Effector-assisted breeding against powdery mildew in grapes.

In last three decades, a great deal of effort and resources have been placed by the grape breeding community on the identification of resistance (R) loci against the grape powdery mildew (GPM) pathogen Erysiphe necator, and their introgression in grapevine varieties of interest. However, the success of these breeding programs can be put at risk, if R-loci that the pathogen can overcome are deployed in the field. Moreover, breeders currently lack the tools that would enable them to swiftly identify new sources of resistance, and predict their durability under field conditions. Effector- assisted breeding has proven a potent contributor to modern breeding for the identification, functional characterization, and deployment of R-genes. The basis of the effector-assisted breeding lays on the principle that dominant R-genes typically mediate resistance through the recognition of matching effector proteins from the pathogen. During the first year of the project, we took the first steps towards the development of an effector-assisted breeding program in grape. Specifically, efforts were made to develop an Agrobacterium tumefaciens-based assay for the transient expression of GPM effectors in select grapevine lines that are currently used at the UC Davis GPM resistance breeding program, thus enabling the functional profiling of matching effector and R- gene pairs. Using as bioreporter the gene encoding for the enhanced green fluorescent protein (eGFP), different combinations of binary plasmid vectors and A. tumefaciens strains were tested for their efficacy to transiently express eGFP in Nicotiana benthamiana, as this is a more amendable and easier to work with system. The best binary vector/A. tumefaciens strains combinations were then tested for their ability to transiently express eGFP in select grapevine lines. A protocol for vacuum infiltrating detached grapevine leaves instead of entire plantlets was developed in parallel, thereby increasing the assay’s practicality and capacity. To this end, we were able to work out the conditions for achieving good levels of agroinfiltration into detached grapevine leaves but the expression level of the transgenes in agroinfiltrated tissue remains low and currently undergoes further optimization. Nonetheless, a set of 35 effector-encoding genes were cloned in one of our select binary vectors that we further modified it to add a 6x-His tag at the C-terminus of each transiently effector and their transient expression was tested in N. benthamaina. For 30 of these effectors we were able to confirm expression in this plant species either by means of a western blot analysis using an anti-6x-His tag antibody or by visual inspection of the plants, as at least six of these effectors triggered cell death in the agroinfiltrated tissue. We are currently completing the cloning of the 35 effectors in a second binary vector and we soon test their transient expression in our select grapevine lines as well.

Furthering Elite Grape Rootstocks With Reduced Nematode Susceptibility

The long-time plantings of vineyards are continuously at risk for infection by soil-borne pests and diseases.  These have ample time and opportunity to establish, build-up in population densities and cause damage.  Depending on the severity of infection, plant-parasitic nematodes can become major yield constraints.  In vineyard soils, soil-dwelling plant-parasitic nematodes can reproduce on susceptible plant roots.  Multiple species are often found at a location.   Meloidogyne spp.  (root-knot nematodes), Mescocriconema xenoplax (ring nematode), Xiphinema americanum or X.  index (dagger nematode), and Tylenchulus semipenetrans (citrus nematode) are most frequent and most damaging.  These can occur in various combinations within at least the upper 5-ft soil layers.  Utility of resistant rootstocks is challenged under these conditions of multiple species being present.  In a prior project, rootstock genotypes that had been selected under greenhouse conditions were tested under field conditions in nematode-infested soils following a >30-year-old planting of ‘Thompson seedless’ grape.  They were grafted to ‘Pinot Gris’ and produced yields in 2022 and 2023.  Twelve genotypes were selected from these studies based on their propagation characteristics, vigor under nematode-infested conditions, and productivity of grape ‘Pinot Gris’.  In the current project year, dormant wood cuttings were made, bench-grafted to ‘Pinot Gris’, and planted to an experimental vineyard in early summer 2023.  Cleanliness from virus (GFLV, GLRaV-3, GRBV) of these cuttings was attested by Plant Foundation Services (FPS) before propagation.

