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.
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 two peptides that appear to effectively protect sweet orange trees from HLB. However, we and other labs continue screening for better genes that more effectively control HLB and can be approved for use in a food crop. In the California lab, we developed methods to rapidly screen anti-bacterial peptides against Ca. L. psyllaurous in tobacco plants. Tobacco plants were either inoculated with Ca. L. psyllaurous by using the tomato psyllid (Bactericerca cockerelli) and challenged one week later with recombinant Tobacco mosaic virus (TMV) expressing the specific peptides, or the plants first were inoculated with recombinant TMV, followed one week later by using B. cockerelli to inoculate Ca. L. psyllaurous. These assays are being analyzed presently. 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. In the last few months, we have continued to screen peptides. We have begun to use new CTV-based vectors that we hope will improve expression of peptides. Additionally, we have built new constructs to test more peptides.
HLB detection using optical sensors: The aerial hyperspectral images acquired in December 2011 were analyzed using different classification algorithms. As a preprocessing step, Savitzky-Golay smoothing was applied to the raw image to remove spectral noise within the data. Then a support vector machine (SVM) was used to build a mask to segment tree canopy from the other background. Vertex component analysis (VCA) was use to extract the pure endmembers of the masked dataset. Then spectral angle mapping (SAM) was applied to classify healthy and the disease infected canopies in the image. To remove false positives, red edge position (REP) was used. The experimental results were compared with another supervised method, Mahalanobis distance (MahaDist), and an unsupervised method, K-means. The detection accuracies for the healthy and HLB infected canopies were 96.1% and 86.7% by SAM, and 92.0% and 86.3% by REP, respectively. Both MahaDist and K-means methods produced an accuracy of 63.6%. Ground-based multispectral with thermal imaging was used to remotely collect the spectral data from the top of the healthy and HLB-infected citrus canopies. The multispectral bands comprised of 12 bands in the range 440-900 nm, while thermal infrared scanned from 7.5-13.5 ‘m. The support vector machine algorithm was able to classify the imaging data reflectance with an overall accuracy of about 87% with HLB samples classification accuracy of about 85%. Few bands such as 560 nm and 710 nm, with thermal band yielded maximum class separability. Moreover, to further expand this work, multispectral images were collected using six-band cameras with a multi-rotor remote sensing (MRRS) platform. Currently, the data is being analyzed. Publications: (1) Li, H., W. S. Lee, K. Wang, and R. Ehsani. 2012. Spectral angle mapper (SAM) based citrus greening disease detection using airborne hyperspectral imaging. 11th International Conference on Precision Agriculture, Indianapolis, Indiana; (2) Katti, A. R., W. S. Lee, R. Ehsani, C. Yang, and R. L. Mangan. 2012. Band selection using forward feature selection algorithm: a study in citrus greening disease detection. Journal of Applied Remote Sensing. Submitted; (3) Sankaran, S., J.M. Maja, S. Buchanon, and R. Ehsani. Huanglongbing (citrus greening) detection using visible-near infrared and thermal imaging techniques. Biosystems Engineering. Submitted. VOC-based detection: The sampling stage of the project has been concluded and the analysis of the data is being carried out. The Twister GC/MS data have been analyzed, and a list of 16 biomarkers of HLB infection has been established. Currently we are testing the hypothesis that these biomarker compounds can also act as ACP attractants by performing additional chemiluminescence experiments using chemical standards and ACP olfactory proteins. The GC/DMS data are currently being analyzed. The data were preprocessed using different chemometrics tools such as baseline correction or Principal Component Analysis for outliers detection and classified using Partial Least Square Discriminant Analysis (PLS-DA). The result shows that it is possible to discriminate healthy and HLB-infected trees with accuracy of at least 75-80% across the entire year. Validation of these results is currently being carried out.
The 2011 trial was conducted in a Ray Ruby grapefruit grove in Vero Beach, FL. Two formula of Cu loaded silica nanogel material (Cankicide pH 7.0 and Cankicide pH 4.0) were used for a comparative field trial along with a number of commercially available Cu compounds (Kocide 2000, Kocide 3000, Cuprofix Ultra 40, Kentan DF, Badge X2, NuCop WP, Nordox 75G and Magna-Bon). The Cankicide pH7 treatment set at the equivalent metallic copper to Kocide 3000 and Cankicide pH4 adjusted to a similar metallic copper rate as Magna-Bon controlled canker on fruit at the same level as the standards. All Cu based treatments were effective despite 45 inches of rain from August to October. The untreated check showed 60-65% disease incidence. Effectiveness of all Cu treatments was due to the presence of windbreaks surrounding the trial block. The 2012 trial at Vero Beach, FL has been initiated. Both the Cankicide pH 7 and Cankicide pH 4 formula have been delivered to the trial site.
