The objective of this project is to characterize the hypI (renamed as hyvI) gene and determine its effects on insect transmission and/or virulence in host plants. Transient expression with alternative expression systems and RT-qPCR, etc., will be used to elucidate the function of the hypI (hyvII) gene of Las and shed light on the molecular mechanism of this “phase variation” phenomenon; thereby developing a novel control strategy for citrus HLB. In addition, antibodies and probes along with standardized protocols developed during this project can be applied for better detection and differentiation of the HLB bacteria. The hyvI and hyvII within two Las prophages were further characterized and some of the results were published in Applied Environmental Microbiology 77:6663-6673, 2011. “Diversity and Plasticity of the Intracellular Plant Pathogen and Insect Symbiont “Candidatus Liberibacter asiaticus” as Revealed by Hypervariable Prophage Genes with Intragenic Tandem Repeats”. We have developed an improved real-time PCR using SYBR Green 1 (LJ900fr) and TaqMan’ (LJ900fpr) protocols with primers and probe targeting the nearly identical tandem repeats of 100bp hyvI and hyvII. The results were published in Molecular and Cellular probes, 26:90-98, 2012. Monoclonal antibodies against the partial HyvI protein (only one repeat) were generated, and their sensitivity and specificity were evaluated for the detection of HyvI protein expressed in E. coli and HLB-infected citrus and psyllids. All antibodies were able to recognize the E. coli expressed HyvI antigen, but were not able to detect the HyvI antigen from HLB-infected plants and psyllids. To determine the cellular localization of the HyvI protein in plant cells and the role of the two putative NLSs in hyvI gene, full-length hyvI and C-terminal region including two putative NLSs were amplified and cloned into pCX-DG vector with GFP driven by CaMV35S promoter. The results indicate that the HyvI protein did not target in plant nucleus but located in cytoplasm (possible in organelle) when transient expression in tobacco. We determined that HyvI targeted tobacco mitochondria by transient expression and MitoTracker Mitochondria-selective probe staining. When the full length of hyvI gene was cloned , and replicated in heterologous hosts, the repeat number of hyvI gene remained the same in E. coli, but varied in Xanthomonas citri. Clones of X. citri containing the hyvI gene displayed different degree of growth retardation, indicating potential toxic effect of hyvI gene to X. citri. HyvI C- terminus and full length HyvII were fused to GFP and expressed in E. coli driven by T7 promoter. Confocal microscopy results show both proteins are localized to the bacterial poles. Protein localization, sequence analysis and protein structure prediction suggest both protein belong to autotransporter family. HyvI and II are unique autotransporters because the non-conserved translocator domain can export both its natural passanger domain and also the GFP fusion domain to E.coli’s cell surface. The protein was determined to localize at cell surface by dot blot, and furthermore the protein surface localization was proved by protease treatment of intact bacterial cells. The immunofluorescence assay to confirm the surface localization of the HyvI protein was unsuccessful. The proteins appear to be on the cell surface but are not folded properly.
We found that the camera’s automatic gain control (AGC) function is designed to maintain image’s intensity and contrast consistently regardless of the illumination conditions and this function would prevent us from identifying the effect of starch accumulation in the images. In order to resolve this problem, a two-step calibration was performed using the background pixel values. At the first step, all the images in each specific illumination and filter direction situation were calibrated separately. Also the actual background’s reflectance in each situation was measured using a portable spectrophotometer and the ratios between them were used for the second step of the calibration. Using this approach the effect of AGC was cancelled and the real reflectance measurement was calculated. Using the image acquisition system, two datasets each including both healthy and HLB infected leaves were created. A 4-class (healthy leaf, healthy vein, HLB leaf and HLB vein classes) training set of 25 healthy samples and 25 HLB samples were selected from one of the datasets. Analyzing the histogram of this training dataset showed that the healthy leaf and HLB leaf classes could be distinguished in the images captured with 591 nm illumination and perpendicular filters. In addition, the healthy vein class is very similar to the healthy leaf class, and the HLB vein class is very similar to the HLB leaf class. This can mean that although healthy vein has a color similar to HLB leaf and vein, however healthy vein has different pixel value in 591 nm with perpendicular filters because there is no starch accumulation in healthy veins. Both datasets were classified using this training set and a linear classifier. The classifier was able to identify the HLB samples with 100% accuracy, but it also misclassified 28% of healthy samples of the first dataset and 36% healthy samples of the second dataset in the HLB class. This might be due to either the classification error or the healthy samples actually being infected by the disease. Furthermore, a dataset containing 4 classes of different kinds of yellow leaves (HLB, young leaves, zinc deficient and unknown symptoms) plus a healthy class as the control class was created to find out the difference between HLB yellowish symptoms and yellowish symptoms with other reasons. The analysis of variance (ANOVA) results showed that all five classes were significantly different. In order to find the effect of starch on polarized light, the absorbance of four levels of starch solutions plus pure water were measured using the spectrophotometer and in three conditions: no filter, parallel filters and perpendicular filters. The results showed that the absorbance increases as the starch concentration increases in the solution. However the Min/Max ratio (the ratio between absorbance data using perpendicular filters to parallel filters) didn’t show any reasonable results.