Evaluating Grape Rootstocks for Nematode Susceptibility

In California grape production is one of the perennial high-value commodities. Because of the traditional long time period a vineyard is cultivated, soil-borne pests and diseases have ample time and opportunity to build-up and be major yield constraints. Once introduced in vineyard soil, these soil-dwelling parasites can reproduce on susceptible plant roots copiously. Typically multiple species including Meloidogyne spp. (root-knot nematodes), Mescocriconema xenoplax (ring nematode), Xiphinema americanum or X. index (dagger nematode), and Tylenchulus semipenetrans (citrus nematode) and others occur in various combinations in vineyard soil and damage the crop. Once they are established in an existing vineyard they are difficult to manage because they colonize at least the upper 5-ft soil layers. Genetic resistance in rootstocks can offer protection from these culprits, and offer sustainable and efficient protection of the risk of nematode damage. The frequent co-occurrence of multiple plant-parasitic nematode species makes attaining highest levels of resistance against such nematode assemblies tedious. Typically, the resistance towards one species has limited to no effect on a different species. In the current project, selections of grape rootstock genotypes selected foremost under greenhouse conditions were exposed to field populations of nematode assemblies. In 2019, one experimental vineyard was planted following a >30 year old planting of Thompson seedless grape. A total of seventeen experimental rootstocks developed in Dr. Andrew Walkers program were planted alongside repeat entries from prior experimentation and longer-tested GRN1, GRN2, GRN4 (A. Walker) and 1017A. In addition, Zinfandel, St. George, Harmony, Flame, and Salt Creek were planted as commercial controls. In mid-June 2019, rootstocks were planted in randomized complete block design with five replications into this nematode-infested site. A month after planting, each planting site receive additional root-knot nematode-infested soil to boost the infestation levels. In 2020, another nine experimental rootstocks with similar controls were planted, and additionally inoculated with infested soil. First nematode assessments were done in the dormant season. Field testing these rootstock candidates will provide important information on the nematological and horticultural status of them. These activities will allow to identify elites that warrant further characterization, and that could add new so urgently needed rootstocks for the industry.

 

Development of next generation rootstocks for California vineyards

Since my last report (June 2019) Nina Romero has made excellent improvements to
our rootstock screening and is currently re-vamping our ring nematode resistance screening, and she has replaced three technicians who departed over the past year. There are 444 genotypes in testing for resistance to nematodes, salt or both. Our 2019 crosses again focused on using fertile and tetraploid VR hybrids to get rotundifolia forms of resistance into better rooting backgrounds, and mostly failed (due to the genetic distance between Vitis grape species and rotundifolia. We have been successful with crosses to two VR hybrids both of which resist phylloxera and combined with rootstocks. Seed from the 2018 crosses are mostly in storage for the next grape breeder, except for the 18113 (GRZN3 x V. acerifolia 9018. Chris Chen is working on his PhD with this population which brings excellent and broad nematode
resistance to our best form of salt tolerance (which also has strong root-knot resistance). We have improved our phylloxera screening in the greenhouse and have verified a number of fertile VR hybrids also have strong phylloxera resistance. A new post-doc (Erin Galarneau from the Baumgartner lab) was hired to direct examinations of phenolic compounds responsible for phylloxera and nematode resistance. They will also assist our efforts to determine how O39-16 induces fanleaf degeneration resistance. We are also making good progress on identifying the basis of red leaf virus tolerance. These efforts are being directed by a visiting scholar from China. We have rootstock examples of strong tolerance (St. George and AXR1) and very sensitive (Freedom and 101-14) and rapid tissue-culture and greenhouse-based
screens that are rapid and mimic field tests.

Evaluation of Grapevine Rootstock Selections

The purpose of this project is to identify selections from a USDA rootstock breeding program that might warrant release as commercial stocks, and to develop useful data on the performance of recently released rootstocks from other breeding programs to aid growers in selecting appropriate stocks for their vineyards. The initial plantings from the USDA rootstock breeding program number over 700 selections. This initial group was grown until maturity, and then evaluated for their ability to be good mother vines. This evaluation identified nearly 150 selections that were good to moderate mother vines. Selections with good mother vines qualities were tested by Dr. Andreas Westphal and Dr. Andrew Walker for resistance to aggressive root-knot nematodes. Selections that performed well in both assessments have been grafted to scions for in field evaluations. Currently six selections (PC0333-5, PC0349-11, PC0349-30, PC04153-4, PC0495-51, and PC0597-13) have been planted in a replicated trial in a commercial winegrape vineyard in Merced. These vines should start production during the 2020 growing season. Five selections had been grafted to a table grape and planted in a replicated trial in a commercial table grape vineyard in Delano, CA. Unfortunately, this trial has run into problems and only limited data will be collected. For the past several years Dr. Gan-Yuan Zhong, USDA-ARS, screened
seedling selections for resistance to aggressive strains of root knot nematodes and shipped cuttings of resistant selections to the UC Kearney Agricultural Center in Parlier, CA, where they were rooted and planted into a vineyard for observation. Since 2016 a total of 165 new selections have been planted. These vines will need to undergo the same testing as the previous rootstocks once they are mature. It is hoped that a new table grape vineyard can be established with these advanced selections. The Merced trial should resolve questions about the potential, if any, of these selections as young grafted vines. The Merced trial is adjacent to another rootstock trial planted by former UCCE advisor Lindsay Jordan in 2016. The second Merced trial includes full rows of 1103P, and more recently released stocks including RS3, RS9, GRN2, GRN3, GRN4, and GRN5 grafted to Malbec, and replicated four times. However, most vines on GRN5 failed, so GRN5 was eliminated from the trial. Vine training was completed during the 2019 growing season.

