Rootstock Tolerance to Soil Salinity: Impact of Salinity on Popular Grape Rootstocks Grown in Contrasting Soil Types

Summary (by Objective): To date, we have accomplished several major goals: Objective 1: Soil Characterization; Monitor the impact of salt accumulation on soil solution chemistry and nutrient balance. The two vineyard sites were selected based on preliminary soil analyses during Year 1. During Years 2 and 3, we performed additional, detailed chemical and physical analyses of the two contrasting soils. The results of these analyses confirmed the differences in morphology and texture between the two soils, and also revealed differences in cation exchange capacity, hydraulic conductivity, and plant available water. In year 4, we applied a saline solution to the soil through the drip system and sampled the soil solution after the salt application. In Year 5, two additional salt applications will be performed; plant tissue and soil solution will be monitored concurrently in order to determine the relationship between soil properties and plant growth and nutrition. Objective 2: Determine the tolerance of a panel of ten rootstocks to irrigation water containing four different salinity levels. (a) Grafting and planting of 10 rootstocks and own-rooted controls at each of the two vineyard sites. The planting scheme was described in section IV above, and a map of one of the vineyard sites is provided in Appendix A. (b) Monitoring of the plants. As expected, some losses among the grafted rootstocks occurred during the first winter. Missing plants were replaced during Year 2 using additional grafted vines provided by the nursery, which had been designated for this purpose. During the warmest weeks of the summer, supplemental irrigation was given to the developing vines to prevent further losses. Subsequent losses, which were minimal, have not been replaced due to the larger size and maturity of the plants. (c) Application of saline irrigation solution. During Year 4, the irrigation system was locally modified to allow the application of solutions with a salinity level of 1.75 mS, 3.5 mS and 4.5 mS. The saline solution was applied using water-powered non-electric chemical injectors coupled with the drip line, during a single, 30-hour irrigation event that was coordinated with the vineyard management staff. The soil solution was sampled following the irrigation event using suction lysimeters buried at depths of 6 and 12 inches, and the chemical analysis of these solutions is ongoing. Leaves and petioles were again sampled at 2, 4 and 6 weeks after the saline solution was applied. The blade tissues are currently being analyzed for chloride ion (Cl-) content, as a marker for salt uptake by the plant tissue. The samples from week 4 are also being analyzed for other macro and micronutrients, as was done for the bloom and veraison samples. Results of these analyses are anticipated in Spring, 2012. In Year 5, two additional applications of saline solution will be performed and the results intensively monitored.

Molecular genetic support to optimize the breeding of fanleaf resistant rootstocks.

This report presents results of Walker lab efforts to optimize the breeding of fanleaf degeneration (fanleaf) resistant rootstocks through molecular genetic methods. These efforts are two-fold: 1) to understand and utilize O39-16?s (a Muscadinia rotundifolia based rootstock) ability to induce tolerance to fanleaf virus infection in scions; and 2) to understand and utilize resistance (XiR1) from Vitis arizonica to the dagger nematode, Xiphinema index, which vectors grapevine fanleaf virus (GFLV) from vine-to-vine by root feeding. We have genetically and physically mapped XiR1 and we have transformed two candidate genes, XiR1.1 and XiR1.2 into V. rupestris St. George, V. vinifera Thompson Seedless, and tomato (all susceptible to X. index feeding) and have produced plantlets that are approaching the size needed to transfer to the greenhouse for X. index inoculation. These studies will determine which of and if these gene candidates controls resistance to X. index. We are also working on ways to rapidly evaluate fanleaf resistance and tolerance through in vitro grafting. We are also preparing to test saps from progeny of the 101-14 x M. rotundifolia population as a means of screening for the ability to induce tolerance to fanleaf disease. We worked with the UC Davis Metabolomics Center director and Doug Adams to identify a group of 5 metabolites and several cytokinin precursors that might be involved in this response based on their levels in infected and healthy O39-16 and St. George. These compounds will be pre-screened in the rotundifolia-based population and about 10 other Vitis species x M. rotundifolia hybrids I have produced. The saps are harvested and are preparing processing at the Metabolomics Center and more will be collected this year.

