The amount of starch in several samples, which had been classified by manual scouting, was measured in Dr. Etxeberria’s lab at the CREC, Lake Alfred, FL. Then the pixel values belonging to the same punched area (which were used for starch measurement) of each sample’s 4 images were compared for the amount of starch. The results showed that there was no significant linear relationship between pixel values and the amount of starch. In order to find an angle in degrees that pure starch rotates the polarization light in our image acquisition system, one of the cameras was replaced with a portable spectrometer to measure the reflectance of the pure starch with a polarizing filter having different angles. The results showed that as the angles between polarizing filters increased, the reflectance also increased. The maximum reflectance occurred at 90 degrees and at 597 nm, while the starch was illuminated with 591 nm LEDs. Another interesting result was the very close similarity between minimum/maximum reflectance ratios at 591 nm, when it was calculated using spectrometer data and the pixel values of the images captured by the cameras. Another dataset of 90 samples including 30 samples of each healthy, HLB infected and zinc deficient leaves were collected from the CREC grove in August, 2012. Starch measurement was conducted in Dr. Etxeberria’s lab, CREC, based on one representative spot of each sample and images of the leaf samples were also acquired. Then, the samples were sent to the United States Sugar Corporation, Technical Operations for a PCR test of the samples. The initial comparison of the amount of starch and the pixel values confirmed that these values were not significantly related with each other. The analysis of the dataset included feature extraction, feature selection, classification and cross validation. Along with pixel values, some textural features were extracted from gray, grey level co-occurrence matrix (GLCM), local binary pattern (LBP) and local similarity pattern (LSP). Each group of features were analyzed separately and also all together using five statistical methods of features’ ranking using Matlab to find the best features which can distinguish HLB-infected leaves from the healthy ones. Also, seven classifiers’ performances were evaluated. Different kernel methods were used to map the training data into kernel space in a support vector machine classifier. Up to now, the accuracy of higher than 90% was obtained using the GLCM features and based on starch measurement results. Once the PCR test will be received, the performance of using all different combinations of features in classifiers will be evaluated to find out the best detection methods.
This work was performed in collaboration with Dr. Nelson Wulff at Fundecitrus. We have completed and annotated the circular genome of Ca. L. americanus (Lam) strain ‘S’o Paulo’ isolated from infected periwinkle from Brazil. The genome size is 1,195,201 bp, with an average GC content of 31.12%, which is somewhat lower than other Liberibacters. There are 1,057 predicted Lam genes, with 1,015 encoding proteins, 9 encoding rRNA genes and 45 encoding tRNAs. Of the protein coding genes, 839 (78.34%) have a predicted function, while 176 (16.43%) have no predicted function. The overall gene organization and structure of the Lam genome is more similar to Ca. L. solanacearum (Lso) than to Ca. L. asiaticus (Las), but Lso and Las are more closely related to each other than to Lam, despite the difference in host range. There are 951 genes common to Lam, Lso and Las, 27 genes found in Lam and Lso but not Las, and only 7 genes common to Lam and Las but not found in Lso. Surprisingly, no novel virulence gene candidates were identified among these 7. Many pseudogenes and truncated genes were found among the unique genes of all 3 species. As with both Liberibacter asiaticus (Las) and L. solanacearum (Lso), two prophage were found in Lam. Also as with Las, two circular forms were confirmed in Lam, with Lam-SP2 being 39,941 bp and Lam-SP1 being 16,398 bp in size. Lam SC1 (and not SC2 as in Las) appears to replicate as a plasmid prophage. The finding of circular forms of both phage indicates that these phage have the potential to become lytic in plant infections. This stable plasmid lysogen can be useful for lysogenic conversion (host range and pathogenicity enhancing), and indeed lysogenic conversion genes are also found on these Lam plasmid prophage—specificially peroxidases and adhesins. Peroxidases are used to reduce plant cell reactive oxygen levels; elevated levels result in plant programmed cell death (PCD). In fact, Las has twice as many peroxidase genes as Lam, possibly resulting in better control of PCD. This might explain why Las reaches higher titers in citrus than Lam, despite the more serious HLB symptoms caused by Lam infections. Surprisingly, although Lam has an outer membrane, Lam is missing nearly all LPS genes, including those involved in making the core, lipidA. The LPS serves a major function in the outer membrane as a protective molecular barrier against salt, detergents and antibiotics, including phytoalexins. This loss of LPS also likely makes the Lam outer membrane more fragile, heat-sensitive, and sensitive to reactive O2. Indeed, Lam is reported to be much less heat tolerant than Las. LPS genes are found in both Las and Lso, and they are not found in contiguous genomic locations in these strains, implying selection for multiple deletion events in Lam. Interestingly, since Las is also missing specific flagellin genes, and since flagella have not been observed with any Liberibacter strains, it indicates that Liberibacters may be under selective pressure to shed PAMPs (Pathogen Associated Molecular Patterns), which are recognized as part of the plant innate ‘immune’ response, that includes reactive oxygen production and can lead to PCD. The very existence of Lam implies adaptive flexibility of two species of Liberibacter in attacking the same host; additional emerging Liberibacter strains may be expected to arise. .
