Objective 1: Potential for soil application of the neonicotinoids, imidacloprid (Admire) and thiamethoxam (Platinum), and acibenzolar-s-methyl (Actigard), to provide long-lived SAR control of canker was evaluated in a trial of 4-yr old grapefruit trees in Ft. Pierce, FL. Despite above average rainfall in May, the disease on the spring flush that emerged in March when it was dry was free of canker. In contrast, spring-summer flushes evaluated in September had 62% incidence of canker diseased leaves. Several of the SAR treatments significantly controlled disease but not as well as with Kocide 3000 and Firewall (streptomycin) sprays at a 21 day interval. SAR treatments that failed to significantly reduce foliar disease were the two applications of Actigard, Platinum split rate and isonicotinic acid treatments. Incidence of canker on the spring- summer-fall flushes evaluated at the end of the season in November had a slightly higher canker incidence than on the earlier set of flushes. By this time all the SAR treatments significantly reduced disease compared to the non-treated check. The most effective treatment was 4 applications of Actigard at 2 oz and least effective treatment was 2 applications of Actigard at 1.0 oz. The best treatment matched the control on the flushes attained by the 11 sprays of Kocide 3000 and/or Firewall. The 4-yr old trees produced enough fruit to evaluate canker incidence. Effectiveness of treatment on incidence of canker fruit varied from moderate for the 4 applications of Actigard at 2 oz, Platinum and Admire to ineffective for isonicotinic acid and Actigard at lesser rates and applications. Kocide 3000 and Firewall were significantly more effective than SAR treatments for reducing fruit disease. Harvestable fruit was reduced by canker’induced premature fruit drop. The non-treated check had the lowest fruit harvested per plot while the 4 applications of Actigard at 2 oz had the highest number of fruit. The number of fruit harvested was significantly negatively correlated with the incidence of disease on the spring summer-fall flush disease, but was not correlated with fruit disease incidence. Control of canker on the leaves apparently reduced the inoculum available for early season infection of fruit. Based on the trial results, Syngenta the manufacturers of Actigard are supportive of seeking a label for soil application of Actigard for SAR control of canker on non-bearing citrus. Objective 2 Integration of soil applied SAR inducers with foliar applications of copper sprays for control of canker on grapefruit was evaluated in the 4 yr-old grapefruit trial above, the best control was Admire applied once at the beginning of the season followed by 11 Kocide 3000 sprays. A trial in 4 yr-old fruiting Hamlin trees was set up in Arcadia, FL to compare trunk and soil applications of Admire at 3x the label rate to account for effect of the larger tree volume. SAR control of canker on foliage and fruit was equal to that of six 21 day sprays of Kocide 3000 starting in March. This suggests that SAR and copper could be used in an integrated program for augmenting canker control for young fruiting trees. However, it is unlikely that either neonicotinoid will be approved for use on bearing trees due to potential risks of increasing rates of soil application leaching into groundwater and residues in the flowers. Because the best SAR treatment for fruit disease control in 4-yr -old tree trial was the 4 applications of the 2 oz rate of Actigard, trials of this SAR treatment for young bearing trees are planned. Objective 3 is to evaluate of the complementation of the use of Actigard and neonicotinoids Platinum and Admire to increase and/or extend canker control in 1-yr-old grapefruit trees. Canker was first observed in the trial area in September 2009 after a very high rainfall period in August (17 inches). The pattern of disease spread was across the trial area from southwest to northeast. Incidence of trees with canker was 56% in the non-treated check trees, whereas in most of the SAR treatment combinations the incidence was less than 10%. This trial will continue in 2010.
