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. 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 and treatments already started. The follow up investigations are ongoing, including monitoring the HLB symptoms, disease incidence and Las titer in leaves. 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. We conducted disease severity survey in Sep. Four compounds were shown to have positive effect on controlling HLB based on two year field test results. We are also testing the mechanism of those compounds showing positive effect on HLB control. Our results indicated that those compounds upregulated defense related genes, e.g., PR2, but not PP2 an callose synthase genes, in both field and greenhouse. Las titers were also lower in treated plants than non-treated controls with the four compounds. We have investigated the effect of those compounds on disease severity, yield, juice content and quality. We will repeat those treatments for one more year. Currently, the treatments are being conducted. SA hydroxylase is being expressed and purified. Multiple SA hydroxylase inhibitors have been identified using molecular modeling. The inhibitory effect of the SA hydroxylase inhibitors have been tested using the purified SA hydroxylase. Three compounds showed inhibitory effect against SA hydroxylase. The three SA hydroxylase inhibitors will be used to control HLB in greenhouse or field trials. For the putative sec dependent effectors, we have identified some putative targets using yeast two hybrid assays. We are currently confirming the protein-protein interactions using immunoprecipitation method.
Our hypothesis is that application of antibacterial-producing bacteria directly to citrus root could suppress Las population in the roots and control Las. Application of antibacterials in this manner will avoid the strict restrictions of application of antibiotics on crops and ease public concerns since those bacteria are naturally present in the soil and are associated with plant roots. In order to achieve the goal, the following objectives will be conducted: Test antibacterial-producing bacteria against Liberibacter crescens and other Rhizobiaceae bacteria which are closely related to Las. We will mainly test the antagonistic effect of Bacillus, Paenibacillus, Streptomyces and Pseudomonas strains Agrobacterium tumefaciens, Sinorhizobium meliloti, and L. cresens; Control HLB using antibacterial-producing bacteria. For the field test, we will investigate how antibacterial-producing bacteria affect HLB disease severity, Las titres, and citrus yield, survival of the antibacterial-producing bacteria in the rhizosphere and expression of the antibacterial biosynthesis genes in vivo. We have isolated Streptomyces spp. Bacillus spp. Paenibacillus spp., and Pseudomonas spp. from Florida groves. Multiple isolates showed antimicrobial production activity. We tested 27 antibacterial compound producing bacteria. These strains had been recovered, purified and confirmed by 16S rDNA sequencing. The antagonistic activity against Agrobacterium, Sionrhizobium meliloti and Xanthomonas citri pv. citri was determined. 5 strains, belonging to Paenibacillus, Burkholderia, Paenibacillus, Streptomyces and Streptomyces showed good antagonistic activity. These strains were inoculated to citrus roots and the colonization was determined by inoculation and recover method in lab condition using small citrus seedlings. Around 10E8 cfu were inoculated to each seedling. Approximately 10E4 cfu were recovered from roots 20 days after inoculation (dpi).
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 (July 1 to September 30, 2014), the following highlights occurred at the Mid Florida Citrus Foundation ‘ A.H. Krezdorn Research Grove: ‘ Plant Improvement Team o Site prep continues for new planting of rootstock and scion cultivars being evaluated for HLB tolerance/resistance o Preparation for NVDMC trees ‘ Dr. Futch evaluations: o Continued evaluations of trifoliate rootstocks for HLB tolerance o Continue evaluating two new Dow herbicide trials ‘ Applications of the ‘Boyd Program’, Keyplex and Ben Hill Griffin programs continued in the ‘commercial scale’ nutritional demonstration. ‘ Conducting fall fertilizer and pest management programs for the groves o Herbicide program on schedule o Psyllid management continued . Participated in coordinated area wide spray in early August o Mechanical weed/middle management evaluation continues o Mechanical vine removal ‘ Continue to maintain Dr. Bowman’s new USDA rootstock plantings ‘ Commercial Trials: o Eurofins evaluations on disease and insect management continue o Keyplex nutritional trial evaluations continue . Initiated soil applied nutritional program evaluating the Roots product o DuPont demonstration for row middles management with Matrix Herbicide continues o Syntech evaluations on insect management and disease management . ACP . Greasy spot and melanose o Florida Ag Solutions continue to make applications to herbicide and insecticide evaluations . Initiated new evaluation in Navel block ‘ Drs. Stelinski and Rogers have continued evaluations of Asian citrus psyllid and citrus leafminer management in their areas
The purpose of the additional funding for this project was to collect additional data from growers so that the efficacy of the enhanced nutrient programs could be assessed more fully after some promising results were obtained from the limited data collected last year. After extensive effort, some data were collected from three growers. However, none of the data sets was complete enough for analysis, and efforts to obtain the additional information failed. Consequently, we will not be able to complete the goals of this project. Because all involved (growers and researchers) were optimistic at the start of the study, some funds were spent so that graduate students would be prepared to conduct the analyses. However, when it became evident that data would not be forthcoming, expenditures stopped. We believe that the growers had full intentions of supplying the data. However, recent events, such as extensive premature fruit drop, have been devastating to the industry, and it is understandable that the growers no longer have the time or energy to help with this project.
