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.
All 4 goals were completed. These were: 1) cloning of previously identified early/late gene promoter regions fused with lacZ as a reporter in E. coli; 2) cloning and expression of both Las and the Lam repressors and determining responsiveness of the lacZ reporter constructs in E. coli, and 3) cloning and expression of all 4 Las and the one possible Lam anti-repressors, and determining responsiveness of the reporter and clones from Milestone 2. The primary goal was Goal 4: development of a assay for Las phage late gene expression. We used quantitative, real time PCR (qRT-PCR) assays to determine levels of expression of the Las SC1 late genes in both citrus and periwinkle. In an effort to functionally confirm that those genes annotated as being lytic cycle genes were in fact lytic cycle (late) genes, we functionally characterized one of them, SC1_gp110, annotated as a holin). Heterologous expression of SC1_gp110 in E. coli revealed bacteriostatic activity and functionally confirmed the annotation of the expressed, predicted protein product as a holin. We used RT-qPCR to monitor the expression of this holin and 4 other putative phage lytic cycle genes in Las- infected periwinkle and citrus. Surprisingly, holin mRNA levels increased significantly in citrus 12 to 24 hours after leaves were detached, indicating a potential lysis triggering mechanism in citrus. In citrus, expression was monitored before and after heat treatment at 42’C for two days, a level demonstrated to cure plants of Las infection. The relative expression of four of the five putative lytic cycle genes examined, SC1_gp025, SC1_gp110, SC2_gp095, and SC2_gp100 were much more highly expressed in periwinkle than citrus. However, heat treatment of citrus leaves failed to increase expression of any of these genes, indicating that lytic cycle induction of these prophages is not tied into a presumed heat stress response of Las in citrus. The possibility of activation by biochemical inducer(s) specific to periwinkles is currently being examined. Conclusions from this year’s funding are: 1) Functional holin activity of the predicted SC1_gp110 gene confirms the annotation. 2) Expression analyses revealed much higher levels of expression of phage late genes, particularly the holin, in periwinkle as compared to citrus, possibly indicating a phage response specific to periwinkle. 3) The significant increase in holin mRNA levels in citrus, 12 to 24 hours after being detached, regardless of temperature, indicates a potential lysis triggering signals in citrus. The holin promoter region is being developed into a reporter gene construct that may be useful for monitoring lytic cycle activation by potential inducers in citrus. 4) The mode of action of thermal therapy (heat curing of Las) in infected citrus does not seem to be connected to phage induction.
Liberibacter crescens BT-1 (Lcr, isolated from mountain papaya), has been cultured and sequenced (GenBank CP003789, Leonard et al., 2012) and has the potential to be developed into a model Liberibacter system. for functional analyses of Las, Lam and Lso genes. To become a model system, BT-1 needs to be 1) reliably and stably transformed with a shuttle vector so that different genes can be added in; 2) gene knockouts in BT-1 must be reliably made, and 3) the strain needs to be introduced into a plant and be shown capable of growth in planta. The first two of these 3 goals have already been met. To determine if BT-1 might be tractable for functional genomics studies, the minimum inhibitory concentrations (MICs) of several antibiotics commonly used for plasmid selection were determined. BT-1 was found to be quite sensitive to: chloramphenicol, < 4 mg L-1; gentamycin, < 1 mg L-1; kanamycin, < 2.5 mg L-1; and tetracycline, < 0.3 mg L-1. BT-1 cells were readily transformed at high frequencies with two different wide host range replicons tested: pUFR071 (RepW; Al-Saadi et al., 2007) and pUFJ05 (Bordatella replicon; Reddy et al., 2007). The transformation frequencies were estimated to be 3000 transformants / mg of DNA for pUFJ05 and ~ 20. greater for pUFR071. Stability of pUFR071 was evaluated; this plasmid was >95% stable, without selection, when grown in BM7 medium for over 20 generations. pUFR071 was extracted from BT-1, retransformed into E. coli and appeared from restriction analysis to be unchanged. BT-1 cells transformed with pUFR071 showed only a marginal loss of plasmid, even when grown for more than 70 generations in the absence of antibiotic selection. The results are in line with the expected stability of RepW origin in the presence of stabilizing partition locus parA (De Feyter et al. 1990). A gene knockout in BT-1 was successfully demonstrated, following the strategy outlined in Casta’eda et al. (2005). Briefly, a partial internal fragment of the BT-1 gene encoding the Type I restriction-modification system restriction subunit R (GenBank B488_07050) was PCR-amplified and cloned into TOPO’ 2.1 (Invitrogen, Carlsbad, CA). The resulting plasmid was used for electroporation of BT-1 cells. A single homologous recombination event at the target site duplicated the cloned region and integrated the vector between incomplete copies of the target gene. The antibiotic resistant colonies were analyzed by PCR to confirm interruption of the target gene and integration of the plasmid backbone. Attempts to artificially inoculate marked BT-1 strains into tobacco, citrus and periwinkle are currently in progress.
