1a. Objectives (from AD-416)
Determine genetic diversity of tristeza and stubborn disease agents in California; 2)Characterize tristeza and stubborn biologically by graft and vector passage; and 3)Examine patterns of spatial and temporal spread of tristeza and stubborn.
1b. Approach (from AD-416)
Isolate, identify, and characterize field strains of tristeza and stubborn in California with respect to phenotype, vector transmissibility, serology, molecular structure and phylogeny, and epidemiology. This will be accomplished by using a combination of the following: enzyme-linked immunosorbent assay (ELISA); reverse transcription (RT) polymerase chain reaction (PCR); conventional PCR; real time PCR; transmission electron microscopy; dark field microscopy; bacterial culturing in cell-free media; cloning; sequencing; spectrophotometry; electrophoresis; insect transmission; geostatistics; and ARC-GIS. Replaces 5302-22000-006-00D.
3. Progress Report
Taqman probes were developed to differentiate MCA13-reactive strains of Citrus tristeza virus (CTV) associated with seedling yellows, stem pitting, decline on sour orange rootstock, and mild phenotypes of CTV. These were used to screen for potentially virulent strains of CTV in a multiplex real-time polymerase chain reaction (PCR) assay. Using this method, ARS Researchers at Parlier, CA identified a virulent T3 genotype of CTV near Exeter, CA. This procedure is now used by the California Tristeza Eradication Agency (CCTEA) to find potentially severe CTV isolates for eradication before natural spread can occur. CTV cross protection experiments continued and sequences thought to be associated with severe CTV strains were down-regulated in the presence of a mild strain. Small RNA profiles were isolated and sequenced and are being analyzed as potential biomarkers for virulence. In a three year study of citrus stubborn disease epidemiology, only a few new trees became infected by Spiroplasma citri, the causal agent of citrus stubborn disease. This suggests natural spread is slow. Graft transmission experiments showed that the disease was easily bud-transmitted by propagation when pathogen titer is high in the donor source tree. Scaphytopius graneticus (Hemiptera: Cicadellidae), a potential new leafhopper vector, was collected and observed breeding on citrus. S. citri transmission tests with S. graneticus are now in progress. This research supports ARS mission to detect and differentiate pathogen populations, understand their biology and population dynamics. Citrus growers and nurserymen and regulatory agencies are the principal stakeholders who will use information and techniques developed in this research program.
1. CTV strain differentiation by real-time polymerase chain reaction (PCR) identifies severe strains. ARS Researchers at Parlier, CA used genotype specific primers in PCR and found trees in commercial citrus groves infected with T3 genotype CTV. These trees were selected for PCR assay after screening and reacting with the strain-discriminating MCA13 monoclonal antibody. Virulence was confirmed by a virus index test in the greenhouse. The Central California Tristeza Eradication Agency (CCTEA) is now using this system to differentiate CTV isolates and removing MCA13-reactive trees.
2. Direct tissue blot immunosorbent assay (DTBIA) to detect CTV. Citrus tristeza virus (CTV) spread is now at a level where eradication has been replaced by a program of selective removal of trees that are positive to a test for severe strains, the vast majority of strains are mild, hence, CTV continues to spread by aphid vectors. This is a major problem for citrus nurseries because the State requires all citrus propagation to be CTV-free, regardless of strain and most nurseries grow trees in an open field. ARS researchers at Parlier, CA teamed up with a company selling serological reagents and a citrus nursery to develop a direct tissue blot immunoassay (DTBIA) kit for a simple, sensitive and cost-effective detection tool which nurseries can use to test their own trees. This test kit was used successfully to monitor CTV infection in thousands of budwood trees and nursery increase blocks. Infected trees were immediately removed by the nursery, thus, meeting the rigorous standard of maintaining and selling virus-free stock and propagations. Since CTV is readily aphid-transmitted, all citrus nurseries in California are vulnerable and can now use the DTBIA kit for this same purpose. Before they had to request regulatory agencies or UC to test their trees, which was expensive and not timely.
3. Small interfering (si) RNAs in CTV-infected plants. ARS Researchers at Parlier, CA have constructed libraries of small RNAs from symptomatic and non-symptomatic sour orange plants infected by seedling yellows strains of CTV in cross-protection experiments. The siRNAs were sequenced by Illumina Genome Analyzer. Forty percent of sequences were associated with the Poncirus trifoliata CTV resistance gene locus. Research is continuing to determine which one of 10 candidate genes is the CTV resistance gene. This research may lead to development of CTV resistance and will provide better understanding of CTV mild-strain cross protection.
4. Rapid assessment of citrus trees suspected of stubborn or Citrus tristeza virus (CTV) infection. Trees with chlorosis, poor yield, off-shaped fruit, and premature fruit drop may be infected by phloem-limited pathogens such as CTV or Spiroplasma citri. ARS is working with Extension agents and growers to determine disease status of suspect diseased trees using real-time polymerase chain reaction (PCR) assays developed by ARS Researchers at Parlier, CA. Previously, management decisions were based solely on symptomotology. Now growers and pest control advisors have useful diagnostic tools available and three different commercial labs have contacted ARS about use of these techniques.
Mello, A.F., Yokomi, R.K., Fletcher, J. 2011. Assessment of Stubborn Disease Incidence in Citrus. In: Proceedings of the International Organization of Citrus Virologists. p. 123-130.