Source: UNIVERSITY OF CALIFORNIA, RIVERSIDE submitted to NRP
PUTTING PHENOTYPIC AND GENOTYPIC TOOLS TO WORK FOR IMPROVING WALNUT ROOTSTOCKS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1016502
Grant No.
2018-51181-28437
Cumulative Award Amt.
$4,031,294.00
Proposal No.
2018-03397
Multistate No.
(N/A)
Project Start Date
Sep 1, 2018
Project End Date
Aug 31, 2023
Grant Year
2019
Program Code
[SCRI]- Specialty Crop Research Initiative
Recipient Organization
UNIVERSITY OF CALIFORNIA, RIVERSIDE
(N/A)
RIVERSIDE,CA 92521
Performing Department
Department of Nematology
Non Technical Summary
Walnuts are an important part of the economy of California and represent a 2 billion dollar a year industry in the U.S.A. Multiple soil-borne pests and pathogens along with numerous abiotic stresses impact this crop negatively. Sustainability-encumbering culprits include: black line disease, crown gall, phytophthora root and crown rot, and plant-parasitic nematodes. Water quantity and quality stresses are also robbing yield. In California, English walnut trees are traditionally, grown on rootstocks with the most common being, to humans and the environment. In addition, use of many of the remaining chemical fumigants is becoming cost prohibitive. In a prior SCRI supported project, progeny from crosses of J. microcarpa mothert trees with J. regia resistance to crown gall, phytophthora root rots, root lesion nematode and root-knot nematode. Breeding populations (>300 offspring) from each of two selected mother trees were generated, genetically characterized, and then examined for their resistance to the diseases mentioned above. By combining the disease resistance data with the genetic characterization multiple regions on chromosome 11 were identified that appear to be associated with the observed disease resistance. Genetic markes are being devloped within these genetic regions and will be used to screen young walut seedings. This process will allow discarding genotypes that do not carry the markers associated with disease reistance. In this process, multiple molecular signals associated with resistance will be tested in a population of >5,000 seedlings and only those with the genetic markers, putatively carrying (mulitple) reistance will be selected. Less than 10% of this population is expected to exhibit resistance to multiple pathogens. These individuals will be tested to confirm disease resistance. In a parallel series of experiments we will examine these off-spring to validate testing systems designed to examine water stress tolerance and tolerance to black line disease. Elite rootstock candidates identified by these procedures will be placed in field trials in different growing regions of California where they will concomitantly serve as outreach teaching tools. Other members of these breeding populations will be used for refinement of spectral analysis for detection of (a)biotic stresses. Comprehensive economic analysis will determine benefits of using new rootstocks in walnut production. Built on previous success with SCRI support, this project will provide urgently needed disease resistant rootstocks and breeding tools for future development of enhanced walnut rootstocks.
Animal Health Component
40%
Research Effort Categories
Basic
30%
Applied
40%
Developmental
30%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011213108140%
2021213112010%
2011213110110%
2011213110010%
2011213110310%
2011213102010%
4041213106010%
Knowledge Area
404 - Instrumentation and Control Systems; 202 - Plant Genetic Resources; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1213 - Walnut;

Field Of Science
1060 - Biology (whole systems); 1101 - Virology; 1103 - Other microbiology; 1120 - Nematology; 1081 - Breeding; 1100 - Bacteriology; 1020 - Physiology;
Goals / Objectives
The majority (ca. 80%) of walnuts are produced on so-called which are hybrids from a cross between J. hindsii and J. regia. Unfortunately, paradox rootstocks are susceptible to a variety of common soil borne pathogens and abiotic stress. In this project, progress made in a prior SCRI-funded project will be extended by increasing knowledge of the biological basis for disease resistance identified in offspring from previous interspecific crosses. The specific objectives are:Identify gene loci (QTL) that confer resistance to the following pathogen systems; crown gall (CG), Phytophthora (PHY), root lesion nematode (NEM), and compatibility to infection with the Cherry Leaf Roll Virus.Determine effects of host genotypic on water relations and disease resistance responses of available breeding material.Pyramiding multiple resistance loci into superior rootstock genotypes by improved selection strategies.Gather information on gene expression of resistant and susceptible infection processes.Further spectral analysis capabilities for detecting specific soil-borne disease impacts on walnuts.Release rootstock genotypes with superior tolerance and resistance characteristics to key soil-borne plant pathogens and abiotic stresses with vetted horticultural traits.Provide economic evaluations for decision support on rootstock use. These activities will improve the sustainability of walnut production by reducing reliance on chemical and agronomic inputs during the production cycle of walnut orchards. The reduced production risk will support agricultural communities by providing a more robust, reliable and environmentally friendly production system.
Project Methods
This project will revolve around the characterization and exploitation of two previously-identified breeding populations for the identification of resistance to multiple pathogens and possibly tolerance to abiotic stresses. These populations have been genotyped, and the phenotyping is being completed. Overlaying the genotypic maps with phenotypic information will allow for the identification of regions in the genotypic map that mediate the positive plant traits, and to identify molecular markers associated with them. These markers will be helpful for identifying genotypes that potentially carry the desirable traits. Concurrently with the ongoing evaluations, new crosses of the preferred mother trees with J. regia pollen will be made. Seed from these crosses will be first screened genetically for the presence of multiple molecular markers, and then the genotypes with the most promising marker combinations will be tested phenotypically.These test genotypes will be entered into the pathogenicity resistance testing pipelines. In greenhouse experiments, walnut selections will be planted into Phytophthora infested soil, or stab-inoculated with Agrobacterium tumefaciens. In field experiments, subsets will be planted into nematode-infested soil, and continually monitored for nematode population density development. Cuttings will be grafted onto CLRV infected rootstocks to examine for blackline tolerance. Saplings also will be entered into the water stress tests. Because these genotypes are comprehensively characterized, additional evaluations will extend into partially known and confirmed aspects. For example, the responses of rootstock with known nematode responses at differing irrigation regiments with and without nematode infection will be examined. This will be done to validate the development and refinement of greenhouse assays for detection of the plants water deficit response.Offspring of the two crosses with known nematode responses will be used to calibrate spectral analysis of the effects of nematode root feeding on foliar reflectance. Tests will include hyperspectral sensing in the range of 190-1750 nm. The large data sets accumulated with these measurements will be thoroughly analyzed to yield the most indicative wavelength to discriminate the plant responses. This in turn will allow us to generate a much simpler, rapid and economically superior sensor for our phenotyping efforts.Genotyping efforts will be increased, and the genotyping-by-sequencing efforts refined plus amended with kompetitive allele specific PCR (KASP). This genotyping effort will result in detection of plant genotypes with combinatory markers of complementary traits. With this refining of the molecular markers, a more favorable chance for marker development exists.Economic data on the cost structure of implementing the novel rootstocks will be collected, and analyzed. This process will consider whether cropping the new rootstocks would allow foregoing soil fumigation, and how manipulations of the irrigation schedule can be sustained.With rootstock trials in place in various areas of the state, the extension programming will take full advantage of these plots as teaching tools. These sites will be the place for educational meetings, and other teaching events. General performance ranking will be supplemented with nematode and other pathogen evaluations.

