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.
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.
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.
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
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.
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.
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.
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.
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.
This report presents results on 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 from Vitis arizonica to the dagger nematode, Xiphinema index, which vectors grapevine fanleaf virus (GFLV) from vine to vine. We are in the process of repeating and clarifying past mapping and xylem sap analysis. We hope to have the previous work verified and corrected by Summer. The field trials we established to study xylem borne compounds with an influence on fanleaf infection will fruit well for the first time this Summer and we have renewed our efforts to determine the basis of induced tolerance. We alsosuccessfully completed a reworking of our fine-scale mapping efforts and that publication is submitted. This work will generate gene candidates for XiR1, the locus we have identified as responsible for X. index, resistance as derived from V. arizonica. This discovery will be followed with transformation experiments to confirm the resistance function of these candidate genes and allow us to use traditional breeding methods more carefully to avoid the breakdown of resistance and might lead to grape rootstocks genetically engineered with grape resistance genes.