The goal of the research is to control citrus HLB using small molecules which target essential proteins of Candidatus Liberibacter asiaticus (Las). In our previous study, structure-based virtual screening has been used successfully to identify five lead antimicrobial compounds against Las by targeting SecA. SecA is one essential component of the Sec machinery. Those compounds showed promising antimicrobial activity. However, further work is needed to apply the compounds. We will evaluate the important characteristics of our antimicrobial compounds including solvents and adjuvants, phytotoxicity, antimicrobial activities against multiple Rhizobia, antimicrobial activity against Las, application approaches, and control of HLB. Those information are critical to for the practical application of those antimicrobial compounds in controlling HLB. We also propose to further optimize the five lead compounds. In addition, we propose to develop antimicrobial compounds against lipid A of Las. The lipid A substructure of the lipopolysaccharides (LPS) of Sinorhizobium meliloti, which is closely related to Las, suppresses the plant defense response. Las contains the complete genetic pathway for synthesis of lipid A. We hypothesized that Las uses lipid A to suppress plant defense. Thus, targeting lipid A could activate plant defense response. Lipid A is also an ideal target and has been targeted for screening antimicrobial compounds for multiple pathogenic bacteria. We are optimizing the compounds in collaboration with IBM. Two compounds with slightly higher binding affinity than C16 were identified. Currently, we are evaluating the best range of composition ratio among each component (%weight) of AIs, solvents and surfactants. The following characteristics are being evaluated: 1) emulsion stability and ease of emulsion; 2) stability of diluted concentrate; 3) freeze-thaw stability; and 4) phytotoxicity to citrus species. We have successfully identified certain combination suitable for all five compounds. Field test is being conducted.
Mid Florida Citrus Foundation (MFCF) a 501c5 not for profit organization which has supported (past 25 years) and currently supports citrus research efforts of scientists from the University of Florida, USDA and private industry. The MFCF supports citrus research through the employment of a full time grove manager whom works closely with researchers to ensure that their projects are handled properly and that the grove is an excellent condition. The management of this grove requires extra financial commitment as grove care costs tend to be higher than commercial groves due to the nature of many of the research projects. Current projects being conducted at the MFCF are Asian citrus psyllid (ACP) pesticide evaluation control trials, low volume applicator trials, windbreak evaluation, HLB nutritional programs, new and existing herbicide trials, variety and rootstock evaluation trials. During the recently completed quarter (October 1 to December 31, 2013), the following highlights occurred at the Mid Florida Citrus Foundation ‘ A.H. Krezdorn Research Grove: ‘ Plant Improvement Team o Scion for HLB tolerance under evaluation o Evaluation of Valencia clones on new rootstocks established o Sugarbelle harvested ‘ Dr. Singh continued to evaluate herbicide tolerance of selected USDA rootstocks to various residual herbicides continues ‘ UAS of America evaluation on supplemental materials applied to the soil and/or foliage to increase tolerance to the affects of HLB and citrus canker continues ‘ Dr. Futch evaluations: o Continued evaluations of trifoliate rootstocks for HLB tolerance ‘ Applications of the ‘Boyd Program’, Keyplex and Ben Hill Griffin programs continued in the ‘commercial scale’ nutritional trial. ‘ Conducting late summer/early fall fertilizer and pest management programs for the groves o Herbicide program on schedule o Dormant psyllid management continued o Initiated mechanical weed/middle management for evaluation ‘ Applications of seven nutritional treatments continue in MFCF replicated nutritional programs evaluation and plant growth data taken in July ‘ MFCF evaluating topping treatments evaluated ‘ Commercial Trials: o Eurofins evaluations on disease and insect management continue o Evaluations of Agri Quest Citrus Root Health Improvement Project continue o Keyplex nutritional trial evaluations continue o DuPont demonstration for row middles management with Matrix Herbicide established o Syntech trial for GLP evaluation ‘ Drs. Stelinski and Rogers have continued evaluations of Asian citrus psyllid and citrus leafminer management in their areas. ‘ Drs. Albrigo and Wong have continued to evaluate antibiotics to manage HLB
