Research Project: Integrated Disease Management Strategies for Woody Perennial Species

Location: Crops Pathology and Genetics Research

2019 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 Sub-objective 1A, almond trees of cultivar Nonpareil with bud failure that had been identified in spring 2018 were unfortunately not available for subsequent samplings due to tree removal by the cooperator; however, two other orchards were identified in Yolo County, California, which had cultivar Carmel trees with bud failure. These trees were sampled, and RNA extracted and submitted for Illumina sequencing at the University of California (UC), Davis. Samples are currently in the queue for sequencing. For Sub-objective 1B, approximately 1,000 Cabernet Sauvignon grapevines planted in Oakville, California, in 2017 were examined for symptoms of Grapevine red blotch virus (GRBV) in the fall and tested for GRBV infection status by quantitative polymerase chain reaction (qPCR). All grapevines tested negative while <0.2% showed symptoms resembling red blotch. This block is in the midst of the red blotch diseased blocks. However, absence of virus infection in the two-year-old block may be due to an extremely low vector population in the vicinity, due to precautionary vector management practices implemented by the premium wine producers in this American Vineyard appellation. ARS scientists, in collaboration with researchers at the University of California, Davis, have located additional vineyards where Grapevine red blotch virus has been spreading. Last year, shot gun sequencing of genomic DNA of four insect morphs encountered in one vineyard, classified as two different Tortistilus species (family Membracidae) previously, confirmed that they belong to a single species of genus Tortistilus. This year, biological data obtained by mating experiments confirmed that they were single species. Insects at two different vineyards were also confirmed to belong to the same species based on the sequence of Cytochrome oxidase 1 gene. Experiments to prove if the insects are capable of transmission of Grapevine red blotch virus are in progress. In Sub-objective 1C, research continued on etiology of Paradox canker disease (PCD) of walnut. ARS scientists conducted graft transmission experiments to test the hypothesis that the disease is caused by a biotic agent. Disks of bark were removed from PCD-affected trees (i.e., from the canker margins on the rootstock; and from above the cankers on the scion) and from apparently healthy walnut trees (from rootstock and scion). Bark disks from each of the four categories (i.e., PCD-affected rootstock, PCD-affected scion, healthy rootstock, and healthy scion) were “patched into” healthy rootstock and scions of additional trees near to those that had supplied the bark disks. Cankers symptomatic of PCD were generated from some of the bark patches originating from the margin of PCD cankers in the rootstock, but none of the other bark patches (i.e., of scion bark on PCD-affected trees or of rootstock or scion bark on healthy trees) resulted in PCD. The results indicate PCD is associated with a graft-transmissible agent. For Sub-objective 1D, microbial community shifts associated with Prunus replant disease (PRD) were monitored in three almond orchard replant trials. The trials included replicated preplant treatments of fumigation and a non-treated control. By early FY19, near optimal growth resulted in almond trees being replanted after preplant fumigation, but the trees replanted in control plots grew significantly less, as is typical in trees affected by Prunus replant disease. Initial bioinformatics analyses were completed for PCR amplicons of ribosomal ribonucleic acid (rRNA) genes extracted from bacteria, fungi, and oomycetes in root and soil samples from the preplant fumigated and control plots. Additional sequencing and bioinformatics are underway to complete assessments of the microbial community shifts associated with PRD. In Sub-objective 1E, a clonal lineage of P. ramorum, NA1 dominates the forests in California, which was believed to be introduced in the 1990s. We previously evaluated in vitro growth patterns and virulence in Rhododendron foliage for 19 isolates belonging to the clonal lineage. ARS scientists scored phenotypes of an additional 42 isolates and determined their genome sequences. Due to the recent introduction and clonality, the observed DNA sequence diversity among Californian isolates is minimal. NA1 isolates have over 230,000 fixed heterozygous single nucleotide polymorphic (SNP) sites, which makes the identification of de novo mutations between clonal isolates difficult. Nevertheless, we established a bioinformatic pipeline to measure genome diversity and identified ca.716 single nucleotide variations as well as 158 structural variant sites. In Sub-objective 2A, DNA samples from cherry trees infected with Western X disease phytoplasma were sequenced. De novo assembly of this metagenome was poor due to poor coverage and use of short-read sequencing technology. New