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

This year we have focused our attention on obtaining a broader genetic base for resistance to virulent pathotypes of root-knot (Meloidogyne spp.) and dagger nematodes (Xiphinema index),broadening our search for resistance to ring nematode (Mesocriconema xenoplax) and to sources of resistance to pin nematode (Paratylenchus sp.). We have invested considerable effort into developing sterile dual-culture techniques which will allow us to understand the mechanisms of resistance provided by various genetic sources. We continue with our efforts to develop culture techniques for Xiphinema americanum and in monitoring the performance of resistant rootstocks in field trials. We continue to expand and to improve accessibility to the database for plant resistance to nematodes and for selection of rotation and cover crops. Further we have disseminated results of our research to end users and in scientific media.

Breeding Rootstocks Resistant to Aggressive Root-Knot Nematodes

Summary:
The USDA Agricultural Research Service (USDA-ARS) grape rootstock improvement program in Geneva, NY has gone through some significant personnel and management changes in the past two years as a result of the resignation of Dr. Peter Cousins from his ARS rootstock breeder position and the abolishment of the vacated rootstock breeder position by ARS due to the recent federal budget cuts. However, there are many promising rootstock selections in the current Geneva rootstock breeding pipeline and it is vital to ensure that these selections are maintained and carried through the breeding process. To meet this challenge, a multi-discipline and –institution cooperative research team has been formed under the encouragement and endorsement of the California grape industry. Dr. Matthew Fidelibus of University of California-Kearney Agricultural Research and Extension Center (UC-KARE) will lead the effort for evaluation of horticultural characteristics and graft performance of the rootstock selections generated from the Geneva rootstock breeding program. Dr. Gan-Yuan Zhong of the USDA-ARS Grape Genetics Research Unit (GGRU) in Geneva, NY, on the other hand, will lead the effort for creating rootstock breeding populations and evaluating these populations and advanced selections for root-knot nematode (RKN) resistance and propagation ability. By forming such a team, we can effectively leverage complementary expertise and capabilities from different organizations to meet the overall project objectives in a timely and efficient manner.

The new team has been in operation since summer of 2013. In spite of the transition, we accomplished all the major goals of 2013. We maintained 687 resistant rootstock selections/mothervines at the UC KARE in Parlier, CA. We worked with nursery representatives and identified 30-40 of the selections for further evaluation on the basis of horticultural characteristics. We screened 854 grape rootstock seedlings for resistance to aggressive root-knot nematodes and retained more than 100 candidate selections for further evaluation. We select only those seedlings which completely suppress nematode reproduction and show zero nematode egg masses. We tested the propagation ability of 49 selections which are currently planted in KARE (already tested once for nematode resistance). We also screened a population, which also is qualified for consideration as rootstocks, for nematode resistance marker development. We continued the effort in evaluating 22 selections, grafted to Syrah, in replicated rootstock trials at the UC-KARE. Matador, Minotaur, and Kingfisher rootstocks, released by this USDA ARS grape rootstock breeding program in 2010, are being distributed by Foundation Plant Services and planted by California nurseries.

Development and Application of Next Generation Sequencing to Facilitate the Release of New Grapevine Accessions in Quaraions in Quarantine and Certification Programs

Recent developments in molecular technology have led to significant improvements in detection and control of many pathogens. The use of those techniques for pathogen detection in quarantine and certification programs has not yet been universally accepted. This is primarily because of the need to validate these techniques and determine their limitations. We are proposing to supply the data for that validation for the case of grapevine registration and certification. We will make a side-by-side comparison of 1) the classical, currently used technique for the analysis of viral pathogens of grapevine, with 2) the more recently developed technology of Next Generation. Sequencing (NGS). In both cases, we will test the same set of fifty selected grapevine accessions infected with one or multiple viruses of importance to the grape industry. In the first case, viral pathogens will be analyzed using biological assays on a standard herbaceous and woody index panel of host plants, as is required by APHIS and CDFA for certification. We will compare the results from that bioassay with a second analysis based on NGS of the total grapevine viruses in each of the selected accessions. The two tests will run concurrently. We expect to show that, for the evaluation of the disease status of grapevine stocks, NGS is superior to biological assay, as well as to ELISA, RT-PCR and real time RT-qPCR in sensitivity, reliability, speed and labor intensity, and cost. We will make the case for the replacement of biological assays with NGS for the certification of novel grapevine accessions. Our data will be useful to federal and state regulatory agencies as evidence supporting the revision of the existing mandated protocols for the testing and release of novel grapevine accessions from quarantine. The improvements brought with the up-date to NGS technology for this application will be of significant benefit to the grape growing industry.

