Citrus canker is a serious disease of most commercial citrus cultivars in Florida.The goal of the proposed research is to identify and characterize novel and critical genes involved in pathogenicity and copper resistance present in X. axonopodis pv. citri (Xac) and related strains. Identification of critical virulence factors is a crucial step toward a comprehensive understanding of bacterial pathogenesis, host-species specificity, and invasion of different tissues thus to design new management strategies for long term control. Treatment of citrus with copper-based bactericides is one of the most common practices used for control. However, there is potential for horizontal gene transfer of copper resistance genes from other closely and distantly related bacterial strains, which will drastically reduce the efficacy of copper bactericides. Currently, copper resistant strains of other xanthomonads, including X. axonopodis. pv. citrumelo, the citrus bacterial spot pathogen, have been isolated from fields in Florida.Understanding the potential mechanisms of copper resistance in Xac and potential horizontal gene transfer of this resistance to Xac is also important for the long-term management of citrus canker.Currently, five Xac related strains are being sequenced with FL-1195 completed, XacAw close in completion (chromosome completed, plasmids in progress), while three more genomes are in the gap closing stage. Genome sequence of Xac strain Aw was completed. It is currently being checked for any problematic regions. The GenePrimp analysis of the annotation showed 1728 anomalies. Manual curation is currently in progress. Multiple gaps still exists for the two plasmids. Primer walking,cloning and sequencing are being used to close those gaps.The genome sequence has been compared to the reference strain XAC A 306 using MAUVE and global rearrangements were observed. FL-1195 was sequenced using 454-pyrosequening, Illumina (Solexa) sequencing and Opgen optical mapping. The finished sequence of XACM (4,967,469bp) was annotated and curated. Our analysis revealed that XACM lacked plasmids, although they are commonly associated with other strains of xanthomonas. Phylogenetic analysis based on housekeeping genes revealed a close relatedness of XACM to Xanthomonas axonopodis pv. citri str. 306 (XAC) causing citrus canker and Xanthomonas campestris pv. vesicatoria str. 85-10 (XCV) causing bacterial spot in tomato and and pepper. Whole genome comparison revealed a gene order similar to both XAC and XCV. Several genome rearrangements and insertion/deletion regions indicating genome plasticity were found. An all against all BLASTP of the complete proteomes revealed a total of 410 coding sequences unique to XACM. Several type III secretion system effector genes of XACM uniquely shared with the citrus or the tomato pathogen were found. Comparative genomics also showed various other changes in genes encoding cell wall-degrading enzymes, lipopolysaccharides, etc.
In Florida, the use of low volume sprayers has been adopted as a cost effective way to reduce Asian citrus psyllid populations. Low volume sprayers are calibrated to deliver 2 to 5 gallons per acre with a volume mean diameter droplet size of 100 microns. This relative measure of droplet size describes the range of droplet sizes, where 50% are smaller than 100 microns and 50% are larger. Smaller droplets are more susceptible to environmental conditions and drift at the time of application. A review of the literature on drift and droplet size identified a number of environmental conditions that could affect the deposition of these small droplets. This study monitored these environmental conditions in a Florida citrus grove from June to December 2010 and provides some insight into the prevalence of the favorable weather conditions for low volume spray applications. A number of environment conditions were monitored during this study. These measured conditions were then used to develop an estimate for the relative favorability for applying a low volume spray application on a given night. The study evaluated wind speed inside and outside a grove, an air dryness factor developed from the difference between dew point and air temperature measurements and the presence of inversion conditions. The trigger values used in the study were when average wind speeds were 0.0 mph or greater than 10 mph, a difference between the air temperature and calculated wet bulb temperature of greater than 140F and wind speeds less than 2 mph with air temperatures higher at the 25 foot level compared to the five foot level (used to determine the potential for a temperature inversion). From the collected weather measurements, the greatest potential threat to low volume spray applications in Florida could be conditions conducive to the development of temperature inversions. During the study period, the potential for temperature inversion existed on 16 to 32% of the nights. It appears in Florida that there is very little potential for evaporation to volatilize low volume spray droplets based on the established criteria. Wind speed conditions were taken from inside the grove at two locations and compared to observations outside the grove. Typically wind speeds fell into the 0.0 to 10 mph range from 82 to 99% of the time. The average wind speed recorded above the tree canopy at a 20 foot level was 8.75 to 20.5 times greater than the wind speed recorded in the tree row within the grove at a level of five-feet. The wind speed above the tree canopy most closely resembled the average wind speed measurements recorded from outside the grove at five and 25 foot levels. Mature hedgerow citrus groves in Florida appear to act as effective windbreaks, thereby significantly reducing wind speed within the block or grove as compared to outside grove observations. Additional activities were also conducted to offer low volume calibration events to Florida citrus growers with 3 events being conducted during the grant period. These events were conducted in conjunction with USDA ARS. These calibrations events assisted growers in adjusting sprayers to provide droplet sizes to be in the 100 micron range. The ability to effectively calibrate low volume spray equipment is not a task that can easily be accomplished without sophisticated and expensive equipment.
