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.

Alternative Trellising Systems for Chardonnay and Merlot Vineyards in the Central Coast

The purpose of this study is to compare the performance of bilateral cordon trained, spur pruned Chardonnay and Merlot grapevines trellised to the vertically shoot positioned trellis system (VSP), the Smart-Henry trellis system (SH) and the Smart-Dyson trellis system (SD) in the Salinas Valley of California. In the first year of the study, VSP vines had less primary, lateral and total leaf area compared to SD and SH vines. Canopies oriented upward on both the SD and SH systems generally had more leaf area than canopies oriented downward. VSP vines had greater leaf layer numbers in the fruiting zone compared to SD and SH vines, and these treatments had greater amounts of sunlight in the fruit zone compared to the VSP treatment. Vine yield components were similar among the treatments in Chardonnay, while SH vines in Merlot produced significantly greater yields compared to the other treatments. In both cultivars, trends toward reduced yields for downward-oriented canopies compared to upward-oriented canopies were observed. Merlot fruit from downward-oriented shoots had lower berry weights, reduced rates of sugar accumulation and higher titratable acidity compared to fruit from upward-oriented shoots. Fruit on downward-oriented shoots on the SH system exhibited this trend more strongly than downward-oriented shoots on the SD system. At harvest, however, no significant difference in combined fruit (upper and lower canopies) soluble solids, titratable acidity and pH were observed among the treatments in either cultivar. Wine lots made from each treatment will be evaluated in late 1999.

Genetic transformation: A Means to Add Disease Resistance to Existing Grape

The aim of this project is to develop a means by which genes can be introduced into existing grape varieties. The genes being used at this early stage are model genes that are easy to detect in the laboratory but ultimately genes will be introduced that are intended to confer new characteristics without otherwise altering the distinctive characteristics of the variety. Genes conferring resistance to specific diseases and pests are of particular interest. During 1998-99, six transformation experiments were initiated and monitored for 6 to 9 months each. The experiments involved Chardonnay, Thompson Seedless, St. George and 11 OR and the experimental factors tested in the experiments included the culture medium, the gene vector and the selection conditions. Two of the experiments have yielded both somatic embryos and a few plantlets that developed after co-cultivation with the gene vector and exposure to selective pressure designed to allow only cells carrying the introduced genes to survive. Some of the embryos exhibit the blue color that indicates expression of the introduced GUS marker gene. However, none of the three plantlets that were tested expressed GUS. Several additional transformation experiments are in progress and it is still too early to evaluate them. During April and May 1999, approximately 8,000 immature grape flowers were dissected in order to culture their anthers to produce new embryogenic cultures. We initiate new cultures each spring so as to always have a supply of relatively young cultures for transformation experiments. Older cultures may accumulate genetic errors and can lose their ability to regenerate plants. Several research groups in other countries and in private companies are already well along in this kind of research, but public research in California has lagged behind because funding was not available for such long-term research. Now that financial support for viticulture research has increased, public research in this field can be undertaken at the University of California. It may lead to better pest and disease management and ultimately to better control of fruit composition.