Location: National Clonal Germplasm Repository for Citrus
2018 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
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
1. 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.
2. 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.
3. 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.
4. 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.
