Report for period ending 9/2015 At the beginning of this reporting period we setup a greenhouse pot study to compare the spatial distribution of insecticide within citrus leaves. Previous preliminary work we conducted suggested that there is likely differences in the distribution of imidacloprid within leaf tissue that may affect the lognevity and amount of control provided by soil drench applications. Here, we examine this further and also examine movement of thiamethoxam and chothianidin as well. The insecticide concentration along the leaf margin was compared to the concentration in the leaf center. Citrus (v. Hamlin / r.s. Swingle) was planted to 3-gal pots containing a custom soil blend (50% sand, 50% potting media). The study was arranged in a randomized complete block design (RCBD) with 4 treatments and 4 replicates. Each plot consisted of 4 citrus trees 3-5 ft in height. A single insecticide application was made to the soil using 8 fl oz of insecticide solution per tree. Leaves were sampled prior to the insecticide application to ensure no insecticide was present, and again every 7 days, until the concentration fell to undetectable levels. At each sampling event, four leaves were removed from the upper canopy of each of the 4 trees within each plot. Leaves will be dissected into two regions: 1. leaf margin region (area 0.5 cm from leaf edge), and 2. leaf center region (area within 0.5 cm on each side of the mid-vein). Concentrations were analyzed using Liquid Chromotography-Mass Spectrometry. Due to number of samples to be analyzed, this experiment will be completed during the next reporting period.
Report for period ending 12/2015 During the previous reporting period, we established an experiment in the greenhouse to compare the spatial distribution of soil applied neonicotinoids within leaf tissues. LC-MS-MS analysis of all of these samples is still ongoing, but we are fairly confident that the preliminary information presented in the following is an accurate representation of what the final results will be. For thiamethoxam, peaked around 2 weeks after application, and slowly declined over the next 8 weeks. The levels of material seen in the plants tissues analyzed across all samples dates was sufficient to control ACP. When tissue samples were separated and analyzed by either leaf margin or leaf center, there was no significant difference between the two for thiamethoxam. For imidacloprid, the concentrations did not peak until 5 weeks after application but even 1 week after application were high enough to control ACP and remained high enough through week 8 of the study. For imidacloprid there was a significant difference in product concentration between the leaf center and leaf margin which we had anticipated based on our previous work. Similar to imidacloprid, clothianidin concentrations peaked 5 weeks after application but were probably not high enough to control ACP 1 week after application. By two weeks after application, concentrations were high enough to have an effect on ACP. Residues remained high enough to control ACP through week 8 of the study. Similar to imidacloprid, clothianidin concentrations were significantly different between the center of the leaf and leaf margins. These results confirmed that large differences do exist between the three soil-applied neonicotinoids in terms of how they move in the plant, become distributed within leaf tissues, and the duration of control. This information will be used to design the next set of experiments examining these and other more pertinent questions under field growing conditions.
Report for period ending 3/2016 During this reporting period we setup an series of growth chamber experiments to evaluate effect of seasonality on neonicotinoid uptake by citrus trees in the laboratory. This is being done to determine if there is any effect of season and/or transpiration rate to neonicotinoid expression in citrus foliage for each of the three neonicotinoid chemistries. Citrus (v. Hamlin / r.s. Swingle) was planted to 3-gal pots containing a custom soil blend (50% sand, 50% potting media). Potted plants were divided between two growth chambers with unique environmental conditions: 1) winter-like conditions characterized by low temperature, short day length, dry soils, and low light intensity (reflect January 30-year average in Immokalee, Florida), and 2) summer-like conditions characterized by high temperature, long day length, wet soils, and high light intensity (reflect August 30-year average in Immokalee, Florida). Plants in each growth chamber were arranged in a randomized complete block design (RCBD) with 4 treatments and 4 replicates. Each plot consisted of three citrus trees. A single insecticide application was made to the soil using 8 fl oz of insecticide solution per tree. Leaf tissue samples (n=6 leaves per tree) were collected weekly until 4 weeks after application. Leaves were excised to differentiate concentrations between the leaf center and leaf margin. In addition to quantifying neonicotinoid expression, the transpiration rate of 4 individual trees were measured in each growth chamber using a Dynamax Sap Flow meter system. A single tree from each treatment was represented in each growth chamber. The initial and final canopy volume and stem diameter for each of the 8 trees was recorded.
