Breeding rootstocks resistant to aggressive root-knot nematodes

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

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

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

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)

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 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

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.

Breeding Rootstocks Resistant to Aggressive Root-knot Nematodes

The objectives for this study are to breed, evaluate, and introduce rootstocks that are resistant to aggressive root-knot nematodes, resulting in improved varieties with adaptation to California viticulture. To achieve this objective, our goal was to evaluate the root-knot nematode resistance of 12,000 grape rootstock seedlings and select resistant seedlings for advancement to the field. We will make crosses specifically for the breeding of rootstocks resistant to aggressive root-knot nematodes.

The USDA grape rootstock improvement program, based at the Plant Genetic Resources 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 2004 we screened 5124 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 81 nematode resistant selections that will be ready for vineyard planting in spring 2004. In 2004 we planted 89 nematode resistant rootstock selections in the vineyard. These selections were identified in nematode resistance screening in 2003 and 2002. In 2004 we pollinated 1500 clusters of crosses specifically aimed at the breeding of improved rootstocks with resistance to aggressive root-knot nematodes.

Developing a Functional Genomics Approach to Berry Ripening and Defense

Our work with fruit ripening and defense related genes is now complete with regard to collecting EST sequences. With the ones we have already collected and the many thousands that are now planned in efforts on this campus and internationally we feel that it will be possible to clone nearly any grape gene we are interested in by application of sequence information and PCR methods. This means that studies where information about gene expression in grape is needed can be undertaken with confidence that most of the genes of interest can be easily cloned. Nevertheless, the picture that has emerged from our work with the veraison stage library is very informative and is a major research accomplishment and result.

We divided the EST sequences we obtained into three classes. Class one are genes for which the role in fruit development or ripening is already known in other systems (e.g. tomato). An example of class-one genes would be polygalacturonase. The second class includes those genes whose function can be known with a high degree of certainty but where its role in fruit development or ripening is unknown. An example of class-two genes would be a tonoplast intrinsic protein. The third class contains those genes that are unknown (a match was found in the data base but no function has been assigned to the protein) hypothetical proteins (e.g. an open reading frame from Arabidopsis) or no match (i.e. no match at all was found in the data base). In our total EST collection 34%of the genes were assigned to class 1; 44%to class 2; and 22%to class 3.

In order to provide a functional classification for the genes in class 1 and class 2 above, we assigned each gene to a functional category. The major group in the functional category contained genes related to stress responses; either oxidative, osmotic or water stress. We found that 22%of the genes were stress related and 20%were related to protein synthesis, processing and degradation. We found that 18%of the genes to which we could assign a function are related to disease resistance, and 8%involved with signal transduction. There were 9%related to RNA processing or were known transcription factors. Other groups with about 5%each were cell wall chemistry, secondary metabolism and photosynthesis.

The view of grape berry ripening that has emerged from our work is much different than we expected when we began to sequence ESTs from veraison berries. The large number of stress induced genes and disease related genes we found was surprising and unexpected. Nevertheless, this result has drawn our attention to the function of stress responses and plant defense gene expression in fruit ripening and berry composition.

PDF: Developing a Functional Genomics Approach to Berry Ripening and Defense

Enhancement of Stress Tolerance in Vitis vinifera

Abiotic stresses affect important aroma, flavor and color components by altering metabolite composition, improving wine quality and human health benefits. Regulated deficit irrigation has been used successfully to grow grapes with less water, an important feature in arid regions such as Nevada. As a first step toward understanding how growth is affected and wine quality improvements might arise following abiotic stress exposure, we have initiated an expressed sequence tag (EST)-based gene discovery program focused solely on stressed plants. We constructed cDNA libraries from mRNA isolated from leaf and berry tissues of Vitis vinifera cv. Chardonnay, exposed to various abiotic stress conditions. To date, we have sequenced over 3000 leaf ESTs and anticipate completing another 5000 berry sequences over the next few months before funds run out this year. Raw sequence data were processed through an automated EST analysis pipeline (ESTAP) developed at the Virginia Bioinformatics Institute (VBI; Blacksburg, VA) in collaboration with UNR and S.R. Noble Foundation (Ardmore, OK). Initial sequence analysis reveals 36%novel genes and a low redundancy of transcripts. All 1878 unique EST data generated to date have been deposited in GenBank and is freely available to the public.

