Since the date of submission of the first report for this project (on 8/7/12), I have learned that progress reports should be written in accordance with the calendar trimesters. For that reason, this report is describing only the period between the second week of August and the end of September. During this time, Citrus Core Transformation Facility maintained its level of productivity and continued to produce transgenic Citrus plants. Number of new orders that were placed was very high for such a short period of time. Altogether, nine orders were placed and all for production of transgenic Duncan grapefruit plants. The binary vectors with the genes of interests were named: pX7, pX11, pX16, pX19, pX20, pX28, pNah, pMED14, and pMED16. All nine vectors were successfully mobilized into appropriate strains of Agrobacterium; glycerol stocks were created, and stored at -80oC. For some of those cultures, the first co-incubation experiments were done. Transgenic plants that were produced during these 7 weeks belonged to some recent orders but also to a few older orders. Altogether, 25 Carrizo citrange plants were produced for the same ‘Y’ order: eleven Y150 plants, six Y102 plants, five Y141 plants, and three Y46 plants. Five Duncan grapefruit plants were produced carrying MYB2 gene from pBCR2 vector and additional six carrying gene from the pAZI1 vector. Mix of 10 Carrizo and five Duncan plants were produced with the BI gene from the pAtBI vector. Three Duncan plants with the gene from pDPR1 vector were made. Finally, a mix of seven additional Duncan plants were produced transformed with the genes from following vectors: three with pEDS5, one with pELP3, one with pWG24-13, and two with pWG25-13. Future work will focus on the newly placed order and on completion of old orders.
IRREC-ACPS project (Barrett Gruber, co-PI) The second objective of this project is to establish an ACPS replant trial at the UF-Indian River Research and Education Center (IRREC, Fort Pierce). The goal of this objective is to evaluate the performance of ACPS for citrus fresh-market citrus varieties and regional disease problems such as canker. After receiving funds in July 2012, RTK-DGPS mapping was used to stake the end-of-tree-rows on 8 bedded acres of research property at IRREC and was done to obtain an estimate of total tree numbers. This mapping was used to plan for two different planting densities: 8′ . 25′ (218 trees / acre, ‘conventional planting’); and, 9′.1/4(3’+19’+3’+25′) (387 trees / acre, ‘higher density planting in double tramlines’), all on standard 50-foot beds. After estimates were obtained, a total of 3,098 new grapefruit trees (1,305 Ray Ruby/US897, 1,305 Ray Ruby/Kuharske, and 488 Ray Ruby/Swingle) were ordered on contract from a certified, commercial nursery. The nursery estimated that these trees would be delivered to IRREC no later than August 2013. CREC-ACPS project at Auburndale The 14-acre ‘Hamlin’ orange project with C-35 and Swingle rootstocks continues to grow well with the in-ground open hydroponics system. Fruit yields from the highest planting density being tested (363 trees/acre) are expected to reach 400-500 boxes/acre in this, the fourth year. Corresponding nitrogen fertilizer rates for that density are expected to reach 180 to 190 lb /acre. Due to the rapidly increasing HLB infection in the block, we decided to lower the priority of high nutrient efficiency in the block because any nutrient stress could adversely exacerbate the development of HLB symptoms. So far this strategy seems to help, and a recent survey of symptom severity on all identified HLB-affected (symptomatic) trees revealed that more than 80% of the tree displayed mild to moderate symptoms, having set and developed a fairly normal crop of fruit. We expect many of the more symptomatic fruits to drop before the harvest in December, but hopefully every HLB-affected tree will still produce a partial yield in order to keep the average yield in the block economically viable. CREC-ACPS project at block 22, CREC A new 4-acre ‘Valencia’ orange experiment using in-ground drip fertigated open hydroponics was planted in August 2012 near the CREC. Rootstocks US897 and Swingle are being compared, as well as a calcium-free versus a calcium-rich hydroponics fertilizer source. The other factor being investigated is the use of alternating double rows or “tramlines” to better utilize grove space in higher planting densities. We are comparing 484 trees/acre in single rows with 538 trees per acre in tramline rows.
