The original smooth-cylinder trap model did not catch many psyllids, <10%, even in high density population exposure; Psyllid trap efficacy also requires correct field positioning ; One design accidentally had a rougher texture on the outside, and it caught more psyllids than similar traps that were smooth; Newer traps were then developed with intentional projections on the outside; Our best trap design so far has yielded a catch rate of 30-40% ACP, compared to psyllid-capture on yellow sticky traps, as determined by a limited number of controlled field trials; 3D-print designs can be modified rapidly from results of field testing, which is currently underway in FL, TX, and CA; 3D-printed traps can also incorporate species cues for light, sound, and odor attractants to overcome field positioning effects. The Asian citrus psyllid is known to obtain nutritional and defensive benefits from its complement of endosymbiotic bacteria. The most abundant of its associated endosymbionts, ‘Candidatus Profftella armatura,’hydroponic nft system has been shown to devote approximately 15% of its predicted genome to polyketide biosynthesis. The interaction between the ACP and Profftella is an ideal target to combat the psyllid since this symbiont is found in all populations of the ACP surveyed thus far and in no other insect.
A dual proteomic-metabolic analysis indicated that the complement of polyketides produced by Profftella plays a significant role in the insect host response to infection with ‘Candidatus Liberibacter asiaticus’ , the bacterial pathogen responsible for Huanglongbing . The primary mechanism of this defense is via production of the cytotoxic polyketide diaphorin, estimated to be produced at 1.3 ± 0.3 mg per 1,000 adult insects. ACP populations reared on CLas-infected citrus trees show significant upregulation of Profftella proteins involved in polyketide biosynthesis as well as quantitative differences in abundance of diaphorin and a related polyketide compared to ACP reared on healthy trees. In this work, we aim to verify previous structural characterizations of diaphorin and probe its potential interactions with receptors in the ACP to gain a better understanding of the interplay between Profftella, diaphorin, and CLas within the insect. We extracted approximately 6,000 adult ACP and verified the presence of diaphorin in the extract using infusion mass spectrometry. Diaphorin is further enriched from the extract using preparative High Performance Liquid Chromatography . Nuclear magnetic resonance is used to evaluate the structure of diaphorin isolated within the fractions, and LC-high resolution mass spectrometry is used to confirm stability of the compound. Future plans include immobilization of purified diaphorin on a solid support resin for use in an affinity purification strategy to identify interacting insect proteins.
This will allow a better understanding of the specific role and localization of diaphorin within the insect, as well as provide insight into the systemic response to CLas acquisition with an aim toward potential control strategies. Citrus Huanglongbing or greening is a devastating bacterial disease that has destroyed millions of trees and is associated with phloem-residing ‘Candidatus Liberibacter asiaticus’ in Florida. In this study, we evaluated the control effect of different antimicrobials including oxytetracycline, streptomycin, and plant defense inducers via trunk injection. Spatiotemporal dynamics of oxytetracycline in planta was evaluated. Las-infected ‘Hamlin’ sweet orange trees on ‘Swingle’ citrumelo root stock at the early stage of decline were treated with oxytetracycline hydrochloride using trunk injection with varying number of injection ports. Spatiotemporal distribution of OTC and dynamics of Las populations were monitored by HPLC method and qPCR assay, respectively. Uniform distribution of OTC throughout tree canopies and root systems was achieved 2 days post injection. High levels of OTC were maintained in leaf and root for at least 1 month and moderate OTC persisted for more than 9 months. Reduction of Las populations in root systems and leaves of OTC-treated trees were over 95% and 99% between 2 and 28 DPI. Conditions of trees receiving antimicrobial treatment were improved, fruit yield was increased, and juice acidity was lowered than water-injected control even though their differences were not statistically significant during the test period. Our study demonstrated that trunk injection of OTC could be used as an effective measure for integrated management of citrus HLB. The Citrus Genome Database is being developed as a one-stop resource for citrus genetics, genomics, and breeding research.