Objective 1: Survey and confirmation of HLB in seedlings from HLB-affected trees. 500 seedlings grown from seed extracted out of mild to severely HLB-affected fruit from Pineapple orange and Murcott tangor groves in Hendry Co. were assayed for HLB detection. All seedlings were negative for HLB detection by PCR in repeated assays in July and Nov. 2009. Objective 2: Thermotherapy of HLB-affected seed for the same seed sources under Objective 1 were treated at 125F, 130F and 135F to test for the effect of heat treatment on HLB detection in seedlings. Thermal treatments were lethal to Murcott, but not Pineapple seed. The emergent seedlings at 125F (142), 130F (98) and 135F (97) were PCR tested. All seedlings were negative for HLB in repeated PCR assay in July and Nov. 2009. Objective 3: In October 2009, seed source trees in two Florida nurseries were found positive for HLB by FDACS-DPI (Nursery 1) or Southern Gardens Diagnostic Lab (Nursery 2). Discovery of infected seed source trees in two Florida citrus nurseries identifies a potential (but unconfirmed) risk of outdoor seed source trees acting as a source of inoculum for introduction into nursery propagations. In Nursery 1, seed was collected from symptomatic branches of two sources of Swingle citrumelo, four sources of ‘Kuharske’ Carrizo citrange and one source of Cleopatra mandarin. In Nursery 2, seed was collected from one source of Sekwasha mandarin. From 63 to 205 seedlings from each source provide enough leaves for testing in February 2010.
Spatial and Temporal Incidence of Ca. Liberibacter in Citrus and Psyllids Detected Using Real Time PCR Objective 1. Assess seasonal patterns of pathogen incidence in citrus trees and psyllids in regions of high HLB incidence. A 12 acre block of ‘Valencia’ orange trees was selected at a commercial grove in 2008. A sample of psyllid adults collected in the block in November 2008 and analyzed at USDA-ARS Riverside labs contained 21% HLB positive psyllids. The block was divided into 16 plots, which receive, two levels of micronutrients+SAR, insecticide treatments, or left as control. Plant and psyllid samples are being collected every four months to test for Candidatus Liberibacter asiaticus using PCR. Insecticide applications significantly suppressed psyllids compared to control. No significant differences in the field distribution of HLB in plants were observed using quantitative geostatistical analysis between November 2008 (40% HLB infection) and April 2009 (33% infection). Results from the fall 2009 and spring 2010 are being currently evaluated. A tree determined to be PCR positive and one PCR negative tree in each plot was trimmed to induce new flush. On 3 June, 10 psyllid adults from HLB negative colony on orange jasmine (Murraya paniculata) were released on a new shoot that had been caged immediately after trimming. An additional cage was placed on a previously uncaged and psyllid-infested shoot on the same tree. All cages along with branches and psyllids were collected 2 weeks later for PCR analysis. Nineteen percent of shoots caged with psyllids from the HLB negative colony tested positive for HLB on trees, regardless of whether trees had previously tested HLB positive or negative. In contrast, shoots that were naturally infested with psyllids when caged were 63% and 56% positive on previously HLB positive and negative trees, respectively. It would appear that infestation with infected psyllids from the field was over 2.5 times more likely to result in a positive plant sample, whereas the previous history of testing had little bearing. However, results were different when the same experiment was repeated in July-August using same plants. Shoots caged with psyllids from HLB negative colony were 64% and 42% HLB positive on previously HLB positive and negative plants, respectively. Whereas, naturally infested caged shoots were 7% and 18% HLB positive on previously HLB positive and negative plants, respectively. Adults emerging from infested shoots caged on HLB symptomatic trees at SWFREC during Feb-March 2009 and analyzed at US Sugar and SWFREC were found to be 5% HLB positive by both labs. Psyllids collected at large at the same time and location were 25% HLB positive, followed by 11% in April. Adults that emerged from infested shoots caged on HLB symptomatic trees were 23% HLB positive according to the USDA-ARS Riverside lab. Adults that emerged in the cages on HLB symptomatic trees from same location during July-August 2008 were 27% positive according to the Riverside lab. Therefore, psyllid origin and time of collection are major sources of variation on PCR results that we will need much more data to sort out. This report contains analyzed data through fall 2009. We currently have additional fall collections of psyllids that are being assayed and data compiled and analzyed. Additional psyllid samples from these experiments up to present 2010 are being processed. As our methods for addressing the first objective are refined as described above, the second objective is being addressed by the identification of additional sites to survey for psyllids which differ with respect to tree age, variety, and rootstock, and block size as described in grant proposal. A survey to administer to growers for tracking HLB and grove management tactics in the selected sites is in preparation and has been administered to a few, but data is still being collected. Despite the delay in receiving funds, the project is progressing in adherence to timeline and objectives.
