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 support of Sub-objective 1A, ARS researchers in Parlier, California, evaluated new citrus tristeza virus (CTV) isolates from declining and dying citrus trees with quick decline (QD) symptoms on sour orange rootstock. The isolates did not react with serogroup MCA13 which has been associated with known severe CTV strains. Further molecular examination showed genome sequences of these isolates aligned with a known mild CTV genotype. Although research is continuing to determine why these isolates are causing tree death, this discovery marks the first observation of CTV isolates with a mild genotype associated with QD. This is important because regulations to control CTV in central California targets only trees infected with CTV with a MCA13 serotype; hence, the new CTV strain is not eradicated and serves as a reservoir for further spread by aphid vectors. 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. In support of Sub-objective 1B, ARS researchers, with University of California collaborators, determined whole genome sequence of seven strains of Spiroplasma citri, causal agent of citrus stubborn disease. The S. citri strains were isolated from different host plants and beet leafhopper vectors. The S. citri chromosome was remarkably similar among strains; however, the number and diversity of extrachromosomal DNA (plasmids) were markedly different from S. citri in leafhoppers. S. citri from citrus and two annual crop hosts (Chinese cabbage and brittle root) had one to three plasmids; whereas S. citri from the leafhoppers contained eight to nine plasmids. The number of plasmids suggested they may play a role in host range extension and leafhopper transmission. This research was also supported by agreement 58-2034-8-011 (Citrus Research Board). Also in support of Sub-objective 1B, ARS researchers continued research to evaluate the economic impact of CTV collected from central California in citrus cultivars on Carrizo rootstock grown in a screenhouse to simulate field conditions. After four years, severe CTV strains (VT) significantly reduced trunk diameter compared to mild CTV strains (T30, T36, RB and S1) in Lisbon lemon and Midknight Valencia, but no statistical differences were observed between CTV strains in Washington Navel, Oro Blanco, W. Murcott, or Minneola. While VT strains produced mild to moderate stunting, seedling yellows, and stem pitting, new foliar growth appeared to compensate for the deleterious effects of the virus, presumably during hot summer temperatures when virus titer of VT strains was reduced, and mild strains dropped to undetectable levels. 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 further support of Sub-objective 1B, ARS researchers continued research on the effect of CTV strains on citrus tree health and found that severe CTV (VT) strains induced higher levels of several stable flavonoids (phytonutrients) in Oro Blanco and all CTV stains reduced total sugar levels in cultivars tested (Lemon, Navel, Valencia, Minneola, Murcott, and Oro Blanco). This data shows that infection by CTV causes physiological changes in citrus. 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 Sub-objective 1D, ARS researchers continue research to develop and multiplex real-time Polymerase Chain Reaction (qPCR) to detect Candidatus Liberibacter asiaticus (CLas) associated with Huanglongbing (HLB), Spiroplasma citri, causal agent of citrus stubborn disease (CSD) and COX (cytochrome oxidase gene of citrus to validate DNA extraction). The multiplex qPCR assay was tested with citrus samples infected with different concentrations if CLas, S. citri, and CLas + S. citri and results were validated by droplet digital PCR. Therefore, this test can now be used in detection programs for HLB or management from samples with HLB-like symptoms to determine if the tree is infected with CLas or S. citri, either singly or double infected, and whether disease synergism occurs. This research is also supported by agreement 58-2034-7-013 and 58-2034-8-011 (Citrus Research Board).
1. Spread of citrus tristeza virus in central California. Quick decline (QD) of citrus on sour orange (SO) rootstock is caused by citrus tristeza virus (CTV) and has killed millions of citrus trees on SO rootstock worldwide. Therefore, the California Department of Food and Agriculture (CDFA), through grower pest control districts, developed quarantines that have evolved into two disparate zones in central California: Zone 1 where abatement including aphid vector control, annual CTV surveys and selective eradication of potentially severe strains of CTV is mandatory; and Zone 2, where no abatement is practiced. ARS researchers in Parlier, California, compared CTV spread and symptoms of disease in both quarantine zones and found annual CTV spread was 0.39 and 1.5%, respectively, in Zone 1 and Zone 2. Spread of mild strains was eleven times greater than severe strains, suggesting that aphid control and abatement of severe strains was limiting spread of severe strains of CTV. This data is being used by CDFA, pest control districts and University of California Lindcove Research and Extension Center on decisions to manage CTV.
2. Whole genome sequence of seven strains of Spiroplasma citri. Spiroplasma citri is a bacterium with a broad host range and is the causal agent of citrus stubborn disease (CSD). CSD is endemic in California and causes symptoms often confused with Huanglongbing (HLB). To improve knowledge and diagnostics of S. citri, seven strains of this pathogen were isolated by researchers in Parlier, California, from different hosts. Whole genome sequence analysis found the chromosomes to be similar except for inserts of repetitive plectroviral sequences in the central region of chromosome. Additionally, S. citri strains had various numbers of extrachromosomal DNA considered to be plasmids. The conserved nature of the multicopy plectroviral sequences were used to design new primers for more sensitive detection of S. citri by real-time Polymerase Chain Reaction (qPCR) assays and are now being used for CSD surveillance.
Hajeri, S., Yokomi, R.K. 2020. Reliable sampling tissue and seasonality for consistent detection of 'Candidatus Liberibacter asiaticus' by qPCR. Current Agriculture Research Journal. 8(1). https://doi.org/10.12944/CARJ.8.1.01.
Yokomi, R.K., Chen, J., Rattner, R., Selvaraj, V., Majeshwari, Y., Osman, F., Pagliacica, D., Vidalakis, G. 2019. Genome sequence resource for Spiroplasma citri, strain CC-2, associated with citrus stubborn disease in California. Phytopathology. 110(2):254-256. https://doi.org/10.1094/PHYTO-08-19-0304-A.
Yokomi, R.K., Sisterson, M.S., Hajeri, S. 2020. Spread of Citrus tristeza virus in citrus orchards in central California. Plant Disease. 104(7):1925-1931. https://doi.org/10.1094/PDIS-08-19-1791-RE.
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