Progress 10/01/22 to 09/30/23
Outputs
PROGRESS REPORT Objectives (from AD-416): 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. Approach (from AD-416): 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 Health 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. This is the final report for project 2036-21000-011-000D, Conservation, Management and Distribution of Citrus and Date Genetic Resources and Associated Information, which has been replaced by new project 2036-21000- 012-000D, Citrus and Date Genetic Resource Conservation and Utilization. During the course of this project, the following progress was made on all objectives and sub-objectives. In support of Sub-objective 1.A, ARS researchers and staff at Riverside, California, acquired 38 new citrus accessions to expand the range of citrus genetic resources maintained. In addition, funding was obtained for a plant exploration in collaboration with the Vietnamese Academy of Agricultural Sciences Plant Resource Center. An initial exploration is planned for Fall 2023 (see ⿿International Collaborations below). Accessions obtained through this collaboration will be sanitized by ARS in Riverside, California, and then incorporated into the United States and Vietnamese germplasm systems. In support of Sub-objective 1.B, accessions maintained in protective structures or in field plantings were regenerated as necessary. During the course of the project, 155 unique accessions of citrus (budwood), 56 citrus budwood repeats, and 39 accessions of date (pollen) were sent to the USDA-ARS National Laboratory for Genetic Resource Preservation in Fort Collins, Colorado, for cryopreservation. This brought the total number of cryopreserved citrus accessions to 461. In support of Sub-objective 1.C, 49 accessions of citrus were sanitized and released from State and Federal quarantine, thus becoming available to the user public. In addition, germplasm distributions of 2,134 accessions were made to 519 requestors during the duration of the project. In support of Sub-objective 2.A, during the life of the project, canines were used to experimentally assess Huanglongbing (HLB) presence in field plantings of citrus and the results compared with various early detection technologies (EDT) as well as two regulatorily accepted laboratory tests. Correlation between the canine ⿿detections and results from the various EDT⿿s were variable within and between EDT. No HLB positive trees were found using the regulatorily accepted tests nor with next generation sequencing (NGS). Regarding Sub-objective 2.B, pathogen tests were developed or implemented for several additional citrus pathogens and an ⿿array for simultaneous detection of over 20 citrus pathogens was developed. Implementation of assays for palm phytoplasmas and the Coconut cadang-cadang viroid were implemented, and an assay for detection of Fusarium in palms was partially implemented (Sub-objective 2.C). In support of Sub-objective 2.D, cryopreservation of tissue cultured date palms was attempted but unsuccessful due to contamination and other issues; however, a technique for cryopreservation of date palm pollen was developed and implemented. In support of Sub-objective 3.A, the Crop Vulnerability Statements (CVC) were updated separately for citrus and date; the two crops had previously been incorporated into a single report. In support of Sub-objective 3.B, the Operations Manual was updated and additional sections added; integration into a single document is pending. In support of Sub-objective 4.A, a rootstock trial to evaluate the performance of USDA-developed, HLB-tolerant rootstocks was planted by ARS researchers and will be evaluated when the trees are established. Research to quantify poly-embryony and zygotic embryo content of citrus accessions was initiated; this will be incorporated into the new project plan. In support of Sub-objective 4.B, 119 database records were added or updated in the Germplasm Resources Information Network (GRIN) database so that additional information was made available via the GRIN public web interface. Artificial Intelligence (AI)/Machine Learning (ML) No Artificial Intelligence or Machine Learning were used to conduct research for this project plan. ACCOMPLISHMENTS 01 Developed a global conservation strategy for citrus. The first ⿿Global Strategy for the Conservation and Use of Citrus Genetic Resources was developed by USDA scientists in Fort Collins, Colorado, and Riverside, California, and a scientist at the University of Florida at Gainesville (DOI 10.5281/zenodo.7757226). An extensive survey distributed to collections of citrus genetic resources from around the world collected data to document the diversity, availability, security and vulnerability of citrus collections. This information was used to propose priority actions that will unify the citrus genebanking community by having access to shared online resources, training opportunities, increased standardization of data collection processes, and identified opportunities to improve the health and security of plant collections representing citrus genetic diversity. This research demonstrates the key role of the USDA National Plant Germplasm System citrus collection with regard to citrus conservation and distribution on an international scale.