Citrus huanglongbing (HLB) is associated with three species of Liberibacter’Candidatus Liberibacter asiaticus (Las), Ca. L. americanus, and Ca. L. africanus. The majority of the testing in Florida is focused on detection of Las, the only bacterium known to be associated with HLB in Florida to date. Over the past four years with funding from Citrus Research Board, we have conducted regular surveys of citrus and citrus relatives in Florida, from various germplasm collections, backyard plants, native and cultivated trees, testing for tolerance to HLB. We have focused on plants showing HLB symptoms but testing negative by standard qPCR tests. A small selected set of symptomatic, qPCR negative samples were analyzed for detection of other genomic regions of Liberibacters by conventional PCR (cPCR), cloning and sequencing. This study confirmed the presence of Liberibacter variants not detectable by standard assays. The purpose of this project is to conduct further research on variants of Liberibacters from citrus and citrus relatives and to develop rapid methods for detection of these variant populations. We also will study the biology of the variants under greenhouse conditions, determine the changes in Liberibacter populations within individual trees over time from analysis of DNA extractions we have made over the past 5 years, and determine if there are interactions among populations of Liberibacter variants which may ameliorate/enhance the symptoms of HLB. Understanding of HLB disease complex caused by all variants of Liberibacters will be useful for developing novel disease management strategies. Research on this project this first quarter has concentrated on the development of a method to ‘trap’ Liberibacter and Liberibacter-like pathogens so that Liberibacter-enriched DNA extractions may be made to facilitate the molecular characterization of the Liberibacter and Liberibacter-like genomes. We anticipate receiving funds soon which will permit hiring of personnel and facilitate the research.
The objective was to evaluate the efficacy of copper loaded silica nanogel (CuSiNG) material against citrus canker. Two CuSiNG formulations (Cankicide pH 7.0 and Cankicide pH 4.0) were prepared at UCF and delivered to Fort Pierce for the 2012 field trial at Vero Beach. The efficacy is being evaluated against a number of commercially available Cu compounds including Kocide 2000, Kocide 3000, Cuprofix Ultra 40, Kentan DF, Badge X2, NuCop WP, Nordox 75G and Magna-Bon. Laboratory research is being continued on CuSiNG materials for testing formulation stability (Shelf-life) and for improving Cu bioavailability. To date, our research data suggest that Cankicide pH 4.0 (highly-transparent) as-synthesized formula is stable for more than 12 months (no settling observed). In contrast, Cankicide pH 7.0 formula is opaque and settling was observed within a couple of weeks. Data from 2011 field trial showed that Cankicide pH 4.0 formula caused very minor injury to plant tissue (but not significant with respect to controls) whereas Cankicide pH 7.0 formula was non-phytotoxic. This is attributed to the availability of some free ionic Cu in the Cankicide pH 4.0 formula. To understand the Cu oxidation states in CuSiNG material, X-ray Photoelectron Spectroscopy (XPS) technique was used for material characterization. XPS data suggest that mixed valence states of Cu exist in CuSiNG material. Improved Cu bioavailability of the CuSiNG material could be correlated to the presence of such mixed valance states. Further studies are in progress to understand the role of mixed Cu valence states in CuSiNG material. To minimize the metallic Cu amount per spray application, we have prepared a novel core-shell nanoparticle and performed systematic laboratory research. The CuSiNG material was coated over the pure silica nanoparticle (that served as non-Cu containing inactive delivery system), thus forming a core-shell nanoparticle with silica core and CuSiNG composite shell. A peer-reviewed research paper entitled, ‘Novel copper loaded core-shell silica nanoparticles with improved copper bioavailability: synthesis, characterization and study of antibacterial properties’ have been published in the Journal of Biomedical Nanotechnology 2012, 8(4), 558-566. Significant improvement of antibacterial efficacy in comparison to Kocide 3000 control was attributed to improved Cu bioavailability in the core-shell nanoparticle. To characterize adherence property of the silica nanogel (SiNG) material to plant tissue, the SiNG material was labeled successfully with fluorescent dyes. However, background fluorescence signal of the plant tissue and the photobleaching of organic fluorescent dyes severely limited our ability to conduct experiments reliably. In an alternative approach, a stable inorganic based near infra-red (NIR) emitting fluorescent label is being developed to address the above limitations. We will disseminate research results on the NIR imaging of CuSiNG materials and their adherence properties to citrus plant surface in future CRDF reports and peer-reviewed journal publications.
