Citrus canker is an economically important bacterial disease of most commercial citrus cultivars resulting in significant losses worldwide. Spread of citrus canker has been a severe problem to the citrus industry of Florida. How bacteria escape from infected plants is underexplored. Understanding the molecular determinants of lesion rupture, how Xcc survives in the intercellular spaces, and how Xcc releases from lesions of host plants will provide many fundamental and practical benefits. Despite the tremendous effort to eradicate citrus canker, the pathogen has spread to most citrus production areas in Florida and continues to spread. Understanding the genetic mechanism of release of Xanthomonas axonopodis pv. citri (Xac) from citrus canker lesions will help develop effective control and containment strategies to stop citrus canker pathogen from spreading. The goal of the proposed research is to understand the genetic mechanism of release of Xac from citrus canker lesions. The specific objectives are to: 1. characterize critical genes involved in release of X. axonopodis pv. citri from citrus canker lesions; 2. understand the release mechanism by studying the host response of citrus upon infection by Xac wild-type strain and mutant strain(s) affected in release from citrus canker lesions. We have identified 12 EZ-Tn5 transposon mutants of Xac with reduced capacities of release from citrus canker lesions. The insertion sites of the 12 mutants have been identified with insertions in 11 different genes including xanA, btuB, gumC, gumB, gumK, gpsA, and several hypothetical genes. Currently, complementation analysis of the mutants is underway. The complementation constructs are being made for the selected mutants using pUFR053. Bacterial growth assays of the mutants and the wild type strain in grapefruit ‘Duncan’and sweet orange ‘Valencia’ have been conducted. EPS and LPS production, capsule assays, and other related assays on the mutants and the wild type strain are being tested. Preliminary tests are being conducted on studying the host response of citrus upon infection by Xac wild-type strain and mutant strain(s) affected in release from citrus canker lesions using quantitative reverse transcription PCR or Affymetrix array.
Xacm strain: Xanthomonas axonopodis pv. citrumelo (Xacm) is a citrus pathogen causing citrus bacterial spot disease that is geographically restricted within the state of Florida. Illumina, 454 sequencing and optical mapping were used to obtain a complete genome sequence of Xacm strain F1, 4.9Mb in size. The strain lacks plasmids. Phylogenetic analysis revealed that this pathogen is very close to the tomato bacterial spot pathogen Xcv 85-10 with a completely different host range. We also compared Xacm to the genome of citrus canker pathogen Xac 306. Comparative genomic analysis showed differences in several gene clusters like Type 3 effectors, Type 4 secretion system, lipopolysaccharide synthesis and others. In addition to pthA, effectors such as xopE3, xopAI and hrpW were absent in Xacm while present in Xac. These effectors might be responsible for survival and reduced virulence of this pathogen on citrus compared to Xac. We also identified unique effectors in Xacm that may be related to the different host range as compared to Xac. Xacm F1 was shown to be pectate lyase deficient in Hildebrand’s medium which is consistent with the sequence analysis. Comparison of the complete genome sequence of Xacm to Xac and Xcv provides valuable insights into the mechanism of bacterial virulence and host-specificity. The complete genome of Xanthomonas axonopodis pv. citrumelo has been deposited at EMBL/DDBJ/GenBank under the accession number NC_016010. The manuscript entitled ‘Comparative genomic analysis of Xanthomonas axonopodis pv. citrumelo F1, which causes citrus bacterial spot disease and related strains provides insights into virulence and host-specificity.’ has been published by Journal of Bacteriology. Xac Aw strain: Whole genome sequencing of Xanthomonas axonopodis pv. citri strain Aw has been completed. The Genome sequence is 5.3 Mb in size. The sequence was annotated using FgeneSB and the annotation curation was done using GenePrimp. The annotated sequence has been reviewed by ICBR and all the doubtful regions were checked and have been confirmed by PCR and sequencing. Plasmids were purified and sequenced separately using 454 titanium sequencing. The sequences for the 2 plasmids were closed using CLCbio Genomics Workbench. Gap closure was done by PCR and sequencing and has been reviewed by ICBR for any doubtful regions. Annotation has been done and curation has been performed using geneprimp. Strain specific genes including XopF1 and avrGf1 gene have been studied for their involvement in virulence and host specificity on different citrus varieties. Xac A*270 and Xacm 1381 (Cu resistant) strain: Plasmids from both the strains were purified and sequenced with Illumina 75 x2 bp sequencing at ICBR. The sequences have been trimmed and De novo sequencing has been performed. Gap closure for the plasmids and chromosomes have been completed. We also characterized two novel virulence related genes gpsX and nlxA which were published on BMC Microbiology and Molecular Plant Pathology, respectively. The gpsX gene encodes a putative glycosyltransferase, which is highly conserved in the sequenced strains of Xanthomonas. Our data indicate that the gpsX gene is involved in EPS and LPS synthesis and biofilm formation in Xac and suggest that the gpsX gene contributes to the adaptation of Xac to the host microenvironments at early stage of infection and thus is required for full virulence on host plants. The nlxA (novel LPS cluster gene of X. citri ssp. citri) gene, in the LPS cluster of X. citri ssp. citri 306, was required for O-polysaccharide biosynthesis by encoding a putativecharacterized. Our results indicate that nlxA plays an important role in extracellular polysaccharide production, biofilm formation, stress resistance, motility, virulence and in planta growth in the host plant.
