Research Project: Integrated Disease Management Strategies for Woody Perennial Species

Location: Crops Pathology and Genetics Research

2022 Annual Report

Objectives
Objective 1: Examine etiology and ecology of key rootstock and scion diseases to enhance sustainability and profitability of tree and vine crops. Subobjective 1A: Conduct transcriptome analysis to identify potential causes of almond bud failure. Subobjective 1B: Determine the epidemiology of Grapevine red blotch-associated virus in California vineyards. Subobjective 1C: Identify potential causes of Paradox canker disease of walnut. Subobjective 1D: Identify soil microbial communities and processes conducive to development of Prunus replant disease. Subobjective 1E: Examine host-induced phenotypic instability in the Sudden Oak Death pathogen Phytophthora ramorum in production nurseries and natural settings. Objective 2: Sequence the genomes of phytoplasmas infecting stone fruit trees in California to enhance development of control and science-based quarantine regulations. Subobjective 2A: Determine the genome sequence of Cherry X disease phytoplasma, Peach yellow leafroll phytoplasma, and Candidatus Phytoplasma pyri. Subobjective 2B: Perform comparative genomics of the Cherry X disease phytoplasma and Peach yellow leafroll phytoplasma with other phytoplasmas. Objective 3: Develop novel amendment-based approaches for the management of soil borne pathogens and diseases. Subobjective 3A: Optimize anaerobic soil disinfestation (ASD) and its effectiveness against key pathogens under in vitro conditions. Subobjective 3B: Enhance and optimize ASD for management of almond orchard replant problems. Subobjective 3C: Characterize microbial community responses to ASD in greenhouse and orchard trials. Subobjective 3D: Quantify greenhouse gas emissions, nitrogen (N)transformations, and inorganic N leachate resulting from ASD. Objective 4: Identify host genotypes that exhibit resistance to key soil borne pathogens. Subobjective 4A: Identify and characterize Juglans rootstock genotypes resistant to Agrobacterium tumefaciens. Subobjective 4B: Identify and characterize Juglans rootstock genotypes resistant to key Phytophthora species. Objective 5: Identify gene and protein targets for use in novel molecular disease management strategies in woody perennial rootstocks. Subobjective 5A: In planta transcriptomic approaches to investigate host-Phytophthora interactions. Subobjective 5B: Examine the feasibility of using RNAi technology to suppress infection by Phytophthora species.

Approach
Objective 1 1A: Collect symptomatic shoots from almond trees exhibiting bud failure (BF) and shoots from non-symptomatic trees. Identify differentially expressed genes in BF trees compared with controls. Validate results of differentially expressed genes to identify BF markers and trees with the genetic potential to exhibit BF. 1B: Monitor grapevines in established plot for the spread of Grapevine red blotch-associated virus (GRBaV). Assess fruit quality of infected grapevines and compare with confirmed non-infected grapevines. Analyze data for variance and spatial and temporal changes in the GRBaV spread. 1C: Examine evidence for host genetic contributions to Paradox Canker Disease (PCD) of walnut. Use established metatranscriptomic libraries to bioinformatically examine signatures of host response to PCD. 1D: Establish plants susceptible to Prunus replant disease (PRD) in replicate plots of soil that induce PRD and replicate plots using the same soil treated so that PRD is not induced. Sample the soil and roots to examine associations of microbial taxa and their activities with PRD induction. 1E: Characterize newly identified plant defense mechanism to explore the feasibility of using nursery ornamentals as a pathosystem. Assess virulence and genetic stability among isolates of P. ramorum. Investigate factors that induce phenotypic instability and reduce aggressiveness towards specific hosts. Objective 2 2A: Purified DNA from petioles of cherry and almond, and the columella of pear fruit will be sheared, barcoded, amplified and sequenced. 2B: Compare annotated genomes to determine quarantine concerns. Examine gene organization by aligning the genomes to visualize regions of synteny and perform other comparative analyses. Objective 3 3A: Perform a series of anaerobic soil disinfestation (ASD) greenhouse trials to screen alternative carbon sources for their ability to generate and maintain anaerobic conditions and for their efficacy in reducing pathogen populations in soil. 3B: Examine efficacy of rice bran and more affordable ASD substrates for control of PRD in a greenhouse soil bioassay. 3C: Characterize microbial community responses to selected ASD carbon sources and organic amendments in the trials described in subobjectives 3 and 3B. 3D: Quantify GHG emissions and nitrate leaching resulting from ASD to facilitate adoption of ASD practices and refine existing biogeochemical models. Objective 4 4A: Produce clonal copies of confirmed interspecific hybrids with resistance to crown gall and Phytophthora. Evaluate clones for resistance. Perform genotyping-by-sequencing (GBS), associated mapping and mapping population analysis. 4B: Produce clonal copies of confirmed interspecific hybrids with resistance to P. cinnamomi and P. citricola. Evaluate clones for resistance. Objective 5 5A: Conduct in planta transcriptomic analyses of P. citricola in walnut and almond. 5B: Select candidate genes from data in subobjective 5A and develop stable host-induced gene silencing lines in walnut.