DNA samples extracted from a cherry tree infected with X disease phytoplasma, along with the DNA infected from an almond tree infected with Peach yellow leaf roll phytoplasma, were prepped and submitted for PacBio sequencing which will facilitate successful genome assembly. For Sub-objectives 3B and 3C, 16S and ITS amplicons of rRNA genes were sequenced to examine root and soil microbial community shifts associated with preplant Anaerobic Soil Disinfestation (ASD) treatments in two almond orchard replant trials. Bioinformatics examinations of bacterial and fungal communities were completed for root and soil samples collected at monthly intervals in the first year after replanting. By early FY19, preplant ASD treatments based on either a rice bran or ground almond hull and shell mixture had performed nearly as well as preplant soil chemical fumigation in stimulating replanted orchard growth. The microbial community examinations indicated that full ASD treatments resulted in similar, root and soil community shifts that differed from those in control and preplant chemical fumigation treatments. The communities in fumigated and control treatments, were similar. ASD treatments without plastic tarp or limited irrigation, were not as effective as full ASD treatments and did not affect root or soil microbial community shifts. In Sub-objective 3A and 3C, greenhouse and field trials of ASD were conducted with rice bran, tomato pomace, mustard seed meal, molasses, and red grape pomace to evaluate the effectiveness of different carbon sources at generating anaerobic conditions and reducing populations of an introduced plant pathogen, as well as to assess changes in soil microbial communities in response to carbon source amendment. In both field and greenhouse trials, tomato pomace was as effective as rice bran at suppressing an introduced plant pathogen and resulted in similar microbial community changes. Bacterial taxa with the potential to produce compounds suppressive to plant pathogens based on in vitro studies grew rapidly (less than 3 days) in response to ASD initiated with rice bran and tomato pomace and persisted throughout the process of ASD (approximately 5 weeks). In Sub-objective 4B, phenotyping of resistance to Phytophthora cinnamomi and P. citricola among clonal hybrid rootstocks from two full-sib families of Juglans microcarpa x J. regia was completed. Over 400 clones (greater than 200 per family), all of which had been genotyped for single nucleotide polymorphisms (SNPs) by colleagues at UC Davis, were represented in the phenotyping, which revealed that clonal individuals in each family were segregating for resistance to each of the Phytophthora species. Bioinformatic analyses that regressed our resistance phenotypes on the SNP genotypes resolved a quantitative trait locus (QTL) for resistance to both pathogens on chromosome 11 of J. microcarpa with greater statistical certainty than our previous examinations had afforded. These findings provided a basis for further genetic resolution of the QTL and development of marker-assisted breeding strategies for resistance to Phytophthora in walnut rootstocks. Sub-objective 5A focused on better understanding the complicated interplays between plants and Phytophthora. Transcriptomes of P. citricola growing in plant tissues and a culture medium were compared. It was discovered that ca. 2000 genes were differentially expressed between P. citricola interacting with plant tissues and that grew on a culture medium. Gene function enrichment analysis showed that genes involved in cellulose catabolic process were overrepresented in P. citricola grown in the plant. On the other hand, proteomic analysis detected 71 P. citricola proteins in plant tissues, and enzymes involved in glycolysis were overrepresented. It is hypothesized that genes and proteins active in plant tissues are essential for parasitic growth of the pathogen, hence chose them for RNAi target for growth suppression in the plant. In the course of data analysis, however, it became clear that P. citricola genome assembly, which was used for the transcriptome and proteome analyses, was contaminated with a fungal genome. We subsequently removed scaffolds derived from the fungal contaminant and cleaned up the P. citricola genome assembly. In Sub-objective 5B, the efficiency of RNAi constructs were evaluated by examining two transient assay methods i.e., virus-induced gene silencing (VIGS) and spray-induced gene silencing (SIGS). Experimental data now indicates that these two methods are unfeasible for the suppression of Phytophthora growth. A third novel approach, host-induced gene silencing (HIGS) technology to evaluate RNAi assays, is being used to suppress Phytophthora inplanta. Since this approach requires stable plant transformants with RNAi construct, we created 11 tomato and 26 Nicotiana benthamiana transgenic lines carrying a total of three RNAi constructs. Currently, these lines are being used to compare the efficiency of three RNAi constructs for Phytophthora disease suppression.

