Analysis of the distribution and diversity of plant pathogens can be informative in understanding epidemiological diseases and the evolution of pathogens in related to their adaptation and genetic diversity. The advances in molecular genotyping and phylogenetics have permitted an increased resolution power in identification and differentiation of the evolutionary relationships among ‘Candidatus Liberibacter asiaticus’ (Las) isolates, allowing inferences to be made regarding the patterns of distribution and sources of invasion. In this project, we have successfully developed and validated a panel of molecular biomarkers for Las genetic analysis. This multilocus simple sequence repeat (SSR, or microsatellite) biomarker system is a powerful tool for genotyping and genetic analyzing Las populations. To better understand genetic relationships among Las populations worldwide, Several thousands of Las isolates representing major HLB populations including South and Southeast Asian countries, South and North American countries were collected and analyzed. The genetic diversity of Las is generally higher in Asia than in the Americas. UPGMA and STRUCTURE analyses identified three major genetic lineages of Las worldwide; India group, East-southeast Asia group and North American group, respectively. Among three lineages, majority of the isolates from Florida were clustered in North American group, but a few of Florida isolates were identified in Southeast Asian group. In contrast, populations from Indian isolates were clustered in a separate group and were genetically more distant in comparison with other two groups. Southeast Asian and Brazilian isolates were generally included in the same group except a few of Brazilian isolates were identified in North American group. While the actual sources of the dominant Las strains in Florida are still in question, the genetic analysis from this study suggested that the introduction of less-pervasive strains may have been introduced directly from Asia or via Brazil based on their genetic similarity. In addition, high level of genetic diversity in Florida populations strongly suggested that recent outbreaks of HLB in Florida likely occurred through multiple introduction events. We also analyzed HLB isolates recently identified in Texas and California. Results showed that all alleles detected in California Las samples matched to alleles recorded in the corresponding loci in Las genotyping database. The California and Texas Las samples appear to be genetically close to Southeast Asian group. The multilocus SSR marker system developed in this study provides adequate discriminatory power for the identification, differentiation of closely-related Las strains, and is a useful tool for tracking Las sources as well as for evolutionary studies of the HLB-associated Liberibacters [1]. To further investigate genetic diversity in whole genomic level, we have obtained complete genome sequences of a Las strain from Guangxi, China, a Laf strain from South Africa and a Lam strain from Brazil. Comparative genome analyses of all three HLB Liberibacter species revealed insights into sequencing variation within and between Liberibacter species. Sequences polymorphisms in virulence gene and phage gene regions were identified. New genomic information facilitated the development of a new set of gene-based biomarkers that advances from current genotyping to phenotyping Las. To confirm the function of selected virulence genes that will be used for new biomarker development. Ten genes were selected for in vitro functional characterization using orthologous gene expression technique. Expressed proteins were purified from expression system. Corresponding bioassays were performed for function analysis of Las virulence gene. Using this expression system, we are able to develop a new set of biomarkers that will be used for phenotyping Liberibacter bacteria. [1] Islam et al., BMC Microbiology 2012, 12:39.
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. Variations of Las populations existed not only in different varieties, but also in different individuals of the same variety. 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 have also revealed only one of the two putative znu operons is responsible for zinc uptake. The results were published in PLoS ONE 7(5): e37340. doi:10.1371/journal.pone.0037340,2012. In addition, we have demonstrated Las encoded a functional flagellin characteristic of a Pathogen-Associated Molecular Patterns (PAMP). The results were published in PLoS ONE 7(9): e46447. doi:10.1371/journal.pone.0046447, 2012. 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 plant was confirmed by PCR using several Las-specific primer sets. Graft transmission of the cutting from this HLB plant confirmed this seed-transmitted HLB. 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 assembled into 1 scaffold. We are conducting the gap closing for each BAC clone and hope to get the full wDia genome soon.
This is a project to examine psyllid inoculation of young citrus flush with HLB. We have found that the young flush can become infected with HLB within a couple of week and then can serve as a source of infection for the progeny of those input psyllids. We have just begun to use this system as an assay to examine genes and RNAi against psyllids.