We have continued to focus on the treatments and analysis of naturally infected trees in the field to evaluate the efficacy of our treatments. Twenty four trees were chosen and first sprayed with our 8 spray treatments that included 6 therapeutic treatments and 2 control treatments. The plot was first sprayed in June 2013 and then again in October 2013 with 3 trees per treatment. The therapeutic treatments included: 2, L- Arginine, 2, gibberellin in combination with 6-benzyl adenine (BA), and 2, atrazine in combination with sucrose. The remaining two treatments were control treatments that accounted for the surfactant Silwet, K-phite and fertilizer LDKP3XTRA that were added to all treatments. After the spray treatments the trees were sampled at day 0, 3 and 6 collecting 6 leaves from around each of the trees. The leaves from each tree was pooled and extracted for DNA, RNA and protein. The first spray yielded 72 samples that have been processed. The analysis of the DNA revealed that 40% of the plants were infected and that infected trees were unfortunately not always randomly distributed among each of the three replicates for each treatment. The RNA were analyzed for 8 genes, which were part of the 30 genes that we had previously shown to be modulated by the treatments also included were 3 housekeeping genes. For most of the genes that responded the activity was highest at day 3 and not day 6. The sprays improved the immune response and this could be seen by an increase in EDS1, PR1 and WRKY70 genes. Also this was observed for the metabolic function where an increase was observed at day 3 for the GA oxidase and GPT2. So these are encouraging indication of a positive and counteracting host response to the pathogen. To overcome the nonrandom distribution problem of infected trees as well as to increase the number of replicates we sprayed an additional 48 trees (6 additional replicates per treatment) so a total of 72rees including the previous 24 trees that we sprayed again for the second time on October, 2013. Samples were collected at 0, 3 and 6 days as we did previously for the June treatment. We have completed a CaLas titer estimation at 3 time points for the 24 trees (June, Aug and Sept) and observed no change in titer of CaLas. The titer of the additional 48 trees was also estimated by a pooling method and most of them are infected, we have isolated RNA from 48 trees 6 tree replicates for each of the 8 sprays. We plan a third spray for Feb 2014. The sprays coincide with the flush observed in this location to have a maximum impact of the spray treatments. We are conducting phenotypic analysis of all trees, initially 24 and now 72. Trees are being rated for the presence of HLB symptoms, stem diameters and canopy height and diameter. These measurements will continue throughout the duration of the experiment and represent a phenotypic analysis of the trees during this experiment.
IRREC-ACPS project (Barrett Gruber, co-PI) The second objective of this project is to establish an ACPS solid-set trial at the UF-Indian River Research and Education Center (IRREC, Fort Pierce). The overarching goal of this component is to develop ACPS for commercial grapefruit on the east-coast. To date, the progress of this project is as follows: -‘Ray Ruby’ grapefruit trees were ordered on-contract from a commercial, disease free certified nursery in August 2012. Currently (September 2013) the trees are near-ready for planting in the field. -The grove space has been prepared for this project: Bed and swale construction/finishing is complete. Herbicide application has been used in-row to suppress weed growth. -GPS was used to mark the ends of tree rows on the bed tops. -The bulk of the necessary irrigation parts/materials have been purchased or have been ordered and are on back-order. -Funds from other sources not related to this project will be used to cover labor costs associated with irrigation installation and tree planting. We are currently conducting a bidding process with multiple vendors for irrigation system installation and tree planting. CREC-ACPS project at Auburndale A second comprehensive survey of HLB incidence in this 14-acre ‘Hamlin’ experiment was completed in September 2013. The total percentage of HLB-symptomatic trees now stands at 62%, up from 44% in May. In general, during September the HLB-symptomatic trees looked better than at any other time of the year, and the condition of the trees actually began improving in June with the beginning of the rainy season and the peak summer temperatures. Leaf nutrient analyses of samples collected in August confirmed that the nutritional status of trees across all treatments was optimal, with perhaps a slight advantage of higher calcium % shown by the fertigated trees versus the conventional dry granular fertilized trees. However by late September the symptomatic fruit was already starting to drop, particularly from the most symptomatic trees with a high fruit set. The early indications are that the fruit drop from HLB-affected trees in this experiment will be as high as during the 2012-13 season. There does not appear to be any correlation of fruit drop with experimental treatments in the trial.
