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Research Project: Improved Pest and Disease Control for Enhanced Woody Perennial Tree Crop and Grapevine Production

Location: Crops Pathology and Genetics Research

2022 Annual Report

Objective 1: Characterize and incorporate resistance to disease into woody perennial crops. Sub-objective 1.A: Characterize and field test crown gall (CG) resistant rootstock genotypes for walnut production systems. Sub-objective 1.B: Identify, develop, characterize, and field test CG resistant rootstock genotypes for almond production systems. Sub-objective 1.C: Identify, characterize, and field test Phytophthora resistant rootstock genotypes for walnut production systems. Sub-objective 1.D: Identify, develop, characterize, and field test Phytophthora resistant rootstock genotypes for almond production systems. Sub-objective 1.E: Develop Phytophthora resistant almond and walnut rootstocks using RNA interference (RNAi). Sub-objective 1.F: Characterize the genetics behind phosphite-induced resistance against Phytophthora in walnut. Sub-objective 1.G: Identify sources of recessive alleles in walnut germplasm that confer resistance to Cherry leaf roll virus (CLRV). Objective 2: Characterize soil/phytomicrobiome communities and targeted phytopathogens to understand their impact on plant and soil health, enhance pathogen diagnostics, and develop optimal disease management strategies for woody perennial crops. Sub-objective 2.A: Characterize potential genes linked to CG and Phytophthora crown and root rot resistance/tolerance in walnut and the in planta gene expression of A. tumefaciens and P. pini. Sub-objective 2.B: Determine antagonistic interactions between A. tumefaciens strains isolated from walnut orchards. Sub-objective 2.C: Characterize populations of Phytophthora in almond and walnut orchards and surface sources of irrigation water. Sub-objective 2.D: Characterize metatranscriptomic and metagenomic profiles of walnut rootstock to which Paradox canker was graft transmitted versus healthy walnut rootstock. Sub-objective 2.E: Develop improved primers for detection of Grapevine fanleaf virus (GFLV) and Grapevine leafroll-associated virus 4 (GLRaV-4). Objective 3: Develop novel and sustainable biologically-based management strategies to control targeted pathogens and replant disorders. Sub-objective 3.A: Examine potential nutritional and microbial contributions of ASD treatment components to PRD induction and management in almond. Sub-objective 3.B: Determine which volatile organic compounds (VOCs) are produced in ASD-treated soils as a function of carbon source. Sub-objective 3.C: Develop sentinel grapevine genotypes for early detection of red blotch virus in vineyards.

Objective 1 1.A: Inoculate walnut rootstocks Agrobacterium tumefaciens and rate crown gall (CG) disease symptoms. Map genetic loci mediating CG resistance. 1.B: Inoculate almond with A. tumefaciens and rate CG severity in greenhouse trials. Evaluate field performance of selected resistant rootstocks. 1.C: Inoculate walnut rootstocks with P. cinnamomi and rate symptoms. Analyze resistance phenotypes and genotyping data to resolve quantitative trait loci. 1.D: Rate experimental and commercial almond rootstocks for resistance to Phytophthora in orchard trials. 1.E: Rate disease symptoms in walnut lines carrying host-induced gene silencing (HIGS) contructs inoculated with Phytophthora. Extract RNA from inoculated and non-inoculated walnut lines and perform RNA-Seq to identify microRNA produced from HIGs vectors. 1.F: Using an in vitro disease assay system, generate and analyze transcriptomes of phosphite-treated walnut inoculated with Phytophthora and controls treated with water treatment and mock-inoculated. 1.G: Graft uninfected walnut trees onto Cherry leaf roll virus-infected ‘Chandler’ trees and monitor for virus infection. Objective 2 2.A: Extract RNA from resistant and susceptible walnut rootstocks inoculated with A. tumefaciens or P. pini. Identify genes mediating CG or Phytophthora resistance in walnut and transcriptional changes in pathogens during infection. 2.B. Assess antagonistic interactions of A. tumefaciens strains with different opine types. Measure growth inhibition zones of A. tumefaciens strains exposed to another strain. Perform Tn5 mutagenesis and tests for phenotype restoration to identify genes involved in antagonism. 2.C: Isolate Phytophthora from almond and walnut orchards with Phytophthora-associated diseases and identify isolates using genotype-by-sequencing. Sample surface water for DNA extraction and sequencing of ITS genes and isolation and identification Phytophthora. Relate Phytophthora populations in orchards and surface sources of irrigation water. 2.D: Extract DNA and RNA from walnut tissue from graft experiments of Paradox canker disease to compare healthy control and infected tissues to identify potential causal agents. 2.E: Using an existing virome database and additional sequences derived from RNA-seq of 576 Vitis vinifera accessions, design with Primer Express 3 and test diagnostic primers and TaqMan probes for Grapevine fanleaft virus and Grapevine leafroll-associated virus 4. Objective 3 3.A: Establish field trial of anaerobic soil disinfestation (ASD) with rice bran- and almond hull and shell-based treatments. Plant almond trees and apply fertilizer treatments of with differing amounts of nitrogen and phosphorus. Profile soil and root microbiomes, soil physicochemistry, nematode populations, and measure tree growth. 3.B: Establish ASD mesocoms with different carbon source treatments. Collect volatiles for GC-MS analysis and soils for microbiome and metabolomic profiling. 3.C: For grapevine, construct synthetic silent expression construct that will result in loss of color upon infection of Grapevine red blotch virus and evaluate performance in greenhouse and field trials.