Our project aims to determine which species and varieties of citrus and citrus relatives are being smuggled into California, with the consequent risk of transmitting exotic pests and diseases; to acquire germplasm of these genotypes if they are not now legally present in California; to make sure this germplasm is pathogen-tested; to make budwood or propagating materials available for distribution through the Citrus Clonal Projection Program; and if feasible and advisable, encourage California cultivation of these commodities, so as to undercut the incentive for their smuggling. In November 2010 we received updated information gathered by the USDA/APHIS/PPQ Smuggling Interdiction and Trade Compliance program listing seizures of citrus and citrus relatives being smuggled from foreign countries, including the plant parts concerned, quantities, countries of origin, and the states where the items were seized. We will use this data to help set our priorities as to which commodities most urgently need to be addressed by our program. The information has prompted further questions regarding its interpretation which we are pursuing in consultation with both USDA and other sources. In November 2010 team member David Karp gave a PowerPoint talk before the California Citrus Nursery Society, explaining the program. He discussed individually with members their perceptions of the horticultural and commercial potential for local cultivation of the various smuggled species. In January 2011 Mr. Karp attended the Huanglongbing Conference held in Orlando, Florida, where he gathered information about recent scientific discoveries germane to the project, and also about Florida sources for, cultivation of, and trade in commonly smuggled citrus and citrus relatives. He then traveled from Orlando to Homestead visiting botanical and germplasm collections, ethnic and specialty farmers, extension agents, backyard farmers, and ethnic stores, to observe, photograph and discuss the germplasm resources and cultivation of these items. Most of the relevant commodities, including curry leaf (Bergera koenigii), bael (Aegle marmelos), and woodapple (Limonia acidissima), grow and fruit more rapidly than in California in the generally warmer and more humid Florida environment. Mr. Karp has also continued to meet with aspiring growers of citron (Citrus medica) for Jewish ritual purposes, in order to guide them to fulfill legal and regulatory requirements in introducing seeds and cuttings of this species into California. We are currently consulting with the Citrus Research Board to determine if a consensus exists favoring distribution of curry leaf seed, in order to help facilitate the growth of California cultivation of this commodity.
The overall goal of this project is to characterize the virulence mechanisms of Candidatus Liberibacter asiaticus, the citrus Huanglongbing (HLB) pathogen, thus to come up with new management strategies by genome sequencing and functional genomics approaches. The original goal of the proposed research is to further complete the genome sequencing of Candidatus Liberibacter asiaticus, for which a draft sequence is available. The goal was modified to meet the current progress in genome sequencing of Ca. L. asiaticus with the advice and permission from FCPRAC. The tile has been changed to the following to better suit the goal: Understand the virulence mechanism of Ca. L. asiaticus by genome sequencing and functional genomics approaches. Major achievements: To understand how Ca. L. asiaticus make a living including nutrients acquisition and overcoming of plant defense in citrus, we first analyzed its metabolic capability and nutrient requirement by conducting curated metabolic reconstruction. Some novel insights were acquired regarding its respiration, amino acids synthesis, Co-factors and nutrient transportation. Our data indicated that Ca. L. asiaticus could rely only on aerobic respiration rather than on anaerobic respiration as reported previously (Duan et al. 2010). Ca. L. asiaticus could not synthesize 12 amino acids and needs external folate, quinines, biotin, thiamine, pantothenate, and pyridoxine for its growth. In addition, genome comparison of Ca. L. asiaticus against Mycoplasma genitalium, one of the smallest bacterial genomes, has identified more than 200 homologs. Those shared genes might be close to the minimal set required for viability of bacteria and good candidates for screening antimicrobial small molecules. Several potential targets including SecA are being used to screen potential antimicrobial small molecules to control HLB. In order to identify the potential virulence genes, real-time quantitative PCR assays using total RNA isolated from infected plants and psyllids, were conducted to test the expression profile of Ca. L. asiaticus. Gene specific primers were used to check the expression of more than 500 genes in Ca. L. asiaticus. The genes showing a differential expression of two fold or more in either the plant or psyllid were selected, and categorized into COG (Clusters of Orthologous Groups of proteins) functional categories. Selected genes that were overexpressed in planta were further studied by expression or screened on Nicotiana benthamiana plants for symptom expression, using transient assays. Totally 28 potential virulence genes were cloned into TMV30bGFP viral vector for transient expression on Nicotiana benthamiana plants. Out of the 28 genes cloned, 13 were successfully assayed on tobacco plants for symptom expression. Three out of the 13 showed interesting symptoms, and hence were selected for further characterization. The following symptoms were observed: LasA1 showed vein clearing after about a week after inoculation, and subsequent wilting and death of the whole plant within 2-3 weeks. LasA2 showed phyllody, stunting and very clear growth defects. The symptoms were significantly different from the infection using the empty vector (TMV 30BGFP). The expression of the genes in planta were confirmed using RT-PCR. Further study is under way to further understand the roles of those two potential virulence factors.
