HLB is characterized by impaired phloem performance, hormonal imbalance, poor root function and architecture, limited nutrient supply resulting in immature fruit drop and ill tree health/death. Our goal is to restore the proper functioning of the phloem tissue and consequently restore tree health by using the newest plant hormone ‘Strigolactones’ (SL). As opposed to other growth regulators, SL are carotenoids derived hormones demonstrated to induce cambial activity, fruit growth and development and symbiotic arbuscular mycorrhizal (AM) fungi association in many crops. SL applications have resulted in substantial increases in vascular tissue including phloem. Besides SL regulates root and shoot architecture by increased formation of primary roots, lateral roots, and elongation of root hairs. Application of SL either foliar and/or drench to citrus trees in nano- to micro- mole quantities, should to induce new phloem, roots in and regulate shoot architecture of HLB trees resulting in restore tree health. month intervals. Strigolactone (SL) application in green house trial resulted in early induction of new flush and flowering in HLB-infected trees in comparison to control tress. Similar results were observed in HLB-infected trees in field experiments. Second, SL spray application was done on green house plants after an interval of three months to observe the effect of SL on fruit retention and development. In addition, SL was also applied on individual branches of Sweet orange ‘Pineapple’ to further confirm the vegetative growth promontory role of SL. Control trees were also sprayed with SL in Wiersdale, Marion County. We are also developing a model system which included one leaf-one stem-one root system of citrus to observe the effect of SL under greenhouse conditions. For all field and greenhouse experiments, measurements of fruit size and petiole diameter have been recorded. In addition, tissue samples were collected for further anatomical studies.
HLB is characterized by impaired phloem performance, hormonal imbalance, poor root function and architecture, limited nutrient supply resulting in immature fruit drop and ill tree health/death. Our goal is to restore the proper functioning of the phloem tissue and consequently restore tree health by using the newest plant hormone ‘Strigolactones’ (SL). As opposed to other growth regulators, SL are carotenoids derived hormones demonstrated to induce cambial activity, fruit growth and development and symbiotic arbuscular mycorrhizal (AM) fungi association in many crops. SL applications have resulted in substantial increases in vascular tissue including phloem. Besides SL regulates root and shoot architecture by increased formation of primary roots, lateral roots, and elongation of root hairs. Application of SL either foliar and/or drench to citrus trees in nano- to micro- mole quantities, should to induce new phloem, roots in and regulate shoot architecture of HLB trees resulting in restore tree health. month intervals. To carry out this project, a Postdoctoral associate was hired. The individual is already established in the lab and commenced with the preparations for all types of experiments. Experimentally, both parts of the project have been started. Greenhouse experiments: For the greenhouse trial,s 50 Valencia on Carrizo trees were purchased. Trees were planted on 3.5 gal pots and placed in a greenhouse. An automatic irrigation system was established and trees will be allowed to acclimate for 30 days prior to the start of treatments. Field experiments: Field grown Hamlin HLB-affected trees have been selected in a block at CREC. Trees have been flagged, area under the tree has been cleaned, fruit per tree counted and experimental fruit flagged. We have purchased the growth regulator strigolactone and are poised to begin the first sprays during the first week in September.
