The objective of this proposal was to translate the analysis of the transcriptome of HLB infected citrus trees to identify key processes connected to the HLB disease phenotype observed in the field in Florida. Based on this analysis two target processes were identified 1) the deficient citrus innate immune response and 2) the sucrose starch metabolism altered by source-sink disruption both caused by HLB. Literature and metadata analysis of our data as well as existing expression data available in NCBI-GEO formed the basis of choosing 6 chemicals that were formulated as treatments. We tested 8 spray treatments that included 6 therapeutic treatments and 2 control treatments. 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 as a nutritional suppliment. These treatments were first successfully tested with greenhouse citrus graft inoculated with HLB. Then these treatments were applied to 24 trees in the field on June 25, 2013 with 3 trees per treatment. In October 2013 these 24 and an additional 48 trees were sprayed with the 8 treatments providing 9 replicate per treatment. The 8 treatments were combined to make 4 , with 2 treatments each containing 4 chemicals and with 2 controls were sprayed on the 72 trees in March 2014. 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 analysis of the DNA was used to determine the bacterial titer. The RNA was used to analyzed 10 biomarkers using qRT-PCR: 5 related to immune responses pathways (EDS1, PR1, WRKY70, WRKY48, WRKY54), 2 involved in gibberellin pathways and 3 related to sucrose and starch metabolism, a pathway highly linked with HLB disease syndrome. As we expected, Gibberellin-2-oxydase was clearly induced with GA + BA treatments. Same trend was observed for one arginine and one atrazine + sucrose treatment. Relating to genes involved in sucrose and starch metabolism, we interestingly observed that at least at one concentration, the six treatments were able to reverse the trend of expression of invertase and sucrose synthase induced by HLB infections. GPT2 is strongly induced by HLB in leaf tissues and it is believed to be strongly associated with the starch accumulation in infected leaves. GPT2 is a transporter present in the chloroplast and responsible of the glucose-6-phosphate transport leading to the HLB-induced starch biosynthesis. Interestingly, we observed a strong down regulation of this gene in all six treatments only at three days after treatments. Data showed that some treatments may allow boosting Citrus immune responses. Indeed, WRKY70, a well-known transcription factor involved in biotic stress response was clearly enhanced by BA + GA spray and one Arginine treatment. Arginine and atrazine treatments induced expression of PR1, a SAR-related protein. Conversely, WRKY48 was not significantly regulated by any six spray conditions. Another key player in biotic stress responses, WRKY54, was in higher abundance in both arginine and atrazine treatments. One concentration of GA + BA showed similar effect. The transcript abundance of EDS1 was unchanged in comparison to both controls. Taken together, these data look promising and indicated that, at least at one concentration, the three therapeutic strategies were able to significantly affect expression of HLB-regulated key biomarkers. Further experiments are needed to be performed with these treatments to evaluate their effect on other important production features like fruit drop, yield and fruit/juice quality.