The main objective of this proposal is the down regulatin of genes of phloem specific Callose synthase andd phloem proteins involved in phloem plugging in citrus infected citrus greening pathogen. Differential gene expression following infection of citrus by Candidatus Liberibacter asiaticus (CLas) bacterium has been documented by microarray and deep transcriptome profiling. CLas infection elicits over-accumulation of callose, and phloem-specific lectin PP2-like protein in the phloem of infected plants, in addition to number of transcripts of starch and sucrose metabolism, hormone biosynthesis, transcription factors etc. Over-accumulation of callose and PP-2-like proteins presumably causes phloem-plugging and consequent impairment in photoassimilate transport and excessive starch accumulation in leaf chloroplasts which results in disease manifestation. Down-regulation of over-expressed genes responsible for phloem-plugging would potentially negate the CLas disease manifestation. Towards this end, we have identified Callose7 as phloem specific callose among many calloses in plants based on their homology to Arabidopsis thaliana. At the same time we have also identified phloem protein B8 and B14 as phloem specific. These have been amplified from the total RNA isolated from healthy citrus tissues by reverse transcription and have been cloned into pGEMT easy vector which will be eventually engineered into the Citrus trizteza virus vector as outlined in the proposal and the details will be presented as they become available in the quarterly report. I am in the process of identifying a suitable post-doctoral candidate to continue with this research.
Understanding foamy bark rot of Fukumoto navel: Preliminary results support the hypothesis that foamy bark rot is initiated by scion-rootstock incompatibility, resulting in nutritional and/or physiological abnormalities, which provide opportunity for infection by certain pathogen(s) and foam production is possibly due to yeast activities. Inoculations with fungi (Fusarium solani, Fusarium sp. and Neofusicoccum mediterraneum) obtained from declining Fukumoto show that most of these fungi are pathogenic on Fukumoto. Bacterial inoculations produced no obvious symptoms on Fukumoto. Analysis of soil samples indicated no significant correlation between foamy bark rot and pH, total N, C, Olsen-P, K, Na, Ca, Mg, and lime. Results from in vitro fungicide screens have identified a number of products currently available that have the potential for use as pruning protectants. Results from this study have been published and can be found at http://www.citrusresearch.org/nov-dec_citrograph Multiple Botryosphaeria species causing ‘dothiorella’ gummosis in citrus: In this survey across six counties, 11 different species of Botryosphaeriaceae, comprising five genera (Diplodia, Dothiorella, Lasiodiplodia, Neofusicoccum and Neoscytalidium), were found associated with cankers of multiple citrus varieties and different ages. All isolates tested were found pathogenic on citrus and further tests on one-year old citrus plants showed the ability of most isolates to produce symptoms of gumming within one to two weeks post inoculation. Results from an in vitro fungicide screen show that a number of commercially available products currently on the market have promise for use as pruning protectants. Results from this study have been published and can be found at http://apsjournals.apsnet.org/doi/pdf/10.1094/PDIS-05-13-0492-RE. The alliance between Fusarium solani and other factors in citrus dry root rot: Results from this study indicate that in addition to F. solani and two species of Phytophthora (P. citrophthora P. nicotiana), F. oxysporum and a new Fusarium sp. are major organisms associated with DRR. Although there was no correlation between season and isolation of Fusarium spp., there was correlation between seasons and the isolation of Phytophthora spp. No correlation between nutrient content and DRR was found. A rootstock susceptibility screen is underway to determine the tolerances of 33 rootstocks to infection by F. solani as well as other Fusaria identified from this study. Results from this study have been published and can be found at http://www.citrusresearch.org/nov-dec_citrograph Investigating Branch Canker and Dieback in Lemon and Grapefruit in the California Desert Region: Surveys conducted across Riverside, Imperial and San Diego counties in Southern California show the presence of Neoscytalidium dimidiatum, Eutypella citricola, E. microtheca and Eutypella sp. associated with citrus branch canker and dieback. Pathogenicity tests on detached shoots show all three species of Eutypella are pathogenic on citrus, however these fungi appear to have a low to moderate virulence. Preliminary results from an in vitro fungicide screen show there are some products currently available that have the potential for use as pruning protectants. Results from this study have been published and can be found at http://www.citrusresearch.org/current-citrograph
