1a. Objectives (from AD-416):
The mission of the National Clonal Germplasm Repository for Citrus and Dates (NCGRCD), Riverside, CA is to access, maintain, preserve, evaluate, and distribute germplasm of Citrus and date palm (Phoenix dactylifera) and their wild relatives. In addition, research is conducted to enhance the viability and impact of the collections. A major focus of research is to reduce vulnerability of citrus and date palm genetic resources to natural disasters and virulent pathogens by devising cryopreservation methods for in vitro preservation and by developing more effective and efficient disease diagnostic procedures. This project’s comprehensive and coordinated genetic resource management efforts will result in ready access to pathogen-tested/pathogen-free propagative and breeding materials for citrus and date palm breeders, research scientists, and producers. Associated information will be made available to customers through the Genetic Resources Information Network, stakeholder meetings, and publications. The specific objectives are: 1) Develop and apply new methods for conserving and propagating citrus and date genetic resources which improve genebank operating efficiency and effectiveness and which enable pathogen-tested materials and associated information to be distributed worldwide. 1A) Efficiently and effectively maintain the National Plant Germplasm System collections of citrus and date palm genetic resources and associated information. Distribute accessions upon request to researchers and breeders world-wide. 1B) Develop and apply cryopreservation, in cooperative research with the National Center for Genetic Resources Preservation, Ft. Collins, as an alternate and efficient means of conserving and distributing citrus, citrus wild relatives, and date palm. 1C) Develop, validate, and apply alternative means, such as cryotherapy, to free citrus germplasm from graft transmissible pathogens. 2) Elucidate patterns of genetic divergence and diversity in citrus, dates, and related taxa, and apply that knowledge to strategically expand the collections and other facets of genetic resource management. Evaluate citrus and date genetic resources for priority genetic and horticultural traits. 2A) Characterize the genetic variability in citrus and citrus wild relatives by DNA sequencing to better classify accessions taxonomically. 2B) Evaluate horticultural quality and other important traits for priority accessions of date palm, citrus and citrus relatives. Incorporate the evaluation and characterization data in GRIN-Global and/or other databases. 2C) Rescue unique and valuable citrus germplasm exposed to Huanglongbing (HLB) and citrus canker for cleanup from known pathogens and incorporation into the collection. Sub-objective 2D. Evaluate citrus and citrus relatives for tolerance or resistance to psyllids and/or HLB. 3) Develop more accurate and high through-put diagnostic methods for priority graft-transmissible pathogens of citrus and for insect-vectored pathogens of dates, to enable the exchange of pathogen-tested accessions, and to more efficiently evaluate accessions for host-plant resistance.
1b. Approach (from AD-416):
New accessions will be acquired through exchange with university breeders, foreign country national programs, botanical gardens, or by plant exploration. New citrus accessions are released from quarantine following comprehensive testing on indicator plants and laboratory tests as required by CDFA and USDA APHIS, to ensure that graft-transmissible pathogens are no longer present. While citrus and date accessions have traditionally been maintained as living plants, methods for cryopreservation will be developed and applied to the 450 accessions held in the citrus protected collection which is used for distribution of budwood. For release from quarantine and before citrus accessions may be distributed, the accessions must be cleaned of graft transmissible pathogens. While the clean-up has traditionally been done by heat treatment or shoot tip grafting, recent research has shown that cryotherapy may be a more efficient method of eliminating graft transmissible pathogens from citrus. The protocols for cryotherapy will be further developed, standardized, and the efficiency of cleaning graft transmissible pathogens by shoot tip grafting and cryotherapy will be compared. In previous studies, the single nucleotide polymorphism of a specific nuclear gene, malate dehydrogenase, has been identified as informative to determine phylogenetic relationships among citrus accessions. This information will be utilized to develop a DNA barcode, based on parsimony-informative characters, to help identify citrus and related genera from the sub-family Aurantioideae. Huanglongbing (HLB) and other invasive diseases threaten germplasm held under unprotected conditions. In Florida where HLB is now endemic and in California where HLB threatens to become established, and other citrus producing states as needed; rare germplasm which is not already backed up elsewhere will be recovered, cleaned, and added to the protected collection following release from quarantine status for distribution upon request. Threatened germplasm will be identified by collaboration with stakeholders. Diverse citrus and citrus relative germplasm will be planted into the field in Florida for screening for tolerance/resistance to HLB and the psyllid vector of HLB. Half of the trees planted in the randomized complete block trial will be inoculated with HLB prior to planting in the field. The plants will be evaluated for HLB, citrus canker, incidence of Diaphorina citri, the psyllid vector of HLB, and other diseases at six month intervals over four years. High through-put methods of nucleic acid extraction will be coupled with high through-put (384-well) qPCR assays to lower the cost per test and to be able to test for multiple pathogens (both RNA and DNA genome pathogens) from one collection/sample using the Taqman™ probes. Taqman™ probes will be developed for the RNA transcripts of the housekeeping gene NADH dehydrogenase (NAD5) gene and for the DNA gene of NAD5 for use as an internal control to monitor the quality of RNA and DNA extractions, respectively.