Progress 09/01/18 to 08/31/23

Outputs
Target Audience:The target audience has not changed throughout the project period. One-on-one meetings and extension group visits constituted the information exchange.Formally, stakeholderrs of the walnut industry steer the project inthe Walnut Rootstock Advisory Panel that hasmet in conjunction with the Walnut Research Conference in January of each project year, and that continues after the project end date. Contact with the stakeholders is more frequent via different commodity-related meertings. Reciprocal information exchange includes visits and discussions at the field trial sites, in nursery settings and other opportunities. Becuase of the field trial network that has developed during the project time, an increasing number of grower collaborators has direct access to the information as it develops. These collaborative effort are extremely valuable because they allow for more feed-back and interactive information delivery. Such hands-on knowledge is extremely important in the implementation of novel rootstocks. The collaborators have grown them on their own farm and can lend their experience toothers. The academic stakeholders of the scientific information gets informed via publications and presentations of the research results. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Houston Saxe (graduate student) and Paolo Zaini (postdoctoral researcher) were trained in genetic studies including transcriptome analysis, complementation assays, QTL analysis, and bioinformatics of sequencing and genotyping data. A postdoctoral researcher and an undergraduate student learned and employed molecular techniques to investigate function of CLRV a difficult to study plant virus. A postdoctoral researcher and a staff research associate (SRA) were trained in nematode phenotyping for nematode diseases in trees. Undergraduate and graduate students learned about the phenotyping for resistance to Phytophthora root and crown rot. 3.5 (1 half time) postdoctoral researchers furthered skills in plant stress physiology and developed knowledge and skills for collaborative work in pathology. Two part time technicians were involved in this work over two summers- one is now in a PhD program at UC Davis and the other has accepted a permanent technician position with USDA-ARS. Three of the 4 post docs that worked on this project have obtained permanent faculty positions, and the other is transitioning into a stable Project Scientist role at UC Davis. How have the results been disseminated to communities of interest?The information of this project was conveyed to the stakeholder community. Formally this was done in the annual Walnut Rootstock Advisory Panel where the different research aspects were reported, and the walnut community made comments on how to fine-tune the research direction. Multiple presentations were made at these meetings that are not listed separately. Reports were also given in oral and written form to a broader audience at the annual Walnut Research Conference. Extension events were given that inculded field days at the various trial locations. Biology and dynamics of pathogens were discussed along with the performance of the novel rootstocks. For example: Hasey, J. - Lake County walnut meeting, "Walnut rootstock field trial update" April 2, 2021 Elizabeth Fichtner and Greg Browne, UCCE Virtual Statewide Walnut Series, "Walnut Rootstocks" Feb. 16, 2021 Hasey, J., Fichtner, E., Milliron, L. UC Cooperative Extension field meetings were held at each field trial site (2016 trials) in June 2022. Tulare County, Sutter County, Glenn County, Lake County. The aspect of walnut rootstocks was also frequently part of presentations of the PD when talking at grower, pest control advisor and other clientele events. Information was delivered tot he scientific community at national and international conferences. For example: Hasey, J., Milliron, L., Fichtner, E., Elkins, R., Lampinen, B., Reyes, C., Lightle, D., Leslie, C., McClean, A., Browne, G., and Kluepfel, D. Horticultural field performance of new putative disease resistant walnut rootstock genotypes compared to standard clonal and seedling Paradox rootstocks. Presented at the IX International Symposium on Walnut & Pecan, Grenoble, France, June 2023. Hasey, J., Milliron, L., Fichtner, E., Elkins, R., Lampinen, B., Reyes, C., Lightle, D., Leslie, C., McClean, A., Browne, G., and Kluepfel, D. 2023. Horticultural field performance of new putative disease resistant walnut rootstock genotypes compared to standard clonal and seedling Paradox rootstocks. Poster presented at the IX International Symposium on Walnut & Pecan, Grenoble, France, June 2023. Jafarbiglu, H., and Pourreza, A. (2022). A comprehensive review of remote sensing platforms, sensors, and applications in nut crops.Computers and Electronics in Agriculture,197, 106844. Jarvis-Shean, K., Hasey, J., Leslie, C., Browne, G., McClean, A., Ott, N., Westphal, A., and Kluepfel, D. Field testing of resistance of novel hybrid clonal walnut rootstocks to soilborne biotic stresses. Presented at the IX International Symposium on Walnut & Pecan, Grenoble, France, June 2023. Omidi, R., Pourreza, A., Moghimi, A., Zuniga-Ramirez, G., Jafarbiglu, H., Maung, Z., & Westphal, A. (2022). A Semi-supervised approach to cluster symptomatic and asymptomatic leaves in root lesion nematode infected walnut trees.Computers and Electronics in Agriculture,194, 106761. Writers of secondary literature articles were supported. For examples: Cecilia Parsons, 2022 Rootstock Trials Providing Data for Disease Tolerance, Production. West Coast Nut, pp 30,32-33, July 2022. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Identify gene loci (QTL) that confer resistance The quantitative trait loci (QTL) for resistance to crown gall (CG), phytophthora (PHY) and root lesion nematodes (NEM) all mapped to the same location on chromosome 4D. Inre-analysis, the QTL ofA. tumefaciensandPhytophthoraspp. phenotypic responses was confirmed.The same QTL was found for reduced susceptibility toP. vulnusand for tree height. Single RNA sequences of five different plant tissues were generated for eight Juglans spp. and deposited in the NCBI bioproject PRJNA758986. Determine effects of host genotypic on water relations and disease resistance RX1 exhibited superior drought stress tolerance own-rooted or grafted to a scion.RX1 was a weaker sink for carbon in the root systems (i.e. smaller but more hydraulically efficient root system) thus allowing more carbon allocation to aboveground parts under drought stress. When grafted, RX1 conferred superior drought resistance through osmotic adjustments in the leaves and canopy conductance. A mini-weighing lysimeter system on load cells could effectively track day vs night water use and determining hydraulic conductance of various parts of the plant system (i.e. root vs. shoot conductance) relative to point measures of water potential, stomatal conductance and photosynthesis. Crown gall- andPhytophthora-resistantaccessions TB33 and AD558were low vigor plants under both control and drought conditions compared tocommercials associated with lower photosynthetic rates and high susceptibility to moderate drought. In factorial drought and pathogen (Crown gall) inoculation experiments (drought +/- and A. tumefaciens +/- and two commercial rootstocks), RX1 responded to drought more effectively than VX211. The dimensions of crown galls were not different between rootstocks nor between water treatments but the infection impacted the ratio of stem diameter above and below the gall under both water treatments suggesting that the gall impacted the carbohydrate distribution and nutrient allocation. In drought and heat tests, J. regia accessions from the National Clonal Germplasm Repository differed in photosynthetic capacity according to their geographic origin associated with inherent anatomical and biochemical differences. One accession, native to the driest and hottest habitats, responded most effectively to both stressors.These accessions hypothetically can serve as source for superior abiotic stress tolerance. Pyramiding multiple resistance loci into superior rootstock genotypes Phenotypic data from screening for CG, PHY and NEM susceptibility plus records of vigor under nematode-infested field conditions revealed one accession with beneficial traits in all four categories, five with three beneficial resistances, four with two, and with vigorous growth under nematode-infested conditions plus resistance to one pathogen. The phenotypic data was prepared for disposition in the Ag Data Commons (https://data .nal.usda.gov). The longevity of these beneficial traits needs to be tested because there is some indication that some of them may only function in the initial cropping years. These superior accessions are produced for further experimentation towards release. Gather information on gene expression of resistant and susceptible infection processes Transcriptome Analysis:In RNAseq analysis of non-inoculated roots of hybrid rootstocks of accessions with distinct responses toA. tumefaciensphenotypic,Phytophthoraspp., to three-year tree height (vigor), and three-yearP. vulnusnematode numbers, cell wall biogenesis-related genes were identified as potentially playing a key role in susceptibility toA. tumefaciens,Phytophthoraspp., and increased vigor, but notP. vulnus. Many genes associated with vigor and susceptibility were associated with the plasma membrane and extracellular space, perhaps connecting these traits to barrier function. RNA processing and splicing, along with predicted targeting to the nucleus, were associated with resistance toA. tumefaciens,Phytophthoraspp., and low vigor, but notP. vulnus. Four genes in theJ. microcarpaQTL region for resistance toA. tumefaciensandPhytophthoraspp. were represented among the transcripts, and two of the genes were differentially expressed in association with resistance toA. tumefaciensand decreased vigor. TheJ. microcarpahaplotype expressed more transcripts associated with resistance toA. tumefaciens,Phytophthoraspp., and low vigor, but notP. vulnusthan theJ. regiahaplotype. CLRVTools for quicker and easier CLRV susceptibility testing were developed. A total of 903 walnut leaf samples from J. hindsii X J. regiabackcross lines andJ. microcarpaandJ. regiahybrid lines were tested for CLRV infection. This process used a grafting assays where test wood was grafted onto infected rootstocks, and the leaves were tested with quantitative real time PCR using CLRV-specific primers. In the development of an in vitro screening assay for hypersensitive response (HR) in walnut lines, candidate molecules of the virus that lead to hypersensitive response, cDNA sequences corresponding to polyproteins 1 and 2 (P1, P2, respectively) of the CLRV genome were amplified. The fragments were assembled to study the role of P1 or P2 in HR induction. P1 appeared to induce cell death inN benthamianaandN tabacumXanthi suggesting it to may be the sought molecule that induces HR. To enrich the public sector (e.g. NCBI) with sequence information on CLRV isolates where the sequences of RNA-1 and RNA-2 of only one CLRV walnut isolate are available, genomes of walnut isolates of CLRV from field samples were sequence using Illumina and Sanger methods. Further spectral analysis capabilities for detecting specific soil-borne disease impacts on walnuts. Spectral analysis:Spectral patterns related to nematode infection were identified in walnut rootstock accessions of different infection levels. VX211 was least affected, followed by MS1 122, and MS1 127 being the most impacted, this aligned with nematological examinations. Spectral analysis may support conventional phenotyping methods of soil and root sampling and nematode counting. High throughput phenotyping by spectral modeling would greatly facilitate the screening process. Radio-frequency analysisReflectance of walnut leaves of trees with different nematode infections was measured with the low-cost proximate radiofrequency tridimensional sensor "Walabot". In machine learning (ML), multiple Scikit-learn classification algorithms, such as k-nearest neighbors, random forest, and Adam optimizer, were applied. The proximate sensor predicted nematode infection levels with 95% accuracy and worked flexibly in the field. Release rootstock genotypes with superior tolerance and resistance characteristics and abiotic stresses with vetted horticultural traits. In data of five field trials, at least one superior rootstock plus two high-level candidates were identified that have beneficial traits that may justify release. One rootstock, initially selected for high levels of CG resistance, showed superior performance in walnut replant situations in the presence of root lesion nematode and the replant problem was considered for release. Others are still discussed. Approximately ten elite clonal walnut rootstock hybrids were selected based on pathogen response, and established in long-term field trials along commercial comparatives. Four experiments were established at commercial sites with natural pathogen infestations (2 RLN, 1 CG, 1 PHY), and two with artificial inoculations on a research farm. These ongoing experiments will determine the disease response under orchard conditions. ?Provide economic evaluations for decision support on rootstock use. A survey on grower attitudes and grower willingness to pay a premium for superior rootstock genotypes was conducted and is in press with California Agriculture - a unique journal that reaches scientists and stakeholders alike.