Breeding grapevine rootstocks for resistance to soil-borne pests and diseases

We made more crosses to increase the number of Muscadinia rotundifolia-based progeny by crossing 101-14 Mgt, 161-49C (V. berlandieri x V. riparia) and 5BB with M. rotundifolia Trayshed pollen to create populations with broad pest resistance and the ability to induce fanleaf tolerance in scions. We have over 200 progeny in the 101-14 x Trayshed population and many are undergoing rooting and horticultural screening this Winter. We have screened subsets of the population for resistance to dagger, root-knot and ring nematodes; they segregate for dagger and root-knot resistance and all resist ring nematodes. To further our study of fanleaf tolerance, we also collected xylem sap from stronger progeny in the 101-14 x Trashed population. We will screen these saps with targeted metabolites from our fanleaf tolerance investigations. We also made crosses to produce PD resistant rootstocks with good nematode resistance and good rooting; better drought and salt tolerance; and crosses to make virus tolerant rootstocks. Cecilia Agüero successfully made constructs of two of the XiR1, Xiphinema index resistance genes we characterized from V. arizonica/girdiana b42-26 (Hwang et al. 2010. Theoretical and Applied Genetics 121:780-799). She has transformed them into St. George and Thompson Seedless, and into tomato (a host of X. index and very easy to transform and study). We should be able to inoculate these plants with X. index later this year to test the function of these gene candidates. This project is primarily funded by the American Vineyard Foundation, and it also supports our efforts to determine which metabolites are responsible for O39-16?s fanleaf tolerance. We will be testing for presence and levels of five metabolites and two cytokinins in saps from O39-16 and about 20 individuals from the 101-14 x Trayshed population. Kevin Fort?s excellent work on the mechanisms of, and screening for, salt tolerance are coming to fruition and four publications are ready for submission. He is now working as a post-doc on generous funding from E&J Gallo who funded a joint project between Andrew McElrone and myself. Kevin is studying interactions between drought and salinity, and working out experiments to accurately screen drought tolerance and to better understand root architecture. He is helping to supervise Claire Heinitz who is studying the eco-genetics of salt tolerance in southwestern Vitis, and Cecilia Osorio who is studying the anatomical basis of drought tolerance. Jean Dodson is also working on drought adaptation by studying the influence of rootstocks on phenological events such as root senescence, leaf drop and harvest dates, which will greatly impact vine water use. Karl Lund is analyzing the feeding behavior of eight phylloxera strains on the root tips of 11 rootstocks and Vitis species. He has also initiated the screening of a V. vinifera x V. berlandieri 9031 mapping population. This V. berlandieri accession has excellent phylloxera resistance and the progeny segregates for resistance, which allows the development of a genetic map for this source of resistance.

Molecular Genetic Support to Optimize the Breeding of Fanleaf Resistant Rootstocks

This report presents results of Walker lab efforts to optimize the breeding of fanleaf degeneration (fanleaf) resistant rootstocks through molecular genetic methods. These efforts are two-fold: 1) to understand and utilize O39-16?s (a Muscadinia rotundifolia based rootstock) ability to induce tolerance to fanleaf virus infection in scions; and 2) to understand and utilize resistance (XiR1) from Vitis arizonica to the dagger nematode, Xiphinema index, which vectors grapevine fanleaf virus (GFLV) from vine-to-vine by root feeding. We have genetically and physically mapped XiR1 and we have transformed two candidate genes, XiR1.1 and XiR1.2 into V. rupestris St. George, V. vinifera Thompson Seedless, and tomato (all susceptible to X. index feeding) and have produced plantlets that are approaching the size needed to transfer to the greenhouse for X. index inoculation. These studies will determine which of and if these gene candidates controls resistance to X. index. We are also working on ways to rapidly evaluate fanleaf resistance and tolerance through in vitro grafting. We are also preparing to test saps from progeny of the 101-14 x M. rotundifolia population as a means of screening for the ability to induce tolerance to fanleaf disease. We worked with the UC Davis Metabolomics Center director and Doug Adams to identify a group of 5 metabolites and several cytokinin precursors that might be involved in this response based on their levels in infected and healthy O39-16 and St. George. These compounds will be pre-screened in the rotundifolia-based population and about 10 other Vitis species x M. rotundifolia hybrids I have produced. The saps are harvested and are preparing processing at the Metabolomics Center and more will be collected this year.