Development of alternative or complementary approaches for effective management of citrus greening is highly desirable and will greatly help the citrus industry due to the difficulty to control the HLB disease. Considering the highly destructive nature of HLB disease and the lack of control measures, there is a huge potential to develop antimicrobial small molecules against the causal agent thus to suppress the population of Las in planta and to reduce the innoculum for psyllid transmission. Treatment of Ca. L. asiaticus infected citrus could be pursued by applying antimicrobials to infected trees. The most common targets for antimicrobial agents development include receptors, proteins and enzymes, DNA, RNA and ribosomal targets. Among them, proteins have become the major target due to their druggable characteristics. In this study, we presented our research on screening small molecule inhibitors against SecA. SecA is one essential component of the Sec machinery which provides a major pathway of protein translocation from the cytosol across or into the cytoplasmic membrane. The Sec pathway was also shown to be required for virulence of Ca. L. asiaticus in our study. SecA is the protein translocase ATPase subunit, which involves in pre-protein translocation across and integration into the cellular membrane in bacteria. First we filtered the structures based on their physico-chemical properties, e.g. Molecular Weight, H-Bond Donor, H-Bond Acceptor & Rotatable bonds and structurally similar to adenine moiety. Approximately 5000 structures were retrieved from ~5 million structures of commercially available databases. The identified data set was used for virtual screening by molecular docking method. Based on the dock scores we eliminated about 4500 low scored structures and selected ~500 (10%) for further molecular docking & minimization to evaluate the scoring functions (Dock glide scores, Hydrophilic, Hydrophobic e.t.c). Based on scoring functions, structural diversity, and our chemical intuition we have chosen forty structures for biological activity studies against purified SecA protein. For that purpose, SecA of Ca. L. asiaticus was expressed in E.coli using the pE-SUMO vector and purified. The inhibition assays of the 40 compounds against SecA was done as described previously by Denis et al. 1978. The IC50 ranged from 0.3’M to 100’M. Those findings were published in article entitled: “Discovery of novel SecA inhibitors of Candidatus Liberibacter asiaticus by structure based design” on Bioorganic & Medicinal Chemistry Letters Volume 21, Issue 14, 15 July 2011, Pages 4183-4188. Using various computational techniques, twenty compounds were selected for biological activity study against SecA of Candidatus Liberibacter asiaticus and Agrobacterium tumefaciens. Five compounds were found to inhibit the ATPase activity of SecA of Las in nano molar concentrations and showed antimicrobial activities against Agrobacterium tumefaciens with MBC ranging from 128 .ug/ml to 256 u.g/ml. The MBC values of all compounds are 2-4 folds higher than streptomycin, which indicates they could probably act as potential antimicrobial agents. These compounds appear to be suitable as lead compounds for further development of antimicrobial compounds against Las. In the optimization step, we have identified fourteen more compounds with potential as antimicrobial compounds. Those compounds are under further study to determine their inhibitory activity against the ATPase activity of SecA of Las and its antimicrobial activities.
The purpose of this project is to preserve citrus germplasm in Florida that is threatened by loss due to huanglongbing and citrus canker. Using input from stakeholders and scientists, threatened germplasm has been identified, collected, and established at Ft. Pierce. Presently 66 accessions are being held at Ft. Pierce. These accessions have been subjected to antibiotic therapy, and are testing negative for Las by qPCR. In collaboration with the Florida Citrus Germplasm Introduction Program, the accessions are beginning thermotherapy, which will be conducted for a 16 week time period. Following the thermotherapy, the accessions will be testing using laboratory methods for Las as well as other graft transmissible diseases of citrus, including viroids. Following the testing and depending on the results, the accessions will be forwarded to the USDA ARS Repository in Riverside for further clean up and testing before release from quarantine. The experiment evaluating the effectiveness of penicillin/streptomycin mixture, cyclohexamide, D4E1 (antimicrobial peptide) and heat treatment (2 weeks of continuous 40 C at 60% relative humidity with 12 hr light/12 hr dark in a growth chamber) as reported on last quarter, is being repeated.