The specific project goals were: 1. Cloning of previously identified early/late gene promoter regions fused with lacZ as a reporter. 2. Cloning and expression of both Las and the Lam repressors and determining responsiveness of the lacZ reporter. 3. Cloning and expression of all 4 Las and the one possible Lam anti-repressors, and determining responsiveness of the reporter and clones from Milestone 2. 4. Development of a chemical assay for Las-responsive SOS. Goal 1 is partially completed and continuing. The project start date was 5/1/12. The pUC19 cloning vector was modified to remove the promoter of lacZ and replace it with a PCR product in a single step, using UDG cloning. In this manner, the intergenic region between the early and late genes of Las phage SC1 and SC2 (regions of both phage between locus tags gp120 and gp125) were cloned in both directions upstream of the lacZ reporter gene in E. coli. When the putatively bidirectionally active SC1 and SC2 promoters were fused with the lacZ gene such that they replaced the early genes in constructs pSZ68 and pSZ64, respectively (early gene direction), both the SC1 and SC2 constructs performed the same and both resulted in light blue color reactions. Conversely, when these promoters were fused with the lacZ gene such that they replaced the late genes (late gene direction), both the SC1 clone (pSZ67) and SC2 clone (pSZ62) resulted in no detected color reaction. These results indicated that, as expected, the early genes of both SC1 and SC2 are constitutively on and the late genes are constitutively off. However, when the bidirectional promoter region was cloned in the late direction, such that it drove expression of the repressor gp125 of SC1, together with the lacZ reporter (in pSZ63), a light to medium blue color reaction was observed. This indicated that gp125 (annotated as a phage C2-like repressor) may be an activator, since it appeared to stimulate its own expression. Alternatively, part of the late gene promoter region may be contained within the gp125 locus. We are testing this idea. In addition, the Las repressor, SC1 gp125 (annotated as a phage C2-like repressor) was cloned into shuttle vector pUFJ5 (Bordatella replicon), forming pUFZ3-4. Finally, the annotated Las anti-repressor (SC1_gp200) was cloned into shuttle vector pUFR047 (forming pSZ77), such that gp200 was constitutively expressed from the lacZ promoter in pUFR047 (oriW). Both the putative repressor and anti-repressor constructs are compatible with pUC19 in the same E. coli cell.
