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.
The main focus is the selection of antibody fragments and peptides against Huonglongbing (HLB), Citrus Variegated Chlorosis (CVC) and the development of immunoassays that incorporate these protein products. We have been able to make significant progress with the identification of new markers and evaluating their application in conventional ELISA immunoassays. We have continued our selection process of scFv antibodies directly against three pathogens, this time we incorporated a subtractive selection through phage display. Last year we were successful obtaining several highly reactive antibody fragments that recognized surface proteins some of them were common between the gamma proteobacteria like Xylella and Xanthomonas both of which express a common though not identical outer membrane protein, MopB. This year we have incorporated a negative selection where the antibody library was bound to the other bacteria first, so if we are interested in CVC we first expose the library to E.coli and processed the phage that did not bind to E,coli. This was then repeated with Agrobacterium and Xanthomonas and then the remaining phage were exposed to Xylella. Those that bound after 2 rounds of selection were then processed to obtain individual clones that can be screened for specificity. Several highly reactive antibodies have been found and new ELISA tests have been performed, and validations are still under biological testing with promising results. Antibody fragments of different sizes were selected and reactivity was highly specific for X. fastidiosa for 13 clones that will expressed, amplified and purified for additional validation assays against infected samples from the field and greenhouse. We have also utilized a new strategy in Brazil, which consisted of a biopanning (selection) of 12-mer random peptides (2 billion clones expressed in M13 vectors) against sap extracts of infected HLB citrus and non-infected samples (controls). The controls were used for subtraction and then for positive selection against infected samples as shown in. We have identified 46 peptide sequences, which were further validated against the infected sap. After identification of sequences, we are getting custom synthesized five biotinylated random peptides with 8 additional residues of the pIII protein of the phage capsid, and with amidation of the carboxy-end. This specific request has not arrived yet, and they will be tested as a preliminary assay in Brazil in August/2012, in order to verify if the peptide design will work.We have also performed a highly sophisticated preparative fractionation Xylella in order to identify additional biomarker proteins. We have isolated proteins present in various subcellular locations in the Xylella bacteria and sequenced the proteins using high resolution proteomics procedures. We used the enzyme trypsin to shave off proteins present on the outermost surface of the cell the outer membrane and then we confirmed the presence and identity of various proteins by high resolution proteomics. We then isolated the membrane fractions to identify all of the proteins present in the outer and inner membranes. We fractionated the secreted proteins present in the periplasmic space as well as outside the cell. A major protein secreted was designated PD1 and we raised antibodies against this protein and found that this protein is present in all fractions and predominantly that are secreted. We compare this distribution of PD1 with that of MopB a protein that has previously shown been shown to be the major outer membrane protein. We are in the process of generating a high titer antibody against PD1. This hypothetical protein is highly conserved 95% between the PD and CVC strains. This antibody will be very useful for detecting the bacteria and possibly the disease at very early stages.
Thus far, 686 media formulations have been tested to support planktonic growth of Candidatus Liberibacter asiaticus (Las). No substantial planktonic growth was observed in any of the media. Various medium additives, such as a lipid mixture, individual amino acids such as asparagine, mineral salts, and peptone preparations appeared to promote biofilm formation of the surface of alimentary canals of Diaphorina citri used as a source of inoculum. Deposition of Las onto the bottom of culture plates by centrifugation in an effort to simulate formation of biofilms appeared to promote grow of Las in some medium formulations. This line of research is being further pursued.
In the present study, performed in collaboration with Dr. Nelson Wulff at Fundecitrus, we have now closed the circular genome of Ca. L. americanus (Lam) strain ‘S’o Paulo’ isolated from infected periwinkle from Brazil. A circular genomic DNA sequence of Ca. L. americanus (Lam) strain ‘S’o Paulo’ isolated from infected periwinkle (Vinca) in Brazil has been completed. The genome size is 1,195,199. The circular genome has been extensively confirmed by PCR. Particular attention has been paid to confirmation of truncated or incomplete proteins and pseudogenes. The average GC content of Lam is 31.12%. So far, 1,071 genes have been called, with 1,003 encoding predicted proteins, and 9 encoding rRNA genes and 45 encoding tRNAs. There is a surprising lack of overall nucleotide similarity among the Liberibacter asiaticus (Las) and L. solanacearum (Lso) sequenced strains. To see large scale conservation of gene organization, the PROmer version of MUMer had to be used. This analysis revealed that the overall gene organization and structure of the Lam genome is more similar to Lso than to Las. There are 851 genes common to Lam, Lso and Las, 27 genes found in Lam and Lso but not Las, and only 8 genes common to Lam and Las but not found in Lso. The latter included a DNA uptake competence gene. As with both Liberibacter asiaticus (Las) and L. solanacearum (Lso), two prophage were found, and with somewhat similar organization. As with Las, two circular forms were confirmed in Lam, with Lam-SC1 being 39,941 bp and Lam-SC2 being 16,398 bp in size. SC1 (and not SC2 as in Las) appears to replicate as an integrative plasmid prophage. This stable plasmid lysogen can be useful for lysogenic conversion (host range and pathogenicity enhancing), and indeed lysogenic conversion genes are found on these plasmid prophage—specificially peroxidases and adhesins. SignalP was used to identify 21 predicted Lam proteins with Type II secretion signals. Since Liberibacters occupy an intracellular niche, Type II secreted proteins would be immediately introduced into the phloem cell cytoplasm. These are currently being further analyzed as potential effectors of disease.