Impacts
(N/A)
Publications
- Garcia-Gonzalez, C., Salomón-Torres, R., Montero-Alpírez, G., Valdez-Salas, B., Coronado-Ortega, M., Curiel-Alvarez, M., Ayala-Bautista, J., Krueger, R., Pérez-Sánchez, A., Torres-Ramos, R., Samaniego-Sandoval, L. 2022. Bioenergy value of seed waste from the Mexican date (Phoenix dactylifera L. ) industry. Biotechnologie, Agronomie, Société et Envrionnement. 26(4):241- 251. https://doi.org/10.25518/1780-4507.20026.
- Krueger, R., Al-Khayri, J., Jain, S.M., Johnson, D.V. 2023. Introduction: The date palm legacy. In: Al-Khayri, J.M., Jain, S.M., Johson, D.V., Krueger, R.R. Date Palm. Oxfordshire, UK: CAB International. p. 1-21. https://doi.org/10.1079/9781800620209.0001.
- Al-Khayri, J.M., Jain, S.M., Johnson, D.V., Krueger, R. 2023. Date Palm. Oxfordshire, UK: CAB International. 648 p. https://doi.org/10.1079/ 9781800620209.0001.
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Progress 10/01/21 to 09/30/22
Outputs
PROGRESS REPORT Objectives (from AD-416): 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. Approach (from AD-416): 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. In support of Objective 1, funding was obtained for a plant exploration proposal with the Vietnamese Academy of Agricultural Sciences Plant Resources Center. This initial plant exploration will take place later in fiscal year (FY) 2022 or in early FY 2023 and should result in the addition of accessions representing new diversity to the citrus collection. Additional citrus accessions have been identified in France and Italy, but covid and administrative issues prevented acquisition in FY 2022. We hope to acquire these genotypes in FY 2023. An additional 30 citrus accessions as budwood and four date accessions as pollen were sent to the National Laboratory for Genetic Resource Preservation (NLGRP) for long-term conservation. Accessions maintained locally in protective structures or in the field were regenerated as needed. A total of 41 citrus accessions were distributed as budwood to 11 requestors, a total of 9,781 seeds representing 56 accessions were distributed to 13 requestors, and 112 fruit representing 19 accessions were distributed to five requestors through June 1, 2022. In support of the citrus sanitation program, three accessions were shoot-tip grafted. For Objective 2, the project involving early detection methodologies for Huanglongbing (HLB) was terminated after testing was completed. Assays for detection of Cadang-cadang viroid and for Fusarium species in date palms were partially implemented. Two new citrus pathogens were acquired to serve as positive controls in the citrus pathogen testing portion of the sanitation process. In support of Objective 3, the Crop Vulnerability Statements for Citrus and Dates were updated, and additional sections of the Operations Manual were added and developed. In support of Objective 4, a rootstock trial to evaluate the performance of USDA-developed, HLB-tolerant rootstocks was planted and will be evaluated when the trees are established. Information on accessions was added to the local database and the GRIN-Global national database. Research was initiated to quantify polyembryony in citrus accessions.
Impacts
(N/A)
Publications
- Keremane, M.L., McCollum, T.G., Roose, M.L., Lee, R.F., Ramadugu, C. 2021. An improved reference gene for detection of ⿿Candidatus Liberibacter asiaticus associated with citrus huanglongbing by qPCR and digital droplet PCR assays. Plants. 10(10). Article 2111. https://doi.org/10.3390/ plants10102111.
- Rivera, D., Bermudez, A., Obon, C., Alcaraz, F., Rios, S., Sanchez- Balibera, J., Krueger, R. 2021. Analysis of ⿿Marrakesh limetta⿿ (Citrus x limon var. limetta (Risso) Ollitrault, Curk & R.Krueger) horticultural history and relationships with limes and lemons. Scientia Horticulturae. 293. Article 110688. https://doi.org/10.1016/j.scienta.2021.110688.
- Krueger, R., Vidalakis, G. 2021. Study and detection of citrus viroids in woody hosts. In: Rao, A.L.N., Lavagi-Craddock, I., Vidalakis, G., editors. Viroids. Methods in Molecular Biology, Volume 2316. New York, NY: Humana Press. p. 3-21. https://doi.org/10.1007/978-1-0716-1464-8_1.
- Lee, R., Keremane, M.L., Ramadugu, C. 2021. Use of young plants for biological indexing of graft transmissible pathogens of citrus. Crop Protection Journal. 143. Article 105524. https://doi.org/10.1016/j.cropro. 2020.105524.
