Copper (Cu) compounds are extensively used as antibacterial/antifungal agents for controlling and prevention of citrus canker and other citrus diseases such as scab and melanose. Most commercially available Cu compounds are water-insoluble (such as copper hydroxide and copper oxychloride) and upon spray application they form films on plant surface (film-forming). Bioavailability of film forming Cu compounds is limited and therefore multiple spray applications (8-10 per season) are needed to achieve good protection. Cu bioavailability is high in water-soluble Cu products (such as Magna-Bon). However, these products have limitations as they cause plant tissue injury (phytotoxity) and exhibit poor rain-fastness. To achieve improved Cu bioavailability and enhanced rain-fastness, we have successfully developed copper loaded silica nanogel (CuSiNG) – a copper-silica composite gel material. In this composite material, Cu is loaded in silica nanogel in two forms, crystalline and amorphous (chelated with silica) as characterized by High-resolution transmission electron microscopy. Moreover, Cu is present in mixed-valence states (+1 and +2 oxidation states) as confirmed by the X-ray photoelectron spectroscopy. Fluorescence based studies indicate that high surface area of silica nanogel indeed improved its retention properties to plant surface. Systematic antibacterial studies of CuSiNG material in two different pH conditions (pH 4 and pH 7) were conducted against E. coli and X. alfalfae (a canker surrogate). Antimicrobial assays including turbidity, growth inhibition assay, MIC, live/dead assays were performed for CuSiNG materials using appropriate controls (Kocide 3000, copper sulfate and silica nanogel). Antimicrobial study results showed improved efficacy of CuSiNG material over Kocide 3000 (insoluble copper) and copper sulfate (soluble copper) at the same metallic Cu concentration. Improved antimicrobial efficacy is attributed to increase in Cu bioavailability and the mixed Cu valence states. Field trials were conducted on 5 yr-old ‘Ray Ruby’ grapefruit trees for three consecutive years, 2010, 2011 and 2012 at Vero Beach, FL (collaborator, James H. Graham, UF-CREC) using two most promising CuSiNG nanoformulations for every year. Several commercially available Cu products (such as Kocide 3000, Nordox 75G, Magna-Bon and Badge X2) were included in the trial as controls. Effect of copper formulations on incidence of canker-infected fruit with old lesions, young lesions and total incidence of lesions were evaluated. In addition, effect of copper formulations on total incidence of copper phytotoxicity, melanose or scab was evaluated on Grapefruit and Hamlin Orange varieties. Two spray rates, 3 lb/acre for CuSiNG pH 7.0 (matched with Kocide 3000 rate) and 0.5 lb/acre for CuSiNG pH 4.0 (matched with Magna-Bon rate) were used. CuSiNG material at both pH 7 and pH 4 conditions did not show any sign of phytotoxiticy at the Kocide 3000 commercial spray rate . Overall protection efficacy of CuSiNG materials against canker was comparable with most commercial Cu products. Interestingly, at Magna Bon spray rate, CuSiNG materials were effective which can be correlated to improved Cu bioavailability. Though only a marginal improvement in canker protection was observed over Kocide 3000, CuSiNG formulations clearly showed superior rain-fastness with no sign of phytotoxicity when compared to Magna Bon. Furthermore, CuSiNG formulations showed comparable efficacy against scab and melanose when compared to commercial products. Research findings have been disseminated through peer-reviewed publications (J. Biomed. Nanotechnol. 2012, 8(4), 558-566; Conference Proceedings/Book chapter in Nanostructured Materials and Nanotechnology VI, John Wiley & Sons, Inc.: pp 55-67, 2012), patents# WO2011126832A3 and several oral (4) and poster (2) presentations. Three relevant manuscripts are under preparation for peer-reviewed publications.
To date, we have tested more than 848 culture medium formulations for their ability to support growth of Candidatus Liberibacter asiaticus. Several formulations support initial increases in population size of the bacterium; however, none have supported sustained growth when fresh medium is added to the culture. Attempts to determine the appropriate carbon source have indicated that alpha-ketoglutarate, malic acid, and fructose have positive effects on growth. Supplementing the medium with biotin, thiamine, choline, and mixtures of essential and non-esential amino acids also appears to have a positive effect on growth. Oxygen levels from normal atmospheric (ca. 20%) to 15, 10, 5, and zero percent have been tested with 10% appearing to be optimal. Attempts are continuing to find the culture conditions that support sustained growth of the bacterium.