Publications

  • Type: Journal Articles Status: Published Year Published: 2023 Citation: Brown, P.J. (2023) Haplotyping interspecific hybrids by dual alignment to both parental genomes. Plant Genome Jun;16(2):e20324. Doi: 10.1002/tpg2.20324. Epub 2023 Apr 14. PMID: 37057366.
  • Type: Conference Papers and Presentations Status: Under Review Year Published: 2023 Citation: Hasey, J., Milliron, L., Fichtner, E., Elkins, R., Lampinen, B., Reyes, C., Lightle, D., Leslie, C., McClean, A., Browne, G., and Kluepfel, D. Horticultural field performance of new putative disease resistant walnut rootstock genotypes compared to standard clonal and seedling Paradox rootstocks. Acta Horticulturae: In review
  • Type: Conference Papers and Presentations Status: Under Review Year Published: 2023 Citation: Jarvis-Shean, K., Hasey, J., Leslie, C., Browne, G., McClean, A., Ott, N., Westphal, A., and Kluepfel, D. Field testing of resistance of novel hybrid clonal walnut rootstocks to soilborne biotic stresses. Acta Horticulturae: In review
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2023 Citation: Liang, J., Levi, A., and Westphal, A. Walnut Growers Preferences Regarding Rootstock Attributes. California Agriculture: In Press.
  • Type: Journal Articles Status: Other Year Published: 2024 Citation: Niu, H. A. Westphal, Y.Q Chen. A low-cost proximate sensing method for early detection of nematodes in walnut using machine learning algorithms. In preparation.
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Omidi, R., Pourreza, A., Moghimi, A., Zuniga-Ramirez, G., Jafarbiglu, H., Maung, Z.T.Z., and A. Westphal. 2022. A Semi-supervised Clustering Approach to Cluster Symptomatic and Asymptomatic Leaves in Root Lesion Nematode Infected Walnut Trees. Computers and Electronics in Agriculture 194  106761.
  • Type: Journal Articles Status: Under Review Year Published: 2023 Citation: Saxe, H., Walawage, S., Balan, B., Leslie, C.A., Brown, P.J., Browne, G.T., Kluepfel, D.A., Westphal, A., and Dandekar, A.M. A Link Between Cell Wall Biogenesis, Pathogenesis, and Vigor in Walnut Root and Trunk Diseases. International Journal of Molecular Sciences: in Review
  • Type: Journal Articles Status: Under Review Year Published: 2023 Citation: Westphal, A., Maung, Z.T.Z., Buzo, T., Brown, P.J., Leslie, C.A., Browne, G.T., Ott, N.J., McClean, A., and Kluepfel, D.A. Identifying walnut rootstocks with resistance to multiple soil-borne plant pathogens. European Journal of Horticultural Sciences (eJHS): In review
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Westphal, A., Buzo, T.R., Maung, Z.T.Z., McKenry, M., Leslie, C.A., Brown, P.J., Kluepfel, D.A. (2022). Identifying walnut (Juglans spp.) rootstocks with resistance and tolerance to plant-parasitic nematodes. Acta Horticulturae 1346. ISHS 2022. DOI 10.17660/ActaHortic.2022.1346.81
  • Type: Websites Status: Published Year Published: 2022 Citation: Hasey, J., Milliron, L., Reyes, C., Selecting the Best Walnut Rootstock for Your Situation Sacramento Valley Walnut News, Spring 2022 edition http://www.sacvalleyorchards.com/walnuts/horticulture-walnuts/rootstock-2022/
  • Type: Journal Articles Status: Under Review Year Published: 2024 Citation: Momayyezi M, Knipfer T, Hernandez P. MI, Kluepfel DA, Albuquerque CP, Bambach NE, DeGrom J, McElrone AJ. Differential impact of commercial walnut rootstocks on the drought response of a common scion. Trees (under revision post-review).
  • Type: Journal Articles Status: Under Review Year Published: 2024 Citation: Momayyezi M, Williams T, Tolentino P, Hammermeister A, Kluepfel DA, Forrestel EJ, McElrone AJ. Some like it hot: Differential photosynthetic performance and recovery of English walnut accessions under emerging California heat waves. Plant Cell and Environment (under review).