Fruit harvesting of the ‘Hamlin’ orange ACPS experiment near Auburndale was completed in December 2013. At that time the trees were exactly 5 years old in the ground. The experiment compares two rootstocks (Sw = Swingle; C35 = C35), two fertigation delivery methods (Drip = 2x 0.5 gph drippers/tree; MS = 1x 10.5 gph microsprinkler/tree) and three planting densities (218, 303 and 363 trees/acre). Cumulative 5-year yields of the best ACPS treatment exceeded 1,300 boxes/acre, while that of the grower control almost reached 800 boxes/acre. Average annual fruit yield from the best treatment was 260 boxes/acre, suggesting that it was rapidly approaching the economic break-even point. This important economic milestone in the life of a new grove strongly depends on the costs of production, the fruit yield, and the price of the fruit sold. The factors under evaluation in the ACPS experiment favored a high, early fruit yield and ensured reasonable limits on the production costs. The price of fruit and fruit revenue were not controlled experimental factors. A moderate production cost with the ACPS was possible despite a higher investment in trees at higher planting densities because the grove care involving sprays was more efficient (more canopy area covered per acre at high densities for broadcast pesticide, fungicide and fertilizer sprays). Water and fertilizer use efficiencies were also up to five times higher for the ACPS treatments than the grower control treatment in the first three years, thus saving fertilizer, fuel, and water. Summary fruit yield data (boxes/acre) for the four years 2-5 are shown in the table: Year 2 Year 3 Year 4 Year 5 STD-Sw (218*) 8.57 91.1 321 370.7 MS-OH-Sw (218) 18.23 93.1 395 339.7 Drip-OH-Sw (218) 17.17 115 388 356.9 Drip-OH-Sw (303) 24.71 159.7 471 489.9 Drip-OH-C35 (303) 34.04 200.9 557 478.1 Drip-OH-C35 (363) 38.52 222.2 622 436.5 * trees/acre Unfortunately the yields in this ACPS experiment have already peaked due to rapidly increasing HLB incidence, with many previously productive trees displaying blotchy mottle leaf symptoms, tree dieback and excessive preharvest fruit drop. At the end of five years, the HLB incidence in the experiment was about 70%.
Experiments to determine the efficacy of different nano-particle systems to deliver nutrients and antimicrobial molecules to citrus leaves: Greenhouse experiments to test the efficacy of nano-particles to deliver Mg to citrus trees are underway. Mg deficient citrus trees were treated with three types of nano-particles containing Mg. The nano-particles being tested are: 1.liposomes, 2. dendrimers, 3. kaolin. As control, we used an aqueous solution of Mg at a concentration commonly used in foliar nutrition programs. Trees are being sampled on a weekly basis for their photosynthetic rates as an indication of the effect of Mg in the reconstitution of the photosynthetic apparatus. Anatomical studies should follow completion of the treatments. Concurrently, polymer based nano-particles and liposomes containing a soluble fluorescent marker have been applied to citrus leaves to study the delivery of antimicrobial substances. Application has been performed on both abaxial and adaxial leaf surfaces. Penetration and movement of the fluorescent probe is being analyzed by fluorescent microscopy and laser scanning confocal microscopy.
Experiments to determine the efficacy of different nano-particle systems to deliver nutrients and antimicrobial molecules to citrus leaves: Greenhouse experiments to test the efficacy of nano-particles to deliver Mg to citrus trees are underway. Mg deficient citrus trees were treated with three types of nano-particles containing Mg. The nano-particles being tested are: 1.liposomes, 2. dendrimers, 3. kaolin. As control, we used an aqueous solution of Mg at a concentration commonly used in foliar nutrition programs. Trees are being sampled on a weekly basis for their photosynthetic rates as an indication of the effect of Mg in the reconstitution of the photosynthetic apparatus. Anatomical studies should follow completion of the treatments. Concurrently, polymer based nano-particles and liposomes containing a soluble fluorescent marker have been applied to citrus leaves to study the delivery of antimicrobial substances. Application has been performed on both abaxial and adaxial leaf surfaces. Penetration and movement of the fluorescent probe is being analyzed by fluorescent microscopy and laser scanning confocal microscopy.
Physiological tests and data collection on the original 36 heat treated trees are being continued. Leaf anatomy samples are being processed and embedded in paraffin for cross-sectioning. Looking at the cross-section of the leaf petiole will show the area of the phloem. An effect of HLB is flattening of the phloem cells which limits nutrient transport and leads to the overall decline of the tree. Measuring the phloem area of the leaf petioles will give insight as to whether the heat treatment process has reduced or rid the tree of the bacteria, allowing the phloem to recover and resume their natural shape. Chlorophyll fluorescence measurements will also be done to further quantify the overall health of the tree. A portable steam generator was used in the design of a new heat treatment system in which a retractable plastic tent has been retrofitted to a fruit hauling truck (goat truck). The steam generator is also part of the design of a heat treatment system for tree roots. Injecting steam into the soil will raise the temperature of the roots in order to kill the bacteria in the root system. The heat treatment system for the roots consists of a array of perforated steel pipes which will be pushed into the soil using the goat’s fruit picking hydraulic arm. Field experiments are planned to evaluate the performance of this system. Preliminary tests will determine the amount of steam that can be applied before permanant damage to the roots occurs.
