Progress 04/09/12 to 04/08/17
Outputs
Progress Report Objectives (from AD-416): Citrus tristeza virus and Spiroplasma citri, causal agent of citrus stubborn disease, are important production-limiting, insect-vectored pathogens in California. Early host response (e.g. over-expressed microRNAs and small interfering RNAs) associated with single and multiple strain graft inoculations of these pathogens in greenhouse tests will be characterized and developed as biomarkers for early-stage pathogen- specific detection. The overall goal is to elucidate mechanism of mild strain cross-protection of Citrus tristeza virus and pathogen genetic bottlenecks resulting from insect vector transmission. New knowledge gained will lead to improved sustainable management strategies for these citrus disease agents and their insect vectors. 1. Develop field deployable systems that provide rapid, sensitive detection of Citrus tristeza virus and Spiroplasma citri in citrus. 2. Determine and characterize genetic variations in Citrus tristeza virus and Spiroplasma citri strains before and after vector passage. 3. Determine changes in host gene expression in Citrus tristeza virus cross-protected citrus that can be used to screen for cross-protective strains of Citrus tristeza virus. Approach (from AD-416): Citrus tristeza virus (CTV) and Spiroplasma citri are both phloem-limited pathogens of citrus and are transmitted by insect vectors. The California Department of Food and Agriculture and stakeholders manage CTV in commercial groves and urban areas by an eradication program and maintenance of pathogen-free budwood sources and citrus propagations in commercial nurseries. CTV eradication was modified in 2009 to detect and eliminate only citrus trees infected with severe strains of CTV. This is achieved by screening field strains of CTV by serology with a monoclonal antibody and polymerase chain reaction (PCR) assays with genotype- specific sequence markers. Genetic diversity of field strains CTV and S. citri will be characterized with respect to the molecular and genetic basis of their host-pathogen-vector relations and disease epidemiology. Further improvement in pathogen surveillance and control requires filling knowledge gaps in host response to pathogen infection as mild CTV strains, which no longer are being eradicated, continue to spread by indigenous aphid vectors. The nature, basis and mechanism(s) of cross-protection will be identified and characterized. CTV symptom phenotype with mixed infections will be monitored for post-translational gene silencing through analysis and characterization of small interfering RNAs, micro RNAs and macro RNAs. New information will facilitate mitigation of losses and misidentifications of CTV and S. citri with the ultimate goal of developing sustainable, integrated management strategies for tristeza and stubborn in California. This is the final report for project 2034-22000-011-00D, "Characterization, Epidemiology and Management Strategies of Citrus tristeza virus and Spiroplasma citri on Citrus in California," which has been replaced by new project 2034-22000D-013-00D, "Characterization and Management of Citrus Pathogens Transmitted by Phloem-Feeding Insect Vectors." For additional information, see the new project report. Under Objective 1, to improve rapid diagnosis of tristeza and stubborn diseases, a user-friendly, field deployable, lateral flow immunostrip (dipstick) test was developed for direct capture of Citrus tristeza virus (CTV) in crude plant extract without need for purification and visualization as a band (colloidal gold particles) at a specific position on the dipstick. Detection of Spiroplasma citri, the microbial pathogen that causes citrus stubborn disease, was improved 1000x from the previously used housekeeping gene of S. citri by targeting a multi-copy gene of the Spiroplasma citri Virus 1 of S. citri in Real Time Polymerase Chain Reaction (qPCR). Because all of these methods provide a relative measure of the population levels of the pathogen in diseased tissues, a droplet digital PCR (ddPCR) procedure was developed for absolute quantitation of CTV and S. citri in the tissue sampled without having to include dilutions standards of the target. These data collectively improved citrus pathogen diagnostics and the ability to evaluate impact of the pathogens on citrus health. Under Objective 2, genetic diversity of CTV was characterized by assembling the full-length genome of several CTV isolates which revealed that the Resistance-Breaking (RB) and a Western sub-group of the VT genotypes of CTV were present in California. CTV RB strains are known to break resistance to CTV in trifoliate orange which has been used as a rootstock and control measure for CTV. Western strains of VT genotype CTV are associated with increased virulence. Hence, these data revealed increased pest status of CTV. However, sequencing also led to the discovery of a new CTV genotype called S1 which was mild and found to be a component of many California CTV isolates and may have potential to ameliorate symptoms of more virulent CTV strains in mixed infections. Separate isolations of S. citri from stubborn infected citrus trees and from its leafhopper vector, were nearly identical with regard to many of the genes used for diagnosis (e.g. spiralin, p58, p89, SPV1 ORF1 and ORF3), hence, any of these genes could be used to monitor S. citri infection in citrus or leafhoppers. Under Objective 3, to examine CTV cross-protection, citrus preinfected with a CTV RB isolate prevented systemic infection of a CTV T36 genotype. This is relevant because previously, CTV cross-protection was thought to result from superinfection exclusion amongst CTV isolates in the same genotype. Characterization of virulent and cross-protective CTV isolates from Peru revealed presence of RB isolates in cross-protected Star Ruby grapefruit trees in Peru and these RB isolates were found to be similar in sequence to those in California. Hence, potentially cross-protective CTV strains against stem pitting CTV may already exist in California. These results are adding to the understanding of how biologically-based integrated disease management by CTV cross-protection may influence disease expression. Accomplishments 01 Biology of RB, S1, and VT genotype isolates of citrus tristeza virus (CTV) in California. CTV-infected citrus trees in CTV suppressive districts are eradicated in commercial citrus regions in central California if they test positive to a presumptive severe-strain discriminating CTV monoclonal antibody, MCA13. A recent increase of MCA13-reactive CTV isolates without concomitant virulent disease symptoms or decreased yields has occurred. ARS researchers in Parlier, California, with the collaboration of the Central California Tristeza Eradication Agency, Tulare, California, tested CTV isolates from central California and found many different strains including RB, S1, and VT reacting to MCA13. RB and S1 isolates were mild in biocharacterization tests, whereas VT strains were severe and induced stem pitting and seedling yellows symptoms in citrus. These data showed that screening for virulent CTV isolates by MCA13 serology is not reliable and would eliminate mild isolates that could be sources for cross-protection. MCA13 tests can be supplemented by molecular tests developed in this research program to avoid needless removal of CTV- infected trees and potential mild, cross-protecting CTV strains. 