Progress on development of an acoustic trap to capture male Asian citrus psyllids (ACP) that are searching for females on citrus tree branches continued on two fronts in the last quarter of 2012. In the third quarter, we had recorded male and female communicatory duets and found that both sexes responded readily to playbacks of recorded signals and to synthetic mimics that had the same temporal and spectral patterns as the recorded calls. The playbacks were produced by a computer that output signals to a commercially available (Bruel and Kjaer) vibration exciter attached to the plant. Both the recorded calls and synthetic mimics attracted males to the signal source, which is a necessary first step in the trapping process. This work was prepared and submitted for publication. Based on the successful results of these studies, we began efforts to develop a less expensive system for detecting and producing the ACP communicatory signals. We purchased an inexpensive Arduino Uno microcomputer system and programmed it to output synthetic mimics of ACP communicatory signals to a piezoelectric buzzer. In addition, we have begun the process of programming the microcomputer to recognize and reply to calls made by males searching for females on a tree. We found that it is possible to use one single buzzer that both produces a female signal and detects the male signal on the plant, which reduces the total cost of the Arduino-buzzer system to less than $50. In comparison, the cost of the signal analysis/vibration exciter system that was used to conduct the initial studies is about $3000. We expect to complete the programming tasks and begin testing the new instrument in the laboratory during the next quarter.
The overall goal of this 3-year research project is to efficiently deliver antimicrobial molecules against HLB bacteria into citrus phloem. This quarterly (Oct.2012 to Jan. 2013) research continued to optimize the W/O (water in oil) and O/W (oil in water) nanoemulsion formulations by evaluating the ideal adjuvant mixtures of solvents (ethanol, acetone, methyl, acetate, and ethyl acetate), oils (soy oil, cremorohor, carvacrol, p-cymemen, orchex796), lipophilic (span80, span85) and hydrophilic surfactants (tween20, tween80) using pseudo-ternary phase diagram and thermodynamic stability methods. Several ideal W/O and O/W nanoemulsion formulations have been optimized for loading the effective antimicrobial compounds. Orchex796, one of the horticultural mineral oils, has been used to control insects in citrus production. The nanoemulsion formulations of Orchex796 will be applied to control both citrus insects and the Las bacteria. Eight compounds from Citrus Research and Education Center, IFAS, UF were tested for their effectiveness against HLB bacteria by the grafted-based chemotherapy method. The results will be showed in next quarterly report. In the next quarter, the research will focus on screening the penetration compounds finalize the applicable formulations.
Ultra High Performance Liquid Chromatography – Pesticide Residue Analysis (Dec 2012 Update) This quarter, use of the equipment purchased focused on validating methods used for analysis of pesticides that we will be working with. We will continue this method development and will begin analyzing research samples in Jan/Feb.
Using highly purified salivary sheaths we were able to study composition of different parts of the sheaths to show that their composition is not homogeneous throughout. This finding has allowed targeted research focused on blocking the initial stages of sheath synthesis as a new strategy for blocking the psyllids ability to transmit the bacterium associated with citrus greening. Research has now been initiated on developing transgenic plants to express genes encoding peptides that could function to block the ability of the psyllid to reach the phloem sieve tube elements with their piercing mouth parts. Because the sieve tube element is the location where the citrus greening associated bacterium resides, it is hoped that by blocking the psyllids form reaching these cells we will block the ability of the psyllid to acquire or transmit the bacterium, thereby blocking the spread of citrus greening disease.
