Location: Crops Pathology and Genetics Research
2024 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 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 2022.
Under Objective 1, ARS researchers in Davis, California, continued greenhouse- and orchard-based evaluations of disease resistance of walnut and almond rootstock genotypes to Agrobacterium (A.) tumefaciens, the causative agent of crown gall (CG); Sub-objectives 1.A and 1.B), and Phytophthora species that cause crown and root rots (PHY); Sub-objectives 1.C and 1.D). Greenhouse experiments were completed to phenotype resistance to CG and PHY in over 300 walnut hybrid seedlings and to re-test putative resistance to these plant pathogens in four elite walnut hybrid genotypes. Orchard-based walnut rootstock evaluations occurred in five continuing trials and included 19 rootstock candidates and three commercial standards. Tree growth and disease incidence and severity indicated overall disease resistance is consistent with results from greenhouse screening for most of the walnut rootstocks. Evaluations of almond rootstock candidates were continued in greenhouse trials for CG and orchard plantings for PHY. Pathogen resistance phenotypes were determined for 65 new and five standard almond rootstocks based on disease incidence and severity in the trials.
In support of Sub-objective 1.E, small RNA sequencing analysis was conducted on two RNAi transgenic walnut lines exhibiting increased resistance to Phytophthora pini in the greenhouse. Small interfering RNAs (siRNA) originating from the RNAi cassettes were detected, supporting the efficacy of our RNAi approach. Concurrently, 19 new transgenic embryonic lines were developed. These lines underwent small RNA analysis to assess the production of siRNAs. Transformant lines showing high siRNA production will be regenerated and subjected to an in vitro inoculation test.
In support of Sub-objective 1.F, a total of 54 walnut tissue culture samples, with and without phosphite treatment at three time points, were subjected to small and messenger RNA sequencing. The effect of phosphite on transcriptomes and disease phenotypes is currently being analyzed.
Under Sub-objective 1.G, the screening of Juglans regia accessions for Cherry leaf roll virus (CLRV) infection has been quite a challenge. Several of the trees which tested positive in 2018 for virus infection could not be confirmed as positive in 2019 or 2023. While our RT-qPCR assay for CLRV detection works well with the controls, the tree samples collected in spring 2018 and 2019 behaved differently from those collected in Fall 2023 indicating the possibility of residual viral RNA in pollen shed in large amounts in spring collected samples. As progress on this sub-objective to identify recessive allele is hindered, identification of a dominant R gene was initiated. Using RNA-sequence data derived from cambial tissue inside the bark close to the graft union of the scion (susceptible) and paradox rootstock (resistant) from blackline affected trees, five putative R-genes that are differentially expressed (greater than 1 fold change and FDR less than 0.05) in the rootstock have been mapped on chromosome 14 on which a sequence-characterized amplified region (SCAR) marker associated with resistance also resides.
For Objective 2, research has continued to address characterizing 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. Under Sub-objectives 2.A.1 and 2.A.2, walnut rootstocks with resistance and susceptibility to crown gall (CG) and Phytophthora crown and root rots (PHY) were received from cooperating commercial nurseries for use in RNA-seq experiments. These experiments included four hybrid walnut rootstock genotypes (AC574, AD253, MS1-86, AD167) and two commercially available rootstocks (RX1 and VLACH) with varying degrees of resistance or susceptibility to both diseases. Initially, we intended to screen MS1-56 only but expanded the list of target rootstock genotypes based on greenhouse and field evaluations of all rootstocks under development and availability of seedlings from cooperating commercial nurseries. All rootstocks were inoculated with Agrobacterium tumefaciens or Phytophthora pini. Samples for RNA extraction were collected prior to inoculation and one-, two-, and eight-days after inoculation for challenge with A. tumefaciens and after flooding events (three time points) for challenge with Phytophthora pini; these samples are undergoing RNA extraction.
In support of Sub-objective 2.B, ARS researchers established a bioassay similar to screening for K84-resistance to test antagonistic interactions between A. tumefaciens strains in our culture collection. Relocation of a lab with equipment needed to set-up and perform bioassays delayed initiation of bioassays by several months. Currently, three A. tumefaciens strains with nopaline biosynthetic genes are being screened for antagonism against 25 other strains with succinamopine or agropine biosynthetic genes in repeated experiments.
Under Sub-objective 2.C, Phytophthora isolates and DNA samples were collected from diseased almond and walnut trees throughout California and from surface sources of irrigation water in the Stockton East Water District (SEWD) in Stockton, California. About 25 isolates of Phytophthora species from the trees were identified based on Sanger sequencing of ITS regions between rRNA genes. Additionally, long-read PacBio sequencing was used to characterize populations of Phytophthora present at 15 distinct locations in the SEWD.
In support of Sub-objective 2.D, ARS researchers collected 18 new walnut samples in 2024 to represent Paradox canker disease (PCD)-affected and healthy walnut trees. This new sampling was conducted because previously extracted RNA samples stored at -80 degrees C were too degraded for sequencing. These samples will be extracted using a protocol that yields high quality and quantity RNA developed under Sub-objective 2.A.