Progress Report
This is the final report summarizing all of the progress made over five years, for project 2032-22000-016-000D, "Integrated Disease Management Strategies for Woody Perennial Species", which has been replaced by new project 2032-22000-017-000D, "Improved Pest and Disease Control for Enhanced Woody Perennial Tree Crop and Grapevine Production". For Sub-objective 1A, ribonucleic acid (RNA) sequencing was done for healthy and bud failure trees. Sequence Data are undergoing bioinformatic analysis. In support of Sub-objective 1B, ARS researchers surveyed vineyards with red blotch disease. Grapevine red blotch virus (GRBV) spread appeared to be quite significant with disease prevalence jumping from 4.74 in 2020 vs. 37.8% in 2021 in Amador County (no rogueing was previously done). Sonoma County incidences jumped from 2.46 to 13.28% despite rogueing diseased grapevines and replanting with GRBV-free grapevines. Results indicate a significant threat of reinfection by GRBV in replanted vineyards. However, at Oakville Viticulture Station, new infections in a replanted block were limited to one vine indicating a manageable level of reinfection. ARS scientists with University of California-Davis (UCD) entomologists, found approximately 13% positivity rate for vines with new girdles caused by treehoppers. The number of girdles were nearly half of that observed in the previous year likely due to smoke from fire in the Sierra affecting treehopper populations/activities. ARS scientists showed, Spissistilus festinus, a treehopper vector of GRBV, only has one generation with adult population peaking in summer. Progress on Sub-objective 1C involved studying Paradox canker disease (PCD) etiology of walnut. The potential for meta-transcriptomics and genomics to detect PCD was validated by application to healthy trees and trees affected by known walnut pathogens (cherry leaf-roll virus affected, Phytophthora (P.), and Brenneria). Approaches failed to associate a potential pathogen with PCD, but detected each of the known pathogens. Graft transmission tests in an affected orchard showed necrotic tissues from canker margins of PCD-affected trees transmit the disease when grafted to healthy trees, while tissues from healthy trees did not. Orchard surveys in Sacramento and San Joaquin Valleys revealed continued statewide importance of PCD and additional symptomology details, such as failure to extend across graft unions with Persian walnut. In support of Sub-objective 1D, ARS researchers in Davis, California, further examined Prunus replant disease (PRD). Twenty-five replant soils from the Central Valley of California were tested for PRD in the greenhouse, analyzed for soil physicochemistry, and characterized for bacteria, fungi, and oomycetes. For 10 of 25 soils, including six that induced PRD and four that did not, communities were examined from corresponding PRD-affected and healthy peach roots. Multivariate analyses, including permutational analysis of variance, ordination, and random forest (RF) classification and regression modeling were used to relate soil physicochemistry and soil and root microbial communities to PRD induction. Among the 25 soils, RF classification modeling effectively discriminated between PRD-inducing and non-PRD inducing soils. Progress on Sub-objective 1E focused on examining host-induced phenotypic changes in Phytophthora ramorum (Sudden Oak Death pathogen). A single clonal lineage, NA1, believed to be introduced in the 1990s, dominates the forests in California and undergoes genome and phenotypic alterations in host plants. Isolates taken from diverse host species in production nurseries and natural ecosystems, and those recovered from artificially inoculated plants, were examined. Various genome alterations such as single nucleotide substitutions and structural variations like duplications, deletions, and translocations of genome segments, were identified. These SVs overlap with genes involved in pathogenicity. SVs and phenotypic changes were associated with host species and forest conditions such as solar radiation and temperature. For Sub-objectives 2A/2B, Illumina sequencing data was obtained for three phytoplasma-infected samples: Western X disease phytoplasma, Peach yellow leafroll phytoplasma, and Candidatus Phytoplasma pyri. Assembly of the genomes resulted in draft assemblies due to gaps. Despite gaps, the genomes were nearly complete based on CheckM analysis. Long read sequencing is required to obtain the complete genomes. For Sub-objectives 3A/3C, anaerobic soil disinfestation (ASD) trials with various carbon (C) sources were performed. More than 15 agricultural by-products were screened in the greenhouse for the ability to induce anaerobic conditions and suppress Agrobacterium tumefaciens populations enabled selection of C-sources that performed as well as rice bran (RB) for studies in orchard or nursery settings. ASD implemented with RB, tomato pomace (TP), mustard seed meal, and