In the context of genetic engineering more cold tolerant grapes, we have successfully transformed and regenerated V. vinifera in our laboratory. Embryo culture was successfully initiated using 0.5 to 1 mm immature anthers that were excised from flowers of Chardonnay plants. The excised anthers were placed on one of the following callus-initiation mediums: NB, PT or PIV medium. Tissues were subcultured to fresh plates every 6-8 weeks. Embryogenic calli suitable for transformation formed on some cultures. Single cell somatic embryos were transformed with CBF1 and CBF3 constructs including the CAMV35S promoter. The CBF/DRE transcriptional activators, CBF1 (DREB1B), CBF2 (DREB1C) and CBF3 (DREB1A) are some of the master switches for drought, salinity and cold tolerance. Eleven transgenic plants have been regenerated after selection on Kanamycin media. Three plants have been positively identified by PCR for transformation with CBF3. Confirmation of transformation by PCR and Southern analysis for the rest of the plants is underway. A second batch of transformed somatic embryos is currently going through the regeneration process. Degenerate primers designed to motifs of the CBF gene family have produced 8 distinct PCR products. These products represent putative grape CBF orthologs. Additional CBF orthologs are expected to be obtained from our on going EST sequencing program.
Must samples were obtained from well-watered and drought-stressed Chardonnay grapes. Standards were developed for gas chromatography/mass spectroscopy in preparation for must analysis.

PDF: Enhancement of Stress Tolerance in Vitis vinifera

Evaluation of Selected Mediterranean Wine Grape Cultivars and Clones in Lake and Mendocino Counties

During the year 2000, phenological and harvest data were taken from three Mediterranean Winegrape Cultivar Test Plots in Lake and Mendocino Counties. Results indicate that there are tremendous differences in budbreak, yield and harvest dates between the cultivars and locations.

In Lake County, all cultivars reached ripeness (23.5 brix) in the Red Hills plot, whereas only the earliest cultivars reached ripeness in the Highland Springs plot. This can be explained partially by the warmer climate of Red Hills, but also by the smaller crop size and lighter soils in that plot compared to larger crops and heavier, cooler soils found in Highland Springs. In general, the ripening sequence is as follows: Pinotage, Barbera, Dolcetto, Grenache, Nebbiolo, Cabernet sauvignon, Sangiovese, Cinsault, Syrah,. Harvest occurred from Sept.15, to Oct. 27th, 2000. At Highland Springs, only Pinotage fully ripened during the 2000 harvest.

At the UC Hopland Research and Extension Center, there is a greater number of cultivars being tested. Ripening sequence this year was Pinot gris, Viognier, Tempranillo, Cinsault, Grenache, Syrah, Sangiovese, Dolcetto, Viognier (?Bonny Doon? clone), Fiano, Freisa, Mourvedre, Nebbiolo, Corvina, and Aglianico. Canailo nero, Montepulciano and Marsanne failed to adequately ripen, and were lost due to rain damage. A research progress report of the UC HREC data is being prepared for a California Agriculture magazine issue featuring the 50th anniversary of the UC Hopland Research and Extension Center.

In nearly all cases, quality of the fruit was quite high, showing adequate sugar (23.5 brix), good acidity, and very little rot. For the combined trials, 22 lots of wine are being fermented. It is clear that there are many cultivar options for quality winemaking in Lake and Mendocino Counties.

We were not able to plant a clonal trial of Syrah during this season. Vines will be planted in 2001. Evaluations of 5 Syrah clones were made from commercial plantings at McDowell Valley Vineyards. Clones include Syrah noir (?Hermitage? clone), Shiraz (UC clone #1), McDowell clone, CTPS #877, and CTPS #174. Significant differences in cluster size, shape and yield per vines were noted, as well as must chemistry. Separate lots of wine were vinified, and there are definite organoleptic differences between the clones. While not statistically valid, these observations are still helpful in assessing differences between the Syrah clones. Information from this survey will be presented at the ASEV Syrah Symposium in San Diego in June, 2001.

PDF: Evaluation of Selected Mediterranean Wine Grape Cultivars and Clones in Lake and Mendocino Counties