02 Identification and distribution of the S1 CTV genotype. Phylogeny of full length genomes of citrus tristeza virus (CTV) differentiates CTV worldwide into seven genotypes. ARS researchers in Parlier, California, in collaboration with the 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 (eighth) CTV genotype (S1) in central California and developed a TaqMan-based probe to rapidly identify this genotype. Comparison of the CTV genomes in the public database indicated that S1 isolates may also be present in Europe and Asia. Direct samples tested using the S1 probe verified that the S1 genotype occurs in Pakistan and Peru. These data demonstrate the value of genome sequencing and bioinformatics to better understand the etiology, genetic diversity, and disease expression of CTV. 03 Detection of Spiroplasma citri and Candidatus Liberibacter asiaticus (CLas) by droplet digital polymerase chain reaction (ddPCR). Citrus stubborn disease (CSD), caused by Spiroplasma (S.) citri, is endemic in California; whereas huanglongbing (HLB), associated with Candidatus Liberibacter asiaticus (CLas), has been detected in ~70+ citrus trees in residential portions of southern California (Los Angeles, Orange, and Riverside counties) but not in any commercial orchards. ARS researchers in Parlier, California, developed a sensitive procedure to identify and quantitate S. citri and CLas using a specialized ddPCR test which partitions a test sample into approximately 20,000 individual droplets. Each droplet undergoes a separate end point amplification by PCR and results in an exact count of DNA target copies in a sample without need of a standard curve. The major advantage of ddPCR over real-time quantitative PCR is its ability to accurately resolve rare events and access targets at low template concentrations despite presence of PCR inhibitors. The ddPCR test for S. citri and CLas was developed as a duplex test since CSD and HLB share many similar symptoms and can be confused with each other. Hence, this test procedure provides a robust method to test the status of symptomatic and non-symptomatic citrus samples for S. citri and CLas in one simultaneous test. 04 Citrus tristeza virus-Resistance-Breaking (RB) genotype interaction with a CTV-T36 genotype. Cross-protection of citrus tristeza virus (CTV) has recently been characterized as superinfection exclusion amongst isolates in the same genotype but not between CTV genotypes. ARS researchers in Parlier, California, found that preinfection of citrus by an RB isolate cross-protected against a T36 genotype but not with isolates from other genotypes (VT, S1, or T30) tested. These results suggested cross-protection can result from CTV isolates from different genotypes in a mixture rather than exclusively from within a single genotype. In addition, they provided new insight on interactions of CTV strains on disease expression.
Impacts
(N/A)
Publications
- Yokomi, R.K., Selvaraj, V., Maheshwari, Y., Saponari, M., Giampetruzzi, A., Chiumenti, M., Hajeri, S. 2017. Identification and characterization of Citrus tristeza virus isolates breaking resistance in trifoliate orange in California. Phytopathology. 107(7):901-908. doi.org/10.1094/PHYTO-01-17- 0007-R.
- Adesemoye, A., Hajeri, S., Yokomi, R.K., Eskalen, A. 2017. Association of Neonectria macrodidyma with dry root rot of citrus in California. Journal of Plant Pathology & Microbiology. 8:391. doi:10.4172/2157-7471.1000391.
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Progress 10/01/15 to 09/30/16
Outputs
Progress Report Objectives (from AD-416): Citrus tristeza virus and Spiroplasma citri, causal agent of citrus stubborn disease, are important production-limiting, insect-vectored pathogens in California. Early host response (e.g. over-expressed microRNAs and small interfering RNAs) associated with single and multiple strain graft inoculations of these pathogens in greenhouse tests will be characterized and developed as biomarkers for early-stage pathogen- specific detection. The overall goal is to elucidate mechanism of mild strain cross-protection of Citrus tristeza virus and pathogen genetic bottlenecks resulting from insect vector transmission. New knowledge gained will lead to improved sustainable management strategies for these citrus disease agents and their insect vectors. 1. Develop field deployable systems that provide rapid, sensitive detection of Citrus tristeza virus and Spiroplasma citri in citrus. 2. Determine and characterize genetic variations in Citrus tristeza virus and Spiroplasma citri strains before and after vector passage. 3. Determine changes in host gene expression in Citrus tristeza virus cross-protected citrus that can be used to screen for cross-protective strains of Citrus tristeza virus. Approach (from AD-416): Citrus tristeza virus (CTV) and Spiroplasma citri are both phloem-limited pathogens of citrus and are transmitted by insect vectors. The California Department of Food and Agriculture and stakeholders manage CTV in commercial groves and urban areas by an eradication program and maintenance of pathogen-free budwood sources and citrus propagations in commercial nurseries. CTV eradication was modified in 2009 to detect and eliminate only citrus trees infected with severe strains of CTV. This is achieved by screening field strains of CTV by serology with a monoclonal antibody and polymerase chain reaction (PCR) assays with genotype- specific sequence markers. Genetic diversity of field strains CTV and S. citri will be characterized with respect to the molecular and genetic basis of their host-pathogen-vector relations and disease epidemiology. Further improvement in pathogen surveillance and control requires