molasses resulted in anaerobic soils, induced similar microbiome changes, and resulted in similar levels of pathogen suppression. Through this work, we identified TP as an ASD substrate that performs equal to rice bran in terms of pathogen suppression, lowering soil redox potential, and shifting microbiome composition. These findings will facilitate adoption of ASD for use in tree crops for nursery and commercial growers. For Sub-objectives 3B/3C, ARS researchers established 10 field trials to test ASD for control of PRD. Ten ASD C substrates were tested, and RB and ground almond hull and shell (AHS) mixture were tested both as ASD drivers and as soil amendments alone (i.e., mixed into soil without added water or tarp). Compared to untreated controls, preplant soil fumigation generally significantly increased tree growth and yields. ASD with RB provided equivalent growth and yield improvements as fumigation, and was the most consistently beneficial of the 10 substrates. ASD using AHS significantly improved growth and yields compared to the control, but less so than RB. Amendment-alone treatments with RB, and to a lesser extent, AHS significantly improved tree growth, compared to the control, but less so than fumigation or full ASD. Microbiome sequencing of soils and roots was completed for several trials to assess microbial community shifts that may mediate treatment effects; analyses of these data are nearing completion. To identify potential metabolism involved in pathogen suppression, metagenomes were collected from RB, AHS, and control treatments. Coassembly and binning yielded 205 metagenomically assembled genomes with many belonging to anaerobic bacteria. For Sub-objective 4A, field trials were used to examine resistance and horticultural characteristics of six previously identified putative disease resistant Juglans hybrid rootstock genotypes. All rootstocks were grafted to a common scion. Initial trunk diameters were recorded and preparations are underway to inoculate with three target pathogens (bacteria, fungal, and nematode). We phenotyped resistance to Phytophthora and Agrobacterium (A.) tumefaciens in more than 500 genotypes of Juglans microcarpa x J. regia hybrids in greenhouse trials. Using phenotyping results, genomic rootstock maps, and genotyping-by-sequencing in collaborations with UCD colleagues, we identified unique genetic regions (QTLs) for resistance to both pathogens and prioritized the clones for potential commercial release. More than 20 clonal rootstocks were advanced for testing of resistance to lesion nematode, A. tumefaciens, and P. cinnamomi. A replicated orchard trial at UCD evaluated resistance to P. cinnamomi in four elite rootstock clones, and a second trial was established in a commercial orchard infested with the pathogen. Additional commercial orchard trials are ongoing to evaluate horticultural performance of elite disease resistant selections of the hybrid clone. To confirm and further resolve the QTL and identify additional elite walnut rootstock genotypes, several 100 additional J. microcarpa x J. regia hybrids were phenotyped and genotyped. ARS scientists have improved our understanding of the genetic bases of resistance to Phytophthora and A. tumefaciens in walnut and have identified elite walnut rootstock clones that are horticulturally acceptable and possess exceptional resistance to these major soilborne pathogens. In support of Sub-objectives 5A/5B, ARS researchers studied the genetic mechanisms underlying infection and disease development in walnut Phytophthora root and crown rots and found that more than 2,000 genes were differentially expressed between P. citricola interacting with plant tissues versus that grown on culture media. Gene function enrichment analysis showed that cellulose catabolic genes were overrepresented in P. citricola grown in the plant. Proteomic analysis detected 71 P. citricola proteins in plant tissues, although enzymes involved in glycolysis were overrepresented. Genes and proteins active in plant tissues are likely essential for pathogen growth; therefore, these genes were chosen for RNA interference (RNAi). 24 genes conserved among diverse Phytophthora species and active during pathogenic growth, were selected and transformation vectors constructed. These vectors were introduced into somatic embryos of walnut, and over 100 transformant lines obtained. Transformant lines exhibiting a high level of transgene activity were induced for shoot regeneration. Shoots from 30 transformant lines were subjected to an in-vitro infection test, which we developed, and two transformant lines showing improved disease resistance. Lines showing enhanced resistance were multiplied as microshoots and rooted. Potted plants were challenged with P. citricola and are currently being monitored for disease development. In addition, transformation protocols for almond were developed, and four transgenic almond lines carrying the RNAi constructs generated.

