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

Location: National Clonal Germplasm Repository for Citrus

2022 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 Heath Inspection Service (APHIS). Seeds are unrestricted and tissue culture is allowed via permit. Pathogen-tested germplasm is maintained in an APHIS approved screenhouse, un-sanitized material is separated in other protective structures, and most accessions are also maintained in a field planting. Cryopreservation as a long-term backup will continue as accessions are released from quarantine and for wild relatives, seeds and pollen. Protocols for the cryopreservation of date palm accessions will be developed and optimized. Therapy and pathogen testing will continue for new citrus accessions and to satisfy quarantine regulations for the protected collection. A pathogen-testing program for date palms will be initiated. We will test whether canines and other early detection technologies can accurately detect Liberibacter-infected trees prior to standard qPCR methods. Dogs trained to alert to HLB-infected citrus trees will return to the Citrus Variety Collection and be shown every tree in the collection. For all dog-alerted trees and adjacents, leaves will be collected and assayed by qPCR using several different primer sets including the standard APHIS primers, tested with other early detection technologies and/or the tree will be pruned for containment in an insect-proof cage. Samples will be collected from caged trees and assayed using qPCR over time. Improvements to the citrus diagnostic protocols used will be implemented based on reports and publications of other researchers to expedite diagnostics. Date palm diagnostic methods developed by others will be optimized targeting phytoplasmas, Cadang-cadang viroid, and Fusarium oxysporum. All SOP’s will be revised. The Crop Vulnerability Statement will be updated. Citrus scion and rootstock germplasm imported from Florida will be evaluated for its horticultural value and reaction to endemic diseases under California growing conditions at two locations. Accessions in the field variety collection will be re-propagated on these tolerant rootstocks. Should HLB become widespread in southern California, we would evaluate for this disease under California conditions. Whether endemic pathogens prevent, mask, or exacerbate the development of HLB will be determined within a contained research facility. All information gathered will be updated and expanded through the GRIN-Global System.

Progress Report
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.

Accomplishments

Review 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.