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.
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 xanA, btuB, gumC, gumB, gumK, gpsA, and several hypothetical genes. Currently, complementation analysis of the 12 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 planta are being tested. EPS and LPS production by the mutants and the wild type strain are being tested.
At the request of the CRB, as of 2011-12, Grafton-Cardwell and Morse merged their core entomology research efforts under a single project, 5500-501 (Morse’s portion of the project is 5500-501b and was funded in the amount of $89,795 replacing his previous CRB project 5500-118). We have always coordinated our research efforts but this arrangement formalizes the situation. This report summarizes Morse’s recent research efforts within this coordinated project. The Success/Delegate chemistry has been the dominant method of citrus thrips control used since Success was registered for use on CA citrus in 1998. We continue to search for non-chemical methods of managing citrus thrips and/or means of reducing the need for treatments but on occasion, chemical control is needed. Citrus thrips has a history of developing resistance and in some areas, populations are fairly tolerant of organophosphates (e.g., dimethoate), carbamates (Carzol), and pyrethroids (Baythroid, Danitol). In 2011, field bioassays confirmed the first case of citrus thrips resistance to Delegate and the situation was discussed with PCAs at the Citrus Roundtable held at Lindcove 9-28-11. As a result, we will accelerate testing for new chemistries useful in control of citrus thrips and will produce an article for Citrograph detailing how growers and PCAs can maintain effective use of Delegate by rotating among other chemistries. Research continues evaluating how to best use Turbocide Gold and Evergreen for post-harvest control of bean thrips on navel oranges shipped to Australia and New Zealand where this insect is considered a quarantine pest. With the assistance of Fruit Growers Supply and the Yokohl Company Packing Co., a new overhead Turbocide Gold application method was tested 11-21-11 and results were quite encouraging. Fuller rose beetle has re-emerged as an export concern due to egg masses being found on shipments of citrus destined for Korea. The CRB funded research on this insect for 6 years, 1985-1991, in relation to shipments of citrus to Japan but this became a non-issue when it was acknowledged that the insect was present in commercial citrus groves in Japan. We have restarted FRB research, looking at possible use of trunk sprays to control the flightless beetles when they walk up the trunk of skirt-pruned trees, evaluating various methods of rearing the weevil, and determining how long beetles which emerge out of the ground can survive and whether they need to feed on foliage prior to laying eggs. Dr. Grafton-Cardwell and Dr. Walse are studying use of foliar pesticides to control adult FRB and post-harvest methods of control, respectively. Through funding obtained via a CDFA Specialty Crop grant, a team of researchers (Kris Godfrey, Jorge Pena, Gary Bender, Jim Bethke, Loretta Bates, and Morse) are importing Aprostocetus vaquitarum, a parasitoid/egg predator from Florida for control of Diaprepes root weevil. Releases are being made in southern California under an APHIS permit and in October we made our first field recoveries of this wasp.
The extension agents are actively educating and assisting growers and urban audiences with information about citrus greening via field visits, educational events (field days, training events/presentations), phone consultations and hosting visitors from around the world. During the last quarter of 2011, over 530 individuals participated in one or more educational events that addressed citrus and/or greening related issues. Efforts have been directed to working with growers in establishing and maintaining CHMAs throughout the state. Currently, 35 CHMAs have been developed with additional areas in the formative stages. CHMAs aid in getting growers to collectively target sprays to achieve greater success in controlling and/or suppressing psyllids as compared to individual psyllid control efforts. All citrus agents continue to promote citrus activities via active citrus newsletters which reach approximately 2,675 growers throughout Florida. Each November, the citrus agents develop and conduct the annual FFA citrus judging contest. This event highlights many of the citrus pests, disorders and varieties, including citrus greening and canker. Teams from 49 schools (25 high schools and 24 middle schools) participated in the annual event. Each team consisted of 4 members for a total of 196 participants plus the 49 chapter advisors. In addition to the citrus newsletters, the agents collectively use their website (http://citrusagents.ifas.ufl.edu) to post their HLB photo series and other citrus related information and happenings. This site received 7,604 web hits related to the HLB photo series and their entire website received 142,499 total hits during the last 3 months of 2011. The use of this electronic media effectively communicates information to growers 24 hours per day at little cost and effectively communicates information to all growers and others interested in citrus.
