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United States Department of Agriculture

Agricultural Research Service

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Location: National Clonal Germplasm Repository for Citrus

2010 Annual Report

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. 3A) Recover citrus germplasm exposed to Huanglongbing (HLB) and citrus canker, and evaluate citrus relatives for tolerance or resistance to psyllids and/or HLB. (NP 301; Component 1; Problem Statement 1A) 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). FY10 Program Increase $112,500.00.(1/10)

3. Progress Report
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, date palms (Phoenix dactylifera), and related taxa, and to conduct research that supports and enhances the objectives of the Repository. 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 (ACP) is now established in Imperial, San Diego, Los Angeles, and Orange counties. 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 evaluations, seed collection, 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 because some accessions flower and fruit better in the desert or the coastal area. A total of 1057 distributions were made in CY2009. Research accomplishments include reporting that the ACP can be transported in trailers of unprocessed fruit in Florida, and that samples of ACP collected from trailers were positive for Candidatus Liberibacter asiaticus (Las) when tested by quantitative PCR, finding bacteria closely related to Las in some citrus relatives in Southern Florida, and using a nuclear gene, malate dehydrogenase, sequence to aid in molecular identification of citrus relatives collected from the landscape.

4. Accomplishments
1. Demonstrating the transport of the Asian citrus psyllid (ACP) and the bacterium associated with huanglongbing (HLB) in fruit trailers in Florida. HLB, also known as citrus greening, is one on the most serious diseases of citrus, and was widely spread in Southern Florida when it was first found in 2005. In cooperation with scientists from Florida Department of Plant Industry (DPI), ARS researchers in Riverside, CA, collected ACP from fruit trailers loaded with oranges waiting at processing plants in Florida and tested for presence of the bacterium associated with HLB, Candidatus Liberibacter asiaticus (Las), using quantitative real-time PCR. About 40 percent of the trailers from which the ACP were collected had ACP which tested positive for Las indicating the bacterium associated with HLB is being transported by movement of the fruit trailers. This finding indicates that fruit coming from areas which have HLB should be subject to a cleaning process to remove plant and leaf debris before being moved into areas where HLB is not present. This information is important for areas, such as Texas, Arizona, and California where the ACP is now present but the HLB disease has not been found.

2. Presence of the bacterium associated with huanglongbing (HLB) in citrus relatives in Southern Florida. Over the past two years in cooperation with scientists from Florida Department of Plant Industry (DPI), ARS researchers in Riverside, CA, have conducted surveys of citrus relatives and citrus from the landscape in Southern Florida, extracted DNA from the samples collected, and analyzed the DNA for presence of HLB using quantitative real-time PCR and also, in cooperation with University of California Riverside, Department of Botany and Plant Sciences, sequenced a portion of the nuclear gene, Malate dehydrogenase, to confirm the taxonomical classification of the plant. A bacterium very close to Candidatus Liberibacter asiaticus (Las), the bacterium associated with HLB, was confirmed to be present in Ceylon Atalantia and boxthorn. This is the first report of these two hosts being naturally infected with Las in the United States. Information on alternate hosts of HLB will be useful in mitigating the effect of HLB.

3. Nuclear gene sequence-based phylogeny of citrus. The taxonomy of the sub-family Aurantioideae, which includes citrus relatives, is based on phenotypic characteristics proposed by Swingle and Reece in 1967. ARS researchers in Riverside, CA, in cooperation with the University of California, Riverside, Department of Botany and Plant Sciences, PCR amplified, cloned, sequenced, and analyzed a portion of the nuclear gene, malate dehydrogenase, from thirty-one genera and seventy-six species to aid in molecular identification of citrus relatives. The sequences were aligned and used to construct a phylogenetic tree. The general pattern of clustering of the species tested was in agreement with the sub-family Aurantioideae proposed by Swingle and Reece in 1967, with some differences in plants which are probably hybrids. While the sequence of malate dehydrogenase may be useful for a quick “bar code” confirmation of taxonomic status, a clearer picture of relationships will likely require analysis of organelle DNA and several nuclear genes.

Last Modified: 06/25/2017
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