This report pertains to the no-cost extension granted by CRDF for project #564 ending 4/30/2014. Work has continued during the no cost extension period on genome-enabled investigation of Diaphorina citri biology and characterization of strain-to-strain variation among Liberibacter asiaticus species. With regard to D. citri biology we are principally interested in the insect response to Liberibacter infection and to this end have mined the scientific literature for insect gene sets linked to bacterial symbiosis and transmission in other species, identified relevant homologs in the D. citri draft genome, and forwarded sequences to collaborators in the Cilia lab (ARS-Ithaca) to use in directed searches of the D. citri proteome in the presence and absence of Liberibacter infection. During the course of these analyses, proteome sequencing has revealed the presence of proteins that appear to lack corresponding genes in the current D. citri draft genome assembly. The absence of experimentally confirmed genes from the current sequence assembly may result from use of a 1 kb cutoff during the assembly process leading to the discarding of ~4000 sequence fragments. These sequences have been retrieved and are currently being used to improve the genome assembly such that we and others who are relying on this draft sequence will have access to the most complete data available. Availability of closely related Liberibacter asiaticus sequences permits analysis of strain-to-strain variation within these species, applicable to both diagnostics and insight into genome evolution. Alignment of Las psy62 with Chinese strains Las gxpsy and the recently released (but not analyzed) Las A4 reveals only limited differences in gene repertoire, with Las gxpsy having eight predicted genes absent from Las psy62 and Las A4, and twelve genes present in both Las psy62 and Las gxpsy that are absent from Las A4. All genes showing variable distribution are either hypothetical or phage-related, reducing the likelihood of significant differences in biological functioning among the strains. Single nucleotide polymorphisms (SNPs) have also been mapped for genes conserved among strains. Las gxpsy was shown to have 1766 SNPs relative to Las strain psy62 and Las A4 to have 1410 SNPs relative to Las psy62. 15-20% of the genes in the Chinese strains account for all of the SNPs relative to Las psy62. As more strain sequence become available, these strategies can be readily applied to determine how patterns of variation observed for the three strains compare with other isolates.
During August and September 2013, three locations (Pico’s Farm, Evan’s Properties, and Blue Goose Groves) were selected for the thermotherapy trial. Roughly 30 HLB-affected Valencia trees were chosen using a random block design at each of the locations. Trees were either subjected to no heat or tented for 96 hours which included 4 full afternoons of summer heat. In late November and December, trees were sampled for 3 months post heat treatment data. Trees were sampled again in March and April 2014 for 6 months post heat treatment data. Trees at Pico’s farm and Blue Goose’s properties were in the process of spring flushing and blossoming. Heated trees had an increase in canopy density. Despite the presence of spring flush, untreated trees were not dramatically thicker. Tree height had no change. Fruit crop was counted. The spring 2014 crop will be compared to spring 2015 crop to understand how thermal therapy affects fruit production, fruit drop, and fruit quality. Leaves were collected and processed to determine the Las titer in specific branches before and after treatment. The trees at Evan’s Properties were 15 years old. Before treatment, the Evan trees had very thin canopies with a large crop of fruit. During the spring sampling period, it was noticed that the heat treated trees contained a dramatic amount of new tender flush. Trees continue to look a bit sparse but there were definite improvement of canopy growth in some regions of the tree canopy observed when comparing photos of trees before and after heat treatment. Evan’s trees were slightly shorter after heat treatment as that the tops of the trees were burnt during treatment. Fruit crop was also counted at this location. Leaves from specific marked branches were collected. At this time, all samples from before treatment and 3 and 6 months later have been processed and analyzed for Las titer. Student t-test and Tukey’s test will be used to determine if changes in Las cells/gram of tissue is statistically less after treatment