Progress 09/01/21 to 08/31/22

Outputs
Target Audience:The target audience has not changed. Formally stakeholderrs of the walnut industry are involved via the Walnut Rootstock Advisory Panel that met in conjunction with the Walnut Research Conference in January 2022. Contact with the stakeholders is more frequent via different commodity-related meertings. Reciprocal information exchange includes visits and discussions at the field trial sites, in nursery settings and other opportunities. The academic stakeholders of the scientific information gets informed via publications and presentations of the research results. Changes/Problems:Covid-19 restrictions continue to restrict travel and meeting opportunities. The necessary changes become the "new normal", and online discussions are similarly fruitful. But the tentative reopening allows more productive in-person meetings. What opportunities for training and professional development has the project provided?Two graduate students in Agricultural Engineering were trained in tree pathology. One of them passed the final Ph.D. defense committee: Haoyu Niu (UC Merced; major professor: YangQuan Chen). March 17, 2022. One MS student was trained in detection, diagnosis and genetic diversity of plant viruses in tree crops using molecular and high throughput RNA sequencing techniques. One postdoctoral researcher furthered skills in molecular biology of Cherry leafroll virus. Two postdoctoral researchers furthered skills in plant stress physiology and developed knowledge and skills for collaborative work in pathology. Two part time technicians were involved in this work over the past two summers and hope to pursue graduate school in similar areas of study based on these experiences. One graduate student furthered genomics approaches to infection processes in walnut pathology examining RNAseq data from non-inoculated plants and comparing them by susceptibility classes. One undergraduate student participated and acquired basic skills in molecular biology. Two undergraduate students participated in the economics portion of the project for academic credit. Taught in the 2022 Principles of Fruit and Nut Tree Growth, Cropping and Management course: "Nematode Biology and Management", and offered a video for this event March 11, 2022. How have the results been disseminated to communities of interest?Westphal presented talk "Walnut rootstock trial for nematode management" at the Nickels Soil Lab Annual Field Day, May 10, 2022. Andreas Westphal presented two posters at the Burchell Nursery 50th anniversary celebration in October 2022. Elizabeth Fichtner (UCCE) held a field day at the Lindcove "Walnut Rootstocks" in fall 2022. Janine Hasey (UCCE) held a field day in Sutter Walnut Meeting, "Walnut rootstock field trial update" in fall 2022. The Walnut Rootstock Advisory Panel (WRAP) met in January 2022. Results were presented and further strategies discussed. Westphal presented strategies for resistance and tolerance development in extension presentations. He organized field days at the Kearney Research and Extension center for key interest groups to review progress of the program under field conditions. What do you plan to do during the next reporting period to accomplish the goals?Walnut Genomics knowledge Base. Algorithms are being developed for easy visualization of data without coding. The phenotypic data of the nematode evaluations and the growth data under nematode-infected field conditions will be analyzed for the presence of putative QTL locations within the genome. A proof-of concept field experiment for resistance to root lesion nematode with open-pollinated offspring of one of the mother trees will be examined. More data of the nematode host response will be collected enabling a more rigorous evaluation of the putative QTLs identified in preliminary evaluation. Putative QTL of the screening efforts will be tested for validity in this tester set. Field trials with trees produced during this grant period will be planted to field experiments at two locations in California. Focus being on root lesion nematode (one location), and one location with artificial inoculations with Agrobacterium tumefaciens (crown gall) and Phytophthora spp. (root and crown rot). Continue taking data in the field trials. The identified genotypes with multiple resistances will be increased in tissue culture and mass produced for future field trial plantings provided their resistance responses can be confirmed. Manuscripts on completed objectives will be prepared and submitted for publication. Preparing manuscripts for publication on the growth, yield and crown gall incidence of seven-year-old trees in the four field trials is planned in 2023. We will also determine whether there is an experimental rootstock that can be released. Stakeholder meetings (WRAP) will be held. Field meetings are planned at the four rootstock trial sites planted in 2016 across the state.

Impacts
What was accomplished under these goals? Almost all edible English walnut in the US are produced in California. Sustainability of production is threatened by Black line, crown gall, Phytophthora rots, and plant-parasitic nematodes as well as water stress. In two breeding populations, multiple genotypes with apparent resistance/tolerance to multiple pathogens were identified. A putative QTL was identified for crown gall and Phytophthora root rots. In gene expression, different patterns formed for different resistant genotypes leading to functional studies. Remote sensing detected soil-borne maladies. Economic parameters and grower perceptions were summarized for publication. Four on-farm rootstock trials were continued, and five new ones started. The field trial data revealed two candidates for rootstock release. Plant propagation Open-pollinated offspring of the mother trees. Seedlings were produced for pathology evaluations. Media for difficult to propagate walnut genotypes was developed. Multi-resistant genotypes were increased in tissue culture and provided to nurseries. Selected genotypes of the seedling screens were cultured. Identify gene loci (QTL) that confer resistance to the following pathogen systems Identification of QTL and predictions of resistance genetics. Flanking markers for resistances to crown gall, and Phytophthora root rots were identified in Juglans microcarpa DJUG 31.01 and DJUG 31.09 on chromosome 4 in the D subgenome. In the QTL, several putative genes for resistance were found in silico possibly corresponding to finds in gene expression studies. Determine effects of host genotype on water relations and disease resistance responses. Interactions of watering and nematode infestation. In a field trial on the interaction of watering and nematode infestation, trees under several years of deficit irrigation remained smaller and had reduced yield potential by an average of 40-50%. Abiotic stress physiology screening. Recent progress included: In drought stress experiments, both TB33 and AD558 (accessions with high resistance levels to Crown gall and Phytophthora root rots) had low vigor with reduced photosynthetic rates under both control and drought conditions compared to commercials and were highly susceptible to moderate drought. When grafted, RX1 responded more gradual to drought stress than VX211 and Vlach. Under heat stress, J. regia accessions from the National Clonal Germplasm Repository had different photosynthetic capacities. Pyramiding multiple resistance loci into superior rootstock genotypes by improved selection strategies. Resistance determination - Phytophthora. From the two breeding populations, four genotypes with higher levels of resistance were forwarded to orchard evaluations. Seedling evaluations were continued to further corroborate and refine the resistance locus. Crown gall resistance. Elites with tolerance to root lesion nematode were similarly susceptible as the industry standards to crown gall. When testing open-pollinated seedlings of five mother trees, about 1/3 of one seedling population fell into a highly resistant category. Low levels of susceptibility was confirmed for three selections from MT 31 09 offspring. Field plantings of these selections were made and inoculated with Agrobacterium tumefaciens. In the consolidated repository of the resistance determination assays of the two breeding populations, two genotypes appeared concomitantly resistant or tolerant to root-knot nematodes, root lesion nematode, crown gall and Phytophthora root and crown rots. Several others had multiple resistances and tolerances. Clonal plants of these multiple resistant genotypes were grafted to the common scion 'Chandler' for horticultural evaluations. Black line disease. A total of 319 samples was tested for Cherry leafroll virus infection in leaf samples from a grafting experiment. A CLRV W-8 isolate, freeze-dried and stored since 1982, was revived on tobacco and partially sequenced to compare with sequences of a CLRV isolate maintained on walnut. The polyprotein region of CLRV RNA-1 and RNA-2 were cloned and are being assembled for identifying determinants of hypersensitivity in black walnuts. Gather information on gene expression of resistant and susceptible infection processes. Transcriptome analysis In seven hybrids, large differences in gene expression correlated with disease resistance/susceptibility. The results suggested that cell wall biogenesis may be a susceptibility factor in these diseases. Inhibition of cellulose synthase (a major contributor to cell wall biogenesis) with the chemical isoxaben conferred Phytophthora resistance to an otherwise susceptible genotype. Assay for Phytophthora pini. In a new in vitro assay, disease responses of known susceptible and resistant genotypes were measurable one week after inoculation with zoospores. In tests of 69 selections in the in vitro assay, most reproduced outcomes as in greenhouse and field trials. Further spectral analysis capabilities for detecting specific soil-borne disease impacts on walnuts. Remote sensing of stress by nematode infection on walnut rootstock genotypes. Spectral data were analyzed. Root lesion nematode-infected trees contained leaves with and without stress signals. A radiofrequency sensor (Walabot) distinguished leaves and roots of infected and non-infected trees. Radiative Transfer Models incorporating reflectance and transmittance of leaves potentially revealed not only the infestation level but also contents of the leaves of protein, chlorophyll, anthocyanin, carotenoid, and other constituents. Release rootstock genotypes with superior tolerance and resistance characteristics to key soil-borne plant pathogens and abiotic stresses with vetted horticultural traits. Field trial evaluations planted in 2016. In four on-farm field trials, commercial and experimental clonal rootstocks were compared across the state. Four experimental genotypes had less crown gall compared to the standards in 2021. Scions on three of the experimental rootstocks grew similarly vigorously as on the commercial VX211 and RX1. Inoculation experiment planted in 2015. Evaluation of the field trial revealed: one of the four experimental lines performed as favorable as RX1 (clonal standard for Phytophthora resistance) and had resistance to crown gall; one elite favorable against crown gall had reduced infections with root rot and root lesion nematode. The latter was also a high-performer in related trials in nematode-infested soils and will be considered for rootstock release. New plantings in 2022. Of 18 genotypes produced, subsets of genotypes promising in regards to key local biotic stress were planted at locations ranging from Butte and Sutter county via San Joaquim county to Fresno county. At one location in Yolo county, Agrobacterium, Phytophthora, and nematode inoculations are scheduled. Two locations aim at nematode evaluations in different soil types. One site was planted to a Phytophthora root rot hot spot, and one to crown resistant genotypes. ?Nursery increase of elites with pyramided resistances. In parallel to plantings in 2022, repeats of some of selected genotypes were nursery-propagated for an additional planting at the Yolo county location in winter 2023. Five genotypes with multiple resistances from the gene-pyramiding efforts were transferred to commercial nurseries for increase and possible field plantings in 2025 or 2026. Provide economic evaluations for decision support on rootstock use. Economic evaluations of rootstock needs and implementation A survey on needs of walnut rootstocks with resistance to soil-borne pathogens was completed by follow-up phone interviews. Data were summarized and presented at the stakeholder meetings (WRAP) and prepared for peer review publication.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Knipfer TK, Reyes CR, Momayyezi M, Brown PJ, Kluepfel DA, McElrone AJ. 2020. A comparative study on physiological responses to drought in walnut genotypes (RX1, Vlach, VX211) commercially available as rootstocks. Trees-Structure and Function 34: 665678; 10.1007/s00468-019-01947-x
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Momayyezi M, Rippner DA, Duong FV, Raja PV, Brown PJ, Kluepfel DA, Earles JM, Forrestel EJ, Gilbert ME, McElrone AJ. 2022. Structural and functional leaf diversity lead to variability in photosynthetic capacity across Juglans regia genotypes. Plant Cell and Environment 45: 23512365.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Knipfer T, Bambach N, Hernandez MI, Bartlett MK, Kluepfel DA, McElrone AJ. 2020. The triphasic relationship of predawn and midday water potential: A predictor for stomata closure, turgor loss and degree of isohydry. Plant Physiology 184(2): 881894
  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Jafarbiglu, H., Pourreza, A. 2022. A comprehensive review of remote sensing platforms, sensors, and applications in nut crops. Computers and Electronics in Agriculture 197 - 106844 https://doi.org/10.1016/j.compag.2022.106844
  • Type: Journal Articles Status: Under Review Year Published: 2022 Citation: Momayyezi M, Knipfer T, Hernandez P. MI, Kluepfel DA, Albuquerque CP, Bambach NE, DeGrom J, McElrone AJ. Differential impact of commercial walnut rootstocks on the drought response of a common scion. Trees (under review).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Westphal, A., T.R. Buzo, Z.T.Z. Maung, M. McKenry, C.A. Leslie, P.J. Brown, and D.A. Kluepfel. Identifying walnut (Juglans spp.) rootstocks with resistance and tolerance to plant-parasitic nematodes. Acta Hortic. 1346. ISHS 2022. DOI 10.17660/ActaHortic.2022.1346.81
  • Type: Theses/Dissertations Status: Submitted Year Published: 2022 Citation: Shaffer, M. 2020. Incidence and Genetic Diversity of Cherry Leaf Roll Virus in Walnuts at the National Clonal Germplasm Repository. Thesis Submitted in partial satisfaction of the requirements for the degree of MS in Plant Pathology, UC-Davis.