Selection of nano-particles (Edgardo Etxeberria, co-PI) Due to its capacity to bind both neutral and charged ions/molecules, we selected PAMAN [poly(amido amine)-tris(hydroxymethyl) amidomethane ] dendrimer as the experimental nanoparticle. This particle can be used to carry any of the ionic or neutral elements components of the foliar fertilization cocktail. Nanoparticle size was also scrutinized in terms of quantitative efficiency to carry nutrient elements and ease of isolation. Due to its size of 5 nm, we selected 4th generation PAMAM dendimers. Isolation of nanoparticles from unbound elements. Separation of element-loaded nanoparticles from unbound elements is a key step in the purification system. We examined three isolation methods using fluorescence-bound nanoparticles: 1. dialysis, 2. ultracentrifugation, 3. low speed nano-filter centrifugation. Nanoparticles were loaded with fluorescent Alexa-488 and recovery determined fluorometrically at 519 nm. We determined that low speed nano-filter centrifugation resulted in higher nanoparticle recovery without loss of volume. The same system was also used to determine the maximum amounts of nanoparticles per filtration step. Efficieny of the system is highest at xxx nanoparticles/mL. Urea as test substrate. Urea was selected as test substance due to its carbon based skeleton that can be followed by 14C scintillation spectroscopy. Binding of urea to nanoparticles. The number of urea molecules carried per nanoparticle was determined using 14C-urea. After a 30 min binding process of urea to nanoparticles consisting of agitation at 30’C and pH 7.0, unbound 14C-urea was removed by low speed filtration. The number of 14C-urea molecules bound to nanoparticles was determined by scintillation spectroscopy. From the standard curve (Fig. 1), it was determined that each nanoparticle binds to approximately 370 molecules of urea. Setup for tests of electrostatically charged spraying and conventional spraying efficacy A computer-controlled motorized turntable was constructed to serve as an infinitely controllable spray platform for potted citrus. The spray nozzles were mounted on a stationary vertical boom to spray the tree canopy on the rotating platform. An ESS single nozzle electrostatic sprayer module was purchased and is being incorporated into the same turntable spray platform. Initial tests with the experimental platform will include 1) nutrient coverage and penetration of citrus leaves with or without electrostatic charging, and 2) with or without commercially available surfactants.
Mid Florida Citrus Foundation (MFCF) a 501c5 not for profit organization which has supported (past 25 years) and currently facilitates 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. Maintenance of this grove requires extra financial commitment as grove care costs tend to be higher and fruit production is lower than commercial groves due to the nature of many of the research projects, especially those involving the management of the HLB vector or other damaging pests. During this funding cycle significant research findings related to Asian citrus psyllid, citrus leafminer and citrus rust mite management were obtained in the MFCF grove and incorporated into the UF/IFAS Extension Florida Citrus Pest Management Guide. Other trials in the grove have been established or continue to evaluate antibiotics for HLB management, nutritional programs for increasing tree health, root and foliar disease management and herbicide efficacy evaluation, to name a few. Also rootstock and cultivar evaluations have continued and been initiated. The Mid Florida Citrus Foundation also serves as a forum to educate growers, scientists, governmental agencies and others on research results. It has been the venue for numerous field days and tours focusing on trials conducted at the site.