In this project we are working on a method for rapid and efficient inoculation of plants with HLB using a Pulse Micro Dose Injection System (PMDIS). Several sets of experiments are being conducted in order to identify 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; to optimize the composition of the extraction buffer used for preparation of the bacterial suspension and the extraction conditions, so they would support high efficiency of the PMDIS-mediated transmission of the pathogen; to optimize the 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. Several sets of plants have been already injected using PMDIS. We are constantly increasing 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. Additionally, we are building a collaboration with Dr. Carlos F. Gonzalez, Professor at the Center for Phage Technology, Faculty of Genetics, Department of Plant Pathology and Microbiology,Texas A&M University. Dr. Gonzalez uses a similar injection system for injection of bacteriophage into grape vines as a part of phage therapy for control of Pierce’s disease. We would like to learn more about their injection system and the injection protocol, so we can transfer the obtained knowledge into our project on the development of a system to inoculate citrus with HLB. Second, we are including Liberibacter crescens as a model system to develop and improve our injection protocol.
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 plants 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. The plants have survived and show various symptoms. The inoculation of the ‘cross protection’ trial is planned to be done in the near future. Selected DNA samples have been sent away for sequencing. We are conducting experiments to develop more sensitive methods of detecting the Ca. Liberibacter species, including the species that are included in our collection. These experiments are on-going.
Aggressive use of Cu pesticides to control citrus cancer and other citrus diseases is a concern due to high level of Cu accumulation in fertile agricultural soil. The primary objective of this research project is to develop an alternative to Cu pesticides. Quaternary ammonium compounds (Quat) are potent antimicrobial agent, however it exhibits strong phytotoxicity. To overcome Quat phytotoxicity while maintaining its antimicrobial efficacy, a series of eight new Fixed-Quat particulate materials have been synthesized by varying the ratio of Quat to silica ingredients. In these Fixed-Quat materials, Quat was immobilized with silica matrix both covalently and electrostatically. Particle size characterization of core-shell Fixed-Quat particulate materials was done using dynamic light scattering and scanning electron microscopy. Particles were in the sub-micron size range (300 – 500 nm). Preliminary antimicrobial studies show significant efficacy against E. coli. Phytotoxicity results indeed demonstrated the role of silica in minimizing phyototoxicity of Quat. A batch of Fixed-Quat formulation has been delivered for field trial this year.
Using the on-the-go HLB detection imaging system, a dataset including the real tree images was created on July 2nd in CREC . The analysis on this dataset showed that the object depth in a frame had a big influence on the object intensity. Since the detection approach in this project is highly dependent on the intensity, the tree images without depth information could not help the detection process. Two lab experiments were conducted on July 19th and September 4th to study the effect of depth on the object intensity in a monochrome image. It was determined that regardless of the camera settings, there was a relationship between the object distance and the object histogram of gray scale pixel values. This relationship was formulated by two functions for the histogram mean and standard deviation. These functions enabled us to calibrate the objects with different distances in an image and remove the effect of depth on the system performance. Analyzing these two datasets showed that the HLB symptomatic areas on the citrus leaf were very obvious and highlighted in the images acquired with the new image acquisition system. A new 3D imaging approach for on-the-go HLB detection system was designed and proposed in which the depth information is measured when the monochrome image is captured, however unfortunately this proposal was not selected for funding extension. As well as the 3D imaging approach for on-the-go HLB detection system, two other proposals were designed and prepared based on the smartphone and tablet application. The other solution was to acquire tree images from the same distance using the current image acquisition system. Another dataset was created for this purpose on September 25th, 2013. This dataset included 20 healthy, 20 HLB, and 20 zinc deficient samples. The leaf images were acquired in three different positioning conditions and with a black panel as the background. The leaves samples were then attached to an artificial tree to evaluate performance of the system in a simulated condition. All these images were captured in three different distances of 60 cm, 80 cm, and 100 cm to determine the best imaging distance. The next step is to analyze this dataset to design and perform an experiment in a real citrus grove. The reviewers’ comments for the ASABE Transaction manuscript were received and the revisions were made. The results of the Valencia variety dataset including an additional class of manganese deficiency samples were also added to the manuscript.
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 currently 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. 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 third time and further analysis of those trees are being conducted. Metagenomic analysis of the sequenced samples is being conducted.
Through the end of the second quarter, SGDL has processed 24,141 samples since the beginning of the funding for this project. The break down of the samples is as follows: Growers 14,811, Research 3,156, Southern Gardens 1,269, and Psyllids 4935.
Through the end of the third quarter, SGDL has processed 35,478 samples since the beginning of the funding for this project. The break down of the samples is as follows: Growers 21,488, Research 5,942, Southern Gardens 1,797, and Psyllids 6,251.