The goals specified in the original 3-year proposal to CATP11 were to: (i) Design and implement a searchable Liberibacter diagnostic sequence database, (ii) Assist with genome sequence assembly and analysis as needed by collaborators, (iii) Generate analyses for newly released Liberibacter genome sequences and incorporate into the existing genome browser, and (iv) Expand the genome browser at the CG-HLB Genome Resources Website to include other bacterial pathogens of citrus. One year of funding was awarded and in that time goals ii-iv have been achieved. The principle features of the CG-HLB Genome Resources web site is a GBrowse genome viewer providing a portal to navigate the genome sequences and search their features. Originally hosting sequences for CLas psy62 and Ca. L. solanacearum, genome sequences have been added during the past year for CLas gxpsy, Liberibacter crescens and Ca. L. americanus PW_SP. Gene features encoded by the various genomes are hyperlinked to tracks detailing functional predictions and cellular locations, with links provided to outside databases such as NCBI. Genome viewing can be problematic for draft genome sequences such as CLam PW_SP. To facilitate easier visualization we have joined the CLam PW_SP contigs into a pseudomolecule to facilitate linear visualization. In addition to adding sequences, the genome viewer is also designed to accommodate links between individual genome features and biological characterization data, as illustrated by inclusion of the in psyllid/in planta expression data generated by Yan et al (2013). Sequences for bacterial citrus pathogens Xylella fastidiosa, Xanthomonas axonopodis citri, and Xanthomonas fuscans aurantifolii have also been added to the browser. Site usage is tracked with Google Analytics. Excluding automatic indexers the site has averaged 1950 visits per year for the past three years with repeat users accounting for 1/3 of the traffic. One of our major goals has been to assist with genome sequence assembly and analysis as needed by collaborators. In light of the increased attention being given to the CLas vector, Diaphorina citri, and the clear need for assistance on the part of researchers involved in D. citri genome analysis, we expanded our bioinformatic efforts to include characterization of both D. citri and its bacterial endosymbionts. This work has resulted in publication of the Wolbachia endosymbiont genome sequence by my group (Saha, S, et al. 2012. Survey of endosymbionts in the Diaphorina citri metagenome and assembly of a Wolbachia wDi draft genome. PLoS One 7:e50067). This publication includes a survey of the D. citri genome assembly for sequences derived from a targeted list of endosymbionts and describes assembly and annotation of the endosymbiont Wolbachia, of particular interest given that it has been successfully manipulated to control the mosquito borne spread of dengue fever. Significant attention was given to ankyrin proteins and other genome features potentially linked to interaction with the insect. The Wolbachia draft genome sequence has been added to the CG-HLB genome browser with candidate host interaction factors included. We additionally contributed to characterization of the D. citri transcriptome, published in Reese et al, 2013. Characterization of the Asian Citrus Psyllid Transcriptome. J Genomics 2:54-58. Recently we have begun work on describing genome features in the most recent D. citri genome assembly and determining sequences of encoded proteins predicted to impact CLas survival and transmission. Sequences are being communicated to collaborators Wayne Hunter (ARS, Fort Pierce) and Michelle Cilia (ARS, Ithaca) for expression analysis.
