Research Project: Integrated Disease Management Strategies for Woody Perennial Species

Location: Crops Pathology and Genetics Research

2021 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 report documents progress of 2032-22000-16-00D entitled, Integrated Disease Management Strategies for Woody Perennial Species. In support of Sub-objective 1A, gene expression profiles were examined in healthy trees and trees exhibiting almond bud failure disease, to better understand the etiology of Almond bud failure which causes major production loss for Almond growers. The project was slowed due to limited tree availability and Covid-19 restrictions. In 2020, the sequence/gene expression data was released from the University of California (UC)-Davis, DNA Core Facility. The data remained pending for bioinformatics analysis due to COVID-19 restrictions by UC-Davis Bioinformatics Services. Research on Sub-objective 1B, focused on characterizing the biology and transmission properties of the Three-cornered alfalfa hopper (Spissistilus festinus, family Membracidae) that has been shown to potentially transmit grapevine red blotch virus (GRBV). Insects belonging to the family Mebracidae are not well-studied in terms of their ability to use grapevines and other plants as reproductive hosts. After establishing the inability of S. festinus to complete their life cycle on grapevines, we examined the ability of the insect to transmit GRBV into the girdle tissue it causes after feeding on the grapevine. Last year, 464 girdle samples from 46 grapevines were collected from July to October and examined for GRBV using real-time quantitative polymerase chain reaction (PCR) tests. We detected GRBV in six girdle samples obtained from six different grapevines in July and August. These data provide evidence for tree hopper insects transmission of GRBV under vineyard conditions. For Sub-objective 1D, analyses of the relationships of soil and root abundance of specific microbial taxa and soil physicochemical properties to the induction of Prunus replant disease (PRD) was revised and enhanced. The revisions used an integrated suite of multivariate analysis tools along with machine learning approaches of “Random Forests”(RF) classification and regression modeling. In soil, using physicochemical and microbial variables and plant response data sets from 25 soils, RF classification modeling effectively discriminated between PRD-inducing and non-PRD inducing soils. Among the 15 most important discriminating soil variables were: relative abundances of 11 bacterial taxa, three fungal and oomycete taxa, and exchangeable potassium (K); low abundances of 14 microbial taxa and high levels of exchangeable K were predictive for PRD induction. In contrast, in roots, abundances of Streptomyces, Sterioidobacter, and Niastella taxa were top RF predictors, and high abundances of these taxa were predictive for PRD induction. In support of Sub-objective 1E, host-induced phenotypic instability in the Sudden Oak Death pathogen Phytophthora ramorum, was examined in production nurseries and natural settings. A single clonal lineage of P. ramorum, believed to be introduced in the 1990s, dominates forests in California. As shown previously, P. ramorum undergoes genome and phenotypic alterations in host plants. We identified diverse genome alterations such as single nucleotide substitutions and structural variations (SVs, e.g., duplications, deletions, and translocations of genome segments) in isolates taken from diverse host species in nurseries and natural ecosystems, as well as those recovered from artificially inoculated plants. 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. Research on Sub-objective 2A focused on sequencing DNA extracted from petioles of almond and cherry leaves collected from trees infected with peach yellow leafroll phytoplasma and Western X disease phytoplasma, respectively, and from the core of pear fruit collected from a pear tree in Solano County, California, infected with Candidatus phytoplasma piri. The sequence data are now being analyzed. For Sub-objectives 3A and 3C, ARS scientists in Davis, California, developed Anaerobic Soil Disinfestation (ASD) as an alternative to pre-plant soil fumigation for the management of soil-borne plant pathogens for woody perennial crops and almond replant problems. To date, numerous greenhouse and field trials of ASD have been conducted using different carbon sources, application rates, and implementation methods (i.e., with and without tarping and irrigating soil), and shifts in the microbiome both during and after ASD have been examined. A manuscript that describes the screening of greater than 15 carbon sources for ASD and their efficacy in maintaining anaerobic conditions in soil and suppressing a targeted plant pathogen has been drafted and two manuscripts describing changes in the soil microbiome that occur in response to ASD using different carbon sources in field and greenhouse trials have been published. In support of Sub-objectives 3B/3C, shotgun metagenomes were sequenced from samples collected in a field trial examining the potential for ASD to control Prunus Replant Disease (PRD). Based on taxonomic profiles from the samples, we detected a shift in the microbial community towards Firmicutes in rice bran and almond hull/shell amended soils. Additional analyses to assemble reads into contigs, retrieve genomes, and reconstruct metabolic pathways are underway. Tree growth response data were collected for analyses and will be used to relate PRD severity to predictors evident in the metagenomic and metabolomic data. Progress on Sub-objective 4A included publishing a manuscript with University of California, Davis, colleagues that identified a genetic region (QTL) that mediates resistance to Agrobacterium tumefaciens, Phytophthora pini, and P. cinnamomi. As well, data analyses from an orchard trial at the University of California, Davis, were completed, evaluating resistance to these three pathogens in four elite experimental walnut rootstock genotypes selected for resistance to the pathogens in greenhouse trials. Three of the experimental rootstocks determined to be as or nearly as resistant to P. cinnamomi as the highly resistant standard rootstock, ‘RX1’. We designed a new orchard trial that will be planted in 2022 and used to validate resistance to P. cinnamomi in four elite walnut rootstock clones. Rootstocks for the trial were selected and are being propagated. We established a field rootstock trial near Parlier, California, to evaluate resistance to two Phytophthora species in 13 commercially available standard rootstocks and 36 experimental rootstocks for almond and other Prunus species; in 2021, plots were re-inoculated, flood irrigated periodically, and evaluated for initial expressions of resistance. To date, both the standard and experimental rootstocks have ranged from highly susceptible to highly resistant, based on Phytophthora crown rot incidence and severity, whereas controls remained free from crown rot. The same experimental and commercial almond rootstock selections were evaluated, under greenhouse conditions, for resistance to crown gall. The majority of genotypes were highly susceptible to Agrobacterium tumefaciens (causative agent for crown gall). Interestingly, several genotypes were found to be resistant to A. tumefaciens. These genotypes are being repropagated for validation under both greenhouse and field conditions. For Sub-objective 5A, In planta transcriptomic approaches were used to investigate host-Phytophthora interactions. The genetic mechanisms underlying infection and disease development in Phytophthora root and crown rots of almond and walnut are poorly understood. It was discovered that approximately 2,000 genes were differentially expressed between P. citricola infecting plant tissues vs P. citricola on culture media. Gene function enrichment analysis showed that genes involved in the cellulose catabolic process were overrepresented in P. citricola grown in the plant while proteomic analysis detected 71 P. citricola proteins in plant tissues, and enzymes involved in glycolysis were overrepresented. Genes and proteins active in plant tissues are likely essential for parasitic growth of the pathogen; therefore, these genes were chosen for RNAi target for growth suppression in the plant (Sub-objective 5B). In support of Sub-objective 5B, research focused on the feasibility of using RNAi technology to suppress infection by Phytophthora species. This project is designed to engineer rootstocks of walnut and almond to confer RNAi-based resistance to Phytophthora. 24 genes conserved among diverse phytophthora species and active during pathogenic growth were selected, and transformation vectors, each containing segments of four of selected genes, were constructed. These vectors were introduced into somatic embryos of walnut; over 100 transformant lines were obtained. Transformant lines exhibiting a high level of transgene activity were induced for shoot regeneration. Shoots from 30 walnut transformant lines were then subjected to an in-vitro infection test; three transformant lines showed improved disease resistance. These lines are being propagated for a greenhouse trial. In addition, transformation protocols for almond were developed, and transgenic almond lines carrying the RNAi constructs were generated.