For the period of time between October 2010 to December 2010, 6140 grower samples have been run, bringing the total for the period of funding under this project to 13,490. In addition, an additional 4,371 samples from Southern Gardens and 1128 psyllid samples have been processed bringing the total number of samples for the period to 21,950.
For the period July 1, 2011 through June 30, 2012 the SGDL processed 39,008 samples. The breakdown of the samples is as follows: Growers 22,352, Research 6,635, Southern Gardens 2454, and psyllid samples 7,567. This level of sample volume is slightly below the previous year. However, it is expected that the number of samples submitted to the laboratory will decline over the next few years. Included in the samples were numerous research samples from USDA, UF, and private research agencies. As HLB has become more established within the state, the trend has been for the submission of less HLB diagnostic samples and for more HLB research samples. This trend is expected to continue. During the period covered by this report, SGDL tested and validated a robotic DNA extraction procedure which has now become the standard extraction protocol in the laboratory. This has resulted in better quality DNA and a reduction in cost for the assay.
Without the ability to culture Candidatus Liberibacter asiaticus (CLas) in vitro, the pathogen can only be studied within the Asian citrus psyllid vector or in the citrus or other host plants. CLas DNA in citrus tissue can be detected with various highly sensitive and robust PCR protocols, however, these methods do not reveal if the DNA target is from living, and pathogenic cells, from dead cells, of from extracellular CLas DNA that may be excreted by the pathogen. Treatment of bacterial cells with DNA intercalating dyes prior to qPCR has promise for distinguishing between live and dead CLas cells in citrus tissues; however, because CLas resides in citrus phloem there are obstacles to this approach. The overall goal of this project is to extend previous findings regarding the use of DNA intercalating dyes and optimize them for quantification of live CLas cells in citrus. During months 1-4 of the project our objectives were to: 1) Determine suitability of PMA-qPCR for distinguishing between living and dead CLas cells in citrus; and 2) validate and compare results of PMA-qPCR with EMA-qPCR. We have made significant progress towards meeting each of these objectives. Specificity and efficacy of EMA- and PMA-qPCR were determined using both purified plasmid DNA containing the CLas DNA target sequence and E. coli cells transformed with the same plasmid. Results with this model system confirm that both EMA and PMA treatments are specific for the CLas target sequence. Amplification of plasmid DNA in qPCR was inhibited 100% by both EMA and PMA. Estimates of live cells using E. coli with EMA or PMA gave similar results of ca. 10% live cells. If cells are heat killed prior to dye treatment, amplification is inhibited 100% . In the course of these experiments we also optimized variables in the protocol to give greatest sensitivity in the assay and the widest working range. Preliminary experiments conducted with DNA extracted from seed coat vascular bundles that had been treated with EMA prior to qPCR protocol showed about 25% of the CLas copy number of that in DNA from non-treated seed coat vascular bundles. We compared results of EMA- and PMA-qPCR with citrus leaf samples. We used leaves that expressed a range of HLB symptoms for these experiments. Samples were collected both from the greenhouse and from the field. Estimates of the number of live CLas cells in leaves treated with EMA were typically less than those obtained using PMA. Over a range of total CLas titers, estimates of live cells averaged 15% based on EMA-qPCR and 50% based on PMA-qPCR. During months 4-8 of this project, based on results of experiments to optimize the EMA- PMA-qPCR protocol, we have conducted extensive experiments to distinguish between live and dead CLas cells in citrus seed coat vascular bundles, a tissue known to contain high titers of CLas, and in citrus leaves, and in Asian citrus psyllids (ACP). Results of these experiments revealed that homogenization method has significant impact on yield of live cells whereas results were not impacted by buffer concentration. Using combinations of filtration and density gradient centrifugation we have obtained enriched fractions of live CLas cell from both seed coat vascular bundles and ACP. Experiments have continued to estimate numbers of live CLas cells in citrus. Considerable variation can result in such experiments and we have begun to identify factors that may impact outcome and interpretation of experiments using DNA intercalating dyes.
