Accumulation of Cu pesticides in soil and development of Cu resistance to citrus canker bacteria demand an alternative that is environmentally-friendly, non-phytotoxic and as effective as Cu compounds. Quaternary ammonium compounds (Quat) are effective antibacterial agent, however they exhibit severe phyto-toxicity to plant species. Therefore, it is not practical to apply quat materials directly to citrus plants. Fixed-Quat is a nanotechnology-enabled formulation that minimizes phytotoxicity of Quat while maintaining its antimicrobial efficacy. Additionally, Fixed-Quat material is designed to exhibit good rainfastness. In this reporting period, optimization of different industrially acceptable synthesis parameters of two previously reported Fixed-Quat nanogel formulations (named hereafter Fixed-Quat NG-A and NG-B) were carried out. Additionally, two new Fixed Quat nanoparticle formulations (named hereafter Fixed-Quat NP-A and NP-B) were synthesized. Fixed-Quat NG formulations are stable thus far (at least 6 months), indicating good shelf-life. Antimicrobial studies of Fixed-Quat NG-A and NG-B were conducted against Xanthomonas alfalfae subsp. citrumelonis (a citrus canker surrogate) using in-vitro microplate Alamar blue assay, bacterial viability (expressed as colony forming units, CFU/mL) and growth curves. Kocide 3000 was used as positive control and silica nanogel was used as negative control. Interestingly, both the optimized Fixed-Quat NG formulations demonstrated complete bactericidal efficacy at concentrations as low as 0.55-1.1 ppm (for Fixed-Quat A) and 3.5-4.4 ppm (for Fixed-Quat B). The MIC values of our previously reported Fixed-Quat NG-A formulation was 2 ppm. Feasibility of producing highly concentrated Fixed-Quat NG formulations has been tested which is industrially attractive for storage and shipping conveniences. We have successfully produced stable Fixed-Quat NG-A and NG-B formulations containing ~ 10000 ppm and ~ 13000 ppm Quat, respectively. Average particle size of Fixed-Quat NG-A and NG-B materials in solution was estimated using Dynamic Light Scattering (DLS) technique. DLS study suggests the formation of sub-micron to micron size hydrophilic NG particles. Fixed-Quat NP-A and NP-B are being characterized. Average particle size of Fixed-Quat NP materials was estimated to be ~450 nm by DLS. Future reports will include research results on Fixed-Quat NP optimized formulations.
Citrus huanglongbing (HLB) is associated with three species of Candidatus Liberibacter: Ca. Liberibacter asiaticus (Las), Ca. L. americanus (Lam), and Ca. L. africanus (Laf). The majority of the testing in Florida is focused on detection of Las as this is the only bacterium known to be associated with HLB in Florida to date, while Lam and Las have both been found in Texas. Nine isolates from citrus and citrus relatives identified as being naturally infected with Ca. Liberibacter species but which would test negative for Las, Lam, and Laf, previously inoculated and established in receptor plants are being challenged by graft inoculation using bark pieces from a lemon plant testing positive for Las. The plants will be monitored for relative titers of the Liberibacter like and Las isolates. In cooperation with ICA in Colombia, we have worked on characterizing a Liberibacter-like bacteria from Colombia using the methods being applied to the isolates in this project and droplet digital PCR. As a result we have announced the finding of a fourth species of Liberibacter, tentatively named ‘Candidatus Liberibacter caribbeanusfrom Colombia. Using the macroarray approach, we are identifying bacterial genome regions which are conserved among the four species of Liberibacter. We are continuing the characterization of the Florida Liberibacter-like isolates using PacBio sequencing, miSEQ and analyses with droplet digital PCR.
