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

Location: Crop Diseases, Pests and Genetics Research

2019 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
Under Sub-objective 1A, ARS researchers in Parlier, California, continued to evaluate spread of Citrus tristeza virus (CTV) strains that reacts to MCA13, a strain-discriminating antibody that detects more virulent strains of CTV. The overall incidence of CTV in MCA13-abatement plots was 5.9 percent with a spread rate 0.2 percent per year between 2008 and 2018 and only two MCA13-positive trees were detected and removed during this 10-year period. In comparison, overall CTV incidence in non-abatement plots over a three-year period (2015 to 2018) was 18.6 percent. MCA13-reactive CTV spreads at a rate of 1.6 percent/per year and comprised 43 percent of the CTV detected. Since trees infected with MCA13-reactive CTV strains are removed when detected, these data suggest roguing of MCA13-reactive CTV reduced the regional inoculum supply resulting in significantly lower rates of spread in abatement orchards compared to that in non-abatement orchards. This research was also supported by agreements 58-2034-5-026 (Tulare County Pest Control District) and 58-2034-8-003 (Citrus Research Board) and supports the California Department of Food and Agriculture (CDFA) Interior Quarantine for CTV. Under Sub-objective 1A, ARS researchers in Parlier, California, continued to evaluate economic impact of CTV collected from local citrus orchards and conducted biological indexing in a greenhouse with the following result: genotypes with T30, T36, S1, and RB were mild; whereas Vermont strains were severe inducing seedling yellows and some stem pitting. Six commercial citrus cultivars grafted on Carrizo rootstock were inoculated with some isolates and planted in a protected screenhouse to simulate field conditions. After three years, VT strains produced mild stunting, some seedling yellows, and a few trees with a low level of stem pitting. Foliar growth, however, was not very different than trees infected with mild strains. Moreover, plant phenolic levels were not appreciably different in response to CTV strains. The results of CTV effects on citrus in screenhouse assays are similar to those observed by growers in orchards. This research was also supported by agreements 58-2034-5-026 (Tulare County Pest Control District) and 58-2034-8-003 (Citrus Research Board). In support of Sub-objective 1C, ARS researchers continued to monitor mixed infections with California strains T30, T36, VT, RB and S1 with T36-GE-CTV from Florida. T30, VT, and S1 did not cross protect against co-infection of T36-GE-CTV but T36 did cross-protect. This was expected because the definition of CTV cross-protection is super infection exclusion by a strain of the same genetic genotype. The RB strain, however, significantly limited co-infection and expression of T36-GE-CTV. Since these strains have different genotypes, it appears that RB is outcompeting T36-GE-CTV in co-infected plants. These data suggest T36-based GE-CTV used as an expression vector to express antimicrobial peptides and RNA Interference constructs in existing citrus plantings may be limited by pre-existing natural infection by T36 and RB CTV strains. This research was also supported by agreement 58-2034-6-010 (Citrus Research Board). In support of Sub-objective 1D, ARS researchers developed duplex real-time Polymerase Chain Reaction (qPCR) and droplet digital PCR (ddPCR) assays to facilitate simultaneous detection of the bacterial pathogens “Candidatus Liberibacter asiaticus and Spiroplasma citri, causal agents of Huanglongbing (HLB) and citrus stubborn disease (CSD), respectively. This test replaces two singleplex assays to detect these pathogens, hence, reducing diagnostic costs and processing time. The duplex qPCR assays are now being used to examine interaction and coinfection of CLas and S. citri in the same citrus plant and has revealed some protection in establishing CLas infection if S. citri is present and vice-versa. Since pathogenicity of S. citri is low in comparison to CLas, it may be worth exploring if S. citri can serve as a protecting agent against CLas infection. This research is also supported by agreement 58-2034-7-013 (Citrus Research Board). Sub-objective 2A, ARS researchers in Parlier, California, continued a collaboration with researchers at University of California, Riverside, to develop California mild strain of Citrus tristeza virus (CTV) as an infectious CTV vector to express antimicrobial peptides (AMPs) and RNA Interference constructs in citrus to help control Huanglongbing (HLB) and the Asian citrus psyllid (ACP). Complexity of sequences at the terminal ends of the CTV genome is an impediment to developing infectious complementary (c) DNA clones of CTV. To this end, heterogeneity of T30-CA and T36-CA termini were determined, facilitating cloning and sequencing T36-CA and T30-CA. This research is key to ongoing studies aimed at engineering a stable CTV-based vector for molecular biology applications. This research was also supported by agreement 2034-22000-013-03R (Citrus Research Board).