In this quarter, we applied three therapeutic strategies to infected and healthy Valencia orange on Kuharske Carrizo rootstocks at Florida. Citrus rootstock trees were HLB inoculated with bark pieces from citrus infected trees using a standard inverted ‘T’ budding technique. The trees were kept in the greenhouse under natural light conditions at 17-25’C until the HLB infection was confirmed by quantitative real-time PCR (qRT-PCR) on August 2012. Eight treatments were applied on 80 healthy and 80 HLB-infected trees, grouped in three experiments. The therapeutic compounds used for the different foliar treatments were: L- Arginine, gibberellin in combination with 6-benzyl adenine (BA), and atrazine in combination with sucrose. The surfactant Silwet, LK-phite and LDKP3XTRA were added to all treatments. All treatments were sprayed on the citrus foliage; the volume sprayed per tree was enough to wet both the upper and lower leaf surfaces just to the point of runoff. Phosphites were added to our therapeutic compound mixtures, since our data mining of HLB infected citrus suggests the presence of genes associated with phosphate deficiency, the symptoms include starch accumulation and collapse of healthy root function. Our rationale is that by eliminating these deficiencies, we will be able to better evaluate if our therapeutic treatments enhance citrus response to HLB, prolong the life of HLB-infected plants, reduce the bacterial titer and counteract the detrimental effects of the infection on citrus production. For the first experiment we used 2 different concentrations of L-arginine dissolved in water with Silwet as surfactant and LK-phite and LDKP3XTRA. We use 10 healthy and 10 HLB-infected trees (1 tree = 1 biological replicate) for each L-arginine concentration. Ten healthy and 10 HLB-infected trees were treated with water containing just the surfactant and used as control trees. Also, 10 healthy and 10 HLB-infected trees were treated with mixture of surfactant, LK-phite and LDKP3XTRA in water and used as control trees. All HLB-infected treated trees and the healthy control sets were sampled. This will provide us 4 treatments x 6 trees x 3 biological replicates X 2 sampling points = 144 samples for analysis (RNA). For the second experiment we used 2 different concentrations gibberellic acid in combination with a single 6-benzyladenine concentration, the solutions were prepared as explained above. We use 10 healthy and 10 HLB-infected trees for each combination. This experiments will provide 2 treatments x 6 trees x 3 biological replicates X 2 sampling points = 72 samples for analysis (RNA). For the third experiment we used 2 different concentrations of atrazine in combination with one concentration of sucrose, solutions were prepared as explained above. We use 10 healthy and 10 HLB-infected trees for each combination. This experiments will provide us with 2 treatments x 6 trees x 3 biological replicates X 2 sampling points = 72 samples for further analysis (RNA). Three leaves that were mid symptomatic based on observation of blotchiness, mottling and hardening of the veins were collected at 0 time and 3 days after treatment from each individual treated or control tree and processed individually. Leaves were immediately frozen in liquid nitrogen and keep at -80’C until extracted. Total RNA was extracted and stored at -80’C. Expression levels of biomarker genes for each treatment will be determined by qRT-PCR.
In the period between the May 1st and the end of July, Citrus Core Transformation Facility continued to offer its services to researchers who needed transgenic Citrus material. Interest for the transgenic Citrus plants remained high. Six new orders were placed: to produce transgenic Carrizo plants transformed with genes from pY48 and pY109 vectors; for transgenic plants of Duncan grapefruit and sweet orange transformed with the genes from pSF1 vector and for transgenic plants of Duncan grapefruit transformed with the genes from pNW3 and pNW4 vectors. Work on these orders has begun immediately upon receipt of DNA material. At the same time facility continued to service old orders. As a result, bulk of the plants produced within this quarter belong to the orders placed previously. Altogether, 106 plants were produced within this quarter: thirty seven Carrizo pY102 plants, fifteen Carrizo pY46 plants, seven Carrizo pY141 plants, thirteen Carrizo pY150 plants, twelve Duncan pBCR2 plants, eighteen Carrizo pAtBI plants, and four Duncan pAZI1 plants. There are about 30 putatively transgenic shoots grafted on rootstock at different stages of early growth before transfer to soil. Productivity of the facility remains on the high level and operation is continuing without any problems.