We have continued a series of olfactometer studies to determine the response of male and female Diaprepes adults to odor sources that include synthetic pheromone [methyl (E)-3-(2-hyroxyethyl)-4-methyl-2-pentenoate], carvacrol, linalool and other compounds previously shown to be active by gas chromatograph-coupled electroantennography. We have also collected volatile compounds in the head space over female Diaprepes in an effort to detect a female-produced pheromone in addition to the male-produced pheromone noted above. That effort has not been successful to date. During this period, effort has been focussed on completion of olfactometer trials; results are not complete as of this writing. A field trial was completed that examined the spatial distribution and movement of individual males and females in a plantation of a eukcalyptus relative, Corymbia torelliana. The trial included mark and re-capture studies. Results are being analyzed and will be provided in our next progress report.
Our group’s efforts are focused on using bioinformatics analyses of CLas strains and the Diaphorina citri in order to make predictions about the biology of these organisms. Regarding D. citri, we have analyzed the new diaci1.1 genome assembly and predictions of encoded proteins to identify a set of sequences for predicted ‘interactome’ proteins likely to be regulated in response to CLas infection and mediating the interaction of CLas and D. citri. These candidates have been identified through analysis of the literature on immune responses and symbiosis related proteins in other insect systems, and their protein sequences used to identify homologs in the daici1.1 assembly. So far we have identified components of the innate immune pathway and proteins with predicted involvement in RNA silencing, as well as cathepsin proteases and cysteine-rich proteins linked to insect interaction with bacterial symbionts. In collaboration with Drs. Wayne Hunter and Michelle Cilia, these sequences have been prioritized and are currently being evaluated for expression in D. citri specimens with and without CLas infection. The outcome of these analyses is expected to reveal useful insights into the insect response to CLas infection. One factor complicating bioinformatic characterization of D. citri is that the diaci1.1 genome data is composed of sequences derived from both the host insect and its endosymbionts. To more clearly distinguish the impact of CLas infection on individual components of D. citri and its microbiome, we have sorted the diaci1.1 data set for sequences derived from bacterial endosymbionts Wolbachia, Ca. Carsonella, and the newly sequenced endosymbiont, Ca. Profftella. Ca. Profftella is of particular interest owing to production of a large polyketide toxin. Protein sequences of biosynthetic genes for this toxin have been extracted from the diaci1.1 data set and are being evaluated for expression by Michelle Cilia in conjunction with her analyses of immunity gene expression. Previous analysis of D. citri gene expression led by Wayne Hunter and conducted with our participation have recently been published (Reese et al. 2013. Characterization of the Asian Citrus Psyllid Transcriptome. Journal of Genomics 2:54-8). Additional ongoing analyses of the diaci1.1 data set includes prediction and comparison of the metabolic capabilities of D. citri and its component endosymbionts to generate hypotheses as to the nutritional relationships of these organisms. Variability among CLas strains has been conducted using the Breseq program to map locations of single nucleotide polymorphisms in strains sequenced by Dr. Robert Shatters relative to the CLas psy62 reference. BLASTP alignment of predicted proteins of the CLas psy62 and CLas gx_psy sequences has also been conducted, leading to identification of 97 proteins with sequence polymorphisms. Those with variability are currently being evaluated for shared structural or functional properties in order to identify features subject to selection pressure from either D. citri or plant hosts.
The overall objective of this research program is to develop an effective and sustainable phage-based biocontrol system for citrus canker. Our current efforts have focused on isolation and characterization of phages for Xanthomonas axonopodis pv. citri (Xac). As reported previously, using direct testing and enrichment of 45 environmental samples with three Xac hosts, we obtained 19 high titer lysates. The Xac panel has now been expanded to include eight additional isolates. Thirty-nine high titer phage lysates were tested using the expanded panel to determine host range. Five host range groups containing multiple phages, and eight phages with unique host range patterns were identified. Groups 1-3 (21 phages), as well as 5 of the 8 phages with unique host range patterns, were determined to be Type IV pilus-dependent. The diverse host range patterns of the Type IV pilus-dependent phages indicate that multiple secondary receptor sites may be involved in the infection process. Electron microscopy studies of plaque-purified phages CCP501-CCP505 showed that the phages had podophage morphology, with short non-contractile tails and exhibited capsids ranging from 52-62nm in diameter. Phages CCP501-CCP505 had genomes that ranged from 40-44.5 kb, as determined by restriction enzyme digest analysis of genomic DNA. The genome of phage CCP501 was sequenced using MiSeq Illumia technology, and preliminary annotation indicates that the genome exhibits phage phiKMV-like architecture and a single subunit RNA polymerase that are indicative of a virulent lifestyle.
