Patent Grant
9247749
Bacterial plant pathogens pose unique problems for disease control. One primary control strategy for bacterial diseases is based on excluding the pathogen through the use of disease free seed, or quarantine and eradication if bacterial pathogens are introduced into an area.
There are only a few chemical control agents for established bacterial diseases, and their use is often limited because of phytotoxicity or pathogen mutations resulting in resistance to the agent. Also, commonly applied protective copper compounds (for example sulfates or oxides) have limited benefit in controlling bacterial diseases because of their poor penetration into plant tissues where bacteria establish themselves and, again, mutations provide bacteria with resistance to these materials.
Unlike the control of disease outbreaks in annual crops that can be remediated in subsequent years through sanitation and the use of bacteria-free seed stocks, replanting of perennial crops such as citrus involves high capital costs to establish the planting, and several years after planting before production is initiated.
Established bacterial diseases such as those caused by Candidatus liberibacter species (citrus greening or Huanglongbing, psyllid yellows and tomato, or purple top and zebra chip of potatoes, etc.) that survive in alternate host plants in the environment and are disseminated by insect vectors that commonly infect throughout the plant life cycle are very difficult to contain because of the wide dissemination range of the insect vector and the long lag time for symptom expression (Bove, J. M. (2006) Huanglongbing: a destructive, newly-emerging, century-old disease of citrus. Journal of Plant Pathology 88:7-37.). Unfortunately, recent attempts to culture the organism were met with limited success (Sechler, A., Schuenzel, E. L., Cooke, P., Donnua, S., Thaveechai, N., Postnikova, E., Stone, A. L., Schneider, W. L., Damsteegt, V. D. and Schaad, N. W. (2009). Cultivation of ‘Candidatus Liberibacter asiaticus’, ‘Ca. L. africanus’, and ‘Ca. L. americanus’ associated with Huanglongbing. Phytopathology 99, 480-486).
Huanglongbing (HLB) disease (also known as citrus greening or yellow dragon disease) is one such disease associated with the fastidious, Gram-negative, phloem-limited bacterial pathogen, Candidatus liberibacter spp. (Las). It is the most destructive citrus disease worldwide (da Graca, J. V. (1991). Citrus greening disease. Ann. Rev. Phytopathol. 29, 109-136; Halbert, S. E., and Manjunath, K. L. (2004). Asian citrus psyllids (Sternorrhyncha: Psyllidae) and greening disease of citrus: A literature review and assessment of risk in Florida. Fla. Entomol. 87, 330-353; and Gottwald, T. R. (2010). Current epidemiological understanding of citrus Haunglongbing. Ann. Rev. Phytopathol. 48, 119-139). The current management strategy of HLB is to chemically control psyllids and scout for and remove infected trees. However, current management practices have not been able to stop the spread of HLB disease (Duan, Y., Zhou, L., Hall, D. G., Li, W., Doddapaneni, H., Lin, H., Liu, L., Vahling, C. M., Gabriel, D. W., Williams, K. P., Dickerman, A., Sun, Y. and Gottwald, T. (2009). Complete genome sequence of citrus huanglongbing bacterium, ‘Candidatus Liberibacter asiaticus’ obtained through metagenomics. Mol. Plant Microbe Interact. 22, 1011-1020).
Candidatus liberibacter species plug the plant's vascular (phloem) tissues to limit nutrient movement. Symptoms of this disease reflect a severe deficiency of essential mineral nutrients (for example copper, manganese, zinc). A temporary masking of symptoms can be achieved by applying high rates of foliar nutrients; however, the bacterial pathogen remains active and infected trees continue to decline in over-all vigor and productivity. The lag time from infection to symptom expression for this disease varies from six months to five years depending on the age of the tree, vigor, and environmental factors (Bove (2006) J Plant Pathology 88:7-37). This lag in symptom expression provides ample time for infection before detection and containment in a new area can be accomplished.