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 on the surface of the CaLas cell and/or related to host pathogen interactions and virulence. These epitopes are found on two flagellar proteins, the major outer membrane protein, two pilus proteins, 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. The visiting scientist who was performing this research returned to China during this reporting period. Due to the short time period remaining on this grant a replacement scientist was not found.
A comparative field trial involving two copper loaded silica nanogel (CuSiNG) formulations (Cankicide-pH7 and Cankicide-pH4) and a series of commercially available Cu biocides (Kocide 2000, Kocide 3000, Cuprofix Ultra 40, Kentan DF, Badge X2, NuCop WP, Nordox 75G and Magna-Bon) was conducted in a Ray Ruby grapefruit grove in Vero Beach FL. The disease incidence in the untreated checks was 60-65%. Overall, all copper based treatments were effective despite 45 inches of rain from August to October due to the presence of windbreaks surrounding the trial block. 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. Detailed materials characterization data using High Resolution Transmission Electron Microscopy (HRTEM), X-Ray Photoelectron Spectroscopy (XPS) and X-Ray Electron Diffraction revealed that copper is embedded in silica matrix in both particulate (<10 nm size; crystalline) form and in complexed (i.e. chelated Cu; amorphous) form. The XPS study indicated the presence of Cu in more than one oxidation states. The HRTEM image of CuSiNG material at low magnification showed micron-size structures. However, a careful investigation of the image at high magnification HRTEM revealed that the CuSiNG material consisted of a gel-like network of copper embedded nanoparticles. The XRD data showed that the material is overall amorphous. These unique features of CuSiNG material are not observed in traditional copper compounds.
A graduate student was hired for developing the proposed HLB detection system using polarized light and conducting experiments for this project. A suitable camera and a frame grabber for this project was identified and purchased. Proper bandpass filters and polarized filters are currently being searched. A relationship between two cameras, their field of view (FOV) angle, and distance between the cameras and leaf sample were investigated, and an overlapped area for the two cameras can be calculated. The overlapped area is where we acquire images of leaf, which will be analyzed for HLB infection status. With a distance of 26.5 cm between the camera and leaf, an overlapped area becomes 4 cm . 6.17 cm, which is similar to leaf size. The actual distance will be determined based on the actual size of an imaging area needed. A leaf measurement system was designed for detecting HLB infection status. The next step will be to acquire necessary filters, and to test the cameras in a laboratory setting.
Pathogenicity islands observed in the CLas genome potentially harbor bacterial effectors required for pathogen virulence, multiplication etc., and probably insect transmission determinants for the horizontal gene transfer. Many of the hypothetical proteins in CLas which do not have any homolog’s in other phytopathogenic prokaryotes potentially may function as effectors of pathogenicity. To functionally identify and express these putative effectors in citrus and study their role in pathogenicity we are using the CTV-vector system which expresses foreign genes in the citrus phloem, where the CLas bacteria are located in the infected plant. We have cloned most of these potential effectors outlined in the proposal into binary vector based CTV vector and have infiltrated Nicotiana benthamiana plants to purify CTV virions carrying putative effector 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 and wild type CTV as control. They have been tested for CTV accumulation (consequent presence of effector) by ELISA and are presently being tested at HLB resistance by Mike Irey at US Sugar Corporation, Clewiston, FL. Other CLas putative effectors in citrus and need to test by ELISA before challenge inoculation include the following; 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 PAMP activity associated with Flagellin domain of HLB.
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