CHARACTERIZATION, EPIDEMIOLOGY AND MANAGEMENT STRATEGIES OF CITRUS TRISTEZA VIRUS AND SPIROPLASMA CITRI ON CITRUS IN CALIFORNIA

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. 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. This is the final report for project 2034-22000-011-00D, "Characterization, Epidemiology and Management Strategies of Citrus tristeza virus and Spiroplasma citri on Citrus in California," which has been replaced by new project 2034-22000D-013-00D, "Characterization and Management of Citrus Pathogens Transmitted by Phloem-Feeding Insect Vectors." For additional information, see the new project report. Under Objective 1, to improve rapid diagnosis of tristeza and stubborn diseases, a user-friendly, field deployable, lateral flow immunostrip (dipstick) test was developed for direct capture of Citrus tristeza virus (CTV) in crude plant extract without need for purification and visualization as a band (colloidal gold particles) at a specific position on the dipstick. Detection of Spiroplasma citri, the microbial pathogen that causes citrus stubborn disease, was improved 1000x from the previously used housekeeping gene of S. citri by targeting a multi-copy gene of the Spiroplasma citri Virus 1 of S. citri in Real Time Polymerase Chain Reaction (qPCR). Because all of these methods provide a relative measure of the population levels of the pathogen in diseased tissues, a droplet digital PCR (ddPCR) procedure was developed for absolute quantitation of CTV and S. citri in the tissue sampled without having to include dilutions standards of the target. These data collectively improved citrus pathogen diagnostics and the ability to evaluate impact of the pathogens on citrus health. Under Objective 2, genetic diversity of CTV was characterized by assembling the full-length genome of several CTV isolates which revealed that the Resistance-Breaking (RB) and a Western sub-group of the VT genotypes of CTV were present in California. CTV RB strains are known to break resistance to CTV in trifoliate orange which has been used as a rootstock and control measure for CTV. Western strains of VT genotype CTV are associated with increased virulence. Hence, these data revealed increased pest status of CTV. However, sequencing also led to the discovery of a new CTV genotype called S1 which was mild and found to be a component of many California CTV isolates and may have potential to ameliorate symptoms of more virulent CTV strains in mixed infections. Separate isolations of S. citri from stubborn infected citrus trees and from its leafhopper vector, were nearly identical with regard to many of the genes used for diagnosis (e.g. spiralin, p58, p89, SPV1 ORF1 and ORF3), hence, any of these genes could be used to monitor S. citri infection in citrus or leafhoppers. Under Objective 3, to examine CTV cross-protection, citrus preinfected with a CTV RB isolate prevented systemic infection of a CTV T36 genotype. This is relevant because previously, CTV cross-protection was thought to result from superinfection exclusion amongst CTV isolates in the same genotype. Characterization of virulent and cross-protective CTV isolates from Peru revealed presence of RB isolates in cross-protected Star Ruby grapefruit trees in Peru and these RB isolates were found to be similar in sequence to those in California. Hence, potentially cross-protective CTV strains against stem pitting CTV may already exist in California. These results are adding to the understanding of how biologically-based integrated disease management by CTV cross-protection may influence disease expression. Accomplishments 01 Biology of RB, S1, and VT genotype isolates of citrus tristeza virus (CTV) in California. CTV-infected citrus trees in CTV suppressive districts are eradicated in commercial citrus regions in central California if they test positive to a presumptive severe-strain discriminating CTV monoclonal antibody, MCA13. A recent increase of MCA13-reactive CTV isolates without concomitant virulent disease symptoms or decreased yields has occurred. ARS researchers in Parlier, California, with the collaboration of the Central California Tristeza Eradication Agency, Tulare, California, tested CTV isolates from central California and found many different strains including RB, S1, and VT reacting to MCA13. RB and S1 isolates were mild in biocharacterization tests, whereas VT strains were severe and induced stem pitting and seedling yellows symptoms in citrus. These data showed that screening for virulent CTV isolates by MCA13 serology is not reliable and would eliminate mild isolates that could be sources for cross-protection. MCA13 tests can be supplemented by molecular tests developed in this research program to avoid needless removal of CTV- infected trees and potential mild, cross-protecting CTV strains. 02 Identification and distribution of the S1 CTV genotype. Phylogeny of full length genomes of citrus tristeza virus (CTV) differentiates CTV worldwide into seven genotypes. ARS researchers in Parlier, California, in collaboration with the Institute for Sustainable Plant Protection, National Research Council, Bari, Italy, and the Department of Soil Plant and Food Science, University of Bari, Bari, Italy, discovered a new (eighth) CTV genotype (S1) in central California and developed a TaqMan-based probe to rapidly identify this genotype. Comparison of the CTV genomes in the public database indicated that S1 isolates may also be present in Europe and Asia. Direct samples tested using the S1 probe verified that the S1 genotype occurs in Pakistan and Peru. These data demonstrate the value of genome sequencing and bioinformatics to better understand the etiology, genetic diversity, and disease expression of CTV. 03 Detection of Spiroplasma citri and Candidatus Liberibacter asiaticus (CLas) by droplet digital polymerase chain reaction (ddPCR). Citrus stubborn disease (CSD), caused by Spiroplasma (S.) citri, is endemic in California; whereas huanglongbing (HLB), associated with Candidatus Liberibacter asiaticus (CLas), has been detected in ~70+ citrus trees in residential portions of southern California (Los Angeles, Orange, and Riverside counties) but not in any commercial orchards. ARS researchers in Parlier, California, developed a sensitive procedure to identify and quantitate S. citri and CLas using a specialized ddPCR test which partitions a test sample into approximately 20,000 individual droplets. Each droplet undergoes a separate end point amplification by PCR and results in an exact count of DNA target copies in a sample without need of a standard curve. The major advantage of ddPCR over real-time quantitative PCR is its ability to accurately resolve rare events and access targets at low template concentrations despite presence of PCR inhibitors. The ddPCR test for S. citri and CLas was developed as a duplex test since CSD and HLB share many similar symptoms and can be confused with each other. Hence, this test procedure provides a robust method to test the status of symptomatic and non-symptomatic citrus samples for S. citri and CLas in one simultaneous test. 04 Citrus tristeza virus-Resistance-Breaking (RB) genotype interaction with a CTV-T36 genotype. Cross-protection of citrus tristeza virus (CTV) has recently been characterized as superinfection exclusion amongst isolates in the same genotype but not between CTV genotypes. ARS researchers in Parlier, California, found that preinfection of citrus by an RB isolate cross-protected against a T36 genotype but not with isolates from other genotypes (VT, S1, or T30) tested. These results suggested cross-protection can result from CTV isolates from different genotypes in a mixture rather than exclusively from within a single genotype. In addition, they provided new insight on interactions of CTV strains on disease expression.