The efficacy of current strategies for management of HLB is limited and no conventional measure has shown to provide consistent and effective suppression of the disease. High cost of frequent insect control and tree removal will eventually render citrus groves unprofitable. In addition, large scale application of insecticides will disrupt the eco-system and pollute the environment (Jun, L. and Xing-Vao. J. (2005). Ecological control of forest pest: a new strategy for forest pest control. J. Forestry Res. 16, 339-342). Frequently, insecticides will become non-effective due to the acquisition of resistance. Insecticides could also kill non-target beneficial insects which disrupt the biological control currently in place.
Antibiotics injected into the tree's vascular system are often toxic to the tree, and previously available surface—applied copper compounds are not mobile enough to inhibit bacterial activity within vascular or other plant tissues. Current HLB control strategies of frequent insecticide sprays to limit populations of the psyllid insect vector, removal of infected trees, and nutrient maintenance to keep existing trees as productive as possible until they die provide little confidence for a sustainable citrus industry or incentive to reestablish it (Bove (2006) J Plant Pathology 88:7-37; UF/IFAS SWFREC, IMMOKALEE IRREC Seminar, 5 Jun. 2009).
Prokaryotes and eukaryotes have evolved numerous systems for the active export of proteins across membranes. In bacteria, the most common form of secretion of peptides with a signal sequence involves the Sec system. SecA is a protein translocase ATPase subunit that is involved in pre-protein translocation across and integration into the cellular membrane in bacteria. It 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 (Manting, E. H., and Driessen, A. J. (2000). Escherichia coli translocase: the unravelling of a molecular machine. Mol. Microbiol. 37, 226-238). Thus, SecA is a promising antimicrobial agent because it is a protein conserved and essential in all bacteria and is absent in humans (Chen, W., Huang. Y. J., Gundala, S. R., Yang, H., Li, M., Tai, P. C. and Wang, B. (2010). The first low microM SecA inhibitors. Bioorg. Med. Chem. 18, 1617-1625; Li, M., Tai, P. C., and Wang, B. (2008). Discovery of the first SecA inhibitors using structure-based virtual screening. Biochem. Biophys. Res. Commun. 368, 839-845; and Jang, M. Y., De Jonghe, S., Segers, K., Anné, J., Herdewijn, P. (2011). Synthesis of novel 5-amino-thiazolo[4,5-d]pyrimidines as E. coli and S. aureus SecA inhibitors. Bioorg. Med. Chem. 19, 702).
SecA cooperates with the SecB chaperone to target pre-proteins to SecYEG as an active ATPase gene to drive protein translocation across the bacterial membrane when it is bound to the SecYEG complex (Economou, A., and Wickner, W. (1994). SecA promotes preprotein translocation by undergoing ATP-driven cycles of membrane insertion and deinsertion. Cell. 78, 835-843). SecA is the peripheral membrane ATPase, which couples the hydrolysis of ATP to the stepwise translocation of pre-proteins (Van den Berg, B., Clemons, W. M. J., Collinson, I., Modis, Y., Hartmann, E., Harrison, S. C., and Rapoport, T. A. (2004). X-ray structure of a protein-conducting channel. Nature. 427, 36-44). The crystal structures of SecA are available for other bacteria such as Escherichia coli (Papanikolau, Y., Papadovasilaki, M., Ravelli, R. B., McCarthy, A. A., Cusack, S., Economou, A., Petratos, K. (2007). Structure of dimeric SecA, the Escherichia coli preprotein translocase motor. J. Mol. Biol. 366, 1545-1557) and the ATPase active site has been clearly defined. This structural information had been utilized for structure based design to identify antimicrobial compounds with IC50 value up to 2.5 μM against SecA of Ca. L. asiaticus (Akula, N., Zheng, H., Han, F. Q., Wang, N. (2011). Discovery of novel SecA inhibitors of Candidatus Liberibacter asiaticus by structure based design. Bioorg. Med. Chem. Lett. 15, 4183-4188).