The objectives of our research project are: Objective 1. Characterize the microbial community of healthy and Liberibacter-infected citrus phloem tissue by serial-section electron microscopy; Objective 2. Localize antigens and DNA sequences specific to Liberibacter in citrus phloem tissues by immunofluorescence microscopy, immunoelectron microscopy, and electron microscopic in situ hybridization. For objective 1, we completed three-dimensional (3D) serial-section electron microscopy analysis of 7 sites in the citrus phloem where Liberibacter cells are detected (One of the sites reconstructed into a 3D model is shown at http://news.ifas.ufl.edu/2009/12/09/uf-researchers-find-lone-culprit-behind-greening). In all the sites, the bacterial cells displayed uniform morphological features including diameter, length, cytoplasmic staining, and cell wall staining, suggesting that Liberibacter is the sole bacterial species in the citrus phloem or it vastly outnumbers other bacteria, if there are any. While we were examining the Liberibacter-infected (Las+) citrus phloem cells by 3D reconstruction, we discovered that the plasmodesmata in the Las+ phloem cells were swollen and heavily labeled by anti callose-specific immunogold particles. In parenchyma cells of Huanglongbing (HLB) symptomatic leaves, chloroplasts are disrupted by massive accumulation of starch granules and the cells eventually die. By fluorescence microscopy with aniline blue staining, we demonstrated that the callose accumulation at the phloem plasmodesmata precedes starch build up in the parenchyma cells of Las+ leaves. Callose deposition in the plasmodesmata reduces their transport efficiency and photosynthetate loading into the phloem is mediated by phloem plasmodesmata. So we tested phloem-loading in the Las-, callose-rich Las+ leaves by injecting carboxyfluorescein dye into their intercellular space. Phloem loading was inhibited in asymptomatic Las+ and symptomatic Las+ leaves while phloem loading was not inhibited in the Las- leaves. This phloem occlusion in the Las+ citrus leaves is likely to interfere with export of photosynthetate from the leaf parenchyma cells and provides an explanation for their excessive starch accumulation. Our results also suggest that the death of non-vascular cells in Las+ leaves is due to an inappropriate plant response rather than damages directly done by the phloem-limited bacterial cells. These results were presented in an international meeting (Plasmodesmata 2010) and a manuscript is being prepared for publication in the Plant Physiology. For objective 2, we have prepared citrus phloem samples ready for immunogold labeling and in situ hybridization. We were able to detect polysaccharides by immunogold labeling but several antibodies against bacterial proteins (from Dr. Duan) have not provided consistent immunogold labeling results. We are testing more antibodies from Dr. Duan. We tried two oligonucleotides for localizing Liberibacter by in situ hybridization but their specificity has not been satisfactory. We are improving our protocols and designing new oligonucleotides. During the first year, we focused on structural characterization of Liberibacter cells in the citrus phloem and elucidating plant responses against Liberibacter infection. In the second year, we will carry out in situ hybridization and immuno-microscopy research using molecular markers of Liberibacter strains as planned in the original proposal. We will also elaborate callose detection/phloem loading research to characterize citrus varieties with different HLB susceptibility. The second year research will complement our structural research of HLB, help understanding the HLB disease development process, and improve current microscopy tools that will be applied not only to HLB but also to the ‘Zebra Chip’ disease, a Liberibacter-associated disease of potato.