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. Goals 1, 2 and 3 are partially completed and continuing. 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. Support for this idea was found when a strongly predicted, bidirectional transcriptional terminator was unexpectedly found within the gp125 locus. Additional constructs are being made to analyze this latter possibility. A second (on SC1) and third (on SC2) bidirectional promoter regions, located between gp175 and gp185 on SC1 and between gp175 and gp180 on SC2 are also being cloned for evaluation as the primary early gene promoter regions. Las anti-repressor gene (SC1_gp200) was then cloned into shuttle vector pUFR047 (forming pSZ77), such that gp200 was constitutively expressed from the lacZ promoter in pUFR047, and subsequently transformed into the strain containing pSZ63. No change in color reaction was observed from that typical of pSZ63, indicating either that the pSZ77 clone was not functional, or that the gp200 locus, annotated as a Bro-N anti-repressor, didn’t affect the function of the predicted gp125 repressor.
In the course of this research project we have developed two libraries of single chain fragment variable (scFv) antibodies of interest to FCPRAC. The first library, was prepared against Xylella fastidiosa strain 9a5c, the causal agent of citrus variegated chlorosis (CVC). This library was prepared to validate and learn the scFv technology while providing an added benefit to FCPRAC, and contained 12 million primary and unique scFv that reacted with strain 9a5c. We have isolated scFv from this library that recognize the CVC strain but not other strain of X. fastidiosa, including the Pierce’s disease strain from grapevine. These will be useful diagnostic tools. The second library was prepared using individual citrus psyllids infected with ‘Ca. Liberibacter asiaticus’ (CaLas) to immunize the mice that were used to make the library. This scFv library contains 20 million unique phage that bind to different antigens present in CaLas and the psyllid vector. We combined the genome sequence data for CaLas with bioinformatics and a bacterial expression vector to clone antigens from CaLas that were used to select scFv from our library. In the course of this work we have developed a protocol to select scFv of interest to every antigen present in the CaLas genome. We focused on proteins of CaLas that are likely to be exposed on the surface of the cell to enable detection assays and/or related to host pathogen interactions and virulence. scFv were isolated that bind to 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 a CaLas infected host plant cell from undergoing programmed cell death by apoptosis. Fifty to 100 scFv were isolated for each antigen. These scFv have been used to detect CaLas in a dot blot format using extracts of infected plants. In order to improve the quality of the scFv, we have made secondary scFv libraries using directed mutagenesis methods to improve the binding affinity of the antibodies to their corresponding antigens. These secondary libraries have more than a million unique scFv for each epitope of CaLas used for selection. We have encountered some challenges which are being addressed in continued research. 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. These include submitting manuscripts to Sigma-tau for review of potential items of interest for patents prior to publication. Further research continues with these scFv on FCPRAC project 551 (detection and diagnostics) and 552 (resistance of citrus through transgenic expression of these scFv). We will also make new scFv libraries in a vector unencumbered by commercial entanglements. This effort will be facilitated by the application of the research protocols developed in the course of this project.
Continuation of diagnostic service for growers for detection of Huanglongbing in citrus and psyllids to aid in management decisions, September 2012. The lab has been in operation for more than 5 years, and as of September 2012, we have processed nearly 30,000 grower samples. Additionally, more than 25,000 samples have been received for research for the entire period of diagnostic service. Numbers specific to this quarterly report are 367 samples received from growers. This number represents a decline from previous years which was expected since incidence of HLB is nearly 100% in southwest Florida citrus. However, it is also representative in that less samples have been historically received during this quarter because the reduction in grove scouting and decline in HLB appropriate field samples during the summer. Typically, there are more samples processed December through June. The HLB Diagnostic Lab webpage was updated to announce the service of detection of CLas in psyllids as funded in this grant.
Continuation of diagnostic service for growers for detection of Huanglongbing in citrus and psyllids to aid in management decisions, December 2012. The lab has been in operation for more than 5 years, and as of March 2012, we have processed nearly 38,000 grower samples. Additionally, more than 29,000 samples have been received for research for the entire period of diagnostic service. Numbers specific to this quarterly report are 489 samples received from growers. This number represents a decline from previous years which was expected since incidence of HLB is nearly 100% in southwest Florida citrus. However, it is also representative in that less samples have been historically received during this quarter because the reduction in grove scouting and decline in HLB appropriate field samples during the summer. Typically, there are more samples processed December through June. Time frame Sample number Growers this quarter (Oct 1-Dec 31) 393 Research samples are not included in this report as those are not funded by this grant.