Comparison of the microbiomes associated with HLB pathogen positive and negative citrus will illuminate the causal agent of citrus greening. Potential beneficial microorganisms could be identified from healthy plants which survived in heavily infected citrus groove with HLB. Beneficial microorganisms have been shown in previous studies to have the capacity to control plant diseases by accelerating seedling emergence, promoting plant growth and development, and preventing the invasion of plant pathogens. Characterization of the beneficial microorganisms associated with citrus in the local environment might identify beneficial bacteria for practical use to promote citrus growth and control HLB. Major achievements: This research has resulted in four publications. 1. A comprehensive study of the bacterial diversity associated with healthy and HLB diseased citrus indicated that Candidatus Liberibacter asiaticus as the pathogen responsible for HLB disease in Florida. Phytoplasma was not found in any of the samples collected from Florida (Sagaram et al. 2009 AEM). 2. We characterized the effect of HLB on the bacterial community associated with citrus roots. This research has been summarized in the following publication ‘Huanglongbing, a systemic disease, restructures the bacterial community associated with citrus roots’ (Trivedi et al. 2010 AEM). 3. Developed a method to quantify viable Candidatus Liberibacter asiaticus with the aid of ethidium monoazide (EMA) which can differentiate live from dead cells (Trivedi et al. 2010 EJPP). 4.Isolation of plant growth promoting bacteria from healthy citrus. Isolation of bacteria with the potential of plant growth promoting and biological control potential might reveal innovative ways of controlling the HLB disease. We specifically focused on delineation of the cultivated endophytic bacterial isolates and characterization of their salient metabolic features. A total of 227 and 125 morphologically distinct colonies were isolated and characterized from HLB asymptomatic and symptomatic trees, respectively. We observed that the frequency of bacterial isolates possessing various plant beneficial properties was significantly higher in the asymptomatic samples. A total of 39 bacterial isolates showing a minimum of 5 beneficial traits related to mineral nutrition [Phosphate (P) solubilization, siderophore production, Nitrogen (N) fixation], development [Indole acetic acid (IAA) synthesis], health [production of antibiotic and lytic enzymes (chitinase)], induction of systemic resistance [salicylic acid (SA) production], stress relief [production of 1-amino-cyclopropane-1-carboxylate (ACC) deaminase] and production of quorum sensing [N-Acyl Homoserine Lactones (AHL)] signals were characterized. A bioassay using EMA-qPCR was developed to select bacteria antagonistic to ‘ Candidatus Liberibacter asiaticus’. Using the modified EMA-qPCR assay, we found 6 bacterial isolates showing maximum similarity to Paenibacillus validus, Lysinibacillus fusiformis, Bacillus licheniformis, Pseudomonas putida, Microbacterium oleivorans and Serratia plymutica could significantly reduce the population of viable ‘Ca. L asiaticus’ in HLB symptomatic leaf samples. In conclusion, we have isolated and characterized multiple beneficial bacterial strains from citrus roots which have the potential to enhance plant growth and/or suppress diseases. Further study is needed to develop appropriate formula to utilize those beneficial bacteria.