The major aim of this research was to develop a method for in vitro culture of ‘Candidatus Liberibacter asiaticus’ (LAS). Specifically, it was hypothesized that microfluidic chambers, by mimicking plant phloem vessels to which LAS is restricted, could be used to facilitate LAS culture. However, liquid culture medium optimization was required prior to successful study of LAS in microfluidic chambers. Therefore, research was focused on systematically quantifying LAS viability over time in different culture media to identify the characteristics of a suitable culture medium for LAS. In the first year of the project, experiments were conducted to compare LAS viability characteristics among different media types over time via EMA-qPCR. In the second year of this project, all data was analyzed from the first year’s experiments. Additional experiments were conducted to analyze the bacterial community composition of the biofilm formed in the media treatments as well as the chemical characteristics of the media tested. The overall finding was that LAS culture media containing citrus juice from different sources promoted cell viability. A peer-reviewed publication of these results has been produced: ‘Viability of ‘Candidatus Liberibacter asiaticus’ prolonged by addition of citrus juice to culture medium’ (2013, Phytopathology). In this publication, we indicate many features of our experiments that will be informative in future design of a successful culture medium for LAS. This publication represents the first significant published contribution to finding a solution for the culture of LAS since the publications from Davis et al. (2008) and Sechler et al. (2009). Future work will focus on refining this liquid medium and testing potential new media formulations both in batch culture as well as in flow conditions in microfluidic chambers.
The main goal of this project is developing of a new method for rapid and efficient inoculation of plants with HLB based on a Pulse Micro Dose Injection System (PMDIS). We are conducting experiments in order to 1) identify what types of tissue within an infected citrus plant can serve as a good resource of the HLB bacteria for preparation of the inoculum by comparing extracts from stems, leaves and seed coats as inoculum sources; 2) examine whether HLB-infected psyllids can be utilized for preparation of the inoculum suspension; 3) 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; 4) 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 for plants being inoculated, types of flushes being inoculated affect efficiency of inoculation. Several sets of plants have been already injected using PMDIS. Those 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 are trying to improve our project by using two strategies. First, 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 have obtained a culture of Liberibacter crescens and are using this culture as a model system to develop and improve our injection protocol. The culture is being used for pulse injection-mediated inoculation of both citrus and papaya (the original host of L. crescens, here is used as a model host).
The objective of this project is to provide an alternative to traditional Cu fungicide/bactericide for protection against citrus canker. Extensive Cu use for plant protection has led to Cu accumulation in fertile soil. It is desirable to develop an alternative to Cu fungicide/bactericide for preventing citrus canker. In this project a new non-phytotoxic quaternary ammonium compounds based nanoformulations (Fixed-Quat) is being developed for canker protection. We have successfully synthesized a few batches of Fixed-Quat formulas using EPA approved quaternary ammonium compounds. Material characterization using HRTEM, SEM, DLS, FTIR etc. is in progress. Antimicrobial efficacy, phytotoxicity and film forming ability of Fixed-Quat formula is being evaluated on Hamlin orange, grapefruit citrus varieties as well as on vinca ornamental species. Future reports will include results from materials characterization results, antimicrobial studies, film-forming capacity and phytotoxicity.