Accomplishments
1. Demonstrated the efficacy of anaerobic soil disinfestation to control soil-borne plant diseases in tree crops. ARS researchers in Davis, California, adopted anaerobic soil disinfestation (ASD) for use in nurseries and orchards. Multiple greenhouse and field trials showed that ASD with various agricultural by-products, such as, carbon substrates effectively suppresses Agrobacterium tumefaciens, oomycete plant pathogens, and Prunus replant disease. Experiments with over 15 carbon substrates identified several less expensive and locally available alternatives to rice bran as ASD carbon sources. These carbon substrates elicit similar soil microbial communities and development of anaerobic soil conditions as rice bran. These more cost-effective carbon substrates should facilitate adoption of ASD as a replacement for pre-plant chemical fumigation of soil.

2. Identified hybrid walnut rootstocks with stacked resistance to key soil-borne plant pathogens. ARS researchers in Davis, California, in cooperation with University of California collaborators have identified multiple hybrid walnut genotypes which are resistant to three key soil-borne pathogens of walnut rootstocks in California. Soil borne pests and pathogens dramatically impact yields and result in millions of dollars of loss each year. Use of these disease resistant genotypes in the industry will reduce use of preplant chemical fumigants and extend the productive life of walnut trees. This will result in a more sustainable and environmentally sound production practice while simultaneously limiting disease induced yield loses.

3. Machine learning approaches identify root microbial community members associated with Prunus replant disease induction and suppression in peach. Prunus replant disease (PRD) is a serious but poorly understood soilborne disease complex that suppresses tree development, efficient water and nutrient use, and crop yield in tens of thousands replanted almond orchards every year. Integrated management of PRD requires a better understanding of factors that drive its occurrence. Using multivariate statistics, followed by machine learning approaches focusing exclusively on root microbial populations, 26 bacteria, two oomycetes, and two fungi were identified as top predictors of PRD induction. Streptomyces scabiei, Steroidobacter denitrificans, Streptomyces bolbili, and Pythium mamillatum were relatively abundant (5-43%) among the top predictors and were positively (former two) and negatively (latter two) associated with PRD induction. The findings will guide future targeted testing of microbial taxa for PRD induction and suppression in roots.

4. Multiple new elite walnut rootstock genotypes were advanced. Several soilborne pathogens, including species of Agrobacterium, Phytophthora, and the lesion nematode cause tree death and serious losses in California walnut orchards. In collaboration with colleagues at University of California-Davis and University of California-Riverside, ARS researchers at Davis selected elite hybrids of walnut rootstocks, which, based on previous replicated evaluations, combine resistances to two or more of the key soilborne pathogens. The selections were advanced to a new series of four walnut orchard trials, all of which will include horticultural assessments and one of which will include soil inoculations to rigorously test resistance to Agrobacterium, Phytophthora, and lesion nematode under orchard conditions. Completion of these trials is expected to result in release of valuable new rootstocks for walnut production.

5. Development of genetic transformation protocol for almond. While genetic transformation system has been available for walnut for ~30 years, Prunus species (e.g., peach, plum, almond) remained recalcitrant until recently. In collaboration with the University of California, Davis, ARS researchers in Davis, California, genetically transformed embryos of almond with RNAi vectors. Transformed embryo lines were then induced to germinate. This was the first demonstration of the genetic transformation of embryogenic lines of almond x peach hybrids, another milestone in generating improved rootstocks for the almond industry. This break-through will facilitate all future genetic manipulations and improvements for both Prunus scion and rootstock genotypes.

6. Confirmed treehoppers that cause girdling on grapevines as vectors of grapevine red blotch virus under vineyard conditions. A treehopper, Spissistilus festinus, Family Membracidae, Order Hemiptera, was shown as a vector of grapevine red blotch virus (GRBV) under laboratory conditions by the ARS unit several years back. Recent work by researchers at Cornell University corroborated this observation. Recently, two other membracids were also shown to be vectors under laboratory/greenhouse conditions. In the study by the ARS unit, evidence was obtained for a role of membracids that cause girdling on grapevines in the transmission of GRBV under vineyard conditions. This information will be useful in developing integrated pest management options to manage the treehopper populations in the vineyards.

Review Publications
Khan, A.R., Wicaksono, W., Ott, N.J., Poret-Peterson, A.T., Browne, G.T. 2021. Characterization of soils conducive and non-conducive to Prunus replant disease. PLoS ONE. 16(12). Article e0260394. https://doi.org/10.1371/journal.pone.0260394.
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.
Hoffmann, M., Talton, W., Nita, M., Jones, T., Al Rwahnih, M., Sudarshana, M.R., Almeyda, C. 2020. First report of grapevine leafroll-associated virus 3 in Vitis vinifera in North Carolina. Journal of Plant Pathology. 103:385-386. https://doi.org/10.1007/S42161-020-00710-3.