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
A quantitative reverse transcription polymerase chain reaction assay (qPCR) was validated to differentiate mild vs. severe strains of Citrus tristeza virus (CTV) from biocharacterized isolates from collections and field samples from California and Florida. CTV isolates were identified to genotype and associated virulence as determined by a citrus host range test. A multiplex direct tissue print immunosorbent assay (DTBIA) was developed to detect CTV infection and reactivity to monoclonal antibody MCA13, which reacts primarily with virulent CTV strains. CTV surveys in Ventura and Riverside Counties indicated most CTV isolates were generally mild and similar to those in the San Joaquin Valley, except that more MCA13 reactive isolates were observed in the southern California and coastal sites. In addition, isolates related to New Zealand CTV isolates capable of breaking CTV resistance in Poncirus trifoliata were identified in Tulare, Ventura and Riverside Counties. For the spiroplasma project, Koch’s postulates were completed for a carrot strain of Spiroplasma citri using transmission by the beet leafhopper. This demonstrated that S. Citri from carrot and citrus were transmitted to each reciprocal host and suggested the same strain could infect both host plants. Symptom severity of citrus stubborn disease in a sweet orange variety was found to be associated with pathogen titer and not to a genetically distinct strain. This research supports ARS mission to detect and differentiate pathogen populations, and to understand their biology and population dynamics. Citrus growers, nurserymen, and regulatory agencies are the principal stakeholders who will use information and techniques developed in this research program.
1. Validation of Citrus Tristeza Virus (CTV) CP intergene sequences to differentiate CTV strains. Virulent strains of CTV cause a scion disease regardless of rootstock and causes economic damage to citrus production. A real time Polymerase Chain Reaction (PCR) method that was developed by ARS scientists in Parlier, CA to differentiate CTV strains into VT, T3 or T36 non-standard genotypes was validated with a worldwide collection of characterized CTV strains and field isolates from California and Florida. CTV isolates with these genotypes are known to cause stem pitting and seedling yellows which results in reduction of fruit quality and yield in many commercial citrus varieties. The Central California Tristeza Eradication Agency (CCTEA) is considering using this system to differentiate CTV isolates and remove only CTV sources that are diagnosed with VT or T3 genotype strains.
2. Development of a duplex direct tissue blot immunosorbent assay (DTBIA) that differentiates Citrus Tristeza Virus (CTV) isolates based on monoclonal antibody MCA13 reactivity. Virulent and decline strains of CTV react in serological tests to MCA13; whereas mild strains typically do not react. A DTBIA test was developed by ARS scientists at Parlier, CA where blotted membranes were exposed first to MCA13 antiserum and then exposed to a broad spectrum CTV antiserum. This user friendly test can assess if sampled trees are infected and if the isolates are MCA13 positive. Using this procedure, the CCCTEA received permission from their Pest Control Districts to adopt this method and remove only potential virulent strains of CTV identified by MCA13 and the real time PCR assay.
3. Validation of Direct Tissue Blot Immunosorbent Assay (DTBIA) as an effective test to detect Citrus Tristeza Virus(CTV) in citrus nurseries and in budwood source trees. A 6 month to 2 year latent period and uneven distribution of initial (CTV) infection is common, hence, a sample in conventional Enzyme-linked immunosorbent assay (ELISA) may not reach the criteria of 2 to 2.5 x healthy control for several years. ARS scientists in Parlier, California found DTBIA (5 to 10 prints per tree) was better than plate ELISA to detect early CTV infection from plots in Ventura, Riverside, and Tulare County, California. All registered budwood source trees in citrus nurseries in California are required to be tested annually for CTV and removed as a source tree if positive. This user friendly test can be performed in part (blotting) or entirely by the nursery operator. The California Citrus Nursery Board will be using this data to request CDFA to certify DTBIA for CTV diagnosis and that results obtained are legally binding.
4. Estimation of citrus stubborn disease incidence. Citrus stubborn disease (CSD) is caused by Spiroplasma citri, a phloem limited prokaryote indigenous in California/Arizona and has some symptom similarity in citrus to Huanglongbing (HLB = greening disease). A high throughput survey was developed by ARS scientists at Parlier, CA where 20% of grove trees were sampled by hierarchical pattern and extracted DNA was assessed for S. citri infection by real time PCR. This is important because HLB surveys are underway in California since the HLB vector, the Asian citrus psyllid, has been found in southern California (San Diego and Imperial Counties). Knowledge of CSD distribution and incidence is important as inspectors are sent to suspect citrus groves to collect samples for HLB testing. Inclusion of S. citri in the panel of probes used in the real time PCR assays for HLB will result in valuable data at no real extra cost for sample collection or processing.
Mello, A.F., Yokomi, R.K., Mulcher, U., Chen, J., Wayadande, A., Fletcher, J. 2008. Genetic Diversity of Spiroplasma citri strains from Different Regions, Hosts, and Isolation Dates. Phytopathology. 98:960-968.