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United States Department of Agriculture

Agricultural Research Service

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Location: National Clonal Germplasm Repository for Citrus

2012 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.

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 Southern California. The NCGRCD contains several collections: The pathogen-tested protected collection with about 460 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, California. In FY12, 1365 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, and use of this information to develop antibodies specific for Candidatus Liberibacter psyllaurous, development of methods to cryopreserve citrus in cooperation with an ARS scientist from Ft. Collins, Colorado, and a full genome comparison of Dweet mottle virus from California with Citrus leaf blotch virus and other members of the Alpha- and Beta-flexiviridae family of viruses.

4. Accomplishments
1. Development of antibodies specific for ‘Candidatus Liberibacter psyllaurous’ (LPS). The bacterium LPS is associated with psyllid yellows of tomato (PYT) and the disease is a useful model system for the study of Huanglongbing (HLB), also known as citrus greening. Using sequence information developed from partial sequencing of the LPS genome, specific genes were identified as possible secretory proteins. These genes were cloned, expressed in E. coli, and the expressed protein used to raise antibodies specific for LPS. Preliminary screening indicates some antibodies are specific for LPS whereas some antibodies also recognize Candidatus Liberibacter asiaticius (LAS), the bacterium associated with HLB. The antibodies may provide an alternative means of identifying plants infected with LPS or LAS and may result in more sensitive detection of the bacteria as the protein may be more uniformly distributed in the plant than the bacteria are.

2. Development of methods to cryopreserve citrus. The occurrence of natural disasters, such as hurricanes, and appearance of devastating diseases of citrus such as citrus canker and huanglongbing, have highlighted the need for secure back up of clonal accessions of citrus. A successful protocol has been developed to store vegetative buds of citrus in liquid nitrogen, and to recover the buds later by thawing and micrografting onto in vitro rootstock seedlings in cooperation with the USDA ARS NCGRP, Ft. Collins, Colarado. Juvenility is avoided in citrus subjected to cryopreservation. Application of the cryopreservation method to citrus accessions present in the USA will enable a secure, long term backup of these important genetic resources.

3. Comparison of the relatedness of Dweet mottle virus (DMV) and Citrus leaf blotch virus (CLBV). Dweet mottle virus (DMV) was first reported from Riverside, California in 1968 during reindexing of a ‘Cleopatra’ mandarin variety introduced from Florida, and sequence analysis showed that DMV has very high sequence homology (over 96%) with the CLBV, which is a concern for seed distributions as CLBV has been reported to be seed transmitted. The biological activities were compared; both DMV and CLBV induce mottling in ‘Dweet’ tangor and stem pitting in ‘Etrog’ citron however, only CLBV causes vein clearing in ‘Pineapple’ sweet orange and bud union crease on trifoliate and trifoliate hybrids rootstocks. Experiments with CLBV infectious clones suggested that the bud union crease and vein clearing symptoms may be caused by a different agent associated with ‘Nagami’ kumquat sources. Analyses of the sequences of DMV and CLBV indicated a close relation between the two viruses. It was concluded that DMV is probably also seed transmitted and testing needs to be performed to ensure it is not present in trees used for seed collection.

Review Publications
Halbert, S.E., Keremane, M.L., Ramadugu, C., Brodie, M.W., Webb, S.E., Lee, R.F. 2010. Trailers transporting oranges to processing plants move Asian citrus psyllids. Florida Entomologist. 93(1)p33-38.

Al-Dous, E.K., George, B., Ai-Mahmoud, M.E., Al-Jaber, M.Y., Wang, H., Salameh, Y.M., Al-Azwani, E.K., Chaludavi, S., Pontaroli, A.C., Debarry, J., Arondel, V., Ohlrogge, J., Saie, I.J., Bennetzen, J.L., Krueger, R., Malek, J.A. 2011. De novo genome sequencing and comparative genomics of the date palm Phoenix dactylifera). Nature Biotechnology. 29:521-527.

Krueger, R. 2011. Date Palm Germplasm. In: Jain, S.M., Al-Khayri, J. M., Johnson, D. V., editors. Date Palm Biotechnology. 1st Edition. New York, NY: Springer. p. 313-336.

Hajeri, S., Ramadugu, C., Keremane, M.L., Vidalakis, G., Lee, R.F. 2010. Nucleotide sequence and genome organization of Dweet mottle virus and its relationship to members of the family Betaflexiviridae. Archives of Virology. 155(9):1523–1527.

Last Modified: 06/24/2017
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