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Progress 10/01/20 to 09/30/21
Outputs
PROGRESS REPORT Objectives (from AD-416): 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. Approach (from AD-416): 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. In support of Objective 1, contact with Vietnamese scientists continued and a plant exploration trip was planned. A proposal to the USDA Plant Exploration funds was made to support plant explorations in Vietnam in fiscal year (FY) 2022 and 2023. Also in support of Objective 1, citrus accessions were sent to the National Laboratory for Genetic Resource Preservation but not in the quantities stated in the milestones. Accessions were regenerated as needed for local maintenance in the field and protective structures. Also, in support of Objective 1, 13 accessions were tested and biologically assayed to request quarantine release. However, this year⿿s index was unsuccessful and will be repeated in FY 2022. Accessions were distributed to cooperators upon request. In support of Objective 2, protected clean citrus accessions were tested for the citrus disease Huanglongbing (HLB) in accordance with the requirements of the USDA-APHIS interstate movement protocol, maintaining the compliance agreement necessary to support citrus germplasm distribution activities. Activities involving advanced detection of HLB by canines continued, and field trees ⿿positive by canine detection were tested using the regulatorily approved quantitative polymerase chain reaction (qPCR) test. Also, in support of Objective 2, the National Clonal Germplasm Repository for Citrus and Dates (NCGRCD) laboratory is now capable of routine testing for palm phytoplasmas. However, Covid restrictions and other factors slowed progress on development or implementation of additional diagnostics for citrus and date palms. In support of Objective 3, Crop Vulnerability Statements for citrus and dates were finalized. Progress was made on the Operations Manual (OM); however, due to a shortage of personnel, the OM has not been finalized. In support of Objective 4, a field trial of new, putatively HLB- resistant rootstocks was planted. Initial evaluations will be made in FY 2022 or FY 2023, depending on plant growth and other biotic factors. In addition, database records were updated. Due to the retirement of the Research Leader, the University of California at Davis contained research facility (CRF) experiment status is unknown. Record of Any Impact of Maximized Teleworking Requirement: The maximized telework requirement had a negative impact on overall on the National Clonal Germplasm Repository for Citrus and Dates (NCGRCD) functioning properly. Due to initial restrictions on on-site work, germplasm accessions were not adequately maintained during the initial period of maximized telework. Later in the maximized telework era, a portion of the staff returned to working on site part-time. However, some staff continued 100% telework and have not been on-site since maximized telework started. Therefore, work that was normally accomplished by 4.0 FTE support positions was covered by 2.0 FTE support positions. In addition, student employees are normally critical for plant maintenance. However, students were not allowed to work by the host University, thus adding more to the on-site staff⿿s burden. Later we were able to hire some graduated students back as Limited Appointment USDA employees, which alleviated some of the pressure. The need to prioritize plant maintenance and care (this is NCGRCD⿿s essential function) also meant that some portion of SY time was spent on technician duties. ACCOMPLISHMENTS 01 Citrus germplasm distributions support critical Huanglongbing research. Huanglongbing (HLB) is a devastating disease of citrus for which there is currently no cure. Research into HLB biology and management is reliant upon citrus germplasm distributed by NCGRCD. In FY 2021, citrus germplasm was distributed by ARS researchers in Riverside, California, to HLB researchers in Riverside, Davis, and Albany, California; Lake Alfred, Fort Pierce, and Waiauma, Florida; Geneva, New York; and New Haven, Connecticut.
Impacts
(N/A)
Publications
- Salomon-Torres, R., Krueger, R., Garcia-Vazquez, J., Villa-Angulo, R., Villa-Angulo, C., Ruiz-Ortiz, N., Samaniego-Sandoval, L. 2021. Date Palm Pollen: Features, Production, Extraction and Pollination Methods. Agronomy. 11(3). Article 504. https://doi.org/10.3390/agronomy11030504.
- Salomon-Torres, R., Ortiz-Uribe, N., Valdez-Salas, B., Rosas-Gonzalez, N., Garcia-Gonzalez, C., Chavez, D., Cordova-Guerreo, I., Diaz-Rubio, L., Haro- Vazquez, M., Mijangos-Montiel, J., Morales-Maza, A., Mahadevan, P., Krueger, R. 2019. Nutritional Assessment, Phytochemical Composition and Antioxidant Analysis of the Pulp and Seed of Medjool Date Grown in Me´xico. PeerJ. 7. Article e6821. https://doi.org/10.7717/peerj.6821.