Progress Report
This report documents progress for project 2032-22000-017-000D, Improved Pest and Disease Control for Enhanced Woody Perennial Tree Crop and Grapevine Protection, which started April 25, 2022 and continues research from project 2032-22000-016-000D, Integrated Disease Management Strategies for Woody Perennial Species. In support of Sub-objective 1A, we established greenhouse trials in July 2022 and are in the process of screening open pollinated seedlings from a Juglans regia scion-breeding orchard. It will take an additional five months to complete this milestone. In support of Sub-objective 1A, a large 2.5-acre field trial at the Armstrong field station in Davis, California, with three clonal industry standards and six experimental putative disease resistant hybrid walnut rootstocks has been established. A second trial has been established with walnut rootstocks in the San Joaquin Valley which is being monitored for disease development. In support of Sub-objective 1B, Prunus hybrid cuttings were collected, rooted, and are ready for grafting by a commercial nursery collaborator. In support of Sub-objective 1C, nine open-pollinated hybrid seedlings were selected for their Phytophthora-resistant phenotypes from among >100 genotypes of Juglans microcarpa x Juglans regia. The elite selections were micropropagated for re-testing of the putative resistance. Also, a new greenhouse experiment was established to phenotype resistance to Phytophthora cinnamomi among an additional 100 genotyped seedlings of the hybrids, which may yield additional elite selections. In support of Sub-objective 1D, orchard trials with almond rootstocks were established and inoculated with Phytophthora cactorum and Phytophthora niederhauserii. In support of Sub-objective 1E, 33 in vitro shoot cultures of walnut transformants were previously screened for Phytophthora (P.) resistance, and two lines, which consistently showed enhanced resistance, were identified. Roots were then induced, and the potted transgenic lines were subsequently challenged with P. pini. Resistance and susceptible walnut lines were also inoculated as a comparison. The inoculation test will take up to three more months. In support of Sub-objective 1F, development of a phosphite fungicide assay system using in vitro culture is largely complete. Owing to the use of aseptic and genetically uniform cultures, the method is highly reproducible and a single spray application of phosphite confers Phytophthora resistance to susceptible cultivars. In support of Sub-objective 1G, accessions will be screened for Cherry leaf roll virus (CLRV) infection in Spring 2023. In support of Sub-objectives 2A.1 and 2A.2, hybrid walnut genotypes have been screened for resistance to bacterial and oomycete pathogens. Resistant genotypes have been selected and placed into in vitro cultures where they were increased in number. These cultures have now been transferred to a commercial nursery cooperator for large scale in vitro propagation to produce rooted plantlets for subsequent greenhouse and field testing to validate the disease resistance phenotype. In support of Sub-objective 2B, laboratory work will begin in October 2022. In support of Sub-objective 2C, diseased root systems and soil samples were collected from several almond and walnut orchards potentially affected by Phytophthora species. From these samples, multiple isolates of Phytophthora were curated and identified based on rRNA gene internal transcribed spacer (ITS) sequencing. In collaborations with University of California (UC) scientists, the isolates will be used to inform evaluations of almond and walnut rootstock resistance to Phytophthora and for assessments of fungicide efficacy for control of Phytophthora. Also, ARS scientists at Davis, collected and processed soil and irrigation water samples from multiple orchards to characterize populations of Phytophthora species in them as a function of irrigation water source being used (i.e., ground water vs. surface water such as canals or rivers). In support of Sub-objective 2D, nucleic acid extractions from Paradox canker disease transmission experiments (healthy and diseased tissues) will begin in late 2022. In support of Sub-objective 2E, sequences of Grapevine fanleaf virus (GFLV) have been downloaded from National Center for Biotechnology Information (NCBI) GenBank and high-throughput sequencing data has been requested from University of California-Davis collaborators. In support of Sub-objective 3A, ARS scientists at Davis, California, treated, planted, and sampled replanted almond orchard plots to examine comparative impacts of: pre-plant anaerobic soil disinfestation (ASD) treatments, preplant soil fumigation treatments, and post-plant phosphorus fertilization treatment for management of Prunus replant disease. The samples were processed to evaluate impacts of the treatments on soil physicochemical properties and soil and root microbial communities. Measurements were taken to assess impacts of the treatments on tree growth. In support of Sub-objective 3C, a synthetic gene sequence to achieve the desired expression of a short hairpin RNA to silence the phytoene desaturase gene was assembled. The sequences need to be custom ordered for synthesis.