The objective of this project is to improve the bioavailability of copper loaded silica nanogel (CuSiNG) material. Further studies of mixed-valence Cu within a CuSiNG system have been performed. Two different mixed-valence Cu systems have been created using different reducing agents to lower the oxidation state of Cu within the Copper silica system. Increases in Cu (I) content have been confirmed using XPS, HRTEM and UV-Vis with the Cu(I) specific chelator neocuproine. HRTEM revealed the presence of cupric oxide, cuprous oxide and metallic Cu crystallites within the mixed valence Cu system. Antimicrobial tests have shown an increase in efficacy over previous CuSiNG materials, along with Kocide 3000, Cu-sulfate and Cu(I)-chloride controls. The presence and action of hydroxyl radicals in the mechanism of action of CuSiNG was indirectly studied using the hydroxyl radical scavengers D-Mannitol and Dimethylthiourea. Phytotoxicity testing was carried out on Hamlin Orange and Vinca sp. Results indicated that Hamlin Orange was resistant to phytotoxicity at all levels tested while Vinca sp was susceptible to plant tissue damage at higher Cu level. No damage was observed at levels used for canker protection in field trials.
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. We are currently testing these inoculated plants to verify the establishment of the Ca. Liberibacter species in the receptor plants. Once this has been verified, the ‘cross protection’ trials will be established. DNA extractions from the isolates has been used for macro array qPCR to identify genomic regions which differ from Las, and selected isolates are being prepared for further sequencing to verify their relationship with Las and Lam and other reported Ca. Liberibacter species.
Based on the PCR results, five classes were defined for the Valencia variety dataset. They included 20 healthy, 20 HLB symptomatic, 20 magnesium (Mg) deficient, 6 zinc (Zn) deficient, and 30 zinc deficient and HLB symptomatic. As explained in the previous report, 30 textural features were extracted from the images. A principal component analysis (PCA) was performed on all 30 features to have a visual characteristic of the samples. The samples were plotted in a two dimensional coordinate system of the first and second principal components (PCs) and a step by step classification model was designed based on this plot. Based on the model, all the samples were classified into two classes of Healthy-Mg deficient class and HLB-Zn deficient class in the first step. Then, the healthy and Mg deficient classes were individually recognized from the Healthy-Mg deficient class. Also, the HLB and Zn deficient classes were identified individually, and the HLB infection within the Zn deficient class was identified as the last step of the model. The best set of features and classifiers was identified for each step by comparing the results achieved using different sets and a 5-fold cross validation model was employed to confirm the results. A programming code for the classification model was prepared and all the dataset was divided randomly into two equal sets. Then the classification model was performed on the first half, while the second half was used as a training set. The classification model was repeated on the second half, and this time the first half was used as a training set. Based on the classification results, all the healthy and HLB symptomatic samples were classified correctly with zero error. Also most of the magnesium deficient samples were classified correctly with a 95% accuracy and only one magnesium deficient sample was misclassified in the HLB symptomatic class. However, the classification accuracies within the zinc deficient class were comparatively lower than other classes because either the zinc deficiency symptoms buried the HLB symptoms or their HLB statuses were questionable. A conference paper was prepared based on these results and submitted for the 2013 ASABE international meeting. The paper was selected from sixty submissions to receive the IET Select Paper Award. An imaging system for the on-the-go HLB detection method was assembled and tested successfully in the lab. A DGPS receiver was added to the system to acquire coordinates of where the images were taken. Software was developed using LabView programing to control the camera, take the images, and log the coordinates. The on-the-go imaging system was tested on July 2nd, 2013 in CREC grove after sunset (9:00 PM to 2:00 AM). A total of 486 images from both sides of 243 trees in two blocks (10A and 2C) were captured and their coordinates were logged. The HLB statuses of these blocks were updated in April, 2013 by a ground inspection crew in CREC who has monitored the grove continually. The imaging system was mounted at the height of 1.6 m on a gator utility vehicle. The vehicle was stopped in front of each tree, the illumination was turned on, tree images and coordinate were stored, and the illumination was turned off. Now the dataset is being analyzed to determine its capability to identify the HLB infection.
Continuation of diagnostic service for growers for detection of Huanglongbing in citrus and psyllids to aid in management decisions, March 2013. The lab has been in operation for more than 5 years, and as of March 2012, we have processed more than 40,000 grower samples. Additionally, more than 30,000 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 941 samples received from growers. This number represents a decline from previous years which was expected since incidence of HLB is nearly 100% in southwest Florida citrus. However, it is also representative in that less samples have been historically received during this quarter because the reduction in grove scouting and decline in HLB appropriate field samples during the summer. Typically, there are more samples processed December through June. The HLB Diagnostic Lab webpage was updated to announce the service of detection of CLas in psyllids as funded in this grant.
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