The objective of this project is to characterize the hypI (renamed as hyvI) gene and determine its effects on insect transmission and/or virulence in host plants. Transient expression with alternative expression systems and RT-qPCR, etc., will be used to elucidate the function of the hypI (hyvII) gene of Las and shed light on the molecular mechanism of this “phase variation” phenomenon; thereby developing a novel control strategy for citrus HLB. In addition, antibodies and probes along with standardized protocols developed during this project can be applied for better detection and differentiation of the HLB bacteria. The hyvI and hyvII within two Las prophages were further characterized and some of the results were published in Applied Environmental Microbiology 77:6663-6673, 2011. “Diversity and Plasticity of the Intracellular Plant Pathogen and Insect Symbiont “Candidatus Liberibacter asiaticus” as Revealed by Hypervariable Prophage Genes with Intragenic Tandem Repeats”. We have developed an improved real-time PCR using SYBR Green 1 (LJ900fr) and TaqMan’ (LJ900fpr) protocols with primers and probe targeting the nearly identical tandem repeats of 100bp hyvI and hyvII. The results were published in Molecular and Cellular probes, 26:90-98, 2012. Working with Mesa Tech International, Inc., we have developed a rapid and simple HLB detection kit for field testing, which only requires less than 30 minutes. Monoclonal antibodies against the partial HyvI protein (only one repeat) were generated, and their sensitivity and specificity were evaluated for the detection of HyvI protein expressed in E. coli and HLB-infected citrus and psyllids. All antibodies were able to recognize the E. coli expressed HyvI antigen, but were not able to detect the HyvI antigen from HLB-infected plants and psyllids. We have characterized two novel autotransporter proteins of ‘Candidatus Liberibacter asiaticus’ (Las), and redesignated them as LasAI and LasAII in lieu of the previous names HyvI and HyvII. Bioinformatic analyses revealed that LasAI and LasAII share the structural features of an autotransporter family containing large repeats of a passenger domain and a unique C-terminal translocator domain. When fused to the GFP gene and expressed in E. coli, the LasAI C-terminus and the full length LasAII were localized to the bacterial poles, similar to other members of autotransporter family. Despite the absence of the signal peptide, LasAI was found to localize at the cell surface by immuno-dot blot using a monoclonal antibody against the partial LasAI protein. Its surface localization was also confirmed by the removal of the LasAI antigen using a proteinase K treatment of the intact bacterial cells. When co-inoculated with a P19 gene silencing suppressor and transiently expressed in tobacco leaves, the GFP-LasAI translocator targeted to the mitochondria. This is the first report that Las encodes novel autotransporters that target to mitochondria. These findings may lead to a better understanding of the pathogenesis of this intracellular ‘energy parasitic’ bacterium and to characterizing new molecular targets for HLB control.
Current methods for inoculation of citrus trees with HLB such as grafting of HLB-containing tissue or psyllid-mediated inoculation have their own challenges and drawbacks. For this reason, the main focus of our work is to develop a new method for inoculation of experimental citrus plants with HLB that would provide an alternative to the existing methods by overcoming the disadvantages of the latter approaches and allowing high throughput inoculations with a greater certainty of pathogen transmission for various research purposes. We are using a Pulse Micro Dose Injection System (PMDIS) to develop a new method for rapid and efficient inoculation of plants with HLB. In our preliminary experiments we have had some success in inoculation of Periwinkle plants using this technique, which suggested a feasibility of further adaptation of the PMDIS system for HLB inoculations. This is a new project and the funds for this project were released. We already have designated personal for conducting the proposed research. The research is in progress. Plant material that will be used in this project is being prepared: new plants of different varieties that will be used as receptor plants for inoculations are being grown in our greenhouse and HLB-infected plants that will be used as an inoculum source are being maintained. Additionally, HLB-infected trees that are present on the field will be also used for inoculum. Currently we are conducting initial trial inoculations. Some plants have been already injected using PMDIS. Those are being maintained in the greenhouse and monitored for the disease development. We are also working on improving the conditions for inoculation by examining how the following parameters affect efficiency of inoculation: age of receptor plants, types of citrus varieties used as HLB bacterium donors as well as for plants being inoculated, types of flushes being inoculated, composition of the buffer used for preparation of bacterial suspension, injection conditions, etc. Those parameters will be examined and analyzed.