Progress continues on the primary objectives of the project:1) Develop a generic epidemiological model that can be used to compare control scenarios and to optimize the probability of controlling and managing high-risk pathogens of agricultural significance. 2) Development and testing of efficient methods of statistical inference to estimate epidemiological parameters from maps of emerging epidemics. 3) Develop user friendly model ‘front ends’ that can be used by researchers and regulatory agencies. During this cycle we have focused primarily on making the models more robust and flexible models to most efficiently analyze and predict the spread of emerging pathogens across a range of scales extending from within-plantation to the landscape and regional scales for both HLB and canker. We are continuing to test the models extensively to assess how host planting age affects the transmission of HLB . We consider the models to be and are developed as part of a flexible tool-box so that they can be readily adapted to new disease threats as well. Work has continued on parameter estimation and writing up the methodology. Removal or treatment of infected trees in a region in which the dispersal and transmission are being estimated for an emerging epidemic can have profound effects in under-estimating some parameter values if not allowed for. Accordingly we have also developed methods to allow for this during parameter estimation. This requires an understanding of how the control is likely to affect epidemiological parameters and where this is unknown how to compare alternative models. Once parameterized, the models allow us to predict where disease is likely to spread most rapidly. We have tested a risk-based strategy that uses epidemiological knowledge on where disease is likely to spread most rapidly to optimize control that involves removal of more susceptible hosts around key infected sites. Using a parameterized model of citrus canker on realistic landscapes, we have shown that the risk-based strategy would outperform a conventional fixed-radius approach when the dispersal and transmission parameters are accurately known. We are testing to what extent performance degrades under varying degrees of uncertainty. Additional work has also been completed in this quarter in collaboration with a graduate student on a model of host-vector systems, considering the spread of HLB within an individual tree in particular. The relative importance of pathogen transmission within the vascular system of the tree and between leaves via psyllid vectors has been studied. Currently, the models are being used to investigate the efficacy of potential methods for the control of HLB, including the use of insecticide, roguing, use of nutritional products and thermotherapy, amongst others. Publications: Cunniffe, N.J., R.O.J.H. Stutt, R.E .DeSimone, T. R. Gottwald, and C.A. Gilligan. 2012. Webidemics: Webbased Interactive Demonstration of Epidemiological Modelling Informing Control Strategies. To be submitted to PLoS One. Gilligan, C. A., F. M. Neri, A.R.. Cook, G. J. Gibson, and T. R. Gottwald. 2012. Bayesian analysis of an emerging epidemic: citrus canker in urban Miami. To be submitted to Journal of the Royal Society Interface. Parry, M.F., G. J. Gibson, T. R. Gottwald, S. Parnell, and C. A. Gilligan. 2012. Spatio-temporal modelling of the spread of HLB/Citrus Greening on orchard scales. To be submitted to Proceedings National Academy Science USA / Journal of the Royal Society Interface.
Xambr’ Brazil Plots: Designed to examine the effect of windbreaks, copper sprays to reduce infection, and leafminer treatments to determine their individual and combined effects on control of citrus canker. Windbreaks were completed and plants were be established in Mid April 2010, but severe winds damaged the windbreaks during two storm events. These windbreaks have been reinforced and rebuilt. From October through December 2012, we completed plot staking and planting of a short line of Casuarina in front of the gates in each block, 7 m outward from the main casuriana perimeter, to allow equipment entry but still adequately break the wind from the interior of the plots. Windbreak and citrus plants will be of sufficient size to begin the experiment in March 2013. New windbreak trials established in Saint Lucie County. Further disease data has been recorded and is awaiting analysis. An additional 10 weather stations were purchased for deployment at a second site (cv. Rubyred grapefruit, Scott Farms, St. Lucie Co.). Weather stations were deployed equidistantly in an East-West and North-South orientation in the citrus block. Data collection has been started. Programmable leaf wetness controller (PLWC): During this quarter we developed a very complex sensor that probably closest emulates a leaf. This sensor, currently named the pin sensor, is a flat surface, made from wax. Contact wires are then connected together in a pattern, so that a drop of water will bridge a ‘+’ to a ‘-‘, creating a connection. The more drops of water, the more connections; the more connections, the more current flow. Currently the sensor is too sensitive and thus we are currently reducing the sensitivity of the electronics, and retesting. Project publications: Bock, C. H., Graham, J. H., Gottwald, T. R., Cook, A. Z., and Parker, P. E. 2010. Wind speed effects on the quantity of Xanthomonas citri subsp. citri dispersed downwind from canopies of grapefruit tree infected with citrus canker. Plant Di Bock C.H., Graham, J.H., Gottwald, T.R., Cook, A.Z., and Parker, P.E. 2010. Wind speed and wind-associated leaf injury affect severity of citrus canker on Swingle citrumelo. Eur J. Plant Path 128:21-38 Bock, CH, Parker, PE, Cook, AZ, Graham, JH and Gottwald, TR. 2001. Infection and decontamination of citrus canker and inoculated the surfaces. Crop Protection 30:259-264. Hall, D.G., Gottwald, T.R. and C.H. Bock. 2010. Exacerbation of citrus canker by citrus leafminer Phyllocnistis citrella in Florida. Florida Entomologist. Florida Entomologist 93:558-566. Bock, C.H., Gottwald, T.R. and Parker, P.E. 2011. Distribution of canker lesions on the surface of diseased grapefruit. Plant Pathology (Accepted). Bock, C.H., Cook, A.Z., Parker, P.E., Gottwald, T.R., and Graham, J.H. 2011. Some characteristics of the dispersal plume of bacteria of Xanthomonas citri subsp citri in wind-driven splash downwind of canker-infected grapefruit tree canopies. (Plant Disease). Bock C.H., Graham J.H., Cook A. Z., Parker P.E., and Gottwald T.R. 2012. Predisposition of citrus foliage to infection with Xanthomonas citri subsp. citri. (Submitted to Plant Disease).