The goal of the proposed research is to understand how Candidatus Liberibacter asiaticus causes Huanglongbing (HLB) disease and how tolerant plant resists Ca. L. asiaticus infection. Citrus HLB is the most devastating disease on citrus. There are very few options for management of the disease due to the lack of understanding of the pathogen and citrus interaction. Understanding the citrus and citrus HLB pathogen interaction is needed in order to provide knowledge to develop sustainable and economically viable control measures. Major achievements: 1. One refereed journal article was published. Kim et al. 2009 Response of sweet orange (Citrus sinensis) to ‘Candidatus Liberibacter asiaticus’ infection: microscopy and microarray analyses. Phytopathology. 99:50-7. Investigation of the host response was examined with citrus microarray. The microarray analysis indicated that HLB infection significantly affected expression of 624 genes related to plant pathogenesis/stress, anthocyanin biosynthesis, cell wall metabolism, cell division, detoxification, lipid metabolism, metabolite transport, metal transport, nucleotide metabolism,phenylpropanoid / flavonoid / terpenoid metabolism, phytohormones, protein kinase, protein metabolism, protein-protein interaction, signal transduction, sugar metabolism, transcription/translation factors and unknown/hypothetical genes. The anatomical analyses indicated that HLB bacterium infection caused phloem disruption, sucrose accumulation, and plugged sieve pores. The up-regulation of three key starch biosynthetic genes including ADP-glucose pyrophosphorylase, starch synthase, granule-bound starch synthase and starch debranching enzyme likely contributed to accumulation of starch in HLB affected leaves. The HLB-associated phloem blockage resulted from the plugged sieve pores rather than the HLB bacterial aggregates since ‘Ca. Liberibacter asiaticus’ does not form aggregate in citrus. The upregulation of pp2 gene is related to callose deposition to plug the sieve pores in HLB-affected plants. The cDNA sequence of PP2 has been requested by four different research groups to use as the target to suppress the HLB symptom development. 2. Leaf samples from different varieties including grapefruit, Murcott, and Hamlin were collected from Florida citrus groves. Both healthy and infected trees were sampled. It is expected gene expression of those trees in the citrus grove will reveal more information when compare with the gene expression profile in greenhouse. Those samples will be used for gene expression analysis using microarray or SSH approaches. 3. Gene expression of Valencia leaf samples (healthy vs infected) in citrus grove are being conducted. Currently, two biological replicates were included in the preliminary test. Further microarray analysis is needed. 4. To further expand our current understanding of Ca. L. asiaticus-host interaction, we are currently comparing two susceptible and two resistant/tolerant cultivars grown in greenhouse. The plants have been inoculated in greenhouse. Finding key genes involved in HLB symptom development will reveal potential management strategy and lead to innovative research to control HLB. Research plan 1.1 Host response in greenhouse condition. Both Carrizo citrange and Poncirus trifoliata exhibited tolerance in the initial test. However, the inoculation has been inconsistent. The tolerance of Poncirus trifoliata to HLB is inconclusive. Thus host response of those varieties to Ca. L. asiaticus infection could not be done. Instead, we have adjusted our plan based on the most recent data on pathogenicity assays of different varieties to Ca. L. asiaticus. Both Persian lime and Eureka lemon are tolerant to HLB. 1.2 Host response in field condition. Due to the differences in greenhouse and field conditions, the disease development in greenhouse and field is quite different. We would to focus on sweet orange. we will use the following samples: leaf, stem, and root.
Work proceeded on several fronts. First, updated budgets that reflect the increase in grove maintenance costs were completed. These budgets indicate that a full program to minimize the effects of greening can increase grove care-taking costs by $450 per acre. Under typical Florida yields, this increase adds more than one dollar per box to the breakeven price. Second, plans are being formulated to hold an “economic summit” to held in April at the CREC. The participants will include economists from both Florida and Sao Paulo. During the last half-day of the meeting, representatives from the citrus industry are invited to listen and respond to the discussion. Third, a model of disease spread through a particular grove that was first developed in Sao Paulo by FUNDECITRUS is being adapted to Florida.
