1a. Objectives (from AD-416):
Citrus tristeza virus and Spiroplasma citri, causal agent of citrus stubborn disease, are important production-limiting, insect-vectored pathogens in California. Early host response (e.g. over-expressed microRNAs and small interfering RNAs) associated with single and multiple strain graft inoculations of these pathogens in greenhouse tests will be characterized and developed as biomarkers for early-stage pathogen-specific detection. The overall goal is to elucidate mechanism of mild strain cross-protection of Citrus tristeza virus and pathogen genetic bottlenecks resulting from insect vector transmission. New knowledge gained will lead to improved sustainable management strategies for these citrus disease agents and their insect vectors. 1. Develop field deployable systems that provide rapid, sensitive detection of Citrus tristeza virus and Spiroplasma citri in citrus. 2. Determine and characterize genetic variations in Citrus tristeza virus and Spiroplasma citri strains before and after vector passage. 3. Determine changes in host gene expression in Citrus tristeza virus cross-protected citrus that can be used to screen for cross-protective strains of Citrus tristeza virus.
1b. Approach (from AD-416):
Citrus tristeza virus (CTV) and Spiroplasma citri are both phloem-limited pathogens of citrus and are transmitted by insect vectors. The California Department of Food and Agriculture and stakeholders manage CTV in commercial groves and urban areas by an eradication program and maintenance of pathogen-free budwood sources and citrus propagations in commercial nurseries. CTV eradication was modified in 2009 to detect and eliminate only citrus trees infected with severe strains of CTV. This is achieved by screening field strains of CTV by serology with a monoclonal antibody and polymerase chain reaction (PCR) assays with genotype-specific sequence markers. Genetic diversity of field strains CTV and S. citri will be characterized with respect to the molecular and genetic basis of their host-pathogen-vector relations and disease epidemiology. Further improvement in pathogen surveillance and control requires filling knowledge gaps in host response to pathogen infection as mild CTV strains, which no longer are being eradicated, continue to spread by indigenous aphid vectors. The nature, basis and mechanism(s) of cross-protection will be identified and characterized. CTV symptom phenotype with mixed infections will be monitored for post-translational gene silencing through analysis and characterization of small interfering RNAs, micro RNAs and macro RNAs. New information will facilitate mitigation of losses and misidentifications of CTV and S. citri with the ultimate goal of developing sustainable, integrated management strategies for tristeza and stubborn in California.
3. Progress Report:
Spiroplasma citri, causal agent of citrus stubborn disease, occurs in low concentration in citrus, making polymerase chain reaction (PCR) detection challenging in a multiplex assay for multiple pathogens. This limitation was overcome by identification of multiple-copy bacteriophage genes integrated into the S. citri genome. Several specific bacteriophage sequences were found to occur as 8 to 140 copies per chromosome. PCR primers were designed for detection of these multiple copy genes. PCR assays tested using the new primers successfully identified the S. citri phage in all of the nine S. citri strains tested as well as from citrus field samples from two Central California commercial citrus groves. Detection of S. citri phage sequences was more sensitive than conventional PCR of a single copy gene (spiralin). In the near future, the bacteriophage sequences from S. citri will be used to detect the citrus stubborn disease pathogen in nursery stock by the California Department of Food and Agriculture Certification Program. Control of Citrus tristeza virus (CTV) in Central California is mandated by the state of California using a serological assay based on reactivity with MCA13, a strain-discriminating monoclonal antibody. Project research determined that many San Joaquin Valley CTV strains that react with MCA13 cause only mild disease. Virulent CTV strains could be differentiated by new primers in a real-time polymerase chain reaction (qPCR) assay. A CTV-strain distinguishing multiplex assay was developed to facilitate rapid identification of virulent CTV strains isolated from field samples. The test incorporates specific primers and probes in individual wells in a 96-well plate using a standardized reaction mix. Test kits are undergoing independent evaluation by different laboratories. Test kits includes custom mixes for CTV strain differentiation and have a versatile format to adapt to testing for other citrus pathogens simultaneously in multiplex assay. The Central California Tristeza Eradication Agency will be using this assay for screening high incidence CTV orchards this year. An alarmingly high proportion of virulent CTV strains were detected in spring of 2013 in Central California. CTV eradication is now impractical. An alternative control for severe CTV strains is cross-protection. The Topara Citrus Nursery in Peru has identified mild CTV strains that protect citrus from severe stem pitting strains of CTV after >20 years of natural field challenge. Extracts from field trees infected with 20 CTV isolates from protected and non-protected trees were obtained from the nursery and examined for strain mixtures and genotype profiling. All protected trees contained a genotype (T36NS) which is found in California. Comparison of the molecular profile of severe and mild Peruvian CTV versus California CTV isolates should provide molecular data to assess and prioritize local CTV isolates for cross-protection trials.