for each time period, as compared between treated and untreated, and between sites. The nine month sampling begins in mid-May 2014. During Spring 2014, a new heat treatment system was developed incorporating a steam generator. A retractable tent was mounted to the side of a fruit hauling truck (Goat truck). The accordion style tent is hydraulically controlled to ease the process of moving from tree to tree. The steam generator along with the electric generator and water tank were mounted into the bed of the truck. A hose is fitted from the steam generator to the side of the goat trailer so that the hose can be placed in the desired position on the ground before the tent is lowered over the tree. A series of trees have been treated at 55C for 1 to 20 minutes. The main objective of these tests was focused on learning the appropriate treatment time and temperature that will not permanently damage young trees. Preliminary results on young small trees (5-6 feet tall) suggested that 10 minutes or higher treatment duration with steam is too harsh for the tree to recover and will kill the tree. The time range was reduced to between 1 and 4 minutes based on tree size and temperature. The temperature around the tree canopy should not exceed 130 ‘F. It was also noticed that placing the outlet of the hose directly against the base of the tree causes too much damage and was later moved to a position in which the hose outlet was facing the tent instead of the tree.
The program research objective is to develop an effective and sustainable phage and/or tailocin (high molecular weight bacteriocin) based biocontrol system for Xanthomonas axonopodis pv. citri (Xac), the causal agent of citrus canker. During the first year of the project, we purified 39 high titer phage lysates and identified 13 phage groups, based on host range studies and type IV pilus dependency for host infection. Groups 1-3 (21 phages), as well as five of eight phages with unique host range patterns, were determined to be type IV pilus-dependent. Purified lysates of 16 phages have been examined using electron microscopy to determine their morphological classification. Electron microscopy results indicate the purified phages active against Xac are from the three families of the Caudovirales. We have identified 11 podophages (7 groups) with short non-contractile tails that exhibit capsids ranging from 52-65 nm in diameter, three siphophages (2 groups) with long non-contractile tails (122-130 nm) that exhibit capsids of 56-57 nm in diameter, and two myophages (2 groups) with long contractile tails (141-149 nm) and capsids of 96 nm in diameter. We have sequenced and annotated five podophage genomes with different host ranges and determined that they exhibit phage phiKMV-like architecture and a single subunit RNA polymerase. This genomic architecture is indicative of a virulent lifestyle, which is a necessary component for the development of functional and sustainable biocontrol program. We are currently sequencing two myophages and three siphophages. We have also identified three tailocins with activity against Xac. Electron micrographs of purified tailocin XT-1 showed a sheathed phage-tail-like structure that resembles an R-type pyocin, with the tailocin having an average length of 135 nm and a width of 18 nm. XT-1 exhibited broad host range activity, killing 13/13 Xac isolates tested. Tailocins XT-2 and XT-3 each had narrow host ranges (killing 2/13 Xac isolates) that overlapped with XT-1, but not with each other. The host ranges of the three XT tailocins indicate receptor site diversity. We have initiated testing of phage using a citrus detached leaf assay. In preliminary studies, the inoculum control panels showed raised, callus-like lesions typical of canker after 4-5 days, whereas the leaf panels challenged with phage (at a multiplicity of infection of 1) 24h post-Xac-inoculation showed no symptoms. qRT-PCR is being used to determine the distribution and persistence of the phage in citrus vascular tissue. To determine the ability of phage to survive in sunlight in the field, UV-C studies were undertaken. Phage CCP504 had a 0.5-, 2- or 4-fold reduction in plating activity when exposed to 10Joules (J), 20J or 40J, respectively, which makes phage CCP504 more resistant than most well characterized phages. UV-A and UV-B studies as well as UV protectant studies are ongoing. First round efficacy greenhouse protection and therapeutic evaluation of a phage cocktail (in cooperation with Dr. Nian Wang, University of Florida) is planned for the next quarter.