Asian Citrus Psyllid (ACP) was first detected in San Diego County, August 29, 2008 (in lemon), and has been slowly moving north. The largest populations of ACP have been discovered in Los Angeles County. The quarantine containment perimeter in Riverside County has remained unchanged since November, 2009; but recently discoveries of ACP have been made in Upland and Redlands. Thus all of the research conducted on the Asian Citrus Psyllid, and its natural enemies, must still be contained within UC Riverside’s certified quarantine facility. Three months into this new project, and we have established good ACP production numbers under the quarantine space and rearing constraints (300 to 400 ACP offspring/0.023 m3 cage). All ACP material must be double caged and all personnel working with ACP production must wear hooded jump-suits. These procedures ensure that no ACP leave the rearing cages or rearing room in quarantine. We communicate to researcher routinely to prioritize utilization of ACP material. Weekly vials of dead adults are provided to the Citrus Research Board (CRB) lab for scout train (e.g. trap card identification of ACP). Other adult specimens (ca. 1,000) were also provided to the CRB lab for special plastic mount curation. We have established quarantine approved methods of removing ACP DNA material for USDA researchers to use as controls for their disease detection research, which they are conducting in collaboration with Florida citrus researchers. This research will commence in April of this year. We continue to provide all of the adult material for the ACP electrophysiology project (Drs. Anand Ray & Robert Luck) and also the associated pheromone work being done in collaboration with Dr. Jocelyn Millar (CRB project). For this same project, we recently provided the material to obtain ACP brains (for brain stain analysis) and developed a collaborative effort to establish protocols that allows the ACP brain material to be removed from quarantine to complete the analysis. We continue to assist CRB and CDFA projects in evaluating the host specificity of Tamarixia radiata, a parasitoid of ACP, by providing adults psyllids and rearing protocols upon request. Our project has also been approached by other researches that may attempt to conduct various pesticide trials in the quarantine facility (e.g., residue & efficacy trials). These projects are currently working on developing protocols that will allow these trials to be conducted in quarantine. There are very strict guidelines for working with pesticides in quarantine. Some of the work may commence this month, for which we will provide the adults. We expect that the demand for ACP material will increase as the year progresses, and will continue to anticipate researchers’ needs so that their research goals can be met in a timely manner under the Federal and State quarantine constraints.
The goals of this ongoing project are to eliminate the incentive, economic and cultural, for smuggling of citrus fruits and plant parts by establishing legal sources of supply for these commodities in California and to provide education about the availability of registered pathogen-tested sources. On July 7, 2011 we received authorization from the California Citrus Research Board to proceed with our proposal to distribute seeds of Bergera koenigii, the curry leaf tree, to California citrus nurseries. This approval was granted after we provided a written description of the seed distribution protocol, which was submitted to CDFA nursery program authorities for comment and approval. Staff at the National Clonal Germplasm Repository for Citrus and Dates (NCGRCD), under the direction of Dr. Richard Lee, are currently testing curry leaf seed source trees at the UC-Riverside Citrus Variety Collection for seed-transmissible pathogens, using real-time PCR tests to test for huanglongbing (HLB), and conventional PCR for citrus leaf blotch virus, psorosis, and xylella (CVC). Approximately 1,000 seeds were previously collected by staff at the NCGRCD, and assuming that the trees test free of seed-transmissible pathogens, the seeds, both stored and freshly collected, will be distributed to several qualified nurseries which have expressed interest in the project. The immediate goal is to facilitate the propagation and cultivation of trees of curry leaf, in order to provide a local source for the leaves, which are in considerable demand from South Asian communities for culinary use. Previously the commercial demand for this commodity was satisfied by growers in Florida and Hawaii, but federal quarantine restrictions now prohibit shipments of curry leaf from Florida, and Hawaii shippers can send to California only product that has been treated with methyl bromide, which drastically curtails shelf life. Because of the unsatisfied demand, curry leaf is being smuggled into California, as attested by official interdictions and media reports; since these leaves are a preferred host for the Asian citrus psyllid, the vector of HLB, such smuggling presents a grave risk of spreading the insect and introducing the disease to this state. It is our intention to facilitate the development of local cultivation of curry leaf that would undercut the demand for smuggled product.