Progress 09/01/20 to 08/31/21

Outputs
Target Audience:The target audience has not changed. Stakeholders of the walnut industry continue to be involved in this project. Formally, the Walnut Rootstock Advisory Panel has met in conjunction with the Walnut Research Conference on January 27, 2021. The panel comprises members from the grower community, processors and the nursery industry. Tree nursery representatives have visited the field trials at the Kearney Agricultural Research and Extension Center (home base of the PD). Changes/Problems:Covid-19 restrictions continue to restrict travel and meeting opportunities. The necessary changes become the "new normal", and discussions are similarly fruitful as is possible in the preferred in-person meeting. What opportunities for training and professional development has the project provided?Two graduate students in Agricultural Engineering were trained in tree pathology. One MS student prepared a thesis in diversity studies of the Cherry Leaf roll virus. One postdoctoral researcher furthered skills in viral molecular biology. One graduate student furthered genomics approaches to infection processes in walnut pathology examining RNAseq data from non-inoculated plants and comparing them by susceptibility classes. Two undergraduate students participated in the economics portion of the project for academic credit. How have the results been disseminated to communities of interest?Elizabeth Fichtner and Greg Browne, UCCE Virtual Statewide Walnut Series, "Walnut Rootstocks" Feb. 16, 2021 Janine Hasey - Lake County Walnut Meeting, "Walnut rootstock field trial update" April 2, 2021 The Walnut Rootstock Advisory Panel (WRAP) met in January 2021. Results were presented and further strategies discussed. Westphal presented strategies for resistance and tolerance development in extension presentations. He organized field days at the Kearney Research and Extension center to review progress of the program under field conditions. What do you plan to do during the next reporting period to accomplish the goals?Walnut Genomics knowledge Base. Algorithms are being developed for easy visualization of data without coding. The phenotypic data of the nematode evaluations and the growth data under nematode-infected field conditions will be analyzed for the presence of putative QTL locations within the genome. A proof-of concept field experiment for resistance to root lesion nematode with open-pollinated offspring of one of the mother trees will be examined. Putative QTL of the screening efforts will be tested for validity in this tester set. Field trials with trees produced during this grant period will be planted to field experiments at various growth environments throughout California. Focus being on root lesion nematode (three locations), crown gall (two locations), and Phytophthora (two locations). The identified genotypes with multiple resistances will be increased in tissue culture and mass produced for future field trial plantings provided their resistance responses can be confirmed. Manuscripts on completed objectives will be prepared and submitted for publication. Stakeholder meetings (WRAP) will be held. Field meetings are planned at the four rootstock trial sites planted in 2016 across the state.

Impacts
What was accomplished under these goals? Most of the US edible walnut are produced in California. Among biotic and abiotic stresses, black line, Crown gall, Phytophthora rots, and plant-parasitic nematodes as well as water stress put sustainable nut production at risk. Two breeding populations of two selected mother trees were tested against these stresses. Tests of the interactions of water stress and pathogen systems were conducted. Genetic markers are proposed, and their validation is ongoing. Consolidating data of the various testing systems yielded multiple genotypes with apparent resistance/tolerance towards multiple pathogens. Studies of gene expression are ongoing. Tools to easier detect soil-borne maladies by remote methods are developed. Economic evaluations and grower perceptions lay the groundwork for economic benefit assessments for the sustainable implementation of these novel rootstocks. Plant propagation Collecting and propagating open-pollinated offspring of the mother trees. Seeds from open-pollinated mother trees were grown into saplings provided for the pathology evaluations. Plant propagation. Improved tissue culture media for cultivation of previously difficult to propagate walnut genotypes was developed. Selected genotypes of the seedling screens were taken into culture. Identify gene loci (QTL) that confer resistance to the following pathogen systems Identification of QTL and predictions of resistance genetics Analyses of resistance response against the crown gall and two Phytophthora root and crown rots revealed the identical QTL for resistance in the genome of Juglans microcarpa DJUG 31.01 and DJUG 31.09 on chromosome 4 in the D subgenome. In both mother trees, 750 to almost 1,000 SNPs were identified as possible markers. Fortuitously, higher frequencies of crossovers were observed in 'Serr' than in the mother trees making this a preferred pollen donor for creating diversity. Flanking markers for resistances to crown gall, and the two Phytophthora rots were identified. This information was published. Determine effects of host genotypic on water relations and disease resistance responses of available breeding material (interaction of biotic and abiotic stresses). Interactions of watering regiment and nematode infestation In field trial on the interaction of watering and nematode infestation, it appeared that trees adopt to the watering regiment to a certain extent. Trees under several years of 60% ET remained smaller but experienced only minimal additional stress measured as SWP in the third year. Screening for stress physiology: 1) assessing inherent, drought- and heat-stressed photosynthetic capacity of Juglans regia accessions from the National Clonal Germplasm Repository; 2) screening advanced accessions identified for Crown gall and phytophthora resistance for drought stres; 3) refining a drought phenotyping platform and application to drought responses of 'Cisco' scion. Significant differences in photosynthetic capacity amongst J. regia accessions from different geographic regions were detected. Significantly different responses to both drought and heat stress were linked to inherent differences in leaf anatomy amongst the accessions. Advanced selections responded differently than commercial standards. Pyramiding multiple resistance loci into superior rootstock genotypes by improved selection strategies. Resistance determination From Phytophthora evaluations of the two breeding populations, four genotypes were forwarded to orchard evaluations. Seedling evaluations were continued to further corroborate the resistance locus and refine its location in the genome. In addition, open-pollinated offspring of the two breeding populations, previously identified elites for tolerance to root lesion nematode were tested for their response to crown gall inoculations and found similarly susceptible as the industry standards. Determination of host plant response to root-knot nematodes and root lesion nematode of the two breeding populations was completed. All plant responses were entered in a draft data repository. In the consolidated repository of the resistance determination assays of the two breeding populations, two genotypes apparently were either resistant or tolerant to root-knot nematodes, root lesion nematode, crown gall and Phytophthora root and crown rots. Several others had multiple resistances and tolerances at lower levels. Genotypes expressed sufficient vigor to enable their transplanting and grafting to the common scion 'Chandler'. Black line disease Diversity of Cherry Leaf roll virus was determined in the efforts of a MS thesis. A postdoctoral researcher examined samples from breeding material of J. microcarpa. Out of 76 samples all were negative for infection. Gather information on gene expression of resistant and susceptible infection processes. Transcriptome analysis In the analysis of seven hybrids, identification of informative biomarkers and molecular mechanisms for resistance was pursued by principal component analysis. PC1 seemed to capture the variation with nematode counts whereas PC2 captured the variation of crown gall size. Assay for Phytophthora pini developed. A new in vitro assay allowed for distinguishing disease responses of known susceptible and resistant genotypes a week after inoculation with zoospores. Further spectral analysis capabilities for detecting specific soil-borne disease impacts on walnuts. Remote sensing of stress by nematode infection on walnut rootstock genotypes Spectral data were analyzed by both groups of agricultural engineers. Trees contained leaves with and without stress signals when infected with root lesion nematodes. A manuscript outlining the machine learning solidifying this information was submitted. In an alternative approach, a radiofrequency sensor (Walabot) distinguished leaves of infected and non-infected trees. Machine learning allowed to classify data to provide them with predictive power of nematode infection. Release rootstock genotypes with superior tolerance and resistance characteristics to key soil-borne plant pathogens and abiotic stresses with vetted horticultural traits. Field trial evaluations. Growth, yield, and crown gall incidence data are collected in four field trials planted in 2016. Clonal rootstocks (commercial and experimental) had lower <6% frequencies of crown gall occurrence than seedling Paradox (50%), which had been the commercial standard for decades. Crown gall infection significantly decreased the rootstock circumference in Paradox seedling and one of the commercial clones (RX1). Scion growth rates of most clones were higher than the seedling Paradox. At three sites, some of the clones had significantly higher yields than seedling Paradox. A field trial planted in 2015 inoculated with Agrobacterium and Phytophthora was completed, final plant health data were taken, and the genotypes classified for their traits. One of the four experimental lines performed as favorable as RX1 (clonal standard for Phytophthora resistance) and had noticeable resistance to crown gall. Two elites had somewhat reduced signs of infections while the fourth had high incidences of Phytophthora rot. Trees for five field trials were produced. Different sets of the total of 18 genotypes will be placed into these trial sites dependent on the key local biotic stress. At one location, Agrobacterium, Phytophthora, and nematode inoculations are planned. At a second location, selected genotypes will be planted into non-fumigated (root lesion nematode infestation), and into fumigated ground to assess the growth potential and competitive benefit of nematode resistance and tolerance. Provide economic evaluations for decision support on rootstock use. Economic evaluations of rootstock needs and implementation A survey on perceived needs of walnut rootstocks with resistance to soil-borne pathogens was completed. Data were summarized and presented at the stakeholder meetings (WRAP).

