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.
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 40,500 grower samples. Additionally, more than 32,000 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 605 samples received from growers. This number represents a decline from previous years which was expected since incidence of HLB is nearly 100% in southwest Florida citrus. However, it is also representative in that less samples have been historically received during this quarter because the reduction in grove scouting and decline in HLB appropriate field samples during the summer. Typically, there are more samples processed December through June. The HLB Diagnostic Lab webpage was updated to announce the service of detection of CLas in psyllids as funded in this grant.
To date, we have tested more than 824 culture medium formulations for their ability to support growth of Candidatus Liberibacter asiaticus. Several formulations have been developed which support initial growth of the bacterium. This is indicated by an increase in the numbers of bacterial cells observed in samples of inoculated medium by epifluorescent microscopy after staining the cells with the DNA-binding fluorochrome SYTO 13. The population of cells reaches a peak after 1-2 weeks. After addition of fresh medium, the population usually diminishes until they no longer can be detected. However, occasionally the population peaks again and then declines to undetectable levels. Our efforts are now aimed at identifying those conditions that will support continuous cultivation of the bacterium.
Nutrient application for maintaining the productivity of HLB-infected orchards is becoming a common practice in Florida, especially with increasing rate of infection. Although the foliar nutritional application program does not remove/reduce the inoculum, it offers the growers an opportunity to maintain productivity and profitability. One of the major challenges in nutrient application is deciding on the sufficient amount or proportion of nutrients to be applied in the field to maintain citrus yield. In this project, we proposed to evalute the potential of a new sensing technology based on laser-induced breakdown spectroscopy (LIBS) for rapid and real-time measurment of foliar nutrient quantity. During the first stage of the project, citrus anomalies with abiotic (iron, zinc, manganese, magnesium) and biotic (HLB, canker) stressed leaf samples were tested with LIBS system. Qualitative analysis using a statistical algorithm (bagged decision tree) indicated that classification of citrus anomalies can be achieved with an accuracy of about 94%. Based on these results, in the second phase of the project, we evaluated the LIBS system for quantification of leaf nutrients. Two sets of data, dataset 1 and dataset 2, were collected during different seasons (fall and spring). The partial least square regression (PLS-R) model with a training dataset was developed to establish a calibration model. Once developed, the calibration model was tested with an independent dataset. In dataset 1, the PLS-R model showed a good correlation between the LIBS spectra and nutrient concentrations during calibration, although the model could not be validated with an independent dataset. However, in dataset 2, amongst the nutrients that were estimated, iron, magnesium, potassium, boron, and calcium showed R2 greater than 0.70. Calcium showed a maximum R2 of 0.80. One of the major challenges during this work was the low variability of foliar nutrient concentrations that limited the robustness of the calibration model. Better results could have been acheived if the concentration of nutrients had had a wider range of variability.
Early detection is critical to delaying and limiting the spread of HLB into California. While commercial growers are inspecting regularly for HLB, approximately half of the citrus trees in California are located in plantings other than commercial groves; many in the yards of homeowners, where regular and comprehensive inspection presents challenges. If HLB infection gains a foothold in non-commercial plantings it could spread widely before being detected in commercial groves. The objective of this task is to complete a proof-of-concept demonstration of the use of airborne surveys with the Airborne Visible and Infrared Imaging Spectrometer (AVIRIS), a high-performance sensor developed by NASA, to improve early detection of huanglongbing (HLB) by mapping the locations of citrus trees in urban and suburban areas as an aid to extension of effective survey coverage to include non-commercial plantings. Since the sensor observes from an aircraft, this approach could regularly and comprehensively survey wide areas to map non-commercial citrus and detect HLB-infected trees. The following milestones have been completed as of the date of this report: 1. A Space Act Agreement for this experiment was drafted, approved, negotiated, and signed by the National Aeronautics and Space Administration (NASA) and the California Citrus Research Board. 2. A Task Plan was written by JPL and approved by NASA. 3. A Task Order was issued by NASA for the Jet Propulsion Laboratory to begin work on the task. 4. A Flight Request was approved by NASA for the use of the AVIRIS Classic instrument in support of this task. 5. AVIRIS flights in support of this task have been tentatively scheduled for late July 2013. 6. The JPL team and CRB representatives met to discuss flight planning, particularly to define the areas of interest for airborne data collection and relative priorities among these areas. 7. Analysis has begun on archival data collected by an instrument similar to AVIRIS over an area that included the main Citrus Variety Collection (CVC) at UC Riverside.