During summer/fall 2012, we completed a gap study trial in St. Lucie Co. We started 2 replicated trials to study efficacy of mating disruption in plots treated with pheromone in rubber septa and 2 validation trials with pheromone loaded in rubber septa in collaboration with ISCA, grove managers, owners, and crop consultants. These trials address pheromone carrier, timing, coverage, formulation, longevity, and efficacy of mating disruption under various crop phenologies. 1. Winter and spring application of SPLAT CLM. We submitted the manuscript ‘Seasonal abundance of Phyllocnistis citrella (Lepidoptera: Gracillariidae) and dormant season application of pheromone in citrus to control overwintering and spring populations’ (Fla Entomol). Winter application of SPLAT CLM did not increase trap catch disruption in spring compared with spring application alone. Moth numbers declined in winter, suggesting that preventive control might help manage this pest. Leaf flushes on south sides of groves may provide favorable habitat during winter. Moth flight in spring preceded mining, suggesting early colonizers arrived from outside the grove. Area-wide management of this pest may be appropriate. 2. Effect of formulation on longevity and efficacy across treatment gaps (St. Lucie Co.). Cooperator: Packers of Indian River. Established 27 April to compare current SPLAT CLM triene pheromone formulation with a formulation that incorporates the ‘natural’ 3:1 triene:diene pheromone blend. Treatments were applied 29 May and 3 June. Mines were evaluated 18 June and 26 July when widespread early-stage leaf mines were present. Data are being analyzed. There appears to be no difference in efficacy between the formulations. 3. SPLAT CLM versus rubber release devices (St. Lucie Co.). Cooperator: Scott Lambeth (Golden River Fruit Co.) An experiment was started 10-13 July to compare trap catch disruption in small plots (0.14 ha) treated with SPLAT versus rubber septa loaded with an equivalent amount of pheromone. Treatments included ‘natural’ 3:1 blend and triene only. In a companion trial, five blocks (71 ha) were treated with SPLAT CLM (10-12 July). In small plots, septa provided superior and long-lasting trap catch disruption. Disruption in SPLAT-treated large blocks was weak and short-lived. We identified a formulation problem in SPLAT. Three of the large blocks (42 ha) were treated (24 Aug) with rubber septa distributed at 318 septa/ha. Septa containing either formulation have provided >95% disruption for 11 weeks in small plots and 5 weeks (so far) in large blocks. Evaluation of leafminer damage is ongoing. 4. Large plot septa trial (St. Lucie Co.). Cooperator: Packers of Indian River. On 7 Sept, an experiment was started to compare trap catch disruption in plots (0.87 ha) treated with rubber septa containing ‘natural’ 3:1 blend (330 septa/ha) versus untreated plots (0.87 ha). 5. Large plot septa trial (Charlotte Co.). Cooperator: TRB Groves. On 20-23 Aug, previous SPLAT CLM grower validation trial was continued with deployment of rubber septa loaded with natural blend. 6. SPLAT grower validation trials. Grower validation trials with SPLAT are complete at 4 sites (Charlotte, DeSoto, Okeechobee and St. Lucie). SPLAT CLM underperformed compared with previous studies due to an apparent interaction between pheromone and a new wax component.
