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ARS Home » Pacific West Area » Riverside, California » National Clonal Germplasm Repository for Citrus » Research » Research Project #434335

Research Project: Conservation, Management and Distribution of Citrus and Date Genetic Resources and Associated Information

Location: National Clonal Germplasm Repository for Citrus

2020 Annual Report

The primary goals of this project plan are to acquire, maintain, preserve, evaluate, and distribute Citrus and other members of the Rutaceae, and date palms (Phoenix dactylifera) and related Phoenix spp. and to conduct research in support of these objectives. Objective 1: Efficiently and effectively acquire citrus and date genetic resources; maintain their safety, genetic integrity, health and viability; and distribute them and associated information worldwide; cryopreserve citrus genetic resources. 1.A. Expand the collection of citrus and date genetic resources, with emphasis on wild relatives and under-represented portions of the collection. Rescue potentially valuable citrus and date germplasm in cultivation areas under threat of genetic erosion. 1.B. Efficiently and effectively conserve germplasm of citrus, citrus relatives and date palm and ensure all current and future accessions are preserved by multiple methods such as field orchards, plantings in screenhouses, and through cryopreservation of seeds, pollen, and clonal tissue. 1.C. Sanitize priority genotypes of citrus and date palms for distribution to users worldwide. Objective 2: Develop more effective maintenance, back-up, pathogen-testing, and pathogen-elimination methods and apply them to priority citrus and date genetic resources. 2.A. Test citrus genetic resources for huanglongbing (HLB)-associated bacteria using recently developed early detection technologies (EDT) such as canines, serology, metabolomics, and spectral responses, and multiple qPCR primer sets. 2.B. Expand the range of pathogens assayed during the screening portion of the sanitation process for citrus and date palms and develop improved diagnostic methods to enhance the efficiency of the sanitation process. 2.C. Develop and implement SOP’s for assaying date palms for the presence of specific pathogens. 2.D. Devise and implement protocols for the cryopreservation of date palm germplasm. Objective 3: With other NPGS genebanks and Crop Germplasm Committees, develop, update, document, and implement best management practices and Crop Vulnerability Statements for citrus and date genetic resource and information management. 3.A. Update crop vulnerability statement. 3.B. Update and expand scope of operations manual. Objective 4: Develop more effective genetic resource characterization and evaluation methods, especially for citrus host-plant resistance to huanglongbing (HLB) and leprosis, and apply them to priority citrus and date genetic resources. Record and disseminate characterization and evaluation data via GRIN-Global and other data sources. 4.A. Strategically characterize and evaluate citrus and date genetic resources for priority traits such as disease, pest, biotic and abiotic stress resistance, quality factors, and other pertinent properties. 4.B. Expand the range of accession information available electronically.