Rootstock Tolerance to Soil Salinity: Impact of Salinity

During Years 1 thru 3 of this project, we made progress towards all three Objectives. For Objective 1, we identified two suitable vineyard plots containing contrasting soils, then sampled and characterized the soil profiles present within these plots. The results of these analyses confirmed the differences in morphology and texture between the two soils, and also revealed differences in cation exchange capacity, hydraulic conductivity, and plant available water. These differences will likely have a significant impact on plant growth and nutrient uptake. For Objective 2, we grafted and planted the various rootstock-scion combinations at both sites. By the end of Year 2, the plants were sufficiently large and well established that we were able to sample petioles and blades at harvest time. Following discussions with the vineyard management staff, the research team decided collectively that the vines were still too small to safely begin treatment with saline water conditions during Year 3. Accordingly, during Year 3 we proceeded with Objective 3, continuing to study baseline differences in plant nutrient uptake, comparing the 10 different rootstocks to one another within a single soil type, and comparing vine replicates grown on identical rootstocks in the two contrasting soil types. There were significant differences between rootstocks for all nutrients studied. In most cases, the rootstock rank order was similar for vines grown on the Alfisol and those grown on the Entisol; however, there were many exceptions to this trend. For the coming year, as described in the renewal application for Year 4, saline irrigation will begin in spring 2011. In Years 4 and 5, plant nutrient uptake and soil solution chemistry will be monitored concurrently in order to determine the relationship 2 between soil salinity and plant nutrition, and to compare rootstocks. This study constitutes a limited rootstock trial employing a panel of 10 rootstocks commonly used in California, comparing two soil types that are also common in California winegrowing regions. This project has high potential for generating information of relevance to California winegrape growers, as it will be one of the most comprehensive studies to date analyzing the effects of soil salinity on specific rootstocks.

Molecular Genetic Support to Optimize the Breeding of Fanleaf Resistant Rootstocks

This report presents results of Walker lab efforts to optimize the breeding of fanleaf degeneration (fanleaf) resistant rootstocks through molecular genetic methods. These efforts are two-fold: 1) to understand and utilize O39-16?s (a Muscadinia rotundifolia based rootstock) ability to induce tolerance to fanleaf virus infection in scions; and 2) to understand and utilize resistance (XiR1) from Vitis arizonica to the dagger nematode, Xiphinema index, which vectors grapevine fanleaf virus (GFLV) from vine-to-vine by root feeding. We made minor adjustments to a genetic map of a small population of vinifera x rotundifolia (VR) hybrids. Dr. Hwang took a new position at Missouri State University and is currently completing a manuscript detailing this map. To better map rotundifolia-based characters in a rootstock background we are turning mapping attention to an expanding population of 101-14Mgt x rotundifolia Trayshed. This population now has about 100 individuals and over 600 viable seed from 2010 crosses are now being germinated to expand the population. Nematode testing has found that the progeny segregate for rooting ability and resistance to dagger and root-knot nematodes, but all are resistant to ring nematode. Once metabolites associated with tolerance to fanleaf are discovered we will examine this population and develop markers to this tolerance. We have completed two years of xylem sap tests from fanleaf infected O39-16 and the highly susceptible St. George compared to uninfected O39-16 and St. George. This analysis was done at the UCD Metabolomics Center and yielded data on thousands of metabolites in grape sap at bud break and bloom. We used the four saps to identify compounds that were at very similar levels in infected O39-16 and uninfected O39-16 and St. George, but different from infected St. George. Dr. Doug Adams helped us identify precursors to phytohormones involved in flowering and other compounds that may play a role in inducing virus tolerance. The final decisions on which of these compounds to pursue this year is waiting for equipment repair at the Metabolomics Center. We do have eight metabolites that appear to be highly associated with rootstock-induced fanleaf tolerance and we will be testing for them in saps from our campus and industry plots this year. The genetic and physical mapping of dagger nematode resistance gene, XiR1, was published after several revisions and submissions (Theoretical and Applied Genetics 121:780-799 (2010)) and we have started testing the first candidate genes (XiR1.1 and XiR1.2) to determine which is responsible for resistance. This process involves engineering the genes into cells from the highly susceptible St. George and testing to see if the resulting transformed plants resist dagger nematodes. The first transformations were recently completed. These tests will not only identify the resistance. We are also working on ways to rapidly evaluate fanleaf resistance and tolerance through in vitro grafting.