- Montazar, A., Krueger, R., Corwin, D.L., Pourreza, A., Little, C., Rios, S. , Snyder, R.L. 2020. Determination of actual evapotranspiration and crop coeffients of California date palms using the residual of energy balance approach. Water. 12(8). Article 2253. https://www.mdpi.com/2073-4441/12/8/ 2253.
- Krueger R.R. 2021. Date palm (Phoenix dactylifera L.) biology and utilization. In: Al-Khayri J.M., Jain S.M., Johnson D.V., editors. The Date Palm Genome, Vol. 1. Compendium of Plant Genomes. Springer, Cham. p. 3-28. https://doi.org/10.1007/978-3-030-73746-7_1_1.
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Progress 10/01/19 to 09/30/20
Outputs
Progress Report Objectives (from AD-416): 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. Approach (from AD-416): 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. 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. Accomplishments 01 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. 02 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. 03 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. 04 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. 05 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. 06 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. 07 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. 08 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.
Impacts
(N/A)
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).
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Progress 10/01/18 to 09/30/19
Outputs
Progress Report Objectives (from AD-416): 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. Approach (from AD-416): 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 continues under Sub-objective 1A, on the acquisition of new germplasm; three date palm accessions were acquired from Date Palm Developments (private company) as tissue culture specimens. These have been propagated but are weak. Ten new citrus accessions were added to the Riverside collection; five were acquired from France, but they arrived in poor condition and only one survived (re-request is planned) and five sanitized accessions were obtained from the Citrus Clonal Protection Program. A proposal was written and submitted for plant exploration in Vietnam. Under Sub-objective 1B, 34 citrus accessions were successfully sanitized, tested and released from quarantine adding to the number of accessions securely maintained within a protective screenhouse in Riverside to 570. Four hundred and six (406) of the 570 sanitized citrus accessions are now cryopreserved and safely secured in Fort Collins. These were either processed in Riverside or sent as budwood to Fort Collins, Colorado. An additional 97 accessions are in the process of viability assessment and if they exceed a threshold of 40 percent viability, they will be placed in long-term storage. In 2018, 26 accessions were sent from Riverside to Fort Collins for processing, 16 accessions were processed in Riverside, and 26 accessions were sent to Fort Collins directly from the Citrus Clonal Protection Program in Exeter, California, where there, are no Huanglongbing Quarantine restrictions at this time. Under Sub-objective 1C, 17 accessions from quarantine which had completed therapy to eliminate citrus pathogens were selected for in depth testing in a biological index. After the completion of additional laboratory testing, these could be released in late 2019 or early 2020. Due to the government shutdown, biological indexing was not carried out optimally this year and not all accessions may qualify for release. A total of 30,435 diagnostic tests were performed on the field, quarantine greenhouse, and protective screenhouse citrus germplasm collections. A total of 37 accessions were subjected to therapeutic procedures: 10 as thermotherapy and 27 as shoot tip grafting. Despite the government shutdown, the repository distributed 426 accessions in the form of budwood, seed, pollen, leaves, and flowers. Under Sub-objective 2A, a variety of field and laboratory testing methods were evaluated to rapidly and accurately test the field germplasm collection while being cost efficient. This was done in collaboration with other USDA and University of California scientists. Early detection technologies (EDT) investigated include: Huanglongbing (HLB) detection canines, serological assays for proteins secreted by the pathogen and bacterial structural proteins, and changes in the metabolomic profile of the host plant. 