We want to obtain an in vitro culture of Candidatus Liberibacter by co-culturing the bacteria with insect cells to study Candidatus Liberibacter physiology, metabolism, virulence and its interactions with the insect vector. We tested various stable insect cell lines to in vitro culture Candidatus Liberibacter asiaticus (LAS), the Asian form of HLB disease, also found in Florida. We tested different inoculums (from various citrus and periwinkle plants) on different insect cell lines. We checked for the presence of LAS in inoculated cell cultures by direct PCR. Current results: – After Mamestra, Spodoptera, Drosophila cell lines, we tested an Aedes albopictus insect cell line. We didn’t detect LAS presence after inoculation. Actually, citrus inoculums have a deleterious effect on Aedes cells and we are now focusing on periwinkles inoculums. – We detected LAS in two lines of drosophila cell cultures by direct PCR. One line lost the detection after 6 transfers. Signal of detection was confirmed to be LAS. For one line, we are currently reaching the 15th culture transfer with a detection by PCR to be confirmed by sequencing. The detection remains very weak and we need to improve the conditions to get a higher titer. – The detection of the bacteria by direct PCR peaks at day 7 after transfer and then declines. The bacteria are multiplying and seem to follow the drosophila cells growth. We began to set up multiplex qPCR assays. Primer sets suitable for qPCR and specific of LAS and drosophila detection were selected and tested with success. – In order to reach higher bacterial concentrations, we tested complements of the culture medium: various sugars, vitamins or trace elements. We analyzed metabolic pathways potentially encoded by the released Liberibacter genome sequences to improve growth conditions and to define limiting factors and/or growth inhibitors. Of the complements tested some had a positive effect on the detection of LAS but negative effects on insect cells survival. With the decline of the insect cell cultures the bacteria was no longer detected. Currently, sodium pyruvate and sterile citrus juice had a positive effect on LAS detection but a lethal effect on insect cells. Proline and fructose had a positive effect on the bacteria detection and are now added to our co-cultures. – We analyzed insect cell culture medium sugar and amino-acids variations over culture time to identify potential LAS growth limiting factors. Rapid depletion of glucose and of some amino-acids by drosophila cells could be limiting factors and are candidates for our medium improvement. Current work: – We are now setting up multiplex qPCR assays to monitor the ratio of LAS and drosophila DNA over culture time. – We are combining complements to the insect cell culture media and looking for new ones to improve LAS concentration – We are analyzing insect cell culture medium minerals and trace elements variations over culture time to identify potential LAS growth limiting or improving factors. – We are testing new insect cell lines to get more consistent presence of the bacteria and higher concentrations. We are reaching our milestones for the first year of this project and we will look into the axenization of our primo-cultures.
The citrus extension agents conducted the “Low Volume Application Technology for Citrus Pests” program in six locations around central and south Florida in October. These programs discussed: low volume labeled materials; low volume application; application considerations; and worker safety. Program attendance exceeded 300 participants. Full program presentations can be viewed by going to the Citrus Agents web site at http://citrusagents.ifas.ufl.edu/events/PDF/LowVolumeApplication/LVA_program.htm . As part of greening control, selected agents are actively involved in assisting with the development of area wide spray events. These targeted sprays are to suppress psyllids over a wider geographic area. In addition to the above program, agents are actively involved in conducting grower programs (6), field visits (47), educating Master Gardeners (2) and the general public in greening identification and control issues.
With the program being recently funded, we are in the process of receiving order items to conduct the study. All materials were ordered in December of 2009 and are expected to be received within the next 30 days. Once delivered, studies will be designed to conduct proposed studies.
Six months after shearing or clipping citrus trees off above the soil, the study has shown that Remedy is effectively controlling stump sprouts at all treatment times of 0, 24, 48 or 72 hours after shearing the tree off above the soil. However, in a few cases lateral roots did exhibit limited sprout formation even with the stump being treatment with Remedy. Complete and thorough coverage of the cut surface is essential for adequate control. Sprouts were produced from the untreated stump as well as on limited lateral roots. The size of stump also impacts sprout formation with larger trunks producing more sprouts and quicker after shearing as compared to smaller stumps. Rootstock also has an impact on sprout formation. Rough lemon rootstock tends to produce more sprouts from the trunk and lateral roots than other rootstocks in this study. However, with limited data the difference between rootstock is not able to be determined. Sprouts from stumps will be collected and tested for HLB (greening) during the next quarter.