Previously, we sequenced and genotyped Las isolates from four major citrus production countries, USA, Brazil, China and India based on the five virulence-related gene loci, ftn (CLIASIA 03035), phoU (CLIASIA 02950), flgH and two genes homologs to pilus assembly proteins (CLIASIA 03575 and CLIASIA 03545). During this report period (April-July, 2012), we extended our sequencing analyses of Las isolates to wider geological locations including Vietnam, Cambodia, Japan, Taiwan, and Thailand. Single nucleotide polymorphism (SNP) in coding regions could result in a change in the amino acid if such a change alters the codon for the amino acid (non-synonymous) or have no effect on the amino acid due to degenerate codon usage (synonymous). Analysis of ftn gene (CLIASIA 03035) showed that while overall sequence similarities among all isolates are 97-98%, one single nucleotide polymorphism (SNP) was consistently identified in Japan and Indian isolates where nucleotide ‘G’ was substituted with ‘C’. This SNP results in the alternation of amino acid from histidines to glutamine. Sequence alignment of pilus assembly gene (CLIBASIA_03075) showed that isolates from Vietnam, Cambodia, Japan, Taiwan, and Thailand have ‘GT’ instead of ‘AC’ which is found most in isolates from USA (Florida), Brazil, Indian, and China, respectively. The switch of GT to AC results in changes of amino acids from serine to alanine and threonine to valine. For pilus assembly gene (CLIBASIA_03045), the DNA sequencies of most isolates are identical except isolates from Cambodia where 24% of them have SNPs for ‘AG’ or ‘GG’ instead of ‘TC’. Such alternations resulted in the changing of amino acid from serine to glycine and arginine to glycine. Analysis of flgH gene sequences indicated that 45% isolates from Cambodia have ‘A’ instead of ‘G’ while 25% isolates from Vietnam have SNP with ‘G’ instead of ‘T’. Nucleotide substitutions result in amino acid changes from proline to leucine and threonine to proline, respectively. Compiling the genotyping of this data with our previous results, we constructed a Las genotyping database based on five putative virulence gene loci. Clearly, that non-synonymous SNPs identified within a gene’s coding region led to the alternation of amino acid sequences that would have potential effects on protein folding, functionality and cellular response to the environment. To further characterize and determine the relationship between the genotype vs. the phenotype, in vitro orthologous gene replacement approach was used. Two pilus genes were first selected to evaluate the possible functions in Las. Comparative analysis of Las and Xylella fastidiosa (XF) genomes revealed that the Las possessed pilus gene that was homologous to the pilus gene of Xf which is a xylem-limited pathogenic bacterium causing citrus and grape diseases. It was reported that the pilus assembly protein accounts for twitching motility in XF which is essential for virulence. Like other intracellular pathogenic bacteria, ability of movement is necessary for Las to causes a systemic infection. To test the function of pilus genes in Las, we created pilus mutant XF by a site specific gene knock-out technique. The DNA sequences (with their own promoters) encoded for two pilus assembly homologue proteins were amplified from different SNP types of Las isolates. The amplified genes were cloned into a protein expression vector which was then transformed into pilus-deficient mutants of Xf via electroporation. The colonies grown from selected medium will be identified and further tested by PCR to confirm successful replacement of Las pilus gene. Currently, we are developing a bioassay to evaluate the mobility of complementary Xf strains.
The main objective of this proposal is to develop a novel method for inoculation of experimental citrus plants with HLB that would provide an excellent alternative to the existing methods by overcoming the disadvantages of the latter approaches and allowing high throughput inoculations with a greater certainty of pathogen transmission for various research purposes. We are using a Pulse Micro Dose Injection System (PMDIS) to develop a new method for rapid and efficient inoculation of plants with HLB. A positive outcome of our preliminary experiments suggested a feasibility of further adaptation of the PMDIS system for HLB inoculations. This is a new project and the funds for this project are currently being released. The research in in progress. We already have designated personal who would conduct the proposed research. Plant material that will be used in this project is being prepared. Using plant material that is already available (existing HLB-infected plants that are used as inoculum source and healthy plants that are used for inoculations) we are setting up initial trial inoculations.
This is a project to find an interim control measure to allow the citrus industry to survive until resistant or tolerant trees are available. We are approaching this problem in three ways. First, we are attempting to find products that will control the greening bacterium in citrus trees. We have chosen initially to focus on antibacterial peptides because they represent one of the few choices available for this time frame. We also are testing some possible anti-psyllid genes. Second, we are developing virus vectors based on CTV to effectively express the antibacterial genes in trees in the field as an interim measure until transgenic trees are available. With effective antibacterial or antipsyllid genes, this will allow protection of young trees for perhaps the first ten years with only pre-HLB control measures. Third, we are examining the possibility of using the CTV vector to express antibacterial peptides to treat trees in the field that are already infected with HLB. With effective anti-Las genes, the vector should be able to prevent further multiplication and spread of the bacterium in infected trees and allow them to recover. We have completed several large screenings of antibacterial peptides against Las in sweet orange trees. About 50 different antibacterial constructs have been tested in trees. We have found only two peptides that appear to give some protection sweet orange trees from HLB. We continue screening for better genes that will more effectively control HLB and can be approved for use in a food crop. We also are improving the CTV-based vector to be able to produce multiple genes at the same time. This could allow expression of genes against HLB and canker or multiple of genes against HLB. Another major goal is to do a field test of the CTV vector with antibacterial peptides, which is an initial step in obtaining EPA and FDA approval for use in the field. After some delays, we have received permission for USDA APHIS and are now establishing the field test.