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Niu, H., Westphal, A., and Y-Q, Chen 2021. A contactless classification method for early detection of nematodes using deep neural networks (DNNS) and tensorflow. Proc. ASME Intern. Design Engineer. Techn. Conf. & Comput. Inform. Engineer. Conf. IDETC/CIE, online: https://github.com/niuhaoyu16/nematodes_detection_using_walabot
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Aradhya, M., Velasco, D., Preece, J., and D.A. Kluepfel 2021. Biogeographic and glacial history of walnut (Juglans regia). Acta Hortic. 1318. ISHS 2021; doi: 10.17660/ActaHortic.2021.1318.2 Proc. VIII International Symposium on Walnut, Cashew and Pecan. (Ed. G. Lemus)
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Leslie, C.A., McGranahan, G., and P. Brown. 2021. Walnut breeding in California. Acta Hortic. 1318. ISHS 2021; doi: 10.17660/ActaHortic.2021.1318.4 Proc. VIII International Symposium on Walnut, Cashew and Pecan. (Ed. G. Lemus)
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Westphal, A., Buzo, T.R., Maung, Z.T.Z., McKenry, M., Leslie, C.A., and D.A. Kluepfel. 2021. Strategies for breeding walnut ((Juglans spp.)rootstocks with resistance and tolerance to plant-parasitic nematodes. Acta Hortic. 1318. ISHS 2021; doi: 10.17660/ActaHortic.2021.1318.5 Proc. VIII International Symposium on Walnut, Cashew and Pecan. (Ed. G. Lemus)
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Kluepfel, D.A., McClean, A.E., Leslie, C.A., Aradhya, M., Luo, M., Ramasamy, R., Dvorak, J. Browne, G.T., and A. Westphal. 2021. Identification and development of crown gall-resistant walnut rootstocks. Acta Hortic. 1318. ISHS 2021; doi: 10.17660/ActaHortic.2021.1318.9 Proc. VIII International Symposium on Walnut, Cashew and Pecan. (Ed. G. Lemus)
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Ramasamy, R.K., Luo, M.C., Leslie, C.A., Velasco, D., Ott, N., McClean, A., Dandekar, A.M., Aradhya, M., Brown, P.J., Browne, G.T., Kluepfel, D.A., Westphal, A. and Dvorak, J. (2021) Co-located quantitative trait loci mediate resistance to Agrobacterium tumefaciens, Phytophthora cinnamomi, and P. pini in Juglans microcarpa x J. regia hybrids. Hortic Res, 8. ARTN 111. Doi:10.1038/s41438-021-00546-7
  • Type: Journal Articles Status: Under Review Year Published: 2021 Citation: Omidi, R., Pourreza, A., Moghimi, A., Zuniga-Ramirez, A., Jafarbiglu, H., Maung, Z.T.Z., and Westphal, A. Hyperspectral Data Analysis Using Unsupervised Learning Methods for Root Lesion Nematode Tolerance Phenotyping In Walnut Trees. Submitted to Computers and Electronics in Agriculture.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Knipfer T, Bambach N, Hernandez MI, Bartlett MK, Kluepfel DA, McElrone AJ. 2020. The triphasic relationship of predawn and midday water potential: A predictor for stomata closure, turgor loss and degree of isohydry. Plant Physiology 184(2): 881⿿894
  • Type: Journal Articles Status: Under Review Year Published: 2020 Citation: Momayyezi M, Brown PJ, Duong FV, Earles JM, Forrestel EJ, Gilbert ME, Kluepfel DA, Raja PV, Rippner DA, McElrone AJ. Structural and functional leaf diversity lead to variability in photosynthetic capacity across Juglans regia accessions from a germplasm collection. Plant Cell and Environment (in review)


