Project : USDA ARS

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Research Project: Integrated Disease Management Strategies for Woody Perennial Species

Location: Crops Pathology and Genetics Research

2020 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
In support of Sub-objective 1A, the transcriptome analysis to examine the potential etiology of noninfectious almond bud failure (NABF) was initially challenging due to non-availability of almond orchards with trees exhibiting bud failure. In due course, we identified a NABF orchard which was sampled and mRNA sequencing has been performed on these samples. Analysis of the sequence data is in progress. In support of Sub-objective 1B, the examination of epidemiology of grapevine red blotch virus revealed a high degree of variability in vector behavior. Artificial field transmission of the virus has not been successful using Three-cornered alfalfa hopper (Spissistilus festinus, family Membracidae). Detection of grapevine red blotch virus in the insect has been inconsistent indicating a population variability over years. Examination of cytochrome oxidase 1 was adopted to determine the species of the prevalent insects (membracids). In support of Sub-objective 1C, Paradox canker disease (PCD) affected walnut trees were identified for future sampling. Plans were made for metagenomic and metatranscriptomic sequencing efforts from samples collected from graft transmission experiments that demonstrated the infectious nature of PCD. In support of Sub-objective 1D, we continued examinations of soil and root microbial communities and physicochemical soil properties associated with Prunus replant disease (PRD). Over the span of this project, five almond orchard replant trials have been established with replicated preplant fumigated and non-fumigated plots. In FY20, tree growth and, in some trials, yield data were collected and used as ongoing measures of PRD severity. The PRD responses are related to significant shifts in the microbial community. Also, we revised bioinformatics analyses and modeling of microbial community shifts associated with PRD in orchard replant soils and a greenhouse bioassay of almond replant soils. For the first time, we applied random forest machine learning approaches to relating soil and root communities to PRD induction. In support of Sub-objective 1E, a total of 78 genomes of Phytophthora (P.) ramorum isolates from diverse hosts and geographical regions were examined for signatures indicating adaptation to the local environment. We showed that since the introduction to California in the 1990s, P. ramorum has accumulated Structural Variations (SVs), such as duplications, deletions, and translocations of genome segments. Occurrence of some SVs were found to be associated with environmental factors such as host density and minimum temperature, implicating local adaptation of the invasive pathogen. In support of Sub-objective 2A, we sequenced the genome of Western X phytoplasma (Candidatus Phytoplasma pruni). Assembly has revealed big gaps in the genome resulting in the need to sequence a second isolate. Research on Sub-objectives 3A and 3C included greenhouse and field trials of anaerobic soil disinfestation (ASD) being conducted to examine soil microbiome changes that occur in response to different carbon sources (rice bran, tomato pomace, red grape pomace, and almond hulls/shells). In both settings, samples were collected within days (2 to 3) of ASD initiation and throughout the treatment period (3 to 5 weeks). Bacterial communities from greenhouse mesocosms that replicate ASD-induced field soil redox conditions were characterized via 16S rRNA gene sequencing and quantitative PCR analysis of genes for nitrogen fixation. We found that within 2 days of ASD initiation, the soil microbiome shifted towards Firmicutes, which remained dominate throughout treatment. Genes for nitrogen fixation also increased over time in soil mesocosms, suggesting a potential for ASD communities to alter soil nitrogen availability. Based on the rapid response and potential functional change of ASD communities in the greenhouse trials, the collected samples were submitted within 3 days of ASD initiation from the field trial for metagenomic sequencing. Large functional differences related to nitrogen cycling are expected between ASD-treated soils and irrigated/tarped controls soils, similar to bacterial communities assessed via targeted gene sequencing. In support of Sub-objective 3B, assessments of anaerobic soil disinfestation (ASD) treatments were continued, with data-driven modifications, for two promising ASD substrates, rice bran and ground almond hull and shell mixture. The latter was of special interest due to its abundance, relatively low cost, and efficacy in some previous ASD trials. Tree growth data were collected from plots in a replanted almond orchard comparing the two substrates and their utilization for ASD under drip irrigation, with and without a tarp covering. In a separate trial at the same orchard, rice bran amendment, without tarp or irrigation, was compared to a non-treated control. The results of the trial to date indicated superior performance of the rice bran substrate, which improved tree growth, compared to non-treated control and water-treated controls. In this trial the almond substrate did not improve tree growth, but rice bran improved tree growth both with and without tarp and with and without drip irrigation. Results of a fertilizer experiment embedded in the ASD trial suggested that a major benefit of the rice bran amendment may have been nutritional, relating to both nitrogen and phosphorus content of the bran. In support of Sub-objective 4A, we phenotyped resistance to Phytophthora and Agrobacterium (A.) tumefaciens in seedling progeny from two Juglans (J.) microcarpa x J. regia breeding populations, thereby augmenting our previous phenotyping efforts in clonal progeny from the same parent trees. For Phytophthora (P.) resistance, approximately 300 of the hybrid seedlings were grown and flooded intermittently in soil infested with P. cinnamomi. Resistance to the pathogen was phenotyped in each plant based on severity of crown and root rot. To screen for A. tumefaciens resistance (i.e. crown gall) 2mm deep wounds were flooded with A. tumefaciens and tumor presence/absence noted 2 and 4 months post inoculation. DNA was extracted from each screened genotypes and analyzed by the University of California Walnut Improvement Program scientists for genotyping single nucleotide polymorphisms. Preliminary modeling of the phenotype and genotype data have confirmed the presence of quantitative trait loci (QTL) for resistance in the case of both P. cinnamomi and A. tumefaciens. In support of Sub-objective 5A, an additional 36 infected and uninfected plant samples with two Phytophthora species were prepared for RNA sequencing analysis. Cross-examination of treatments, disease phenotypes, and transcriptomes will give insight into genetic mechanisms of pathogenicity as well as host defense. However, due to the Covid-19 lockdown, RNA-sequencing has been postponed. In support of Sub-objective 5B, three transformation vectors, each containing segments of four Phytophthora protein-coding genes, were constructed. These vectors were then introduced into somatic embryos of walnut, and over 100 transformant lines were obtained. Transformant lines exhibiting a high level of transgene activity were afterwards induced for shoot regeneration. Resulting shoot cultures from 30 walnut transformant lines were then subjected to an in vitro infection test, which we developed in this rating period, and consequently, three transformant lines showing improved disease resistance were identified. In a subordinate project in support of Objective 4, ARS scientists in Davis, California, have sequenced, assembled and annotated 29 A. tumefaciens isolates collected from crown gall affected trees throughout the major walnut growing regions of the Central Valley of California.