Objective 1. Localization of Liberibacter asiaticus (Las) in the Asian citrus psyllid (ACP). For a persistent pathogen to be transmitted, it has to be ingested, pass through the alimentary canal cells and membranes to the hemolymph, move into the salivary glands, and exit through the salivary gland membranes into salivary secretions, so that it can be inoculated into host plants during feeding. 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, in press). 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 apparently 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). ACP adults from an infected colony were tested for inoculativity by caging them singly or in small groups on detached healthy citrus leaves for 1-2 weeks, and the leaves assayed by qPCR 1-2 weeks later by two Las primers (Li and LJ900). Our results, using the more commonly used but less sensitive Li primers, are largely comparable to those obtained by previous workers using whole citrus seedlings for Las-inoculation by ACP from Florida. This new method can save considerable time, material and greenhouse space, and may hopefully enhance vector-relation studies on Las and other Liberibacter spp. associated with HLB in other parts of the world. [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. Also the relative Las titer (compared to that of S20 psyllid gene) was generally higher in nymphs than in adults and higher with longer AAP in both life stages. However, we are still analyzing the results of these experiments that will be repeated for confirmation and further statistical analysis. [C] Using confocal and epifluorescence microscopy on sections of citrus leaves in which ACP eggs were laid indicated that the stalks of ACP eggs are only embedded in the first few layers of the epidermis, far away from the phloem tissue, which refutes previous claims that ACP females may be able to transmit HLB during oviposition on host plants. [D] A study on the ultrastructure and morphometrics of ACP stylets, and the food and salivary canals inside the maxillary stylets, is underway using confocal and transmission/scanning electron microscopy. This study is expected to reveal the negative and/or positive roles of these canals in Las transmission by ACP nymphs and adults, and it may also explain some differences in feeding behavior between psyllid nymphs and adults.
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 Liberibacter. Five HLB-infected priority breeding selections of different parentage were budded onto Volkamer lemon with 20 trees of each 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. Most cyclohexamide treated buds died, but overall bud take wasn’t too bad. However a much higher percentage of buds never emerged or didn’t grow than is normally when using buds from healthy tissue. Most buds that didn’t emerge are still alive and green. One of the candidate clones (a complex pummelo hybrid) had a much worse bud emerging than the other clones being tested. Leaf samples were taken from the shoots developed from the emerged buds from all plants in the test before the heat treatment part of the test was started. New growth resulting from the heat treated trees will be propagated on clean rootstocks and tested as soon as the tissue is mature enough. Another complete set of leaf samples will be taken from all surviving trees in the test before the termination of the trial.
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 huanglongbing (HLB) for researchers, growers, and students throughout the world. The database was designed to be a centralized source of relevant information with an accessible, user-friendly interface. Entries represent over 100 years of worldwide research on the various aspects of HLB: 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 2076 citations, 86% of which are linked to their original sources. While the majority of the entries are in English (86%), as the intention of the database is to serve the international research community, the remaining 14% of the entries are in Spanish, Portuguese, Afrikaans, Japanese, Chinese, French, German, Vietnamese, Dutch, Farsi, Arabic, Czech, Thai, and Hebrew. There were 796 page visits during Jan-Sept, 55% were new visitors from 29 countries, 48% from the US according to Google Statistics. During this past quarter, July-September 2011, we have continued to add new information to the database as well as develop the official Facebook page as a place to share citrus news and events in real time and invite fellow researchers and growers to HLB related events occurring throughout the world. The page is updated daily. We have also continued to develop the HLB_database listserv. The listserv adds a dynamic interactive component to the project by providing a forum for open communication among individuals around the world who have an interest in sharing information on Citrus greening related issues. We also use the listserv as a platform to distribute a weekly news summary of the most current citrus headlines. In addition to maintaining the most relevant and accurate information, we have begun exploring alternative housing and platform options that would be better suited for long term management of the database. This project has been presented to researchers and students in several national and international meetings in the U.S, Germany, and in 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 content and marketing of the HLB/Greening Facebook page and the listserv service, 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) FCPRAC request for proposals 2009 [www.fcprac.com/proposals-2009.html] (3)Florida Entomological Society. [www.flaentsoc.org/]. (4) The Grower’s Citrus Greening Resource Center. [www.growermagazine.com/CitrusGreeningResearchCenter] (5) University of Florida Entomology and Nematology Pest Alert. Gainesville FL. [www.entnemdept.ufl.edu/pestalert/] (6) University of Florida- IFAS- Extension CREC. [www.crec.ifas.ufl.edu/extension/greening/links.htm] (7) Southern Plant Diagnosis Network [http://www.sepdn.org/webfm_send/158] (8) Facebook:HLB (greening) database [http://www.facebook.com/pages/HLB-Greening-Database/164498020244442]