Progress 09/01/19 to 08/31/20

Outputs
Target Audience:The target audience for this project has not changed. Stakeholders of the walnut industry are foremost involved in this porject. During this project period, members of the Walnut Research Advisory Panel were informed on progress and future strategy of the project at the annual Walnut Reserach Conference, Bodega Bay, CA January 29-31, 2020. This panel has memebrship among the grower community, prosessors, and nurserymen. Discussions helped with the fine-tuning of the research direction. In-person meeting for reaching higher proportions of the target audienece had to be scaled back becuase of the pandemic. Yet, information was shared via newsletter and trade journal articles as well as webinar particpation of project participants. Changes/Problems:This project year was overshadowed by the Covid-19 pandemic. Based on restrictions to curtail the spread of Covid-19, the campuses where researchers of the project are stationed were closed. Ongoing field activities received exemption from shut-down so that critical and seasonal work could continue. Covid-19 rules for social distancing resulted in reduced laboratory occupancy, and revised work schedules. This restrictions have slightly delayed activites but for the majority of project objectives, activities are on track. Under strict guidelines, field work continued. Outreach activity that typically include traditional Extension tools of in-person meetings and twilight meetings at respective trials to illustrate the scientific progress had to be minimized. Activities were shifted to online events where rootstock information was included in the presentations. Trade journal artciles were published to spread the topic of walnut rootstocks further. As a group, we do not anticipate Covid-19 restrictions to greatly delay progress being made. Researchers have adopted to it, and for most part activites are ongoing. What opportunities for training and professional development has the project provided?The program employs several postdoctoral students who aspire new skills in the walnut system. Often from other disciplines, they learn about specifics of the walnut system. For example, one postdoctoral researcher who had worked in nematode diseases of annual crops before working on walnuts receives additional training. Time horizons and crop specifics, e.g., irrigation strategies are quite different from the prior experience, and current project objectives enlarge the postdocs experiences. Haoyu Niu is engineering graduate student with YangQuan Chen at UC Merced. The world of nematode diseases of walnut has been novel to him. In his graduate project, he can combine engineering skills with biological questions, so truly work multidisciplinary. This has improved his skill set, and his "engineering look" at biological questions has been enriching to the project. How have the results been disseminated to communities of interest?Information enters the peer-reviewed publications (see respective section). Due to the nature of the long-term crop, several projects are only now entering the phase of data collection and analysis. Walnut rootstocks and the clonal walnut research trials were discussed at Tri County Walnut Day in the southern San Joaquin Valley and at Sutter-Yuba-Colusa Walnut Day in the Sacramento Valley inFebruary 2020. Therootstock survey developed by Annette Levi was presented and distributed at the many UCCE Walnut meetings throughout the central valley in early 2020. It was also advertised through the "UCCE Sacramento Valley Walnut Newsletter" reaching walnut growers in Yolo, Solano, Sutter, Yuba, Colusa, Butte, Glenn, and Tehama counties and through UCCE newsletters in the southern San Joaquin Valley reaching growers in Tulare, Kings, Kern, Fresno and Madera Counties. The survey will also be advertised in UCCE newsletters in the northern San Joaquin Valley this November. The PD, entered and became semifinalist in the project of the American Phytopathological Society-sponsored competition in the International Year of Plant Health to be allowed to participate in the US Congressional reception for research. This included an abstract, and a video that is available per request. In May 2020, an article was published in West Coast Nut -- Economic Benefits of Using Improved Walnut Rootstocks with the purpose to inform growers about the importance of rootstock selection; to update growers about this funded USDA and Walnut Board research program focused on walnut rootstock breeding, and to encourage walnut growers to take the survey. What do you plan to do during the next reporting period to accomplish the goals?The coming project year will be used for in-depth analysis of data generated in the current project period. Large data sets have been generated in the different aspects of the project. Offspring of both mother trees will be produced and entered into the disease testing pipelines. Proof-of-concept for the tentatively identified QTLs as predictors for plant resistance will be conducted. Such data are slated to become available for the pathogen systems in place. Molecular responses to infections by the different pathogens will be documented in resistant and susceptible genotypes to elucidate the genetic response. Elite genotypes will be advanced through the nursery process to field plantings of rootstock trials. Economic implications of rootstock choice will be refined by completing the reported survey, and by economic analysis.

Impacts
What was accomplished under these goals? US production of edible walnuts almost exclusively in California contributes to the economy and rural communities. Its sustainability is threatened by biotic stresses, e.g., crown gall, phytophthora crown and root rot, plant-parasitic nematodes, and black line disease, and abiotic stresses, e.g., scarcity of water. Traditionally used rootstocks are susceptible to these stresses. Breeding populations (>300 each) from two mother trees are used to determine the genetics of resistances against these stresses. Studies on the interaction of the soil-borne maladies and irrigation practices are being conducted. Genetic markers are being developed to identify superior walnut saplings among new crosses. Using clonal propagation, many plants for disease testing and for production of superior elites for advanced testing in on-farm rootstock trials are made. In rootstock trials from a prior SCRI, the number of elites that warrant commercial release is validated. In a survey, grower preferences on rootstock characteristics plus the willingness to accept higher prices for such improved rootstocks were determined. Subsets of the rootstock breeding populations are used to identify the parameters to adopt remote sensing methods. Identify gene loci (QTL) that confer resistance to the following pathogen systems In a submitted manuscript, the putative QTL for resistance to crown gall and Phytophthora rots were reported on chromosome 4D. In phenotyping of plant response to nematodes for QTL analysis, one trial will be evaluated in the upcoming dormant season (2020/2021). In a field study planted in 2020, 250 seedlings from the 31.09 MT were inoculated with nematodes, and will be evaluated in 2021/2022. Tentative markers will be tested using phenotypic and genotypic data of this experiment. Determine effects of host genotypic on water relations and disease resistance responses of available breeding material. Two trials on the interactions of pathogen × water treatment × rootstock genotype were conducted. In a lathhouse study with the crown gall pathogen, two clonal susceptible rootstocks (RX1, VX211) were exposed to different watering regiments, and inoculated with the crown gall pathogen or left uninoculated. No reduction of waterflow due to the crown galls was detected. The stem ratio above and below the gall was lower in the less water-efficient rootstock VX211 then in the more efficient RX. Anatomical validation is still ongoing. A field experiment with P. vulnus and different watering regiments planted in 2018, was evaluated in 2019 with repeat measurements in 2020. Two experimental hybrid rootstocks grafted to 'Chandler' scion were cultivated along with Chandler own-rooted. One water treatment received 100% ET, and the second only 60%ET. In 2019, treatments with nematode infestation appeared to have smaller canopies in the ET60 treatments than the ET100. Analysis of the 2020 data is still ongoing. Pyramiding multiple resistance loci into superior rootstock genotypes by improved selection strategies. Mass crosses were made by grafting scions of the mother trees 31.01 and 31.09 into an orchard of English walnut to create diversity for identifying genotypes with multiple resistances. At flowering, catkins were removed from the mother trees, and the female flowers allowed to open-pollinate with J. regia pollen. In Fall 2019, ca. 10,000 nuts were harvested from the mother trees. In Spring 2020, the nuts were germinated, and only hybrids continually propagated. The overall success rate (% germination × % hybrid) averaged 40% across mother trees. At the Davis Genome Center, GBS libraries were constructed using a set of 768 DNA barcodes, and 768-plexed samples were sequenced on an Illumina Hiseq instrument (SR100 reads). Approximately 2,500 genotyped hybrids from the 2019 harvest were distributed to team members in spring/summer 2020 for disease screening. Gather information on gene expression of resistant and susceptible infection processes. MicroRNAs (miRNAs) are important factors for the post-transcriptional regulation of protein-coding genes. In a sequence-homology-based computational approach for miRNAs and their target genes in Juglans regia, and J. microcarpa, 119 and 121 miRNA precursors, respectively, were detected. In the J. regia genome, 325 different genes were targets (87.08% transcript cleavage; 12.92% translation repression). In the J. microcarpa genome, 316 different genes were targets (88.92% transcript cleavage; 11.08% translation repression). While only minor differences were detected in pre-miRNAs between the J. regia and J. microcarpa genomes, about one-third of the pre-miRNA loci were not conserved between homoeologous chromosome within each genome. Gene expression profiles of candidate resistant or susceptible rootstock genotypes were compared by RNA sequencing. Genotypes with resistance or tolerance to crown gall and phytophthora clustered while genotypes with nematode resistance/tolerance clustered with genotypes susceptible to the other two diseases. Further spectral analysis capabilities for detecting specific soil-borne disease impacts on walnuts. Spectral analysis of experimental orchards has been conducted. Data are currently being analyzed. In a low-cost proximate sensing method, signals of a "walabot' were analyzed in relation to nematode infection levels. This inexpensive sensor uses radio frequency for the measurement of environmental signals. Computer learning algorithms allowed classification of nematode responses from the walabot data. Details of this study were published as proceedings article. Release rootstock genotypes with superior tolerance and resistance characteristics to key soil-borne plant pathogens and abiotic stresses with vetted horticultural traits. A total of 1,138 in vitro cultures of all hybrid genotypes from controlled crosses using J. regia 'Serr' pollen onto Juglans microcarpa mother trees are maintained, have been genotyped and serve as source material for field trials. Four genotypes, JMS13, K3, 29JM12, and STJM6 of superior performance in intermediary grafted rootstock trials were increased further for nursery cultivation and expansive field trials in nematode-infested replant soil. Forty-one genotypes are cultivated in a commercial nursery. During the first year of growth, vigorous plants could have been "June-budded to a nut-bearing scion 8 months after planting but many genotypes were still too small, and the grafting was delayed to the upcoming dormant season. Excessively poorly growing genotypes were eliminated. Five ongoing rootstock field trials with four elite experimental genotypes planted in 2015 and 2016 are curated in this project. A 2015 planted trial was inoculated with Agrobacterium tumefaciens and Phytophthora. In 2019 in the four June-budded trials, the standard clones VX211 and Vlach grew the most vigorous, but no yield differences were ascertained among rootstocks across three sites. The two putatively Agrobacterium-resistant experimental genotypes had 0% crown gall infection at ground level compared to 25% infection for the highly susceptible paradox seedling rootstock. Provide economic evaluations for decision support on rootstock use. A IRB-approved survey for grower preferences of walnut rootstocks was vetted by members of the Walnut Research Advisory Panel, and then administered to growers. Data of 57 returned surveys will be further validated by an intensive phone survey. Some preliminary results were: Crown gall was the most reported issue by 75% of respondents, followed by Root rot (50%,) and Replant problems (43%). In some areas, blackline disease and drought tolerance were ranked as the most significant problems. Growers reported to be 'very interested' in having resistance to Crown gall (84%), Root rot (64%) and Root lesion nematodes (54%). They stated to be willing to pay 20% or more for resistance to Crown Gall and Root rot.