The first year objectives of this project have been met. We have identified the first batch of 20 grapevine accessions that carry infections of agronomic importance, for use in the comparative demonstration of the effectiveness of the two techniques evaluated in this project. We have   chipbud grafted material from each of those to the standard four bioassay index hosts, and begun their two year incubation period toward symptom scoring. We have also made total RNA extractions from those infected plants and begun NGS analysis, using BLAST sequence comparisons to subtract the host coded sequences from those of the pathogens of interest. This progress will generate the data for the comparisons between the techniques, which will meet the subsequent objectives of this proposal.

Development of Next Generation Rootstocks for California Vineyards

The 2013 crosses focused on developing rootstocks with deeper root systems, the genetics of root architecture traits, and introgressing the excellent soil pest resistance from rotundifolia into rootstocks using semi-fertile vinifera x rotundifolia (VR) hybrids (see Table 1). This may also be a way to incorporate fanleaf tolerance and allow improvement of O39-16. VR hybrids are normally sterile but a few were selected by Olmo to have some fertility. Unfortunately they are also crosses with vinifera so we must be assured of their phylloxera resistance (studies underway).

GRN Field Trials – This was the first year data was gathered from GRN rootstock trials; most of which are being overseen by farm advisers and Constellation. We took crop yields at a trial in Dunnigan with Franzia and another in Lodi with Gallo. This data will be combined with pruning weights (not yet taken) and presented with the next report and as a bulletin to nurseries and cooperators.

Nematode testing – We work closely with Howard Ferris and his technician to evaluate the nematode resistance of rootstock breeding populations. Nin Romero (my chief greenhouse and field technician) propagated and assisted with the nematode resistance screening of hundreds of seedlings this year. Nina and I first examined the populations and evaluated them for brushy growth, internode length, and vigor. Most were also evaluated for their ability to root from dormant cuttings. They were tested for resistance to the Harmony/Freedom aggressive root-knot strains (HarmA and HarmC) and Xiphinema index, and many were also screened for ring nematode resistance. The best 21 are shown in Table 2 and will be advanced to field testing on the UC Davis campus with 101-14 and 1103P comparison controls.

Fanleaf – We continue to make progress on identifying and verifying the function of the Xiphinema index resistance gene from V. arizonica b42-26, and it resistance locus XiR1. Two gene candidates are members of the NB-LRR (nucleotide binding-leucine rich repeat) resistance gene family that control recognition of pests and diseases and the triggering of a defense reaction. These two candidates were transformed into St. George and Thompson Seedless and they reduced susceptibility to X. index resistance, but the transformed plants were still susceptible. There are more lines to test and we are examining gene expression with qPCR and will pursue native promoters to determine if they can increase resistance.

Breeding Rootstocks Resistant to Aggressive Root-Knot Nematodes

The USDA Agricultural Research Service grape rootstock improvement program, based at the Grape Genetics Research Unit, is breeding 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. We screened 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. Selected seedlings are propagated and then planted into the vineyard. We tested the propagation ability of 168 selections (already tested once for nematode resistance). We also screened a population, which also is qualified for consideration as rootstocks, for nematode resistance marker development. We evaluated 22 selections, grafted to Syrah, in replicated rootstock trials at the University of California Kearney Agricultural Research and Extension center. We pollinated 661 clusters of crosses in more than 60 unique combinations specifically aimed at the breeding of improved rootstocks with resistance to aggressive root-knot nematodes and collected rootstock cross seeds. Matador, Minotaur, and Kingfisher rootstocks, released by this USDA ARS grape rootstock breeding program in 2010, are being distributed by Foundation Plant Services and planted by California nurseries.

