Location: National Clonal Germplasm Repository for Citrus
2023 Annual Report
Objectives
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
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.
Progress Report
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.
Accomplishments
1. 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.
Review 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.