In this presentation we highlight CGD features that provide the citrus community with data and tools to combat Huanglongbing . The database has been redesigned and features a streamlined user interface that allows for quick access to data and tools and is also mobile friendly. It contains curated citrus genetic marker, map, and QTL data, genome data for clementine and sweet orange, as well as annotated reference transcriptomes generated by analysis of published RNA-Seq and EST datasets. The database also has the most up-to-date version of CitrusCyc v3.0 which includes metabolic maps for the C. clementina and C. sinensis genomes. Tools such as BLAST for searches against the genome sequences, GBrowse/JBrowse for viewing genomes, and CMap/TripalMap for viewing and comparing genetic map data are also available on CGD. In addition to the tools to view and search published data in CGD, the Breeding Information Management System allows breeders to upload their data via the web, or directly from the Field Book app, to a private account. The private data than can be analyzed in conjunction with the public data. In addition to citrus data, the genomes of the different ‘Candidatus Liberibacter’ species and psyllid vector are available in CGD in JBrowse and the sequence data is searchable with the BLAST tool. As a community resource, CGD is being developed based on user feedback with the goal of providing the data and tools that will enable citrus crop improvement. CGD is supported by USDA-NRSP10, NSF-PGRP, and USDA-SCRI. First report of delivery of two synthesized oligo nucleotide products into plants. Subsequent feeding on treated plants by insects resulted in ingestion and systemic movement of the two gene-based targeting products: FANA_ASO, _; and a PPMO . Topical sprays onto citrus leaves, root absorption, and tree trunk injections, resulted in cell delivery of the molecules which were designed to: Asian citrus psyllid; Plant pathogenic bacteria of citrus, Candidatus Liberibacter asiaticus , Psyllid endosymbionts, and Diaprepes abbreviatus, weevil.Treatments reduced CLas bacteria within infected citrus trees,nft channel reduced Wolbachia in cell cultures, the insects, while resulting in increased insect mortality. This is the first evidence for successful delivery of FANA_ASOs and PPMO into plants, and use of these molecules for plant delivered strategies to reduced plant pathogens in citrus, and to manage insect pests. Molecules were shown to move systemically through plant tissues as visualized with Confocal microscopy and spectrophotometry.Adult insects showed systemic movement through hemolymph and organs: supra and sub esophageal ganglions, fatbodies, nerves, and alimentary tract. Results suggest a role for these products in the reduction of plant pathogens like CLas, associated with Citrus greening disease; and reduction of insect vectors of pathogens on citrus and other agricultural crops.Two circular prophage genomes have previously been described integrated in Ca. Liberibacter asiaticus strain UF506 genome, and all Florida Las strains have both prophages. Either one or rarely, both, prophages were reported absent in certain recently sequenced Chinese and Japanese Las strains. The SC1 lytic cycle, marked by upregulation of several late genes including a functionally lethal holin , is activated when Las is in planta, but not when infecting the Asian citrus psyllid host. The holin promoter was cloned into the wide host range vector pUFR071, structurally replacing the native lacZ promoter to drive the beta-glucuronidase reporter gene. The holin promoter exhibited very strong and constitutive expression of the GUS reporter gene in Liberibacter crescens , a culturable proxy for Las. We previously reported the presence of an unidentified, heat labile, protease-sensitive repressor in aqueous psyllid extracts that quenches the expression of the holin:GUS reporter. Tandem LCMSMS analyses were used to identify ca. 25 peptide fingerprints in the DNA-binding protein eluate captured by the holin promoter DNA immobilized on magnetic DynaBeads.
