Location: Crops Pathology and Genetics Research
2023 Annual Report
Objectives
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.
Approach
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 fiscal year (FY) 2023 progress for project 2032-22000-017-000D, titled, "Improved Pest and Disease Control for Enhanced Woody Perennial Tree Crop and Grapevine Production", which started in April of 2022 and continues research from project 2032-22000-016-000D, titled, "Integrated Disease Management Strategies for Woody Perennial Species."
In support of Sub-objective 1.A, ARS researchers in Davis, California, established several greenhouse and field trials to screen open pollinated seedlings for disease resistance (e.g., crown gall and Phytophthora crown and root rot). In summer 2022, researchers established a greenhouse trial to screen seedlings from a Juglans regia scion-breeding orchard and crown gall disease resistance ratings are still underway. They also established 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. A second field trial has been established with walnut rootstocks in the San Joaquin Valley, California, which is being monitored for disease development.
ARS researchers have largely completed Sub-objective 1B. Prunus hybrid cuttings were collected, rooted, and are ready for grafting by a commercial nursery collaborator.
In support of Sub-objective 1.C, nine open-pollinated hybrid seedlings were selected for their Phytophthora and Agrobacterium 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 1.D, ARS researchers established orchard plots with almond rootstocks, and inoculated the plots with Phytophthora cactorum, and Phytophthora niederhauserii and disease monitoring is ongoing.
In support of Sub-objective 1.E, 33 in vitro shoot cultures of walnut transformants were 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 the pathogen, P. pini. Resistance and susceptible walnut lines were also inoculated as a comparison. In addition, in order to improve silencing efficiency, and to optimize target genes, RNAi vectors were redesigned and approximately 20 walnut transformants were generated.
In support of Sub-objective 1.F, a phosphite fungicide assay system using in vitro culture was developed and optimized. 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.
For Sub-objective 1.G, in summer 2022, ARS researchers tested 10 Juglans (J.) regia accessions for natural infection by Cherry leafroll virus (CLRV). About 300 of the remaining J. regia accessions are being sampled for CLRV infection status. Due to a prolonged rainy season and delay in hiring of a Biological Science Technician, the testing of the remaining accessions will be completed in early summer 2024.
For Sub-objectives 2.A.1 and 2.A.2, hybrid walnut genotypes have been screened for resistance to bacterial and oomycete pathogens. Resistant genotypes and susceptible controls were selected for gene expression assays. The selections were transferred to a cooperating commercial nursery where they were propagated and are being grown to size for testing and gene expression assays. To optimize RNA extraction and sequencing protocols for gene expression assays, a pilot study with Agrobacterium (A.) tumefaciens (bacterial plant pathogen) and two walnut rootstocks (MS1-56 and STJM4) was conducted. The rootstocks were inoculated with A. tumefaciens or water as a control and tissue was sampled for RNA extraction at 24- and 48-hours post inoculation. RNA extraction was successful based on high yield and quality. The extracts have been submitted for RNA sequencing to profile walnut and A. tumefaciens gene expression.
In support of Sub-objective 2.B, ARS researchers selected an initial set of Agrobacterium tumefaciens strains to test for antagonistic interactions based on their genomic similarity to other strains and opine biosynthesis genes. These strains are being grown to establish working stocks for the screening assays. The initial screening for antagonistic interactions will consist of testing the ability of three strains that produce different opines (nutrients taken up by A. tumefaciens) to inhibit 27 other strains. Progress on this objective was delayed due to a Biological Science Technician being on leave.
In support of Sub-objective 2.C, diseased root system and soil samples were collected from multiple 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 collaboration with University of California, Davis, 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, California, 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 2.D, exploratory RNA extractions were conducted from Paradox canker disease samples from disease transmission experiments (healthy and diseased tissues). Results of the extractions indicated that RNA had been degraded over the years of storage. Resampling will be required but has been delayed due to a Technician position vacancy.
In support of Sub-objective 2.E, sequences of 18 RNA1 and 20 RNA2 sequences of Grapevine fanleaf virus (GFLV) have been downloaded from the National Center for Biotechnology Information (NCBI) GenBank and aligned using ClustalW Omega. High-throughput sequencing data has been requested from the University of California, Davis, collaborator at the Foundation Plant Services. Based on the previous RT-PCR data obtained from table and wine grapes, we have identified 50 samples for high through-put sequencing (HTS). Samples will be collected and subjected to HTS in summer of 2024.