filling knowledge gaps in host response to pathogen infection as mild CTV strains, which no longer are being eradicated, continue to spread by indigenous aphid vectors. The nature, basis and mechanism(s) of cross-protection will be identified and characterized. CTV symptom phenotype with mixed infections will be monitored for post-translational gene silencing through analysis and characterization of small interfering RNAs, micro RNAs and macro RNAs. New information will facilitate mitigation of losses and misidentifications of CTV and S. citri with the ultimate goal of developing sustainable, integrated management strategies for tristeza and stubborn in California. Under Objective 1, a field deployable system that provided to be a rapid and sensitive method of detection of Citrus tristeza virus (CTV) was developed. This system used a lateral flow immunostrip test kit which was CTV-specific, accurate to a sap dilution of 1/80 to 1/160, rapid (10 minutes), inexpensive, and required no equipment, expertise, or training to use. This technology was developed to allow user-friendly, economic in- field detection for CTV disease management and/or exclusion purposes by citrus growers, nurserymen, regulatory agencies, and researchers. Specifically, antibodies were developed against CTV virions produced in tobacco plants (Nicotiana benthamiana) infected by a recombinant infectious cloned DNA of CTV. Under Objective 2, great progress was made in determining CTV genetic diversity. Full length sequences of eight California CTV isolates were completed. Analysis with 46 other full length CTV sequences showed three isolates had a RB genotype that breaks CTV resistance in trifoliate orange. Two isolates constituted a new genotype called CA-S1 which differed by > 12% of sequence identity to all other known CTV genotypes. The other genotypes verified by full length sequencing were T36, T30, and VT. Sequencing was conducted using Next Generation Sequencing of small RNAs isolated from CTV-infected sweet orange plants. All isolates except T30 reacted with a severe-strain differentiating monoclonal antibody, MCA13. Greenhouse biocharacterization showed the VT strain induced a strong seedling yellows reaction indicative of a more severe strain of CTV. However, all other isolates sequenced were mild and did not induce symptoms in field citrus grown on non-sour orange rootstocks. These data indicated that the VT sequence group, often associated with virulent stem pitting of citrus, was present and surveys showed VT incidence was increasing in Central California. Therefore, screening for severe strains of CTV requires more than MCA13 reactivity to support management of CTV as mandated by California Department of Food and Agriculture�s Interior Quarantine for CTV in California. Under Objective 3, systemic infection of a modified, recombinant CTV in citrus was achieved. Plasmids carrying a recombinant cDNA clone of a Florida infectious CTV strain were used to transform Agrobacterium tumefaciens which was, in turn, used to inoculate tobacco (Nicotiana benthamiana) plants with genetically modified (m) CTV. mCTV virions produced in tobacco were harvested and inoculated into citrus to produce a systemic infection. The infected citrus was then used to graft inoculate other citrus plants to proliferate the mCTV lines. This procedure was standardized as a routine procedure to be used to create hybrid lines of CTV encoding antimicrobial peptides or small RNAs for interference against insect pests or down regulation of citrus genes as therapies to manage Huanglongbing. Accomplishments 01 A resistance-breaking genotype and a new CTV genotype called CA-S1 were identified by full length sequencing. Under California Department of Food and Agriculture�s Citrus tristeza virus (CTV) Interior Quarantine, suppression of economically important severe strains of CTV in Central California is based on serological reaction to MCA13, a monoclonal antibody developed against a severe quick decline CTV strain. ARS researchers in Parlier, California, in collaboration with scientists in the Institute for Sustainable Plant Protection, National Research Council, Sezione di Bari, Bari, Italy and the Central California Tristeza Eradication Agency, conducted Next Generation Sequencing of small RNAs from citrus infected with MCA13- reactive California CTV isolates. Three isolates were found with a resistance-breaking (RB) genotype that replicated in CTV-resistant trifoliate orange. Two isolates had genomic differences greater than 7% (sequence identity) to constitute a new genotype which was called CA-S1. The RB and CA-S1 isolates were mild in virus indexing and showed that MCA13-reactivity was insufficient to support suppression of economically important CTV strains in Central California. This determination can be used to exempt trees infected with RB and S1 genotypes from eradication and save the grower�s cost and time needed to replant virus-free trees and waiting 3 years to regain fruit production. 02 Silencing of a citrus gene with a recombinant cloned DNA of a Citrus tristeza virus vector carrying a truncated citrus gene. Huanglongbing (HLB) is a new and destructive invasive disease of citrus associated with an uncultivable bacterium that inhabits phloem and blocks or limits phloem transport. ARS researchers in Parlier, California, in collaboration with scientists at the Central California Tristeza Eradication Agency, Tulare, California and the Citrus Research and Education Center, Lake Alfred, Florida successfully showed silencing of the citrus endogenous gene phytoene desaturase (PDS) induced by a CTV vector containing a truncated PDS gene. This is an important proof of concept that a modified CTV vector can be engineered to downregulate expression of citrus genes. Target citrus genes include those involved in HLB-induced premature fruit drop or callose and phloem protein 2 which are involved in phloem plugging. In addition, the CTV vector can be further modified to produce antimicrobial peptides and RNA interference in phloem against the HLB pathogen and its psyllid vector. If regulatory approval is obtained, this approach can be used for short to mid-term control of HLB and reduce reliance of insecticides to manage the psyllid vector.
Impacts
(N/A)
Publications
- Wu, F., Deng, X., Wallis, C.M., Trumble, J., Prager, S., Yokomi, R.K., Liang, G., Cen, Y., Chen, J. 2016. The complete mitochondrial genome sequence of Bactericera cockerelli and its comparison with three other psylloidea species. PLoS One. 11(5):e0155318. doi: 10.1371/journal.pone. 0155318.