Report for period ending 6/2016 Over the past year, studies using electropenetrography (EPG) studies have been conducted with the goal of determining how much imidacloprid is needed in leaf tissues to control ACP, in particular to disrupt psyllid phloem-feeding behaviors. During these studies, we have continued to increase the dose of imidacloprid delivered to plants in order to reach the point where 100% of psyllids are incapable of reaching the phloem. However, the results obtained to date using EPG suggest that even at unrealistically high levels of imidacloprid applied to the plant, an average of 2% of the psyllids are still likely to perform some feeding behaviors in phloem. However, our data suggests that because the proportion is so low, and not every feeding bout by a psyllid results in infection, this use of imidacloprid to reduce infection in young tree plantings is still a very useful tool. However, some results obtained here suggest that the soil-applied neonics ability to prevent infection may be due more to feeding deterrence rather than direct mortality. Thus, we initiated experiments using an artificial diet-based bioassay to develop a dose-response curve for the three neonics versus the asian citrus psyllid. The majority of time spent this quarter was in perfecting the artifical diet system building upon the success of other researchers who have used such diets for studying ACP. Our goal here is to develop a feeding-based LC50 and LC90 for imidacloprid, clothianidin and thiamethoxam. THese LC50/90 values will then be compared to those previously reported values for contact bioassays using either leaf dip or vial assays. Concurrent with this work, we are continuing to analyze the large backlog of leaf samples gathered from our ongoing field studies where we are investigating the uptake of the three neonics at different times of the year, distribution of the three neonics within a tree, and appropriate rate of product applied based on tree size. We literally have thousands of samples in the freezer awaiting analysis. We continue to run samples as fast as the machine can analyze them.
Report for period ending 9/2016 In the last reporting quarter we began work with artificial diets to develop a dose response curve to help determine the amount of the three neonicotinoids that are needed in order to control ACP. We were able to master the use of an artificial diet to deliver varying concentrations of imidacloprid, thiamethoxam and clothianidin to psyllids as they feed to model the toxicity and behavioral changes that would occur when feeding on treated plants. The results from this study corroborated our findings last reporting period where we used EPG to demonstrate that the soil-applied neonics primarily control psyllids through feeding deterrence as it took large levels of all three insecticides to kill 90% of the population. Of special interest, we found that the LC50 and LC90 values for feeding exposure to neonics were far greater than the values obtained for contact assays. This discovery has led us to pursue two additional studies this next quarter where we will use our feeding bioassay against psyllids from different parts of the state to see if the results from our lab colony are consistent with the wild populations of psyllids in Florida. We will also begin using the artificial diet bioassay in our EPG studies to get a better gauge of exactly what concentrations of insecticides psyllids must be exposed to to quit feeding…but not necessarily die from that exposure. Previously this could only be estimated in past studies where plants were treated with varying doses of insecticides. Now, with the artificial diet assay, we can more reliably determine the amount required to cause psyllids to withdraw their mouth parts from treated plants. Concurrent with this work, we are continuing to analyze the large backlog of leaf samples gathered from our ongoing field studies where we are investigating the uptake of the three neonics at different times of the year, distribution of the three neonics within a tree, and appropriate rate of product applied based on tree size. We literally have thousands of samples in the freezer awaiting analysis. We continue to run samples as fast as the machine can analyze them.
Report for period ending 12/2016 During this quarter we surveyed ACP populations across the state to compare the wild populations of ACP with our laboratory colony in terms of amount of the three neonics required to control ACP based on method of delivery. Sites chosen to collect psyllids included locations in Vero Beach, Lake Placid, Lake Alfred and LaBelle FL. In all cases, the wild psyllid populations responded in a similar manner to our laboratory colony with the LC50 and LC90s for ingestion being far greater than those values for the contact assays. We also did note some variation in the amount of product required to reach an LC90 value. This results suggests that more monitoring should be conducted for potential shifts in psyllid susceptibility to the nenicotinoid insecticides. We have also been making great progress with our EPG studies of psyllid feeding on artificial diets to determine the amount that is needed to cause psyllids to quit feeding (not necessarily die) and thus reduce transmission probabilities. Here we have been able to identify the waveforms produced that are drastically different from those in plants and then begin record and analyze psyllid feeding on artifical diets containing varying levels of iinsecticides to develop the LC50/90 data for mouthpart withdrawal. As previously reported…we are continuing to analyze the large backlog of leaf samples gathered from our ongoing field studies where we are investigating the uptake of the three neonics at different times of the year, distribution of the three neonics within a tree, and appropriate rate of product applied based on tree size. We literally have thousands of samples in the freezer awaiting analysis. We continue to run samples as fast as the machine can analyze them.