Gaps in the citrus and date palm germplasm collection are identified by taxonomy, geographic origin, characterization data, and stakeholder input. New germplasm is acquired by exchange with cooperating scientists or industry personnel and by plant exploration for wild germplasm. Areas for acquisition of new citrus germplasm include Australia and Vietnam. A trip to Vietnam will be planned and contact made with Botanic Gardens in Australia that have a good representation of native Rutaceae and are open to exchange germplasm. Native Rutaceae have shown potential resistance and/or tolerance to huanglongbing (HLB) making these vital gene sources for breeding purposes. Areas for date palm acquisition are primarily in the Middle East and North Africa, however, importation of offshoots is prohibited by the Animal and Plant Heath Inspection Service (APHIS). Seeds are unrestricted and tissue culture is allowed via permit. Pathogen-tested germplasm is maintained in an APHIS approved screenhouse, un-sanitized material is separated in other protective structures, and most accessions are also maintained in a field planting. Cryopreservation as a long-term backup will continue as accessions are released from quarantine and for wild relatives, seeds and pollen. Protocols for the cryopreservation of date palm accessions will be developed and optimized. Therapy and pathogen testing will continue for new citrus accessions and to satisfy quarantine regulations for the protected collection. A pathogen-testing program for date palms will be initiated. We will test whether canines and other early detection technologies can accurately detect Liberibacter-infected trees prior to standard qPCR methods. Dogs trained to alert to HLB-infected citrus trees will return to the Citrus Variety Collection and be shown every tree in the collection. For all dog-alerted trees and adjacents, leaves will be collected and assayed by qPCR using several different primer sets including the standard APHIS primers, tested with other early detection technologies and/or the tree will be pruned for containment in an insect-proof cage. Samples will be collected from caged trees and assayed using qPCR over time. Improvements to the citrus diagnostic protocols used will be implemented based on reports and publications of other researchers to expedite diagnostics. Date palm diagnostic methods developed by others will be optimized targeting phytoplasmas, Cadang-cadang viroid, and Fusarium oxysporum. All SOP’s will be revised. The Crop Vulnerability Statement will be updated. Citrus scion and rootstock germplasm imported from Florida will be evaluated for its horticultural value and reaction to endemic diseases under California growing conditions at two locations. Accessions in the field variety collection will be re-propagated on these tolerant rootstocks. Should HLB become widespread in southern California, we would evaluate for this disease under California conditions. Whether endemic pathogens prevent, mask, or exacerbate the development of HLB will be determined within a contained research facility. All information gathered will be updated and expanded through the GRIN-Global System.