The goal of this project is first to identify a Bacillus thuringiensis (Bt) crystal toxin with basal toxicity against Asian citrus psyllid (ACP). The toxicity of the selected toxin will then be enhanced by addition of a peptide that binds to the gut of ACP. This modification of the toxin is expected to enhance both binding and toxicity against ACP. The proteolytic profile of 25 Bt isolates have been characterized at Iowa State University (ISU) by Teresa Fernandez-Luna so far. Toxin samples provided by Teddi Mitchell and Mike Blackburn, USDA ARS, MD were solubilized (sodium carbonate pH 10.5, 10 mM DTT, lysozyme 200 mg per ml) for 3 to 16 hours at 37’C, according to the solubility of each strain. Samples are then dialyzed for 22 hours against 50 mM Tris-Cl pH 8.5. To determine the proteolytic profile and protein stability, 10 ug of each sample was incubated with bovine trypsin at a final concentration of 10% at 37 ‘C for 1 or 2 hours. Finally samples were activated for 1 hour to obtain at least 2 mg per sample for ACP toxicity assays conducted by USDA ARS, FL. Trypsin was removed from samples using Benzamidine sepharose with a 30 min incubation at room temperature and removal of the sepharose by centrifugation. Eight groups of Bt strains were identified based on the number of protein bands (between 1 and 8) seen on SDS-PAGE following toxin activation. ACP bioassays were conducted in the lab of David Hall at USDA ARS, FL to test 13 Bt isolates for the presence of ACP-active toxins. Two of the 13 isolates showed toxicity against ACP, and an additional four isolates identified with potential activity against ACP. One of the toxic strains was selected for purification of individual toxins by ion exchange and gel filtration column purification at ISU. These purified toxins will then be tested individually against ACP. A series of experiments is underway at ISU to standardize procedures for screening of a phage display library for identification of an ACP gut-binding peptide. Following confirmation of binding of the selected peptide to the ACP gut, this peptide will be used for Bt toxin modification.
At the request of the CRB, Grafton-Cardwell and Morse merged their core entomology research efforts under a single project, 5500-501 (Morse’s portion of the project is 5500-501b). We have always coordinated our research efforts but this arrangement formalizes the situation. This report summarizes the Morse lab’s recent citrus research under this coordinated project (all arthropod research except ACP which is a separate project, i.e. 5500-189). Our major efforts this last year have focused on helping the industry deal with Fuller rose beetle (FRB) in relation to citrus exports to Korea. For the last several years, Korea has put increasing pressure on the CA industry to reduce egg mass levels on export fruit and there is a concern that the situation will become quite severe in 2014-15. To better advise growers and pest control advisors what can be done, 10 industry presentations on Fuller rose beetle have been made over the last year (since 10-1-13) and a Korea pre-season export meeting will be held in Tulare 9-16-14. Grafton-Cardwell and Morse have also updated Fuller rose beetle pest management guidelines recently to reflect all recent information. In addition, statistical analyses were recently completed to relate egg mass infestation levels of fruit with the probability of passing phytosanitary inspection given the number of fruit that are inspected. These data have been presented to industry and will be discussed at upcoming conference calls. 2013 field FRB control trials were run at Lindcove by Grafton-Cardwell (15 treatments evaluated) and in Pauma Valley by Morse (10 treatments evaluated). None of the treatments results in 100% control but it appears that 2 applications are relatively effective — either 2 ground sprays of bifenthrin, 2 trunk sprays of bifenthrin, or 2 foliar sprays of combinations of carbaryl, cryolite, and/or thiamethoxam. Based on these studies, two new field trials were set up in 2014 at the same field sites as in 2013 (at Lindcove, the same trees were used so as to look at FRB reduction after 2 years of treatments; at Pauma, we selected new trees to obtain high resolution between treatment efficacy). Citrus thrips resistance to Delegate has been confirmed on citrus in the San Joaquin Valley and more recently, we have investigated several reports of high levels of citrus thrips resistance to both Delegate and Success on blueberries in Kern and Tulare Co. Our concern is that citrus thrips from blueberries might be selected with these products in late summer and fall and then fly into citrus with further selection in spring. MGK recently released an organic formulation of Veratran D for citrus thrips control. A lab trial showed that this new formulation is just as effective, if not more so, against citrus thrips as the old non-organic formulation produced by Dunhill (unopened bag from 2008). In addition, it was confirmed that acidification of the spray tank to pH 4.5 before adding the Veratran D improves performance. Growers now have two effective organic options for citrus thrips control – Entrust SC + oil and Veratran D. We are continuing to examine contaminants of export citrus using 10 randomly selected cartons per load from a variety of citrus packing houses. To date, we have processed 41 such loads with a diversity of commercial varieties examined. We plan to continue this work through Sept. 2015.