The goal of the project is to identify a Bacillus thuringiensis (Bt) toxin with toxicity against Asian Citrus Psyllid (ACP) and to improve its toxicity by genetic modification with a psyllid gut binding peptide. The peptide will be identified by screening a phage display library for peptides that bind to the gut of the ACP. Addition of the selected peptide to the identified Bt toxin will result in significant enhancement of toxicity of the modified toxin relative to the wild type toxin toward the ACP. Durign the current reporting period, the focus was on trypsin activation of the partially purified Bt toxins from selected Bt strains provided by USDA ARS, MD. Trypsin activation of Bt toxins was carried out at Iowa State University. Briefly, Bt toxins were solubilized using sodium carbonate pH 10.5 + 10mM DTT for three hours at 37’C and dialyzed against 50 mM Tris-Cl pH 8.5. Bt toxins were then incubated with bovine trypsin at a final concentration of 10% of the toxin concentration at 37 ‘C for 1 h. Removal of trypsin was carried out using benzamidine sepharose. The samples were boiled in denaturing SDS sample buffer for 5 min, separated on 10% (wt/vol) SDS/PAGE and stained with Coomassie blue. Different SDS-PAGE profiles of trypsin-treated Bt toxins were apparent with multiple molecular weight protein bands ranging from ~18 kDa to ~150 kDa. Based on the similarity of the molecular mass of the toxin profiles, eleven Bt strains are classified into the following six groups. These groups are distinct from the five groups reported in the previous update. Group one: Five Bt strains, each with three activated toxin bands of ~60, ~65 and ~70 kDa. Group two: Two Bt strains, each with two activated toxin bands of ~60 kDa. Group three: One Bt strain with one activated toxin band of ~60 and ~65 kDa. Group four: One Bt strain with one activated toxin band of ~70 and~73 kDa. Group five: One Bt strain with activated toxin bands of ~70, ~90, and ~150 kDa. Group six: One Bt strain with activated toxin bands of ~18, ~20, ~22, ~28,~30, ~35, ~49, ~60, and ~70 kDa. Two milligrams of each of four trypsin-activated Bt toxin samples were sent to Dr. David G. Hall, USDA-ARS for ACP membrane feeding assays for toxicity analysis.
ACPS -grapefruit research at IRREC: The overarching goal of this component is to develop ACPS and high-density plantings for commercial grapefruit on the east-coast. To date, the progress of this project is as follows: Irrigation installation was completed in October 2013. We are currently testing and evaluating the irrigation systems to fine-tune their performance and eliminate inefficiencies and any errors made during installation. The first experimental block of citrus was planted in November 2013. ‘Ray Ruby’ grapefruit trees on ‘Sour orange’ and ‘US-897’ rootstocks were planted on 8 acres on the citrus research grove at the UF-IFAS-IRREC station in Ft. Pierce. ‘Ray Ruby’ / ‘Sour orange’ trees were planted at a density of 152 trees/acre with microsprinklers and will be fertilized with granular, dry fertilizer: these plantings will serve as the ‘grower standard’ control treatment. ‘Ray Ruby’ trees on both ‘Sour orange’ and ‘US-897’ rootstocks were planted in staggered-set, tramline configurations at a high-density of 421 trees/acre: half of these plots will be fertigated with microsprinklers and half will be fertigated with in-line drip tubing. Funds from this project were also used to partially support the construction of a 5-acre high-density block of ‘Ray Ruby’ / ‘Kuharske’ grapefruit at the IRREC. In this block, all trees are being irrigated with microsprinklers but are planted at 3 different densities: 126, 189, and 421 trees/acre. Funds from other sources were used to cover the costs to complete this research block. These trees were also planted in November 2013. Data collection on tree growth, psyllid abundance, and CLas presence will begin in late December 2013. We plan to hold a field day for stakeholders in late winter/early spring of 2014. This field day will exhibit the two trials listed above. A Ph.D. graduate student was recruited and hired through the UF-Horticultural Sciences Department to assist in executing this objective. Currently, this student is in Gainesville completing her required coursework. The student will be available to monitor the progress of the research blocks full-time starting in the summer of 2014. A full-time OPS worker was hired and began work in August 2013. The balance of this worker’s wages came from other funding sources. This employee has been extremely instrumental in completing the irrigation installation and maintenance of the research block. This employee will continue to help maintain these research blocks and assist the graduate student (described above) with data collection.