Early detection using cost-effective surveillance techniques is crucial to successfully fighting the spread of HLB. The strategy of early detection of HLB focuses on the analysis of host VOC responses that are triggered early in the infection cycle as part of the plant innate immune responses. Based on previous CRB-funded effort, there is strong evidence that VOC analysis of citrus trees can lead to early detection of the HLB and other citrus diseases. VOC field testing is performed using EZKnowz’ instruments supplied by EZDiagnostix (EZDx), the sensor commercialization arm of Applied Nanotech, Inc. (ANI). The EZKnowz’ trace chemical analyzer uses a gas chromatograph (GC) combined with a differential ion mobility spectrometer (DMS). Our effort in this program is the following: ‘ Reduce sampling and analysis time from 10 minutes (currently) to < 1 minute: Several GC columns have been tested. We have achieved about 3 minute analysis time but we are still targeting 1 minute. ' Develop a VOC sampling method to collect VOCs from a significant portion of the tree. We have separated the VOC collection from the analysis, which will lead to reduction in time as collection and analysis can be done in parallel. A hand-held sniffer prototype is under development. ' Develop an algorithm for identification of HLB (Year 1) based on the modified tool: A bread-board instrument with the modifications described above is ready for characterization in the field. We are ready to begin developing a library based on the modified tool. ' Develop software to implement the disease detection algorithms: Analysis is intended to be directly on the device for rapid feedback. We have purchased a Trimble YUMA-2 which will be the platform on which analysis will be performed in the field. This will be the interface to the analyzer, the sniffer and the operator. The UC Davis team is developing the scripts that will perform the algorithm analysis in the field. We continue to test the VOC algorithm using standard devices in orchard fields in south Texas. We have completed 4 months of orchard testing, about 60 trees/month. We are expanding the libraries of citrus varieties beyond sweet orange. Expected Outcomes and/or functional product/solution The potential value of this early-detection solution on the citrus industry is tremendous. By 'flagging' infected trees at the asymptomatic stage, eradication would be both more effective, and kept to the minimum necessary, since it would take place well before other trees become infected and enter the latent period. This would interrupt the deadly infestation cycle at the source, significantly reduce the heavy costs of losing trees and citrus produce for a period of three to five years and cut down the costs of planting new trees.
Our results indicate that ‘Ca. Liberibacter asiaticus’ cells can be extracted and gradient purified from both plant tissues (citrus seed coats), but there still are significant difficulties to overcome. Two standard methods of tissue disruption, polytron homogenization and bead-beater based tissue pulverization, released bacteria from plant tissues but filtration and real-time PCR (qPCR) analysis of plant material trapped on filters (40, 20 and 10 micron) indicated significant levels of bacterial DNA (interpreted to mean bacterial cells) were associated with tissue fragments in excess of 10 micrometers, suggesting physical disruption was not adequate to release a large portion of bacteria from tissue. We adapted protocols to use Percoll gradients to concentrate bacteria released from disrupted tissues; after filtration through the 10 micrometer filter, cells were pelleted and concentrated by low speed centrifugation prior to application to the gradient. Real-time PCR analysis of the supernatant from the low speed centrifugation (which should be depleted of bacterial cells) still had significant amounts of bacterial DNA. This result indicated that a significant portion of bacterial DNA in these preparations is extracellular and that bacterial cell numbers estimated from qPCR data may be significant overestimates of the numbers of intact bacterial cells in tissue preparations. Our experimental design did not allow us to conclude if this extracellular DNA was a result of the physical disruption of bacterial cells in the tissue or if it is an aspect of bacterial colonization of the phloem sieve tubes, such as its being a component in a bacterial biofilm and is released upon tissue disintegration. We used qPCR to determine that fractionation of tissue extracts on Percoll gradients concentrated bacterial cells at a position equivalent to cultured E. coli cells. Regardless of the number of cells estimated by qPCR to be present in the tissue extract at the start of the purification, the estimated numbers of purified cells recovered from the gradients were disappointingly low, usually in the low thousands out of an estimated starting number as high as 30 million. Percoll fragments were washed from purified cells prior to qPCR, so it is possible more cells were concentrated in the Percoll fraction but were lost during the washing process. Evaluation of cells by Fluorescence In Situ Hybridization microscopy (FISH) showed rounded bodies of a size likely for bacteria, but not the more familiar bacilliform shapes recognized from electron micrographs. We analyzed fractions from the different purification steps for bacterial cell viability using ethidium monozaide (EMA) treatment prior to qPCR; EMA prevents amplification of DNA from bacterial cells with damaged membranes, i.e. non-viable cells. For unknown reasons, the estimated cell viability was ‘1-2% at nearly all stages of the purification, including the initial extract. The percentage of viable cells was consistently higher (5-6%) for the Percoll fractions, additional evidence that the Percoll gradient was concentrating bacterial cells. We will try to elevate this level by altering components of the isolation protocol.
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. We are using this system to screen RNAi constructs and different peptides against psyllids. Preliminary results are encouraging. We also are attempting to adapt the system into a method to screen peptides against HLB more quickly.
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