A heat treatment machine using steam as a source of supplemental heat was developed. The system is able to heat treat single citrus trees up to the height of six feet one tree at a time. The system consists of a hydraulically controlled enclosure which descends to cover the entire canopy. A steam generator, together with a water tank was mounted on the bed of a ‘goat’ truck. Steam from the steam generator is streamed into the enclosure. The system was used to test steaming scenarios using multiple combinations of temperature and heating duration. More than 800 trees were heat treated in five different groves. From the experiment, we determined that applying steam at temperatures above 60 C (140 F) are likely to cause damage to the trees. When testing treatments with a temperature of 65 C (149 F), we discovered that in most cases the trees did not recover. There were two scenarios which we tested using the steaming system and they both seemed to work equally well. The first one was steam treating the tree at 55 C (131 F) for 3 minutes and the second was steam treating the tree canopy up to either 60 C (140 F) or 30 seconds after reaching a temperature of 55 C (131 F), whichever came first. These two scenarios were selected after several trial runs of different temperature and heating duration combinations. From the trial runs, these two scenarios seem to provide sufficient heat without damaging trees or causing significant leaf lost which delays the recovery of the trees. The tested trees are still under observation and the effectiveness of the treatment will be studied over a longer period to evaluate the performance of heat treatment. At this time, three, six, and nine months post heat treatment samples have been collected from HLB-positive Valencia at three grove locations. Samples have been processed and analyzed for Las bacteria. In addition, fruit harvest for the 2013-2014 growing year has been determined for each tree in the study. Fruit set and first fruit abscission for the 2014-2015 year have also been counted. These counts will be compared to fruit counts of heat treated and control trees of the harvest in spring 2015. Initial results show that four-year-old Valencia trees responded better (increased canopy growth, decreased Las bacterial titer) than 15-year-old Valencia. Additionally, the treated HLB positive four-year-old Valencia located in a well-managed commercial grow (excellent fertilizer and insect management program) visually outperformed the other block of Valencia located in a grove with a weaker management program. In depth statistics of the three randomized block design experiments will be performed after one year samples are taken (August-September 2014).
The overall objective of this study was to deliver antibiotics into HLB infected citrus plants using nanoparticulate carrier systems such as emulsions, liposomes and polymer nanoparticles. During initial proof of concept experiments, dye-doped liposome and polymer nanoparticles encapsulating water-soluble dyes as surrogate for antibiotics have been prepared. In addition, double emulsions of water-in oil-in water have been developed with a stable inner aqueous phase for the delivery of the water-soluble antimicrobials to plants. Liposomes particles have been prepared using e.g 1,2-dioleoyl-3-trimethylammonium-propane , 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphocholine, and cholesterol through the thin lipid film hydration, reverse phase evaporation and ethanol injection methods. Constituent lipids amounts were varied to encapsulate water-soluble dyes such as 9-aminoacridine, sulforhodamine B and fluorescein. In order to minimize the leaching of the dyes, cationic lipids were incorporated in the lipid composition. The particle size was restricted to less than 100nm by the use of appropriate filtration membrane and the choice of hydrating solvent. Cryo transmission electron microscopy of Liposomes prepared via thin film hydration (before extrusion) demonstrated particle size to be around 200nm. Dye-doped polymeric nanoparticles comprising of environmentally safe and biodegradable materials such as poly (lactic acid) (PLA) and encapsulating fluorescent dyes have been prepared using double emulsion method. Polymer particles were characterized for their particles size distribution, absorbance, and fluorescence properties. PLA particles mixed with adjuvants were sprayed on abaxial and adaxial sides of citrus leaves and analyzed for leaf uptake by IVIS spectrum imaging system. Whole leaf