Mid Florida Citrus Foundation (MFCF) a 501c5 not for profit organization which has supported (past 25 years) and currently supports citrus research efforts of scientists from the University of Florida, USDA and private industry. The MFCF supports citrus research through the employment of a full time grove manager whom works closely with researchers to ensure that their projects are handled properly and that the grove is an excellent condition. The management of this grove requires extra financial commitment as grove care costs tend to be higher than commercial groves due to the nature of many of the research projects. Current projects being conducted at the MFCF are Asian citrus psyllid (ACP) pesticide evaluation control trials, low volume applicator trials, windbreak evaluation, HLB nutritional programs, new and existing herbicide trials, variety and rootstock evaluation trials. During the recently completed quarter (January 1 to March 31, 2012), the following highlights occurred at the Mid Florida Citrus Foundation ‘ A.H. Krezdorn Research Grove: ‘ A trial/demonstration involving the application of DuPont Vydate’ Insecticide/Nematicide to improve the root health of young Valencia orange trees was initiated. DuPont staff will assist in evaluations of this grower trial. ‘ Dr. Singh initiated a trial to evaluate a pre-mix product containing Oryzalin and Glyphosate for efficacy in young citrus plantings. ‘ Applications continued and harvest of the Midsweet and Valencias occurred in the large scale nutritional demonstration/trial where four different commercial programs are under evaluation. ‘ BASF is reporting positive response on melanose and greasy spot control in their ongoing Headline’ evaluations. ‘ Drs. Stelinski and Rogers have continued evaluations of Asian citrus psyllid and citrus leafminer management in their areas. ‘ Drs. Albrigo and Wong have continued to evaluate antibiotics to manage HLB. ‘ The Plant Improvement Group is reporting observations of differences in foliar HLB symptom expression and fruit drop among approximately 20 clones of Valencia being evaluated. ‘ Additional small plantings have been made: o Approximately 1 acre for Dr. Futch for herbicide studies o Additional selections for the New Varieties Development and Management Corporation ‘ The Sugar Belle’ area will be hedged to reduce crop load and improve fruit size and quality. ‘ A field day attended by 71 growers was held on March 1, 2012 and focused on: o Large scale commercial nutritional program demonstration/trial o Asian citrus psyllid management o Leafminer monitoring and management o USDA citrus rootstock evaluations o Critical temperatures in citrus grove canopy during freeze events
We obtained 27 healthy, 3 yr, Valencia grafted on Cuban rootstocks to evaluate 3 foliar treatments to be initiated in May 2012: 1) L- Arginine to stimulate the innate immune response; 2) Gibberellin in combination with 6-benzyl adenine to improve innate immunity and to inhibit pathogen mediated source-sink relationship in favor of the plant and 3) Sucrose plus the herbicide atrazine to improve the photosynthetic capacity of the leaf tissues. We intend will evaluate the efficacy of the treatments, ensure they are not phytotoxic or cause any stress to the trees but enhance immune response and improve plant health. L- Arginine, will be sprayed to enhance the innate immune response of citrus plant tissue to HLB through the induction of disease resistance pathways via NO biosynthesis, promoting the activities of plant defensive enzymes, like phenylalanine ammonia-lyase (PAL), chitinase (CHI), polyphenol oxidase (PPO) and .-1,3-glucanase (GLU). Zheng et al (2011) reported that endogenous NO concentrations were positively correlated with an increase in defensive enzyme activity, when postharvest disease resistance was induced after L-arginine treatment at pre-harvest stage in tomatoes. Genes encoding pathogenesis-related protein 1(PR1), aconitase, mitogen-activated protein kinase (SIPAK) and PAL will be used as biomarkers to evaluate this treatment. A mixture of Gibberellin (GA3) and 6-benzyladenine (BA) is included in our therapeutic strategy because of the important role they play in modulating the complex hormone-crosstalk and signaling pathways. We expect the treatment to enhance the modulation of jasmonic acid-salicylic acid (JA-SA) crosstalk and systemic acquired resistance (SAR) and the regulation of sugar metabolism, starch accumulation, glycolysis and minor carbohydrates metabolism. Genes encoding gibberellin regulated family proteins, gibberellin 20 oxidase 2, gibberellin-regulated protein 4 precursors and protein gast1 precursor will be used as biomarkers for this treatment. The third foliar treatment combines sucrose and the herbicide atrazine to jump start photosynthesis in source leaves producing a flow of carbon in HLB infected plants. Atrazine a well-known photosystem II inhibitor affects gene expression of plants, seedling physiology, potentiality impairs protein translation and reactive-oxygen species (ROS) defense mechanism (Ramel, et al 2007). However when applied in combination with sucrose, induces a protection against atrazine suggesting an important interaction between sucrose and xenobiotic signaling or/and sucrose and ROS signaling. Genes encoding glutathione S-transferase, bZIP transcription factor protein, granule-bound starch synthase, starch branching enzyme 2.2, invertase, acid .-fructofuranosidase, non-expressor of PR gene 1, glucose-1-phosphate adenyltransferase family protein and carbohydrate trasmembrane trasporter will be used as biomarkers for this treatment. Expression levels of each of these biomarker genes will be determined by qRT-PCR. Literature cited: Zheng Y., Sheng J., Zhao R., Zhang J., Shengnan L, Lingyi L and Shen L. Preharvest L-arginine treatment induced postharvest disease resistance to Botrytis cinerea in tomato fruits. J. Agric and Food Chem 59: 6543-6549. Ramel F., Sulmon C., Cabello-Hurtado F., Taconnat L., Martin-Magniette M-L., Renou J-P., El Amrani A., Couee I., and Gouesbet G. 2007. Genome-wide interacting effects of sucrose and herbicide-mediated stress in Arabidopsis thaliana: novel insights into atrazine toxicity and sucrose induced tolerance. BMC Genomics 8:450.