During this period (January- March, 2012), we continue characterizing sequencing variation of five additional virulence-related genes of Las. We investigated 24 Las isolates from four different geological locations: USA (Florida), China (Guangxi), Brazil (S’o Paulo), and India (Andhra Pradesh). Five genes selected for this study were based on the putative roles of pathogenicity. The gene ftn, (CLIASIA 03035) is coded for enzyme, ferroxidase for ferrous iron uptake. Analysis showed that while overall sequences among all isolates are 98-99% similar, one single nucleotide polymorphism (SNP) was consistently identified in all Indian isolates where nucleotide ‘G’ was replaced with ‘C’. This SNP results in the substitution of amino acid from lysine to Arginine. In addition, two SNPs were identified in 98% of Brazilian Las isolates, which resulted in changes of serine to proline and glycine to alanine. The phoU gene is identified for putative phosphate transport in this bacterium. The deficiency of phosphorus (P) in citrus was reported to be correlated with the symptom expression and development of citrus Huanglongbing (HLB). The supplementation of inorganic phosphate (Pi) alleviates HLB symptoms and improves fruit yield. The sequences analyses of phoU gene (CLIASIA 02950) showed that 78% of isolates from USA (Florida) and 55% of isolates from India (Andhra Pradesh) have ‘CC’ instead of ‘AT’ or ‘TT’ which is prevalently found in Brazil and China, respectively. Interestingly, the type of SNP in this gene appears to be somehow correlated with geographic origin. Two nucleotide substitutions in this gene result in amino acid changes from isoleucine to proline and glycine to alanine. It is not clear the impact of such an alteration. Two pilus assembly homologue proteins (CLIASIA 03575 and CLIASIA 03545) were also included in this study. It was reported that the function of pilus assembly protein is essential for virulence. For example, pilus assembly protein accounts for twitching motility in Xylella fastidiosa, a xylem-limited pathogenic bacterium that causes grape Pierce’s disease. Sequence alignment of Pilus assembly gene (CLIBASIA_03075) among all Las isolates identified 5 SNPs in China, 3 SNPs India, and 3 SNPs in Brazil samples, resulting in substitution of amino acid from histidine to glutamine, asparagine to threonine, and valine to proline. For pilus assembly gene (CLIBASIA_03045), most DNA sequences from China, Brazil and India are identical. However, 91% isolates from Brazil have SNP for ‘A’ instead of ‘G’ as compared with the isolates from USA, India and China, which resulted in the changing of amino acid from serine to glycine. The flgH is the fifth gene selected in this study. Like intracellular pathogenic bacteria, ability of movement is necessary for Las cause systemic infection. Phloem-limited Las appears to manage movement via use of a flagella-like structure. Annotation of asiaticus genome revealed that bacterium possessed genes associated with assembly of flagella apparatus. The flgH gene product involves the assembly of flagella. Analysis of flgH gene sequences indicated that all isolates from China, 97% of isolates from Brazil, and 68% of isolates from India shared nucleotide sequence ‘AAGG’ in this locus. In contrast, most isolates from US Florida process ‘CGAT’ instead. Four nucleotide substitutions result in amino acid changes from leucine to serine, arginine to glycine, valine to alanine, and proline to serine. The significance of such a sequence variation is unknown. Presumably, sequence variation in this coding region could affect selective advantage among genotypes. With the identification of sequencing variations amongst genotypes, functional characterization of these pathogenicity genes will be carried out. We will use the in vitro heterogous gene expression system to determine and characterize the functions of these virulence-related genes in next period of research. The results from these studies will facilitate the development of function-based phenotyping for Las.