imaging allowed detection of the uptaken particles by leaves. Confocal experiments on select leaves showed the presence of the particles in the leaf surface. Future experiments will be planned to determine the particle and adjuvant conditions for increasing the uptake. In addition, double emulsion particles, i.e. water-in-oil-in-water or w/o/w were also developed. These emulsions contain an encapsulated aqueous phase suspended in bulk water were stabilized by varying the surfactants and relative ratio of the amounts of the internal and external aqueous phases. Emulsions containing oxytetracycline and dye in the internal aqueous phase were developed to be used for future experiments. Highlights: In these experimental trials three types of particles were developed for the delivery of water soluble compounds (antimicrobial agents) to citrus plants. ‘ Liposomes encapsulating water soluble dyes in the size range 100-200nm have been prepared and characterized. ‘ Polymer particles encapsulating dyes were prepared using safe and biodegradable polymers. The dye-doped polymer particles were applied to citrus plants and imaged after incubation. Whole leaf imaging and confocal imaging indicated the presence of the fluorescence particles near the surface. ‘ Double emulsions systems containing water soluble compounds in the internal phase were developed and analyzed using optical imagining. Emulsions exhibiting stability for about three weeks and encapsulating active ingredient were prepared for future studies.
Construction of the imaging system: An optical sensing system based on a modified DSLR camera is now largely constructed and is able to acquire images of isolated leaves and small citrus seedlings. At present, the device can take photos of fluorescent emissions at the following excitation and emission wave lengths: UV LED-364nm (emission filters: 500nm, 690nm and 730nm; Blue LED-470nm (emission filters: 500nm, 690nm and 730nm); Green LED-527nm (emission filters: 690nm and 730nm). In addition, the camera system can take images based on the optical rotation of polarized light by starch grains. Image analysis: Images of seedlings have been acquired and manipulated by focus stacking to produce a high resolution macro-image at visible wavelengths. The focus stacking approach will be applied to fluorescence and polarized images. In the initial stages, images are being taken of individual leaves from HLP-infected and non-infected sweet orange trees. Fluorescence images are combined as ratios to identify regions of leaves that show chlorophyll loss. The Optical rotation images are being used to map the location of starch accumulation within infected leaves. Psyllid-inoculation of citrus seedlings: Sweet orange seedlings are being germinated and are ready to be exposed to feeding by HLB-positive psyllids. However, these experiments have been temporarily delayed due to low population numbers in the infected psyllid colony. It is anticipated that psyllid feeding studies will be initiated within the next reporting period.
The project entitled ‘Further characterization of HLB resistant clones of selected citrus varieties’ (project no. 758) is in its second year. The aim of the project is to test plants that have exhibited tolerance/resistance to HLB in a previous field trial. After this confirmation, we want to conduct experiments to understand the basis of HLB tolerance. Research conducted this quarter (from April 2014 to June 2014) consisted of: a) As a part of the greenhouse evaluation trial, we have already challenged 66 selected plants by feeding them with Liberibacter positive Diaphorina citri in a no-choice situation. We are testing DNA extracts made from these plants at various time intervals to measure the Liberibacter titer in all the plants. We have also been collecting samples and extracting them to prepare RNA for future use in conducting RNAseq experiments designed to see differences between susceptible and resistant cultivars. b) During the spring of 2014, we conducted over 1000 pollinations using HLB susceptible and resistant cultivars belonging to the genera Citrus, Eremocitrus, Microicitrus and Poncirus. We have collected seeds from 2013 pollinations and we are testing them for the presence of genetic material from the pollen and seed parents. c) At this time we are in the process of psyllid challenge of another set of 66 plants in Fort Pierce. These plants were also selected based on the results of the previous field trial. We are on track as per our proposed milestones.