Continuation of diagnostic service for growers for detection of Huanglongbing in citrus and psyllids to aid in management decisions, March 2013. The lab has been in operation for more than 5 years, and as of March 2012, we have processed nearly 40,000 grower samples. Additionally, more than 28,000 samples have been received for research for the entire period of diagnostic service. Numbers specific to this quarterly report are 865 samples received from growers. This number represents a decline from previous years which was expected since incidence of HLB is nearly 100% in southwest Florida citrus. However, it is also representative in that less samples have been historically received during this quarter because the reduction in grove scouting and decline in HLB appropriate field samples during the summer. Typically, there are more samples processed December through June. The HLB Diagnostic Lab webpage was updated to announce the service of detection of CLas in psyllids as funded in this grant.
To date, 733 culture medium formulations have been tested in attempts to grow Liberibacter asiaticus in axenic culture. Efforts to further optimize media that appear to support initial growth of the bacterium are under way. Replacing the mixtures of essential and non-essential amino acids in the media with more complex sources of amino acids and oligopeptides, such as Trypticase Peptone (BBL) or Casamino Acids (Difco), did not result in improved growth. Supplementing the medium with complex tissue culture media was examined. Insectagro DS2 medium (Cellgro) at 10% appeared advantageous. Further supplementing the medium with 10-20% Medium 199 (Sigma) also appeared advantageous, but CMRL medium 1066 (Gibco) at 4% produced inconclusive results. The addition of Lipid Mixture 1 (Sigma) at 0.5% appeared helpful. As previously reported, zinc sulfate, betaine and taurine appeared deleterious to growth and were consequently eliminated from the medium. Addition of fetal bovine serum at 0-30% to the medium produced equivocal results and is presently being added at 15%. Numbers of Liberibacter asiaticus cells increase in some media after 6-14 days. Fourfold dilution with fresh media sometimes promotes additional growth. However, bacterial numbers eventually decline even following addition of fresh medium. Efforts are underway to optimize the medium and growing conditions to support continuous growth of the bacterium.
Volatile organic compounds (VOC)-based HLB detection: We have completed the study of the correlation of health status of a plant with its released VOC ‘fingerprint profile’ for the HLB pathogen. It is demonstrated that an early (even at asymptomatic stage), rapid and non-invasive means of detecting the HLB pathogen is possible via analysis of the VOCs emitted by citrus plants as measured using a portable Gas Chromatography/Differential Mobility Spectrometer (GC/DMS) due to their metabolism changes post-infection. Application of GC/DMS allowed us to longitudinally monitor VOC ‘fingerprint profile’ differences in-vivo throughout an entire year to account for seasonal variations of the disease and plant’s life cycle. A mathematical model was developed to classify the diseased and control groups based on GC/DMS data, with discrimination accuracy of 80-90% and close to 100% under optimal conditions. However, while using conventional GC with either flame ionization (FI) or mass spectrometry (MS) detectors, we were unable to consistently differentiate volatiles, due to very low concentration levels of leaf volatiles. Initial testing of SPME fibers with exposure times from 1-8 hrs lead to some identifications but tree to tree variation was as big as healthy to HLB. Purge and trap devices employing Tennax and combinations with charcoal improved sample amounts, but we were never successful in getting consistent results due to sample carryover and apparent breakdown volatiles from the trap itself. Sampling volumes ranged from 12-48 liters of air sampled within 5 mm of the leaf surface. The major limitation was the inability to use the purge and trap samples on the MS detector. We were only set up to use FI detection with these collection tubes. HLB detection using optical sensors: With the aerial hyperspectral images acquired in December 2011, a novel method named ‘extended spectral angle mapping (ESAM)’ was developed to detect the citrus greening disease. Firstly, Savitzky-Golay smoothing filter was applied to remove spectral noise within the data. Then support vector machine was used to build a mask to segment tree canopy from the other background. Vertex component analysis was chosen to extract the pure endmembers of the masked dataset. Then spectral angle mapping (SAM) was applied to classify healthy and HLB infected areas in the image. Finally, red edge position was used to filter out false positive detections. The experimental results were compared with other methods, Mahalanobis distance and K-means. The ESAM performed better with a detection accuracy of 86% than those two methods which yielded accuracies of both 64%. In regard to the ground-based optical sensing, several methods such as visible-near infrared spectroscopy (Vis-NIR), mid-infrared (MIR) spectroscopy, fluorescence spectroscopy, thermal imaging, and laser-induced breakdown spectroscopy (LIBS) were tested. Each of these methods showed unique capabilities, having their own advantages and limitations. Although MIR spectroscopy and LIBS are destructive methods that require leaves to be removed from the trees; these methods provide chemical signature that are robust and remains unaffected by the incident light source. Moreover, MIR spectroscopy could be used for detecting starch accumulation, which begins even before the symptoms appears in the leaves. Similarly, Vis-NIR spectroscopy, fluorescence spectroscopy and thermal imaging are non-destructive methods and can be applied for aerial sensing as well. Based on the evaluation studies, we achieved a classification accuracies of 90% and higher. Total number of peer-reviewed publications: 12+, Presentations: 10+, Patents: 1.