The ultimate objective of this project is the development of a phage-based biocontrol system for citrus greening. ‘Candidatus Liberibacter asiaticus’ (Las), the causal agent of citrus greening, has not been successfully cultured. However, a member of the same genus, Liberibacter crescens BT-1, has recently been cultured under laboratory conditions. The current focus of our project is to develop a detection system for bacteriophage (phage) and/or phage components (tailocins; high molecular weight bacteriocins) using L. crescens strain BT-1. We have developed an efficient overlay assay that has been used to test 307 individual phage lysates from diverse hosts including Burkholderia, Pseudomonas, Clavibacter and Xanthomonas species, as well as 11 broad host-range tailocins. Utilizing the same system, 41 plant extracts (weed, citrus, alfalfa, rice, fresh papaya pulp and seed), 27 citrus psyllid extracts, and 38 water samples were assayed for the presence of phage/ tailocin active against L. crescens BT-1. No phage or tailocin active against L. crescens BT-1 have been detected to date. Additionally, mitomycin C and/ or UV-induced filtrates from 24 diverse bacterial isolates were tested for phage, tailocin, and/or bacteriocin activity against L. crescens BT-1 with no activity detected. However, during our survey, we identified two bacterial isolates (Microbacterium strain TM-313 and Brevibacillus strain TB-205) that exhibit antimicrobial activity against L. crescens BT-1. We have undertaken preliminary steps to purify and characterize the antimicrobial factor from Microbacterium strain TM-313. To isolate the compound, TM-313 cultures and control medium were extracted with non-polar to polar solvents. The methyl tert-butyl ether extract of the TM-313 medium produced a zone of growth inhibition on L. crescens BT-1 overlay, with no activity observed in the control medium extract. Preliminary characterization of the active compound(s) [AC313] indicates that it is protease (Proteinase K, Trypsin, alpha-chymotrypsin) and heat (100.C/10min) insensitive, as well as being < 10kDa in mass; these are indicative of a non-proteinaceous small molecule. Further characterization is ongoing. Bioinformatic analyses of L. crescens BT-1 prophage regions (LC1 and LC2) and the Las prophage regions (SC1 and SC2) have identified putative tail fiber genes that can be used to modify an existing tailocin in the laboratory. In cooperation with laboratories in Florida we are testing extracts from L. crescens BT-1 and Las infected plants for phage activity. We are continuing to collect and test environmental samples to isolate phage.
The primary goal of this project is to develop cryotherapy as a practical, reliable method of eliminating graft transmissible pathogens from Citrus and citrus relatives without inducing juvenility. Permits have been obtained to ship pathogen infected citrus material from Riverside, CA and from the Exotic Disease Quarantine Laboratory, Beltsville, MD to Ft. Collins, CO, and materials from Ft. Collins, CO can be sent under permit to Riverside for lab testing. In the first 18 months of the project, we have developed a cryotherapy protocol which works well with any type of citrus or near citrus relative. Two technicians from Riverside and one technician from Beltsville have been trained in Dr. Volk’s lab on the technique. Initially we concentrated on therapy from citrus viroids and Citrus tatterleaf virus, and were able to eliminate these pathogens, the most difficult to remove by traditional shoot tip grafting (STG) using the cryotherapy protocol. Presently we are testing the protocol for elimination of huanglongbing, Citrus mosaic virus, and citrus variegated chlorosis (in Beltsville), citrus stubborn, Citrus psorosis virus, Citrus leaf blotch virus, Dweet mottle virus, citrus concave gum disease, and Citrus vein enation virus. Efficiency of therapy with cryotherapy is being compared with results obtained by shoot tip grafting with most pathogens. For Citrus tatterleaf virus, we have recovered 32 plants which are clean from 32 recovered plants; viroids 11 clean from 14 recovered plants; Citrus leaf blotch virus 6 clean of 6 recovered plants; Citrus psorosis virus 25 clean of 25 recovered; and Citrus Stubborn 10 clean of 10 recovered. Because we wait at least 12 weeks to conduct lab testing of recovered plants, we have a constant backlog of plants to be tested. In Riverside, we are testing recovered plants after they have been grafted from the test tube to a rootstock, thus representing plants that would be continued through the indexing required for a variety release. The research is continuing.