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 was placed on cryotherapy with conventional shoot tip grafting being used for comparison purposes. Application of crytherapy to recover plants free of HLB from budwood approached 95 percent efficiency. The project also included determining the effectiveness of using young indicator plants for biological indexing to verify elimination of graft transmissible pathogens. Infected material was sent to Ft. Collins, CO under permit for cryotherapy treatment. Recovered plants were allowed to grow for 12-14 weeks following therapy before testing for the presence of HLB or other pathogens as appropriate. Personnel were trained in the cryotherapy technique at Riverside, CA and at Ft. Pierce, FL. With pre-treatment and cryotherapy, results indicated the procedure was very effective at eliminating Citrus tatterleaf virus and citrus viroids. These pathogens are the most difficult to eliminate by thermaltherapy and/or by shoot tip grafting. Forty USDA Pummelo hybrids were passed through the standard cryotherapy treatment. The treated buds were recovered, and trees grown from this treatment have been maintained in a greenhouse and monitored for Las. To date none of these trees have tested positive for Las, even though the material which supplied the buds for the cryotherapy treatment did test positive. Approximately 75 advance USDA scion selections that existed in the field and were infected with HLB have been rescued by collecting budwood from the least symptomatic branches on the trees and subsequently grafting onto rootstocks in the greenhouse. As the trees grew in the greenhouse, they were monitored for Las at intervals and trees testing Las positive were destroyed. The trees that test Las negative will be maintained and testing will continue. In Riverside, a system has been developed using Jiffy pots for the seedling used as indicator plants, allowing for a grow out of about 75-90 days after the seed was sown until the young plants can be inoculated and used as indicators. The inoculum bud survival is very high, greater than 98 percent. The entire indexing procedure can be done with the plants in the Jiffy pots. A trial was performed to compare the results obtained by using the mini-plant biological indexing compared with the traditional standard indexing protocol, using indicator plants 10-14 months old, for 15 accessions. The results from the mini-plant index revealed the presence of Citrus vein enation virus in one accession which had been missed using the traditional standard indexing protocol. The advantage of using the mini-plants for biological indexing is that 35 indicator plants may be contained in the space normally occupied by three traditional indicator plants; quicker turnover of the small plants for indexing means more accessions may be tested per year; biological indexing for pathogens expressing only under cool temperatures may be indexed for year-round as the smaller plants may be placed close to the cool pads in the greenhouse, and because they occupy less space, the whole trial can be done utilizing the cooler temperatures. This project was to have been funded beginning April 2012 and to continue for two years, ending April 30, 2014. The agreement was not executed until September 28, 2012, and funding allocated on December 18, 2012 but due to inefficiencies in the various systems, it was not possible to submit an invoice for funds for the research performed the first year. A request for a no-cost extension was denied at the end of the second year, so the results reported are the result of of one year’s funding for what was to be a two year project.
The goal of the proposed study is to characterize the effect of application of beneficial bacteria (MICROBE Program) on management of HLB. In the enhancement project, we are expanding the field test to include two more field trials in two more locations, larger scale of field test, test more beneficial microbes, and test different approaches to enhance the survival of the beneficial microbes in the soil. The field trial has been done for 9 nine months for one experiment site and 5 months for a second experiment site. For one site, nine applications were conducted. We have surveyed the disease incidence twice for this site. For another site, the first application was conducted in February, 2014. The experiment is ongoing with monthly treatment. We conducted the background survey regarding HLB disease incidence, severity, Las titers, Phytophthora, nematodes, root (root density and health status), and microbial diversity right now. We will be collecting most meaningful data including disease incidence, Las titers, gene expression of defense genes, survival of beneficial bacteria around 12 months after first application. For a better understanding of the Microbe Program, all the tested products were analyzed for the microbes in the products. The survival of the tested microbes are being investigated.
The overall objective of this research project is to develop an effective and sustainable phage/phage component-based biocontrol system for Xanthomonas axonopodis pv. citri (Xac), the causal agent of citrus canker. We currently have 39 Xac high titer phage lysates that are assembled into 13 groups based on host range and type IV pilus dependency for host infection. First round efficacy protection and therapeutic evaluation of a phage cocktail was conducted in cooperation with Dr. Nian Wang (University of Florida-Lake Alfred). A cocktail composed of four characterized phages from four different groups was used. Hamlin sweet orange plants grown in a citrus canker quarantine greenhouse were trimmed and fertilized to induce new growth. Plants were spray treated with sterile tap water (STW), Xac or Xac/phage cocktail mix. Xac inoculated plants were treated with the cocktail at a multiplicity of infection (MOI) of 20 or 0.5, 6 h-pre- or 6 h-post-Xac inoculation (5 X 10^6 or 5 X 10^8 