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United States Department of Agriculture

Agricultural Research Service

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Location: National Clonal Germplasm Repository for Citrus

2011 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).

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, 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 Imperial, San Diego, Los Angeles, and Orange counties. The NCGRCD contains several collections: The pathogen-tested protected collection with about 450 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, located at CVARS near Thermal, CA. In FY11, 1294 distributions were made. Research accomplishments include the construction of metagenomic BAC libraries using the genomic DNA of Bactericera cockerelli, the psyllid vector of Candidatus Liberibacter psyllaurous, evaluation of 87 genotypes of citrus and citrus relatives for colonization by Diaphorina citri, and characterization of the Alpha Proteobacteria in Citrus and Citrus relatives.

4. Accomplishments
1. Construction of Metagenomic BAC libraries of Bactericera cockerelli, the psyllid vector that transmits the bacterium ‘Candidatus Liberibacter psyllaurous’ (LPS) associated with psyllid yellows of tomato (PYT). PYT is a useful model system for the study of Huanglongbing (HLB), also known as citrus greening. Because LPS is unculturable, to sequence its genome, we had to start with the psyllid carrying LPS. In cooperation with University of California entomologists, ARS scientists at Riverside, CA, used an insect colony with 100% infection of LPS for the construction of three large DNA libraries. Using the genome sequence of a closely related bacterium, Ca. L. asiaticus (LAS), associated with HLB, as reference, a panel of 384 primer pairs were made and screened against LPS-infected tomato psyllids as well as plants. The library of 57,600 clones contained about 12-18x coverage of the LPS genome. Size estimation of the clones was done using pulsed field gel electrophoresis. Overlapping clones were identified by testing the selected clones with multiple sets of primers. Genome-wide clones were identified and sequencing of 80 percent of the LPS genome is completed. The results demonstrate this approach is useful, especially for characteraztion of the unculturable pathogens/symbionts.

2. Evaluation of 87 genotypes of citrus and citrus relatives for colonization by Diaphorina citri. HLB, spread by its insect vector, D. citri, is a devastating disease of citrus, limiting citrus production in areas where it occurs. Control of Huanglongbing (HLB) and the vector, D. citri, is inadequate, but incorporation of D. citri resistance traits from uncultivated citrus and citrus relatives is a potential management strategy. ARS scientists in Riverside, CA, with the help of USDA ARS scientists at the USHL, Ft. Pierce, FL, planted seedlings of 87 different genotypes of citrus and citrus relatives in the field in a random block design plot where they were exposed to D. citri. The seedlings were observed and rated for presence of eggs, nymphs, and adults four times from June-September 2010. One citrus relative, Casimiroa edulis, was avoided by all three life stages of D. citri, two selections of Poncirus trifoliata had very low levels of D. citri. The identification of partial resistance to D. citri will be useful to direct future citrus breeding efforts intended to reduce the effect of HLB, and has provided useful information to establish future germplasm screening trials.

3. Characterization of the alpha proteobacteria in citrus and citrus relatives. Huanglongbing (HLB), first reported in Florida in 2005, is a devastating disease of citrus, and is now present in many countries in the Americas and the Caribbean Basin. From surveys of citrus and citrus relatives in South Florida, we have identified a bacterium in some citrus relatives which give a qPCR positive reaction for Candidatus Liberibacter asiaticus (Las), but sequencing of the 16 S RNA region indicates only about 92-94 percent homology with Las. A macro array for several areas of the Las genome indicates some areas are conserved and other areas have low or no homology with Las or other Ca. L. species. These alpha proteobacteria may be useful to outcompete or ameliorate symptoms caused by Las, the bacterium associated with HLB.

Review Publications
Barkley, N.L., Krueger, R., Federici, C.T., Roose, M.L. 2009. What phylogeny and gene genealogy analyses reveal about homoplasy in citrus microsatellite alleles. Plant Systematics and Evolution. 282:71-86.

Hajeri, S., Ramadugu, C., Keremane, M.L., Ng, J., Lee, R.F., Vidalakis, G. 2011. In vivo generated Citrus exocortis viroid progeny variants display a range of phenotypes with altered levels of replication, systemic accumulation and pathogenicity. Virology. 417(2):400-409. DOI:10.1016/j.virol.2011.06.013

Chen, J., Civerolo, E.L., Lee, R.F., Jones, J., Deng, X., Hartung, J.S., Keremane, M.L., Brlansky, R. 2011. “Candidatus liberibacter sp.”, without koch's postulates completed, can the bacterium be considered as the causal agent of citrus Huanglongbing (yellow shoot disease)?. Acta Phytopathologica Sinica. 41(2):113-117.

Westbrook, C.J., Hall, D.G., Stover, E., Duan, Y.P., Lee, R.F. 2011. Colonization of citrus and citrus-related germplasm by Diaphorina citri (Hemiptera: Psyllidae). HortScience. 46(7):997-1005.

Last Modified: 05/27/2017
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