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. Use of tissue blots to extract nucleic acids for diagnostic assays and preparing reagents in-house rather than purchasing commercial nucleic acid extraction kits which are very costly, generated a cost savings of 90 and 65 percent, respectively. Project results were reported as an oral presentation at the International Organization of Citrus Virologists in March. Significant progress has been made regarding Sub-objective 2B, three additional citrus pathogens have been acquired for use as positive diagnostic assay controls. Published diagnostic protocols are being optimized and validated under Riverside conditions for newly described citrus pathogens. As intended in Sub-objective 2D, the cryopreservation of date palm accessions as tissue culture has been challenging and therefore, focus has been placed on the cryopreservation of pollen. An ARS Scientist from Fort Collins visited the date palm collection and collected pollen from 13 accessions. Under Sub-objective 3A, it was decided to separate the previously unified citrus and date crop vulnerability statements (CVS). Some preliminary information for revision and updating of the crop vulnerability statements was gathered. The template for the CVS was sent to the chair of the citrus and date Crop Germplasm Committees for distribution to members as a way of soliciting information not directly available to the curator. Under Sub-objective 3B, the revision of the repository Operations Manual is progressing; an outline of contents has been developed and assignments have been delegated to staff. Under Sub-objective 4A, three accessions with multiple plants per accession (over 100 total seedings) of Australian finger limes have been evaluated. Rootstock seed reported to have tolerance to HLB have been obtained from ARS in Fort Pierce, Florida. A subset of these seeds has been tested for citrus greening-associated pathogens (both seed coat and embryo) as the seeds were collected from field-grown trees that are subjected to natural infection. Seeds have been germinated, grown and have been grafted with three commonly used scions: Washington navel, Limoneira 8-A lemon, and Tango mandarin in preparation for a field evaluation trial using Carrizo (trifoliate hybrid) rootstock as a standard for comparison. These will be planted in 2020. Accomplishments 01 Increased number of citrus accessions released from quarantine. The National Clonal Germplasm Repository (NCGR) in Riverside, California, completed therapy to eliminate citrus pathogens for 34 citrus accessions. All accessions were tested extensively by laboratory assays and biological indexing to confirm that the therapeutic methods were successful. The request submitted to the Animal and Plant Health Inspection Service (APHIS) and the California Department of Food and Agriculture (CDFA) for the release of these accessions from quarantine status was submitted and approved by both agencies. NCGR technicians have successfully propagated new plants of these accessions and the plants are now securely maintained in the APHIS-inspected, protective screenhouse, increasing the total number of citrus accessions to 570. All pathogen-tested germplasm is available to researchers, breeders, certification programs, and the public upon request. 02 Cryopreservation of citrus germplasm continues. Scientists at the National Clonal Germplasm Repository (NCGR) in Riverside, California, continue to collaborate with scientists at the National Laboratory for Genetic Resources Preservation (NLGRP) in Fort Collins, Colorado, to provide an additional back up of the crucial citrus germplasm collection through cryopreservation methods. Fifty-two citrus accessions were sent to the NLGRP; 16 of the 52 accessions were cryoprocessed in Riverside and sent to Fort Collins in liquid nitrogen. Ten accessions were sent from Riverside to Fort Collins as budwood for cryoprocessing. Due to the Huanglongbing Quarantine and repository Hold Order, 26 accessions were sent as budwood by University of California collaborators in Exeter, California, for cryoprocessing in Fort Collins. Cryopreservation provides for an additional and secure backup of the germplasm collection for the long-term. 03 Increased diagnostic efficiency for citrus pathogens. The use of commercial nucleic acid extraction kits is convenient, but very costly. ARS scientists at the National Clonal Germplasm Repository (NCGR) in Riverside, California, in collaboration with ARS scientists in Beltsville, Maryland, devised and evaluated two methods to extract nucleic acids from citrus plants. Leaf tissue collected in the field was directly blotted onto nitrocellulose membranes. The nucleic acids were eluted from the membrane and used in the polymerase chain reaction (PCR) diagnostic assay for a 90 percent cost savings without losing accuracy. The second method, which involves the in-house preparation of reagents for nucleic acid extraction, is a bit more time consuming, but resulted in a 65% cost savings. 04 Development of information on water use in date palm. Water use in date palm is not well documented. ARS scientists at the National Clonal Germplasm Repository (NCGR) in Riverside, California, are involved in two projects to address this issue. In collaboration with a scientist at the University of California - Riverside, NCGR scientists developed new information on water use in date palm production. The effects of regulated deficit irrigation (RDI) of date palms was developed, and the effect of RDI on the market defect ⿿puffy skin was evaluated. A second project using a different methodology to determine date palm water use at the canopy level has been initiated with University of California Cooperative Extension personnel. Ultimately, date growers will use irrigation water more efficiently and produce a higher quality crop. 05 Evaluation of early detection technologies and survey of the field germplasm collection for huanglongbing-associated pathogens. Because