Research Project: Characterization and Management of Citrus Pathogens Transmitted by Phloem-Feeding Insect Vectors

Location: Crop Diseases, Pests and Genetics Research

2022 Annual Report

Objectives
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.

Approach
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.

Progress Report
This is the final report for project 2022-21000-013-000D, Characterization and Management of Citrus Pathogens Transmitted by Phloem-Feeding Insect Vectors. This project completed National Program 303 Office of Scientific Quality Review and has been replaced by new project 2034-22000-015-000D, Identifying Vulnerabilities in Vector-host-pathogen Interactions of Grapevine and Citrus Pathosystems to Advance Sustainable Management Strategies. Under Sub-objectives 1A, 1B, and 1C, ARS researchers in Parlier, California, monitored spread of citrus tristeza virus (CTV) in over 5000 citrus trees in four commercial orchards where CTV eradication was abandoned after 1995, with a CTV spread rate in non-abatement plots of 0.2% per year and overall CTV incidence of 5.9%. Test of CTV isolates using MCA13, a strain-discriminating antibody that detects more virulent (VT) strains of CTV, showed that MCA13-reactive CTV strains spread at a rate of 1.6%/per year and comprised 43% of the CTV populations. The MCA13-reactive strains were characterized molecularly, and most were found to be VT strains. Although VT strains are often phenotypically associated with virulent stem pitting strains of CTV that cannot be controlled by rootstock, the field trees remained asymptomatic with normal yields. Subsequent metabolite studies with citrus cultivars infected by mild and severe strains of CTV revealed that individual amino acids, terpenoids, or phenolics successfully linked leaf samples to specific citrus varieties and identified infection status with good accuracy. Collectively, this study indicated biochemical patterns associated with severity of CTV infections that can potentially be utilized to help identify in-field CTV infections of economic relevance. This research was supported in part by Agreements 58-2034-5-026 and 58-2034-8-003. Under Sub-objective 1B, ARS researchers examined the genetic diversity of CTV and Spiroplasma citri, causal agent of citrus stubborn disease. Whole genome sequences of eight isolates of CTV were ascertained including discovery of two isolates of S1, a new CTV genotype, and three new Resistance-Breaking (RB) isolates and their mild phenotypes. Seven strains of S. citri, isolated from various hosts at different times were fully sequenced and results showed generally low sequence variations among strains with some sequence rearrangements predominantly in the center of the chromosome. In addition, one to nine plasmids were identified in the extrachromosomal DNA and were associated with expanded host range and vector transmissibility of the bacterium. Improved detection of CTV VT strains was accomplished as well as absolute quantification of pathogens by droplet digital polymerase chain reaction and recombinase polymerase amplification and lateral flow detection of ‘Candidatus Liberibacter asiaticus’ (CLas). Under Sub-objective 1C, ARS researchers sequenced mild CTV cross-protective strains and found RB strains in productive star ruby grapefruit trees in Peru, where virulent stem pitting CTV and the brown citrus aphid, Toxoptera citricida, are prevalent, which requires use of mild strain CTV cross protection as the only way to economically produce grapefruit. Our discovery of RB strains in local citrus suggests that California may have excellent CTV cross protection against virulent stem pitting. Under Sub-objective 1D, ARS researchers showed pre-infection of citrus with S. citri resulted in very limited graft transmission of CLas, suggesting pathogen interaction may slow spread of one or both bacterial pathogens. However, when co-infection occurred, the citrus plant died. Under Sub-objective 2A, ARS researchers selected the T30 and T36 genotypes of CTV and characterized their mildness in a citrus host range test, which allowed sequencing their whole genomes through collaboration with the University of California-Riverside, in order to bioengineer CTV for use as virus vector (CTVvv). Once developed, CTVvv could include genes coding for CLas antimicrobial and RNA interference (RNAi) molecules to control Huanglongbing and the Asian citrus psyllid (Diaphornia citri), which vectors CLas. Under Sub-objective 2B, ARS researchers working with collaborator from University of Florida-Lake Alfred, inoculated California wild type CTV strains (T30, T36, RB, S1 and VT) with CTVvv T36-FL. T36-FL readily co-infected T30, S1 and VT but not T36 and was limited by RB. Ample expression of GFP and phytoene desaturase activity from T36-FL showed stability of this CTVvv in T30, S1, and VT. Superinfection exclusion prevented co-infection of T36-FL and wild type California T36. T36 may be responsible for RB strain competition resulting in limited expression of the T36-FL viral vector. This research shows the complexities that may be expected in the use of CTVvv expression in commercial citrus previously infected with endemic strains of CTV. Under Sub-objectives 2C and 2D, ARS researchers in Parlier, California, continued efforts to express antimicrobial peptides (AMPs) in citrus tristeza virus (CTV) to control CLas and induce RNA interference (RNAi) against the Asian citrus psyllid insect vector. These efforts will contribute to novel approaches for controlling Huanglongbing disease in citrus.