Experiments to determine the efficacy of different nano-particle systems to deliver nutrients and antimicrobial molecules to citrus leaves: The objective of this study is to deliver water-soluble antimicrobial compounds into citrus plants affected by greening disease. The delivery of the antimicrobial compounds will be accomplished using nanoparticulate carrier systems (e.g. liposomes and polymer nanoparticles). In the first quarter of this project, dye-doped lipid and polymer nanoparticles that have encapsulated water-soluble dyes as surrogate for antibiotics have been prepared. Liposomes have been prepared using e.g. 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-3-trimethylammonium-propane, and cholesterol through the thin lipid film hydration method. Initial studies have been made to encapsulate water-soluble dyes such as sulforhodamine B and fluorescein. In order to minimize the leaching of the dyes, cationic lipids are incorporated in the lipid composition. Primarily, the particle size is being restricted to less than 100nm by the use of appropriate filtration membrane and the choice of hydrating solvent. Characterization of the developed dye-doped lipid structures as well as optimization to minimize leaching is under progress. In addition, dye-doped polymeric nanoparticles comprised of environmentally safe materials such as poly (lactic-co-glycolic acid) have also been prepared. Initial studies have been performed to encapsulate hydrophilic dyes in the polymer matrix. Using the double emulsion method, polymer nanoparticles that have successfully encapsulated fluorescent dyes have been synthesized. The size of the dye-doped particles lies between 200-300 nm. The engineered particles are being characterized for their particles size, distribution, absorbance, and fluorescence properties. Future studies involve the encapsulation of select dyes that have low interference from the auto-fluorescence of citrus plants and to study their uptake.
Experiments to determine the efficacy of different nano-particle systems to deliver nutrients and antimicrobial molecules to citrus leaves: The objective of this study is to deliver water-soluble antimicrobial compounds into citrus plants affected by greening disease. The delivery of the antimicrobial compounds will be accomplished using nanoparticulate carrier systems (e.g. liposomes and polymer nanoparticles). In the first quarter of this project, dye-doped lipid and polymer nanoparticles that have encapsulated water-soluble dyes as surrogate for antibiotics have been prepared. Liposomes have been prepared using e.g. 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-3-trimethylammonium-propane, and cholesterol through the thin lipid film hydration method. Initial studies have been made to encapsulate water-soluble dyes such as sulforhodamine B and fluorescein. In order to minimize the leaching of the dyes, cationic lipids are incorporated in the lipid composition. Primarily, the particle size is being restricted to less than 100nm by the use of appropriate filtration membrane and the choice of hydrating solvent. Characterization of the developed dye-doped lipid structures as well as optimization to minimize leaching is under progress. In addition, dye-doped polymeric nanoparticles comprised of environmentally safe materials such as poly (lactic-co-glycolic acid) have also been prepared. Initial studies have been performed to encapsulate hydrophilic dyes in the polymer matrix. Using the double emulsion method, polymer nanoparticles that have successfully encapsulated fluorescent dyes have been synthesized. The size of the dye-doped particles lies between 200-300 nm. The engineered particles are being characterized for their particles size, distribution, absorbance, and fluorescence properties. Future studies involve the encapsulation of select dyes that have low interference from the auto-fluorescence of citrus plants and to study their uptake.
A new high-capacity indirect hot air portable heater was purchased for further study on the effect of heat treatment of HLB inoculum as well as tree health. The heater works by convection, heating the air as it passes over a flame while a fan moves the heated air to the heat treatment tent as opposed to the electric heaters used in the previous treatments which use radiant heat transfer. The goal of using the dry hot air heater was to provide supplementary heat to increase the temperature more rapidly and test the ability of heat treatment late in fall and winter as well as to resolve the issues that were created by electric heaters used in the first part of this study. In November, five trees were tested at temperatures ranging from 50-55’C (122-131’F). A new portable tent was built to accommodate the new hot air heater and the larger trees subjected to the test. In all five tests, it was observed that the temperature within the tent rose much more rapidly than when the electric heaters were used. However, at this point, it is too early to assess the differences in leaf damage due to heat stress. It seems that hot air can increase the temperature more uniformly and rapidly than electric heaters, but it also increases the amount of leaf drop in the canopy after treatment. The original 36 trees that were heat treated in the summer are still undergoing physiological tests to evaluate the overall health of the trees. Six month post-treatment tests are currently underway. The goal of these tests is to quantify the change in health of the tree due to the heat treatment. This data will be used to support evidence of fruit yield and fruit quality improvement. A mathematical model was developed using the finite difference method to simulate heat transfer in the citrus canopy. This model will help to predict the amount of heat needed for different sizes of trees. Two types of heat transfer, conduction and convection, were applied within the model. The conduction heat transfer model will simulate heat transfer through the trunk and branches, while the convection heat transfer will simulate heat transfer around the canopy under the enclosure. The input parameters included the trunk diameter, thermal conductivity of the trunk, heat transfer coefficient of air, and air velocity from the fan. The output parameters were temperature and heating duration, in which a mapping of temperature with heating duration was produced. Regressions and model fitting was done based on the plots generated by the data. Model calibration and validation were done by comparing the data from the model with experimental data obtained from the preliminary results.