1. QuantiGene Luminex-based assay for multiplexed citrus pathogen detection. 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, developed a multiplex hybridization assay which can detect and differentiate between 9 citrus RNA pathogens or strains in one simultaneous test. Citrus pathogens from the Citrus Clonal Protection Program and USDA-ARS at Beltsville, Maryland; Riverside, California; and Parlier, California, were detected accurately using crude plant samples. The assay was robust, user-friendly and cost-effective. The method may be used by the California Department of Food and Agriculture and USDA for citrus disease-free certification programs.
2. Robotic high-throughput extraction procedures for citrus pathogens. Rapid and standardized methods are needed for detection of citrus pathogens for certification and disease management programs. ARS researchers at Parlier, California, in collaboration with scientists at the University of California Riverside, University of Bari in Bari, Italy, and, Consiglio Nazionale Delle Ricerche Bari (National Research Council), adapted an automated procedure for nucleic acid extraction from citrus tissue. Homogenization and reagent concentrations were optimized for pathogens from citrus using a robot with magnetized beads. Resultant samples contained ample pathogen RNA and DNA to allow reliable detection in PCR assays. The new extraction method is being used by the California Citrus Research Board’s Dimitman Laboratory for diagnosis of the pathogen associated with huanglongbing (HLB, also known as citrus greening) as well as the University of California Citrus Clonal Protection Program (CCPP) in Riverside, California for citrus nursery pathogen free budwood certification.
3. Improved detection of Spiroplasma citri in citrus samples. Examination of S. citri genomes revealed that the bacterium harbors multiple copies of prophage genes. Two primer sets (Php-orf1 and Php-orf3) were developed from prophage sequences and used with a cyanine dye (SYBR-Green)-based real-time polymerase chain reaction to evaluate detection sensitivity with 18 S. citri cultures isolated from different hosts. Compared with primer sets based on single-copy genes, the prophage primer set increased detection sensitivity by approximately 1.8 to 4.9 log units. Significant improvement of detection sensitivity was shown for 252 field samples from two citrus orchards in Central California from 2007 to 2011. Validation for the assay will lead to certification and routine use prophage primer sets for detection of S. citri. The increased sensitivity of the assay will benefit citrus nursery certification programs.
4. The beet leafhopper is a single dispersed population in Central California. The beet leafhopper is an important vector of curtovirues, spiroplasmas and phytoplasmas associated with disease agents of curly top, citrus stubborn and potato purple top, respectively. Because these pathogens are from different pathogen taxa, a question arises if different genetic populations of the leafhopper exist which may be predisposed to a specific vector-pathogen association. Circulifer tenellus virus 1 (CiTV1), a dsRNA virus associated with the beet leafhopper, was isolated from 33% of the beet leafhoppers collected from 35 locations in spring and fall along a 100 mile corridor of Highway 99 (central region) and Interstate 5 (western region) in Central California. Incidence and sequence comparison among three CiTV1 strains (identified on the basis of the CiTV1 RdRp gene) indicated a lack of spatial or temporal population structure of the insect virus. This suggests that CiTV1 represents a single population and, similarly, infers only one population of beet leafhopper in Central California. This information is relevant for beet leafhopper control strategies directed at targeting specific populations or ecological habitats of the leafhopper vector as implemented by the State of California.
Loconsole, G., Onelge, N., Potere, O., Giampetruzzi, A., Bozan, O., Satar, S., De Stradis, A., Savino, V., Yokomi, R.K., Saponari, M. 2012. Identification and characterization of Citrus yellow vein clearing virus, a putative new member of the genus Mandarivirus infecting Citrus spp. Phytopathology. 102: 1168-1175.