Quarterly report for July-September 2011 The citrus extension agents work collectively to educate growers on HLB and production practices to minimize psyllids in groves with the use of citrus health management areas (CHMAs). The agents were actively involved in developing additional CHMA areas throughout all citrus production regions and to support those already developed areas with the local team captains. CHMAs offer the greatest opportunity to manage psyllid within a larger geographical area as compared to individual management efforts. The HLB photo series is being hosted on the agents’ website (http://citrusagents.ifas.ufl.edu/hlb_photos/index.htm) whereby monthly photos are posted to allow growers to view the progression of HLB over time. The agents made 29 grove visits, conducted 7 local educational programs, in addition to assisting with the Citrus Expo that is conducted annually in Ft. Myers. All events are designed, developed, and conducted to maximize educational opportunities to enhance grower knowledge within the citrus industry to ensure all are aware of HLB and suggested management strategies. Three local field days were conducted during the period to enhance surveying tasks for HLB or psyllid monitoring. Agents are also actively involved in assisting county extension offices with Master Gardener trainings or to assist with urban citrus issues and problems.
Quarterly report Jan. – March 2011 To assist in answering growers questions and to identify grove specific problems, the citrus agents conducted 45 field visits during the period of January ‘ March 2011. The citrus agents were actively involved with the Florida Citrus Show (formerly Indian River Citrus Seminar) whereby over 500 attendees gained knowledge via the 11 presentations related to HLB, canker or black spot identification. On a local level, 9 production schools or meetings were conducted to disseminate educational information to the local growers on a wide range of topics that have been determined to local grower needs or advisory committees. All citrus agents participated in the International HLB conference hosted in Orlando to gain information and then share with growers throughout the citrus industry. To ensure that low volume sprayers are delivering the proper spray droplet size as required on some pesticide labels, a sprayer rodeo calibration event was hosted in Bartow. This event utilized resources provided by the USDA researchers and provided printed reports to each participant to document droplet size. Programs were also offered to enhance pesticide and equipment safety to area citrus workers. These programs offer printed certificates to document training as required by worker protection standards or various insurance agencies. HLB photo series is available to all growers and interested parties via the web at: http://citrusagents.ifas.ufl.edu/hlb_photos/index.htm. This site allows interested individuals to view the condition of selected citrus trees that have been confirmed to have HLB over time.
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. In another 3-4 weeks, the thermotherapy treatment will be finished. Experience has indicated that laboratory testing before 12 weeks post treatment will result in false negative results. Thus in September, 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. In Riverside, the penicillin/streptomycin mixture, cyclohexamide, D4E1 (antimicrobial peptide) treatments used in Florida are being evaluated for elimination of Candidatus Liberibacter associated with tomato psyllid yellows and citrus stubborn from tomato and citrus, respectively.
The purpose of this project is to determine methods to easily eliminate Candidatus Liberibacter asiaticus (Las), the bacterium associated with huanglongbing (HLB) in Florida, from citrus with emphasis on cryotherapy, and to determine the effectiveness of using young indicator plants for biological indexing to verify elimination of graft transmissible pathogens. In this first quarter of the project, we have begun activities while waiting for the funds. In Riverside, young plants are being evaluated for use for biological indexing of Citrus tatterleaf virus, citrus vein enation, and citrus viroids. The results obtained from using young indicator plants (less than 4 months old) are being compared to the conventional method of biological indexing for these pathogens. In Colorado, permits have been obtained to permit shipment of huanglongbing infected material from Florida so that cryotherapy protocols can be done. In Florida, elite breeding material has been identified and prioritized for testing/treatment.