Aquaporin-Regulated Response of Grapevine Roots to Salinity and Role in Inherent Differences Among Rootstocks

Soil salinization is an emerging problem in California vineyards. Research is needed to more fully understand the physiological response of grapevine roots to salt stress in order to develop cultural strategies that improve in-field management and to facilitate breeding of tolerance. Upon exposure to salinity, roots often exhibit a rapid decrease of water uptake capacity caused by inhibition of water-channel proteins called aquaporins. Aquaporins are found throughout fine root cellular membranes and can control the efficiency of water extraction from the soil. Prevention and/or alleviation of salinity-induced aquaporin inhibition have been demonstrated for some plants using calcium supplements in experimental conditions. Such a mechanism may contribute to the success of gypsum (i.e. calcium sulfate) applications used to lessen the detrimental effects of vineyard salinity.

In the original grant, we proposed to address the following short-intermediate term goals: 1) to quantify aquaporin response to salinity and the ameliorative effects of calcium in a suite of grapevine rootstocks using both hydraulic physiology and molecular probes under hydroponic and soil growth conditions; and 2) to investigate the role that aquaporins play in grapevine rootstock physiological responses to other abiotic factors (i.e. drought) and their contribution to vine vigor.

Our results indicate that aquaporins play a role in water uptake across numerous Vitis rootstocks. We documented significantly higher inherent aquaporin expression in high vigor and drought resistant rootstocks (e.g. 140Ru, 1103P and 110R) compared to those with low vigor and drought intolerance ratings (e.g. 420A and 101-14). These inherent differences may explain the known variation in vigor among these rootstocks, likely play a role in divergent patterns of drought tolerance, and represent potential target genes for breeding similar traits. In more recent efforts, we characterized anatomical, molecular, and biophysical aspects of fine roots impacting water uptake in Vitis, a woody perennial. This study provides one of the few quantitative analyses of tissues specific aquaporin expression in roots, and the first in a woody species. The study revealed strong parallels in developmental anatomy, distribution of aquaporins, and relationships with Lpr between herbaceous and woody fine roots within the meristematic/elongation and maturation zones. These similarities suggest a common foundation likely underlies the integration of root development and water uptake across plants. Fine root hydraulic permeability along the root length was positively correlated with aquaporin gene expression and negatively correlated with suberin deposition.

For the salinity response of aquaporins, we consistently found a dramatic upregulation of aquaporin gene expression for both the PIP1 and PIP2 aquaporin families. This initial response dampened over time as expression patterns returned to near pre stress conditions. Patterns of expression were similar across rootstocks (i.e. patterns of response were not clearly and consistently associated with resistance or susceptibility to salinity stress among rootstocks). Salinity experiments were done using a variety of experimental procedures and always showed similar results. Hydraulic conductivity of fine roots did not show a concomitant increase after salt stress initiation. This suggests that the increase in aquaporin expression increase following salt stress likely played a more local role on the cellular basis (i.e. by affecting local cell to cell water relations) rather than affecting the bulk tissue conductivity. Similar gene expression patterns were found in our newly developed tissue culture method when roots were transferred to saline media. This method was very successful in enabling us to track growth rate of individual roots over time and after exposure to salt. Fine root growth slowed abruptly upon transfer to saline media, but this effect was ameliorated if gypsum was present in either the establishment media or in the transfer media. These results suggest that gypsum applications commonly used in vineyards to lessen the effects of soil salinity are not only affecting the ion exchange of the soil column, but also having a direct impact on grapevine physiology by enabling the roots to maintain growth despite the saline conditions. More work is needed to explore this in detail and Drs. Walker and McElrone have recently initiated efforts to use the tissue culture system for evaluating Boron toxicity.