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Progress 10/01/14 to 09/30/15
Outputs
Progress Report Objectives (from AD-416): Citrus tristeza virus and Spiroplasma citri, causal agent of citrus stubborn disease, are important production-limiting, insect-vectored pathogens in California. Early host response (e.g. over-expressed microRNAs and small interfering RNAs) associated with single and multiple strain graft inoculations of these pathogens in greenhouse tests will be characterized and developed as biomarkers for early-stage pathogen- specific detection. The overall goal is to elucidate mechanism of mild strain cross-protection of Citrus tristeza virus and pathogen genetic bottlenecks resulting from insect vector transmission. New knowledge gained will lead to improved sustainable management strategies for these citrus disease agents and their insect vectors. 1. Develop field deployable systems that provide rapid, sensitive detection of Citrus tristeza virus and Spiroplasma citri in citrus. 2. Determine and characterize genetic variations in Citrus tristeza virus and Spiroplasma citri strains before and after vector passage. 3. Determine changes in host gene expression in Citrus tristeza virus cross-protected citrus that can be used to screen for cross-protective strains of Citrus tristeza virus. Approach (from AD-416): Citrus tristeza virus (CTV) and Spiroplasma citri are both phloem-limited pathogens of citrus and are transmitted by insect vectors. The California Department of Food and Agriculture and stakeholders manage CTV in commercial groves and urban areas by an eradication program and maintenance of pathogen-free budwood sources and citrus propagations in commercial nurseries. CTV eradication was modified in 2009 to detect and eliminate only citrus trees infected with severe strains of CTV. This is achieved by screening field strains of CTV by serology with a monoclonal antibody and polymerase chain reaction (PCR) assays with genotype- specific sequence markers. Genetic diversity of field strains CTV and S. citri will be characterized with respect to the molecular and genetic basis of their host-pathogen-vector relations and disease epidemiology. Further improvement in pathogen surveillance and control requires filling knowledge gaps in host response to pathogen infection as mild CTV strains, which no longer are being eradicated, continue to spread by indigenous aphid vectors. The nature, basis and mechanism(s) of cross-protection will be identified and characterized. CTV symptom phenotype with mixed infections will be monitored for post-translational gene silencing through analysis and characterization of small interfering RNAs, micro RNAs and macro RNAs. New information will facilitate mitigation of losses and misidentifications of CTV and S. citri with the ultimate goal of developing sustainable, integrated management strategies for tristeza and stubborn in California. Citrus tristeza virus (CTV) is a regulated plant pathogen in central California and disease suppression is supported by removal of trees infected with potentially severe strains of CTV as detected by MCA13 monoclonal CTV antiserum. CTV surveys in commercial orchards in Tulare County in spring 2015 identified groves with CTV incidence up to 21%. These assays showed that 39% of the CTV isolates reacted with MCA13, a severe-strain discriminating monoclonal antibody. Since CTV is transmitted by aphid vectors, this was an indication that potentially severe CTV strains may be increasing. These data suggest rouging of MCA13 isolates may help to suppress the impact of CTV in the quarantine area. This research supports Objective 2. Citrus is a perennial crop, and repeated inoculation by viruliferous aphids gives rise to multiple infections and CTV variants. Genetic analysis of MCA13-reactive isolates from the field surveys of CTV showed 90% of these isolates had either VT or T3-like genotypes which are associated with potentially virulent CTV strains. Ground truth surveys of individual trees showed no visual differences amongst CTV-infected and non-infected trees. Representative field isolates were collected and are being indexed in the greenhouse. Visual inspection and fruit production of trees will be evaluated annually to determine the impact of these MCA13-reactive CTV isolates in these plots. This research supports Objective 2. Disease suppression of stem pitting CTV can be achieved by mild strain cross-protection, however, the mechanism of cross protection is unknown. To better understand this phenomenon, construction of an infectious cDNA clone of CTV has begun. Full-length DNA binary constructs of California CTV strains T30 and T36 are nearing completion. Infectious full-length CTV clones will allow studies to determine which genes play a role in cross-protection. Infectious CTV clones can also be used as transient expression vectors of therapeutic genes that produce antimicrobial peptides (AMPs) to control the bacterial disease agent huanglongbing (HLB) (aka citrus greening) or produce RNA interference (RNAi) against Asian citrus psyllid, vector of the HLB pathogen. These therapies can be used on existing citrus trees without need for generation of transgenic plants. This research is in support of Objective 3. Accomplishments 01 Sensitive non-PCR method for multiplex detection of citrus pathogens. ARS researchers in Parlier, California, in collaboration with scientists at the University of California, Riverside, developed a QuantiGene Luminex-based 10-plex assay for simultaneous detection, identification, and quantification of nine RNA citrus pathogens. A housekeeping citrus gene was used as an internal control. Pathogen targets attached to Luminex beads can be multiplexed to detect up to 80 RNA targets. Targets hybridize to specific sequences; the signal is amplified by branched DNA amplification. This detection platform can use crude tissue extracts, without prior nucleic acid purification for high throughput pathogen detection. This assay is an efficient, cost- effective diagnostic suite for citrus certification and clean stock programs. 02 Genotype classification of Citrus tristeza virus (CTV) in California. Symptoms caused by CTV range from symptomless to rapid tree death of citrus depending on the strain of the virus and citrus cultivar. ARS researchers in Parlier, California, working with the Citrus Pest Detection Program of the Central California Tristeza Eradication Agency (CCTEA), Tulare, California, characterized the molecular genotype of approximately 300 CTV isolates collected in Tulare County in 2015 that reacted with severe-strain discriminating MCA13 monoclonal antibody. A high percentage of isolates were classified with a VT or T3 genotype which are associated with higher CTV virulence due to stem pitting. These isolates are undergoing further biological characterization in the lab, greenhouse and field to determine economic impact. This is in contrast to data from prior years which showed MCA13-reactive CTV isolates were rare and suggests changes in regulatory priorities in the grower-funded CTV suppression program in California may be needed in the foreseeable future. 03 Confirmation of resistance breaking (RB) CTV isolates in California. ARS researchers in Parlier, California, identified CTV isolates collected from southern and central California, with an RB genotype, which suggests that these isolates can infect CTV-resistant citrus germplasm. Isolate inoculation into CTV-resistant Poncirus trifoliata showed CTV replication in both roots and leaves, confirming that these isolates are RB. These RB isolates were mild in virus indexing tests so efforts are underway to construct an infectious cDNA clone with this isolate. This clone can be engineered and used as a CTV vector in roots and shoots of citrus to express foreign genes that produce antimicrobial peptides to control the bacterial disease agent of huanglongbing (HLB) or produce RNA interference against the Asian citrus psyllid (ACP) vector of the HLB disease agent.
Impacts
(N/A)
Publications
- Wang, X., Doddapaneni, H., Chen, J., Yokomi, R.K. 2015. Improved real-time PCR diagnosis of citrus stubborn disease by targeting prophage genes of Spiroplasma citri. Plant Disease. 99:149-154.
- Saponari, M., Loconsole, G., Cornara, D., Yokomi, R.K., De Stradis, A., Boscia, D., Bosco, D., Martelli, G., Krugner, R., Porcelli, F. 2014. Infectivity and transmission of Xylella fastidiosa Salento strain by Philaenus spumarius L. (Hemiptera: Aphrophoridae) in Apulia, Italy. Journal of Economic Entomology. 107(4):1316-1319.
- Loconsole, G., Giampetruzzi, A., Saldarelli, P., Onelge, N., Yokomi, R.K., Saponari, M. 2015. Deep sequencing of small RNAs from citrus affected by graft-transmissible diseases of unknown etiology leads to discovery of two novel viruses. Acta Horticulturae. 1065(2):817-824.
- Cheung, W.H., Zhao, W., Pasamontes, A., Kapaun, T., Yokomi, R.K., Swe, M., Fiehn, O., Davis, C.E. 2015. Volatile Organic Compound (VOC) profiling of Citrus tristeza virus (CTV) infection in sweet orange citrus varietals using thermal desorption gas chromatography time of flight mass spectrometry (TD-GC/TOF-MS). Metabolomics. doi: 10.1007/s11306-015-0807-6.
