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

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

Location: National Clonal Germplasm Repository for Citrus

2019 Annual Report

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

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

Progress Report
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.

1. 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.

2. 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.

3. 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.

4. 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.

5. 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.

6. 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.

Review 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.