Our overall goal is to better understand how California mandarins respond to the postharvest environment and how handling practices influence the consumer experience. Our objectives for the 2011-2012 fiscal year are: 1. Screen CA mandarins for postharvest characteristics including susceptibility to chilling injury and alteration in sensory characteristics; 2. Test alternative technology for quick determination of internal citrus quality (SSC, TA) ‘ collaboration with D. Slaughter; 3. Screen mandarin varieties and the M. Roose Fortune x Fairchild mapping population (at UC Riverside) for off-flavor development and postharvest characteristics. During the last 6 months we have mostly met our proposed milestones for this time period. Commencing in November 2011 we began harvesting Satsuma mandarins for determination of waxing-induced anaerobisis and enhanced ethanol production. We have been harvesting fruit approximately every 2 to 3 weeks for a total of 8 sampling times as of 2/28/2012. We harvest 3 varieties per collection. We plan to have several more samplings. Unfortunately, the fruit load on the mapping population at UC, Riverside is low for the current year. Therefore we have had only 1 harvest of fruit from this trial. This was done on 2/13/2012. The analysis of this fruit is underway. We are also conducting more in depth evaluations of different fruit waxing strategies. We completed a test using Owari Satsuma mandarin that was harvested in November 2011. We have also completed a test using Daisy mandarin (harvested January 2012). Tests for Gold Nugget and Tango mandarin are underway. We plan to do 1 additional test using Tango in April. We are in the midst of sampling fruit for David Slaughter and this will be completed in May 2012. We conducted a consumer panel using Owari Satsuma in December 2011 at the annual open house at UC Lindcove Research and Extension Center.
Website development: The Genome Viewer on the HLB-Citrus Greening Genome Resources Website has been upgraded to GBrowse version 2.4, providing users with a smoother interface for accessing genome data and bioinformatic characterization. A short series of instructions for use of the primer design tool has been added to the web site to aid users in navigation of the recently installed Primer3 module for design of PCR primers to be used for cloning and diagnostic purposes. Bioinformatic Analyses: In collaboration with Manjunath Keremane (ARS-Riverside) approximately 0.38 MB of assembled sequence has been generated from reads of Ca. Liberibacter psyllaurous (LPS) generated by Manjunath’s group. Alignment of the 16 LPS contigs with the Ca. L. asiaticus and Ca. L. solanacearum genome sequences has revealed unique genes in the different strains with potential relevance to differences in the interactions of these strains with their respective plant hosts. As a reduced genome bacterium, Ca. Liberibacter has lost many of the metabolic functions characteristic of free-living bacteria and so is highly dependent on the nutritional environment provided by the insect vector and plant host in order to thrive. Wayne Hunter (ARS-Ft. Pierce) has led a collaborative effort directed toward sequencing the metagenome of the Asian citrus psyllid. There is evidence supporting the presence of a wide variety of bacteria living in association with the psyllid vector and potentially contributing to a nutritional environment favorable to Liberibacter. Sequence reads derived from Wolbachia, a confirmed psyllid endosymbiont, have been pulled from the metagenome dataset, assembled into a draft genome sequence, and compared with other sequenced Wolbachia strains. To identify less well characterized endosymbionts using the sequence data and to compare the predicted repertoire with those identified using other strategies, several approaches are being applied including screening of the metagenome dataset for reads corresponding to bacterial ribosomal RNA sequences, sorting of the short raw reads according to sequence properties, and screening of short sequence assemblies for sequence similarity to bacterial sequences in public databases. While endosymbiont populations in insects have long been known, more recent availability of plant genome sequences is revealing that many plants contain bacterial and fungal endosymbionts with potential to impact interactions between the plants and their pathogens. Strategies are currently being explored for creation of a computational pipeline that could be used to examine sequence data for plants hosts of citrus greening for the presence of endosymbiotic microbes. Identification of endosymbiots in both the insect vector and plant host is an important first step to characterizing the nutritional relationships that allow Liberibacter to survive in these environments.