The objectives of this project are to improve Cu bioavailability and increase retention of the Cu loaded silica nanogel (CuSiNG) materials to plant surfaces. Our 2011 canker field trial data (Ray Ruby grapefruit grove in Vero Beach, FL) showed that the efficacy of the CuSiNG material in preventing fruit infection was comparable to Kocide 3000, Badge X2 and Magna Bon formulations despite 45 inches of rain fall. In trial, amount of residual Cu present on fruit surface was estimated using Atomic Absorption Spectroscopy (AAS) to indirectly support the retention of the CuSiNG materials. To better understand the adherence property of the CuSiNG material to plant surfaces, the CuSiNG material was chemically labeled with Tris(bipyridine)ruthenium(II) chloride (Rubpy; a metallorganic fluorescence dye) for imaging purposes. However, we could not collect reliable data based on our imaging studies due to several problems. (i) Cu reduced fluorescence of Rubpy through quenching, (ii) photostability of the dye was not enough to withstand sunlight, resulting in fluorescence fading (note that Rubpy is photostable in comparison to traditional organic based fluorescent dyes such as rhodamine 6G and fluorecein) and (iii) background fluorescence signal from plant surfaces. To overcome these limitations, we have synthesized red-emitting CuInS2/ZnS core-shell fluorescent labels (known as quantum dots, Qdots). CuInS2 are extremely photostable, ultra-small (~3.5 nm size semiconductor crystals; about the size of a Green Fluorescent Protein, GFP). These CuInS2 Qdots are not expected to exhibit Cu ion induced fluorescence quenching as they contain Cu. We will be reporting adherence and retention properties of Qdot labeled CuSiNG materials to plant surfaces in future reports. Shelf-life of the CuSiNG – transparent formula has been tested. Our results show that this formula is stable for over two years without loss of antibacterial efficacy. In general, CuSiNG material show improved antibacterial efficacy against X. Alfalfae and E. Coli in comparison to Kocide 3000 and Cu sulfate under laboratory conditions. Improvement of antibacterial efficacy of CuSiNG formulations in comparison to Cu sulfate and Kocide 3000 controls has been attributed to increase Cu bioavailability. However, an increase of overall efficacy due to the presence of mixed valence Cu in CuSiNG material (as observed from our XPS studies) can not be ruled out. Research is underway to evaluate the role of mixed valence Cu in CuSiNG materials.
Citrus huanglongbing (HLB) is associated with three species of Liberibacter’Candidatus Liberibacter asiaticus (Las), Ca. L. americanus, and Ca. L. africanus. The majority of the testing in Florida is focused on detection of Las, the only bacterium known to be associated with HLB in Florida to date. Over the past four years with funding from Citrus Research Board, we have conducted regular surveys of citrus and citrus relatives in Florida, from various germplasm collections, backyard plants, native and cultivated trees, testing for tolerance to HLB. We have focused on plants showing HLB symptoms but testing negative by standard qPCR tests. A small selected set of symptomatic, qPCR negative samples were analyzed for detection of other genomic regions of Liberibacters by conventional PCR (cPCR), cloning and sequencing. This study confirmed the presence of Liberibacter variants not detectable by standard assays. The purpose of this project is to conduct further research on variants of Liberibacters from citrus and citrus relatives and to develop rapid methods for detection of these variant populations. We also will study the biology of the variants under greenhouse conditions, determine the changes in Liberibacter populations within individual trees over time from analysis of DNA extractions we have made over the past 5 years, and determine if there are interactions among populations of Liberibacter variants which may ameliorate/enhance the symptoms of HLB. Understanding of HLB disease complex caused by all variants of Liberibacters will be useful for developing novel disease management strategies. Research on this project this second quarter has included conducting a survey of South Florida to re-locate isolates previously identified. Molecular assays will be conducted to confirm the nature of the isolates, and budwood has been secured for establishment of the isolates within protected greenhouses to enable further study under controlled conditions. Further research has been conducted on development of methods to ‘trap’ Liberibacter and Liberibacter-like pathogens to facilitate molecular characterization of the genomes. The funds were received in October which will enable hiring of personnel to facilitate this research.
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.
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