Accomplishments
1. Huanglongbing in Southern California have different origins. “Candidatus Liberibacter asiaticus” (CLas) is a bacterium associated with Huanglongbing (HLB) and was first detected in Florida in 2005 and in California in 2012. HLB has now devastated citrus throughout Florida and has now been detected in over 1,494 infected citrus trees in urban southern California properties. The only control for HLB is removal of infected trees or repeated insecticide application to kill Asian Citrus Psyllid (ACP), the insect that is a vector for HLB, is not economically nor environmentally or biologically sustainable. ARS researchers in Parlier, California, along with scientists from California Department of Food and Agriculture, Sacramento, California; USDA Animal and Plant Health Inspection Service Plant Protection and Quarantine, Beltsville, Maryland, and South China Agricultural University, Guangzhou, Guangdong, China, examined genomic diversity of 10 CLas strains from southern California by Next Generation Sequencing (NGS) and found diverse prophages were detected in all CLas strains. The California strains formed four prophage groups associated with different collection sites and were more closely related to strains in Asia, rather than Florida which indicated CLas pathway of entry was from independent introductions at multiple times. The information is important to formulate grower HLB management strategies for different pathogen strains and for regulatory agencies to prioritize and optimize pathogen interdiction surveys.

2. Rapid and field deployable diagnosis of citrus stubborn disease. Citrus stubborn disease (CSD) is caused by the bacterium Spiroplasma (S.) citri which causes stunted and low yielding citrus trees. S. citri has a wide host range including ornamentals, cruciferous crops and weeds as well as citrus. CSD is present in temperate and semi-arid citrus-growing regions where its leafhopper vector thrives. ARS researchers in Parlier, California, along with scientists from CIHEAM - Istituto Agronomico Mediterraneo, Valenzano, Italy and Tuscia University, Viterbo, Italy developed a Loop-Mediated Isothermal Amplification (LAMP) technique using spiralin primer sets that efficiently detected S. citri from California, Algeria, Turkey and Syria. Although the LAMP technique was nine-fold less sensitive than real-time polymerase chain reaction (PCR), it was effective in detecting S. citri-infected trees in the citrus orchards tested. The Android-based LAMP device is field deployable and simple to use, thus, enabling growers, pest control or diagnostic service providers to rapidly test for S. citri in the field without DNA purification and providing a tool for disease management.

3. Temporal and spatial distribution of Citrus tristeza virus is suppressed by disease management. Citrus tristeza virus (CTV) is one of the most destructive viruses of citrus and is transmitted in nature by aphids and all citrus varieties are susceptible. CTV is comprised of genetically diverse population of variants and strains that are characterized by symptoms in specific citrus cultivars and rootstocks. Because CTV is a regulated pathogen in California, ARS researchers in Parlier, California, along with a scientist from the Central California Tristeza Eradication Agency, Tulare, California, examined CTV spread in plots with abatement (removal) of strain-selective monoclonal antibody MCA13-reactive CTV strains and mandatory aphid control versus plots in an adjacent non-abatement CTV district under an insect control program required for fresh citrus export to Korea (NAVEK). The overall incidence of CTV in the abatement plots was 5.9% with a spread rate 0.2 percent over a 10-year period from 2008 to 2018 versus an incidence of 18.6 percent and a spread rate of 1.6 percent in non-abatement plots over a three-year period from 2015 to 2018. MCA13-reactive CTV in non-abatement plots comprised 43 percent of the CTV detected. Data suggested roguing of MCA13 positive CTV has reduced the regional inoculum supply, resulting in significantly lower rates of spread among orchards compared to that of MCA13-negative CTV.