The objectives of this project are to characterize the molecular interactions between the effectors and the host mitochondrial proteins; to screen for molecules that inhibit the effector functions; and to control HLB using the inhibitor(s) and/or other related molecules. Although we previously reported that LasAI and LasAII target host mitochondria, we had to co-inoculate the gene silencing suppressor P19 to have a detectable expression of LasAI and LasAII (PLoS ONE8:e68921, 2013). After optimization for a variety of parameters that are critical for efficient gene expression in plants, high expression level of LasAI/LasAII were detected without co-inoculation of P19. Transgenic Arabidopsis plants expressing lasAI or lasAII showed a different degree of impaired growth. In particular, the LasAI contains domains responsible for abnormal growth of the root and/or meristem. Transient expression of LasAI and three different LasAI domains, LasAI-N-terminal, LasAI-repeat, LasAI-C-terminal allowed us to visualize the sub-cellular localizations of different domains. Because of high level expression of these effector proteins, we developed a novel in vitro screening system that can evaluate small molecules against these Las effectors. We are currently screening potential chemicals that could interfere LasAI localization in the mitochondria. The library consists of more than 30 million compounds obtained from the small molecule libraries of the TPIMS (Torrey Pines Institute for Molecular Studies). Interestingly, a few groups of compounds showed interference activity against the mitochondrial localization of LasAI, indicating that some potential inhibitors against these Las effectors may be screened out in the following screening process. Meanwhile, to study the function of LasAI in citrus, transgenic citrus were generated to express LasAI, LasAI N-terminal, LasAI C-terminal, LasAI repeat region, LasAII and GFP control, respectively. Transgenic citrus expressing different domains of LasAI are under evaluation. In addition, another hypothetical protein has been expressed in planta via transient and stable transformation, and founded to affect host resistance to a bacterial pathogen. The antibody against this protein was able to detect this antigen both in the transgenic plants and in the Las-infected plants. Meanwhile, the Western blot results revealed unique formation of this protein in E. coli and plants. Citrus plants with high level expression of this gene displayed HLB-like symptoms, yellow shoot and impaired growth. Further characterization of this effector revealed its unique sub-cellular localizations. Understanding the molecular mechanism of how the effector induces HLB-like symptom is underway. Polyclonal antibodies against LasAI and LasAII were generated and tested fro their sensitivity and specificity. The detection of LasAI protein transiently expressed in Nicotiana benthamiana using Western blot was well established. However, the detection of LasAI from infected citrus and psyllid hosts remains to be optimized.
The purpose of this project is to determine methods to effectively eliminate Candidatus Liberibacter asiaticus (Las), the bacterium associated with huanglongbing (HLB) in Florida, from citrus. Emphasis is being placed on cryotherapy with conventional shoot tip grafting being used for comparison purposes. The project also includes determining the effectiveness of using young indicator plants for biological indexing to verify elimination of graft transmissible pathogens. During this past quarter, 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. Bud survival is very high, greater than 98 percent. The entire indexing procedure can be done with the plants in the Jiffy pots. Another trial has been initiated whereby the results from using the mini-plant biological indexing will be compared with the traditional standard indexing protocol, using indicator plants 10-14 months old, for 15 accessions.