Accomplishments
1. Host mediated genomic reconstruction of Phytophthora ramorum in California. Researchers in Davis, California, recently confirmed that some host plant species permanently alter the P. ramorum genome and reduce its aggressiveness. Forty-two P. ramorum genomes, isolated from diverse plant hosts, were sequenced which allow the scientists to quantify genome changes using a variety of computational techniques. Consistent with the recent introduction of the P. ramorum, only 716 single nucleotide mutations were identified among the 42 genomes. In addition, 158 changes in chromosomal structure were identified which were found to be associated with growth rate and aggressiveness of the pathogen. Understanding this new relationship between isolates will enhance epidemiological studies and development of management strategies for Sudden Oak Death.

2. Impacts of soil fumigation and anaerobic soil disinfestation on soil and root microbial communities in replanted almond orchard. Anaerobic soil disinfestation (ASD) is being developed as an alternative to preplant soil fumigation for management of almond replant problems. ARS scientists in Davis, California, determined that ASD based on either rice bran or ground almond hull and shell mixtures is nearly as effective as soil fumigation for management of Prunus replant disease (PRD). They determined that despite their similar levels of effectiveness, ASD and soil fumigation resulted in significantly different soil and root microbial communities that persist at least through the first year of orchard development. These differences may affect long-term orchard health and performance and therefore are of keen interest to tree crop growers.

3. Long-term assessment of improved walnut rootstocks with resistance to key plant pathogens. Walnut rootstocks selected for resistance to soil borne pathogens under greenhouse conditions can benefit from extended validations under orchard conditions. ARS scientists in Davis, California, in collaboration with University of California, Davis researchers and commercial walnut growers, assessed long-term orchard survival of walnut rootstock hybrid, RX1, which was previously selected for resistance to Phytophthora in greenhouse trials. In an orchard infested with Phytophthora cinnamomi, the most aggressive cause of crown and root rot on California walnuts, more than 30% of trees on standard Paradox seedling rootstock had died within two years after planting, whereas no trees on RX1 rootstock had died after eight years of the ongoing trial. The tree losses on Paradox rootstock were associated with infection by the resident pathogen. The findings validate superior resistance of the RX1 rootstock under challenging commercial conditions.

4. Characterization of soil microbial communities responsive to Anaerobic soil disinfestation (ASD). ARS researchers in Davis, California, profiled microbial community changes in response to different ASD carbon source amendments. Results from replicated greenhouse and field trials identified a core group of microbial responders to rice bran and tomato pomace that have the genomic potential to produce organic acids known to inhibit the growth of plant pathogens and perform nitrogen fixation and denitrification. Tomato pomace was equally effective as rice bran at controlling populations of target plant pathogens. ASD using tomato pomace is more cost-effective than rice bran and offers an alternative to chemical soil fumigants known to be harmful to human health and the environment. Development of a chemical-independent form of preplant soil fumigation (i.e. ASD) will have significant positive impacts in both nursery and production agriculture. In addition, wide spread use of ASD will reduce the use of harmful chemicals which negatively impact both the environment and human health across the U.S.

5. Graft transmission indicates biotic cause of a walnut Paradox rootstock canker disease. Paradox hybrid has become the dominant rootstock for U.S. English walnut production due to its tendency to support good yields, yet it suffers from Paradox canker disease (PCD), an emerging malady of unknown cause. ARS researchers in Davis, California, cultured tissues and isolated nucleic acids from affected tissues in efforts to identify causal pathogens, yet none were detected, leading to conjecture of a possible abiotic cause for the disease (e.g., metabolic dysfunction, chemical or environmental injury, etc.). As an alternative approach, the researchers attempted to graft transmit the canker disease from affected Paradox rootstock tissues to healthy Paradox rootstock trees. Some of the graft transmissions were successful, indicating canker disease is caused by a pathogen instead of an abiotic factor. These findings will facilitate development of effective PCD control strategies. California walnut growers impacted by PCD statewide will benefit from this research.

6. Involvement of a virus which limits graft compatibility of common grape scions on rootstock widely used in the industry. Grapevine rupestris stem pitting-associated virus (GRSPaV) is involved in graft incompatibility reaction of Pinot Noir clones on 110 Richter rootstock. In previous projects, a disease of Pinot Noir clones on 110 Richter (110R) rootstock described as ‘Grapevine necrotic union’ was hypothesized to be due to infection of the scion by a latent virus that elicited a hypersensitive reaction from the rootstock leading to the disease. Previous attempts had failed to establish the causal agent of the disease. By deep sequencing of dsRNA or total RNA from diseased grapevines from different locations over years and subsequent molecular tests, ARS scientists in Davis, California, discovered that GRSPaV is the likely etiological agent. This virus is currently not regulated in California, but due to increasing reliance on 110R rootstock because of its drought tolerance, there may soon be a need to regulate the presence of the virus in grafts made with this particular rootstock. Beneficiaries of this finding will be growers of Pinot Noir in California and Oregon who may like to use 110 Richter rootstock and nurseries producing grafted grapevines on 110 Richter rootstock.

7. Black walnuts serving as asymptomatic reservoirs for an important viral pathogen of English walnuts. Cherry leafroll virus can infect black walnut species. All black walnuts (Juglans hindsii, J. major, J. nigra) were believed to be resistant to infection by Cherry leafroll virus (CLRV) that causes blackline disease of English walnut (J. regia) trees grafted on black walnut derived rootstocks. Last year, using molecular approaches, ARS scientists in Davis, California, discovered that several asymptomatic black walnut accessions at the USDA, ARS, National Clonal Germplasm Repository in Davis, California, were infected by CLRV. ARS scientists were able to confirm the CLRV infection in some of these trees. Seedlings from these lines will be tested by challenging with CLRV by approach grafting followed by molecular tests for virus infection, a screening method developed by ARS scientists in Davis, California, in collaboration with walnut breeders at University of California, Davis.