The objective of this research project is to investigate and develop a potential non-phytotoxic, environmentally-friendly film-forming ACP repellent solution for preventing HLB infection. In the last reporting period, OS-SG 15 and OS-SG 16 were tested for their plant safety and surface coverage , along with material characterization using infrared spectroscopy. Testing of those formulations were compared to commercial control, Surround WP. Preliminary results for this testing revealed comparable, but no significant improvement over commercial control for surface coverage. With surface coverage being of paramount importance in preventing infection via ACP-leaf interaction, further material development was needed. During this current reporting period , a new formulation, OS-SG 17 is being developed to overcome the weaknesses found in previous versions of the material. A new, all natural EPA approved silica source (“Fumed Silica”) was used as a silica substrate to create a multi-layered silica gel matrix when combined with our previous silica composite. The hydrophobic nature of the “fumed silica” is expected to increase the surface coverage of the new OS-SG 17 composite. Different iterations of OS-SG 17 are being studied as controls using our previously used EPA approved polymer to improve stability, dispersion and rainfastness. This new formulation is expected to display high colloidal stability in aqueous solution, high surface coverage and moderate rain-fastness properties. It was characterized using UV-Vis and FTIR spectroscopy. The colloidal stability of the formulation was checked via measuring %Transmittance (%T) of the supernatant collected from the solution left undisturbed. The formulation revealed less than 50 % transmittance up to 8+ hours which was found to be better compare to commercial control -Surround WP. Testing of this new version is in progress to ascertain the extent of its surface coverage and confirm that it does not create an unfavorable temperature increase on leaf surfaces. Phytotoxicity studies were conducted using a Panasonic Environmental Test Chamber (Model MLR- 352H) to control light intensity, humidity and temperature cycling to simulate summer conditions (85% RH, 32 Celsius). OS-SG 17 formulation did not cause any plant tissue damage at the applied rates, matching the commercial control. Next we will conduct ACP repellent studies using psyllid containment cages which have been acquired recently.
Irrigation water acidification (target pH, 7.5, 6.0, 5.0, and 4.0) continues at two citrus groves (one a 20 year-old Hamlin sweet orange trees predominately on Swingle rootstock and the second a three year old Hamlin sweet orange trees on Swingle rootstock). The last sulfur application in this study will be made to selected treatment blocks in January 2017. Soil samples taken prior to the summer 2016 sulfur application in June indicated that plots receiving both acid injection and sulfur had soil pH significantly lower than plots receiving only irrigation water acidification. Soil samples taken in December indicated that soil pH in plots receiving both irrigation water acidification and sulfur application had similar soil pH. These results would indicate that the relatively slow release sulfur product (Tiger 90) reduced soil pH below that achieved by irrigation water acidification only but lasted less than six months. Root density samples taken in June indicate a significantly greater root length density with lower soil pH. These results indicate a positive correlation between root density and reduction in soil pH from greater than 7.0 to less and 5.0. Leaf Ca, Mg, Mn, and Zn in November samples were greater for trees treated with both irrigation water acidification and sulfur application compared with irrigation water acidification only. These results verify previous finding that leaf nutrient status is negatively correlated with soil pH. Thus, tree nutrient status is increased with lower soil pH. Tree size and fruit drop measurements indicate significant growth with reduced soil pH to approximately 5.0 with no additional decrease below that level. A talk on methods of irrigation water acidification and expected improvements on citrus nutrient status will be given on January 19, 2017 to growers attending a nutrient BMP meeting at the SWFREC. Since previous grower presentation, numerous personal contacts have been made with individual citrus growers to continue, alter or initiate soil acidification projects in groves throughout the state.