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 genera closely related to Citrus like Microcitrus, Eremocitrus and Poncirus. This quarterly research (from Oct 2013 to Dec 2013) consisted of: a) Conclusion of pollination experiments conducted during the spring of 2013: Out of 1000 pollinations conducted, we obtained about 50 usable fruits of pummelos, mandarins and Microcitrus (crosses were made between different HLB tolerant/resistant varieties with susceptible cultivars). As expected, some of the intergeneric hybrids did not set fruit. We are now collecting the seeds of the pollinated fruits and will be ready to test for the parental genotypes using a qPCR assay developed in our laboratory. b) Progress in greenhouse experiments for further testing and evaluation: Batch one plants consisting of HLB tolerant and susceptible citrus and relative accessions that were raised in October 2013 are now ready for psyllid challenge under greenhouse conditions in Fort Pierce. We have made arrangements for sample collection at various time points and subsequent RNA seq analysis as proposed. Since germination of certain genotypes was poor in the previous batch, fresh seed was collected and germinated in Fort Pierce greenhouses. We have good germination from the current batch and would include these in the HLB challenge assays as soon as they attain an acceptable size. c) Metagenomic analysis of several HLB resistant field plants has been conducted. The results of 16S rRNA sequencing from resistant and susceptible plants is now being examined to understand the role of the microbiome in the HLB tolerance phenomenon.
The research program objectives are to develop an effective and sustainable phage-based biocontrol system for Xanthomonas axonopodis pv. citri (Xac), the causal agent of citrus canker. During the third phase of year 1, we have expanded the phage bank with activity against Xac to 13 groups based on host range studies and type IV pilus dependency for infection. Host range diversity is indicative of multiple receptor sites being targeted by phages. Such diversity is a necessary component for the development of functional cocktails and for the prevention of phage resistant derivatives in the field when phage therapy is applied. Although type IV pili have not been implicated as virulence factors in Xac, their role in the pathogenic process is well documented in plant, human and animal bacterial pathogens. Electron microscopy studies of 16 purified phages from 10 groups indicate that we have isolated and purified 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 had previously reported that phages CCP501-CCP505 had genomes that ranged from 40-44.5 kb, as determined by restriction enzyme digest analysis (REDA), and that phage CCP501 exhibited phage phiKMV-like architecture with a single subunit RNA polymerase indicative of a virulent lifestyle. We now report that phages CCP504 and CCP507 also have similar architecture and are therefore virulent. Preliminary genomic analysis using multiple restriction enzymes indicates at least two to three REDA types in host range groups 1-5, and four unique REDA types in groups 6-10. Ongoing sequencing, annotation, and abortive lysogeny experiments will confirm the virulence of phages in the expanding phage bank. The availability of a large bank of virulent phages with a diversity of attachment sites is a necessary first step in developing a sustainable phage based control strategy. We have recently obtained a USDA-APHIS permit to conduct detached leaf assays with citrus in the laboratory. We will conduct population dynamics studies to monitor the effect of phages on Xac populations, which have been introduced to detached leaves. In the next period, we will conduct first round efficacy greenhouse protection and therapeutic evaluation (in cooperation with Dr. Nian Wang, University of Florida) and UV sensitivity and protectant studies.
‘Candidatus Liberibacter asiaticus’ (Las), the causal agent of citrus greening, has not been successfully cultured. However, one species of the genus, Liberibacter crescens, has recently been cultured under laboratory conditions. The focus of our project is to develop a detection system for bacteriophage (phage) and/or phage components (tailocins) using L. crescens strain BT-1. It is hypothesized that once Las is successfully cultured, the protocols developed for L. crescens will be adaptable to Las. During the second phase of the project we have developed a protocol to obtain reliable and uniform bacterial lawns in overlays; a necessary first step for the detection, purification and propagation of phage. Standardization of the assay now allows us to move forward with the evaluation of environmental extracts (plant, water, soil or insect) for phage, testing of a large archive of on-hand phage stocks, and screening for tailocins and/ or temperate phages that may have activity against L. crescens. Using the overlay assay, we have initially tested 272 individual phage lysates from diverse hosts and seven broad host tailocins with no activity against L. crescens detected. Utilizing the same system, 37 plant (weeds, citrus, alfalfa, rice, fresh papaya pulp and seed) and 21 citrus psyllid extracts, as well as 27 water samples and supernatants of four UV-induced Rhizobium species were evaluated for phage that replicate and lyse L. crescens; no phage plaques were detected. Classical bacteriocins and tailocins production assays using diverse bacterial isolates from our large collection have also been initiated. We have identified two bacterial isolates that exhibit antimicrobial activity against L. crescens. The nature of the activity is currently under investigation. Exploiting the developed detection system, we will expand our screening for phages, tailocins and microbial activity against L. crescens.