We proposed to identify and assess gene sequences for their negative effects on sap-sucking Hemipteran insects via RNAi using both in vitro and in planta dsRNA feeding assays. To date, we have cloned sequences from nine homologous D. citri and M. persicae transcripts. In addition, we have carried out artificial feeding assays on M. persicae using dsRNA derived from the salivary gland-specific Coo2, midgut-specific glutathione-S-transferase S1 (GSTS1) and constitutively expressed S4e ribosomal protein from M. persicae, as well as a control derived from green fluorescent protein (GFP) sequence. Since recent evidence suggests that RNAi in sap-sucking insects may operate more effectively in planta than in vitro, we evaluated the RNAi strategy in planta for its effects against our model insect, M. persicae (objective 2). In this objective, Gateway-based vectors were used to express the selected insect dsRNA (Coo2, GSTS1 and S4e) either constitutively (35S promoter) or in a phloem-specific manner. Our results suggest that the M. persicae-specific dsRNA expressed in planta has a negative effect on both the lifespan of the insects and the number of offspring generated. In the fall of 2010, we began working on objective 3: to transform citrus with RNAi-inducing transgenes against D. citri. Previously, we conducted 3′ rapid amplification of cDNA from vacuolar ATP synthase subunit G, S4e, and .-tubulin transcripts from D. citri. We have now inserted sequences of the aforementioned transcripts into Gateway-based vectors downstream of both the constitutive 35S and our novel phloem-specific citrus CsSUS1 promoters. To date, we are in the process of transforming and regenerating citrus with the D. citri-specific gateway vectors for evaluation and use by the Florida citrus industry. Initial attempts to transform and regenerate Citrus sinensis ‘Valencia’ and ‘Hamlin’ containing reporter gene constructs were successful. Currently we have completed transformation of citrus callous tissue using gateway vectors with the vacuolar ATP synthase subunit G or S4e transcripts inserted downstream of a phloem-specific citrus CsSUS1 promoter. We have regenerated at least one line for each of the constructs of interest, and we are in the process of in vitro micro propagation to produce additional copies of each line. We are still regenerating other transformed lines, and are preparing to generate additional lines with the other transcript/promoter combinations.
The questions to be addressed by this research project are 1) how does Imidicloprid move in the sandy soils of south Florida, 2) how long does imidicloprid persist in sandy soils, and 3) how much Imidicloprid leaches below the root zone of citrus trees. Soil sample analysis and field shylid populations counts have been completed for all studies except these ending in the past quarter. Two field studies were conducted the spring and summer of 2012. Initial analysis of soil samples from the spring study indicate that Imidicloprid concentrations decreased rapidly in the soil and was either taken up by the plants or leached within 5 to 10 days at recommended microsprinkler irrigation rates, however reduced irrigation following applications allowed for greater soil residual time and uptake. During these initial application studies, adult psyllid populations per tree decreased from approximately 1.7 for both treated and non-treated trees to 0.1 psyllids per tree for the treated trees and remained suppressed greater than 50 days after application. These data would indicate great persistence of the insecticide in citrus tissue and is supported by elevated leaf tissue concentrations. Isotherms studies to determine the sorption rate and persistent concentration in soil with time was conducted. The sorption studies, relating Imidicloprid concentrations in soil solutions with Imidicloprid concentrations in the solid phase of sandy soil were conducted using Immokalee fine sand. Imidicloprid decreased in soil concentrations due to biodegradation at a rate of 0.013 .g g-1 d-1. Imidicloprid was found to penetrate to a depth of 45 cm and have greatest affinity in the 0-15 cm soil depth due to higher soil organic matter at that depth. These data were supported by soil partition coefficients (Kd) of 1.68 mg of Imidicloprid. in one liter of soil solution at the 0-15 cm depth and decreases to 0.33 and 0.25 mg l-1 at 15-30 and 30-45 cm, respectively. These data suggest that Imidicloprid is leached rapidly from the soil if not taken up by the target plant.