Development of alternative or complementary approaches for effective management of the disease 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 plants and to reduce the inoculum for psyllid transmission. Development of antimicrobial small molecules may provide economic and ecological benefits by reducing producing costs, decreasing insecticide application, preserving the natural habitat and populations of beneficial insects, and enhancing productivity of citrus in the presence of HLB.
There is thus a need for antimicrobials that (i) are not subject to the types of antibiotic resistance currently hampering antibiotic treatment of bacteria, (ii) can be developed rapidly and with some reasonable degree of predictability as to target-bacteria specificity, (iii) are effective at low doses, meaning, in part, that they are efficiently taken up by wild-type bacteria or even bacteria that have reduced permeability for antibiotics, and (iv) show few side effects. In particular, there is currently a need in the art for an effective antimicrobial compound to target Ca. L. asiaticus.
The present invention provides novel compositions and methods for improving plant health and controlling phytopathogenic bacteria and endophytic microorganisms on or within plant tissue.
The subject invention provides antimicrobial compounds that interrupt, in bacteria, the ATP-hydrolysis process in a Sec system and disrupt pre-protein translocation. In one embodiment, SecA inhibitors are provided that interfere with the ATP binding pocket of SecA, thus affecting protein translocation, including potential signal peptide dependent virulence factors. In a specific embodiment, the SecA inhibitors affect protein translocation and potential signal peptide dependent virulence factors of Ca. L. asiaticus.
In accordance with the subject invention, SecA inhibitors are administered to plants to contact bacteria. In one embodiment, an SecA inhibitor is directly injected into the plant vascular system.
The compounds of the invention can be used against bacterial diseases of annual as well as perennial crops and ornamental plants. In preferred embodiments, the SecA inhibitors are particularly effective against Ca. L. asiaticus in citrus plants.
The invention provides SecA inhibiting compounds that have antimicrobial activities. The term “inhibiting” indicates a reduction in the rate or amount of a measurable interaction. An SecA inhibiting compound of the present invention inhibits SecA activity in a Sec system, preferably in a bacterial Sec system, as measured by a block or delay in the ATP-hydrolysis process in a Sec system.
The SecA inhibiting compounds of this invention can be used in treating bacterial diseases of annual as well as perennial crops and ornamental plants. Preferably, the SecA inhibiting compounds are used with citrus plants; however, in other embodiments, the compounds of the invention can be used with a variety of plant species including, for example, trees, vines, forage, and annual plants.
The SecA inhibiting compounds of the invention are active against a variety of bacteria, including Ca. L. asiaticus. According to the subject invention, the SecA inhibiting compound interferes with the ATP binding pocket of SecA, thus affecting pre-protein translocation, including potential signal peptide dependent virulence factors of Ca. L. asiaticus and other bacteria.
Examples of SecA inhibiting compounds of the invention are as follows:
Their potent antimicrobial action is evident from their antimicrobial activities against Agrobacterium tumefaciens with MBC ranging from 128 μg/ml to 256 μg/ml.
In view of the aforementioned antimicrobial activities, the subject invention provides compositions comprising SecA inhibiting compounds, and salts thereof, as the active ingredient in anti-microbial compositions. The subject invention further provides methods of combating microbial growth through the use of an effective amount of the SecA inhibiting compounds, or salts thereof.
The subject compounds can be used in suitable solvents or diluents, in the form of emulsions, suspensions, dispersions, on suitable solid or semi-solid carrier substances, if desired, together with other compounds having antimicrobial activity.
Solid carrier substances that are suitable for the preparation of compositions in powder form include various inert, porous and pulverous distributing agents of inorganic or organic nature, such as, for example, tricalcium phosphate, calcium carbonate, in the form of prepared chalk or ground limestone, kaolin, bole, bentonite, talcum, kieselguhr and boric acid; powdered cork, sawdust, and other fine pulverous materials of vegetable origin that are also suitable carrier substances.
