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.
In Sub-objective 1A and agreements 58-2034-5-026 (Tulare County Pest Control District) and 58-2034-8-003 (Citrus Research Board), ARS researchers in Parlier, California, monitored an increase in trees infected with citrus tristeza virus (CTV) isolates that reacts to MCA13, an antibody that identifies virulent strains of CTV, which may result in further spread, increased production losses, and trigger new mitigation efforts. They showed that, in 2018, overall infection level of CTV in three plots, totaling 3,705 bearing commercial citrus trees, was 18.6% with 6.6% reacting to MCA13, a strain-discriminating CTV monoclonal antibody associated with virulence. However, in the recent three-year period ending in 2018, CTV spread rate in these plots was only 1.7%/year, and MCA13-reactive isolates spread at a low rate of 0.2%. In contrast, CTV incidence in a nearby organic young citrus grove increased at an annual rate of 5.3% in 2018. This difference indicated different vector control measures and horticultural conditions greatly influence spread of CTV. In Sub-objective 1B and agreements 58-2034-5-026 (Tulare County Pest Control District) and 58-2034-8-003 (Citrus Research Board), research was conducted to evaluate economic impact of MCA13-reactive strains collected from local citrus orchards as part of a California Department of Food and Agriculture (CDFA) suppressive quarantine program for CTV in central California. CTV-VT strains reduced growth and plant phenolic to levels that reduce plant defenses and contributed to severe symptom development. However, MCA13-reactive strains S1, and RB did not induce these symptoms in greenhouse and screenhouse biocharacterization tests. In Sub-objective 1C and agreement 58-2034-6-010 (Citrus Research Board), research was conducted on CTV control by cross-protection, a natural phenomenon by which citrus protects itself from some strains of CTV. CTV-RB strain was identified to suppress and cross-protect against systemic infection of a CTV T36 genotype. This is contrary to the belief that cross-protection occurs only within the same CTV genotype. This knowledge can be used to improve durability and sustainability of CTV cross-protection, and suppress and cross protect against systemic infection of a different genotype of CTV (T36). In Sub-objective 1D and agreement 58-2034-7-013 (Citrus Research Board), it was not known if preinfection of tristeza or citrus stubborn disease, both endemic diseases of citrus in California, would impact infectivity and symptom development of “Candidatus Liberibacter asiaticus" (CLas), the pathogen associated with Huanglongbing (HLB). Researchers found that stubborn disease somewhat inhibited infection by CLas; whereas CLas greatly inhibited stubborn infection. Tristeza did not impact infection by CLas or visa-versa. Research in Sub-objective 2A and agreement 2034-22000-013-03R (Citrus Research Board) was conducted to develop an infectious full-length clone of a mild CTV strain from California that could be used as a transient vector to express control factors in citrus phloem to mitigate HLB and the Asian citrus psyllid in California. This method has shown promise in Florida against HLB. A full-length cDNA of CTV-RB strain was assembled in pCambia 1380 binary vector and transformed in Agrobacterium tumefaciens and will soon be tested for infectivity in Nicotiana benthamiana.
1. Biology of RB, S1, and VT genotypes isolates of citrus tristeza virus (CTV) in California. CTV has killed hundreds of millions of citrus trees on sour orange rootstock worldwide. In Central California, CTV screening is applied to identify potentially virulent CTV isolates with severe-strain discriminating MCA13 monoclonal antibody. Researchers in Parlier, California, in collaboration with the Central California Tristeza Eradication Agency, Tulare, California, recently found an increase in occurrence of MCA13-reactive CTV isolates, sequenced full-length genomes of several MCA13-reactive isolates, and found VT, RB, and S1 (a new genotype) were present since before 1968 in Central California. RB and S1 isolates were mild, whereas VT strains were severe, inducing stem pitting and seedling yellows symptoms in sweet orange and grapefruit. These data showed screening for virulent CTV isolates by MCA13 serology is not sufficient to identify virulent CTV isolates and should be supplemented by strain-specific molecular tests.
2. Detection of Spiroplasma citri and ‘Candidatus Liberibacter asiaticus’ (CLas) by droplet digital PCR (ddPCR). Spiroplasma citri causes citrus stubborn disease (CSD), causing symptoms similar to and easily mistaken for Huanglongbing (HLB). CSD is fairly widespread in California but is a manageable disease of citrus; whereas HLB is a devastating citrus disease subject to quarantine and immediate removal of infected trees. Researchers in Parlier, California, developed a sensitive procedure to identify and quantitate S. citri and CLas from citrus and vector tissue using droplet digital PCR (ddPCR), which was more sensitive and reliable than the standard qPCR at low pathogen titer. The ddPCR test was developed in a duplex test for S. citri and CLas detection saving time and significant cost. The ddPCR test provided a robust method to test symptomatic and non-symptomatic citrus samples for CSD and HLB in a single test and may be used by regulatory agencies as an improved testing procedure to differentiate between deadly HLB and the more benign CSD.
3. Identification and distribution of a new genotype of citrus tristeza virus (CTV) called S1. Citrus tristeza virus (CTV) is an economically damaging disease of citrus, and strains of the virus can induce a range of disease symptoms from asymptomatic to debilitating stem pitting. Researchers in Parlier, California, with collaboration with 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 CTV genotype in California called S1 and developed a sensitive molecular method to identify the S1 genotype. Multiple S1 isolates were found in California CTV collections going back to 1968. Analysis of public nucleotide sequence databases further indicate CTV-S1 presence in Mediterranean and Asian regions. These data document genetic diversity in California and worldwide and contribute to a greater understanding of the etiology, genetic diversity, and disease expression of CTV.
Selvaraj, V., Maheshwari, Y., Hajeri, S., Chen, J., Mccollum, T.G., Yokomi, R.K. 2018. Development of a duplex droplet digital PCR assay for absolute quantitative detection of "Candidatus Liberibacter asiaticus". PLoS One. 13(5):e0197184. https://doi.org/10.1371/journal.pone.0197184.
Yokomi, R.K., Selvaraj, V., Maheshwari, Y., Chiumenti, M., Saponari, M., Giampetruzzi, A., Weng, Z., Xiong, Z., Hajeri, S. 2018. Molecular and biological characterization of a novel mild strain of citrus tristeza virus in California. Archives of Virology. 163(7):1795-1804. https://doi.org/10.1007/s00705-018-3799-5.
Maheshwari, Y., Selvaraj, V., Hajeri, S., Yokomi, R.K. 2017. Application of droplet digital PCR for quantitative detection of Spiroplasma citri in comparison with real time PCR. PLoS ONE. 12(9):e0184751. https://doi.org/10.1371/journal.pone.0184751.