Citrus blight has imposed consistent losses and challenges to citrus industry since the causal agent of the disease remains unknown. The present study would be instrumental in knowing the mysterious pathogen causing citrus blight and pave way for devising efficient management or control methods to help citrus industry to tackle citrus blight. We will characterize the microbiomes of the blight diseased and healthy citrus roots through metagenomic approaches. We have surveyed three groves at Lake Alfred, Auburndale, and Haines city. Citrus blight trees at different development stages and healthy trees are being confirmed based on symptoms, water injection, and P12 antibody that have been known as the diagnosis tools for citrus blight. We selected the blight diseased and healthy citrus trees to be used for sampling. Root samples were collected from 24 trees. The first set of DNA and RNA samples have been purified and sent for deep sequencing to identify the microbes associated with blight diseased and healthy citrus. We have received the sequencing result for the first batch of samples and are almost done with analyzing the data. The publication of Sweet orange genome significantly helps our analysis. Now we are aligning the reads from DNA samples to sweet orange genome and C. clementina genome (V1.0), about 30%-40% reads could not mapped on these three citrus genomes. Those unmapped reads which are not citrus sequences are being used for metagenomic analysis. We also analyzed the RNA-seq data. Totally 2383 citrus genes were down-regulated while 2017 genes were up-regulated by citrus blight. Meanwhile, two methods were used to analyze these differentially expressed genes: GSEA (Gene set enrichment analysis) which is Gene ontology based method and Mapman-Mapman pathway based method. Root samples were collected again from 12 trees in the selected citrus grove at St. Cloud in March 2014. Interestingly, further test in April indicated that two previous healthy trees became citrus blight positive. Further analysis of those trees are being conducted. All the sequencing data have been uploaded to public database. We analyzed the hormones in the blight diseased trees and healthy trees. Quantitative reverse transcription PCR was used to further compare the gene expression of selected genes of citrus. We sampled for the fourth time and further analysis of those trees are being conducted. Metagenomic analysis of the sequenced samples is being conducted. In addition, the release of 8 citrus genomes including one sour orange, 2 pummelo, 4 mandarin and 1 sweet orange in the database has facilitated our analysis of the metagenomic data. Two manuscripts are being prepared for publication.
We have made progress with the scFv library made with the earlier grant from CRDF. We had previously used the scFv when expressed as part of the M13 phage vector particle in ELISA and dot blot formats. Our efforts in the past quarter have built on that work, and now we are using the scFv alone in tissue print assays of citrus plants to detect ‘Ca. Liberibacter asiaticus’. scFv are expressed and purified from from E. coli cells using a 6X His tag incorporated in the scFv protein. We have produced purified scFv at concentrations in the mg/ml range. Differences are observed among different scFv clones. Results from SDS-PAGE gels are consistent with post translational folding being problematic for some scFv as has been reported in the literature. The tissue print assays continue on nitrocellulose membranes. Color development is observed in the vascular cylinder (phloem) of HLB infected petioles but not in comparable petioles from healthy trees. In some tissue prints, color development is observed in discrete spots outside of the phloem cylinder. Similar results are obtained with all scFv that were selected to bind to proteins expressed on the surface of ‘Ca. Liberibacter asiaticus’. These targets include an ATPase associated with the type IV pilus, a pilus assembly protein, two flagellar proteins, the major outer membrane protein OmpA, and the efflux protein TolC. These results have been attributed to non specific cross reactions of the commercial monoclonal antibody directed at the 6X His TAG. The His tag will not be very useful for tissue printing. In continuing work, we have recently made tissue prints with a modified technique, using ‘Super block’, a commercial product used in Northern and Southern blotting on nitrocellulose membranes and detection with a monoclonal antibody directed at the FLAG epitope on the scFv. This protocol produces remarkably sharper tissue prints with dramatically reduced background, and color tightly focused as a ring in the phloem cylinder of HLB infected, but not healthy petioles. These results vary by manufacturer of the monoclonal anti-FLAG antibody conjugate. We have identified the best supplier. We have also prepared rabbit polyclonal antibodies against the major outer membrane protein (OmpA) and detected them with alkaline phosphatase labeled goat anti rabbit monoclonal antibody. The polyclonal antibodies produce distinct spots of color corresponding to individual phloem cells infected with ‘Ca. Liberibacter asiaticus’. This is a useful assay for ‘Ca. Liberibacter asiaticus’, and we are using it to describe the distribution of ‘Ca. Liberibacter asiaticus’ in infected citrus trees, fruit and seed. We have conducted research with the anti OmpA antibodies and have demonstrated that they are at least as effective as qPCR for the detection of ‘Ca. Liberibacter asiaticus’ in plant samples when used in a tissue print format. We have used these antibodies to characterize the distribution of Ca. Liberibacter asiaticus’ in infected citrus and can unambiguously detect it in all plant and seed parts tested. We have combined this antibody with PCR and have developed a sensitive immunocapture-PCR (iPCR) protocol for the detection of Ca. Liberibacter asiaticus’. Three manuscripts are in draft form related to this work.