The purpose of this project is to determine methods to effectively eliminate Candidatus Liberibacter asiaticus (Las), the bacterium associated with huanglongbing (HLB) in Florida, from citrus. Emphasis is being placed on cryotherapy with conventional shoot tip grafting being used for comparison purposes. The project also includes determining the effectiveness of using young indicator plants for biological indexing to verify elimination of graft transmissible pathogens. During this past quarter, selections of mandarin and sweet orange materials have been forwarded to Ft. Collins for therapy using cryotherapy and shoot tip grafting. Recovered plants are allowed to grow for 12-14 weeks following therapy before testing for the presence of HLB. There are nearly 200 plants being held waiting for the growout period before they are tested. In Riverside, we are investigating alternative media and potting methods for growing and handling young plants used as indicators. Ninety day old Mexican limes have been used to index for Citrus tristeza virus successfully. Seed has been sown to allow to the complete biological indexing of 12 clones which have been therapied; this requires a total of 396 indicator plants. The results will be compared to the results from biological indexing using the traditional method with 10-14 month old indicator plants.
The aim of our project entitled ‘Further characterization of HLB resistant clones of selected citrus varieties’ (project no. 758) is to conduct experiments to understand the basis of HLB tolerance in citrus relatives like Microcitrus, Eremocitrus and Poncirus. This quarter (July 2013 to September 2013) we focused on two aspects: a) During the spring of 2013, we conducted over 900 crosses using HLB susceptible mandarins and pummelos and HLB tolerant/resistant citrus relatives. The seed parents used were: Microcitrus, Eremocitrus, three mandarins-Encore, Fortune and Wilking and one pummelo, Hirado Buntan. At present, after the initial fruit drop that occurs during June in California, and after some bad weather including storms and gusty winds, we still have about 90 fruits of mandarin and pummelo that have been pollinated with various pollen parents known to have HLB resistance/tolerance. We also have about 100 fruits of Microcitrus crossed with various pollen parents. After the fruits ripen, we will harvest, evaluate the seeds and determine the genotypes of hybrid plants. We are currently developing qPCR primers to differentiate the hybrids based on the sequence of a nuclear gene, malate dehydrogenase. b) We have raised seedlings of putative HLB tolerant and susceptible plants in Fort Pierce (with collaboration from Ed Stover). At present the seedlings are about 8-10 inches tall. In the next quarter, we will be able to challenge these plants via psyllid feeding and collect tissue for analysis of various HLB tolerance mechanisms. We are on track as per our proposed milestones.