The goal of the proposed study is to characterize the effect of application of beneficial bacteria (MICROBE Program) on management of HLB. Currently, we are setting up the experiments to test different Microbe Products in management of HLB. We have developed a culture collection of approximately 400 bacteria initially isolated from the root and rhizosphere of citrus. These bacterial isolates have been screened for various beneficial traits . We are also evaluating the antagonistic activity of these bacterial strains against some well-known plant pathogenic fungi. Especially we have screened a bacterial isolate designated as 43A which possess multiple plant growth promoting activity and is also able to antagonize different fungi. We are also testing the plant growth promoting activity of 24 isolates using seed germination pouch in greenhouse. We have also selected several Bacillus spp. possessing multiple beneficial traits to develop bacterial consortium which can be further developed as carrier based bioformulation. Assay for compatibility between isolates using antagonistic survival tests showed that all the selected beneficial bacteria are compatible with each other. Plant growth promoting activity of six selected isolates was evaluated using the model plant Arabidopsis grown in vitro. The results suggested that three isolates could promote plant growth. The plant growth promoting activity of these six isolates was tested using citrus (grapefruit) seedlings in greenhouse. Greenhouse assays suggested that a consortium of three Bacillus and relative isolates (AY16, PT6 and PT26A) may delay the development of both HLB symptoms and pathogen population on citrus leaves after root inoculation. The potential of the consortium to recover the tree decline from HLB infection is being evaluated in greenhouse. The growth conditions of the three strains were optimized using a small fermenter. Three antifoam agents, A204, PPG200 or M-Oil did not affect the growth of the three bacterial strains. The initial neutral to alkaline pH values (7.0 ~ 8.0) favor growth of the three bacteria in LB, while acidic pH (5.0 ~ 6.0) suppress bacterial growth. The optimal cultural temperature was determined to be around 30C with average bacterial population of 109-1010 cfu/ml after 20-hour incubation, although the bacteria may grow slowly under room temperature (~ 23C). The shelf life of three different formulations of the bacterial culture is being evaluated under room temperature. In a six-month time course, the bacterial populations in LB broth, OPB broth and tape water are comparatively stable with initial and final both at ~ 108cfu/ml. Following up study is being conducted. Two more field trials are being conducted including more beneficial bacteria.