The project has two specific aims. We outline below the progress made for each of them. Specific Aim 1: Identify sweet orange responses to Huanglongbing disease (HLB) through deep transcriptome profiling using next generation DNA sequencing technologies. Transcriptome analysis was conducted using Illumina Genome Analyzer II. We obtained read lengths up to 125 bp pair-ended for the entire set of mature fruits analyzing the four types of samples: symptomatic, asymptomatic and apparently healthy from infected locations and healthy plants from disease-free location. 25 and 31 million reads/sample were obtained and 12,8-18.99 million reads were mapped to the NCBI citrus Unigene set (15808). Using velvet assembly we obtained 158,656 contigs that were BLASTed to the following databases : JGI 1.2x citrus genomic sequences (78.6% of contigs had hits), NCBI C. sinensis Unigene set (45.7% of contigs had hits), TAIR9 protein sequences (53.6% of contigs had hits) poplar protein sequences (58.7% of contigs had hits). About 1156-1734 of the citrus unigenes were differentially regulated in pair wise comparisons of symptomatic, asymptomatic, and apparently healthy fruits with control fruits. We have also completed construction of three of the four cDNA libraries from immature leaves. We are planning to obtain the last sample (control) to soon run this set of samples before the analysis of the mature leaves and immature fruits. Additional RNA was isolated from both mature fruits and leaves for the validation of the results using qRT-PCR. Specific Aim 2: Define and validate gene networks and identify host (sweet orange) response biomarkers regulated by HLB at different stages of infection. We have analyzed transcriptome data using BLAST2go, mapman, pathexpress software. A gene set enrichment analysis has revealed the following HLB-regulated pathways: starch metabolism, lignin, stilbene biosynthesis, phenylpropanoid, sucrose metabolism, coumarin, salicylic acid and hormone metabolism. A pair wise comparison between the four types of samples has identified several genes that can be used to distinguish infections before the symptoms appear. After qRT-PCR validation these genes can be confirmed as biomarkers for asymptomatic diagnosis. A list has been made including genes belonging to sucrose metabolism, photosynthesis, cell wall metabolism, lipid metabolism and certain volatile metabolite pathways. Terpene metabolism was highly regulated in apparently healthy compared to HLB-infected fruits. Jasmonate, salicylic acid, ethylene and auxin were the key hormones involved in host response to HLB-infection in the fruit peel tissues. Among transcription factors, WRKY, NAC, MYB, EREBP, bZIP were mostly highly regulated after the infection proceeding from the asymptomatic stage. With respect to cell biology processes the most significant up regulation was observed in pathways related to, protein modification, protein degradation, transport, metal handling pathways, while cell division and cell cycle and organization were down regulated. Up regulation was observed in HLB symptomatic fruits for genes involved in Photosystem II and redox chain reactions as well as ubiquitin dependent protein degradation. The following receptor like proteins were found to be involved in the host responses in the fruit: leucine-rich-repeat protein receptors, DUF26, WAK, thaumatin. We are conducting a network analysis of citrus responses based on a Arabidopsis knowledge base of protein-protein interactions to determine which genes play key roles in the cascade of HLB-regulated pathways (hubs or connecting hubs). Such genes will be excellent candidates as biomarkers for diagnosis and as targets for therapeutics. Currently, qRT-PCR has been performed to validate 6 genes belonging to starch, sucrose, hormone metabolism and additional analysis will include up to 20-30 genes to identify biomarkers for asymptomatic host responses in fruit to HLB.
