The three pathogens in this Project Plan cause diseases in citrus ranging in severity from tree death; greatly reduced fruit quality and production; and differential symptoms depending on host cultivar and pathogen strains. The pathogens are CLas; presumed causal agent of Huanglongbing (HLB) (aka citrus greening); CTV, causal agent of tristeza quick decline (QD) and stem pitting (SP); and S. citri, causal agent of citrus stubborn disease (CSD). Key characteristics shared among these pathogens include: i) phloem-association; ii) transmission by phloem-feeding hemipterans; iii) graft-transmissible; iv) incurable by practical means; and v) management practices include removal of infected trees to reduce inoculum and limit pathogen spread by insect vectors. This project plan is designed to evaluate mixed infections of these pathogens on citrus production and pathogen diagnosis, and explore improved management of these diseases. To manage HLB, research will explore using a modified genome of a mild California recombinant CTV (CA-rCTV) isolate to deliver antimicrobial peptides (AMPs) and induce RNA interference (RNAi) against CLas and ACP, respectively, in inoculated plants. Information from this research will assist regulatory agencies, growers, diagnostic laboratories, and integrated pest management practitioners to significantly improve citrus disease management. This research also will improve disease diagnostics and lead to a better understanding of CTV cross-protection. During the next five years, focus will be on the following Objectives and Sub-objectives. Objective 1: Identify the genetic diversity of ‘Candidatus Liberibacter asiaticus’, Citrus tristeza virus, and Spiroplasma citri in California and their impact on epidemiology, disease synergism, cross protection, and diagnosis. • Subobjective 1A: Capture pathogen targets from field trees and/or insect vectors. • Subobjective 1B: Examine phenotypes of California strains of CTV, S. citri, and CLas and improve pathogen detection. • Subobjective 1C: Examine cross-protection within and between genotypes of CTV. • Subobjective 1D: Examine if disease synergism occurs when CLas co-infects citrus with CTV and/or S. citri. Objective 2: Develop and evaluate the potential of mild California Citrus tristeza virus expression vectors for delivery of antimicrobial peptides and RNAi for control of HLB and its respective psyllid vector. • Subobjective 2A: Develop a CA-rCTV infectious cDNA clone. • Subobjective 2B: Examine phenotype and stability of the CA-rCTV singly and in mixed infections with California wild-type CTV isolates. • Subobjective 2C: Incorporate AMPs against CLas and RNAi against ACP in the CA-rCTV vector. • Subobjective 2D: Develop the CA-rCTV as a virus-induced gene silencing (VIGS) vector to downregulate citrus host genes to ameliorate HLB disease symptoms.
Objective 1: The hypothesis to be tested is that CTV, S. citri and CLas, which inhabit the same tissue in citrus, will interact when doubly or triply infected and the disease phenotype will be affected. It is unknown if the result will be neutral, synergistic or cross-protective. Field citrus trees in California will be tested by Enzyme-linked immunosorbent assay (ELISA), lateral flow immunoassay (LFIA), polymerase chain reaction (PCR), real-time PCR (qPCR) or loop-mediated isothermal amplification (LAMP) to diagnose infected trees. Pathogens identified from these assays will be graft propagated into different potted citrus cultivars and grown in the greenhouse to examine symptoms to determine disease phenotype. Inter and intra level competition between pathogens or their strains will be examined by symptom expression, pathogen titer determined by qPCR and droplet digital PCR (ddPCR). Vector transmission profiles (acquisition, latent period, transmission) will be determined for vectors exposed to coinfected plants: aphids for CTV, leafhoppers for S. citri, and psyllids for CLas. Genetic diversity will be determined by PCR, Reverse Transcription PCR, TaqMan probe assays, cloning, sequencing as well as Next Generation Sequencing. Sequences from unique conserved gene regions will be selected and strain specific primers and probes developed for PCR detection. Objective 2: The hypothesis is that an infectious recombinant CTV can be used as a transient expression vector to express foreign therapeutic genes in citrus against HLB and/or the Asian citrus psyllid (ACP). This strategy can be used to express antimicrobial peptides and RNA interference (RNAi) constructs to manage or control HLB and/or ACP in existing citrus without the need of transgenic citrus or replacing trees. Recombinant DNA technology will be used to develop full length infectious cloned DNA (cDNA) to California strains of CTV. Gene replacement of a Florida rCTV will be performed in step-wise fashion from the 3’UTR to the 5’UTR region with genes from a mild California CTV isolate. In case common restriction sites are not found between isolates, other restriction sites will be explored or PCR fragments will be amplified by overlap-PCR. The full length cDNA clone of California recombinant CTV will be sequenced to confirm accuracy. Clones will be transformed into Agrobacterium tumefaciens and incorporated with silencing suppressors and agroinfiltrated into Nicotiana benthamiana. Virions produced in tobacco will be harvested, purified and inoculated into citrus to produce citrus systemically infected with rCTV. rCTV can be readily increased by graft propagation to new citrus plants. Infectious California rCTV can then be manipulated by inserting antimicrobial peptides and RNA interfering constructs for the Asian citrus psyllid, vector of CLas. Application of this technology will be to inoculate existing field trees with the rCTV as a biocontrol agent against HLB/ACP without the use of transgenic plants.
This report documents progress for the project 2034-22000-013-00D, which started in April 2017 and continues research from project 2034-22000-011-00D, "Characterization, Epidemiology and Management Strategies of Citrus Tristeza Virus and Spiroplasma citri on Citrus in California." Under Sub-Objective 1A, approximately 5,000 citrus trees were sampled for Citrus tristeza virus (CTV) in four orchards in Tulare County, California. Sixteen percent were found to be infected with CTV. These isolates were further screened with MCA13, a severe strain discriminating monoclonal antibody, and indicated that 6.2% of these trees may be infected with a severe CTV strain; however, no field symptoms or reduction of yield were observed. Under Sub-Objective 1B, the virulence and genetic diversity of selected MCA13-positive CTV isolates were examined by Next Generation Sequencing (NGS) to further differentiate each isolate’s specific genotype. These isolates were then inoculated in six citrus cultivars on Carrizo citrange rootstock in a quarantine screenhouse to evaluate virulence by monitoring visual symptoms and measuring plant biomass. Under Sub-Objective 1C, cross-protection CTV was observed when the CTV RB strain reduced or prevented systemic infection of a T36 strain of CTV but not with other CTV genotypes. This is new information since cross-protection was previously thought to occur within the same genotype, not between different genotypes. Under Sub-Objective 2A, progress was made toward developing an infectious clone of an RB strain of CTV that would make an improved expression vector to mitigate huanglongbing (HLB) by expressing foreign genes to combat HLB in trifoliate rootstock, the most popular citrus rootstock in the U.S.
Maheshwari, Y., Selvaraj, V., Hajeri, S., Ramadugu, C., Keremane, M.L., Yokomi, R.K. 2017. Novel development of a lateral flow immunoassay for rapid field detection of citrus tristeza virus. Molecular Biotechnology. doi: 10.1007/s12600-017-0591-0.