Progress continues on the primary objectives of the project:1) Develop a generic epidemiological model that can be used to compare control scenarios and to optimize the probability of controlling and managing high-risk pathogens of agricultural significance. 2) Development and testing of efficient methods of statistical inference to estimate epidemiological parameters from maps of emerging epidemics. 3) Develop user friendly model ‘front ends’ that can be used by researchers and regulatory agencies. During this cycle we have focused primarily on making the models more robust and flexible models to most effeciently analyze and predict the spread of emerging pathogens across a range of scales extending from within-plantation to the landscape and regional scales for both HLB and canker. We are continuing to test the models extensively to assess how host planting age affects the transmission of HLB . We consider the models to be and are developed as part of a flexible tool-box so that they can be readily adapted to new disease threats as well. The principal modeling results for this quarter relate to further testing and refinement of a tool box to estimate parameters for dispersal and transmission using citrus canker and HLB. We now have a set of rigorous Markov-chain Monte Carlo (MCMC) methods to estimate parameters for dispersal and transmission rates of emerging epidemics from successive mapped snapshots of disease. Specifically, we have developed data augmentation to infer the sequence of infections between successive snapshots of disease and used reversible jump MCMC methods to compare alternative models, for example when it is unclear how important a particular biological process is likely to be. MCMC methods were combined with techniques from time series analysis (such as sliding windows estimates) in order to track the value of time-varying parameters such as the transmission rate. We also developed and applied statistical methods to test the goodness-of-fit of the model using inferred parameters against the observed temporal and spatial patterns. We believe this to be the most rigorous analysis of how to fit epidemiological models to emerging epidemics. We have also made significant progress in the implementation of Approximate Bayesian Computation (ABC) algorithms for stochastic, spatially explicit epidemics as an alternative method suited to some epidemics that if successful is likely to be easier to use by non-specialists. We have successfully tested our algorithms on synthetic datasets and small real datasets (including citrus canker data). Work is currently under way to test the robustness of the method, and to apply it to large epidemic datasets. Publications: Cunniffe, N.J., R.O.J.H. Stutt, R.E .DeSimone, T. R. Gottwald, and C.A. Gilligan. 2012. Webidemics: Webbased Interactive Demonstration of Epidemiological Modelling Informing Control Strategies. To be submitted to PLoS One. Gilligan, C. A., F. M. Neri, A.R.. Cook, G. J. Gibson, and T. R. Gottwald. 2012. Bayesian analysis of an emerging epidemic: citrus canker in urban Miami. To be submitted to Journal of the Royal Society Interface. Parry, M.F., G. J. Gibson, T. R. Gottwald, S. Parnell, and C. A. Gilligan. 2012. Spatio-temporal modelling of the spread of HLB/Citrus Greening on orchard scales. To be submitted to Proceedings National Academy Science USA / Journal of the Royal Society Interface.
Objective 1 is to evaluate the potential for soil application of the neonicotinoids, imidacloprid (IMID) and thiamethoxam(THIA), and the commercial SAR inducer, acibenzolar-s-methyl (ASM)(Actigard’, Syngenta), to provide long-lived systemic control of canker caused by Xanthomonas citri subsp. citri (Xcc) in young trees. Objective 2 is to integrate SAR with foliar applications of copper sprays for control of canker on the most canker susceptible cultivar in Florida, grapefruit. Objective 3 is to investigate the integrated use of neonicotinoids THIA (which is more soluble, rapidly taken up and translocated), IMID (which moves more slowly in the soil and plant) and ASM to increase and/or extend disease control activity. Under Objective 1 and 2 soil applications of inducers of SAR at various rates and application frequencies were evaluated for control in a field trial of 3- and 4-year-old ‘Ray Ruby’ grapefruit trees in southeastern Florida. Reduction of foliar incidence of canker produced by one, two or four soil applications of IMID, THIA, and ASM was compared with 11 foliar sprays of copper hydroxide and streptomycin applied at 21-day intervals. In 2008 and 2009 crop seasons, canker incidence on each set of vegetative flushes was assessed as the percentage of the total leaves with lesions. By the end of the 2008 season, despite above average rainfall and a tropical storm event, all treatments significantly reduced foliar incidence of citrus canker on the combined Spring-Summer-Fall flushes. Sprays of copper hydroxide and streptomycin were effective for reducing canker incidence on shoot flushes produced throughout the season compared to the untreated control, whereas soil applied SAR inducers reduced foliar disease