We have focused on immature citrus seeds as sources of ‘pure’ Liberibacter asiaticus (Las) cells. During early summer, seeds are immature, generally soft and easily homogenized. We dissect the vascular bundles from the seed coats since these contains the phloem which contains the Las cells. Homogenized extracts were passed successively through 20 and 10 micron filters to remove tissue fragments larger than Las cells; this filtrate was centrifuged to concentrate bacterial cells, suspended in a small volume of buffer and loaded onto pre-formed Percoll gradients. We have treated isolated vascular bundles with Macerozyme, a commercial enzyme preparation sold for dissolution of plant tissues and compared these results with those from vascular bundles not treated with Macerozyme. Without treatment two main bands are visible in the gradient and real time PCR (qPCR) assays indicate that the topmost band contains the most Liberibacter DNA. This band is higher up in the gradient than we expect for bacterial cells not associated with plant tissue and FISH microscopy studies showed Las cells were still associated with plant tissue fragments. With Macerozyme treatment this upper band was not seen and the qPCR assays showed the majority of the Las DNA was associated with the band lower down in the Percoll gradient; this is significant since this lower location in the gradient is where cultured E. coli cells band in Percoll, suggesting that the Macerozyme treatment digested the vascular bundles and released Las cells. FISH microscopy on the material in this lower and showed numerous individual bacteria; based on the conditions of the work and appearance of the bacteria we feel certain these are Las cells and not a different bacteria which is present as a contaminant. The current results suggest we are improving our isolation protocol and we are continuing this work with seeds and wills start initial experiments with foliar tissue to see if we obtain similar results.
In previous reports we have described the preparation of a scFv library prepared in phagemid vector pKM19. The basic scFv library contains 2 x 10_7th unique phage that bind to different antigens present in ‘Ca. Liberibacter asiaticus’ (CaLas) and the psyllid vector. We have also reported that we have isolated scFv from this library that bind to epitopes contained in proteins of CaLas that are likely to be related to host pathogen interactions and virulence. These epitopes are found on two flagellar proteins, the major outer membrane protein, a pilus protein, a protein believed to polymerize the capsular polysaccharide surface layer of the bacterium, the TolC protein required for survival in a plant host, and InvA, the invasiveness protein that prevents an infected cell from undergoing programmed cell death by apoptosis. The vector and expression system that we have used for this project allows selection of the scFv antibody expressed from a phagemid genome but packaged on the surface of an M13 particle. This facilitates selection, but large scale expression of the scFv requires cloning of the scFv gene into a cognate plasmid expression vector. We have had problems with many of our scFv at this step, because ‘stop’ codons can accumulate in the phagemid without affecting the selection process, but prevent expression from the plasmid vector. During this reporting period we have repaired the improper ‘stop’ codons for several of our scFv to allow full expression of the scFv. These scFv were selected because they showed the desired specificity when selected in the phagemid format, but failed to produce scFv when used in the expression vector. The repairs were done by sequencing the defective scFv and identifying the stop codons, designing primers for a series of PCR that allowed amplification of the scFv while replacing the incorrect codons with corrected sequence, and transforming the corrected plasmid with the scFv into E. coli for expression. We have corrected the sequences for scFv that bind FlhA (scFv B947, B1096, B1072); KpsA (B520, B1199, B1202); the major outer membrane protein OmpA (B743); Pilus protein (B556, B557). These scFv are now available in pKM16, our expression vector for testing. The next steps will require expression of the scFv, purification of the scFv and testing it for yield and specificity against purified antigens. These scFv will be added to our inventory of multiple scFv for each target of interest to improve our chances of finding scFv that will be extremely useful for detection assays and for labeling cells for scientific studies. In the current reporting period a great deal of effort was directed at negotiating with Sigma Tau Pharmaceutical of Rome Italy in an attempt to establish parameters for commercial development of the single chain antibodies developed by this project. Sigma Tau owns the vector used to isolate these scFv and has an ownership interest in the scFv. Mutually agreeable terms for commercialization of the scFv were not found. However arrangements were agreed to enable continued research with the scFv.