Progress Report
Due to the upsurge in efforts to apply CRISPR technology to citrus, requests for seeds have significantly increased this year. Additional progress in Objective 1 included, for the first time, nucleic acid extractions which were performed and distributed to scientists evaluating citrus accessions for disease resistance genes, particularly for huanglongbing (HLB). Germplasm distributions for the year also included budwood, leaves, and fruit. In support of Sub-objective 1B, pollen collected from 15 date palm male accessions was sent to ARS in Colorado for cryopreservation. To demonstrate to stakeholders that the cryopreservation process does not alter the morphological and taste characteristics of citrus, germplasm that has been preserved in liquid nitrogen for at least five years is being regenerated by ARS scientists in Fort Collins, Colorado, and sent back to the Riverside, California, repository in agar medium. The repository scientists and technicians have acclimated the plants back to soil medium and will repropagate trees that will be planted into a field evaluation plot. Fruit will be evaluated and compared to non-cryopreserved germplasm. Genomic analyses will be performed to determine whether cryopreservation causes any changes to germplasm at the genomic level. Research in support of Sub-objective 1C included two new citrus pathogens which were acquired from the USDA ARS Exotic Pathogens of Citrus Collection (EPCC) to be used as positive controls in the pathogen-testing program. These include: Citrus yellow mosaic virus (CYMV) and Citrus chlorotic dwarf virus (CCDV). Diagnostic protocols for these pathogens were optimized in the ARS Riverside, California, laboratory and implemented as routine assays. Federal quarantine regulations require that new date palm accessions from outside the United States must be obtained under a Plant Controlled Import Permit (PCIP). This permit, issued in 2020 requires testing for specific pathogens (Phytoplasmas, Cadang-cadang viroid, and others) before date palm accessions can be released from quarantine. Seven new date palm accessions currently being held in quarantine were tested for Phytoplasmas. A diagnostic protocol and a nucleic acid extraction of Cadang-cadang viroid to be used as a positive control were obtained from an ARS scientist in Beltsville, Maryland, and assay optimization is in progress. Sixteen citrus accessions were successfully sanitized, tested, and released from Federal and State quarantine adding to the number of accessions securely maintained within a protective screenhouse in Riverside to 603. Eighty-one citrus accessions were cryopreserved in 2020; 74 were sent as budwood and processed by ARS scientists in Fort Collins, Colorado, and 7 were processed in Riverside, California. Five hundred and thirty-four (534) of the 603 sanitized citrus accessions are now cryopreserved and safely secured in Fort Collins, Colorado. In 2020, the final biological index was initiated on 17 citrus accessions for anticipated release from quarantine in FY 2021. Research goals for Sub-objective 2A have experienced a few delays due to the pandemic, including a delay in the transference of the huanglongbing-detection canines from Florida to California and ARS scientists not being able to collect plant samples for pathogen testing this spring; however, much progress has been accomplished, including all fall/winter plant samples being collected and tested using quantitative polymerase chain reaction (qPCR). The results were all negative. In pursuit of a more cost and labor effective alternative testing method for huanglongbing, ARS staff evaluated the use of nitrocellulose membranes. Instead of using expensive, commercial nucleic acid extraction kits, bark tissue collected from field trees was blotted onto nitrocellulose membranes. The blots were cut from the membrane, the nucleic acids were eluted from the membrane, and qPCR was performed using the crude nucleic acid extractions. Although not approved for regulatory analyses, this method enabled ARS staff to test a large number of field trees at a cost savings of 45% with no loss of accuracy. This method was also shown to be effective when testing field trees for viroid pathogens. In June 2020, canines trained to detect HLB, alerted on approximately 500 trees on the research station. Depending on re-opening status, samples will be collected from canine-alert trees in late summer or early fall. ARS technicians are learning to perform next generation sequencing (NGS) to verify infection status of the field germplasm collection, especially when qPCR results are inconclusive or when citrus relatives are being analyzed. In support of Sub-objective 2B, two new citrus diseases were recently identified, including Citrus yellow mosaic virus (CYMV) and Citrus chlorotic dwarf virus (CCDV). Positive controls have been obtained and diagnostic protocols have been optimized for the Riverside, California, Laboratory. The testing of all accessions for these pathogens is in progress. A diagnostic assay, “RNArray” developed by a repository scientist, allows for the simultaneous detection of multiple RNA citrus pathogens. This assay will increase the speed of screening the citrus germplasm collection, not only for quarantine release, but also for periodic checks of the entire protected collection. Progress on Sub-objective 2C included the release of new date palm accessions, achieved only after confirming that the germplasm tested negative for Phytoplasmas, Cadang-cadang viroid, and fungi. Phytoplasma-infected date palm germplasm was obtained from a Florida scientist for use as positive controls in diagnostic assays. Laboratory testing protocols have been optimized for the detection of palm Phytoplasmas and all palm germplasm being held in quarantine has been tested and found to be Phytoplasma-free. Living germplasm containing the Cadang-cadang viroid is not allowed in California, but a nucleic acid extraction of the pathogen can be used in molecular-based assays as a positive control. This and a testing protocol were obtained from an ARS scientist in Beltsville, Maryland. The pandemic closure of the repository has halted the progress of this activity. Good progress has been made on Sub-objective 2D, in the development of a protocol for the cryopreservation of date palm pollen; pollen from 12 male accessions was cryopreserved last year, and an additional 15 accessions in 2020. Cryopreservation of tissue cultured date palms has proven more challenging due to various factors. Recently, the tissue culture medium has been changed and bacterial inhibitors added, with promising results. Work in support of Sub-objective 3A focused on drafting Crop Vulnerability Statements and presenting them to the Citrus and Date Germplasm committees. Crop Vulnerability Statements for citrus and date palms were drafted and presented to the Citrus and Date Crop Germplasm Committees. Progress for Sub-objective 3B included drafting, revising, and reviewing Standard Operating Procedures (SOP’s) for inclusion in an updated Repository Operations Manual. The repository is using the National Plant Diagnostic Network template to standardize the format of all SOP’s. This template will be used by all Citrus Centers in the United States that receive funding from the National Clean Plant Network. In support of Sub-objective 4A, pigmented seedlings of Australian finger limes were evaluated in collaboration with scientists in Riverside, California, for horticultural characteristics in anticipation of release from quarantine to stakeholders. In cooperation with scientists in Riverside and Davis, California, precision agriculture techniques are being developed or adapted to citrus production. A mandarin and a navel orchard in Tulare County, California, are being monitored by hyperspectral measurements and the results compared with leaf mineral nutrient levels and fruit quality measurements with the goal of developing predictive models. In cooperation with scientists in Riverside and Imperial, California, in support of Sub-objective 4A, actual water consumption of mature date palms was measured using various techniques. Results indicate that although there is some variability in date palm water use, actual use is substantially lower than water applications by many growers. As water supplies become more restricted in the desert area, this information will be valuable in conserving water in date production. International collaborations with scientists in Qatar and Abu Dhabi have resulted in multiple sites of domestication within east/west genepools.