Diagnostic service for growers for detection of Huanglongbing to aid in management decisions, April 2012. As we did in previous reports, this update covers the entire period that Huanglongbing Diagnostic Laboratory has been in service because one of the objectives for the funding is for continued, uninterrupted diagnostic services to growers while expanding our ability to provide diagnostics quickly and assist with research efforts. The HLB Diagnostic Laboratory has been operational at UF-IFAS-SWFREC since February 2008. Since the opening of the lab, there has been continued development of techniques, protocols and efficiency. The lab has been in operation for nearly four years, and as of mid-April, 2012, we have processed approximately 27,500 grower samples, with approximately 600 arriving since the last report, January 2012. Additionally, over 22,000 samples have been received for research, of which more than 4,000 were within that same time frame. Techniques, Protocols and Research For DNA extractions, we continue to use the magnetic particle based system, which has proved both reliable and fast. Current methods of sample processing have become streamlined and therefore seen little change. We have recently introduced the use of TaqMan Fast Advanced MasterMix for real-time-PCR reactions as this is more economical and has shown comparable-to-superior amplification and detection of gene(s)-of-interest when compared to the TaqMan Fast Universal PCR MasterMix. We now lyophilize all plant samples prior to BeadBeating, which enables superior sample maceration when compared to use of liquid nitrogen. Protocol for the detection of HLB in Asian Citrus Psyllid has been validated, including quantification of HLB in both plant and psyllid samples, with the primary goal of serving research projects within the entomology and plant pathology departments that also contribute funds from their research grants to support the labor and supply costs for research samples. The protocol established in 2010 for the quantification of the HLB bacteria in both the psyllid and host tissue using a standardize curve is being used for research and extension samples. The basic diagnostic service remains available to growers, researchers, extension faculty and dooryard citrus growers. However, we are also expanding the data analysis of PCR-processed-samples to include data from individual groves that consented to have their data used. In conjunction with an epidemiologist and computer mathematician, the spread of the disease will be modeled. These studies are not supported by lab funds but are an offshoot of the database collection. The intent is to have additional tools for looking at the spread of HLB in sites where incidence is still relatively low.
We have cloned the potential effectors outlined in the proposal into binary vector based CTV vector at a position that maximizes the expression of the effectors.These clone have been infiltrated into Nicotiana benthamiana plants to purify CTV virions for inoculation of citrus. The following potential effectors have been already in citrus; CLIBASIA_05165 at position 1134356- 1133904, CLIBASIA_05605 at position 1216188- 1215856, CLIBASIA_01555 at position 334228- 333266, CLIBASIA_05195, CLIBASIA_05200, CLIBASIA_05620, CLIBASIA_05635, CLIBASIA_05665, CLIBASIA_05130, CLIBASIA_05155, CLIBASIA_05265, CLIBASIA_05560, CLIBASIA_05150, CLIBASIA_05180, CLIBASIA_05245, CLIBASIA_02250, CLIBASIA_03020, CLIBASIA_03025, CLIBASIA_02090 and CLIBASIA_02905. Additionally, we are also testing full-length Flagellin gene of CLas and bacterial Flagellin for possible effector function in citrus because of our earlier observation of the Pathogen-associated Molecular Patterns (PAMP) activity associated with Flagellin domain of HLB. These plants have been tested by ELISA for CTV and consequently for the presence of the effectors. Initially for citrus inoculations we use Citrus macrophylla which is a preferred host for CTV. Subsequently a large batch of either sweet orange and /or grapefruit citrus varieties will be used for graft inoculation from CTV infected C. macrophylla, since we have to screen for HLB resistance with sweet orange and /or grapefruit. These plants are presently being tested at HLB resistance by Mike Irey at US Sugar Corporation, Clewiston, FL, and at the DR. Dawson green house facility at CREC, Lake Alfred, FL. We also want to screen for HLB resistance reciprocally by graft inoculation of HLB positive citrus plants with buds from citrus plants expressing effectors.