The active ingredient is mixed with these carrier substances by, for example, being ground therewith. Alternatively, the inert carrier substance can be impregnated with a solution of an SecA inhibiting compound of the invention in a volatile solvent and the solvent is thereafter eliminated by heating or by filtering with suction at reduced pressure. By adding wetting and/or dispersing agents, such pulverous preparations can also be made readily wettable with water, so that suspensions are obtained.
Inert solvents used for the production of liquid preparations should preferably not be flammable and should be as far as possible odorless and as far as possible non-toxic to warm-blooded animals or plants in the relevant surroundings. Solvents suitable for this purpose are high-boiling oils, for example, of vegetable origin, and lower-boiling solvents with a flash point of at least 30° C., such as, for example, polyethylene glycol isopropanol, dimethylsulfoxide, hydrogenated naphthalenes and alkylated naphthalenes. It is, of course, also possible to use mixtures of solvents. Solutions can be prepared in the usual way, if necessary, with assistance of solution promoters.
Other liquid forms that can be used include emulsions or suspensions of the active compound in water or suitable inert solvents, or also concentrates for preparing such emulsions, which can be directly adjusted to the required concentration. For this purpose, an SecA inhibiting compound of the invention can be, for example, mixed with a dispersing or emulsifying agent. The active component can also be dissolved or dispersed in a suitable inert solvent and mixed simultaneously or subsequently with a dispersing or emulsifying agent.
Furthermore, it is possible for the SecA inhibiting compounds of the invention to be used in the form of aerosols. For this purpose, the active component is dissolved or dispersed, if necessary, with the aid of suitable inert solvents as carrier liquids, such as difiuorodichloromethane, which at atmospheric pressure boils at a temperature lower than room temperature, or in other volatile solvents. In this way, solutions under pressure are obtained which, when sprayed, yield aerosols which are particularly suitable for controlling or combatting fungi and bacteria, e.g., in closed chambers and storage rooms, and for application to vegetation for eradicating or for preventing infections by bacteria.
The subject compounds and compositions thereof can be applied by conventional methods. For example, a plant to be treated or to be protected against attack by bacterium can be treated with the subject compounds and the compositions thereof by dusting, sprinkling, spraying, brushing, dipping, smearing, impregnating, injection into the vascular system, application to root system or other suitable means. In a preferred embodiment, the SecA inhibiting compounds of the invention are directly injected in the vascular system of a plant.
When the subject SecA inhibiting compounds are employed in combination with suitable carriers, e.g., in solution, suspension, dust, powder, ointment, emulsion, and the like forms, a high activity over a very high range of dilution is observed. For example, concentrations of the SecA inhibiting compounds can range from 100 μM, 500 μM and 1 mM. Of course, higher or lower concentrations may also be employed as warranted by the particular situation. Moreover, the subject SecA inhibiting compounds can be employed with other programs, such as commercial fertility programs.
Following are examples which illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.
Materials and Methods
Computational Methodology
Homology model of SecA was built with Prime structure prediction of Schrodinger software (Schrödinger, LLC (2005) Schrödinger Suite 2006. Induced Fit Protocol, Prime version 1.5, Schrödinger, LLC, New York). Reported X-ray crystal structures in Protein Data Bank (PDB ID: 2VDA, 2FSF & 2FSG) (Papanikolau, Y., Papadovasilaki, M., Ravelli, R. B., McCarthy, A. A., Cusack, S., Economou, A., Petratos, K. (2007). Structure of dimeric SecA, the Escherichia coli preprotein translocase motor. J. Mol. Biol. 366, 1545-1557; and Gelis, I., Bonvin, M. A., Keramisanou, D., Koukaki, M., Gouridis, G., Karamanou, S., Economou, A., Kalodimos, C. G. (2007). Structural Basis for Signal-Sequence Recognition by the Translocase Motor SecA as Determined by NMR. Cell. 131, 756-769) of E. coli SecA homodimer bound with ATP was used to build SecA homology model of Las. The coordinates for all reported proteins were obtained from the PDB.