3. Progress Report:
This is a new project which began in March of 2013 and continues research from project 5310-21000-009-00D, "Management of Citrus and Date Genetic Resources and Informaton". The National Clonal Germplasm Repository for Citrus and Dates (NCGRCD) is both a service and research unit with a mission to collect, maintain, preserve, evaluate, and distribute germplasm of citrus, citrus relatives, and date palm (Phoenix dactylifera), and to develop sensitive and better diagnostic methods to promote exchange of pathogen tested stock, recover citrus germplasm exposed to Huanglongbing (HLB) and citrus canker, and evaluate citrus relatives for tolerance or resistance to psyllids and/or HLB. The NCGRCD is located on the campus of the University of California, Riverside (UCR) and supports NP 301 Plant Genetic Resources, Genomics, and Genetic Improvement. The NCGRCD is playing an important role in preserving, maintaining and cleaning germplasm from Florida where the germplasm is threatened by huanglongbing (HLB) and citrus canker and also in California where the Asian citrus psyllid is now established in Southern California. The NCGRCD contains several collections; the pathogen-tested ‘Protected Collection’ with about 470 accessions which are used for distributions of budwood, the citrus variety collection with over 1,100 accessions (owned by UCR but utilized by NCGRCD for seed collection, evaluation, DNA extractions, and pollen collection), the citrus relatives collection, and the date palm collection which is located near Thermal, California. Research accomplishments include the identification of varying degrees of tolerant to huanglongbing (HLB)from a four year field trial in Florida where 100 accessions of citrus and citrus relatives were evaluated in severe natural challenge conditions and the development of a home-based detection kit for use by growers and nurserymen that enables detection of the bacterium associated with HLB in the psyllid vector in less than 30 minutes without the need for elaborate, expensive equipment.
1. Identification of tolerance to huanglongbing (HLB, citrus greening) in citrus and citrus relatives. Huanglongbing (HLB, citrus greening) disease is associated with a fastidious prokaryote, Candidatus Liberibacter asiaticus (Las) in Florida, and is one of the most destructive diseases of citrus in the world. No sources of resistance have previously been identified in commercial citrus and many citrus relatives have not been evaluated. From a four year field trial in cooperation with USDA ARS, Fort Pierce, Florida, in which 100 accessions of citrus and citrus relatives were evaluated under natural challenge conditions, various levels of tolerance to HLB was identified. Some tolerance is in accessions which are sexually compatible with Citrus, and some citrus relatives displayed resistance to HLB. The identification of tolerance to HLB in accessions that are sexually compatible with Citrus should make it easier for citrus breeders to incorporate tolerance to HLB into commercial citrus.
2. Development of a 30 minute detection method for detection of huanglongbing (HLB, citrus greening) by growers and nurserymen. Huanglongbing (HLB, citrus greening) disease is devastating to citrus production, and management of the disease requires timely detection of the bacterium associated with HLB in the psyllid vectors. A rapid, field deployable detection system has been developed for the detection of the bacterium associated with HLB in 30 minutes without the need for skilled technical personnel or expensive equipment. The technique will be useful for citrus growers, nurserymen, extension personnel, researchers, and regulatory personnel in situation where quantitative PCR testing is not feasible. Test samples can be forwarded to a diagnostic laboratory for confirmatory tests, if needed.
3. Development of methods for cryotherapy of citrus. Before citrus germplasm may be distributed to nurserymen and growers, it must be free of graft transmissible diseases. In cooperation with the USDA, ARS National Center for Genetic Resources Preservation (NCGRP), Ft. Collin, Colarado, we have developed a protocol of freezing vegetative buds of citrus in liquid nitrogen to eliminate graft transmissible pathogens. The protocol has been demonstrated to eliminate Citrus tatterleaf virus and citrus viroids. The technique does not require the high level of skill needed to eliminate the pathogens by shoot tip grafting, and has been demonstrated to be effective at eliminating two pathogens (Citrus tatterleaf virus and citrus viroids) that are not eliminated by traditional thermal therapy.