depending on rate, frequency and timing of application. Except for the treatment of four applications of ASM at 0.2 g a.i. per tree or two applications of IMID, SAR inducers were ineffective for reducing foliar disease on the flushes that were present during the tropical storm. In 2009, all treatments significantly reduced the incidence of foliar canker on the combined Spring-Summer-Fall flushes but not all treatments of Spring-Summer flushes with SAR inducers were effective compared to the untreated control. Hence, depending on rate, frequency and timing of application, soil-applied SAR inducers reduced incidence of canker on foliar flushes of young grapefruit trees under epidemic conditions. Under Objective 2 integration of soil applied SARs with foliar applications of copper sprays for control of canker, the efficacy of the inducers for control of citrus canker on young fruiting Red grapefruit and Hamlin orange trees was evaluated. To keep the chemicals out of the soil, SAR inducers were applied as sprays on the trunk as well as soil drenches. Trunk application of IMID, THIA and ASM at 3-3.5x the label rate to account for greater tree volume was as effective as soil drench for canker control on fruit but somewhat less effective than 21 d copper sprays on young fruiting trees. Because these trials involve testing of non-registered ASM and the insecticides IMID and THIA at non-labeled rates, fruit from the treated trees must be dropped and destroyed (‘crop destruct’ of nonregistered chemical treatments). Recently, Syngenta was granted an Experimental Use Permit (EUP) by EPA that permits testing of Actigard on up to 300 acres of grapefruit without crop destruct. The trials will act as the first step toward registration of Actigard on grapefruit in Florida. Under Objective 3, the integrated use of ASM, THIA and IMID soil applications to increase and/or extend canker control in 3-yr-old grapefruit and 2-yr-old Vernia orange trees was evaluated. The highest incidence of disease was in the untreated check in each trial compared with a very low incidence of canker in the integrated SAR treatments. Field trials with soil applied neonicotinoids in Parana, Brazil evaluated IMID (Confidor) as a soil drench and IMID (Winner) a formulation for trunk application as well as THIA (Actara) and Clothianidin (Poncho). These neonicotinoids provided canker disease control on 2-yr old Valencia and 3-yr old Pera orange trees. ASM as a single drench application was less effective on the larger 3 yr-old trees. Clothianidin is now registered for use on citrus in Florida, hence all soil applied neonicotinoids used for insect control in Florida have been demonstrated to have SAR activity against canker.
In field trials with Red grapefruit and Hamlin oranges, the non-insecticidal SAR-inducer acibenzolar-S-methyl (Actigard) and the neonicotinoids imidacloprid (Admire Pro) and thiamethoxam (Platinum) have been applied by soil drench or trunk application to evaluate their efficacy for canker control on young bearing trees in St. Lucie Co. (grapefruit) and Hardee Co. (Hamlin) with canker epidemics. For evaluation of contact activity, copper hydroxide (Kocide 3000) or other film forming coppers have been sprayed every 21 days with a handgun (grapefruit) or airbast sprayer (Hamlin) to compare with soluble copper formulations, Magna-Bon (copper sulfate pentahydrate), Cop-R-Quik (Cu nitrate), or novel soluble CuSi NP formulations. For both sets of trials, fruit infection and fruit drop data for the treatments on grapefruit and Hamlin orange have been completed and data analysis is underway.
Objective 1: Preliminary trials conducted with potted nursery and young field trees indicate that soil drench is an effective and consistent application method for increasing copper status of leaves up to several fold over the baseline concentration in leaves. Hence, in a trial in Picos Farm at USDA-Ft. Pierce, Hamlin on Swingle citrumelo trees were root drenched in late spring with the copper bactericides EXP, Cop-R-Quik, (CQ) Magna-Bon (MB), and copper phosphite (CP) and a zinc phosphite (ZP). Spring flush leaves were collected in July to measure copper and zinc concentration in leaves to test for systemic movement of the metals. Overspray of micronutrients obscured detection of treatment differences. Leaf samples for PCR testing were also collected to track Las infection progress. So far incidence of infected trees is less than 1%. To further evaluate soil drenches of the two commercial bactericides containing copper chelate (CQ and MB), three locations were selected. Two trials are in blocks with 1 year old trees that are apparently healthy and a third trial in a block of 4-yr old trees with symptomatic and pre-symptomatic HLB infection as determined by PCR status. Hence, the preventative and curative activity of the copper bactericides are being followed in pre- and early stage HLB infected trees. In the two trials with 1 yr-old young trees, a wide range of copper concentrations in flush leaves has been established. Samples for PCR status of the trees in all trials will be collected and analyzed later this fall.