Project title: 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 2009 we screened 5126 candidate grape rootstock seedlings (representing 69 different populations) for resistance to aggressive root-knot nematodes. We select only those seedlings which completely suppress nematode reproduction and show zero nematode egg masses. Selected seedlings are propagated and then planted into the vineyard. We screened an additional 420 seedlings for nematode resistance genetics studies. We tested the propagation ability of 190 selections (already tested once for nematode resistance) and of these retested 80 selections to confirm nematode resistance in replicated trials. We planted eleven selections, grafted to Syrah, into a new rootstock trial at the University of California Kearney Ag Center and identified eleven more selections to be grafted to Syrah for a rootstock trial to be planted in 2010. We pollinated 317 clusters of crosses in 55 unique combinations specifically aimed at the breeding of improved rootstocks with resistance to aggressive root-knot nematodes. Virus testing is complete for our most elite selections and several of these are candidates for possible variety release.

Search for, and Development of, Nematode Resistance in Grape Rootstocks

This year we have focused our attention on sources of resistance to the ring nematode. Criconemoides xenoplax; and to obtaining a broader genetic base for resistance to root-knot (Meloidogyne spp) and dagger nematode (Xiphinema index). We have also initiated and are repeating trials to find resistance to the citrus nematode, Tylenchulus semipenetrans, and the root-lesion nematode, Pratylenchus vulnus.

Aquaporin-regulated response of grapevine roots to salinity

Soil salinization is an emerging problem in California vineyards. Research is needed to more fully understand the physiological response of grapevine roots to salt stress in order to develop cultural strategies that improve in-field management and to facilitate breeding of tolerance. Upon exposure to salinity, roots often exhibit a rapid decrease of water uptake capacity caused by inhibition of water-channel proteins called aquaporins. Aquaporins are found throughout fine root cellular membranes and can control the efficiency of water extraction from the soil. Prevention and/or alleviation of salinity-induced aquaporin inhibition have been demonstrated for some plants using calcium supplements in experimental conditions. Such a mechanism may contribute to the success of gypsum (i.e. calcium sulfate) applications used to lessen the detrimental effects of vineyard salinity. In our grant, we proposed to address the following short-intermediate term goals: 1) to quantify aquaporin response to salinity and the ameliorative effects of calcium in a suite of grapevine rootstocks using both hydraulic physiology and molecular probes under hydroponic and soil growth conditions; and 2) to investigate the role that aquaporins play in grapevine rootstock physiological responses to other abiotic factors (i.e. drought, anoxia, nutrient status) and their contribution to vine vigor. We are on target to achieve these goals over a two year funding period (the current Research Support Agreement between USDA-ARS and UCDavis expires 31 September 2010). Our results from 2008 and 2009 indicate that aquaporins play an integral role in water uptake across numerous Vitis rootstocks and exhibit a strong and rapid response to salinity stress. In 2008, we documented significantly higher inherent aquaporin expression in high vigor and drought resistant rootstocks (1103P and 110R) compared to those with low vigor and drought intolerance ratings (420A and 101-14). In additional experiments conducted during 2009, we continued to find this pattern regardless of the growing conditions (i.e. soil or hydroponics). These inherent differences may contribute to the known variation in vigor and drought tolerance among these rootstocks, and represent potential target genes for breeding similar traits. In 2009, we continued to assess the response of roots to salinity stress and the ameliorative affects of calcium. Aquaporin gene expression in numerous rootstocks (Ramsey, Riparia, French Colombard, Thompson seedless, 420A, 101-14, 110R, 039-16, 5BB, 1103P) was highly responsive to salt stress. In most of the rootstocks studied, expression increased significantly under salinity stress across all PIP1 and PIP2 aquaporin genes. In a subsequent hydroponic study, we tracked the hydraulic conductivity of fine roots under control, NaCl, or NaCl plus calcium treatments. We found no ameliorative effects of calcium, but did find that conductivity under salinity was maintained similarly to controls for some of the rootstocks. An up-regulation of aquaporin gene expression under salinity conditions likely plays a role in this response (gene expression for these roots will be completed by Spring 2010). We have begun additional experiments to determine if long term growth in calcium enables tolerance to future exposure to salt stress.