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Progress 10/01/13 to 09/30/14
Outputs
Progress Report Objectives (from AD-416): Citrus tristeza virus and Spiroplasma citri, causal agent of citrus stubborn disease, are important production-limiting, insect-vectored pathogens in California. Early host response (e.g. over-expressed microRNAs and small interfering RNAs) associated with single and multiple strain graft inoculations of these pathogens in greenhouse tests will be characterized and developed as biomarkers for early-stage pathogen- specific detection. The overall goal is to elucidate mechanism of mild strain cross-protection of Citrus tristeza virus and pathogen genetic bottlenecks resulting from insect vector transmission. New knowledge gained will lead to improved sustainable management strategies for these citrus disease agents and their insect vectors. 1. Develop field deployable systems that provide rapid, sensitive detection of Citrus tristeza virus and Spiroplasma citri in citrus. 2. Determine and characterize genetic variations in Citrus tristeza virus and Spiroplasma citri strains before and after vector passage. 3. Determine changes in host gene expression in Citrus tristeza virus cross-protected citrus that can be used to screen for cross-protective strains of Citrus tristeza virus. Approach (from AD-416): Citrus tristeza virus (CTV) and Spiroplasma citri are both phloem-limited pathogens of citrus and are transmitted by insect vectors. The California Department of Food and Agriculture and stakeholders manage CTV in commercial groves and urban areas by an eradication program and maintenance of pathogen-free budwood sources and citrus propagations in commercial nurseries. CTV eradication was modified in 2009 to detect and eliminate only citrus trees infected with severe strains of CTV. This is achieved by screening field strains of CTV by serology with a monoclonal antibody and polymerase chain reaction (PCR) assays with genotype- specific sequence markers. Genetic diversity of field strains CTV and S. citri will be characterized with respect to the molecular and genetic basis of their host-pathogen-vector relations and disease epidemiology. Further improvement in pathogen surveillance and control requires filling knowledge gaps in host response to pathogen infection as mild CTV strains, which no longer are being eradicated, continue to spread by indigenous aphid vectors. The nature, basis and mechanism(s) of cross-protection will be identified and characterized. CTV symptom phenotype with mixed infections will be monitored for post-translational gene silencing through analysis and characterization of small interfering RNAs, micro RNAs and macro RNAs. New information will facilitate mitigation of losses and misidentifications of CTV and S. citri with the ultimate goal of developing sustainable, integrated management strategies for tristeza and stubborn in California. Decline and death of young (3- to 10-year old) citrus trees on trifoliate- type rootstocks are increasing in central California. This problem, described here as trifoliate decline, is of unknown etiology and is thought to be due to a genetic incompatibility between scion and rootstock. Molecular assays conducted on some diseased trees showed infection by a severe genotype of Citrus Tristeza virus (CTV) plus a cachexia strain of a citrus viroid. Pathogen isolates from these trees were propagated in the greenhouse to reproduce symptoms under controlled conditions. If CTV and cachexia are found to play a role or otherwise intensify symptom development of trifoliate decline, the disease can be mitigated by cultural practices such as sanitation to prevent mechanical transmission of viroids or removal of CTV-infected trees. The bacterium, Spiroplasma citri, causal agent of citrus stubborn disease, is difficult to detect in citrus due to low concentration and erratic distribution. Greater detection sensitivity was achieved using polymerase chain reaction (PCR). The new PCR procedure was 10-fold more sensitive than the previous PCR method and was used to show that nearly 2% of trees previously diagnosed as negative for infection by the less sensitive PCR method were, in fact, positive for S. citri. The new PCR method was more accurate and will be used to detect the bacterium in budwood source trees in California�s citrus nursery certification program. Citrus tristeza virus (CTV) is a California Department of Food and Agriculture regulated plant pathogen and is controlled in central California by removal of trees infected with severe CTV strains as detected by reaction with monoclonal antibody 13 (MCA13). Greenhouse biological assays showed only a small portion of the MCA13-reactive isolates in central California caused severe disease symptoms. Therefore, molecular methods targeting multiple genomic regions associated with severe biological symptoms were used as a secondary screen to discriminate potentially virulent strains. The Central California Tristeza Eradication Agency, a grower-funded organization that surveys for CTV, now sends MCA13 reactive CTV isolates to ARS in Parlier, California, for secondary screen by molecular marker assays to identify potential severe strains of CTV, thus, reducing the number of trees that need removal. CTV infection produces viral components that contribute to gene silencing, a cellular process controlling gene expression and defense against viral infection. Gene silencing is regulated by small interfering (si) RNAs. Citrus infected with severe and mild CTV strains were characterized for siRNAs. The CTV-derived siRNAs were mapped onto the CTV genome which revealed a close association with the CTV.20 gene in the CTV resistance locus of the citrus genome. Resistant/tolerant cultivars (trifoliate orange and Carrizo) versus susceptible citrus cultivar (sour orange) were then challenge-inoculated with CTV. The CTV.20 gene was examined and found to be differentially methylated (methylation affects gene expression). Studies are continuing to confirm the role of methylation on host response to infection by different strains of CTV. Gene expression in pathogen-infected plants is modulated by host micro (mi) RNAs induced by the pathogen. Therefore, miRNAs from citrus infected with a severe-strain versus a mild strain were examined by Next Generation Sequencing. Results