Publications

  • Type: Journal Articles Status: Submitted Year Published: 2020 Citation: Ramasamy, RK, Luo, M-C, Leslie, CA, Velasco, D, Ott, N., McClean, A, Dandekar, AM, Aradhya, M, Brown, PJ, Browne, GT, Kluepfel, DA, Westphal, A, J Dvorak. 2020. Co-located quantitative trait loci mediate resistance to Agrobacterium tumefaciens, Phytophthora cinnamomi, and P. pini in Juglans microcarpa x J. regia hybrids. Horticultural Research
  • Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Haoyu Niu, Tiebiao Zhao, Andreas Westphal, YangQuan Chen, "A low-cost proximate sensing method for early detection of nematodes in walnut using Walabot and scikit-learn classification algorithms," Proc. SPIE 11414, Autonomous Air and Ground Sensing Systems for Agricultural Optimization and Phenotyping V, 114140K (21 April 2020); doi: 10.1117/12.2558214
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Knipfer T, Reyes C, Momayyezzi M, Kluepfel D, McElrone AJ (2020) A comparative study on physiological responses of commercially available walnut genotypes (RX1, Vlach and VX211) to drought stress. Trees (Berl) 34: 665678
  • Type: Journal Articles Status: Accepted Year Published: 2020 Citation: Wang, L, Zhu, TT, Deal, KR, Dvorak, J, M-C Luo 2020. Computational identification and comparative analysis of conserved miRNAs and their putatice target genes in the Juglans regis and J. microcarpa genomes. Plants 2020, 9, 1330; doi:10:3390/plants9101330


Progress 09/01/18 to 08/31/19

Outputs
Target Audience:This project is in collaboration with walnut stakeholders who are informed on progress and provide input at the annual stakeholder meeting at the Walnut Research Conference, Bodega Bay, CA, January 23-25, 2019. There is ample interaction with certain portions of the walnut industry. For example, a field tour of UC Cooperte Extension personnel (farm advisors and CE Specialists plus Experiment Station scientists) also involves nurserymen. In this feed-back loop, information is shared, discussed and strategies refined. The on farm field trials are teaching tools for advisory groups and end users of the information (pest control advisors, industry representatives, walnut producers and other interested parties) who participate in these events. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This is the initial year of the project. Several postdoctoral students and graduate students are involved in the project. A typical lag-time at project initiation until first involvement of these budding scientists was observed. Now, a postdoctoral researcher is working on the water relations bioassays and physiology alongside with a field trial to determine the interactions of water relations and plant-parasitic nematodes. The workgroups on spectral analyses involve two graduate students who part-time work on this proejct. The UC Merced Co-PI made some aspects of the spectral analysis project an undergraduate class where in a cape stone-similar project, agricultural engineering students learn about walnut cultivation and soil-borne diseases of this crop. This expands the typcial exposure circle to undergraudte students to this interdisciplinary project. How have the results been disseminated to communities of interest?Information on this project was disseminated in various avenues by project personnel. For example, the PI gave six formal presentation the rootstock project reaching scientific departments, undergraduate classes, and stakeholders at scientific and extension meetings. Co-PIs who oversee the on farm rootstock trials conducted in-field meetings illustrating the importance fo rootstock choice. In the Walnut Rootstock Advisory Panel (stakeholder committee overseeing the project), aspects of the project were presented by members of the research group and openly discussed. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Walnut production is an important contributor to the Californai economy. Sustainable production is threatened by biotic and abiotic stresses. Previously, soil fumigation alleviated these challenges. With increasing concerns for environmental and human health, more environmentally benign methods are urgently needed. Resistant and tolerant rootstocks offer promise for a more sustainable cultivation of this important specialty crop. Traditionally, hybrids of crosses of English and black walnut ("Paradox") have been used as rootstocks for English high quality nut-producing scions. Such seedling rootstocks carry genetic variability from tree to tree, and are susceptible to soil-borne maladies. As a result of the technological break-through of being able to regenerate clonal plants from interspecific crosses via embyro rescue and tissue culture regeneration of plants, it has become possible to exploit genetically uniform rootstocks with improved disease resistance. Two breeding populations of a prior SCRI-supported project are being characterized for response to a bacterial disease (crown gall), a watermold malady (Phytophthora crown and root rot), plant-parasitic nematodes (root lesion and root-knot nematodes), and response to black line disease (viral disease). In addition, these breeding populations and a wide diversity of walnut genotypes are investigated for response to water deficits. Cutting-edge technology is used to elucidate the genetic background for the observed phenotypes. Such studies are facilitated by the availability of several sequences of walnut genomes from precursor projects. This genome resource allows identifying the genetic basis for resistance to multiple diseases, and will allow to molecularly identify desirable hybrids of the various crossing experiments. With the availability of molecular markers, much larger breeding populations that are currently generated will be quickly screened for more favorable hybrids allowing for combining multiple positive traits in single trees that then can be quickly increased in numbers. Remote sensing technologies assist in monitoring these beneficial traits under field condtions. Owed to the changing regulatory framework and the societal expecations of agricultural production, socio-econmic evaluations will determine the cost-benefit values of these improved walnut rootstocks. During this first year of the project the following steps have been accomplished. *Identify gene loci: For all three pathogens systems testing for plant resistance, about half to two-thirds of the genotypes of each of the breeding populations have been characterized (phenotyped). This allowed for detailed statistical testing of the resistance loci. For crown gall and Phythtophthora, tentative loci were identified a statistically further ascertained on chromosome 11. For nematodes, sufficient phenotypic data has just become available to warrant statistical analysis. *Determine genotypic effects on water relations: three commercial clonal rootstocks were used to determine basic physiological responses to water stress. In these studies one rootstock was identified as much more efficient in water use, and less demanding in resource consumption. This characteristics made this rootstock prime candidate for deficit irrigation situations. This project part allowed identifying the measurable parameters that can distiguish genetic differences influencing the repsonse to water deficit. Designing assays for testing hybrids form the two breeding populations for water relations was enabled. *Pyraminding multiple resistance loci: this project part is initiated by testing tentative molecular markers for identifying the loci responsible for CG or PHY resistance in mass crosses of the two mothertrees with random English pollen. Plants are currently regenerated from such crosses and examined for the presence of positive molecular markers. *Gene expression in resistant and susceptible infection: Analyses of the constitutively (without pathogen infection) expressed genes have been conducted, and will be extended to gene expression studies during pathogen infection. *Spectral analysis: Studies on the detection of spectral signals of nematode-infected plants are being conducted. Data are frequently collected to determine the necessary collection conditions with potential for differential detection of infected and non-infected plants. *Release of new, improved rootstocks: On farm-rootstock trials are ongoing. A first harvest was made at one of the five locations where novel rootstocks from the prior SCRI project were planted in randomized complete block design for horticultural evaluations. Exposure to these different agro-ecological environments will further identify beneficial rootstocks. *Economic analysis: A survey for walnut stakeholders was drafted, refined with the help of the Walnut Research Advisopry Panel (stakeholder committte overseeing the project). This survey will be distributed electronically and in paper format to reach as many growers as possible to assess their plans to adopt novel rootstocks with the underlying economical relations.

Publications