This project is a continuation of a previous project #95 “PREPARATION OF ANTIBODIES AGAINST CANDIDATUS LIBERIBACTER ASIATICUS”. Progress reports for the previous project are on file. The reimbursable agreement with CRDF was established on September 5, 2012. We continue to study the literature to identify vectors to use for a future scFv library made as part of this project. The goal is to find a suitable vector that is not encumbered by intellectual property and patent issues. I have written twice to a laboratory in Germany which has published results with a suitable vector but have had no reply. We are also optimizing the cloning strategies that will be used to move already selected scFv into transgenic plants. We have obtained the vector, pUSHRL-26, to be used for plant transformation of the scFv constructs from Ed Stover at Fort Pierce and the plasmid has been purified. We have purchased the restriction enzymes and designed primers to be used for PCR to amplify the cloned scFv encoding inserts from vector pKM19. The cloned inserts will be sequenced to confirm that they are correct and then cloned into the transformation vector. The scFv have been modified by the addition of a four amino acid leader sequence (KDEL) and both Sma I and Spe I cloning sites. The KDEL sequence is expected to stabilize the concentration of scFv in phloem cells by facilitating proper folding of the protein in the microtubules and thereby protecting the ScFv from proteolytic digestion. Eleven scFv inserts have been sequenced to be sure that the expected sequences are correct, and five ScFv sequences have been successfully cloned into the recombinant vector pUSHRL-26 for transformation of citrus rootstocks. These inserts include three different scFv that bind to the protein InvA and two that bind to the protein TolC. The protein InvA is produced by CaLas and secreted into the host to prevent the infected host cells from entering into apoptosis, and the protein TolC targeted by the scFv, is in the external membrane and is essential for the removal of antimicrobial substances produced by the plant. The vector is designed to direct expression of the scFv into the phloem cells of citrus, where CaLas grows, and the vector encoding the scFv genes is being introduced into rootstock varieties by Agrobacterium mediated transformation. In the period just ending we have purified 9 scFv genes and cloned them into the plant transformation vector developed by Ed Stover at Fort Pierce. Sequencing confirmed the clones were correct. The Stover laboratory has transformed the constructs into Agrobacterium and the Agrobacterium has been used to transform Carrizo seedlings. Nine transgenic lines have been established with between 150-400 epicotyl explants for each line. These explants include scFv for both TolC and InvA, and are being grown at Fort Pierce for subsequent evaluation.
We have made progress with the scFv library made with the earlier grant from CRDF. We had previously used the scFv when expressed as part of the M13 phage vector particle in ELISA and dot blot formats. Our efforts in the past quarter have built on that work, and now we are using the scFv alone in tissue print assays of citrus plants to detect ‘Ca. Liberibacter asiaticus’. scFv are expressed and purified from from E. coli cells using a 6X His tag incorporated in the scFv protein. We have produced purified scFv at concentrations in the mg/ml range. Differences are observed among different scFv clones. Results from SDS-PAGE gels are consistent with post translational folding being problematic for some scFv as has been reported in the literature. The tissue print assays continue on nitrocellulose membranes. Color development is observed in the vascular cylinder (phloem) of HLB infected petioles but not in comparable petioles from healthy trees. In some tissue prints, color development is observed in discrete spots outside of the phloem cylinder. Similar results are obtained with all scFv that were selected to bind to proteins expressed on the surface of ‘Ca. Liberibacter asiaticus’. These targets include an ATPase associated with the type IV pilus, a pilus assembly protein, two flagellar proteins, the major outer membrane protein OmpA, and the efflux protein TolC. These results have been attributed to non specific cross reactions of the commercial monoclonal antibody directed at the 6X His TAG. The His tag will not be very useful for tissue printing. In continuing work, we have recently made tissue prints with a modified technique, using ‘Super block’, a commercial product used in Northern and Southern blotting on nitrocellulose membranes and detection with a monoclonal antibody directed at the FLAG epitope on the scFv. This protocol produces remarkably sharper tissue prints with dramatically reduced background, and color tightly focused as a ring in the phloem cylinder