4. Rapid and field deployable diagnosis of VT strains of Citrus tristeza virus. Citrus tristeza virus (CTV) is one of the most destructive viruses of citrus and is transmitted by aphids and all citrus varieties are susceptible. Virulent CTV strains induce stem pitting in twigs, stems, branches and trunk regardless of rootstock, and weakens the citrus tree, resulting in a significant reduction of fruit quality and production. VT strains have much greater sequence variation at the 5’ end of its genome than non-VT strains, which typically have a mild phenotype. ARS researchers in Parlier, California, along with a scientist from the Central California Tristeza Eradication Agency, Tulare, California, developed a Loop-Mediated Isothermal Amplification (LAMP) technique to rapidly detect VT strains of CTV. This procedure was supplemented with an initial immunocapture step that eliminated the need for nucleic acid purification while homogenizing leaf samples to trap CTV which were then directly used in the LAMP assay. This method can be used by growers and diagnostic service providers with a field deployable Android-based LAMP device to test for presence of a VT strain in citrus trees, which then can be targeted for roguing by the grower or regulatory agency.

5. Absolute detection of “Candidatus Liberibacter asiaticus” in low titer samples. “Candidatus Liberibacter asiaticus” (CLas), vectored by the Asian citrus psyllid, is the bacterium associated with devastating citrus disease, Huanglongbing (HLB). Eradication of CLas inoculum sources is the first line of defense against spread of HLB but erratic distribution and low titer of the pathogen in citrus limits efficacy of diagnosis using real-time PCR and the regulatory detection target, 16S rRNA gene of CLas. ARS researchers in Parlier, California, along with a scientist in the Central California Tristeza Eradication Agency, Tulare, California developed duplex real-time polymerase chain reaction (PCR) and droplet digital PCR tests for absolute detection of low titer CLas samples utilizing two different genetic targets (16S and RNR) in the chromosome of CLas. Sensitivity of the duplex assay was shown to be down to 2 DNA copies per typical PCR reaction volume. Using low titer (marginally positive) samples of CLas in leaf tissue and ACP, detection of CLas was greater with the RNR than the 16S gene target. Therefore, the duplex ddPCR described can be used to confirm qPCR results have high or marginal Ct values by the regulatory standard 16S target.

6. Complete genome sequence of Citrus tristeza virus strains T36-CA and T30-CA. Infectious cDNA clones of Citrus tristeza virus (CTV) has been shown to be a stable viral vector to express foreign genes into citrus to help control Huanglongbing (HLB) associated with the bacterium, “Candidatus Liberibacter asiaticus” and its vector, the Asian citrus psyllid. This strategy is of interest to citrus growers in California to help mitigate HLB disease. ARS researchers in Parlier, California, along with scientists at the University of California, Riverside, determined complexity of sequences at the termini of California CTV strains T36-CA and T30-CA which facilitated establishing full-length genome sequences and successful cloning of these two strains. Although systemic infection of cDNA from T36-CA and T30-CA was established in tobacco, these GE-CTV has not been successfully established in citrus. This research supports ongoing studies aimed at engineering a stable CTV-based vector for molecular biology applications.

Review Publications
Dai, Z., Wu, F., Zheng, Z., Yokomi, R.K., Kumagai, L., Cai, W., Rasco, J., Polek, M., Deng, Z., Chen, J. 2019. Prophage diversity of “Candidatus Liberibacter asiaticus” strains in California. Phytopathology. 109(4):551-559. https://doi.org/10.1094/PHYTO-06-18-0185-R.
Drais, M.D., Maheshwari, Y., Selvaraj, V., Varvaro, L., Yokomi, R.K., Djelouah, K. 2019. Development and validation of a Loop-Mediated Isothermal Amplification technique (LAMP) for the detection of Spiroplasma citri, the causal agent of citrus stubborn disease. European Journal of Plant Pathology. 155(1):125-134. https://doi.org/10.1007/s10658-019-01755-6.
Chen, A.Y., Watanabe, S., Yokomi, R.K., Ng, J.C. 2018. Nucleotide heterogeneity at the terminal ends of the genomes of two California Citrus tristeza virus strains and their complete genome sequence analysis. Virology Journal. 15(1):141. https://doi.org/10.1186/s12985-018-1041-4.
Saponari, M., Giampetruzzi, A., Selvaraj, V., Maheshwari, Y., Yokomi, R.K. 2019. Identification and characterization of RB isolates of citrus tristeza virus. In: Catara A., Bar-Joseph M., Licciardello G., editors. Citrus Tristeza Virus. Methods in Molecular Biology. New York, NY: Humana Press. p. 105-126.
Yokomi, R.K. 2019. CTV vectors and interactions with the virus and host plants. In: Catara, A., Bar-Joseph, M., Licciardello, G., editors. Citrus Tristeza Virus. Methods in Molecular Biology. New York, NY: Humana Press. p. 29-53.