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 third quarter has involved molecular characterization of samples collected in October in Southern and Central Florida. Research has included development of a unique methodology to selectively prepare DNA from Liberibacter and Liberibacter-like pathogens for genome sequencing. Observations are being made of symptoms produced on citrus in the field and greenhouse conditions.
The 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. At this stage we are studying the interaction of the plants expressing HLB effectors and their response to HLB infection through psyllids. A potential useful outcome of this project is the finding that CTV-vector can be used as an RNAi vector. This has been demonstrated to silence citrus endogenous Phytoene desaturase (PDS), delta amino levulinic acid dehydratase (d ALA dehydaratase) genes, and has also be used to potentially silence endogenous genes of psyllids (vecotr of HLB) in co-operation with scientists at CREC and USDA, Fort Pierce. We have positive feedbacks from these groups on the successful silencing of psyllid endogenous genes. This line of research potentially holds great promise in mitigating the spread of HLB by psyllid vector. An oral presentation based on this investigation will be presented in the upcoming 2013 HLB conference in Orlnado. Shubash Hajeri, Choaa El-Mohtar, William O. Dawson and Siddarame Gowda. Citrus tristeza virus-based RNA-interference (RNAi) vector and its potential in combating citrus Huanglongbing (HLB).
To date, 793 medium formulations have been tested for their ability to support growth of Candidatus Liberibacter asiaticus. Growth has been observed over 2-3 low level passages to fresh media for periods of over two months. Attempts are now aimed at obtaining vigorous growth. Insect tissue culture media have been used as the source of the basic nutritional requirements of the bacterium. DS2 tissue culture medium (Mediatech) has appeared advantageous; however, the formulation of this medium is proprietary. So attempts are presently being made to find a suitable replacement for DS2 medium that has a freely known formulation. The effects of complex reagents, such as Bacto Proteose Peptone No.3, Phytone Peptone, and Brain Heart Infusion Broth, are also being evaluated. Presently, a medium formulation containing Graces tissue culture medium instead of DS2 medium, and supplemented with, among other ingredients, a mixture of lipids, sterol, and pluronic acid, has produced good early results and is being evaluated for continuous growth support.
PCR test results for the 90 leaf samples collected in Aug. 2012 were received and compared with starch measurement results. The cycle threshold (CT value) which is the number of necessary cycles for the fluorescent signal to cross the threshold was used to decide the samples’ infection status. The CT value below 33 showed HLB infection of the samples. The PCR results were used as a reference for defining training and validation sets. The starch concentration for healthy samples should not exceed 5 ‘g/mm2, however, there were some healthy samples with starch density of more than 5 ‘g/mm2 which means that the reason of starch accumulation was not HLB infection. There was also one HLB infected sample with less than 5 ‘g/mm2 of starch density which could be considered as an experimental error. A pixel based analysis was performed to determine the HLB detection ability in each imaging condition. Two clusters were defined for a K-means algorithm, and the clustered pixels were evaluated to determine whether the pixels belonging to the same class (with prior knowledge) were clustered in the same cluster as well. The investigation showed that only the 591 nm (Min) images contained very useful information and classes were distinctive, however, for some samples, the general average intensity of the image was higher and led to a misclassification of some healthy pixels in other classes, and still the healthy and not-healthy (including HLB infected and zinc deficient) areas were distinguishable, when the clustering was performed for individual samples separately. Then, the same textural features (as reported previously) were extracted only from 591 nm (Min) images and a step by step classification method was implemented, in which at the first step zinc deficiency was detected and HLB infection was identified at the second step. A best set of textural features was determined separately for each classification step using Bhattacharyya distance feature selection method. Also performances of seven classifiers (as reported previously) were evaluated to determine the best classification approach for each step. From the analyses results, zinc deficient samples were detected with an accuracy of 96.7%. HLB infection was also detected with average accuracies of 90% and 96.5% for zinc deficient and non-zinc deficient classes, respectively. However, the main purpose in this study was to detect HLB infection and not the zinc deficiency. Therefore, when only the HLB detection was considered, an average classification accuracy of 93.1% was obtained, including 97.9% and 88.4% accuracies for healthy and HLB infected leaves, respectively. A manuscript is currently being prepared based on the above results to be published in the Transactions of the ASABE journal. An abstract was submitted for the 2013 ASABE annual meeting in July 2013. In Dec. 2012, a new dataset of 96 samples containing 20 healthy, 20 magnesium deficient, 20 zinc deficient, 20 HLB infected, and 16 HLB infected & zinc deficient samples in a Valencia variety were collected from the CREC grove to be evaluated and compared with the previous dataset from a Hamlin variety. Starch measurement for these samples were completed and the samples were sent for a PCR test.