We continue to make progress in our goal to identify proteins that are specific to Liberibacter-infected citrus. These proteins, and the antibodies made against them, will be used in the development of a rapid diagnostic tool for the disease in the field. We have previously reported that all IgYs that will be necessary to perform pCMAT are in hand. Briefly, IgYs were raised in chickens against diseased plants at 3 months, 1 year and 2 years post-infection. The material from each time point was injected to two hens for IgY production. One of the two hens used for the 3 months time point unfortunately entered molting and had to be discarded. The last batch of antibodies was shipped to Oragenics on October 7th. It was demonstrated by Western analysis that the IgYs that were raised against diseased plants are broadly immunogenic. All pools of hen serum had a strong reactivity against plant extracts. Several hundred bands were observed as well as a smear, which indicates a broad and measurable reactivity against a large number of plant (and presumably pathogen) proteins. The next phase of this study aimed at optimizing the steps of IgY adsorption using healthy plant material. This was to deplete the IgYs from those antibodies that are reactive with proteins constitutively expressed in healthy and in diseased plant tissues. This was performed using a proprietary method called PCMAT. Briefly, IgYs were adsorbed against insoluble fractions of homogenized and sonicated healthy plant tissues. To monitor the adsorption process, sequential absorption of pooled IgYs was performed using PCMATTM against extracts from healthy plant tissues. The IgYs that did not bind to healthy plant tissue were collected and purified, and used to probe cellular extracts in an ELISA format. Clearly, the adsorption process is not yet complete after 5 rounds of adsorptions with insoluble material, yet resulted in a significant decrease of reactivity against healthy plant extracts. We are currently optimizing the conditions necessary to bind soluble healthy plant extracts to solid matrices, which will be necessary for further absorb the IgYs; until the reactivity against healthy plant extracts reaches background levels. Thoroughly adsorbed IgYs which specifically retain their reactivity against extracts from diseased specimens should be available by the end of the month. In summary, we are on schedule to perform the experiments described in the original application. The next few months will be spent on refining the adsorptions process with soluble plant extracts. The resulting IgYs will then be used as a probe to isolate proteins of citrus that are specifically induced during infection as well as gene products of the bacterial pathogen produced during that process. This will be performed by immunoaffinity and LC-MS/MS, as originally proposed.
Fall tests of herbicide applications to trunks continued into the winter. Efficacy of alternatives to 1 1/2 inch hatchet blade cuts are being evaluated. Spring and Summer tests, when trees are more difficult to kill, are critical for this testing. An electro-hydraulic mechanized tree trunk incision clamp system was developed and tested first on a 4 X 4 ATV, and later transferred onto a utility tractor to assist with weight distribution and hydraulic power source issues. Also, to deal with operating stresses, the clamp system was revised to incorporate a larger bore cylinder and a lesser resistant serrated clamping mechanism. The increased force from the larger cylinder made it necessary to build components from heavier materials. Also, due to fluctuating tree trunk shapes and sizes, the clamping mechanism wasn’t able to consistently contact the tree without shearing pivot point components. Further testing identified the need to decrease the system weight and to create a lesser resistant means for making tree trunk incisions, in order to become an efficient system. A less-resistant methodology consisted of changing the system function for the cutting mechanism. These changes compensate for the trunk size and shape variances and will allow reductions in system weight. Recent orchard tests have indicated the cutting mechanism functions with less resistance and it is more consistent in performance and efficiency. The spray system was added to provide a complete prototype and several trees were treated with either undiluted or 1 to 1 diluted Arsenal solution. The efficacy of the complete system in these treatments will be evaluated over the next 4 weeks. Full evaluation of the system needs to occur during the spring-summer when trees are more difficult to kill. We are now asking CRDF for guidance on issues about patents and a manufacturing agent for the machine. The results of the 600 acre aerial spray test applied by South Florida were reported last quarter. The one area not completely killed will be evaluated this quarter. Aerial sprays treatments to the additional 40 acres of abandoned grove at this site has been delayed until some issues on chemicals to be tested are resolved. It is still planned to have 4 treatments with 2 replications (5 acre plots) in order to evaluate selected chemicals. Work on use of the soil fumigant, Midas, has also been delayed until the parent company can make their representatives available to continue the tests. Apparently, they are currently involved in testing their product on vegetable sites. They expect to be available by early spring.