The objective of this project is 1) to complete the Las genome sequence and conduct comparative genomics studies on the Liberibacter species; 2) to explore the potential role of the microbial community and genetic diversity of Las bacteria in HLB development; 3) to confirm if Las bacteria are seed-transmissible and their role in HLB development. A complete circular genome of Candidatus Liberibacter asiaticus (Las) was obtained using a metagenomics approach and published in MPMI 22:1011-1020, 2009. In collaboration with Dr. Hong Lin at the USDA-ARS in Parlier, California, we have obtained and published a complete genome sequence of Ca. L. solanacearum and published in PLoS ONE 6(4): e1913, 2011. All BAC clones of Las were sequenced, and sequence analyses revealed a potential mechanism of GENOME REDUCTION. Based on the variations within the Las prophages, eight different populations (genotypes) have been identified. In addition, a major chromosomal deletion/insertion of Las was also identified, and their correlations with phenotypes and disease severity of HLB are under investigations. We have characterized the ATP translocase from Las and proved its function using a heterologous E. coli system. This data was published in J. Bacteriol. 192:834-840, 2010. We are currently developing an antibody-based “drug” to target this protein, aimed at disrupting ATP import, which may be important for its survival. We have also characterized the individual genes of two putative zinc operons in Las, confirming only one operon responsible for zinc uptake. Seed transmission of Las was tested in grapefruit, sweet orange, sour orange and trifoliate orange. Relatively high titers of Las were detected from both seed coats and inner seed coats collected from HLB-affected citrus plants. A very low titer of Las was detected from the embryos and seedlings using nested PCR and real-time PCR. Most, if not all the seedlings did not show typical HLB symptoms and contained a relatively low Las bacterial titer for HLB, even in the three to four year old seedlings. The results indicated that the seed-transmitted Las could not cause typical HLB disease by themselves, which suggested “Detection of Candidatus Liberibacter asiaticus was NOT necessarily equal to the presence of “HLB disease” in plants.” Psyllid transmission study on the Las-positive seedlings was performed. High percentage of psyllids acquired Las bacterium but did not have the same bacterial levels as those from HLB-affected citrus plants. However, it is FIRST TIME that ONE SEED-TRANSMITTED HLB SEEDLING was confirmed by PCR using several Las-specific primer sets. Graft transmission of the cutting from this HLB plant confirmed this seed-transmitted HLB. Progress on culture of Las bacterium in vitro has been made. Up to 1,000,000 to 100,000,00 cells/ml were obtained within 48 hrs based on qPCR estimation. The Las bacterial growth reached Stationary Phase and Death Phase in 48-72 hours in the liquid cultures. We are looking into factors affecting further growth. Fifty-one BAC clones with overlapped Wolbachia endosymbiont of Diaphorina citri (wDia) genome sequences were screened using wDia specific primers from BamHI BAC library and were sequenced. The average size for the 51 clones was 85.4kb with 95-100% coverage and the average GC content is 34.3%. Assembly results indicated that due to large amount of repeat elements, such as transposase, only 13 BAC clones were able assembled into 1 scaffold. We are conducting the gap closing for each BAC clone and hope to get the full wDia genome soon.
Single color light emitting diodes (LEDs) were decided to be used in the leaf measurement system instead of regular light source and band-pass filters for cameras. By using the LED-illumination, the problem with low transmission efficiency of band-pass filters was resolved. LEDs in 591 nm (target wavelength) and 400 nm (reference wavelength) as well as a LED driver were purchased and their responses were evaluated and confirmed using a mobile spectrometer. An electronic circuit was designed for LED illumination system which is able to switch between two wavelengths and also control the amount of light in each wavelength separately. In order to use the maximum light of LEDs, the angle of LEDs’ light were narrowed down using proper lenses with a field of view of 24′. A box containing two cameras, LEDs, control circuit and also the imaging platform was design and created. As this box covers the leaf during the imaging and only the LED’s light is illuminated on the platform, the effects of other light sources in the grove can be eliminated so that imaging during the day is also possible. Also the distance between cameras and leaf can be kept fixed during the imaging. A visible linear polarizing laminated film was purchased and cut to be placed in front of the cameras and LEDs. The polarizing film in front of the LEDs and one of the two cameras was set to be in the same direction, however a polarizing film was installed in a perpendicular direction in front of the second camera. The frame grabber’s software was customized so that it can capture two snapshots from two channels at the same time. The preliminarily evaluation of leaf measurement system was performed in the Lab with some citrus leaves in different conditions. Four images were captured from each leaf, two images at 591 nm with parallel and perpendicular polarizing directions with light source and also two images at 400 nm with the same directions. The visual evaluation of images confirmed that there are differences between the images from the same scene when they were captured with different situations. The leaf measurement system is now ready for an experiment in a citrus grove.
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