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 continuing experiments to find ways to quickly detect psyllid reproduction as a method to detect early infections. We are developing this system into a method to screen RNAi constructs against psyllids. Preliminary results are encouraging. We also are attempting to adapt the system into a method to screen peptides against HLB more quickly.
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 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, lemon 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. Of the 50,000+ available operational taxonomic units (OTUs, “Species”) on PhyloChip’ G3, 7,028 known OTUs were present in citrus leaf midribs. These OTUs were from 58 phyla, and five, Proteobacteria (44.1%), Firmicutes (23.5%), Actinobacteria (12.4%), Bacteroidetes (6.6%) and Cyanobacteria (3.2%), contained 100 or more OTUs. The results illustrate that the low Las level was seen as a both a seasonal fluctuation, part of the bacterial population dynamics, and as a response to the antibiotic treatments, The results are going to be published in BMC Microbiology. Using Las-specific molecular markers, we are able to differentiate the Las populations with different pathogenicity, and differentiate psyllid-transmissible and non-transmissble populations.
The objective of this project was to characterize the hypI gene and determine its effects on insect transmission and/or virulence in host plants. Transient expression in alternative expression systems, RT-qPCR, and other molecular tools were used to elucidate the function of the hypI gene of Las. HypI has been redesignated as LasAI. A second prophage gene, hypII (now LasAII), was identified and both genes were characterized in global Las isolates. This revealed sequence conservation within the individual repeats but an extensive variation regarding repeat numbers, their rearrangement, and the sequences outside of repeat region. Detailed results have been published in Applied Environmental Microbiology, 2011: 6663-6673. We developed real-time PCR protocols using SYBR Green 1 (LJ900fr) and TaqMan’ (LJ900fpr) with primers and probe targeting nearly identical tandem repeats of 100 bp within the two Las prophage genes. Because of the higher copy number of the tandem repeats per bacterial genome, these methods significantly improved the detection capacity for the HLB bacterium, especially for the detection of extreme low titer. The results were published in Molecular and Cellular probes, doi:10.1016/j.mcp.2011.12.001. Working with Mesa Tech International, Inc., we have also developed a rapid and simple HLB detection kit (tree-side detection kit) for field-testing, which requires less than 30 minutes. 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. Using the LasAI-specific marker, we were able to associate better insect transmission and pathogenicity with wild-type LasAI, but variations with LasAI mutants. Using transient expression of the LasAI in tobacco leaf, the dissociation of the cell wall and cytoplasm in affected cells was observed under electron-microscope. Further characterization of the LasAI and LasAII are underway so as to have a better understanding of the pathogenesis of this intracellular ‘energy parasitic’ bacterium, and to control HLB by blocking the functions of the LasAI and LasAII.
We are developing a new method for rapid and efficient inoculation of plants with HLB based on a Pulse Micro Dose Injection System (PMDIS). 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. Our goal now is to optimize the protocol for PMDIS-based inoculation of citrus hosts with HLB. 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. During this funding period we added another direction. In collaboration with Dr. Dean Gabriel we are testing our inoculation approach using the culture of Liberibacter crescens, a bacterium that belongs to the same genus as HLB causing agent, Candidatus Liberibacter asiaticus. We believe that this would allow us to better make necessary improvements to the inoculation conditions and to assess whether citrus could be a host for this bacteria. Successful inoculation of Liberibacter crescens into citrus would suggest that this bacterium could be used as a model in various experiments to better understand the biology of HLB agent.
Citrus huanglongbing (HLB) is associated with three species of Candidatus Liberibacter: Ca. Liberibacter asiaticus (Las), Ca. L. americanus (Lam), and Ca. L. africanus. 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, although recently Lam has been reported in Texas detected only from psyllids, not plants. In October 2012, we conducted a wide survey of citrus and citrus relatives in the landscape and in collections in Central and South Florida. The DNA extracts were forwarded to Riverside and analyzed. Based on these results, 12 specific plants that had Ca. Liberibacter-like infections were identified and recollected in March 2013. These specific isolates were graft inoculated into four plants each in an isolated greenhouse at Ft. Pierce. One of these isolates originated from pummelo and has undergone treatment to eliminate Las. These plants will be tested in late May ‘ early June to determine if the HLB-like pathogen has been established, and the isolates that have become established will be utilized to establish trials to establish ‘cross protection’ trials. The DNA extracts collected from samples collected in March 2013 are being analyzed by the macro array qPCR.