Hardware components for the proposed sensing system were searched. Different monochrome cameras, bandpass filters, polarized filters, and a frame grabber were found from various information sources, and their characteristics were compared. One of the difficulties was to find spectral sensitivity of cameras, since many of them did not specify the information. Most fiber optic cameras were for medical applications. A graduate student was hired to conduct the search and design the sensing system.
The aim of this project was the attempt to in vitro culture the bacterium associated with the Citrus Greening disease:Candidatus Liberibacter asiaticus (LAS). The strategy was anchored on the use of insect cells cultures as feeder cells for the primocultures of LAS. The infected plant material originated from Vietnam and maintained in greenhouse by grafting and by Diaphorina citri transmission. This LAS strain was identical to the LAS strain occuring in Florida according to its 16s rDNA. Another aspect of the strategy was the use of LAS infected periwinkles in which LAS was transferred by D. Citri and maintained by grafting. The highest concentrations of LAS were obtained in periwinkles. We used several cell lines from Lepidoptera (Mamestra brassicae, Spodoptera littoralis, S. frugiperda, Lymantria dispar), Diptera (Aedes albopictus, Drosophila melanogaster) and Hemiptera (D. citri). For Spodoptera and Drosophila, “commercial lines” adapted for recombinant protein production and “laboratory lines” were used. The main culture media were: Grace insect cell medium, LM 15 and Drosophila medium. The first step concerned the inoculum. This was obtained from Citrus or periwinkle after surface sterilization with ethanol and 1% sodium hypochlorite, and addition of proline (10mM) or/and sodium pyruvate according to the cell line used and 5 fluorocytosine. The best plant for primocultures depended of each cell line (e.g. periwinkle for A. albopictus). Lepidoptera cells did not provide transferable cultures. The same for D. citri cells (no more than 3 transfers). Diptera cells allowed the best results. LAS could be maintained for 4 to 8 passages (every 7-10 days) in “commercial” Drosophila cells, but LAS was lost, overgrown by the fast growth of the insect cells. “Laboratory line” D-S2 allowed much better results: up to 20 transfers. From 10 to 19 transfers of LAS could be obtained when using Aedes cells for primocultures. After successive dilutions we could get rid of of insect cells without losing LAS detection. To verify that the LAS culture was axenic, we checked by PCR if LAS was the only bacterium. Other bacteria were detected in several cultures when using Aedes/periwinkle system (mainly, actinobacteria and Delftia acidivorans). Strong antibiotic selection was applied to remove the contaminants but this resulted in the loss of LAS signal. No contaminants were identified when using Drosophila/Citrus system and we conclude that we obtained a true axenic culture of LAS. Our strategy based on feeder cells proved to be very reproducible. We tried to freeze the obtained cultures with addition of glycerol or DMSO. Unfortunately it has been impossible so far to freeze and thaw the cultures. Recently, in order to fulfil the Koch’s postulates, we inoculated healthy Citrus plantlets with LAS from “axenic cultures” (cultures from the Drosophila system at a stage when no other bacteria was detected) and with culture orginating from Aedes cells system even if we knew there was a contaminant. Two protocols were used: mechanical inoculation despite the fact that never a phloem-restricted microorganism has been re-introduced mechanically in the sieve tubes of phloem by this way; or by D. citri, after acquisition through membrane. These inoculated plants are under observation for HLB symptoms in the greenhouse and will be checked by PCR from time to time.