Potential effectors of HLB engineered into Citrus Tristeza Virus (CTV) vector have been transferred into citrus as outlined in the previous research updates and are being screened for their response to HLB pressure. They include the following; CLIBASIA_05165, CLIBASIA_05605, CLIBASIA_01555, 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. 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. We use Citrus macrophylla which is a preferred host for CTV for expression of putative effectors and subsequently transferred into sweet orange and /or grapefruit citrus varieties by graft inoculations. These plants are being challenged inoculated with HLB by exposing them to infected psyllids. Additionally, we have used a faster approach using Potato virus x (PVX) vector system to screen for virulence genes of HLB. Transient expression of prophage-related gene cluster of HLB in PVX vector did not induce obvious phenotype changes in tobacco plant. However, transient expression of HLB-flagellin, and flg22, HLB flagellin domain, induced cell death and callose deposition in N. benthamiana. Substitution experiments revealed that the 38th and 39th amino acid residues were essential for callose induction. However, the synthetic peptide (DRVSSGLRVSDAADNAAYWSIA) could not induce cell death, but retained the ability to induce callose deposition at the minimal concentration of 20 ‘M. These results demonstrated that Flg22Las has PAMP activity, and may play an important role in determining citrus resistance to HLB. These results have been submitted for publication in PLoS One. Progress is also being made in cloning some these potential CLas effectors into a universal secretion study vector with MYC tag to determine if any of these effectors are secreted using western analysis and eventually study the citrus plant response to the secreted proteins.
To date, 716 culture medium formulations have been tested in attempts to grow Liberibacter asiaticus in axenic culture. Some attempts looked promising. The bacterium was still detected in one medium after five transfers and a total of 50 days after the culture was initiated. Efforts to further optimize this medium are under way. Although genomic information indicates that the bacterium has a transport mechanism for zinc, addition of zinc to the culture medium appears to inhibit growth. Similar results were obtained for betaine and taurine. Fetal bovine serum appears to be inhibitory at 30% in the medium, but at 15% or less results have been uncertain. Promising results have been obtained under both aerobic and microaerophilic conditions, but not under anaerobic conditions. Some medium formulations support the development of biofilms of the bacterium on the alimentary canal of Diaphorina citri in culture but not planktonic growth of the bacterium, while other formulations appear to support both. Efforts to culture the bacterium continue.
Diagnostic service for growers for detection of Huanglongbing to aid in management decisions, July 2012. This report covers the entire period that Huanglongbing Diagnostic Laboratory has been in service. The objectives for the funding were to provide 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, we have processed approximately 31,000 grower samples Additionally, more than 40,000 samples have been received for research. Only the equipment is used for the research samples because PI are paying for the cost of materials and personnel on a per charge basis to keep separate from the grower’s service. Techniques, Protocols and Research For DNA extractions, the final protocol was as follows using 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 used the 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 was 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 was being used for research and extension samples. The basic diagnostic service remains available to growers, researchers, extension faculty and dooryard citrus growers. However, we expanded 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.
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 they also feed on older leaves. We are examining whether and how well the psyllid can transmit the disease when feeding on non-flush leaves. 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 are being tested in trees. We have found only two peptides that appear to give some protection sweet orange trees from HLB. We are examining whether they can be used to treat infected trees. We are preparing inoculum for a field test. 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. This USDA and EPA approved field test is underway.
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, FP1 (CP001677.5) and FP2 (JF773396.1), twelve (A to H) different populations (genotypes) have been identified. Type A and B are located in FP1 and FP2, respectively. Typing revealed A, B and C as the three most abundant groups in libraries from psyllid, citrus and periwinkle, although psyllid contained much more type A sequence than the plant hosts. Typing also revealed periwinkle and dodder contained all population types, but psyllids did not. They had low to no titers of four types, including type D. In citrus a high titer of type D was associated with typical blotchy mottle symptoms. We are continuing to studies the different population types and their correlations with HLB phenotypes and disease severity. We have characterized the ATP translocase from Las and proved its function using a heterologous E. coli system (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. Seed transmission of Las was tested in grapefruit, sweet orange, sour orange and trifoliate orange. 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 Las 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. 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.
(863) 956-8817
(863) 956-5894
700 Experiment Station Road
Lake Alfred, FL 33850
Email Us