Accomplishments
1. Development of Phytophthora-resistant rootstocks of walnut and almond via RNAi-based method. Phytophthora root and crown rot is a major threat to almond and walnut production in California. In collaboration with the University of California, Davis, ARS researchers in Davis, California, employed RNA interference (RNAi)-based strategies to accelerate genetic improvement of almond and walnut rootstocks. Using RNAi transformation vectors three transformant lines were generated which exhibited enhanced disease resistance. For almond, a transformation protocol, which was not available prior to this work, was developed, and transgenic lines carrying RNAi constructs were generated. The RNAi-based plant disease resistance will minimize, and in some cases, eliminate the need for soil fumigation and fungicide applications. This will dramatically enhance the sustainable production of nut tree crops while reducing its environmental impact and human health risk for the public and agricultural workers.

2. Demonstrated efficacy of anaerobic soil disinfestation (ASD) to control soil-borne plant pathogens in nurseries and orchards. ARS researchers in Davis, California, optimized ASD for use in almond and walnut production. Multiple greenhouse and field trials identified alternative carbon sources to rice bran for ASD. These carbon substrates stimulated similar shifts as rice bran in soil microbial communities while reducing targeted plant pathogens in soil and remediating Prunus replant disease. The identification of more cost-effective ASD carbon sources will facilitate ASD adoption as an alternative to pre-plant chemical fumigation of soil.