Management of phloem-limited bacterial diseases is very challenging. These bacteria employ unusual and sometimes unique strategies by which to optimize their niche occupation and obtain their nourishment from the host plant. Their location within the living (sieve tubes) plant cells, rather than in the intercellular spaces, offers different challenges and opportunities for them to avoid the host plant’s defense system. Phloem is also difficult for any bactericides to reach to control the pathogen population. Among the phloem-limited bacterial diseases, citrus Huanglongbing (HLB, greening) is one of the most devastating diseases. The current management strategy of HLB is to chemically control psyllids and scout for and remove infected trees. However, the current management practices have not been able to control HLB and stop spreading of Candidatus Liberibacter asiaticus (Las). The goal of the proposed study is to develop HLB management strategies which boost plant defense to protect citrus from HLB by exploiting the interaction between Las and citrus and understanding how Las manipulates plant defense. Recently, we compared the gene expression of PR1, PR2 and PR5 in healthy trees and Las infected citrus plants. The expression of PR1, PR2 and PR5 was significantly reduced in HLB diseased grapefruit as compared to healthy grapefruit after inoculation with Xac AW. We also tested whether infection by Las can make citrus more susceptible to infection by Xanthomonas citri subsp. citri. We also sprayed four times with different chemicals in 17 different combinations on citrus to test their effect in controlling HLB in one grove. Multiple compounds showed control effect. To further test those compounds, we have selected two more groves to expand the field test. The disease index of the two groves have been investigated. We compared the SA levels in HLB infected and healthy grapefruit after the inoculation with Xac AW. We also compared the SA levels in HLB infected and healthy Valencia citrus. We are continuing to evaluate the effect of different compounds on management of HLB both in greenhouse and in citrus grove. We have applied different compounds at three separate field trials. Four compounds were shown to have positive effect on controlling HLB based on two year field test results. To confirm this result, two more field trails were initiated in Lake Wales to further test the effect of these chemicals on HLB, and the follow up investigations are ongoing, including monitoring the HLB symptoms, disease incidence and Las titer in leaves. We are also testing the mechanism of those compounds showing positive effect on HLB control. We have investigated the effect of those compounds on disease severity, yield, juice content and quality. We will repeat those treatments for one more year. We are characterizing the two putative virulence genes sndA and stbA of Las, e.g. subcellular localization and host proteins interacting with them using yeast two hybrid system. Interestingly, some targets identified are transcription regulators, transporters, zinc ion binding proteins.
The goal of this study is to understand the role of biofilm formation and quorum sensing (QS) in X. citri ssp. citri infection of citrus fruit and to prevent its infection by interfering with biofilm formation and QS. Recently, we compared the attachment of the QS mutants on the citrus fruit surface. Compared with wild type stain Xac 306, the quorum sensing mutant ‘rpfF showed significantly reduced attachment to the fruit surface as revealed by CLSM (confocal laser scanning microscopy) observation with the GFP-labeled bacterial strains. We also evaluated the effect of nine compounds on Xac biofilm formation on abiotic surfaces using the crystal violet staining method. The data obtained showed that three compounds were active in inhibiting Xac biofilm formation in NB liquid medium at. Three compounds exhibited a significant reduction in biofilm formation both on polystyrene surface and in glass tubes compared to the untreated control, where the level of biofilm formation were reduced to 50% and 60% of control, respectively. Plant test in greenhouse showed that treatment with the three compounds prior to infection could reduce biofilm formation of Xac on leaf surface, reduce the formation of canker lesions on spray-inoculated grapefruit leaves with the wild-type strain. Effects of the three compounds on Xac on detached immature citrus fruit were also tested using spray inoculation. Preliminary results showed that these small molecules affected Xac 306 infection of unwounded and wounded citrus fruits at sub-inhibitory concentrations. We have completed testing the effect of those compounds in different combinations with copper based bactericides in controlling Xac infection of grapefruit plants in the greenhouse. The sensitivity of biofilm and planktonic cells of Xac 306 to copper (copper sulfate) were evaluated by measuring the MICs. Biofilms are less susceptible to copper than planktonic cells. Effect of the selected compounds on sensitivity of Xac planktonic cells and biofilm cells to copper sulfate was also investigated. In the NB medium, planktonic cells exhibited a MIC of 0.50 mM CuSO4 without biofilm inhibitor. In the presence biofilm inhibitors at sub-MIC concentrations , the MICs of CuSO4 against Xac 306 planktonic cells were decreased to 0.25 mM. In a in vitro biofilm system test, the combined use of copper sulfate and the compounds individual or both resulted in significantly increased killing compared to killing by copper sulfate alone. The results have been accepted for publication consideration by Phytopathology. One patent is filed based on the results. We also identified multiple new biofilm inhibitors. The effect of those biofilm inhibitors to control citrus canker is being investigated. We tested the survival of both biofilm deficient and QS mutants on fruit surface. Effects of biofilm formation inhibitors on Xac infection on detached immature citrus fruit were tested using spray inoculation. The inhibitors affected the infection of Xac on both unwounded and wounded citrus fruits. We are testing more potential biofilm inhibitors. We are setting up the field trial to test the effect of the identified biofilm inhibitors to control citrus canker. 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.
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