1. Support of critical research on Huanglongbing. Support of critical research on Huanglongbing. Huanglongbing (HLB) remains a threat to commercial citrus production. Researchers at ARS, land grant universities, and other entities are actively working on solutions. The ARS Citrus and Date Repository in Riverside, California, supplies citrus germplasm to researchers in Ithaca, New York; Wimauma, Fort Pierce, and Lake Alfred, Florida; New Haven, Connecticut; West Lafayette, Indiana; Beltsville, Maryland; and elsewhere supporting HLB-related research. This germplasm is both tested for susceptibility and used for making crosses to create new Citrus hybrids with greater resistance to HLB.

2. Provides clean source citrus propagative material. As a component of the National Clean Plant Network – Citrus, ARS researchers in Riverside, California, supplied clean propagative materials to the minor citrus producing states that do not have a State-level program (Arizona, Louisiana, Alabama, Georgia). ARS in Riverside, California, also provided clean source propagative materials to programs in other states and countries. This material is used for making crosses to develop improved cultivars. This will improve the overall plant health of the global citrus industry.

3. Evaluation of early detection technologies for huanglongbing (HLB)-associated pathogens. Evaluation of early detection technologies for huanglongbing (HLB)-associated pathogens. If HLB can be detected early, infected trees can be removed quickly, which will slow the spread of this disease and help save the Citrus industry. Early detection technologies (EDT) investigated include: HLB detection canines, serological assays for proteins secreted by the pathogen and bacterial structural proteins, changes in the metabolomic profile of the host plant, and next generation sequencing (NGS). These methods were compared to the standard regulatory quantitative Polymerase Chain Reaction (qPCR) diagnostic method. While the EDT’s have identified suspect positive trees, extensive qPCR assays have negative results. Canines continue to be the most rapid detection method and research continues, to determine the time between canine detection and a positive qPCR result for the same tree.

4. Development of tissue blot method for citrus pathogen detection. Development of tissue blot method for citrus pathogen detection. Citrus germplasm collections in both the field and within protective structures are routinely screened for pathogens. Nucleic acid extraction kits are costly. A forty-nine percent reduction in cost of supplies was realized by blotting fresh bark tissues onto nitrocellulose membranes, cutting out the blots, and eluting the nucleic acids from the membrane. Quantitative polymerase chain reaction was performed using the crude nucleic acid extraction. This method has proven accurate for the detection of citrus viroids and huanglongbing, which is necessary to determine if the collection remains free from these pathogens.

5. Selection of pigmented Australian finger limes. Selection of pigmented Australian finger limes. Pigmented seedlings of Australian finger limes were evaluated by scientists in Riverside, California, for horticultural characteristics in anticipation of therapy and release from quarantine in the next few years. Currently, only one lightly pigmented selection is available to stakeholders and the industry desires more options.

6. Estimation of actual water use in date palm production. Estimation of actual water use in date palm production. In cooperation with scientists in Riverside and Imperial, California, actual water consumption of mature date palms was measured using various techniques. Results indicate that although there is some variability in date palm water use, actual use is substantially lower than water applications by many growers. As water supplies become more restricted in the desert area, this information will be valuable in conserving water in date production.