of HLB infected, but not healthy petioles. These results vary by manufacturer of the monoclonal anti-FLAG antibody conjugate. We have identified the best supplier. We have also prepared rabbit polyclonal antibodies against the major outer membrane protein (OmpA) and detected them with alkaline phosphatase labeled goat anti rabbit monoclonal antibody. These polyclonal rabbit antibodies are very useful in a tissue print detection format. The tissue print assay also preserves the anatomical distribution of CaLas in plant tissues. The sensitivity of the assay is comparable to qPCR but interestingly is more successful for detection of CaLas in asymptomatic plant tissues than is qPCR. This is because although the concentration of CaLas may be locally high in individual phloem cells, the overall concentration of CaLas in tissues can be low in these presymptomatic leaves. The ease and cost of the dot blot assay is also much less than qPCR. We have used the assay to follow the distribution of CaLas in infected trees. The assay shows the distribution of CaLas in roots, stems, leaves, peduncles and seed of citrus samples collected from HLB affected groves in Florida. The results of the tissue print assays have been correlated with results from qPCR.
Sept 18,2014 The objectives of this proposal are 1) to conduct a statewide survey of tangerine and tangerine hybrid groves to determine the proportion of strobilurin resistant Alternaria alternata isolates along with the identification and characterization of resistance-causing mutations; 2) establish the baseline sensitivity of Alternaria alternata to the SDHI class fungicide, boscalid and characterize field or laboratory SDHI resistant mutants to determine the likelihood of SDHI resistance development in Florida tangerine production and 3) Develop an accurate and rapid assay to evaluate sensitivity to DMI fungicides. During this quarter we accomplished: ‘ Boscalid paper (revisions completed and final acceptance in Plant Disease) ‘ Finished isolate screening for DMI baseline project
June 4,2014 The objectives of this proposal are 1) to conduct a statewide survey of tangerine and tangerine hybrid groves to determine the proportion of strobilurin resistant Alternaria alternata isolates along with the identification and characterization of resistance-causing mutations; 2) establish the baseline sensitivity of Alternaria alternata to the SDHI class fungicide, boscalid and characterize field or laboratory SDHI resistant mutants to determine the likelihood of SDHI resistance development in Florida tangerine production and 3) Develop an accurate and rapid assay to evaluate sensitivity to DMI fungicides. During this quarter we accomplished: ‘ QoI fitness paper (accepted in Plant Disease) ‘ Boscalid paper (submitted to and accepted in Plant Disease) ‘ Assay for DMI screening developed and isolates are being screen for baseline assay.
This is a project to continue one of the most fruitful leads that accidentally resulted from our previously funded work. We have found that citrus becomes a source of Huanglongbing (HLB) inoculum for spreading the disease to other plants much earlier than previously thought. The working hypothesis is that the female psyllid finds an area of new flush to lay her eggs. As she is laying eggs, she probes the phloem to feed and transfers Candidatus Liberibacter asiaticus (Las) to the tree. As the eggs develop into nymphs, Las begins to multiply in that localized area of the plant, where the new nymphs then feed and acquire Las. Thus, infection of only a micro area of flush tissue where the nymphs develop is sufficient for the first generation of psyllids to become infected and to be vectors to spread the disease to other trees. Thus, the time-period after a tree becomes infested by infected psyllids until it is a donor for other trees could be as short as 15-30 days or less. The limitation is actually the time for the second generation of psyllids to develop. We are working with a group in the Math Department of UF to develop a model of spread of HLB in new planting of citrus. A manuscript is being prepared reporting these results. We are using this rapid screen to determine whether a peptide can inhibit Las multiplication within 60 days instead of approximately one year. However, this rapid screen measures resistance, but not tolerance. We are still screening using infected psyllids to inoculate plants, but this information allows us to know which plants are inoculated with Las and is greatly improving those assays also. This work is continuing as described above. Our major emphasis is to use this system to screen for the effect of specific RNAi constructs against psyllids. Preliminary results suggest that it is possible that RNAi can reduce the number of psyllids produced and/or reduce the number of psyllids that become infected with Las.