This grant started in May 2009. Three peer-reviewed journal articles have been published and three others submitted for publication so far. Following are some of the key findings in this project on the interactions of the Asian citrus psyllid (ACP) and the HLB-associated bacterium, Candidatus Liberibacter asiaticus (Las): (1) Transmission barriers and localization of Las in organs and tissues of ACP: Our results show the near systemic infection of ACP organs and tissues by Las. Additionally, the proportion of Las-infected salivary glands was significantly lower than that of infected alimentary canals or other organs suggesting that the salivary gland constitutes a major barrier to Las transmission by ACP (Ammar et al. 2011, Ann. Entomol. Soc. of Amer. 104:526-533; Ammar et al. 2011, J. Phytopath. 159:726-734). Furthermore, ultrastructural studies indicated that the food and/or salivary canals in the maxillary stylets of ACP may be also a barrier to Las transmission especially in younger nymphs (unpublished results). (2) We developed a new ‘excised-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 excised citrus leaves (abstract: Ammar et al. 2011, Proc. of 2nd Inter. Res. Conf. on HLB, Orlando, FL, Jan. 2011). We demonstrated that excised citrus leaves can be successfully used to assay the rate of inoculativity in ACP adults, and that such assays can save considerable time, material and greenhouse space, and may hopefully enhance vector-relation studies on Las and probably other Liberibacter species associated with HLB. The percentage of Las-positive leaves, when single ACP adults were tested per leaf, ranged from 2-6% using the more conventional HLBaspr primers, and 10-20% using the more sensitive LJ900 primers. Higher proportions of Las-positive leaves, however, were obtained with the following: a. increasing the number of inoculating psyllids (to 5-10 adults) per leaf, b. longer inoculation access periods, and c. incubation of the excised leaves for 1-wk post-inoculation before being processed for qPCR. Furthermore, logistic regression analysis indicated a positive correlation between Las titer in the ACP adults tested singly and the probability of detecting Las in the inoculated excised leaves, which can be very useful in epidemiological studies, e.g. to predict the rate/proportion of ‘Las inoculative’ psyllids from that of ‘PCR-positive’ ones in field samples collected in various locations or at various times of the year, which can provide timely and valuable information for monitoring and/or combating HLB (full paper submitted for publication). (3) Las replication in ACP nymphs and adults: We conducted three large experiments to study the effects of various acquisition access periods (AAP) on Las-infected citrus plants by ACP nymphs and adults on Las acquisition, transmission and replication in psyllids. In the first two experiments, using qPCR, the relative Las titer was generally higher in ACP nymphs than in adults and higher with longer AAP in both life stages. We are currently finishing and analyzing the third experiment by qPCR to confirm these results. (4) Development of new techniques: In addition to the ‘excised-leaf assay method’ mentioned above, we developed new and simple methods for rearing ACP (Ammar & Hall 2011, Florida Entomol., 94:340-342), and for studying the stylets, salivary sheaths and bacteriomes (organs containing symbiotic bacteria) of ACP and other Hemiptera, which can enhance various studies on vector relations of HLB and other hemipteran-borne pathogens (submitted for publication).
In April, the citrus agents conducted the annual Citrus Growers Institute in Avon Park with over 300 participating in the event. The program consisted of 12 presentations with a major focus related to HLB and management of psyllids. Presentations from the 2012 event (as well as the 2008, 09, 10 and 11) are posted to the citrus agents website at: http://citrusagents.ifas.ufl.edu/events/GrowersInstitute2012/GrowersInstitute2012.htm . Presentations as well as oral recordings can be viewed via internet connections from any location worldwide. These recorded programs serve as an archive of progress within the industry to manage HLB and psyllids. Citrus agents have also been assisting growers with 34 field visits to discuss citrus production issues and offer possible solutions. Grove visits are an essential tool in increasing knowledge and participation, especially with smaller growers. Citrus agents are providing CHMA assistance to aid in adoption of area wide spray events targeting psyllids over a regional effort instead of individual efforts. These regional efforts appear to be providing greater psyllid control as compared to areas where regional sprays are not being conducted. Seven citrus grower meetings were conducted around the state to address educational needs and enhance participation in various production related issues including HLB and psyllid management. The citrus agents are also involved in: hosting international visitors; assisting youth with 4-H citrus tree projects; conducting master gardener trainings; and making presentations at Florida State Horticultural Society. In addition to the above activities, a new foliar nutritional trial has been established in 2 plantings (Immokalee and Arcadia) where young citrus trees (3 years old and recently planted trees) are being grown. Trees are sprayed on a monthly basis (during the growing season) to determine the impact of applying foliar nutritional materials on the growth of young trees in areas where HLB is endemic. The study consists of 9 nutritional treatments that are similar to those being studied in mature trees that are HLB positive. The 3-year old block is virtually 100% HLB infected.