Structures were prepared using the Maestro (Schrödinger, LLC (2005). Maestro, Version 7.5, Schrödinger, LLC, New York) software package and aligned using the Protein Structure Alignment module in Prime. If a PDB structure was missing side-chain atoms, Prime was used to predict their locations. Water molecules in all structures were removed. A brief relaxation was performed on each starting structure using the Protein Preparation module in Maestro with the “Refinement Only” option. The modeled protein structure was prepared with appropriate bond orders and formal charges by protein preparation wizard of Maestro module. Then ATP ligand was manually docked as reported (Akula, N., Zheng, H., Han, F. Q., Wang, N. (2011). Discovery of novel SecA inhibitors of Candidatus Liberibacter asiaticus by structure based design. Bioorg. Med. Chem. Lett. 15, 4183-4188) against the homology model and ligand-protein complexes were energy minimized. The receptor grid file was generated by excluding ATP and defining 8 Å radius from ATP without any constraints.
Structure-based virtual screening was used to screen putative SecA inhibitors from approximately 11 million small molecule compounds available from ZINC11 database (Irwin and Shoichet, (2005). ZINC—A Free Database of Commercially Available Compounds for Virtual Screening. J. Chem. Inf. Model. 45, 177-182). To reduce the workload into the pipeline, twenty thousand structures were selected based on the physicochemical properties e.g. Net charge (−1 to +1), H-Bond donor/acceptor (2-6), and molecular weight (300-600 Da). Glide program (Friesner, R. A., Banks, J. L., Murphy, R. B., Halgren, T. A., Klicic, J. J., Mainz, D. T., Repasky, M. P., Knoll, E. H., Shelley, M., Perry, J. K., Shaw, D. E., Francis, P., Shenkin, P. (2004). Glide: A New Approach for Rapid, Accurate Docking and Scoring. 1. Method and Assessment of Docking Accuracy. J. Med. Chem. 47, 1739-1749) was used to build receptor grid file and for the docking studies.
Molecular minimization of ATP, highly active ligands with protein complexes were performed by Macromodel suite program (Mohamadi, F., Richards, N. G. J., Guida, W. C., Liskamp, R., and Lipton, M. (1990). MacroModel—an Integrated Software System for Modeling Organic and Bioorganic Molecules Using Molecular Mechanics. J. Comput. Chem. 11, 440-467). OPLS (Jorgensen, W. L., Maxwell, D. S., and Tirado-Rives, J. (1996). Development and Testing of the OPLS All-Atom Force Field on Conformational Energetics and Properties of Organic Liquids. J. Am. Chem. Soc. 118, 11225-11236) force field and “distance-dependent” dielectric constant were used during minimization. All the minimizations were carried out by means of 5000 iterations of Polak-Ribiere conjugate gradient method and followed by converge on gradient until a convergence threshold of 0.05 kJ/mol·Å. All the molecular modeling studies have been performed on HP ProLiant, RedHat Linux operating system and docking postures were taken by PYMOL program (DeLano, L. L. (2002).
Inhibition Assay Against SecA of Las
Cloning, expression, and purification of SecA were conducted as described previously (Akula, N., Zheng, H., Han, F. Q., Wang, N. (2011). Discovery of novel SecA inhibitors of Candidatus Liberibacter asiaticus by structure based design. Bioorg. Med. Chem. Lett. 15, 4183-4188). Quantichrom ATPase/GTPase kit (Bioassay Systems, Hayward, Calif.) was used to test the inhibitory effect of different compounds on SecA of Ca. L. asiaticus as described previously (Denis, L., Guy, G. P., and Andrien, R. B. (1978). A convenient method for the ATPase assay. Anal. Biochem. 85, 86-89; and Richard, D. H., John, L. V., and Richard, A. W. (1998). A microassay for ATPase. Anal. Biochem. 169, 312-318). All these assays were done in triplicate and repeated three times with similar results. The OD values are measured from Benchmark plus ELISA micro plate spectrophotometer (Bio-Rad Hercules, Calif.). ATPase activities were determined by the release of phosphate ion (Pi) detected spectrophotometrically, using malachite green and inhibition was calculated by showing the percentage (%) of the remaining ATPase activities (Akula ibid).