indicated at least six specific miRNAs were up-regulated in asymptomatic plants infected by the mild isolate. Specific miRNAs may be useful as biomarkers for diagnosis of CTV infection and screening of CTV isolates for virulence or mildness. Successful mild-strain cross-protection to control virulent CTV strains has been monitored for the past twenty years in Peru. Therefore, Peruvian CTV isolates were selected and imported to a quarantine facility in Beltsville, Maryland, and analyzed. Full-length genome sequencing was achieved and showed the severe Peruvian isolate was identical to a severe CTV strain from Japan, whereas the protective mild Peruvian strain was identical to a CTV resistance-breaking (RB) isolate from New Zealand. Controlled greenhouse cross-protection experiments are being established in Beltsville, Maryland, to confirm Peruvian field observations. This is relevant because ARS in Parlier, California, has shown some mild California strains have similar RB genotypes, and research is now planned to test these California mild CTV strains for cross-protection in Beltsville. Huanglongbing (HLB), also known as greening disease, was detected in one residential property in Los Angeles and eradicated on site by the California Department of Food and Agriculture. The disease is associated with an uncultivable bacteria that multiplies to high titer in the Asian citrus psyllid (ACP) vector. Since there is no known cure for or resistance to HLB, progress was made to develop an infectious complementary (c) DNA of a mild California strain of Citrus tristeza virus (CTV). So far, approximately 25% of a cDNA of a mild California CTV isolate has been successfully cloned. Once fully cloned, the infectious cDNA of CTV can be engineered to express anti-microbial peptides in phloem to control the HLB bacterium and RNA interference to control the ACP. This therapy can be applied to existing citrus trees without use of transgenic plants. Accomplishments 01 Detection of Citrus Tristeza virus (CTV) in trees. Non-destructive field sampling for early diagnosis of citrus trees is urgently needed to manage high priority diseases such as Huonglongbing (HLB) and CTV. ARS researchers at Parlier, California, in collaboration with scientists at the University of California, Davis (UCD) used a Twister- sorbent system for in-field passive volatile organic compound (VOC) sampling. VOC analysis identified specific compounds statistically associated with CTV-infected trees. Thus, VOC profiling and multivariate analyses were used to distinguish CTV-infected from healthy citrus trees. A commercial company, in collaboration with UCD, has developed a prototype portable, battery-operated unit to conduct chemical analysis and VOC profiling directly in the field. 02 Classification of Citrus Tristeza virus (CTV) in a 100 year-old germplasm collection. Symptoms caused by CTV range from symptomless to catastrophic rapid tree death. ARS researchers at Parlier, California, in collaboration with scientists at the University of California, Riverside conducted a molecular analysis of 48 CTV isolates which had been collected over a span 100 years from all citrus-growing regions of California. Data analysis provided a historical perspective with regard to introduction, movement, and genetic diversity of CTV in California. CTV diversity was greater prior to the 1960�s than after this period, which was attributed to implementation of CTV quarantines, eradication, and disease management strategies. This research demonstrated the importance of regulatory control and grower cooperation with regard to controlling invasive, insect-vectored disease agents such as CTV. 03 Principal vector of Xylella fastidiosa in Italy identified. Olive trees in Apulia, southern Italy began to decline and die in 2010 with Olive Quick Decline Syndrome, and in 2013, the bacterium Xylella fastidiosa was detected in symptomatic trees. ARS researchers at Parlier, California, in collaboration with scientists at the University of Bari, Bari, Italy, and, Consiglio Nazionale Delle Ricerche Bari (National Research Council), surveyed diseased orchards for insect vectors and found that the meadow spittlebug was the most prominent xylem-fluid feeding insect present. PCR showed that a high percentage of field-collected spittlebugs were positive for Xylella fastidiosa, and confirmation of transmission was achieved by spittlebug transmission tests. This research identified the principal vector of Xylella fastidiosa in Italy. 04 Technical assistance to APHIS PPQ. Export of fresh cherries to Japan without post-harvest fumigation is supported by a bilateral agreement between Japan�s Ministry of Agriculture, Forestry and Fisheries (MAFF) and the Animal and Plant Health Inspection Service (APHIS) Plant Protection and Quarantine (PPQ) program through a systems approach involving maintaining zero to extremely low populations of codling moth. ARS researchers in Parlier, California, provided molecular diagnostic identification of larval codling moth in support of this program. They also supplied technical expertise in response to inquiries from APHIS and MAFF on protocols for identification of codling moth larva. Export of California cherries was fully successful in 2014 because all program criteria were met.
Impacts
(N/A)
Publications
- Spear, A., Yokomi, R.K., French, R.C., Stenger, D.C. 2013. Occurrence, sequence polymorphism and population structure of Circulifer tenellus virus 1 in a field population of the beet leafhopper. Virus Research. 176:307-311.
- Saponari, M., Loconsole, G., Liao, H., Bo, J., Savino, V., Yokomi, R.K. 2013. Validation of high-throughput real time polymerase chain reaction assays for simultaneous detection of invasive citrus pathogens. Journal of Virological Methods. 193:478-486.
- Loconsole, G., Onelge, N., Yokomi, R.K., Abou-Kubaa, R., Savino, V., Saponari, M. 2013. Rapid differentiation of citrus Hop stunt viroid variants by use of real-time RT-PCR and high resolution melting analysis. Molecular and Cellular Probes. 27:221-229.
- Wang, J., Bozan, O., Kwon, S., Rucker, T., Yokomi, R.K., Lee, R.F., Folimonova, S., Greer, G., Krueger, R., Bash, J., Vidalakis, G. 2013. Past and future of a century old Citrus Tristeza Virus collection: A California citrus germplasm tale . Frontiers in Microbiology. 4:366. DOI: 10.3389/ fmicb.2013.00366.