3. Identified and validated in field trials, disease resistant (both Crown Gall and Phytophthora species) walnut rootstock genotypes. ARS researchers in Davis, California, in collaboration with University of California, Davis, walnut breeders have identified walnut hybrids between Juglans regia and Juglans microcarpa which exhibit elevated levels of resistance to key soil borne pathogens. These disease resistant genotypes also exhibit commercially acceptable horticultural traits that will facilitate their use in the walnut industry which will dramatically reduce the need for the use of ozone depleting soil fumigants.

4. Tree hopper insects (membracids) responsible for transmitting grapevine red blotch virus in a vineyard. ARS researchers in Davis, California, examined girdles apparently caused by tree hoppers (membracids) on healthy grapevines. They found that girdles from six out of 46 grapevines collected in early summer through August, tested positive for grapevine red blotch virus. This is a crucial piece of information needed to understand the epidemiology of this virus and develop effective control strategies.

5. Machine learning approaches identify microbial and physicochemical variables related to induction to Prunus replant disease. 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 induction. Using multivariate and machine learning approaches with soil and root data sets, ARS scientists at Davis, California, determined that exchangeable K and pH, as well as abundances of multiple bacterial, fungal, and oomycete taxa were strong predictors for PRD induction. The findings will guide targeted testing of microbial taxa and soil properties for their roles in PRD induction and value in integrated PRD management strategies.

6. Developed an orchard screening procedure for evaluating resistance to Phytophthora in almond and other stone fruit rootstocks. Species of the soilborne water mold Phytophthora commonly kill up to several percent of trees among almond and other stone fruit orchards, which now total well over a million acres in California. Use of rootstocks with genetic resistance to the pathogens is the most economical and strategic approach to managing these disease losses, but reliable assessments of the resistance have been a challenge, especially under greenhouse conditions. ARS researchers based at Davis, California, developed a field-based method of testing the resistance, which uses artificially infested soil and managed periods of soil water saturation. The procedure has discriminated between high and low levels of resistance and will accelerate breeding of almond and other stone fruit rootstocks with improved resistance to species of Phytophthora.

Review Publications
Kasuga, T., Hayden, K., Eyre, C., Croucher, P., Schechter, S., Wright, J., Garbelotto, M. 2021. Innate resistance and phosphite treatment affect both the pathogen’s and host’s transcriptomes in the Tanoak-Phytophthora ramorum pathosystem. The Journal of Fungi. 7(3). Article 198. https://doi.org/10.3390/jof7030198.
Thompson, C., McCartney, M., Roubtsova, T., Kasuga, T., Ebeler, S., Davis, C., Bostock, R. 2021. Analysis of volatile profiles for tracking asymptomatic infections of Phytophthora ramorum and other pathogens in rhododendron. Phytopathology. https://doi.org/10.1094/PHYTO-10-20-0472-R.
Ramasamy, R.K., Luo, M., Leslie, C.A., Velasco, D.M., Ott, N.J., McClean, A.E., Dandekar, A.M., Aradhya, M.K., Brown, P.J., Browne, G.T., Kluepfel, D.A., Westphal, A., Dvorak, J. 2021. Co-located quantitative trait loci for resistance to Agrobacterium tumefaciens, Phytophthora cinnamomi, and P. pini in Juglans microcarpa × J. regia hybrid rootstock. Horticulture Research. 8. Article 111. https://doi.org/10.1038/s41438-021-00546-7.
Pereira, G.E., Padhi, E.M., Sudarshana, M.R., Fialho, F.B., Plaza, C.M., Girardello, R.C., Tseng, D., Bruce, R.C., Erdmann, J.N., Slupsky, C.M., Oberholster, A. 2020. Impact of grapevine Red Blotch Disease on primary and secondary metabolites in ‘Cabernet Sauvignon’ grape tissues. Journal of Food Chemistry. 342. Article 128312. https://doi.org/10.1016/j.foodchem.2020.128312.
Browne, G.T., Hasey, J.K., Ott, N.J., Forbes, H., Arnold, K., Milliron, L.K. 2021. Flooding by California rivers results in walnut scion infections by species of Phytophthora. Plant Health Progress. https://doi.org/10.1094/PHP-02-20-0011-RS.
Knipfer, T., Bambach, N., Hernandez, M.I., Bartlett, M.K., Sinclair, G., Duong, F., Kluepfel, D.A., McElrone, A.J. 2020. Predicting stomatal closure and turgor loss in woody plants using predawn and midday water potential. Plant Physiology. 184(2):881-894. https://doi.org/10.1104/pp.20.00500.