Antimicrobial Activity Against A. tumefaciens
A. tumefaciens was obtained from the culture collection of citrus associated bacteria initially isolated from the roots and rhizosphere of citrus and maintained in our laboratory as glycerol stock stored at −80° C. Minimum bactericidal concentrations (MBC) of the selected compounds against A. tumefaciens were determined by the broth microdilution method. Streptomycin was used as a positive control. Each small molecule compound was dissolved in DMSO and two-fold serially diluted with LB medium to give final concentrations of 8 μg/ml to 512 μg/ml. Aliquots of 100 μl were poured into 96-well microplates. A. tumefaciens was grown overnight and diluted with LB medium to give final concentrations 5×105 colony-forming unit (CFU) ml−1. Samples of 100 μl were inoculated onto LB microdilution plates containing the tested compounds and incubated at 28° C. for 24 h. After 24 h of incubation at 28° C., 100 μl samples from different dilutions were transferred onto antibiotic-free LB agar plates, which were then incubated at 28° C. After incubation for 2 days, the number of colonies recovered was counted. Survival rate was determined as the percentage of recovered cells compared to viable cells in the initial inoculum. MBC was defined as the lowest drug concentration producing a survival rate of <0.1%. Experiments were carried out in triplicate and repeated three times.
Results and Discussion
Homology Modeling & Optimization of ATP Binding Site
Previously, only one PDB structure (2FSG) was used as a template to build the SecA homology model (Akula, N., Zheng, H., Han, F. Q., Wang, N. (2011). Discovery of novel SecA inhibitors of Candidatus Liberibacter asiaticus by structure based design. Bioorg. Med. Chem. Lett. 15, 4183-4188). In this study, three PDB structures (2VDA, 2FSF and 2FSG) were used as templates to further optimize the homology model of SecA. The precise positions, orientations/conformations of the helices in the homology modeled SecA are slightly different from the reported templates. The homology model of SecA was used to superimpose on the reported PDB (2FSG) structure to identify the ATP binding site on SecA protein of Ca. L. asiaticus (
Molecular Docking: High Throughput Virtual Screening/SP/XP
High throughput screening (HTVS) by molecular docking was performed and calculated docking scores to filter the structures, and the filtrates were subjected to standard and extra precision docking to evaluate the scoring functions. Approximately, 20,000 structures identified from the physicochemical properties were subjected to glide HTVS to filter the low scoring ones. The initial step to screen the small molecule databases is grid generation. Receptor grid file was generated that enabled Glide (Friesner, R. A., Banks, J. L., Murphy, R. B., Halgren, T. A., Klicic, J. J., Mainz, D. T., Repasky, M. P., Knoll, E. H., Shelley, M., Perry, J. K., Shaw, D. E., Francis, P., Shenkin, P. (2004). Glide: A New Approach for Rapid, Accurate Docking and Scoring. 1. Method and Assessment of Docking Accuracy. J. Med. Chem. 47, 1739-1749) to search for favorable interactions between the ligand and ATP binding-site region. The shape and properties of the receptor are represented on a set of grids for positioning and scoring ligand poses. From HTVS, approximately 5000 structures were selected based on the docking scores against SecA. The resulting poses of the docked compounds were ranked according to their Glide standard and extra precision docking scores. Compounds detected by 2D and 3D ligand-based searches were analyzed with the binding modes, intermolecular interactions, and with the chemical intuition twenty compounds selected for biological activity studies (
Inhibitory Assay Against SecA ATPase Activity of Ca. L. asiaticus and Antimicrobial Assay Against A. tumefaciens
The selected twenty compounds were tested for their inhibition against SecA ATPase activity. Among the twenty compounds, only five of them showed greater than 50% inhibition at 1 μM (
The minimal bactericidal concentrations (MBCs) of the five identified compounds and streptomycin were determined by broth microdilution method. The MBC values of five compounds C16, C17, C18, C19, C20 and streptomycin are 256, 256, 256, 128, 256 and 64 μg/ml, respectively. The MBC values of all compounds are 2-4 fold higher than streptomycin.