Evaluation of grapevine rootstock selections

The purpose of this project is to identify selections from a USDA rootstock breeding program that might warrant release as commercial stocks, and to develop useful data on the performance of recently released rootstocks from other breeding programs to aid growers in selecting appropriate stocks for their vineyards. For the past several years Dr. Gan-Yuan Zhong, USDA-ARS, screened seedling selections for resistance to aggressive strains of root knot nematodes and shipped cuttings of resistant selections to the UC Kearney Agricultural Center in Parlier, CA, where they were rooted and planted into a vineyard for observation. Thirty selections were sent in 2016, 40 in 2017, and the remaining 112 selections in 2018. The vines will be grown at Kearney for three years before being screened for their potential as rootstock mother vines. Cuttings of vines with excellent traits will be challenged with a multi-species inoculum of plant parasitic nematodes, in collaboration with Dr. Andreas Westphal. Genotypes showing excellent mothervine traits and superior resistance to root knot nematode and/or broad resistance to multiple parasitic nematode species, will be advanced to replicated field trials as grafted vines. Stocks that show excellent potential as grafted vines will be referred to the USDA for consideration of release.

Currently five selections (PC0333-5, PC0349-11, PC0349-30, PC04153-4, and PC0597-13), have advanced to a replicated trial in a commercial table grape vineyard in Delano, and six selections (the same five from Delano plus PC0495-51) are in a replicated trial in a commercial winegrape vineyard in Merced. The vines in Delano were planted in summer, 2016, and grafted to Autumn King in 2017, but many of the PC selections failed after budding and were thus replanted in 2018 and are becoming established. The same rootstock selections were planted in Merced in September 2016, but will not be grafted until 2019. The replanting in Delano, and the grafting in Merced, should resolve questions about the potential, if any, of these selections as young grafted vines. The Merced trial is adjacent to another rootstock trial planted by former UCCE advisor Lindsay Jordan in 2016. The second Merced trial includes full rows of 1103P, and more recently released stocks including RS3, RS9, GRN2, GRN3, GRN4, and GRN5 grafted to Malbec, and replicated four times. However, most vines on GRN5 failed, so GRN5 was eliminated from the trial. Vine training in this trial was mostly completed in 2018, and petiole and nematode samples were collected. These preliminary data suggest the various stocks may affect vine nutrition and that they might differ with respect to nematode resistance. It is anticipated that the first fruit quality and yield data will be collected from the Merced trial in 2019.

Evaluation of Grapevine Rootstock Selections

The primary purpose of this project is to identify selections from a USDA rootstock breeding program that might warrant release as commercial stocks. Dr. Gan-Yuan Zhong screened seedling elections for resistance to aggressive strains of root knot nematode shipping 30 such selections to Kearney in 2016, 40 in 2017, with the remaining 100 selections to be shipped in 2018. Selections are  rooted and outplanted at Kearney and, when established, will be screened for their potential as rootstock mother vines. Those with excellent traits will be advanced to replicated field trials as grafted vines. Any stocks that show excellent potential as grafted vines will be referred to the USDA for consideration of release. Currently five selections (PC0333-5, PC0349-11, PC0349-30, PC04153-4, and PC0597-13), have advanced to a replicated trial in a commercial table grape vineyard in Delano, and six selections (the same five from Delano plus PC0495-51) are in a replicated trial in a  commercial winegrape vineyard in Merced. The vines in Delano were planted in summer, 2016, and grafted to Autumn King in 2017, but many of the PC selections failed after budding. It is not clear whether the failures were due to the stocks per se, or for some other reason. Vines on Freedom and own rooted vines in that trial have done much better than the vines on PC stocks, but the vines on Freedom were planted at a different time, and not budded in the field. Therefore, the PC selections will be replaced in 2018, and more closely monitored. The same rootstock selections were planted in Merced in September 2016, but will not be grafted until 2018. The replanting in Delano, and the grafting in Merced, should resolve questions about the potential, if any, of these selections as young grafted vines. The Merced trial is adjacent to another new rootstock trial planted by Lindsay Jordan in 2016. The second Merced trial includes full rows of 1103P, RS3, RS9, GRN2, GRN3, GRN4, and GRN5 grafted to Malbec, and replicated four times. Jordan’s resignation in 2017 could have led to the abandonment of the trial. We collected petiole data from those vines last year, as Jordan planned, and decided to include the trial in this proposal, at least for the 2018 season. If this additional objective is supported, it will provide an opportunity to acquaint Karl Lund, Jordan’s replacement, with the trial. It will be decided later if it should be separated from the others. Petiole analyses from the Malbec trial revealed that the stocks differed with respect to uptake of nitrate, phosphorus, potassium, boron, calcium, zinc, and magnesium. It was also observed that most vines on GRN5 failed, whereas most of the other vines are becoming established.  Therefore, GRN5 will be eliminated from the trial. It is anticipated that preliminary fruit quality and yield data may be collected from the second trial in Merced in 2018.