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Progress 10/01/12 to 09/30/13
Outputs
Progress Report Objectives (from AD-416): Citrus tristeza virus and Spiroplasma citri, causal agent of citrus stubborn disease, are important production-limiting, insect-vectored pathogens in California. Early host response (e.g. over-expressed microRNAs and small interfering RNAs) associated with single and multiple strain graft inoculations of these pathogens in greenhouse tests will be characterized and developed as biomarkers for early-stage pathogen- specific detection. The overall goal is to elucidate mechanism of mild strain cross-protection of Citrus tristeza virus and pathogen genetic bottlenecks resulting from insect vector transmission. New knowledge gained will lead to improved sustainable management strategies for these citrus disease agents and their insect vectors. 1. Develop field deployable systems that provide rapid, sensitive detection of Citrus tristeza virus and Spiroplasma citri in citrus. 2. Determine and characterize genetic variations in Citrus tristeza virus and Spiroplasma citri strains before and after vector passage. 3. Determine changes in host gene expression in Citrus tristeza virus cross-protected citrus that can be used to screen for cross-protective strains of Citrus tristeza virus. Approach (from AD-416): Citrus tristeza virus (CTV) and Spiroplasma citri are both phloem-limited pathogens of citrus and are transmitted by insect vectors. The California Department of Food and Agriculture and stakeholders manage CTV in commercial groves and urban areas by an eradication program and maintenance of pathogen-free budwood sources and citrus propagations in commercial nurseries. CTV eradication was modified in 2009 to detect and eliminate only citrus trees infected with severe strains of CTV. This is achieved by screening field strains of CTV by serology with a monoclonal antibody and polymerase chain reaction (PCR) assays with genotype- specific sequence markers. Genetic diversity of field strains CTV and S. citri will be characterized with respect to the molecular and genetic basis of their host-pathogen-vector relations and disease epidemiology. Further improvement in pathogen surveillance and control requires filling knowledge gaps in host response to pathogen infection as mild CTV strains, which no longer are being eradicated, continue to spread by indigenous aphid vectors. The nature, basis and mechanism(s) of cross-protection will be identified and characterized. CTV symptom phenotype with mixed infections will be monitored for post-translational gene silencing through analysis and characterization of small interfering RNAs, micro RNAs and macro RNAs. New information will facilitate mitigation of losses and misidentifications of CTV and S. citri with the ultimate goal of developing sustainable, integrated management strategies for tristeza and stubborn in California. Spiroplasma citri, causal agent of citrus stubborn disease, occurs in low concentration in citrus, making polymerase chain reaction (PCR) detection challenging in a multiplex assay for multiple pathogens. This limitation was overcome by identification of multiple-copy bacteriophage genes integrated into the S. citri genome. Several specific bacteriophage sequences were found to occur as 8 to 140 copies per chromosome. PCR primers were designed for detection of these multiple copy genes. PCR assays tested using the new primers successfully identified the S. citri phage in all of the nine S. citri strains tested as well as from citrus field samples from two Central California commercial citrus groves. Detection of S. citri phage sequences was more sensitive than conventional PCR of a single copy gene (spiralin). In the near future, the bacteriophage sequences from S. citri will be used to detect the citrus stubborn disease pathogen in nursery stock by the California Department of Food and Agriculture Certification Program. Control of Citrus tristeza virus (CTV) in Central California is mandated by the state of California using a serological assay based on reactivity with MCA13, a strain-discriminating monoclonal antibody. Project research determined that many San Joaquin Valley CTV strains that react with MCA13 cause only mild disease. Virulent CTV strains could be differentiated by new primers in a real-time polymerase chain reaction (qPCR) assay. A CTV- strain distinguishing multiplex assay was developed to facilitate rapid identification of virulent CTV strains isolated from field samples. The test incorporates specific primers and probes in individual wells in a 96- well plate using a standardized reaction mix. Test kits are undergoing independent evaluation by different laboratories. Test kits includes custom mixes for CTV strain differentiation and have a versatile format to adapt to testing for other citrus pathogens simultaneously in multiplex assay. The Central California Tristeza Eradication Agency will be using this assay for screening high incidence CTV orchards this year. An alarmingly high proportion of virulent CTV strains were detected in spring of 2013 in Central California. CTV eradication is now impractical. An alternative control for severe CTV strains is cross-protection. The Topara Citrus Nursery in Peru has identified mild CTV strains that protect citrus from severe stem pitting strains of CTV after >20 years of natural field challenge. Extracts from field trees infected with 20 CTV isolates from protected and non-protected trees were obtained from the nursery and examined for strain mixtures and genotype profiling. All protected trees contained a genotype (T36NS) which is found in California. Comparison of the molecular profile of severe and mild Peruvian CTV versus California CTV isolates should provide molecular data to assess and prioritize local CTV isolates for cross-protection trials. Accomplishments 01 QuantiGene Luminex-based assay for multiplexed citrus pathogen detection. Improved methods are needed for sensitive and economic detection of multiple citrus pathogens. ARS researchers at Parlier, California, in collaboration with scientists at University of California Riverside, developed a multiplex hybridization assay which can detect and differentiate between 9 citrus RNA pathogens or strains in one simultaneous test. Citrus pathogens from the Citrus Clonal Protection Program and USDA-ARS at Beltsville, Maryland; Riverside, California; and Parlier, California, were detected accurately using crude plant samples. The assay was robust, user-friendly and cost- effective. The method may be used by the California Department of Food and Agriculture and USDA for citrus disease-free certification programs. 02 Robotic high-throughput extraction procedures for citrus pathogens. Rapid and standardized methods are needed for detection of citrus pathogens for certification and disease management programs. ARS researchers at Parlier, California, in collaboration with scientists at the University of California Riverside, University of Bari in Bari, Italy, and, Consiglio Nazionale Delle Ricerche Bari (National Research Council), adapted an automated procedure for nucleic acid extraction from citrus tissue. Homogenization and reagent concentrations were optimized for pathogens from citrus using a robot with magnetized beads. Resultant samples contained ample pathogen RNA and DNA to allow reliable detection in PCR assays. The new extraction method is being used by the California Citrus Research Board�s Dimitman Laboratory for diagnosis of the pathogen associated with huanglongbing (HLB, also known as citrus greening) as well as the University of California Citrus Clonal Protection Program (CCPP) in Riverside, California for citrus nursery pathogen free budwood certification. 03 Improved detection of Spiroplasma citri in citrus samples. Examination of S. citri genomes revealed that the bacterium harbors multiple copies of prophage genes. Two primer sets (Php-orf1 and Php-orf3) were developed from prophage sequences and used with a cyanine dye (SYBR- Green)-based real-time polymerase chain reaction to evaluate detection sensitivity with 18 S. citri cultures isolated from different hosts. Compared with primer sets based on single-copy genes, the prophage primer set increased detection sensitivity by approximately 1.8 to 4.9 log units. Significant improvement of detection sensitivity was shown for 252 field samples from two citrus orchards in Central California from 2007 to 2011. Validation for the assay will lead to certification and routine use prophage primer sets for detection of S. citri. The increased sensitivity of the assay will benefit citrus nursery certification programs. 04 The beet leafhopper is a single dispersed population in Central California. The beet leafhopper is an important vector of curtovirues, spiroplasmas and phytoplasmas associated with disease agents of curly top, citrus stubborn and potato purple top, respectively. Because these pathogens are from different pathogen taxa, a question arises if different genetic populations of the leafhopper exist which may be predisposed to a specific vector-pathogen association. Circulifer tenellus virus 1 (CiTV1), a dsRNA virus associated with the beet leafhopper, was isolated from 33% of the beet leafhoppers collected from 35 locations in spring and fall along a 100 mile corridor of Highway 99 (central region) and Interstate 5 (western region) in Central California. Incidence and sequence comparison among three CiTV1 strains (identified on the basis of the CiTV1 RdRp gene) indicated a lack of spatial or temporal population structure of the insect virus. This suggests that CiTV1 represents a single population and, similarly, infers only one population of beet leafhopper in Central California. This information is relevant for beet leafhopper control strategies directed at targeting specific populations or ecological habitats of the leafhopper vector as implemented by the State of California.