The purpose of this project is to preserve citrus germplasm in Florida that is threatened by loss due to huanglongbing and citrus canker. Using input from stakeholders and scientists, threatened germplasm has been identified, collected, and established at Ft. Pierce. Preliminary clean up is being done on the accessions while they are being held at Ft. Pierce before the remainder of the plants will be forwarded to the USDA ARS Repository in Riverside for further clean up and testing before release from quarantine. An experiment was conducted to compare recently disclosed methods to reduce or eliminate Candidatus Liberibacter asiaticus (Las), the bacterium associated with HLB in Florida. Five HLB-infected priority breeding selections of different parentage were budded onto Volkamer lemon. Sixteen trees of each of four genotypes moderately symptomatic for HLB were subjected to the following treatments: Control; Penicillin/ streptomycin mixture; cyclohexamide; D4E1 (antimicrobial peptide); and heat treatment. The antibiotic mixture, cyclohexamide, and D4E1 were all vacuum infused into the pre-cut buds, then rinsed and budded. Heat treatment, consisting of 2 weeks of continuous 40 C at 60% RH with 12/12 hours light/ dark in a growth chamber, was conducted after scions from the control, penicillin/streptomycin mixture, cyclohexamide, and D4E1 antimicrobial peptide treatments had grown and hardened. Controls remained on the greenhouse bench the entire time. The percent of plants with Ct values = 40 (e.g. no Las detectable) and mortality of the plants, respectively, undergoing the treatment after about three months were: Control treatment, 29%, 11% mortality; Heat Treatment, 35%, 2% mortality, D4E1 antimicrobial peptide, 52%, 6% mortality; Penicillin/streptomycin, 57%, 9% mortality; and cyclohexamide, 23%, 80% mortality. Six clones of accessions from Florida are currently undergoing final biological indexing for release from quarantine status at the Repository.
Objective 1. Localization of Liberibacter asiaticus (Las) in the Asian citrus psyllid (ACP). This objective has been achieved and two papers have been published on it. Using quantitative PCR (qPCR) and fluorescence in situ hybridization (FISH), we detected Las in the alimentary canal, hemolymph, salivary glands, and other organs/tissues of ACP (Ammar et al., 2011, Ann. Entomol. Soc. Amer. 104:526-533 & Ammar et al., 2011, J. of Phytopathology, 159:726-734). Using both qPCR and FISH, the proportion of Las-infected salivary glands was significantly lower than that of infected alimentary canals or other organs/tissues. Using qPCR in combination with a ‘detached-leaf assay’ method, only 4-10% of the psyllids were able to inoculate Las into detached citrus leaves, although 46-78% of these psyllids were Las-infected (PCR-positive). Our results show the near systemic infection of ACP organs/tissues by Las and suggest that both the alimentary canal and salivary gland constitute infection and/or transmission barriers to Las in this vector. In the salivary glands both entry and exit barriers are proposed, whereas in the alimentary canal only an entry barrier is indicated. Objective 2. Elucidation of various acquisition and transmission parameters between ACP and Las. [A] We continued developing a new ‘detached-leaf assay’ method that potentially speeds up Las-inoculativity tests on ACP from the current 6-12 months (when using whole citrus seedlings for inoculation) to only 2-3 weeks (when using detached citrus leaves). This new method (Ammar et al. 2011, Proceedings of 2nd Inter. Res. Conference on HLB, Orlando, FL, Jan. 2011) can save considerable time, material and greenhouse space, and may hopefully enhance vector-relation studies on Las and other Liberibacter spp. associated with HLB. [B] We conducted two large experiments to study the effects of various acquisition access periods (AAP) on Las-infected citrus plants by nymphs and adults on Las acquisition and transmission as well as on Las replication in ACP. Following 1-7-day AAP by nymphs, 34-52% of ACP became infected, whereas only 11-23% were infected after 1-7-day AAP by adults. Using qPCR with specific primers to Las and to a psyllid gene, the relative Las titer was generally higher in nymphs than in adults and higher with longer AAP in both life stages. We are currently repeating these experiments to confirm these results. [C] Since nymphs are known to be much more efficient than adults in Las acquisition and/or transmission, a study on the feeding behavior as well as stylet ultrastructure and morphometrics in ACP nymphs and adults is underway. Our early results indicate that the stylet length of first instar nymphs averaged 259 ‘m (80% of the body length) whereas that of the 5th (last) instar was more than twice as long (614 ‘m, 34% of the body length). The stylet length of the latter instar, however, was not significantly different from that of the adults. Nymphs feed only on young citrus leaves, especially on the sides (rather than the top) of the midrib, whereas adults can feed on both the sides and top of young and older (mature) leaves. Cross sections in healthy and Las-infected citrus leaves indicated that the distance to the phloem is shorter from the sides of the midrib compared to that from the top, and is considerably shorter in young citrus leaves compared to that in mature leaves. These results at least partly explain the preference of nymphs to feed on the sides, rather than the top, of the midrib as well as their inability to feed on older/mature leaves. Las bacterium is known to reside in the phloem of infected citrus plants. Transmission and scanning electron microscopy of the stylets of ACP nymphs and adults is underway, which may reveal the negative and/or positive roles of the salivary and food canals of the maxillary stylets in Las transmission by ACP nymphs and adults from the phloem of diseased plants.