For Sub-objective 2.E, ARS researchers have mined sequence data in the public domain and from in-house datasets from our cooperator at the University of California Davis (UCD) in Davis, California. Primers have been designed for Grapevine fanleaf virus (GFLV) detection: one set with degeneracy and another set that use dITP for RT-qPCR and TaqMan assays. Our primers can detect four GFLV sources which could not be detected by an unpublished new assay developed by UCD cooperator; however, our primers failed to detect six sources that their assay could detect. The sequence spanning the primer binding region from the six sources is being determined to re-design the primers and increase the efficiency of the assay. Publicly available sequences of Grapevine leafroll-associated virus 4 (GLRaV-4) isolate sequences have been downloaded and a phylogenetic tree has been constructed to determine clades. It is difficult to identify highly conserved regions due to the high variability of this RNA virus and closely related clades are being aligned to target the primers.
Under Objective 3, ARS researchers are continuing to develop novel and sustainable biologically-based management strategies to control targeted pathogens and replant diseases.
Under Sub-objective 3.A, second-year tree growth and third-year nut yields were collected and analyzed in response to pre-plant anaerobic soil disinfestation (ASD) treatments, pre-plant soil fumigation treatments, and post-plant nitrogen and phosphorus fertilization treatments for management of Prunus replant disease (PRD).
In support of Sub-objective 3.B, ARS researchers have successfully generated metagenomes and metabolomic profiles (primary metabolites, lipids, and short chain fatty acids) of control soils and soils subjected to anaerobic soil disinfestation (ASD) with carbon sources of different efficacy. Based on metagenomic analysis of these samples, they obtained over 500 metagenomically-assembled genomes (MAGs) that were at least 50% complete with approximately a third representing bacterial taxa previously identified through amplicon-sequencing to be core responders to ASD. Correlation analysis of metagenomes and metabolomes linked several metabolites (e.g., lipids with antimicrobial properties, fermentation products, and short-chain fatty acids) to MAGs enriched under ASD. Additional progress on this sub-objective has been hindered by difficulties in collection of volatile organic compounds (VOCs) and inconsistent extraction of intact RNA from soil to profile active microorganisms. We are re-designing microcosms used for ASD to avoid moisture loss and reintroduction of oxygen during headspace sampling for volatile organic compounds (VOCs). These microcosms will consist of nested glass jars with standing water and the use of gas-tight stainless tubing and fittings/connectors. If this approach fails, we will test solid-phase micro-extraction (SPME) fibers to capture VOCs (this approach has not been pursued due to the high cost of the fibers).
For Sub-objective 3.C, the construction of the expression vector for grape phytoene desaturase gene has been delayed due to inability to hire a Biological Science Technician and complete the work for other milestones. This work will be done before the end of the current fiscal year and transformation will be outsourced.
Accomplishments
1. Quantified yield impacts of soil-amendment-based alternatives to pre-plant soil fumigation for almond. Prunus replant disease (PRD) is a poorly understood soil-borne disease complex that suppresses early years of tree growth and yield in tens of thousands of acres of replanted almond orchards every year. Pre-plant soil fumigation effectively prevents PRD but faces serious regulatory challenges; non-fumigant approaches for PRD prevention are needed. Assessments of non-fumigant treatment efficacy are often limited to tree growth parameters because yield assessments are expensive and require three years to begin. ARS scientists in Davis, California, continued an almond replant study near Parlier, California, through the first year of nut yields and determined that pre-plant anaerobic soil disinfestation (ASD) increased kernel yields by 192 to 352 percent with rice bran and 102 to 211 percent with almond hull and shell amendments compared to non-treated controls, depending on almond cultivar; while pre-plant soil fumigation increases were 114 to 262 percent and soil amendment with rice bran alone increased kernel yields by 111 to 124 percent. Yield data generally indicate technical efficacy of the non-fumigant treatments, and combined with additional years of yields, will facilitate detailed economic assessments of fumigant alternatives.
2. Improved methods for monitoring Phytophthora populations in irrigation water sources. In California, surface sources of irrigation water such as rivers and canals contribute vitally to crop irrigation and thereby partially offset ground water overdrafts, a chronic problem in the state. However, surface water, unlike groundwater, can contain Phytophthora species, some of which are lethal root and crown pathogens affecting orchards, and growers’ use of surface water is therefore tempered by perceived disease risk. Because of technical challenges of characterizing Phytophthora populations in irrigation water, little is known about the actual disease risk. ARS scientists in Davis, California, applied high-throughput, long-read DNA sequencing and associated bioinformatic technologies to characterize Phytophthora populations at 15 locations in a major California irrigation district. The methods detected 39 species of Phytophthora, and compared to previous shorter-read sequencing approaches, provided superior species resolution. The long-read methods are now being applied to examine impacts of surface water use on orchard soil populations of Phytophthora species to further examine disease risk.