Development of Next Generation Rootstocks for California Vineyards

The 2012 crosses focused on drought and salt resistance from newly screened highly resistant accessions of southwestern Vitis species; mapping of 101-14 x 110R; introgression of M. rotundifolia into 161-49C, and 5BB; GRN rootstock crosses with better rooting rootstocks and lower vigor; Ramsey x 1616C for mapping root architecture and salt tolerance; Freedom x St. George to allow study of virus tolerance; 101-14 x SG for virus and salt tolerance; and combining nematode and salt with GRNs and southwestern Vitis spp;

Fanleaf – We have made some progress on confirming the role of cytokinins and their precursors in O39-16’s ability to induce tolerance to fanleaf infection. We have tested a subset of the 101-14 x rotundifolia ‘Trayshed’ population for their cytokinins and there is variation. We will begin applying these potential biomarkers for fanleaf tolerance to this population and field trials. Our Xiphinema index resistance gene (XiR1) gene candidates have been transformed into the susceptible St. George and we should have results on the effect of these XiR1 candidates on preventing feeding by Summer. We have also launched a project to examine O39-16’s effect as a bionematicide. We have identified a field site at Niebaum Coppola; developing potted collections of common cover crops and weeds for X. index feeding studies; and healthy and infected O39-16 root systems to determine whether X. index can reacquire GFLV from them.

Salt and Drought Resistance – we made good progress on a root architecture x drought resistance assay, and have almost finished examining 30 rootstocks with a rhizotron to examine rooting angles and architecture, and confirmed that rooting angles from herbaceous cuttings are an effective trait and that these angles segregate in several potential mapping populations. A second generation cross involving V. berlandieri c9031 and vinifera now established to test this apparently single gene source of chloride exclusion from c9031. A subset will soon be tested and if salt exclusion varies it will be used as a mapping population. Extensive tests documentedthat relative growth rate has a large impact on salt uptake and extra controls are now included on all tests to assess relative growth rate.

Southwest Vitis and Salt Tolerance – Three collection trips were made in 2012 – the Red River of northern Texas and southern Oklahoma; the east flank of the Rockies in Colorado and New Mexico; a transect from Las Vegas to St. George Utah – with the goals of acquiring more highly chloride resistant germplasm and to complete a wide

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.

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

This year we have focused our attention on obtaining a broader genetic base for resistance to virulent pathotypes of root-knot (Meloidogynespp.) and dagger nematodes (Xiphinema index),broadening our search for resistance to ring nematode (Mesocriconema xenoplax) and to sources of resistance to pin nematode (Paratylenchus sp.). We have invested considerable effort into developing sterile dual-culture techniques which will allow us to understand the mechanisms of resistance provided by various genetic sources. We continue with our efforts to develop culture techniques for Xiphinema americanum and in monitoring the performance of resistant rootstocks in field trials. We continue to expand and to improve accessibility to the database for plant resistance to nematodes and for selection of rotation and cover crops. Further we have disseminated results of our research to end users and in scientific media.

Breeding Rootstocks Resistant to Aggressive Root-knot Nematodes

The USDA Agricultural Research Service grape rootstock improvement program, based at the Grape Genetics Research Unit, is breeding 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. We screened 5212 candidate grape rootstock seedlings (representing 34 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. The nematode resistance evaluation total includes 494 seedlings of a genetic study population that also are qualified for consideration as rootstocks. Selected seedlings are propagated and then planted into the vineyard. We tested the propagation ability of 123 selections (already tested once for nematode resistance). We evaluated 22 selections, grafted to Syrah, in replicated rootstock trials at the University of California Kearney Agricultural Research and Extension center. We pollinated 412 clusters of crosses in 48 unique combinations specifically aimed at the breeding of improved rootstocks with resistance to aggressive root-knot nematodes and collected 33,530 rootstock cross seeds. Matador, Minotaur, and Kingfisher rootstocks, released by this USDA ARS grape rootstock breeding program in 2010, are being distributed by Foundation Plant Services and planted by California nurseries.

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

This year we have focused our attention on obtaining a broader genetic base for resistance to root-knot (Meloidogyne spp.) and dagger nematode (Xiphinema index) and broadening our search for resistance to the root lesion nematode (Pratylenchus vulnus), and the citrus nematode (Tylenchulus semipenetrans). We tested the durability of resistance to root-knot nematodes in the UCD-GRN series rootstocks under fluctuating soil temperature conditions. We have continued our tissue culture studies for better understanding of the nature of resistance to nematodes in different rootstocks. We have commenced monitoring ongoing field trials with the UCD-GRN series rootstocks to determine their performance in diverse situations. We have considerably expanded and improved accessibility to the database for plant resistance to nematodes and for selection of rotation and cover crops.