Progress 08/18/03 to 05/30/07
Outputs
Progress Report Objectives (from AD-416) 1)Enhance management of citrus tristeza based on disease suppression; 2) Determine diversity of CTV strain; and 3) Develop improved methods to detect, identify, and distinguish CTV strains. Approach (from AD-416) 1) Isolate, identify and characterize CTV isolates in California with respect to pathogenicity vector and transmissibility, serology, and molecular structure and organization; and 2) Adapt existing and develop new biological, serological, and molecular-based methods to detect, identify, and distinguish CTV strains in California. Documents SCA with Citrus Research Board. Formerly 5302-22000-006-01S. Significant Activities that Support Special Target Populations This report serves to document research conducted under a Specific Cooperative Agreement between ARS and the Citrus Research Board for research on citrus tristeza virus. Additional details of the research can be found in the report for the parent project 5302-22000-009-00D, Characterization & Epidemiology of Citrus Tristeza Virus and Other Invasive and Emerging Graft-transmissible Diseases of Citrus in California. Madam Vinous sweet orange grafted on two separate transgenic Carrizo lines containing CTV constructs (CTV-1 = CP-P20-3�UTR; and CTV2 = CP-P23-3�UTR), were graft-challenged with P100 and P81 CTV isolates to determine if the small RNAs produced by the transgene(s) was effective in the scion and could interfere and prevent virus infection. Plants were challenged, and after a suitable incubation period, ELISA will be performed to determine if plants are infected. Prior to virus challenge inoculation, Northern blot analysis was conducted to show which rootstocks were expressing the foreign genes. This research was conducted by the Cooperator. The Central California Tristeza Eradication Agency (CCTEA), Tulare, CA continued to conduct host range biological characterization of CTV isolates collected from their surveys in California. The CCTEA is presently assessing the host range virulence of CTV isolates obtained in May 2007 from the Lindcove Research and Extension Center, Exeter, CA in collaboration with ARS, Parlier. ARS is assessing the same isolates for aphid transmissibility, genotype, and molecular sequencing analysis. Research progress was monitored by meetings, site visits, email exchanges, phone calls, reports, and writing and editing manuscripts on research completed.
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Progress 10/01/05 to 09/30/06
Outputs
Progress Report 4d Progress report. This report serves to document research conducted under a specific cooperative agreement between ARS and the Citrus Research Board, Visalia, California. Additional details of the research can be found in the report for the parent project 5302-22000-006-00D, Characterization and Epidemiology of Citrus Tristeza in California. Two artificial Citrus tristeza virus (CTV) RNA silencing resistance genes were made and engineered into Nicotiana benthamiana and Carrizo citrange in the Plant Pathology Department at UC Davis. Both genes were confirmed to provide resistance against CTV sequences in N. benthamiana plants and showed that the resistance is effective against a number of different CTV sequence variants. Molecular markers associated with CTV resistance in some of the Carrizo plants were identified. To test resistance of the transgenic plants, all of the Carrizo plants were vegetatively propagated and grafted with various
rootstock/scion combinations using the transgenic Carrizo, Madam Vinous, Washington Navel, and Mexican lime plants. After several graft and aphid challenges by CTV, the plants are under evaluation for resistance to CTV. The greenhouse facility at the Central California Tristeza Eradication Agency in Tulare houses the most extensive collection of California Citrus tristeza virus isolates. The majority of the 339 isolates maintained in the collection were collected in the San Joaquin Valley. However, isolates from southern California including Riverside and Ventura counties are also represented. Over 250 of the isolates have undergone bio-characterization.
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Progress 10/01/04 to 09/30/05
Outputs
4d Progress report. This report serves to document research conducted under a Specific Cooperative Agreement between ARS and the Citrus Research Board, Visalia, California. Additional details of the research can be found in the report for the parent project 5302-22000-006-00D, Characterization and Epidemiology of Citrus Tristeza in California. The Falk and Dandekar labs at UC Davis produced engineered Carrizo plants with the CTV1 artificial chimeric Citrus Tristeza Virus (CTV) resistance gene and are awaiting challenge (transmission) tests to determine if transformed plants have CTV resistance. The Ding lab at UC Riverside showed that CTV encodes for three suppressors of RNA silencing and that the best use of RNA silencing may require incorporating all three in transformed citrus. The Ullman lab at UC Davis in collaboration with the Yokomi lab at the San Joaquin Valley Agricultural Sciences Center, Parlier, Calif., have shown the brown citrus aphid efficiently
transmitted Calif. CTV isolates that are poorly transmissible by A. gossypii. The Polek group at the Central California Tristeza Eradication Agency, Tulare, Calif., continues to biocharacterize CTV isolates and maintains a collection of CTV isolates for researchers. Collectively, these projects assess severity and spread potential of existing isolates while developing RNA silencing as a new strategy for CTV. The outcome of this research is to obtain genetically modified citrus with durable resistance to CTV which will be needed when the brown citrus aphid arrives in California.
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Progress 10/01/03 to 09/30/04
Outputs
4. What were the most significant accomplishments this past year? 4d. Progress Report This report serves to document research conducted under a specific cooperative agreement between ARS and the Citrus Research Board (California) . Additional details of the research can be found in the report for the parent project 5302-22000-006-00D, Characterization and Epidemiology of Citrus Tristeza in California. Transgenic Nicotiana benthamiana plants containing chimeric, self- complementary constructs of citrus tristeza virus (CTV) sequences were generated and analyzed for post transcriptional gene silencing (PTGS)- mediated resistance. Recombinant potato virus X (PVX) constructs containing CTV sequences to the transgene constructs were used to inoculate plants. Most test plants developed typical PVX symptoms but a few lines showed a portion of non-symptomatic plants. Small interfering RNAs (siRNAs) were detected by northern hybridization. This suggests that PTGS can be
generated by this approach. Direct comparison of vector transmissibility of 6 California CTV isolates was conducted in a containment facility at Fort Detrick, Maryland. Preliminary analysis shows that two of our most poorly transmitted isolates are highly transmissible by the brown citrus aphid (BrCA). Highly transmissible California isolates were not well transmitted by the BrCA .
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