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

Location: Crop Diseases, Pests and Genetics Research

2021 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
In support of Objective 1, ARS scientists in Parlier, California, continued research to characterize Citrus tristeza virus (CTV), Spiroplasma citri, and ‘Candidatus Liberibacter asiaticus” (CLas) collected in California. Ten new CTV quick decline strains were obtained and whole genome sequence studies were initiated. Eight strains of S. citri, causal agent of citrus stubborn disease, were fully sequenced and published. Detection of S. citri and CLas was improved by developing multiplexed real time polymerase chain reaction (qPCR) and droplet digital PCR (ddPCR) assays that combined reaction mixtures of both pathogens to allow simultaneous testing for S. citri and CLas from suspect samples in a single, rather than multiple tests, saving time and resources. The improved detection of citrus pathogens is important to citrus growers and regulators. This research was also supported by agreements 58-2034-8-003 (Citrus Research Board), and 58-2034-0-012 (California Citrus Nursery Board). In further support of Objective 1, ARS scientists in Parlier, California, with a collaborator in Tulare, California, continued to examine the impact of different strains of CTV on different citrus cultivars grafted on the CTV-tolerant rootstock, Carrizo citrange, and grown in-ground in a screenhouse to simulate field conditions. Many strains of CTV were mild (genotypes T30, T36, RB, S1) and did not significantly affect growth or key physiological metabolites (carbohydrates, sugars, amino acids, or phenolic compounds). However, CTV with a VT genotype induced significant reduction in growth and changes in some critical metabolites (e.g. phenolic compounds) depending on the citrus cultivar. Moreover, VT strains induced low to moderate levels of stem pitting which can decrease fruit quality and cause branches to become brittle and break easily. The impact of CTV strains on commercial citrus cultivars are important for growers and regulators since CTV is rated as a regulated pathogen by the California Department of Food and Agriculture. This research was also supported by agreement 58-2034-8-003 (Citrus Research Board). In additional support of Objective 1, ARS scientists in Parlier, California, developed a fast (seven minutes), portable assay to detect Huanglongbing (HLB) in crude citrus extracts without need to purify DNA. This method uses Recombinase Polymerase Amplification (RPA) at a constant temperature rather than complicated cyclical temperatures. The resultant amplified product is detected by a specifically labeled fluorescent probe which is detected in a hand-held lateral flow device that allows HLB detection to be performed in the field as a point-of-use assay. The RPA assay yielded results comparable to quantitative real-time PCR to a level of Ct 34. The method is user friendly and can be used by growers and pest control advisors with minimal training and does not require a laboratory or sophisticated equipment. This device is a great tool for citrus farmer to be able to make quick management decisions regarding HLB mitigation. This research was also supported by agreement 58-2034-0-012 (California Citrus Nursery Board). In support of Objective 2, an ARS scientist at Parlier, California, with a collaborator from the University of California, Riverside, assisted in the development of an infectious T36 strain of CTV originally collected in Porterville, California, from Washington Navel in 2010. This was accomplished by the collaborator by substituting key amino acid bases in the CTV genome to suppress gene silencing mechanisms and develop an infectious strain of a California T36 strain. This research is of great interest as a potential new strategy of inserting foreign genes in genetically engineered CTV to express genes in citrus phloem where CLas grows to control HLB. This research was also supported by agreement 58-5302-4-011 (Citrus Research Board).

Accomplishments
1. New technology detects two different citrus pathogens in one test. Huanglongbing (HLB) is the most destructive citrus disease and, in California, is managed by intensive surveys and immediate eradication when detected. However, symptoms of Citrus stubborn disease, an endemic citrus pathogen in California, can easily be mistaken for HLB and complicates HLB eradication efforts. ARS researchers in Parlier, California, developed a duplex real time polymerase chain reaction (PCR) test and a precise duplex droplet digital PCR assay to rapidly differentiate the two pathogens. These methods provide unambiguous quantitation of the pathogen(s), which is critical when target DNA is low in concentration, and is critically important in regulatory programs involving mandatory eradication.

2. Chromosome organization of Spiroplasma citri strains and number of plasmids. Spiroplasmas represent a complex of bacterial endophytes in diverse hosts/habitats including plants, insects, ticks, crustaceans and vertebrates and have host interactions ranging from symbiotic, commensal, and/or pathogenic. Spiroplasma citri is the causal agent of citrus stubborn disease, which causes significant damage to citrus trees and can result in reduced fruit production and quality. Spiroplasma citri strains are similar in chromosome organization but greatly differ in number of plasmids. ARS researchers in Parlier, California, found that Spiroplasma citri has remarkably similar chromosome organization among diverse S. citri strains with gene rearrangements predominantly in the center of the chromosome. The greatest genetic difference found was the number of extra chromosomal DNA (presumptive plasmids) which are involved in host range expansion and leafhopper transmission. This information is of scientific importance to epidemiologists and pathologists dealing with Spiroplasma-host interactions as well as diagnosticians developing sensitive pathogen detection methods.

Review Publications
Rattner, R., Thapa, S.P., Dang, T., Osman, F., Selvaraj, V., Maheshwari, Y., Pagliaccia, D., Espindola, A.S., Hajeri, S., Chen, J., Coaker, G., Vidalakis, G., Yokomi, R.K. 2021. Genome analysis of Spiroplasma citri strains from different host plants and its leafhopper vectors. BMC Genomics. 22:373. https://doi.org/10.1186/s12864-021-07637-8.
Maheshwari, Y., Sevaraj, V., Godfrey, K., Hajeri, S., Yokomi, R.K. 2021. Multiplex detection of “Candidatus Liberibacter asiaticus” and Spiroplasma citri by qPCR and droplet digital PCR. PLoS ONE. 16(3):e0242392. https://doi.org/10.1371/journal.pone.0242392.