the citrus germplasm field collection at the National Clonal Germplasm Repository (NCGR) in Riverside, California, is threatened by endemic Asian citrus psyllid (ACP) populations which potentially carry huanglongbing-associated pathogens, vigilant survey and testing is required. The Animal and Plant Health Inspection Service (APHIS) diagnostic method of polymerase chain reaction (PCR) does not always detect these pathogens before the ACP can transmit them to healthy trees; therefore, additional methods are necessary for detection. With researchers from cooperating institutions, NCGR scientists are evaluating new technologies including HLB-sniffing canines, serological assays to detect bacterial proteins, and metabolic changes in the host plant to find the disease as early as possible and with increased efficiency and to ultimately develop management strategies that can be used by citrus stakeholders. These early detection technologies (EDT) have identified suspect positive trees that are continuously monitored using PCR by NCGR personnel. Some of these suspect trees have been placed under insect-proof cages to prevent further feeding by ACP and to determine the length of time from the EDT determination to a positive PCR result. Over 500 trees in the germplasm collection have been extensively tested using the PCR method, but no positive trees have been confirmed. 06 Compliance Agreement executed. Scientists and technical staff at the National Clonal Germplasm Repository (NCGR) in Riverside, California, cooperated with the Animal and Plant Health Inspection Service (APHIS) and the California Department of Food and Agriculture (CDFA) to complete the required testing of the protected citrus germplasm collection. Two subsampling collections and plant tissue analysis for huanglongbing-associated pathogens, six months apart, of the protected screenhouse germplasm collection was completed. As a result, the Hold Order placed on the NCGR for citrus due to the implementation of the Huanglongbing Quarantine in Riverside was lifted. A new Compliance Agreement was executed and the distribution of germplasm has resumed.
Impacts
(N/A)
Publications
- Ortiz-Uribe, N., Salmon-Torres, R., Krueger, R. 2019. Date palm status and perspective in Mexico. Agriculture. 9(3):46.
- Shimwela, M.M., Halbert, S.E., Keremane, M.L., Mears, P., Singer, B.H., Lee, W.S., Jones, J.B., Ploetz, R.C., Van Bruggen, A.H. 2019. In-grove spatio-temporal spread of citrus huanglongbing and its psyllid vector in relation to rain. Journal of Phytopathology. 109(3):418-427.
- Mahamoud, Y.A., Mathew, L.S., Torres, M.F., Younuskunju, S., Krueger, R., Suhre, K., Malek, J. 2019. Novel subpopulations in date palm (Phoenix dactylifera) identified by population-wide organellar genome sequencing. BMC Genomics. 20:498.
- Dai, Z., Wu, F., Zheng, Z., Yokomi, R.K., Kumagai, L., Cai, W., Rasco, J., Polek, M., Deng, Z., Chen, J. 2019. Prophage diversity of ⿿Candidatus Liberibacter asiaticus strains in California. Phytopathology. 109(4):551- 559.
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Progress 10/01/17 to 09/30/18
Outputs
Progress Report Objectives (from AD-416): 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. Approach (from AD-416): 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. This is the first report for this new project which just began in March 2018 and continues research from the previous project, 2036-21000-010-00D, �Management and Characterization of Citrus and Date Genetic Resources and Associated Information�. Please see the report for the previous project for additional information. Sub-objective 1A: Because of increased research on Citrus tristeza virus (CTV) and new information regarding genotypes, seven new isolates of CTV were added to the citrus pathogen collection. All known CTV genotypes are now represented and will be used as positive controls for biological indexing, laboratory assays, and ongoing research. An isolate of citrus leaf rugose virus was also added to the pathogen collection. Additional contacts have been made with scientists and programs in Vietnam and Australia in preparation for a planned exchange and exploration to likely take place in 2020. An application was submitted for a Plant Controlled Import Permit (PCIP) to potentially receive new date palm accessions from the center of origin in the Middle East and North Africa. Sub-objective 1B: Now that a new compliance agreement has been executed and the Hold Order placed on the repository has been lifted, germplasm can be distributed. Four citrus accessions have been processed for cryopreservation and sent to Fort Collins, Colorado, for long term storage in liquid nitrogen. Sub-objective 1C: The sanitation of citrus accessions currently held under quarantine continues into the new project plan; nucleic acids were extracted from 287 citrus inventory items held in quarantine and 8,115 polymerase chain reaction (PCR) tests were conducted to determine pathogen status. This determined the method of therapy that was necessary to clean up each accession. One hundred and twenty-eight shoot tip grafts have been completed on 14 accessions and seven citrus accessions have undergone thermotherapy. Nucleic acids were extracted from the