Continuation of diagnostic service for growers for detection of Huanglongbing in citrus and psyllids to aid in management decisions, March 2014 The lab has been in operation for more than 7 years, and as of March 2014, we have processed nearly 623,000 grower samples. Additionally, more than 78,000 samples have been received for research for the entire period of diagnostic service supported by grant funding of individual researchers. Numbers specific to this report are 1,000 samples received from growers. This number represents an increase from previous years which was expected since disease incidence of HLB is near100% 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.
The goal of this project is to develop a method for efficient inoculation of plants with HLB using a Pulse Micro Dose Injection System (PMDIS). We have several sets of plants that we have injected with the HLB bacterium-containing extracts using different conditions. In these inoculations we tested different types of tissue (stems, leaves, seed coats within an infected citrus plant or HLB-infected psyllids) that can serve as resources of the HLB bacteria for preparation of the inoculum, different composition of extraction buffers for preparation of the bacterial suspension and different parameters of injection. We are also evaluating how age of receptor plants, types of citrus varieties used as HLB bacterium donors as well as types of flushes being inoculated affect efficiency of inoculation. Plants that have been injected are now being tested by PCR with HLB bacterium-specific primers to evaluate what proportion of plants became infected. We have done a large number of inoculations in which we test different conditions. Those plants are being maintained in the greenhouse and monitored for the disease development. Some successful infections of citrus plants using PMDIS were achieved, however infection rates were less than those seen upon graft-inoculation of plants with HLB-containing tissue. Currently we are working on improvement of PMDIS-based inoculation procedure. We included plants of many species into our experiments: citrus, tobacco, periwinkle, papaya. In addition, we are also testing the ability of Ca. L. asiaticus (as well as Liberibacter crescens that we are also using in our inoculations as discussed below) to survive in planta after injection. Dr. Carlos F. Gonzalez, Professor at the Center for Phage Technology, Faculty of Genetics, Department of Plant Pathology and Microbiology,Texas A&M University uses a similar injection system for injection of bacteriophage into grape vines as a part of phage therapy for control of Pierce’s disease. Craig Davis, a scientist from my lab who is involved in this project, visited Gonzalez’s lab and participated in injection system-based inoculations in order to learn the protocol that Dr. Gonzalez’s lab uses. Craig Davis brought back some new nozzles that Dr. Gonzalez uses with his injection device. We are testing those nozzles to see if they would increase the efficiency of our inoculation procedure. Now we are looking to see if we can improve our method by doing some modifications. We also try our injection system with Liberibacter crescens culture. We obtained a USDA-APHIS permit for working with this bacterium. Liberibacter crescens culture was received from Dr. Triplett’s lab and has been to inject several different plant hosts. We also have been assisting Dr. Dean Gabriel with injections of his L. crescens culture into citrus plants using our device.
The goal of this project is to develop a method for rapid and efficient inoculation of plants with HLB using a Pulse Micro Dose Injection System. We are evaluating several sets of plants that we have injected with the HLB bacterium-containing extracts using different conditions. Dr. Carlos F. Gonzalez, Professor at the Center for Phage Technology, Faculty of Genetics, Department of Plant Pathology and Microbiology,Texas A&M University uses a similar injection system for injection of bacteriophage into grape vines as a part of phage therapy for control of Pierce’s disease. Craig Davis, a scientist from my lab who is involved in this project, visited Gonzalez’s lab and participated in injection system-based inoculations in order to learn the protocol that Dr. Gonzalez’s lab uses. Craig Davis brought back some new nozzles that Dr. Gonzalez uses with his injection device. We are testing those nozzles to see if they would increase the efficiency of our inoculation procedure. Now we are looking to see if we can improve our method by doing some modifications. We also try our injection system with Liberibacter crescens culture. We obtained a USDA-APHIS permit for working with this bacterium. Liberibacter crescens culture was received from Dr. Triplett’s lab and has been to inject several different plant hosts. We also have been assisting Dr. Dean Gabriel with injections of his L. crescens culture into citrus plants using our device