During this quarter, the young ‘Hamlin’ orange trees in the Ridge experiment were harvested in December 2010, by which time they were exactly two years old. Large significant differences in fruit yield and external and internal fruit quality were measured at that time between the conventional practice treatment and the six advanced production (ACPS) treatments. Notably the percentage orange peel color was more than doubled , brix/acid ratio was 33% higher, fruit yield per tree was up to 186% higher, fruit yield per acre was up to 375% higher, and soluble solids production per acre was 364% higher in ACPS than in conventional plots. The highest average fruit yield at two years age reached 38.5 boxes per acre. The yield in the microsprinkler fertigated treatment was not significantly different from the drip fertigated treatment on the same Swingle rootstock, but was significantly higher than yield of the conventional production treatment. Although this yield is above average for such early production in Florida, there is still scope for improvement by stimulating more rapid canopy production in the early years. In the first year of this experiment, the tree canopy’s young emerging flush was repeatedly damaged by citrus leaf miner larvae, which restricted the canopy development and fruit setting capacity in year two. Better pest control is essential for the success of the ACPS. Additional gains could also be made through more refined nutrient application timing, dosing, and composition. The first four HLB-positive trees were identified during this quarter. The first of those trees has been protected with a psyllid-proof screened enclosure for continued observation and study of the disease, but the other three trees were removed and subsequently infected trees will also be eradicated. The difficulties experienced with pest control in the first year were thought to have contributed to this early appearance of HLB, but in reality there are infected backyard trees receiving minimal to no care within 300 feet of the experiment. Summary points: 1. Fertigate ‘daily’ at 100 to 150 ppm N concentration in final water volume; use calcium nitrate to provide ample Ca (see recipe on WWW) 2. Inject the actual fertilizer as a pulse and follow with water; Inject from tanks 1 and 2 sequentially 3. Fertigate to keep the top 0-6 inches of soil at about field capacity. Monitor moisture at 18 inches depth to avoid excess. Use sensors. 4. Fertigate in the morning after sunrise when photosynthesis and transpiration are at their peak 5. During long dry periods in the first year, flush salts from the root zone with plain irrigation (3 to 4x normal irrigation amount) every 2 weeks For all the latest up-to-date information on the ACPS research, especially for photos and calculator which cannot be displayed here, browse to http://128.227.177.113/ACPS/index.html The water calculator is on the Data/Reports page.
As we approach the end of our third year of evaluating the Boyd cocktail of macro-nutrients, micro-nutrients, phosphite, SARs, and hydrogen peroxide we are collecting data that is showing the importance of macro and micro-nutrients, and the phosphite. The ‘Hamlin’ trial was harvested in January and yield results will be available for the next report. The two ‘Valencia’ trials will be harvested in April. The rehabilitation trial begun this year to evaluate the value of buckhorning existing HLB declining trees to rebalance the shoot/root ration and applying foliar nutrients to the regrowth has gotten a lot of interest from growers. The idea of of salvaging existing trees over removal, replanting, and bringing a new planted tree into production has a lot of advantages. One advantage is the cost and another is having the tree out of production only one or two years. Another advantage is having a tree with vigorous regrowth and fully productive canopy. Growth measurements of shoot length and leaf size of the regrowth have shown shoot length to be twice the length of unpruned trees receiving the same nutrients on every flush during the first year. The pruned trees have reached more than half their original height and tree volume in one growing season. Leaf nutrient analysis data has been collected to monitor the status of the vigorous growing trees. The trees in both the Boyd cocktail trials and the rehabilitation trial all look good considering the leaf drop experienced this past October statewide in groves. In addition, we have experienced similar leaf and fruit drop from the freezes in December that other growers have had. We have documented greater leaf and fruit drop from HLB infected trees where the added stress makes the trees more susceptible. HLB makes the citrus tree less tolerant to any stress factor like drought, poor nutrition, freezes, disease, insects, etc., and can result in leaf and fruit drop. We are in the third year of a replicated experiment in a 12-acre experiment commercial block of 8-year-old ‘Valencia’ oranges on ‘Swingle’ to test effects of micro-nutrients + systemic acquired resistance inducers, and Asian citrus psyllid (ACP) chemical control on ACP populations on Can. Libericacter asiaticus (CLas) titer, and plant yield. Since our last report we have conducted two dormant sprays on the treated plots. We are conducting preparation for the third harvest in these plots possibly towards the end of March beginning or April 2011. This third harvest is considered to be key to confirm the observed tendencies from years past. In our last harvest, trees in plots where ACP was managed using insecticides produced significantly more Lb-solid per tree (5.76’0.36 Lb-solid/tree) than trees in untreated plots (4.17’0.65 Lb-solid/tree), continuing a trend seen last year. Also, trees treated with Micro+SAR produced more (5.26’0.55 Lb-solid/tree) compared with untreated trees (4.55’0.64 Lb-solid/tree), following trends seen from the first harvest in the 2008-2009 season. Neither the insecticide treatment nor the nutrients have slowd down the spread of HLB (now virtually 100%), Treatments where ACP is being managed have had consistently lower titer over the two year study, except in our May 2010 where the CT values were not different Treated (24.0’0.3), Untreated (24.1’0.2). In January we are conducting the plant samples for HLB detection. Simultaneously, we are analyzing the data from the last plant sample collection.
