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.
3. Progress Report:
Spiroplasma citri, causal agent of citrus stubborn disease, has a wide host range which includes citrus, annual weeds, ornamentals and crops, such as carrots and sesame. Graft transmission to citrus is inefficient and natural pathogen spread to citrus and other hosts is by leafhopper vectors. Only partial gene sequences of S. citri are in the public database which limits genetic comparisons between S. citri populations or strains. DNA libraries from 8 strains of S. citri were prepared and subjected to deep sequencing. Greater than 90% of the genome sequence of each strain was obtained and used to compare sequences from putative functional genomic regions among strains as well as orthologus sequences from other organisms. Gene regions that differed among strains were selected for analysis. These data were used to develop sequence-specific primers (markers) for polymerase chain reaction assays to rapidly detect S. citri and distinguish strains. Studies are now underway to determine genetic relationships between S. citri strains from different plant hosts and leafhopper vectors. A Citrus tristeza virus (CTV) survey, conducted in 2012, at the Lindcove Research and Extension Center (LREC), Exeter, CA, identified 17 CTV-infected trees out of 12,000 sampled (0.14%). Half of the LREC trees were tested for CTV by direct tissue blot immunoassay (DTBIA) and the other half by standard enzyme-link immunosorbent assay (ELISA) to compare cost and efficacy of the two methods. DTBIA was found to be more reliable and economical than ELISA for CTV detection. Rapid and accurate detection is critical because the virus has a long latent period during which the virus is transmissible by aphid vectors. All CTV-infected trees at LREC were removed to eliminate inocula. CTV from all infected trees was characterized for serological reactivity and with molecular markers targeting different CTV genome regions to determine genotype. Although all strains collected were classified as genotypes associated with mild strains, they still pose a threat as LREC houses the UC Citrus Clonal Protection Program which maintains virus-free citrus budwood source trees for the citrus industry. The mild strains collected are being evaluated in greenhouse tests to determine if they confer cross-protection against severe CTV strains.
1. Multiplex citrus pathogen detection assay. Improved methods are needed for sensitive and economic detection of multiple citrus pathogens. ARS researchers at Parlier, California, in collaboration with scientists at University of California Riverside and ARS-Riverside developed a multiplex assay system which can simultaneously detect and differentiate 9 citrus RNA virus pathogens or strains in a single test. The test is based on sequence-specific hybridization to microspheres labeled with pathogen-specific tags that allow signal amplification. The resultant assay was robust, user-friendly and cost-effective for multiple pathogen detection in a high through-put system. The new method is suitable for use in regulatory and certification programs as well as disease diagnosis in pest management programs.
2. Robotic high-throughput extraction procedures for citrus pathogens. Improved methods are needed for sensitive and economic detection of multiple citrus pathogens. ARS researchers at Parlier, California, in collaboration with scientists at UC Riverside developed an automated procedure for nucleic acid extraction from citrus tissue. Homogenization and reagent concentrations were optimized for pathogen target molecule extraction from citrus using a robotic high throughput process providing high quantity and quality nucleic acids. Resulting samples were suitable for use as Polymerized Chain Reaction (PCR) templates. The new extraction method will be used by the Citrus Research Board to process citrus tissue for detection of 'Ca. Liberibacter' associated with huanglongbing disease.
3. Genome sequence analysis of Spiroplasma citri allows strain differentiation. Understanding of the genetic basis of disease severity and leafhopper transmission of S. citri is needed to develop better control measures for citrus stubborn disease. ARS researchers at Parlier, California, in collaboration with scientists at University of Iowa, used deep sequencing to sequence 90% of the genome for each of 8 S. citri strains. Genome comparisons resulted in selection of specific genome regions for strain differentiation and PCR primer design. The resultant PCR assay was used to show presence of genetic variation between laboratory field strains. The genetic diversity detected will be correlated with leafhopper transmissibility, plant host range and disease virulence.
Vidal, E., Yokomi, R.K., Moreno, A., Bertolini, E., Cambra, M. 2012. Calculation of diagnostic parameters of advanced serological and molecular tissue-print methods for detection of Citrus tristeza virus. A model for other plant pathogens. Phytopathology. 102:114-121.