The primary objective of this research is to identify diverse germplasm which has a tolerance to huanglongbing (HLB) and its psyllid vector, Diaphorina citri. An earlier germplasm screening block containing 87 different accessions of citrus and citrus relatives and planted in the field at Ft. Pierce for four years provided evidence of different types of tolerance to HLB. As a result of the first trial, this is a second trial which includes accessions of Poncirus trifoliata, hybrids with P. trifoliata, sour orange types, and kumquats. For this present trial, 97 different accessions were selected based on the population structure which suggested that the accessions contained genetic material from P. trifoliata, kumquat, or sour orange. Seed was collected from the University of California Citrus Variety Collection; heat and fungicide treated using standard protocols, and shipped to Ft. Pierce where the seedlings were grown under greenhouse conditions. The trees in Riverside used for seed collection were tested for Citrus leaf blotch virus, Citrus psorosis virus, HLB, and Xylella fastidiosa prior to shipment of the seed. Seven seedlings of each accession were inoculated with HLB while in the greenhouse in March 2013, seven seedlings remained un-inoculated. The seedlings were planted in the field in late spring 2013 and the first visual assessment was made in November 2013. Sampling for HLB will continue biannually and incidence of Asian citrus psyllid will be made this summer season.
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. Application of crytherapy to recover plants free of HLB from budwood approaches 95 percent efficiency. 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, additional 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. With pre-treatment and cryotherapy, results indicated the procedure is very effective at eliminating Citrus tatterleaf virus and citrus viroids; pathogens most difficult to eliminate by thermaltherapy and by shoot tip grafting. Additional selections of several varieties of citrus infected with huanglongbing have been forwarded to Ft. Collins for therapy, and will be tested in another 6-8 weeks. In Riverside, a system has been developed using Jiffy pots for the seedling used as indicator plants, allowing for a growout of about 75 days from the seed planting 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 3 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.
The project entitled ‘Further characterization of HLB resistant clones of selected citrus varieties’ (project no. 758) is aimed at conducting experiments to understand the basis of HLB tolerance in Aurantioideae with genera sexually compatible with citrus like Microcitrus, Eremocitrus and Poncirus. Research conducted this quarter (from Jan, 2014 to March, 2014) consisted of: a). Second batch of pollinations conducted in Riverside (Citrus Variety Collection) using HLB tolerant and susceptible accessions. This year we have utilized certain mandarin cultivars known to be more tolerant to HLB than others based on field trials in Fort Pierce by collaborators. The crosses between these ‘tolerant’ mandarins and ‘resistant’ citrus relatives (as determined by our previous trial) is likely to yield useful resistance/tolerance to HLB. We have now performed over 1100 crosses using mandarin and pummelo as seed parents. Based on previous year’s results, we were able to select seed parents that are likely to set fruit when crossed with the pollen from HLB resistant citrus relatives. b). Seeds collected from previous year’s pollinations were germinated in the greenhouses in Riverside for confirmation of genotypes. These experiments are in progress. A part of the seeds will be sent to collaborators in Fort Pierce in the next few weeks for field evaluation of resistance. c). One batch of selected HLB tolerant and susceptible seedlings that were psyllid challenged in Fort Pierce (Hall lab) were used for making RNA extractions in Fort Pierce. The samples are now being processed and analyzed. The experiment continues in Fort Pierce and sampling will be done periodically according to our original plan. We are on track as per our proposed milestones.
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 field trial 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. Repeated experiment in greenhouse is underway.
The goal of the proposed study is to characterize the effect of application of beneficial bacteria (MICROBE Program) on management of HLB. In the enhencement 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 site were identified. For one site, six applications were conducted. For another site, the first application was conducted in February, 2014. 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. The following up study is ongoing.
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