One of the most recent challenges with deploying DMDS in the ISCA release device called SPLAT (specialized pheromone lure application technology) has been the phytotoxicity of the treatment to leaves and branches. A substantial amount of the DMDS active ingredient is needed in order to affect psyllid population densities. Unfortunately, we have found the DMDS active ingredient burns tree foliage and can even kill entire small tree branches, if applied directly to the wood surface. Therefore, we have been developing alternative release devices that would allow deploying the DMDS active ingredient within trees without touching tree surfaces. Our most recent prototype is a sachet that is hung in trees with a wire hanger. The sachet contains the SPLAT-DMDS, which is allowed to evaporate through a porous membrane. Therefore, the DMDS dispenser is deployed in trees; however, the active ingredient does not come in direct contact with tree branches or leaves. We are currently investigating whether these devices will be effective. The experiment was initiated in September and we are still analyzing data. The results of an earlier experiment conducted in August with a newer formulation of SPLAT-DMDS have been analyzed. The results of this test did not replicate the success we observed with the same formulation last fall. Psyllid populations were not significantly reduced by deployment of this formulation in this mid-summer test (as compared with control plots)as was observed last fall. We are unsure why we were unable to replicate the earlier success. We are trying to determine if the inconsistent results are because of differences in psyllid population densities between these tests or differences in environmental conditions when the different tests were conducted. It is possible that the formulation is not holding up to the intense temperatures and rainfall experienced in the summer as compared with cooler and dryer conditions in the fall. Finally, we have initiated testing of three new formulations of SPLAT that contain repellents other than DMDS that have proven effective against psyllids in laboratory tests. These experiments have only recently been initiated and we should have initial results in 3-4 months.
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. 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. Among the twenty compounds, five were found at IC50 nano-molar and MIC50’s are in 1’3. ug/ml range against A. tumefaciens. Molecular docking and minimization studies were used to identify the structural difference between higher & lower activity compounds. Based on these results new compounds are being designed by adding suitable structural fragments to enhance the potency of the identified five active lead compounds. Currently, the five compounds have been submitted for patent application. The most active five compounds are being used to test their effect against Ca. L. asiaticus Mutational studies have been performed by computationally on SecA to identify the critical amino acid residues which are involving in ATP binding. Subsequently experimental studies will be designed to mutate the critical residues in order to verify the reduced level of ATPase activity.
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 accepted by Journal of Bacteriology. Xac Aw strain Whole genome sequencing of Xanthomonas axonopodis pv. citri strain Aw was completed. The doubtful regions were checked and have been confirmed by PCR and sequencing. The annotation curation is done. Plasmid data for the strain was not obtained with high confidence level from initial sequencing. Hence, plasmids were purified from the genome and sequenced separately using 1/8th plate of 454 titanium sequencing. The preliminary sequences for the 2 plasmids have been generated using CLCbio Genomics Workbench. Gap closure was done by PCR and sequencing. Initial analysis of T3SE revealed presence of XopF1 and avrGf1 gene and absence of HpaA as different from Xac A 306. Genetic analysis of those genes are underway. Xac A44 (Cu resistant), Xac A*270 and Xacm 1381 (Cu resistant) strain: The chromosomes were completed while the plasmids were still not complete and gap closing is underway for those three strains. The genome sequencing has provided valuable information regarding the virulence mechanism and copper resistance of citrus canker pathogen and related species.
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. 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 xanA, btuB, gumC, gumB, gpsA, and 7 hypothetical genes which are putative virulence genes. Currently, complementation analysis of the 12 mutants is underway.
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