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.

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.

 

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.