Objective 1. Localization of Liberibacter asiaticus (Las) in the Asian citrus psyllid (ACP). This objective has been achieved and two papers have been published on it. Using quantitative PCR (qPCR) and fluorescence in situ hybridization (FISH), we detected Las in the alimentary canal, hemolymph, salivary glands, and other organs/tissues of ACP (Ammar et al., 2011, Ann. Entomol. Soc. Amer. 104:526-533 & Ammar et al., 2011, J. of Phytopathology, 159:726-734). Using both qPCR and FISH, the proportion of Las-infected salivary glands was significantly lower than that of infected alimentary canals or other organs/tissues. Using qPCR in combination with a ‘detached-leaf assay’ method, only 4-10% of the psyllids were able to inoculate Las into detached citrus leaves, although 46-78% of these psyllids were Las-infected (PCR-positive). Our results show the near systemic infection of ACP organs/tissues by Las and suggest that both the alimentary canal and salivary gland constitute infection and/or transmission barriers to Las in this vector. In the salivary glands both entry and exit barriers are proposed, whereas in the alimentary canal only an entry barrier is indicated. Objective 2. Elucidation of various acquisition and transmission parameters between ACP and Las. [A] We continued developing a new ‘detached-leaf assay’ method that potentially speeds up Las-inoculativity tests on ACP from the current 6-12 months (when using whole citrus seedlings for inoculation) to only 2-3 weeks (when using detached citrus leaves). This new method (Ammar et al. 2011, Proceedings of 2nd Inter. Res. Conference on HLB, Orlando, FL, Jan. 2011) can save considerable time, material and greenhouse space, and may hopefully enhance vector-relation studies on Las and other Liberibacter spp. associated with HLB. [B] We conducted two large experiments to study the effects of various acquisition access periods (AAP) on Las-infected citrus plants by nymphs and adults on Las acquisition and transmission as well as on Las replication in ACP. Following 1-7-day AAP by nymphs, 34-52% of ACP became infected, whereas only 11-23% were infected after 1-7-day AAP by adults. Using qPCR with specific primers to Las and to a psyllid gene, the relative Las titer was generally higher in nymphs than in adults and higher with longer AAP in both life stages. We are currently repeating these experiments to confirm these results. [C] Since nymphs are known to be much more efficient than adults in Las acquisition and/or transmission, a study on the feeding behavior as well as stylet ultrastructure and morphometrics in ACP nymphs and adults is underway. Our early results indicate that the stylet length of first instar nymphs averaged 259 ‘m (80% of the body length) whereas that of the 5th (last) instar was more than twice as long (614 ‘m, 34% of the body length). The stylet length of the latter instar, however, was not significantly different from that of the adults. Nymphs feed only on young citrus leaves, especially on the sides (rather than the top) of the midrib, whereas adults can feed on both the sides and top of young and older (mature) leaves. Cross sections in healthy and Las-infected citrus leaves indicated that the distance to the phloem is shorter from the sides of the midrib compared to that from the top, and is considerably shorter in young citrus leaves compared to that in mature leaves. These results at least partly explain the preference of nymphs to feed on the sides, rather than the top, of the midrib as well as their inability to feed on older/mature leaves. Las bacterium is known to reside in the phloem of infected citrus plants. Transmission and scanning electron microscopy of the stylets of ACP nymphs and adults is underway, which may reveal the negative and/or positive roles of the salivary and food canals of the maxillary stylets in Las transmission by ACP nymphs and adults from the phloem of diseased plants.