Huanglonbing (HLB) or ‘citrus greening’ is widely considered to be caused by ‘Candidatus Liberibacter asiaticus’ and is currently the most serious disease of citrus worldwide. Existing control methods for HLB include removal of HLB-infected trees to decrease inoculum sources, application of chemical insecticides or biocontrol methods to reduce psyllid populations and deployment of disease-free certification programs for budwood sources. Since ‘Ca. L. asiaticus’ is currently non-culturable, we intend to use Liberibacter crescens strain BT-1 as a model bacterium for our studies. L. crescens BT-1, first isolated from papaya, has been cultured. The L. crescens BT-1 genome was found to share 75.5% sequence similarity with that of ‘Ca. L. asiaticus’, and analyses of both the ‘Ca. L. asiaticus’ and strain BT-1 genomes predict bacteriophage susceptibility. We have proposed to develop a functional plate assay for the detection of bacteriophage (phage) and/or high molecular weight bacteriocins (tailocins) active against L. crescens strain BT-1, that may target other Liberibacter species. During the first phase of the project, we have focused on optimizing growth conditions for strain BT-1 in broth and solid media with modifications to existing growth media. To support bacterial lawn uniformity and consistency, we have evaluated different gelling agents for use in semi-solid overlays. Additionally, we have assessed the time-temperature exposure limits and oxygen requirements of strain BT-1. We observed no significant difference in growth of strain BT-1 when incubated under atmospheric or reduced (8-10%) oxygen conditions. Soil, plant and citrus psyllid extracts are being collected as potential sources of phage and will be tested once screening conditions are optimized.
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. All the 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. We are repeating this assay to eliminate any false positives. Pectate lyase assay, proteinase assay and motility tests, and EPS assay have been completed for all the mutants. Characterization of the mutants on LPS, capsule and biofilm formation are complete. Currently, we are studying the release process 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. Anatomical analysis of the inoculated tissues were conducted using transmission electron microscopy. The total RNA of grapefruit infected with wild-type Xac and mutant were extracted and microarray analysis was conducted. We are analyzing the data using Mapman and GO based approaches. Quantitative reverse transcription PCR was conducted to study expression of selected plant genes. Among the mutants affected in bacterial release from lesions, pthA4 mutation has the most significant effect. We have identified the target of PthA4 and studied its mechanism. We plan to modulate the target of PthA4 to control citrus canker in the future. Currently, we are working on two manuscripts to report the final findings of this project.
Management of phloem-limited bacterial diseases is very challenging. These bacteria employ unusual and sometimes unique strategies by which to optimize their niche occupation and obtain their nourishment from the host plant. Their location within the living (sieve tubes) plant cells, rather than in the intercellular spaces, offers different challenges and opportunities for them to avoid the host plant’s defense system. Phloem is also difficult for any bactericides to reach to control the pathogen population. Among the phloem-limited bacterial diseases, citrus Huanglongbing (HLB, greening) is one of the most devastating diseases. The current management strategy of HLB is to chemically control psyllids and scout for and remove infected trees. However, the current management practices have not been able to control HLB and stop spreading of Candidatus Liberibacter asiaticus (Las). The goal of the proposed study is to develop HLB management strategies which boost plant defense to protect citrus from HLB by exploiting the interaction between Las and citrus and understanding how Las manipulates plant defense. As requested by CRDF and SAB, we have revised project and will focus on the following two objectives: to characterize how Las causes HLB disease symptoms and how Las manipulates plant defense response by investigating the roles of putative virulence factors; to test different compounds in controlling HLB and characterize their mechanisms in controlling HLB. Recently, we compared the gene expression of PR1, PR2 and PR5 in healthy trees and Las infected citrus plants. The expression of PR1, PR2 and PR5 was significantly reduced in HLB diseased grapefruit as compared to healthy grapefruit after inoculation with Xac AW. We also tested whether infection by Las can make citrus more susceptible to infection by Xanthomonas citri subsp. citri. We also sprayed four times with different chemicals in 17 different combinations on citrus to test their effect in controlling HLB in one grove. Multiple compounds showed control effect. To further test those compounds, we have selected two more groves to expand the field test. The disease index of the two groves have been investigated. We compared the SA levels in HLB infected and healthy grapefruit after the inoculation with Xac AW. We also compared the SA levels in HLB infected and healthy Valencia citrus. We are continuing to evaluate the effect of different compounds on management of HLB both in greenhouse and in citrus grove. We have applied different compounds at three separate field trials. Following up study is ongoing. Four compounds were shown to have positive effect on controlling HLB based on two year field test results. To confirm this result, two more field trails were initiated in Lake Wales to further test the effect of these chemicals on HLB, and the follow up investigations are ongoing, including monitoring the HLB symptoms, disease incidence and Las titer in leaves. We are also testing the mechanism of those compounds showing positive effect on HLB control. We are characterizing the two putative virulence genes sndA and stbA of Las, e.g. subcellular localization and host proteins interacting with them using yeast two hybrid system. Interestingly, some targets identified are transcription regulators, transporters, zinc ion binding proteins.