Management of citrus Huanglongbing (HLB), which is associated with Candidatus Liberibacter asiaticus, could be achieved by application of antimicrobials and stopping the spread of HLB pathogen. Curing Ca. L. asiaticus infected citrus trees is one attractive goal due to the high value of citrus trees and the high cost of citrus tree removal and replanting. Wide spread of HLB throughout Florida renders curing Ca. L. asiaticus infected citrus trees necessary. Treatment of Ca. L. asiaticus infected citrus could be pursued by applying antimicrobials to infected trees. The most common targets for antimicrobial agents 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. Currently, we are testing the antimicrobial activities of the top 10 compounds against Sinorhizobium meliloti, Agrobacterium tumefaciens, and E.coli. In addition, those compounds will serve as leads to develop new compounds.
Citrus canker is a serious disease of most commercial citrus cultivars in Florida.The goal of the proposed research is to identify and characterize novel and critical genes involved in pathogenicity and copper resistance present in X. axonopodis pv. citri (Xac) and related strains. Identification of critical virulence factors is a crucial step toward a comprehensive understanding of bacterial pathogenesis, host-species specificity, and invasion of different tissues thus to design new management strategies for long term control. Treatment of citrus with copper-based bactericides is one of the most common practices used for control. However, there is potential for horizontal gene transfer of copper resistance genes from other closely and distantly related bacterial strains, which will drastically reduce the efficacy of copper bactericides. Currently, copper resistant strains of other xanthomonads, including X. axonopodis. pv. citrumelo, the citrus bacterial spot pathogen, have been isolated from fields in Florida.Understanding the potential mechanisms of copper resistance in Xac and potential horizontal gene transfer of this resistance to Xac is also important for the long-term management of citrus canker.Currently, five Xac related strains are being sequenced with FL-1195 completed, XacAw close in completion (chromosome completed, plasmids in progress), while three more genomes are in the gap closing stage. Genome sequence of Xac strain Aw was completed. It is currently being checked for any problematic regions. The GenePrimp analysis of the annotation showed 1728 anomalies. Manual curation is currently in progress. Multiple gaps still exists for the two plasmids. Primer walking,cloning and sequencing are being used to close those gaps.The genome sequence has been compared to the reference strain XAC A 306 using MAUVE and global rearrangements were observed. Analysis of T3SS effectors revealed presence of unique XopF1 and avrGf1 effector genes which are absent from Xac A 306 strain. Mutant of xopF1 and double mutant of xopF1/avrGf1 are being constructed to determine their roles in host specificity. Complete genome sequence of X. axonopodis pv. citrumelo FL-1195 was completed. At least 48 regions, which were found incorrect during annotation analysis, were rechecked and corrected. Correction was done using the 454 and Illumina read data as well as PCR sequencing of the questionable regions. The CDS prediction was curated using the GenePrimp pipeline. 1485 anomalies were checked and 570 CDS were corrected after being identified as long, short, broken or new genes. The genome sequence has been compared to the reference strain Xac A 306 using MAUVE and global rearrangements were observed. In detail analysis of T3SS, other secretion systems, effectors, LPS, EPS, motility clusters and unique regions different from Xac A 306 strain is currently in progress.