Molecular Docking and Minimization: Comparative Study of ATP Binding of High and Low Activity Compounds
The optimized homology model of SecA was used for molecular docking study. To validate the generated grid model and molecular docking methodology, the ATP structure was docked to the active site of SecA of Las. Glide Xtra-Precision software was used to rigidly dock the ligand at the binding site of the Ca. L. asiaticus SecA without any constraints and water molecules. The obtained results indicate that the binding mode of ATP inside the receptor is similar and in agreement with the experimental mode of the crystal structure (
To further understand the difference between high and low activity structures, energy minimization studies were performed after the molecular docking analysis. While the docking methodology facilitates studying the binding of the Ca. L. asiaticus SecA inhibitors with flexible and rigid biding modes, it does not allow the protein mobility. In order to give free molecular motion of SecA protein along with ligands, the ligand protein complexes were minimized and the binding energies calculated (
Conclusions
Structure based virtual screening was performed to identify the novel SecA inhibitors of Las. Five compounds were found to inhibit the ATPase activity of SecA of Ca. L. asiaticus in nano molar concentrations. These compounds showed antimicrobial activities against A. tumefaciens with MBC ranging from 128 μg/ml to 256 μg/ml. Molecular docking and minimizations studies showed the correlation between the experimental and theoretical studies.
Numbers with the same letter in a column indicates no significance at P<0.05; Numbers with the different letters in a column indicate significance at P<0.05. The number in each table cell is the mean Ct value for a sample (a quantitative PCR procedure to determine the CLAS populations); the statistical test used is Fisher's LSD method.
Conclusion:
There was significant effect (P<0.05) of CLAS population reduction in the treatments of both C16 and oxytetracycline as compared to water-treated control (Table 1).
Plant Materials
HLB-infected budwoods were collected in HLB infected grove in teaching block at CREC. The presence of CLAS titers in budwood was confirmed by qPCR.
Treatments
C16 and streptomycin were applied at the rate of 500 μg/ml; water-treated budwood was used as check. The budwoods were placed in a glass bottle containing either treatment or water as check. The glass bottles were kept in a ventilation hood with light on to increase the evaporation rate, therefore, promoting the uptake of antibiotics. Leaf and bark samples were collected from each budwood and 100 mg were accurately weighed and used for DNA extraction with Promega genomic DNA extraction kit. CLAS populations in each sample were determined using standard qPCR protocol. Each DNA sample was run in triplicate wells.
After overnight treatment, the antibiotic-treated budwood was grafted onto 2-3 years old Valencia trees. A total of 15 trees were grafted for each treatment. Three and five months after grafting, all 45 trees were sampled to determine whether CLAS has been transmitted through antibiotic-treated scion. Four leaves from the main stem of Valencia trees were sampled for DNA extraction and determination of CLAS populations.
The number in each table cell is the mean Ct value for a sample (a quantitative PCR procedure to determine the CLAS populations); the statistical test used is Fisher's LSD method.
Conclusion
C16 and streptomycin has reduced the CLAS transmission rate by 20% to 35% as compared to water control, which had 100% transmission rate of CLAS (Table 2).
Bacterial Strains and their Sub-Culturing Conditions
Cultures of Agrobacterium tumefaciens and Escherichia coli strain DH5α were maintained in LB medium at 28° C. and 38° C., respectively; Liberibacter crescens and Xanthomonas citri subsp. citri was grown in BM7 medium (Leonard, 2012) and nutrient broth at 28° C. Rhizobium etli (ATCC 51251), Bradyrhizobium japonicum (ATCC 10324), Mesorhizobium loti (ATCC 700743), Sinorhizobium meliloti (RM1021) were grown in Yeast-Mannitol broth at 28° C. Bacterial culture was grown to the logarithmic-phase to match the turbidity of 0.5 McFarland standard (Andrew, 2001). Thereafter, the bacterial suspensions were adjusted to approximately 106 CFU/mL with appropriate medium. A total of 100 μl of inoculum were added to a well in MIC plates.