7. Development of an array for simultaneous detection of 15 RNA viruses and viroids. Development of an array for simultaneous detection of 15 RNA viruses and viroids. A novel array method was developed for the simultaneous detection of two targets each from 15 different citrus ribonucleic acid (RNA) pathogens in addition to two reference gene targets. The 96 well assay plate can be used for assaying two plant samples (RNA extractions) along with one pooled positive control RNA sample in about 75 minutes. The array consists of 32 sets of primers and probes along with stabilizers and reporters placed in individual wells, lyophilized and stored frozen. The platform was used to detect actual pathogen status (15 RNA pathogens) of about 50 accessions in the citrus pathogen inventory. This method was validated by the ARS, Riverside, laboratory personnel in independent tests.

8. Use of Citrus tristeza virus (CTV) as a vector for antimicrobial peptides and ribonucleic acid interference (RNAi). Use of Citrus tristeza virus (CTV) as a vector for antimicrobial peptides and ribonucleic acid interference (RNAi). Similar to the Citrus tristeza virus (CTV)-vector developed in Florida, mild California CTV isolates collected by the Central California Tristeza Eradication Agency were cloned and transformed by collaborating scientists at the University of California - Riverside. ARS scientists and technicians provided healthy citrus plants, inoculated plants with purified virus, and assayed for CTV. After three years, a stable CTV vector was developed and is now ready to use as a delivery mechanism for therapeutic antimicrobial peptides and RNAi strategies against pathogens such as Huanglongbing-associated bacteria and insect pests.

Review Publications
Hazzouri, K.M., Gros-Balthazard, M., Flowers, J.M., Copetti, D., Lemansour, A., Lebrun, M., Masmoudi, K., Ferrand, S., Dhar, M.I., Fresquez, S., Roas, U., Zhang, J., Talag, J., Lee, S., Kudrna, D., Powell, R.F., Leitch, I.J., Krueger, R., Wing, R., Amiri, K.M., Purugganan, M.D. 2019. Genome-wide association mapping of date palm fruit traits. Nature Communications.
Ollitrault, P., Curk, F., Krueger, R. 2020. Citrus taxonomy. Book Chapter.
Ramsey, J.S., Chin, E., Chavez, J., Saha, S., Mischuk, D., Mahoney, J., Mohr, J., Robison, F., Godfrey, K., Levesque, C., Foster, L., Xu, Y., Strickler, S., Fernandez, N., Polek, M., Giovannoni, J.J., Mueller, L., Slupsky, C., Bruce, J., Heck, M.L. 2020. Longitudinal transcriptomic, proteomic, and metabolomic analysis of Citrus limon response to graft inoculation by ‘Candidatus Liberibacter asiaticus’. Journal of Proteome Research. 19:2247-2263.
Salomon-Torres, R., Sol-Uribe, J., Valdez-Salas, B., Garcia-Gonzalez, C., Krueger, R., Hernandez-Balbuena, D., Norzagaray-Plascenci, S., Garcia-Vazquez, J., Ortiz-Uribe, N. 2020. Effect of four pollinating sources on nutritional properties if ‘Medjool’ date (Phoenix dactylifera L.) seeds. Agriculture 2020, 10 (45).
Gottwald, T.R., Luo, W., Posny, D., Poole, G.H., Louws, F., Mccollum, T.G., Hartung, J.S., Bai, J., Duan, Y., Taylor, E.L., Da Graça, J., Schneider, W., Polek, M., Hall, D. 2020. Canine olfactory detection of a vectored phytobacterial pathogen, Liberibacter asiaticus, and intergration with disease control. Proceedings of the National Academy of Sciences. 117(7)3492-3501.
Padhi, E.M., Maharaj, N., Lin, S., Mishchuk, D.O., Chin, E., Godfrey, K., Foster, E., Polek, M., Leveau, J.H., Slupsky, C. 2019. Metabolome and microbiome signatures in the roots of citrus affected by Huanglongbing. Phytopathology, 109 (12).