Website Creation and Development A genome viewer feature has been added to the CG-HLB Genome Resources website for the purpose of providing a convenient interface for accessing Ca. Liberibacter genome features and analysis of predicted proteins (http://www.citrusgreening.org/HLB-GBrowse.html). The initial version of the viewer, brought online in the previous quarter has now been moved to a faster and more flexible server hosted by the Cornell Center for Advanced Computing. New features include explanatory pop-up windows to aid novice users and a greater variety of imbedded hyperlinks. Users can search the genome by gene name, locus tag, feature type, or coordinates. Previously implemented tracks show predicted functions and cellular locations of the encoded proteins as well as genome structural features such as predicted operons and repeat sequences. More recent additions to the genome viewer include direct hyperlinks to precomputed lists of similar proteins at NCBI. Links to the metabolic pathways at the BioCyc Database are currently being installed to provide ready access to predicted metabolic roles of Liberibacter proteins. Addition of new tracks and links is ongoing as more bioinformatic analyses are performed. Bioinformatic Analyses Bioinformatic analyses are focused on genome sequence comparison with other bacteria. Of highest priority is comparison of the Las psy62 sequence to the recently released genome sequence of L. solanacearum CLso-CZ1. The two sequences are highly similar and we are exploring different approaches for representing both SNPs (single nucleotide polymorphisms) and insertions and deletions that occur between the two sequences. Our goal is to develop an effective means of visually representing sequence variation that can be scaled up as more Liberibacter sequences become available, providing a tool for rapid design of diagnostic primers that distinguish among strains. Genome sequence comparisons are also being conducted with related free-living bacteria as well as bacteria occupying analogous biological niches. By distinguishing between conserved core regions and those regions or genes that either exhibit faster rates of evolution or are more similar to genes from bacteria in analogous niches, genes critical to adaptation to the insect/phloem niche may be identified. Outreach An announcement of the genome viewer has been sent to all 76 registered users of the CG-HLB Genome Resources web site together with a survey soliciting comments on additional features that researchers might find of use. Information on the CG-HLB Genome Resources web site and associated genome viewer is being presented and additional feedback sought at the upcoming research conference on HLB in Orlando Florida.
The Citrus Greening Bibliographical Database [ http://swfrec.ifas.ufl.edu/hlb/database/ ] managed by the Entomology group at the University of Florida – IFAS in Immokalee with cooperation from the Florida Center for Library Automation in Gainesville has become a widely used source for information on Huanglongbing (HLB) for researchers, growers, and students throughout the world. Entries represent worldwide research on the various aspects of HLB: the associated bacteria (Candidatus Liberibacter spp.), the vectors [Diaphorina citri Kuwayama and Trioza erytrea (Del Guercio)], the effects of the disease on plants and vectors, and the most current management tactics. The database was designed to centralize relevant information in an accessible, user-friendly interface. This past quarter, October to December 2010, we continued to add and update information to the database and cross-reference all information for accuracy. We now have 1968 citations, 81% of which are linked to their original sources. Eighty-six percent of the entries are in English, the remaining 14% are in Spanish, Portuguese, Afrikaans, Japanese, Chinese, French, German, Vietnamese, Dutch, Farsi, Arabic, Czech,Thai, and Hebrew. We have also created an interactive Official Facebook Page [ http://www.facebook.com/pages/HLB-Greening-Database/164498020244442 ] with which you can become a ‘friend’ from the database webpage. This will serve as a forum in which researchers and growers can exchange HLB related information and discuss topics of interest and/or concern, post news and events related to HLB. The development and use of this project has been presented to researchers, students and growers in several national and international meetings in the U.S and in Mexico and has continued 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 upcoming quarter is to continue to improve this service by updating existing documents to ensure accuracy, searching for the most current information from researchers around the world, continuing to link existing entries to their original documents, and promoting participation in the interactive Facebook forum (1) FCPRAC request for proposals 2009 [www.fcprac.com/proposals-2009.html] (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) USDA- APHIS. Plant Health – Citrus greening [http://www.aphis.usda.gov/plant_health/plant_pest_info/citrus_greening/links.shtml ] Arevalo, H.A., A.B. Fraulo, and P.A. Stansly. 2010. The HLB Bibliographical database: an information tool for growers and researcher. Citrus industry. 91:6. 22-23
