1a. Objectives (from AD-416)
1) Strategically expand and improve collections of priority genetic resources of citrus and date palm and associated information. 2) Conserve and propagate citrus and date genetic resources efficiently and effectively, and distribute pathogen-tested samples and associated information worldwide. 3) Strategically characterize (“genotype”) and evaluate (“phenotype”) citrus and date palm genetic resources for priority genetic and horticultural traits. 4) Develop more rapid and accurate diagnostic methods for priority graft-transmissible pathogens of citrus to promote exchange of pathogen-tested stock and efficiently screen 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 accessions will be quarantined, indexed and therapied before being available for distribution, and they will be characterized using the ‘Descriptors for Citrus’ published by IPGRI. In addition to distribution of germplasm, information on the accessions is disseminated via the GRIN website/server and the Repository website. The ‘genotype’ and ‘phenotype’ of citrus and date palm genetic resources will be characterized for priority genetic and horticultural traits, such as level of antioxidants and the tolerance/resistance to selected diseases. Using SSR markers and by sequence analyses of selected regions of the chromosomal DNA, the genetic variability of core accessions of orange hybrids and major groups will be examined and used to determine ancestral origin. Laboratory-based diagnostic methods will be developed for citrus vein enation and citrus concave gum disease, presently detectable only by biological indexing on indicator plants. Diagnostic tests for huanglongbing disease will be utilized to screen subsets of citrus genetic resources so as to identify new sources of host-plant resistance/tolerance to this disease. Replaces 5310-21000-008-00D (3/08).
3. Progress Report
The Repository is both a service and research unit with a mission to collect, maintain, preserve, evaluate, and distribute germplasm of Citrus, date palms (Phoenix dactylifera), and related taxa, and to conduct research that supports and enhances the objectives of the National Clonal Germplasm Repository for Citrus and Dates (NCGRCD). 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 since the finding of the Asian citrus psyllid in September 2008. The NCGRCD contains several collections: the pathogen-tested protected collection which is used for distributions of budwood, the citrus variety collection (owned by University of California, Riverside but utilized by NCGRCD for seed collection, evaluations, DNA extractions, and pollen collection), the citrus relative collections, and the date palm collections. The citrus relatives are maintained at three field locations because of their susceptibility to cold and some accession flower and fruit better in the desert or the costal area. A total of 564 distributions were made in CY2008. Research accomplishments include a genetic characterization of citrus relatives, identification of new hosts of HLB among the citrus relatives, and identification of a variety which repels the Asian citrus psyllid, and progress on development of a single extraction method which allows for testing of viroids, RNA viruses, and systemic prokaryotic pathogens. The development of such a method will help speed the process of cleaning germplasm and making it available for distribution.
1. Application of quantitative real time Polymerase Chain Reaction (PCR) for early detection of Huanglongbing (HLB) in new areas and to monitor effectiveness of management approaches for the disease. Testing of plants for presence of the bacterium associated with HLB in new geographical areas not previously reported to have the disease or to monitor the effectiveness of different management approaches to ameliorate the effect of HLB is not very effective because of irregular distribution in the plants and lack of sensitivity in the detection methods. ARS scientists at Riverside, CA developed a quantitative assay to detect the bacterium associated with HLB in psyllids, previously reported, as the bacterium multiplies in the insect vector allowing for earlier detection of the bacterium as compared to testing for the bacterium in plants. This assay has been utilized for early detection of HLB in areas where the disease has not been previously reported and also to monitor the effectiveness of various management approaches to HLB in Brazil. In April, 2009, the Repository found the first batch of psyllids from Belize with the Candidatus bacterium Liberibacter associated with HLB, the confirmation of HLB in plants has now been confirmed by USDA APHIS and Belizean scientists. The results validate the usefulness of monitoring the psyllid vectors for presence of the bacterium associated with HLB.
2. Determine genetic relationships of citrus relatives by sequencing a fragment of the plant genome. A comprehensive study of the genetic relationship of citrus relatives by the use of molecular methods has not been reported although some limited studies have been done using regions of the chloroplast DNA. ARS scientists at Riverside, CA in cooperation with the University of California, Riverside Department of Botany and Plant Sciences, studied the genetic relationship of 35 genera with 61 species by sequencing a nuclear gene, present in all plants. This approach has been previously applied to the citrus core collection to assess genetic relatedness as compared to information derived by use of Simple Sequence Repeat (SSR) markers. The phylogenetic information generated provides a source of single nucleotide polymorphism (SNP) markers and aids in a better understanding of the relationships between the different accessions studied.
3. Spread of Huanglongbing (HLB) in fruit trucks in Florida. Huanglongbing (HLB,citrus greening) is one of the most serious of citrus diseases. Movement of the disease occurs as a result of natural vector-borne infection and by movement of plant material. ARS scientists at Riverside, CA demonstrate here that the Asian citrus psyllid, (vector of citrus greening pathogens) can be transported in trailers of unprocessed fruit. Several samples of psyllid collected from trailers of oranges were positive for the bacterium associated with HLB by quantitative real time Polymerase Chain Reaction (PCR), indicating that the pathogen also can travel with fruit. This information is important for areas, such as Texas, Arizona, and California where the Asian citrus psyllid is now present but the HLB disease has not been found.