2012 LAS experiments have been completed and data analysis is being conducted. These experiments were similar in design to 2011 experiments, with the treatments being 1/3 King’s B (K) medium and commercial grapefruit juice (G) medium. However, the focus was shifted to analyses of the biofilm-like substance formed at the air-liquid interface of culture flasks. 2011 experiments showed that increased formation of this substance was found in juice-containing media, which correlated with prolonged LAS viability. In 2012 experiments, slides with the biofilm-like substance formed on them were stained with Calcofluor (stains exopolysaccharides white) and LIVE/DEAD BacLight (stains live cells green and dead cells red). Micrographs of stained slides showed that exopolysaccharides were present, indicating that the substance was a true biofilm. Results also showed that the majority of cells in biofilm from medium K were dead after several weeks, while the majority of cells were alive in medium G. Analyses are currently underway to determine the percentages of viable and nonviable LAS cells in this biofilm, as well as determine the presence of other potential “helper” bacteria. Analysis of Illumina MiSeq high-throughput sequencing data of 16S amplicons from experimental samples will soon be completed. Data from the initial citrus-seed inoculum showed that LAS was the largest bacterial component of the inoculum, but significant populations of several other bacterial endophyte species were also present in the seed coats. These other populations, if they also grow in the cultures, could have effects (either positive or negative) on the culture of LAS. The 16S amplicon data from biofilm samples will soon be analyzed. This will indicate if any of these other endophytes were able to grow in the LAS cultures. A draft of the publication for these data has been written. When the rest of the 2011 and 2012 biofilm bacterial community data is analyzed (qPCR and Illumina data) it will be added to the publication. The data will be presented at the 3rd International Conference on Huanglongbing to obtain any additional insights/comments from other researchers in the field. Subsequently, the publication should be ready for submission.
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. We are currently conducting functional analysis and determination of the LasAI and LasAII functional domain(s) via mutagenesis.
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. We are currently conducting functional analysis and determination of the LasAI and LasAII functional domain(s) via mutagenesis.
During summer, seeds generally are immature, relatively soft and easily homogenized. We tested two experimental approaches: 1) enzyme treatment without subsequent physical pulverization of the tissue and 2) enzyme treatment followed by physical pulverization of the tissue. For immature seeds, enzyme treatment followed by only extensive vortexing could cause extensive degradation of the tissues and release bacteria for further purification by filtration and centrifugation on a Percoll gradient. Enzyme treatment followed by physical disruption of tissue by treatment with a Polytron apparently gave at least equivalent tissue disruption if not slightly better based upon the calculated number of bacterial cells determined to be in the different portions of the Percoll gradient. Percoll fractions that were indicated to contain the highest numbers of cells as determined by qPCR were assayed with Fluorescence In Situ Hybridization (FISH) microscopy. The most intense fluorescence was observed with spherical bodies (not seen in negative control preparations); the bacilliform shapes seen in fresh tissues disrupted and chemically fixed in place (paraformaldehyde) were infrequent, suggesting a change in the shape of the bacteria occurs during extraction from the tissue. Also, the numbers of cells calculated from qPCR values indicated more cells should be present than were observed the microscope. We do not know the basis of this discrepancy.
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.
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