CFU/ml, respectively). Control plants were inoculated with STW or Xac. Symptom development was monitored and the Xac population was quantified for a 21-day period. In in two independent experiments, when phage was applied, pre, post or at the time of the inoculation of Xac, reduced canker lesions numbers were observed, as compared to the Xac only inoculated plants. The most effective canker lesion control was observed when the phage cocktail was applied simultaneously with Xac at an MOI of 20, with the same treatment at an MOI of 0.5 resulted in reduction too. Treatment of plants 6 h pre- or post-Xac inoculation at an MOI of 20 or 0.5 also resulted in a reduction in lesions. The bacterial populations recovered from the leaves correlated with the visual observations over the 21-day post-inoculation period. In a separate study, phage movement experiments were conducted using Hamlin plants grown in the TAMU greenhouse facility. Plants were trimmed and fertilized to induce new growth. Phage CCP504 [8 X 10^8 plaque forming units (PFU)] was injected into each of two sites/stems, inoculating multiple stems with branching to determine phage movement and/or persistence over a three week period. Samples were obtained at time of inoculation and at 1, 2, and 3 weeks post inoculation, all in duplicate. Time point samples were sub-divided into 2-inch segments below and above the point of inoculation (POI) and homogenized in buffer. The samples were processed and the PFU/g of plant tissue determined both by direct plating and qRT-PCR. Initial studies indicated that phages survived three weeks in citrus plants without a host and moved from the POI into leaves. We have also identified two new tailocins, XT-4 and XT-5, the former exhibiting an active broad host range (killing13/13 Xac isolates-similar to XT-1) and the later a narrow host range (4/13). However, the host range of XT-5 does not overlap with that of the previously reported XT-2 or XT-3 tailocins. In our continued expansion of the phage pool, we have isolated 11 new potentially unique phages that are currently being characterized.
The focus of this project is to develop a detection system for bacteriophage (phage) and/or phage components (tailocins) using Liberibacter crescens strain BT-1 as the host. It is hypothesized that once ‘Candidatus’ Liberibacter asiaticus (Las) is successfully cultured, the protocols developed for L. crescens will be adaptable to Las. We developed an overlay assay for L. crescens that to date has been used to test 329 individual phage lysates from diverse hosts, as well as 12 broad host-range tailocins. Utilizing the same system, 131 plant extracts, 33 citrus psyllid extracts, and 55 water samples have been assayed for the presence of phage/tailocins active against L. crescens BT-1. No phage or tailocins active against L. crescens BT-1 have been detected to date. Additionally, liquid cultures of 32 diverse bacterial isolates were mitomycin C and/or UV-induced and tested for phage, tailocin, and/or bacteriocin activity against L. crescens BT-1, with no activity detected. Previously we reported antimicrobial activity against L. crescens by a Microbacterium isolate. We expanded our search to include a wide range of bacteria, including alpha, beta, and gamma proteobacteria, as well as actinobacteria; with most microorganisms exhibiting no antibacterial activity when tested against L. crescens using the overlay assay. However, several Actinomycetales produced large zones of growth inhibition against L. crescens. The diameters of the zones produced are indicative of small molecular weight compounds. To identify the compound(s) responsible for the observed inhibitory activity, strains producing inhibitory zones were grown using a range of conditions. Minimal or no production was achieved for most strains using standard liquid production techniques. Production conditions for the Actinomycetales antimicrobials are currently being optimized. Fractionation of the previously reported AC-313 inhibitory compound from Microbacterium (strain TM-313) indicates that the active compound is a mixture of three compounds. Large-scale production using strain TM-313 (10 liters) has been initiated to obtain quantities of the AC-313 compound(s) required for structural characterization. We are continuing the search for environmental phage(s) active against L. crescens, while initiating a recombinant approach. Sequences encoding for putative phage tail fibers have been identified in the L. crescens BT-1 genome. Experiments will be initiated to use sequence of identified phage tail fibers in the construction of a tailocin with activity against L. crescens BT-1.
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. 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 published by Phytopathology in a manuscript entitled: Foliar application of biofilm formation-inhibiting compounds enhances control of citrus canker caused by Xanthomonas citri subsp. citri. 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 are testing more potential biofilm inhibitors. We continue characterizing how quorum sensing and biofilm formation contribute to Xac infection of citrus fruit. Multiple virulence genes involved in quorum sensing and biofilm formation are being investigated. The involvement of ColR, RpfF, and three more genes involved in biofilm formation or quorum sensing is studied in details. Two manuscripts are under preparation. The field trial (two different sites) is ongoing to test the effect of the identified biofilm inhibitors to control citrus canker. One new compound is able to inhibit QS at a concentration of 100 .M based on observation of bacterial phenotype of aggregate formation. Plant test in greenhouse showed that the QS inhibitor (100 .M) treatment could reduce the formation of canker lesions and bacterial population on spray-inoculated grapefruit leaves simultaneously with the canker bacterium Xcc 306.
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