Objective 1. Localization of Liberibacter asiaticus (Las) in the Asian citrus psyllid (ACP): [A] We continue our studies to improve the sensitivity and specificity of several FISH protocols to localize Las in hemolymph smears and dissected organs of ACP, and in leaf sections from HLB-infected plants as positive controls. So far, Las has been detected by FISH in the hemolymph, filter chamber and midgut of HLB-infected ACP from our laboratory colonies or from the field. [B] Using Q-PCR on dissected insect organs, Las was detected in the salivary glands, alimentary canals and other body parts of HLB-infected ACP adults. Our results suggest that the salivary glands constitute a major transmission barrier to Las in ACP, and that Las may replicate or accumulate in both the alimentary canal and salivary glands of its vector. Objective 2. Elucidation of various acquisition and transmission parameters between ACP and Las. [A] We have started two large experiments to compare between young (2nd-3rd-instar) nymphs and adults having various acquisition access periods (AAPs) on HLB-infected citrus plants. Previous reports have indicated that ACP nymphs are more efficient in Las acquisition than adults, and that Las probably multiplies in ACP nymphs (not adults), but the effects of various AAPs on nymphal and adult stages in this regard have not been carefully investigated. Our experiments are designed to study the effects of various AAPs on both acquisition and transmission of Las by ACP, as well as on Las replication in both nymphs and adults. We are now analyzing with Q-PCR hundreds of individual ACP from these two experiments. Results will be discussed in coming reports. [B] Since using whole citrus plants for maintaining psyllids when studying pathogen-vector relations takes considerable space, time and other resources, we have developed a new and simpler method for short-term rearing of ACP in conical 50-ml plastic tubes using detached citrus leaves for adults and detached young terminal shoots for nymphs. Survival of young adults was 89, 80 and 75% after 2, 3 and 4 weeks, respectively, on detached leaves changed weekly. Survival and development of 2nd-3rd-instar nymphs reared on detached terminal shoots in these tubes were largely comparable to those previously reported for ACP nymphs reared on whole citrus seedlings. ACP excretion droplets fell down from the leaves and accumulated in the conical bottom of the rearing tubes. Thus, in addition to its potential use for collecting psyllid excretions, this new method allows closer observation and photography of psyllid nymphs and adults, and can save time, space and other resources in various studies on the biology, behavior, management and pathogen-vector relations of ACP and probably other citrus psyllids. [C] We used the above method in devising a new “Detached Leaf Assay” to test the inoculativity of ACP with Las. Our results indicate that after feeding 10, 5 or 1 infected adult ACP/leaf on detached leaves of sweet orange for 7 days, the percentages of Q-PCR-positive leaves were 40, 18.8 and 4.4%, respectively, using Li primers, and 60, 40.6 and 11.1%, respectively, using the more sensitive LJ900 primers. These results, even using the less sensitive but more commonly used Li primers, are largely similar to those reported earlier on inoculativity/transmission tests of Las-infected ACP using whole citrus seedlings. Using more sensitive primers, however, can increase the usefulness of this method. We suggest that this new ‘Detached-leaf assay’ method can potentially speed up Las-inoculativity tests on ACP from 3-12 months to only 2-3 weeks, which can greatly enhance pathogen-vector relation studies on Las and ACP.
The intent of this study is to examine the effect of windbreaks, copper sprays to reduce infection, and leafminer treatments to determine there individual and combined effects on control of citrus canker in Brazilian commercial citrus and the applicability of this strategy to the US commercial citrus industry. Via a USDA/ARS specific cooperative agreement with the University of Sao Paulo, and the Brazilian cooperator, new replicated plots have now been established at the IAPAR farm, in Xambr’, Parana state, located 350 km west from Londrina and 250 km west from Maring’. The plots consist of the cultivar P’ra on Rangpur lime, two years of age at the beginning of the experiment. Windbreaks have been completed and plants were be established in Mid April 2010. Plots are progressing and the following treatments are being applied: 1) no sprays (control), 2) Cu++ sprays to reduce citrus canker incidence, and 3) insecticide sprays to inhibit infestations of Asian leafminer (secondary effects). Main effects are windbreak versus no windbreaks. Citrus canker incidence is being estimated on multiple branches on each tree treated as the number of leaves per branch infected. Data collection is currently underway. We anticipate running these plots for 2-3 more years. The development of the Programmable leaf wetness controller (PLWC) software was written, debugged, is complete, and the control program is working well. New leaf wetness sensors were designed and constructed. Calibration has been tricky as this is a new type of sensor we are developing and has never been used before by any other research group. The new design of the leaf wetness controller has much greater sensitivity and provides better environmental control, but with these benefits comes added complexity in electronics. A circuit to control the leaf wetness sensor and another to control fans that facilitate wind generation in ambient environments have been completed, tested and calibrated. Initial trials have demonstrated some glitches that we are currently addressing. Once completed, we will continue with studies to examine the survival characteristics of bacterial pathogens under field conditions.
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