Objective 1 is to conduct a field evaluation of nutritional sprays for control of HLB and HLB symptom expression and yield. The field study was set up May 2010 in Southern Grove, Hendry Co., FL. Six treatments were located in 4 plots of 150 trees per treatment (interior 10 trees in each block were identified for PCR, leaf nutrition sampling, tree health and yield evaluation). Trees were 8 years old at the initiation of the trial. Treatments were 1) non-treated check; 2) Nutri-Phite sprayed 4 times bimonthly; 3) N-Sure sprayed bimonthly; 4) Agra Sol Mn/Zn/Fe plus Nutri-Phite plus triazone urea sprayed bimonthly; 5) Keyplex 1400 DP plus Nutriphite plus triazone urea sprayed bimonthly; 6) Wettable powder nutrients (Diamond R #2) plus Nutri-Phite P+K sprayed bimonthly. The materials were applied to both sides of the tree in 125 gallons per acre with an airblast sprayer driven at 2 mph to obtain thorough coverage. Five disease ratings have been taken so far with the latest in June 2012 and a slight decline in tree health has been observed, but no significant treatment effects have been detected. The 2011 harvest experienced a 27% yield increase in all treatments compared with the 2010 harvest, but no significant treatment effects were observed. The 2012 harvest will be collected in December. Disease ratings and PCR samples will be taken by the end of this month Objective 2 is to determine the mechanism of HLB symptom suppression by foliar nutritional application, Rep 1 using Hamlin sweet orange trees inoculated with HLB and treated bimonthly with the nutritional sprays treatments 1, 2, 3, and 5 from objective 1 has finished and results are in previous reports. Rep 2 continues with monthly samplings of leaf and root tissue are underway. Root samples are split for qPCR Las quantification and starch analysis for a quantitative measure of phloem function throughout the plant. Microscopy will be continued, however the high variability of phloem plugging and collapse even within the same midrib from a symptomatic leaf makes interpretation of results difficult. In the trial 72 of the 75 inoculated trees are now Las positive with disease symptoms. No treatment effects have been observed, except that phosphite alone results in higher than expected infection of roots within 4 months of inoculation and continues to be the case at 6 months post inoculation. Thin sections of root samples are currently being visualized for potential detection of treatment differences. Foliar symptoms are slightly more apparent on control trees, but no difference in tree vigor as measured by size and frequency of flush has been observed. Multiple trees in all treatments are showing significant decline of fibrous roots health. Final data collection of data from Rep 2 will begin in two weeks when tree vigor will be determined by biomass of leaves and roots and mineral and carbohydrate status of roots and leaves will be analyzed to determine treatment effects on overall tree health and phloem function.
In 2012, Florida field trials of the rates and and timing of soil drenches and trunk treatments with neonicotinoids imidacloprid and thiamethoxam that are congruent with recommendations for use of neonicotinods for systemic control of psyllids are under evaluation on 3-5 yr old bearing Red grapefruit and Vernia orange trees. Actigard, a commercial SAR product, and two novel non-neonicotinoid compounds with demonstrated systemic activity against canker in greenhouse trials are also under field evaluation. SAR activity is also being integrated with foliar copper sprays for evaluation of canker control on non-bearing and young bearing trees. The results from the Florida trials will be summarized in the final report within the next 30 to 45 days. Results from 2011-12 trials conducted in Parana State Brazil confirm the findings in Florida thus far. Soil drenches and trunk treatments of the imidacloprid on two year old Pera orange trees on Rangpur lime rootstock were equally effective for control of canker disease incidence on foliage compared with thiamethoxam and Actigard which were less effective.
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