Copper continues to be used as antibacterial/antifungal agent for citrus crop protection due to its effectiveness and affordability. However, prolonged and aggressive use of copper compounds has raised concerns due to copper accumulation in agricultural soil and development of bacterial resistance. This demands a search for suitable Cu alternatives. The goal is to develop Quaternary ammonium compound (Quat) based bactericide as an alternative to Cu compounds. Quat exhibits strong antimicrobial properties. However, direct foliar spray of Quat to plant is not practical as it causes severe phytotoxicity. Moreover, Quat is highly soluble in water, therefore exhibits poor rainfastness. This project is focused on developing Fixed-Quat formulations which will overcome phytotoxicity and rainfastness issues of Quat material without compromising its strong antimicrobial properties. A series of Fixed-Quat materials are being developed using three different EPA approved Quat materials. In this reporting period, two most promising Fixed-Quat nanogel and a Fixed-Quat nanoparticle formulations have been synthesized which remained stable for over two months. Scanning Electron Microscopy (SEM) characterization of Fixed-Quat nanogel material has revealed the formation of highly polydispersed submicron to micron size silica-gel particles. Phytotoxicity studies have been carried out on Vinca sp. (known to be highly susceptible to phytotoxicity and often used in agricultural industry as model test plants) to observe any potential plant tissue damage from Fixed-Quat materials. Phytotoxicity results (observed for 72 hours) have shown zero plant tissue damage when applied at rates as high as 900 ppm. Antibacterial studies have been conducted using in-vitro microplate alamar blue assay, bacterial viability expressed as colony forming units (CFU/mL) and growth curves using gram-negative model bacterial system, E coli. Optimized versions of these formulations showed complete bactericidal efficacy at concentrations as low as ~ 2ppm over our previously reported ~8ppm concentration. One of the Fixed-Quat nanogel materials (containing all EPA approved ingredients) has been delivered for 2014 field trials. Field efficacy will be evaluated at two different foliar spray rates.
Citrus huanglongbing (HLB) is associated with three species of Candidatus Liberibacter: Ca. Liberibacter asiaticus (Las), Ca. L. americanus (Lam), and Ca. L. africanus (Laf). The majority of the testing in Florida is focused on detection of Las as this is the only bacterium known to be associated with HLB in Florida to date, while Lam and Las have both been found in Texas. In March 2013, twelve different isolates from citrus and citrus relatives identified as being naturally infected with Ca. Liberibacter species but which would test negative for Las, Lam, and Laf, were inoculated into receptor plants in a greenhouse at Ft. Pierce. From the twelve isolates which were inoculated into receptor plants in the greenhouse, nine isolates have been established. The isolates which were not recovered came from citrus relatives that are not highly graft compatible with citrus. The nine isolates which have been recovered and established have been grafted into plants for the cross protection trial. DNA extracts from these nine isolates are being sequenced using miSEQ and PacBio approaches.
The goal of this project is to generate antibodies of selected secreted proteins from Ca. Liberibacter and use them as detection markers for HLB. This method is more reliable than examining host (i.e. citrus) changes (including DNA, RNA and protein) in the infected plants, which may not be specific for HLB. The basic idea is to generate antibodies targeting selected proteins secreted from Ca. Liberibacter asiaticus (CLas), which are relatively independent on live pathogen cells and can spread out systemically in the infected trees through phloem flow. Using these secreted proteins as detection markers, we have a better chance to detect Ca. Liberibacter in a direct and highly specific manner by overcoming the large variability in titer and distribution of the pathogen within citrus trees, types of tissue tested and degrees of disease progression. We have made significant progress and obtained exciting results. We identified CLas secreted proteins that are highly expressed in citrus, generated antibodies against four unique CLas secreted proteins, and developed detection assays using direct tissue imprints. Our results suggested that these antibodies were able to specifically detect HLB from infected citrus trees. We are now in the process of extensively evaluating these four antibodies using HLB-infected field trees from California and Florida. We will also monitor the presence of movement of the secreted proteins using graft-inoculated citrus seedlings generated at the Contained Research Facility at the University of California Davis. Our results will be compared to other detection methods targeting plant metabolites and CLas DNA. This method holds promise for allowing early detection on newly infected trees and large-scale field surveys.
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