Goals 1, 2 and 3 were recently completed. These were: 1) cloning of previously identified early/late gene promoter regions fused with lacZ as a reporter; 2) cloning and expression of both Las and the Lam repressors and determining responsiveness of the lacZ reporter constructs, 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. None of the genes annotated as repressors or anti-repressors in the Las or Lam genomes functioned in these assays as repressors or as activators. The primary goal was Goal 4: development of a chemical assay for Las-responsive SOS. In order to accomplish this, we developed and tested an entirely different assay approach; instead of using artificial promoter and reporter constructs in E. coli, we used semi-quantitative, real time PCR (qRT-PCR) assays to determine levels of expression of the Las SC1 late genes in citrus and periwinkle. The qRT-PCR methodology turned out to be a surprisingly successful approach, and obviated the need for E. coli reporter constructs. In citrus, relative expression levels of SC2-gp095 (peroxidase), SC2-gp100 (glutathione peroxidase), SC1-gp110 (‘holin’) although nominally placed in the ‘late’ gene regions of the phage, were much higher than expression levels of SC1-gp025 (‘tail fiber’). These results, initially performed using cyber green, were repeated and confirmed in citrus using gene-specific probes, and indicated that the promoter regions P0, P1 and P2 that we examined as predicted ‘late gene’ promoters, are likely not late gene promoters, nor repressed. These results were expanded to include the late gene SC1-gp035 (“endolysin”), heat treatment of Las infected citrus, and Las-infected periwinkle. 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 three of the three lytic cycle genes examined, SC1_gp025, SC1_gp110 and SC2_gp095, were much more highly expressed in periwinkle than citrus. However, heat treatment of citrus leaves failed to increase expression of these genes, indicating that lytic cycle induction of these prophage is not tied into a presumed heat stress response of Las in citrus. In addition to use of qRT-PCR in Las-infected citrus and periwinkle, we began to develop Liberibacter crescens as a model Liberibacter species. To determine if L. crescens might be tractable for functional genomics studies, the minimum inhibitory concentrations (MICs) of several antibiotics commonly used for plasmid selection was determined. BT-1 was found to be quite sensitive to: chloramphenicol, < 4 mg/L; gentamycin, < 1 mg/L; kanamycin, <2.5 mg/L, and tetracycline, < 0.3 mg/L. Both the repW (on pUFR071) and Bordatella replicons (pUFJ05, derived from pBBR1MCS5) were transformed by electroporation at high frequencies into BT-1. 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. Attempts to artificially inoculate marked strains into tobacco, citrus and periwinkle are currently in progress, as well as attempts to reconstruct the replicating form of phage SC2 in L. crescens, for use in chemical treatment assays.
The objective of this project is to improve the bioavailability of copper loaded silica nanogel (CuSiNG) material, as well as increase retention of the spray formulation to plant surface. Our studies have shown that both Cu(I) and Cu(II) co-exist in CuSiNG material. The presence of mixed-valence Cu has been correlated to improved antimicrobial efficacy of CuSiNG material over Kocide 3000 and Cu-sulfate controls based on both laboratory based test results and field trials. To further confirm the role of mixed-valence Cu in CuSiNG material, we have synthesized a series of CuSiNG nanoformulations with varying Cu(I)/Cu(II) ratios. Our laboratory test results have shown that a higher ratio of Cu(I)/Cu(II) improved antimicrobial efficacy in comparison to our previous formulations. Characterization of the material was done using HRTEM, XPS, UV-VIS and DLS. The two most promising CuSiNG mixed-valence formulations with increased Cu(I) amount over Cu(II) were prepared and delivered for 2013 field trial. We have also conducted an adherence property study of CuSiNG material using CuInS2/ZnS red-emitting quantum dots (Qdots) as planned and discussed in our previous reports. The adherence study was carried out using Hamlin Orange leaves and red-emitting Qdots. Red-emitting Qdots were quite visible when exposed to a hand-held 366 nm multi-band UV excitation source and easily seen after being applied to the plant surface. After washing and comparing with controls, more Qdots remained on the plant surface that was treated with CuSiNG material. This study suggests that the CuSiNG material adheres well to the citrus leaf surface. In future reports, we will include phytotoxicity results, antimicrobial studies from mixed-valence CuSiNG materials along with SEM material characterization data.
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