The Citrus Greening Bibliographical Database is managed jointly by the Entomology group at the University of Florida – IFAS in Immokalee and the Florida Center for Library Automation in Gainesville and continues to be a widely used source for information on citrus greening (HLB) for researchers, growers, and students worldwide. The database was designed to be a centralized source of current, relevant information with an accessible, user-friendly interface. Entries represent research from around the world on the various aspects of HLB, including the associated bacteria (Candidatus Liberibacter spp.), the vectors [Diaphorina citri Kuwayama and Trioza erytrea], effects of the disease on plants and vectors, and management tactics. We now have 2087 citations, 86% of which are linked to their original sources. While the majority of the entries are in English (86%), the remaining 14% of the entries are in Spanish, Portuguese, Afrikaans, Japanese, Chinese, French, German, Vietnamese, Dutch, Farsi, Arabic, Czech, Thai, and Hebrew, as the intention of the database is to serve the international research community. During the 2011 calendar year, there were over 100,000 articles downloaded from the site which has been accessed by users in 37 countries. This past quarter, October- December 2011, there were 382 visits to our front page and 9743 articles were accessed and downloaded. We have continued to increase the user base by expanding outreach efforts to Germany and Brazil. We have added new information to the database as well as continued to develop the official Facebook page as a place to share citrus news and events in real time. We update the page daily and our fan base has been increasing by five new users each month. We have also continued to develop the HLB database listserv to encourage open, dynamic communication among individuals around the world who have an interest in sharing information on Citrus greening related issues. This project has been presented to researchers and students in several national and international meetings in the U.S, Germany, Brazil, and Mexico and continues to have increased exposure within the research community through citrus research and extension web pages that have published links to our database (see partial list below). Our goal for the next quarter is to continue mining and uploading the most current HLB related information, enhancing the relevance and visibility of the HLB/Greening Facebook page and the listserv service, increase our efforts to connect the international citrus/HLB community, and continue exploring the most appropriate solution for a more powerful and permanent platform and location for this service. Selected external links to the database: (1) Strategic planning for the Florida Citrus Industry: Addressing citrus Greening [http://www.nap.edu/openbook.php?record_id=12880&page=217] (2) Florida Entomological Society. [www.flaentsoc.org/]. (3) The Grower’s Citrus Greening Resource Center. [www.growermagazine.com/CitrusGreeningResearchCenter] (4) University of Florida Entomology and Nematology Pest Alert. Gainesville FL. [www.entnemdept.ufl.edu/pestalert/] (5) University of Florida- IFAS- Extension CREC. [www.crec.ifas.ufl.edu/extension/greening/links.htm] (6) Southern Plant Diagnosis Network [http://www.sepdn.org/webfm_send/158] (7) Importance and management tendencies of citrus psyllids in the US and Brazil. Global insecticide Fall meeting BASF. Limburgerhof. Germany (October 2011) (8) Meeting to study the IRAC recommendations to reduce the risk of insecticide resistance in Asian citrus psillid. Campinas, Brazil, November 2011
Xacm strain: Xanthomonas axonopodis pv. citrumelo (Xacm) is a citrus pathogen causing citrus bacterial spot disease that is geographically restricted within the state of Florida. Illumina, 454 sequencing and optical mapping were used to obtain a complete genome sequence of Xacm strain F1, 4.9Mb in size. The strain lacks plasmids. Phylogenetic analysis revealed that this pathogen is very close to the tomato bacterial spot pathogen Xcv 85-10 with a completely different host range. We also compared Xacm to the genome of citrus canker pathogen Xac 306. Comparative genomic analysis showed differences in several gene clusters like Type 3 effectors, Type 4 secretion system, lipopolysaccharide synthesis and others. In addition to pthA, effectors such as xopE3, xopAI and hrpW were absent in Xacm while present in Xac. These effectors might be responsible for survival and reduced virulence of this pathogen on citrus compared to Xac. We also identified unique effectors in Xacm that may be related to the different host range as compared to Xac. Xacm F1 was shown to be pectate lyase deficient in Hildebrand’s medium which is consistent with the sequence analysis. Comparison of the complete genome sequence of Xacm to Xac and Xcv provides valuable insights into the mechanism of bacterial virulence and host-specificity. The complete genome of Xanthomonas axonopodis pv. citrumelo has been deposited at EMBL/DDBJ/GenBank under the accession number NC_016010. The manuscript entitled ‘Comparative genomic analysis of Xanthomonas axonopodis pv. citrumelo F1, which causes citrus bacterial spot disease and related strains provides insights into virulence and host-specificity.’ has been published by Journal of Bacteriology. Xac Aw strain: Whole genome sequencing of Xanthomonas axonopodis pv. citri strain Aw has been completed. The Genome sequence is 5.3 Mb in size. The sequence was annotated using FgeneSB and the annotation curation was done using GenePrimp. The annotated sequence has been reviewed by ICBR and all the doubtful regions were checked and have been confirmed by PCR and sequencing. Plasmids were purified and sequenced separately using 454 titanium sequencing. The sequences for the 2 plasmids were closed using CLCbio Genomics Workbench. Gap closure was done by PCR and sequencing and has been reviewed by ICBR for any doubtful regions. Annotation has been done and curation is being performed using geneprimp. Initial analysis of T3SS effectors revealed presence of XopF1 and avrGf1 gene and absence of HpaA as different from Xac A 306 effectors. The mutant of xopF1 did not show any difference on the grapefruit plants. Double mutant of xopF1 and avrGf1 has been made. A complementation with HpaA is being done to determine its role in host specificity. All the mutants and control are being tested on Key Lime, Grapefruit and Valencia for virulence and host specificity change. Xac A*270 and Xacm 1381 (Cu resistant) strain: Plasmids from both the strains were purified and sequenced with Illumina 75 x2 bp sequencing at ICBR. The sequences have been trimmed and De novo sequencing has been performed. The results show presence of 3 and 1 plasmid in XacA*270 and Xacm1381 strain respectively. Gap closure for the plasmids and confirmation is in progress along with the genome sequence of both the strains. We also characterized two novel virulence related genes gpsX and nlxA. Our result indicate that they are involved in Xac EPS and LPS synthesis, and biofilm formation, virulence and in planta growth in host plant grapefruit.