In support of Sub-objective 3.A, ARS researchers treated, planted, and sampled replanted almond orchard plots to examine comparative impacts of: pre-plant anaerobic soil disinfestation (ASD) treatments, pre-plant soil fumigation treatments, and post-plant phosphorus fertilization treatment for management of Prunus replant disease. The samples were processed to evaluate the 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 3.B, soil microcosms were fabricated, and samples were submitted for gas chromatography-mass spectrometry (GC-MS) analysis of volatile organic compounds (VOC). Two sizes of soil microcosms (60 and 750 mL) were fabricated from PVC tubes and capped with lids containing butyl rubber stoppers. A flow through system is in the process of being tested that is intended to capture headspace gas in the PVC tubes and the gas samples will be analyzed for background VOC levels to decide if proceeding with plastic-based soil microcosms is acceptable for this work, as VOCs released form the plastic may contaminate samples. GC-MS analysis of anaerobically disinfested and control (no carbon added) soils successfully detected six short chain fatty acids (volatile organic compounds), of which three were present at higher abundances in ASD-treated soils.
In support of Sub-objective 3.C, a synthetic gene sequence to achieve the desired expression of a short hairpin RNA to silence the phytoene desaturase gene was assembled. A synthetic gene has been custom ordered. Overlapping genomic fragments of grapevine red blotch virus genome have been amplified to make infectious clones from three field isolates.
Accomplishments
1. An in vitro assay system for the study of phosphite-induced resistance against Phytophthora in walnut. Phosphite fungicides have been used to control Phytophthora diseases such as Sudden Oak Death and root and crown rot of almond and walnut. As observed in the field, a single spray application of phosphite protects susceptible plants, including almond and walnut in commercial orchards, from Phytophthora infection. The mode of action is, however, not well understood. ARS researchers in Davis, California, developed and optimized an in vitro disease assay system using shoot tissue culture of walnut. Because aseptic and genetically uniform cultures are used, the method is highly reproducible and suitable for high-throughput mRNA and small RNA analyses. The information obtained in this research, such as phosphite-activated RNA species, will aid the development of rootstocks that show Phytophthora resistance without phosphite application.
2. Juglans hybrids (J. acrocarpa x J. regia) exhibit commercially acceptable levels of resistance to Agrobacterium tumefaciens (Crown gall disease). Crown Gall disease is one of the top soil-borne diseases of walnut (Juglans species) rootstock genotypes in California. All current commercially available walnut rootstocks are susceptible to this disease. ARS researchers in Davis, California, have identified Juglans genotypes which exhibit high levels of crown gall resistance and are horticulturally acceptable for widespread use in the industry. These new and novel disease resistant rootstock genotypes are performing well in numerous field trials across the California walnut region and could help offset the estimated annual $241 million of walnut industry revenue lost to soil-borne pathogens.
3. Soil amendment and soil fertilization practices contribute to management of Prunus replant disease. Prunus replant disease (PRD) is a serious, but poorly understood soil-borne disease complex that suppresses tree development, efficient water and nutrient use, and crop yield in tens of thousands replanted almond orchards every year. ARS researchers in Davis, California, determined that pre-plant soil amendment with rice bran alone (9 tons/treated acre) and post-plant fertilization with phosphorus at (elemental P at 2.6 oz/tree) each significantly improve tree growth in replanted almond orchards. In a replanted almond orchard subject to PRD, soil amendment with rice bran alone, phosphorus fertilization alone, and the combination of both treatments resulted in 81%, 62%, and 82% of tree growth generated by pre-plant soil fumigation, whereas trees in non-treated plots (the control) produced only 51% of the growth generated on soil that received pre-plant fumigation. The findings will support non-fumigant- based approaches for managing replant disease of almond. This will improve air quality and reduce potential for exposure to chemical fumigants. The almond industry has engaged ARS researchers in extending economic evaluations of the practices through field days and a formal presentation planned for the 2023 Almond Research Conference.
Review Publications
Rumbaugh, A.C., Medina-Plaza, C., Sudarshana, M.R., Oberholster, A. 2023. Grapevine red blotch virus alters grape skin cell-wall composition impacting phenolic extractability during winemaking. Journal of the Science of Food and Agriculture. 103(7):3457-3467. https://doi.org/10.1002/jsfa.12481.
Khan, A.R., Wicaksono, W., Ott, N.J., Poret-Peterson, A.T., Browne, G.T. 2022. Random forest analysis reveals taxa predictive of Prunus replant disease in peach root microbiomes. PLOS ONE. 17(10). Article e0275587. https://doi.org/10.1371/journal.pone.0275587.
Rumbaugh, A.C., Sudarshana, M.R., Oberholster, A. 2021. Grapevine red blotch disease etiology and its impact on grapevine physiology and berry and wine composition. Horticulturae. 7(12). Article 552. https://doi.org/10.3390/horticulturae7120552.