Review Publications
Diaz-Lara, A., Klaassen, V., Stevens, K., Sudarshana, M.R., Rowhani, A., Maree, H.J., Chooi, K.M., Blouin, A.G., Habili, N., Song, Y., Aram, K., Arnold, K., Cooper, M.L., Wunderlich, L., Battany, M.C., Bettiga, L.J., Smith, R.J., Bester, R., Xiao, H., Meng, B., Preece, J.E., Golino, D., Alrwahnih, M. 2018. Characterization of grapevine leafroll-associated virus 3 genetic variants and application towards RT-qPCR assay design. PLoS One. 13(12):e0208862. https://doi.org/10.1371/journal.pone.0208862.
Rojas, M.R., Macedo, M.A., Maliano, M.R., Soto-Aguilar, M., Souza, J.O., Briddon, R.W., Kenyon, L.A., Rivera Bustamante, R.F., Zerbini, M.F., Adkins, S.T., Legg, J.P., Kvarnheden, A., Wintermantel, W.M., Sudarshana, M.R., Peterschmitt, M., Lapidot, M., Martin, D.P., Moriones, E., Inoue-Nagata, A.K., Gilbertson, R.L. 2018. World management of geminiviruses. Annual Review of Phytopathology. 56:637-677. https://doi.org/10.1146/annurev-phyto-080615-100327.
Preto, C.R., Sudarshana, M.R., Bollinger, M.L., Zalom, F.G. 2018. Vitis vinifera (Vitales: Vitaceae) as a reproductive host of Spissistilus festinus (Hemiptera: Membracidae). Journal of Insect Science. 18(6):1-7. https://doi.org/10.1093/jisesa/iey129.
Preto, C.R., Bahder, B.W., Bick, E.N., Sudarshana, M.R., Zalom, F.G. 2019. Seasonal dynamics of Spissistilus festinus (Hemiptera: Membracidae) in a Californian vineyard. Journal of Economic Entomology. 112(3):1138-1144. https://doi.org/10.1093/jee/toz022.
Dalton, D.T., Hilton, R.J., Kaiser, C., Daane, K.M., Sudarshana, M.R., Vo, J., Zalom, F., Buser, J.Z., Walton, V.M. 2019. Spatial associations of vines infected with grapevine red blotch virus in Oregon vineyards. Plant Disease. 103(7):1507-1514. https://doi.org/10.1094/PDIS-08-18-1306-RE.
Poret-Peterson, A.T., Albu, S., McClean, A.E., Kluepfel, D.A. 2019. Shifts in soil bacterial communities as a function of carbon source used during anaerobic soil disinfestation. Frontiers in Environmental Science. 6:160. https://doi.org/10.3389/FENVS.2018.00160.
Zhu, T., Wang, L., You, F.M., Rodriguez, J.C., Deal, K.R., Chen, L., Li, J., Chakraborty, S., Balan, B., Jiang, C., Brown, P.J., Leslie, C.A., Aradhya, M.K., Dandekar, A.M., Mcguire, P.E., Kluepfel, D.A., Dvorak, J., Lou, M. 2019. Sequencing a Juglans regia × J. microcarpa hybrid yields high-quality genome assemblies of parental species. Horticulture Research. 6:. https://doi.org/10.1038/s41438-019-0139-1.
Malar C, M., Yuzon, J.D., Panda, A., Kasuga, T., Tripathy, S. 2019. Updated assembly of Phytophthora ramorum pr102 isolate incorporating long reads from PacBio sequencing. Molecular Plant-Microbe Interactions. 32(11):1472-1474. https://doi.org/10.1094/MPMI-05-19-0147-A.
Malar, M.C., Yuzon, J.D., Das, S., Das, A., Panda, A., Ghosh, S., Tyler, B.M., Kasuga, T., Tripathy, S. 2019. Haplotype-phased genome assembly of virulent Phytophthora ramorum isolate ND886 facilitated by long-read sequencing reveals effector polymorphisms and copy number variation. Molecular Plant-Microbe Interactions. 32(8):1047-1060. https://doi.org/10.1094/MPMI-08-18-0222-R.
Browne, G.T., Ott, N.J., Poret-Peterson, A.T., Lampinen, B.D., Metcalf, S.G., Yaghmour, M.A., Holtz, B.A., Culumber, M., Brar, G., Doll, D.A. 2018. Anaerobic soil disinfestation as an alternative to soil fumigation for management of Prunus replant disease: effects on tree growth and yield. Acta Horticulturae. 1219:287-294. https://doi.org/10.17660/ActaHortic.2018.1219.44.