Mid Florida Citrus Foundation (MFCF) a 501c5 not for profit organization which has supported (past 25 years) and currently supports citrus research efforts of scientists from the University of Florida, USDA and private industry. The MFCF supports citrus research through the employment of a full time grove manager whom works closely with researchers to ensure that their projects are handled properly and that the grove is an excellent condition. The management of this grove requires extra financial commitment as grove care cost tend to be higher than a commercial grove due to the nature of many of the research projects. Current projects being conducted at the MFCF are Asian citrus psyllid (ACP) pesticide evaluation control trials, low volume applicator trials, windbreak evaluation, HLB nutritional programs, new and existing herbicide trials, variety and rootstock evaluation trials. 2011 MFCF expansion projects included establishment of an economic study of high density citrus rooted cuttings for early production of citrus in the presence of greening, remedial and preventive tests for HLB infection, new herbicide trials, variety evaluation with HLB tolerance in Florida and ACP studies. A total of 31 acres has been and planted this year. This large planting of young trees also requires added young tree care expenses and an increase in grove care costs without any fruit to support continuing operations. In 2011 with FCPRAC support include the following purchases; a new laptop for farm manager, a slightly used utility vehicle, the improvement of interior roads with clay. Additionally the MFCF made another annual payment for the used tractor/sprayer combination acquired in 2009. Additionally private companies with a vested interest in the citrus industry have made considerable donations of materials and irrigation equipment for these new projects. This is a leverage of research dollars that does not get recognized. Between private donations and EQIP grant monies, the CRDF is getting approximately 2.00+ dollars towards research projects for every 1.00 dollar in grant monies awarded.
Development of alternative or complementary approaches for effective management of citrus greening is highly desirable and will greatly help the citrus industry due to the difficulty to control the HLB disease. Considering the highly destructive nature of HLB disease and the lack of control measures, there is a huge potential to develop antimicrobial small molecules against the causal agent thus to suppress the population of Las in planta and to reduce the innoculum for psyllid transmission. Treatment of Ca. L. asiaticus infected citrus could be pursued by applying antimicrobials to infected trees. The most common targets for antimicrobial agents development include receptors, proteins and enzymes, DNA, RNA and ribosomal targets. Among them, proteins have become the major target due to their druggable characteristics. In this study, we presented our research on screening small molecule inhibitors against SecA. SecA is one essential component of the Sec machinery which provides a major pathway of protein translocation from the cytosol across or into the cytoplasmic membrane. The Sec pathway was also shown to be required for virulence of Ca. L. asiaticus in our study. SecA is the protein translocase ATPase subunit, which involves in pre-protein translocation across and integration into the cellular membrane in bacteria. First we filtered the structures based on their physico-chemical properties, e.g. Molecular Weight, H-Bond Donor, H-Bond Acceptor & Rotatable bonds and structurally similar to adenine moiety. Approximately 5000 structures were retrieved from ~5 million structures of commercially available databases. The identified data set was used for virtual screening by molecular docking method. Based on the dock scores we eliminated about 4500 low scored structures and selected ~500 (10%) for further molecular docking & minimization to evaluate the scoring functions (Dock glide scores, Hydrophilic, Hydrophobic e.t.c). Based on scoring functions, structural diversity, and our chemical intuition we have chosen forty structures for biological activity studies against purified SecA protein. For that purpose, SecA of Ca. L. asiaticus was expressed in E.coli using the pE-SUMO vector and purified. The inhibition assays of the 40 compounds against SecA was done as described previously by Denis et al. 1978. The IC50 ranged from 0.3’M to 100’M. Those findings were published in article entitled: “Discovery of novel SecA inhibitors of Candidatus Liberibacter asiaticus by structure based design” on Bioorganic & Medicinal Chemistry Letters Volume 21, Issue 14, 15 July 2011, Pages 4183-4188. Using various computational techniques, twenty compounds were selected for biological activity study against SecA of Candidatus Liberibacter asiaticus and Agrobacterium tumefaciens. Five compounds were found to inhibit the ATPase activity of SecA of Las in nano molar concentrations and showed antimicrobial activities against A. tumefaciens with MIC50 ranging from 1.2 ug/ml to 2.5 ug/ml. These compounds appear to be suitable as lead compounds for further development of antimicrobial compounds against Las. These five compounds are being evaluated for antimicrobial activity against Las. In addition, optimization of those lead compounds is underway to further increase their antimicrobial activities.
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