Accomplishments
1. Improved regulatory sampling for Huanglongbing (HLB) infection. HLB is a devastating disease of citrus associated with the bacterium Candidatus Liberibacter asiaticus (CLas), which is efficiently transmitted by the Asian citrus psyllid and has been found in urban settings in southern California. The first line of defense to control spread of HLB is to eradicate infected trees. ARS researchers at Parlier, California, working with California Department of Food and Agriculture and Central California Tristeza Eradication Agency, improved detection of CLas by sampling peduncles for consistent isolation and detection of CLas DNA by real-time polymerase chain reaction. Improvement of CLas detection and rapid eradication of infected trees reduces CLas spread.

2. Incidence of Citrus tristeza virus (CTV) in commercial citrus in California estimated. Use of Citrus tristeza virus (CTV) virus vector (CTVvv) to express antimicrobial proteins and RNA interference to mitigate Candidatus Liberibacter asiaticus (CLas) and Asian citrus psyllid is a novel method to help control Huanglongbing, a devastating disease of citrus. Genetic engineering of CTV requires recombinant technology to incorporate foreign genes into the CTV genome. Therefore, field release requires development of an environmental assessment and environmental impact statement and approval by USDA Animal and Plant Health Inspection Service, Plant Protection and Quarantine, the U.S. Environmental Protection Agency and the Food and Drug Administration, which regulate plant incorporated protectants. ARS researchers in Parlier, California, conducted surveys of commercial citrus in nine major citrus-growing counties in California, excluding the San Joaquin Valley. The surveys showed that the overall estimate of CTV-infected trees was 27% and was limited to T30 genotype in San Bernardino, Riverside, San Diego and Ventura County. Sweet orange had the highest infection rate followed by grapefruit, and CTV was rare or absent in other cultivars. This information will be used in the environmental assessment and environmental impact statement for field release application for CTVvv in California.

Review Publications
Hajeri, S., Vidalakis, G., Yokomi, R.K. 2021. Detection of viroids using RT-qPCR. In: Rao, A.L.N., Lavagi-Craddock, I., Vidalakis, G., editors. Viroids. Methods in Molecular Biology, vol 2316. New York, NY: Humana. p. 153-162. https://doi.org/10.1007/978-1-0716-1464-8_14.
Yokomi, R.K., Rattner, R., Osman, F., Maheshwari, Y., Selvaraj, V., Pagliaccia, D., Chen, J., Vidalakis, G. 2020. Whole genome sequence of five strains of Spiroplasma citri isolated from different host plants and its leafhopper vector. BMC Research Notes. 13. Article 320. https://doi.org/10.1186/s13104-020-05160-9.
Hajeri S., Yokomi R. 2021. Immunocapture-reverse transcriptase loop-mediated isothermal amplification assay for detection of plant RNA viruses. In: Wang, A., Li, Y., editors. Plant Virology. Methods in Molecular Biology, vol 2400. New York, NY: Humana. p. 245-252. https://doi.org/10.1007/978-1-0716-1835-6_23.
Wallis, C.M., Gorman, Z.J., Rattner, R., Hajeri, S., Yokomi, R.K. 2022. Amino acid, sugar, phenolic, and terpenoid profiles are capable of distinguishing Citrus tristeza virus infection status in citrus cultivars: Grapefruit, lemon, mandarin, and sweet orange. PLoS ONE. 17(5) Article e0268255. https://doi.org/10.1371/journal.pone.0268255.
Rattner, R., Godfrey, K., Hajeri, S., Yokomi, R.K. 2022. An improved Recombinase polymerase amplification coupled with lateral flow assay for rapid field detection of "Candidatus Liberibacter asiaticus". Plant Disease. https://doi.org/10.1094/PDIS-09-21-2098-RE.
Yokomi, R.K., Delgado, J.K., Unruh, T.R., Barcenas, N.M., Garczynski, S.F., Walse, S., Perez de Leon, A.A., Cooper, W.R. 2021. Molecular advances in larval fruit moth identification to facilitate fruit export from western United States under systems approaches. Annals of the Entomological Society of America. 115(1):105-112. https://doi.org/10.1093/aesa/saab040.