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 UC Davis, ARS researchers in Davis, California, employed RNA interference (RNAi)-based strategies to accelerate genetic improvement of almond and walnut rootstocks. RNAi transformation vectors containing selected Phytophthora gene segments were constructed, delivered to plants, and 100 transformants were created. ARS researchers developed a disease resistance bioassay to identify three walnut transformant lines showing improved Phytophthora resistance. Almond transformants have also been generated and are being examined in disease resistance bioassays. 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 the environmental impact and human health risks of the public and agricultural workers.

2. Characterized the genetic diversity of representative Agrobacterium tumefaciens collected from crown galled trees throughout the central valley of California. Crown gall is the number one disease throughout the walnut growing region of California. The genomes of 29 Agrobacteruim tumefaciens collected from across the central valley were sequenced, assembled and annotated. The genetic and phenotypic diversity of these isolates were characterized. This is facilitating the efforts of ARS scientists to develop both crown gall resistant rootstock genotypes and widely effective crown gall management strategies.

3. Characterization of soil microbial communities responsive to anaerobic soil disinfestation (ASD), an alternative to chemical-based soil fumigation. ARS researchers in Davis, California, assessed soil microbiome shifts in response to different ASD carbon source amendments. Results from field and greenhouse trials showed rice bran and tomato pomace elicit highly similar microbial communities and exhibit similar efficacy at controlling targeted plant pathogens. Analyses of these communities point to the potential importance of nitrogen fixation and denitrification in altering nutrient dynamics in ASD-treated soils. Tomato pomace is a more cost-effective carbon source for ASD than rice bran, and this may facilitate ASD adoption as an alternative to pre-plant chemical fumigation of soil to control plant pathogens.