The new variable rate controller continues to perform well in the approximately 50 units currently deployed in Florida citrus groves. Testing is continuing on the addition of a liquid crystal display, with the current focus being to implement all the user input and operation status functions via a single color touchscreen and rugged weatherproof enclosure. The elimination of all physical switches and pilot lights from the enclosure will increase the durability of the controller and make it easier to upgrade all of its functions by using virtual switches and status lights on the display. We have completed tests on the latest range of ultrasonic and optical sensors with the variable rate controller in order to offer alternative sensor packages to the users. The different sensor beam widths and speeds now available could have advantages in different grove situations (e.g. bedded flatwoods versus ridge) and the controller should be able to accept an array of different sensors, including optical, ultrasonic and microwave (radar). Effective systemic insecticides like imidacloprid have a labeled annual application rate limit of active ingredient (0.56 kg/ha) which constrains their application frequency to about 4.5 per year when broadcast with air-blast sprayers. Variable rate technology and band application can extend the efficacy of such rate-limited agrochemicals per planted hectare by precision placement of active ingredient only to the tree canopies. A sprayer used in young groves must have a very fast response speed because the tree canopies and adjacent spaces are small. A precision row sprayer with a hoop-shaped boom was developed for young citrus, using the prototype variable rate control system. By using a fast solenoid valve (<0.2 s), the sprayer is capable of real-time pesticide flow control according to the presence of citrus tree canopies. The sprayer system consists of a WAAS GPS receiver, near-infrared diffuse reflectance optical sensors, electric solenoid valve and two embedded microcontrollers with synchronized look-ahead and adjustable buffering. Spray savings were estimated to exceed 90% in the first year, and average 50 to 75% by the time the trees become productive, thus allowing more efficient and flexible use of rate-limited pesticides and reducing production costs for greater profitability. A video of this sprayer is available at http://128.227.177.113/pa/Video.html We are conducting research on finding ways to change the pesticide spray flow rate in real time instead of the simple on/off actions currently employed. In conjunction with sensor information about canopy density, the proportional flow control would allow customization of spray amounts as needed per unit of canopy and its condition. So far the stepper motor valves tested have shown potential for effective proportional flow control, with response times better than 0.5 s.
The two primary objectives of the current proposal are: (1) determine and compare the metagenomes of healthy and infected citrus phloem, and (2) continue to develop and apply AthenaBio’s cultivation technologies towards producing a pure culture of Ca. Liberibacter. Objective 1 Discussion. Laser cutting microdissection (LCM) will be developed to purify phloem-containing sieve cells from both healthy and infected citrus samples. DNA extracted from the purified cells will be analyzed by 16S rRNA sequencing to evaluate the contained microbial diversity. The most diverse DNA samples will be selected for large-scale metagenomic analysis. All DNA extraction and sequencing work will be performed by Dr. Ravel’s group (U. of Maryland) Objective 2 Discussion. AthenaBio will continue to develop and apply its cultivation technologies to isolate a pure cultures of Ca. L. species. In addition to Ca. Liberibacter species, we aim to cultivate other phloem endophytes to provide reference genomes for metagenomic analysis. Two approaches will be employed to disrupt quorum sensing (QS) and thereby enhance growth: (1) physical methods (i.e. a ‘flow cell’) will be used to wash QS signals away from cells, and (2) enzymes will be used to inactivate QS signals through chemical modification. The advantage of the flow cell approach is that knowledge of the chemical structure of the QS signal is not required, and this approach is therefore expected to work on a broad variety of QS systems. Both approaches (flow cell and enzyme) will be combined with various media formulations and incubation conditions. Work has continued in method development for LCM of citrus phloem cells. Healthy leaf tissue was dissected into small pieces (~5mm), immediately placed in tissue freezing medium and placed on dry ice. Various thickness levels and staining procedures were tested. We believe we have a working method but the instrument was out of order for a period of time. The instrument is now fully functional and we are anticipating DNA analysis to begin by mid-February.
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