inventory items that had undergone therapy during the former project plan and 690 PCR tests were conducted to determine pathogen status. Culturing for Spiroplasma citri and testing for citrus viroids were completed for accessions that had been biologically indexed as clean under the previous project plan. A request has been submitted to regulatory officials for the release of 17 citrus accessions from quarantine. Sub-objective 2A: The field planting of citrus accessions is systematically being tested for Huanglongbing (HLB)-associated pathogens. This effort began under the former project plan and will continue through the duration of the new plan. Approximately 25 percent of the field planting has been surveyed with all trees testing negative for HLB- associated pathogens. A new survey and testing method is being evaluated based on research reported by ARS scientists in Beltsville, Maryland. Plant tissue sap is blotted onto nitrocellulose membranes in the field. Instead of laborious nucleic acid extractions, the nucleic acids are eluted from the membrane and used in polymerase chain reaction assays. ARS scientists extracted nucleic acids from 157 positive control tree inventory items and performed 960 PCR assays. Sub-objective 2B: Application was submitted and received to obtain and maintain new pathogens to be used as positive controls for diagnostics. Discussions were conducted regarding sources of these pathogens. Sub-objective 2C: New research was initiated regarding date palms and the development of disease testing protocols. ARS researchers in Riverside, California, extracted nucleic acids from 78 trees from the date palm collection and performed 466 PCR assays. Extraction protocols are being evaluated and optimized. Nucleic acid extracts have been obtained for use as positive controls for phytoplasma-caused diseases. Sub-objective 2D: Date palm pollen has been sent to ARS scientists in Fort Collins, Colorado, to initiate the development of cryopreservation protocols. Sub-objective 3A: Some additional information on citrus genetic resources has been accumulated to be incorporated into an updated Crop Vulnerability Statement. Sub-objective 3B: Additional Standard Operating Procedures (SOP�s) have been drafted or modified and will be incorporated into the Operations Manual when finalized. Sub-objective 4A: A cooperative experiment to determine actual water use by date palms and the effect of regulated deficit irrigation on date fruit quality was initiated. Data on date palm mineral nutrition was analyzed and a submission started. Various declining date palms were cooperatively evaluated for nutrient and pathogen status. Sub-objective 4B: Twenty-five accession records in the Genetic Resource Information Network (GRIN) Global were entered, updated, or expanded. Accomplishments 01 Collection development. ARS scientists in Riverside, California, acquired 8 new citrus pathogen accessions. Seven of these accessions are new genotypes of Citrus tristeza virus (CTV). One is citrus leaf rugose virus, a pathogen important in nursery settings due to its transmission by grafting and contaminated pruning tools. These accessions will improve the repository�s diagnostic capabilities in both biological indexing and laboratory assays. 02 Quarantine processing and release. ARS researchers in Riverside, California, extracted nucleic acids from 287 citrus inventory items held in quarantine and conducted 8,115 polymerase chain reaction (PCR) tests to determine pathogen status. One hundred and twenty-eight shoot tip grafts were completed on 14 accessions and 7 citrus accessions have undergone thermotherapy. Nucleic acids were extracted from the inventory items that had undergone therapy and 690 polymerase chain reaction (PCR) tests were conducted to determine pathogen status. The ARS researchers completed all required testing including culturing for Spiroplasma citri and citrus viroids for accessions that had been biologically indexed as clean. A request has been submitted to regulatory officials for the release of 17 citrus accessions from quarantine. 03 Protected, pathogen-tested collection. A requirement of the California Interior Citrus Tristeza Virus (CTV) Quarantine is to test citrus accessions annually for CTV. ARS researchers in Riverside, California, tested 1,139 trees maintained in the protective screenhouse using the Enzyme-Linked Immunosorbent Assay (ELISA). All trees tested negative for the virus. Four citrus accessions have been processed for cryopreservation and sent to ARS Fort Collins, Colorado, for long term storage in liquid nitrogen. 04 Diagnostic improvements. ARS scientists in Riverside, California, in collaboration with ARS scientists in Beltsville, Maryland, evaluated the use of plant sap tissue blots in polymerase chain reaction (PCR) to improve labor efficiency and decrease diagnostic costs. This method has proven to be useful when the objective is to quickly screen germplasm collections. A cost analysis was performed to compare commercial extraction kits with the nitrocellulose membrane extraction. The cost savings is approximately 90 percent.
Impacts
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Publications
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