX) vector as a gene knockout system. These cDNA clones have been amplified by PCR to sub-clone into the PVX binary vector pGr106. The cell death relevant genes will be identified using VIGS as a functional assay to be followed by northern blot analysis to confirm induction of selected clones.

Research progress in non-model plants like grapevine is contingent upon the effectiveness of plant transformation technology. An important tool for root biologists is the Agrobacterium rhizogenes-derived composite plant, which has made possible genetic analyses in a wide variety of transformation recalcitrant dicotyledonous plants. To further test nematode resistance in transgenic roots harboring genes of interest, we have adapted an ex vitro protocol to evaluate the potential of this technique to produce transgenic composite grapevine plants. We need to alter and improve this technique to generate transgenic grapevine roots for future study. In addition, we found that kanamycin is not a suitable selection marker for composite plant regeneration because too many putative transformants are not affected and escape detection. Efforts to optimize A. rhizogenes-mediated transformation using embryogenic tissue of various grape cultivars is underway in our laboratory.

Characterization and Utilization of Rootstock that Promote Normal Scion Fruit Set, Despite Presence of Grapevine Fanleaf Virus (GFLV)

AVF Proj. ID: 315 / /Cat.: ,
Primary Investigator(s): Andrew Walker, Brady Smithby

This research was directed at determining how the Muscadinia rotundifolia based rootstock O39-16 induces tolerance to grapevine fanleaf virus (GFLV), and developing genetic markers associated with this trait to facilitate breeding of fanleaf degeneration resistant rootstocks. O39-16 is resistant to GFLV?s dagger nematode vector (Xiphinema index), but the nematode?s test feeding is able to inoculate GFLV which moves freely into the scion. GFLV affects crop yields by disrupting pollination and greatly reducing berry set.

However, if a scion is grafted to O39-16 it maintains normal crops even while infected. Other dagger nematode resistant rootstocks do not possess O39-16?s ability to induce fanleaf tolerance. This effect is likely due to O39-16?s M. rotundifolia parentage and that a flowering-associated phytohormone generated by its root system is compensating for GFLV?s impact on flowering. The most obvious phytohormone is cytokinin, which is produced primarily by the root system and known to be involved in flowering.

In previous studies we determined that rootstocks capable of inducing GFLV tolerance do not impede the virus movement or alter the virus foliar disease symptoms. Various grafted combinations of O39-16, O43-43 (a sibling rootstock with the same fanleaf tolerance), St. George and Cabernet Sauvignon (both highly susceptible to both GFLV and X. index) have been created in standard, reverse, and interstock arrangements. These combinations were bench grafted with both infected and healthy Cabernet Sauvignon. ELISA data indicate that GFLV is able to move freely across the graft union of all genotypes, regardless of positioning of VR rootstock in the grafted vine. Additionally, tissue samples isolated from scions where M. rotundifolia served as an interstock grafted to a GFLV infected rootstock, generated foliar symptoms in the GFLV herbaceous indicator, Chenopodium quinoa. The presence of the portion of the viral genome that encodes for the GFLV coat protein has been verified in scions of vines grown on both St. George and O39-16. Based upon these results, we believe that induced tolerance is best explained by a mechanism which does not directly interact with the virus.

A modified phytohormone separation protocol for HPLC was adopted, this method allows for the separation of auxin, abscisic acid, and cytokinin from a single sample. The initial results of HPLC separation of cytokinin into the nucleoside, riboside and base conformation were verified through competitive ELISA. This method of analysis will be used to track the phytohormonal status of both rootstock and scion of an 80 vine experimental plot planted this year at UC Davis. Samples will be collected throughout the growing season, with particular focus on the ratio of cytokinins to abscisic acid in the developing inflorescence and flowers of GFLV infected vines.

We have begun the process of generating simple sequence repeat (SSR) based linkage map of F1 hybrid of Vitis vinifera x M. rotundifolia. Previous work by H.P. Olmo found this population segregates for resistance to phylloxera, root and dagger nematode, as well as many morphological traits. It is predicted that this population will also segregate forinduced tolerance to fanleaf degeneration.

Evaluation of Zinfandel Heritage Selections

AVF Proj. ID: 318 / /Cat.: ,
Primary Investigator(s): James Wolpertby

Data were taken for the first time in 2005 on ?Phase 2? of the Zinfandel Heritage Vineyard which is the large, replicated group of 22 clones taken from the Phase 1 vineyard.

Objective(s) and Experiments Conducted to Meet Stated Objective(s):

  1. Viticultural evaluation of Zinfandel Heritage selections
  2. Evaluation of wine made from a select number a Zinfandel Heritage Selections.
  3. Viticultural and enological evaluation of Zinfandel Heritage Selections in the new replicated trial.

Summary of Major Research Accomplishments and Results (by Objective):

In consultation with Zinfandel Advocates and Producers? (ZAP) Research Committee, a decision was made to set aside objectives 1 and 2 in preference to Objective 3. Additional investment in data from the unreplicated vineyard and of small lot fermentations did not make sense in light of the better data and larger wine lots available. Objective involved the viticultural and enological evaluation of Zinfandel Heritage Selections in the new replicated trial.

Twenty-two selections were evaluated for yield components. Yields varied from 2.8 to 6.0 kg/vine. While clusters per vine are somewhat regulated (particularly in young vines relative to vine vigor) they still varied from a low of 14 to a high of 18. The

greatest difference in yield (as is typical for clones) was due to cluster weight, varying about two-fold, from 182 g to 374 g. Berries per cluster varied by about two fold as well, from a low of 89 berries to a high of 181. Berry wt values varied remarkably little from 2.0 to 2.2. Clones were picked on two dates September 9 and September 20. Berry sample Brix at harvest varied from a low of 22.6 to a high of 25.4. An attempt was made to harvest the plots at lower sugar values because of previous years? difficulties completing fermentations in high Brix juices. Values for pH with the Zinfandel at the Oakville Station Vineyards, were low ranging from 3.08 to 3.23 which was consistent with historical values, and TA levels ranged from 6.63 to 7.56.

Wines were made at the Ravenswood winery under the direction of Don Williams and Joel Peterson. Wine data will be available later in the spring. Pruning weight data will be taken later in the winter.

This larger trial will be the source of the most important data from the Heritage Vineyard, as the wine lots will be large enough to give more relevant experience with wines. However, it is too early to place a great deal of value on this data. As vines mature and additional years of data are collected, we will be better able to develop stronger conclusions about the zinfandel selections from the larger replicated trials with greater confidence.