The goal of this study is to understand the role of biofilm formation and quorum sensing (QS) in X. citri ssp. citri infection of citrus fruit and to prevent its infection by interfering with biofilm formation and QS. Recently, we compared the attachment of the QS mutants on the citrus fruit surface. Compared with wild type stain Xac 306, the quorum sensing mutant ‘rpfF showed significantly reduced attachment to the fruit surface as revealed by CLSM (confocal laser scanning microscopy) observation with the GFP-labeled bacterial strains. We also evaluated the effect of nine compounds on Xac biofilm formation on abiotic surfaces using the crystal violet staining method. The data obtained showed that three compounds were active in inhibiting Xac biofilm formation in NB liquid medium at. Three compounds exhibited a significant reduction in biofilm formation both on polystyrene surface and in glass tubes compared to the untreated control, where the level of biofilm formation were reduced to 50% and 60% of control, respectively. Plant test in greenhouse showed that treatment with the three compounds prior to infection could reduce biofilm formation of Xac on leaf surface, reduce the formation of canker lesions on spray-inoculated grapefruit leaves with the wild-type strain. Effects of the three compounds on Xac on detached immature citrus fruit were also tested using spray inoculation. Preliminary results showed that these small molecules affected Xac 306 infection of unwounded and wounded citrus fruits at sub-inhibitory concentrations. We have completed testing the effect of those compounds in different combinations with copper based bactericides in controlling Xac infection of grapefruit plants in the greenhouse. The sensitivity of biofilm and planktonic cells of Xac 306 to copper (copper sulfate) were evaluated by measuring the MICs. Biofilms are less susceptible to copper than planktonic cells. Effect of the selected compounds on sensitivity of Xac planktonic cells and biofilm cells to copper sulfate was also investigated. In the NB medium, planktonic cells exhibited a MIC of 0.50 mM CuSO4 without biofilm inhibitor. In the presence biofilm inhibitors at sub-MIC concentrations , the MICs of CuSO4 against Xac 306 planktonic cells were decreased to 0.25 mM. In a in vitro biofilm system test, the combined use of copper sulfate and the compounds individual or both resulted in significantly increased killing compared to killing by copper sulfate alone. The results have been accepted for publication consideration by Phytopathology. One patent is filed based on the results. We also identified multiple new biofilm inhibitors. The effect of those biofilm inhibitors to control citrus canker is being investigated. We tested the survival of both biofilm deficient and QS mutants on fruit surface. Effects of biofilm formation inhibitors on Xac infection on detached immature citrus fruit were tested using spray inoculation. The inhibitors affected the infection of Xac on both unwounded and wounded citrus fruits. We further tested three potential biofilm inhibitors. We are setting up the field trial to test the effect of the identified biofilm inhibitors to control citrus canker.
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. 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 is involved in pre-protein translocation across and integration into the cellular membrane in bacteria. In our study, we 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. We are evaluating different solvents and adjuvants for the five compounds. Their antimicrobial activities against Liberibacter crescens, which is closely related Ca. L. asiaticus and could be cultured, have been tested. Currently, we are optimizing these five antimicrobial compounds to identify compounds higher antimicrobial activity. Fourteen new small molecule inhibitors were identified. Further studies are being conducted on those 14 compounds. Currently, one manuscript is in preparation to report the latest findings. We are also in collaboration with IBM to further expand this study. Optimization of C16 was conducted. Extension of this project was approved by CRDF and we are continuing this project.
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