Determination of Minimum Inhibitory Concentration (MIC)
The MIC is defined as the lowest concentration of a compound that inhibits visible growth of an organism after overnight incubation (this period of 20-24 h is extended for slowly growing organisms such as anaerobes that require longer incubation for growth) (Andrew, 2001; Wallace J R. et al, 1986). In our case, appropriate incubation period was chosen for each bacterial species according to their varying doubling time (Table 3). The broth microdilution method (Andrew, 2001; Jorgensen, 2009; Wallace J R. et al, 1986) was used to test a microorganism for its ability to produce visible growth. MIC plates were prepared with a 96-well microplate containing eight different levels of twofold dilution series of antimicrobial compounds. Antimicrobial compounds were dissolved in the formulation MM1 and twofold dilution series starting from 1024 μg/ml to 8 μg/ml were made in appropriate broth. A total of 100 μl were added to a well and each concentration was replicated in 4 wells. For each bacterial species, streptomycin and oxytetracycline was included as check. In addition, four wells containing only 200 μl broth and four wells containing broth and inoculum were also included to each MIC plate. A total of 23 antimicrobial compounds were subjected to test in this study. After incubating for appropriate period, MIC plates were examined for visible bacterial growth as evidenced by turbidity. Turbidity of MIC plates was also recorded in a microplate reader at a wavelength of 630 nm.
Escherichia coli strain
Agrobacterium tumefaciens
Xanthomonas citri subsp.
citri
Sinorhizobium meliloti
Mesorhizobium loti
Rhizobium etli
Bradyrhizobium japonicum
Liberibacter crescens
Determination of Minimum Bactericidal Concentration (MBC)
MBC is defined as the lowest concentration of a compound with the reduction by 99.9% of the initial inoculum of multiplying bacteria within the incubation period. While the MBC test is not well established and less reproducible (Taylor, 1983), the majority of literature on MBC measurement followed the procedure as follows: after MIC reading, 100 μl of bacterial suspension samples was taken from the first well with turbidity and all clear wells and inoculated on a 9-cm agar plate containing appropriate medium without antimicrobial compound. The inoculated plates were incubated under conditions as described for MIC test. Plates with colony forming number less than 50 were considered as MBC. Alternatively, MBC was determined by centrifuging the MIC plate at 4000 rpm for 8 min, removing supernatant (containing antimicrobial compounds), adding 200 μl of appropriate broth and incubating under conditions as described for MIC test. After incubating the plate for appropriate period (Table 3), the plate was examined for visible bacterial growth as evidenced by turbidity; MBC is the lowest concentration in wells that stayed clear.
MIC and MBC tests of various compounds against the bacteria tested according to the claimed invention are provided in Tables 4-10. Concentration of the compounds is in μg/ml and the solvent contains N-methyl-2-pyrrolidone:Hallcomide M 8-10:Tergitol L-61:Rhodafac RS 610:Emulpon CO-360 in the ratio of 20:10:8.8:1.6:59.6.
All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.
It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application.
This application is a continuation in part of International Patent Application No. PCT/US2013/0338710, filed Mar. 26, 2013; which claims the benefit of U.S. Provisional Application No. 61/615,555, filed on Mar. 23, 2012, all of which are incorporated herein by reference in their entirety.
| Number | Name | Date | Kind |
|---|---|---|---|
| 20040009972 | Ding et al. | Jan 2004 | A1 |
| 20040170978 | Schmidt et al. | Sep 2004 | A1 |
| 20050272723 | Glick | Dec 2005 | A1 |
| Entry |
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| Number | Date | Country | |
|---|---|---|---|
| 20150087512 A1 | Mar 2015 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61615555 | Mar 2012 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2013/033871 | Mar 2013 | US |
| Child | 14498454 | US |