4. Walnut rootstocks selected for resistance to Phytophthora advanced for nursery propagation and commercial orchard testing. Phytophthora is a watermold that causes crown and root rots and contributes significantly to an estimated $ 29 million annual revenue loss due to soilborne diseases in the California walnut industry. Preplant soil fumigation is increasingly regulated and not completely effective for management of the soilborne disease complex, so walnut rootstocks resistant to multiple pathogens are needed. ARS scientists in Davis, California, selected multiple rootstocks that exhibited resistance to Phytophthora and one or more of the other major soilborne pathogens of walnut. The advanced selections are being propagated at a commercial nursery for testing in commercial orchards. The rootstock resistance, once validated in commercial settings, will contribute valuably to walnut industry sustainability.

5. Walnut and Prunus rootstocks selected for resistance to Agrobacterium tumefaciens (crown gall) were advanced and delivered for nursery propagation and commercial orchard testing. Agrobacterium tumefaciens is a soil borne bacterium which causes crown gall disease on commonly used walnut and almond rootstocks contributing to the estimated $29 million annual loss due to soil borne diseases in California. There is an urgent need for disease resistant rootstocks. The crown gall resistant genotypes identified by ARS scientists in Davis, California, have been commercially propagated and will be prepared for distribution for reexamination in field trials.

6. Phytophthora species identified as contributing to walnut tree death in orchards subjected to river flooding and seepage. Phytophthora is a soilborne watermold that can infect trees in water-saturated soil and reside in rivers and canals that are used to irrigate orchards. In 2017, catastrophic high water flows associated with exceptional spring storms occurred in the Feather and Stanislaus Rivers, inundating adjacent walnut orchards directly and by seepage through levees. Bleeding cankers, which typically appeared to start in the English walnut scions above the graft union, formed in many but not all of the flooded orchards. ARS scientists from Davis, California, sampled the cankers, identified the species of Phytophthora, and confirmed their walnut pathogenicity. It was concluded that tree death and decline associated with the cankers resulted from infection by Phytophthora, whereas tree loss in the flooded orchards without cankers may have resulted from root oxygen deprivation and/or as yet unexplored root disease. Application of systemic fungicides that suppress Phytophthora may be justified in walnut orchards inundated by flooding.

7. Confirmation of Cherry Leafroll virus infection of black walnut trees maintained in germplasm collection. Blackline disease is exclusive to black walnut rootstocks in California. The disease is due to hypersensitive response of black walnut rootstocks to virus-infected scions. Cherry leafroll virus is spread by pollen shed by infected trees and thus a rootstock tolerant to the virus is of huge value in managing black line disease in California. An accession each of Juglans (J.) hindsii and J. microcarpa have been confirmed to be infected by cherry leafroll virus isolates. J. microcarpa hybrids have already been confirmed to be resistant to crown gall and infection by Phytophthora and thus having tolerance to Cherry leafroll virus would be a highly desirable trait in the advancement of hybrids of J. microcarpa as rootstocks. These rootstocks are expected to have potential for a huge impact in managing the most important diseases of walnuts in California by walnut growers.

8. Study of genetic diversification of the Sudden Oak Death (SOD) pathogen Phytophthora ramorum. Phytophthora (P.) ramorum is an invasive pathogen that causes Sudden Oak Death in coastal forests in the western United States and ramorum blight in nursery ornamentals. It is not clear whether the dominant original single clonal lineage NA1 has diversified and adapted to local environments or particular host species since its introduction in the 1990s. ARS researchers in Davis, California, examined a total of 78 P. ramorum isolates from diverse hosts and geographical regions for signatures indicating adaptation to the local environment and discovered that the NA1 lineage had accumulated Structural Variations (SVs), such as duplications, deletions, and translocations of genome segments. Several SVs were found to be associated with environmental factors such as host density and minimum temperatures, which implies local adaptation of the pathogen. The information on genetic diversification and local adaptation, as we have shown, is useful for the management of this devastating disease. For instance, SOD can now be considered as a chronic disease caused by locally adapted P. ramorum lineages. For the restoration of forests affected by SOD, tree seedlings for replanting should be evaluated for disease resistance using P. ramorum individuals infested locally under local environmental conditions.

U.S. DEPARTMENT OF AGRICULTURE

Crops Pathology and Genetics Research: Davis, CA