A putative repressor protein was identified that was unique to the Wolbachia bacterial endosymbiont found in psyllids and absent in Wolbachia found in the fruit fly . This putative Wolbachia repressor gene was cloned in an expression vector and used for in vitro cell-free protein synthesis. The in vitro translated Wolbachia protein partially repressed the holin::GUS reporter in a dose-independent manner, as compared to the aqueous extract from the psyllid, thus indicating that complete suppression of holin promoter requires an additional partner. Heat-inactivated psyllid extract was unable to enhance the Wolbachia repressor- induced suppression of holin promoter activity, confirming the heat labile proteinaceous nature of the additional psyllid-sourced partner. A conspicuous feature of plant defense reactions in response to an attempted pathogen invasion is the engagement of an oxidative burst. We have previously shown that an SC2 prophage-encoded peroxidase in Ca. Liberibacter asiaticus strain UF506 represents an important lysogenic conversion gene whose expression may increase bacterial fitness and delay symptom development in the host plant. However, absence of either one or both prophages in certain recently sequenced Chinese and Japanese Las strains, prompted a review of H2O2-detoxification mechanisms conserved amongst Liberibacters. Two putative peroxiredoxin genes have been annotated on the Las chromosome, which are conserved and identical among all the sequenced Las strains. The BCP-like peroxiredoxin is predicted to be nonclassically secreted. The expression levels of Las PR2 in both citrus and psyllid were equivalent. Notably, however, the expression of the predicted secreted BCP-like peroxiredoxin was similar to that of PR2 in citrus but nearly undetectable in psyllids, reminiscent of the low level of SC2_gp095 peroxidase expression in psyllids. Despite carrying a native ortholog, Lcr transformed with the BCP-like peroxiredoxin showed a significant increase in tolerance to 100 µM H2O2, as compared to a marginal 4-5 fold tolerance provided by either the PR2 peroxiredoxin or SC2 peroxidase. Most importantly, BCP-like peroxiredoxin augmented Lcr tolerance to 50 µM tert-butyl hydroperoxide by nearly 1000 fold. We hypothesize that both the Las peroxiredoxins may be implicated in alleviating the oxidative burden originating from physiological electron transport reactions. However, the extracellular BCP-like peroxiredoxin may be essential for Las survival in an oxidative host cell environment through a) direct detoxification of host-generated H2O2, b) limiting the catastrophic chain reaction spread of a lipid peroxidation event initiated by H2O2 permeating the bacterial cell membrane, and c) most importantly, akin to the secreted SC2_gp095 peroxidase, dampening the systemic propagation of H2O2-mediated defense signaling in the host plant following an initial colonization event. Las BCP-like peroxiredoxin may represent a critical secreted effector conserved across all pathogenic Liberibacter species that functions to suppress host symptoms, a tactic used by most biotrophic plant pathogens. Detection of HLB-associated Candidatus Liberibacter asiaticus by real time PCR has been widely used in citrus industry since its development a decade ago. However, the methodology is not adaptable to quantitation by digital droplet PCR since it uses a multicopy mitochondrial gene as an internal control. Since there are hundreds of copies of COX gene per cell, inefficient amplification of the low copy target gene can occur. In this study, we sequenced a single copy nuclear gene, malate dehydrogenase from over 100 accessions including several citrus and citrus relatives in the subfamily Aurantioideae. The plant samples selected are known to be hosts of both HLB and the psyllid vector. Primers and probe were developed using conserved regions of MDH gene for Taqman® based qPCR and also for ddPCR analyses. We conducted qPCR assays using various dilutions of HLB infected plant samples to detect CLas in duplex reactions using two different internal controls – COX and MDH in separate reactions. ddPCR assays were conducted using a BioRad QX200 droplet digital PCR System. The advantages of using MDH as an internal control gene in both qPCR and ddPCR will be discussed. Since confirmatory tests to detect the presence CLas cannot be done with plant samples having low bacterial titers, additional confirmatory assays by ddPCR of other regions of CLas genome were also developed. Detection of CLas in samples with low titers is critical for both prevention and management of HLB, especially during early stages of the epidemic, and to slow down the spread of disease. Testing of psyllid vectors for the presence of huanglongbing-associated Liberibacters is known to be an effective method for early detection of HLB in citrus groves.