3. Developed walnut transgenic RNAi lines with improved resistance to Phytophthora. ARS researchers in Davis, California, developed two walnut transformant lines with improved resistance against Phytophthora pini using RNAi based on both in vitro and greenhouse pathogenicity tests. In these lines, small RNAs (sRNAs) derived from transformation cassettes were observed, which indicates inverted gene segments were transcribed and processed properly to generate small interfering RNAs. This work demonstrates the effectiveness of RNAi for control of Phytophthora crown and root rots and serves as a basis for developing stacked resistance in walnut to additional plant pathogens (e.g., Agrobacterium tumefaciens and nematodes).
4. Documented temporal transitions in walnut transcriptomes in response to phosphite fungicides. Phosphite fungicides have been employed to manage Phytophthora diseases, though their exact mode of action remains unclear. It is suggested that phosphite might boost plant innate immunity in addition to its direct toxic effects on Phytophthora pathogens. Therefore, understanding gene regulations linked to phosphite application could potentially enhance plant immunity even without phosphite treatment. Previous studies conducted by ARS researchers in Davis, California, demonstrated that a single spray application of phosphite could protect susceptible tissue cultures from Phytophthora infection. In this reporting period, they found that phosphite can alter the mRNA and small RNA transcriptomes of walnut tissue cultures for at least two weeks.
5. Reconstructed genomes of plant pathogen-suppressing bacteria in anaerobically-disinfested soils. Anaerobic soil disinfestation (ASD), an organic amendment-based pre-plant treatment that offers an alternative to the use of chemical fumigants, relies on the activity of microbial communities to control soil-borne plant pathogens. Using genome-resolved metagenomics, ARS researchers in Davis, California, retrieved 506 metagenomically-assembled genomes (MAGs) that were at least 50 percent complete with less than 10 percent contamination from unamended and ASD-treated soils. Abundance analysis identified numerous MAGs affiliated with Bacillota and Bacteroidota that were enriched under ASD and representative of bacterial taxa previously identified as core responders to ASD using amplicon-based sequencing. Metabolic pathway predictions indicated these bacteria were capable of producing compounds known to inhibit plant pathogens, including acetate, butyrate, p-cresol, and dimethyl sulfide. These MAGs likely represent key mediators of plant pathogen suppression in ASD-treated soils and additional work is underway to directly link these taxa to the production of inhibitory compounds.
Review Publications
Cochetel, N., Minio, A., Guaraccino, A., Garcia, J., Figueroa-Balderas, R., Massonnet, M., Kasuga, T., Londo, J., Garrison, E., Gaut, B., Cantu, D. 2023. A super-pangenome of the North American wild grape species. Genome Biology. 24. Article 290. https://doi.org/10.1186/s13059-023-03133-2.
Cai, Y., Anderson, E., Xue, W., Wong, S., Cui, L., Cheng, X., Wang, O., Mao, Q., Liu, S.J., Davis, J.T., Magalang, P.R., Schmidt, D., Kasuga, T., Garbelotto, M., Drmanac, R., Kua, C., Cannon, C., Maloof, J.N., Peters, B.A. 2024. Assembly and analysis of the genome of Notholithocarpus densiflorus. G3, Genes/Genomes/Genetics. 14(5). Article jkae043. https://doi.org/10.1093/g3journal/jkae043.
Gordon, P.E., Ott, N.J., Brar, R.K., Holtz, B.A., Browne, G.T. 2024. Phosphorus fertilization can improve young almond tree growth in multiple replant settings. HortTechnology. 34(2):161-171. https://doi.org/10.21273/HORTTECH05143-22.
Westphal, A., Maung, Z., Buzo, T., Brown, P., Leslie, C., Browne, G.T., Ott, N., McClean, A.E., Kluepfel, D.A. 2024. Identifying walnut rootstocks with resistance to multiple soil-borne plant pathogens. European Journal of Horticultural Science. 89(2):1-10. https://doi.org/10.17660/eJHS.2024/008.
Ott, N., Nouri, M., Browne, G.T. 2024. Full-length ITS amplicon sequencing resolves Phytophthora species in surface waters used for orchard irrigation in California’s San Joaquin Valley. Plant Disease. https://doi.org/10.1094/PDIS-09-23-1991-RE.
Saxe, H., Walawage, S., Balan, B., Leslie, C., Brown, P.J., Browne, G.T., Kluepfel, D.A., Westphal, A., Dandekar, A.M. 2024. Transcriptomic evidence of a link between cell wall biogenesis, pathogenesis, and vigor in walnut root and trunk diseases. International Journal of Molecular Sciences. 25(2). Article 931. https://doi.org/10.3390/ijms25020931.
Wang, Z., Wang, Y., Kasuga, T., Hassler, H., Lopez-Giraldez, F., Dong, C., Yarden, O., Townsend, J. 2023. Origins of lineage-specific elements via gene duplication, relocation, and regional rearrangement in Neurospora crassa. Molecular Ecology. https://doi.org/10.1111/mec.17168.