This research agreement became effective 1-Jun-11. A GS5-7 biological support technician was in place the first week of December, 2011. Preliminary results of foliar and seed coat tissue fractionation and filtration experiments were obtained both before and after the effective date of the Research Agreement. Analysis suggests that Liberibacter cells reside in a matrix within the phloem sieve elements and that a portion of this matrix is composed of bacterial DNA. Results from microscopy with Fluorescence In Situ Hybridization (FISH) indicated that individual bacteria or clumps of bacteria could be liberated by mechanical pulverization of source tissues, but numbers of cells were less than expected based on real time PCR (qPCR) data, and cell morphology appeared to be altered, which is a result consistent with published reports on purification of phytoplasma cells (also phloem colonizing bacteria) from plant tissue. A small library of prokaryote 16S rRNA gene sequences generated with degenerate primers indicated the prokaryote population in seed coat vascular tissue is 100% Liberibacter asiaticus. Experiments indicated that tissue pulverization methods which employ large or small steel ball bearings to dissociate tissue may not pulverize tissue adequately to release bacterial cells; a laboratory tissue homogenizer seems to be a better choice for tissue disruption. Experiments with fractionation of homogenized Liberibacter-containing foliar and seed coat tissues on Percoll gradients yielded distinct profiles of distribution of Liberibacter DNA (cells) for the two tissue sources, with seed coats providing a cleaner preparation. While real-time PCR (qPCR) assays indicate that ~35% of Liberibacter cells remain within seed coat tissue that moves only a short distance into the Percoll gradient as a highly visible band of debris, qPCR assays showed that ~15% of the Liberibacter DNA bands further down in the gradient at a position equivalent to where cultured E. coli cells band. This material is being examined with microscopy to ascertain the presence and character of bacterial cells. A portion of this research is being submitted for publication in Phytopathology and will be a poster presentation at the general meeting of the American Society for Microbiology, June 19-24, 2012.
This report was inadvertently not submitted at the appropriate time. This research agreement (418) became effective 1-Jun-11. A term GS-5 Biological Science Labortory Technician was in place as of Dec. 5, 2011. We have continued and confirmed results from previous analyses that suggested that Liberibacter cells reside in a matrix within the phloem sieve elements and which may contain bacterial DNA. We have additional results from microscopy with Fluorescence In Situ Hybridization (FISH) confirming that individual bacteria or clumps of bacteria could be liberated by mechanical pulverization of source tissues; efforts will be made to improve the recovery of bacterial cells from seed coat tissue. With technical support in place we will expand experiments to include different methods of tissue homogenization and fractionation on Percoll gradients.
We are continuing to examine the interactions between the psyllid, the plant, and the greening bacterium. We are examining the disease epidemic under confined conditions. We have developed a containment plant growth room to examine natural infection of citrus trees by psyllid inoculation. We have made several significant observations: First, we have found that the time period between when plants first become exposed to infected psyllids and the time that new psyllids can acquire Las is much shorter that we expected. In our population of psyllids in the containment room, the proportion of infected psyllids born on newly inserted healthy plants starts increasing after about 30 days suggesting that the receptor plants begin becoming donors at about that time. We are examining this process in more detail now. It is clear that psyllids reproduce on new flush, but feed on older leaves. We are examining whether and how well the psyllid can transmit the disease in the absence of flush. We also have developed methods to greatly speed up results of field tests for transgenic or other citrus trees or trees being protected by the CTV vector plus antibacterial or anti-psyllid genes. In order to interpret results of a field test, most control trees need to become diseased. Under natural field pressure in areas in which USDA APHIS will allow field tests, this level of infection could take 2-3 years. By allowing the trees to become adequately inoculated by infected psyllids in a containment facility, we can create the level of inoculation that would naturally occur in the field within 2-3 years in 2-5 months in the containment room, after which the trees are moved to the field test site. Trees are not being examined in the field that first were maintained under heavy inoculation pressure by infected psyllids for several months. Other peptide protected plants are being prepared for field testing. Another objective is to provide knowledge and resources to support and foster research in other laboratories. A substantial number of funded projects in other labs are based on our research and reagents. We supply infected psyllids to Mike Davis’s lab for culturing of Las and plants containing potential anti-psyllid genes for Kirsten Pelz-Stelinski’s lab and for Bob Shatters et al. lab in Fort Pierce. We routinely screen citrus genotypes or transgenic citrus for other labs for tolerance or resistance to greening or psyllids. We have found poncirus/sweet orange hybrids that are tolerant to HLB and are looking at possibilities of quickly getting sources of trees that can be productive in the field in the presence of HLB.
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