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. Currently, complementation analysis of the mutants is underway. Bacterial growth assays of the mutants and the wild type strain in grapefruit ‘Duncan’and sweet orange ‘Valencia’ have been conducted. Three mutants were tested for affect in release from citrus canker lesions (dispersal assay) on grapefruit for 5, 7, 10 and 14 days as compared to wild type strain. The preliminary assay showed differences in growth rate (total count) and dispersal rates (surface count). The dispersal will thus be calculated according to percentage point difference to negate any effect due to growth changes. Pectate lyase assay, proteinase assay and motility tests (both swimming and swarming) have been completed for all the mutants. Further characterization of the mutants using EPS, LPS, capsule and biofilm assays, is in progress.
Recent activities have involved a multi-pronged effort focused on bioinformatic characterization of genome sequence data for the D. citri insect vector, bacterial endosymbionts in the insect, and Liberibacter asiaticus, the pathogenic agent of citrus greening. Using data provided by Wayne Hunter and other members of the International Psyllid Genome Consortium, sequences derived from bacterial endosymbionts in the currently available D. citri sequence data have been characterized and a draft genome sequence for the Wolbachia endosymbiont generated. Wolbachia is of particular interest owing to reports of successful control of other diseases through Wolbachia-based manipulation of the biology of the insect vector. In addition to supporting the presence of Wolbachia, the D. citri genome data provides support for the presence of an enteric bacterium most closely related to Salmonella. Phylogenetic analysis of several Wolbachia gene sequences from the Florida D. citri isolate supports an origin of the insect distinct from China and consistent with a Southwest Asian origin. The genome has been manually curated and closely analyzed for features potentially linked to interaction with the host insect including ankyrin proteins, secretion pathways, and potential sources of nutritional provisioning of the insect. Comparison with sequenced Wolbachia isolates from other insects have also been generated. We are additionally involved in the effort to characterize features in the D. citri sequence itself, with particular focus on identification of repeats that can interfere with correct assembly of the genome. Strategies developed for analysis of the Florida D. citri isolate and its component endosymbionts are readily applicable to sequence data for other isolates as more data becomes available. Comparison of Liberibacter strains is ongoing, information on which will be presented by collaborators at the upcoming Citrus Health Research Forum in Fort Collins, CO.
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 Ca. L. asiaticus 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. In our recent study, we further expanded our previous study in identifying lead antimicrobial compounds with higher activities by targeting SecA using various computational techniques like homology modeling, virtual screening, molecular docking & minimization. Due to the uncultivable nature of Ca. L. asiaticus, we tested the potential inhibitory effect of the selected compounds against Agrobacterium tumefaciens, which is phylogenetically related to Ca. L. asiaticus. Twenty compounds were selected for biological activity study against SecA of Ca. L. asiaticus and A. tumefaciens. Five compounds were found to inhibit the ATPase activity of SecA of Ca. L. asiaticus in nano molar concentrations and showed antimicrobial activities against A. tumefaciens with MBC ranging from 128 ug/ml to 256 ug/ml. These compounds appear to be suitable as lead compounds for further development of antimicrobial compounds against Ca. L. asiaticus. Those findings were published in the article entitled: Identification of small molecule inhibitors against SecA of Candidatus Liberibacter asiaticus by structure based design. on Eur J Med Chem (http://dx.doi.org/10.1016/j.ejmech.2012.05.035). To test the application potential of those compounds on plants, the phytotoxicity studies were performed on the five compounds against citrus. At higher concentrations (0.25mg/ml), all five compounds showed phytotoxicity. When the concentrations were diluted to 0.025mg/ml, the toxicity went down to mild to low. Currently, we are testing the inhibitory effects of the five compounds again Ca. L. asiaticus in planta.
(863) 956-8817
(863) 956-5894
700 Experiment Station Road
Lake Alfred, FL 33850
Email Us