Impacts
(N/A)
Publications
- Loconsole, G., Onelge, N., Potere, O., Giampetruzzi, A., Bozan, O., Satar, S., De Stradis, A., Savino, V., Yokomi, R.K., Saponari, M. 2012. Identification and characterization of Citrus yellow vein clearing virus, a putative new member of the genus Mandarivirus infecting Citrus spp. Phytopathology. 102: 1168-1175.
- Zhao, H., Sun, R., Albrecht, U., Padmanabhan, C., Wang, A., Coffey, M.D., Girke, T., Wang, Z., Close, T.J., Roose, M., Yokomi, R.K., Folimonova, S., Vidalakis, G., Rouse, R., Bowman, K.D., Jin, H. 2013. Small RNA profiling reveals phosphorus deficiency as a contributing factor in symptom expression for Citrus Huanglongbing Disease. Molecular Plant. 6(2):301-310.
- Satar, S., Kersting, U., Yokomi, R.K. 2012. Presence of two host races of Aphis gossypii Glover (Hemiptera: Aphididae) collected in Turkey. Annals of Applied Biology. 162:41-49.
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Progress 10/01/11 to 09/30/12
Outputs
Progress Report Objectives (from AD-416): Citrus tristeza virus and Spiroplasma citri, causal agent of citrus stubborn disease, are important production-limiting, insect-vectored pathogens in California. Early host response (e.g. over-expressed microRNAs and small interfering RNAs) associated with single and multiple strain graft inoculations of these pathogens in greenhouse tests will be characterized and developed as biomarkers for early-stage pathogen- specific detection. The overall goal is to elucidate mechanism of mild strain cross-protection of Citrus tristeza virus and pathogen genetic bottlenecks resulting from insect vector transmission. New knowledge gained will lead to improved sustainable management strategies for these citrus disease agents and their insect vectors. 1. Develop field deployable systems that provide rapid, sensitive detection of Citrus tristeza virus and Spiroplasma citri in citrus. 2. Determine and characterize genetic variations in Citrus tristeza virus and Spiroplasma citri strains before and after vector passage. 3. Determine changes in host gene expression in Citrus tristeza virus cross-protected citrus that can be used to screen for cross-protective strains of Citrus tristeza virus. Approach (from AD-416): Citrus tristeza virus (CTV) and Spiroplasma citri are both phloem-limited pathogens of citrus and are transmitted by insect vectors. The California Department of Food and Agriculture and stakeholders manage CTV in commercial groves and urban areas by an eradication program and maintenance of pathogen-free budwood sources and citrus propagations in commercial nurseries. CTV eradication was modified in 2009 to detect and eliminate only citrus trees infected with severe strains of CTV. This is achieved by screening field strains of CTV by serology with a monoclonal antibody and polymerase chain reaction (PCR) assays with genotype- specific sequence markers. Genetic diversity of field strains CTV and S. citri will be characterized with respect to the molecular and genetic basis of their host-pathogen-vector relations and disease epidemiology. Further improvement in pathogen surveillance and control requires filling knowledge gaps in host response to pathogen infection as mild CTV strains, which no longer are being eradicated, continue to spread by indigenous aphid vectors. The nature, basis and mechanism(s) of cross-protection will be identified and characterized. CTV symptom phenotype with mixed infections will be monitored for post-translational gene silencing through analysis and characterization of small interfering RNAs, micro RNAs and macro RNAs. New information will facilitate mitigation of losses and misidentifications of CTV and S. citri with the ultimate goal of developing sustainable, integrated management strategies for tristeza and stubborn in California. Research in this project was initiated in April 2012 to replace the previous project (5302-22000-009-00D) which terminated. FY12 milestones are, therefore, in progress. Success was achieved in Milestones 1 (pathogen target capture to membranes); Milestone 2 (cDNA and small RNA libraries for Tristeza-infected cross-protection tests); Milestone 5 and 6 (New tristeza isolates or strains were collected in 2012 characterizations and incorporation into cross-protection tests initiated. For Milestones not met, aphid and leafhopper vector colonies are being developed for vigor and abundance to start vector transmission tests in fall when greenhouse temperature is more favorable for pathogen multiplication (Milestone 3 & 4) and new pathogen collections can be made from field sources (Milestone 4). Accomplishments 01 Cross-protection of Citrus tristeza virus. The mechanism and duration o effective ross-protection (where a mild isolate of the virus can protect plant against disease development caused by a severe isolate of the viru of tristeza are unknown. ARS researchers at Parlier, California, in collaboration with scientists at Consiglio Nazionale Delle Ricerche, Bar Italy, examined RNA interference (RNAi) as a natural plant defense syste for cross-protection of a severe virus strain. Profiling of small RNAs from sour orange plants infected by different combinations of mild and severe tristeza strains revealed defined small RNAs were correlated with plants showing mild or no symptoms. Further profiling revealed 40 microR families with different levels of expression in plants and suggested degradation of tristeza strain. These data suggest RNAi was active in tristeza cross-protection and that small viral RNAs or plant microRNAs m be useful as biomarkers to identify cross-protective tristeza strains. 02 Next generation sequencing of Citrus Tristeza Virus. Cross-protection o tristeza that occurred with a mixture of tristeza genotypes was differen from cross-protection by exclusion of a strain within the same genotype. ARS researchers at Parlier, California, in collaboration with scientists at Consiglio Nazionale Delle Ricerche, Bari, Italy, achieved ~95% full- length genome sequencing of a mild cross-protective Citrus Tristeza Viru (CTV) strain (Dek-NS) using deep sequencing technologies. Complete sequence of mild cross-protective tristeza strains complements studies o RNAi and host microRNA-mediated regulation of gene expression in relatio to disease symptomology. Indepth analysis of molecular interactions between citrus and tristeza strain(s) should lead to improved selection cross-protective tristeza strains to control severe strains of CTV.
Impacts
(N/A)
Publications
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