Trees and branches to monitor for vegetative and reproductive bud development were selected in the test blocks and initial ratings were established. The Flowering Monitor System provided an initial flowering wave full bloom date of February 11 to 20 depending on the location within the Florida citrus production regions. A second wave of flowering wave projected to occur from March8 to 11 depending on location. Data collection is now started with bud break estimated to occur at the time of this report. Dr. L. Stelinski has agreed to cooperate in evaluating psyllid control when a block is sprayed at the beginning of spring budbreak rather than later after feather leaves are present. Two locations will be evaluated until full bloom, one near Frostproof and another near Lake Alfred. We will have our third year of data on the days before full bloom that bud break occurs, which presently is about 56 days.
The objective of this project was to identify a Bacillus thuringiensis (Bt) crystal toxin with basal toxicity against Asian citrus psyllid (ACP) and to enhance the toxicity of the selected toxin by addition of a peptide that binds to the gut of ACP. The added peptide is expected to enhance both binding and toxicity of the toxin against ACP. Having identified two Bt toxins with toxicity to ACP, the most toxic of these ACP-active toxins was modified with gut binding peptide 18. The sequence encoding ACP gut binding peptide 18 was introduced into the toxin at four different sites. However, some of the modified toxins did not express well in E. coli. To identify the optimal system for toxin expression, ACP-active and wild type toxins are being expressed in Bt acrystalliferous strains 4Q7 and 78/11 using E. coli – Bt shuttle vectors. Once toxin expression has been optimized, toxins will be purified for use in ACP bioassays to test for enhancement of toxicity.
We have continued to investigate movement of Asian citrus psyllid (ACP) as it relates to biotic and abiotic factors. We have continued to investigate how previous experience and learning affects ACP movement behavior as it relates to developing practical pest management tools for managing this pest with behavior modifying chemicals. We designed three experiments to test whether males learn about female odor, specifically in the context of mating, where copulation acts as a biologically significant unconditioned reinforcer. First, we compared the responses of mated and virgin males to female odor (proxy for volatile cuticular hydrocarbons thought to function as sex pheromone). If male attraction is experienced-dependent, we expected only mated males to show preference for female odor. Then, we compared the acquisition of response of a novel olfactory stimulus, vanillin, in males exposed to the odor under different conditions: as an environmentally derived odor, an odor associated with females directly, and an odor associated with a food source. If the learned responses were explicitly linked to mating experience, we speculated that only the males exposed to vanillin associated with females directly should demonstrate a learned response. Finally, we compared antennal responses of virgin and mated males to female cuticular extracts electrophysiologically to determine if changes in male response to female odor are caused by peripheral sensitization or true learning. In the ACP stimulatory cuticular hydrocarbons act as sex pheromone attractants. Male psyllids locate aggregations of females using those olfactory cues, as well as vibrational communication on the plant surface. Although previous research has indicated that learning plays a role in modulating female reproductive behaviors in psyllids, it is unknown whether males similarly use learning to increase fecundity. We used an olfactometer-based bio-assay to study the effects of experience on male response to female odor. First, we compared male attraction to female odor in virgin and previously mated males. Second, we tested the effect of several modes of experience with a novel odor, vanillin, to determine whether mating, feeding, or general environmental exposure elicited a learned response. We found that male attraction to female odor significantly increased after mating experience. In addition, we found that males learn about odor specifically in the context of mating, rather than feeding or general exposure. Electrophysiological measurements of antennal response to odorants confirmed that mating status did not affect the sensitivity of the peripheral nervous system to volatile stimuli implicating learning at the level of the central nervous system. These results suggest that male response to female odor is not an innate behavior. Males require mating experience with female conspecifics to develop attraction to those olfactory cues in the environment. This adaptive plasticity may allow males to detect females in an ever-changing environment and promote diversification and further specialization on different host genotypes. Our results may have implications for development of behaviorally based management tools for ACP. Semiochemical-based management tools for ACP are currently under development. Non-traditional control methods are also being explored, given the importance of this pest. If male attraction to female odor is experience-dependent, it may be possible to manipulate male behavior in such a way that mate detection and reproduction is suppressed. For example, appetitive learning in insects is specifically mediated by octopamine (RS-4-(2-amino-1-hydroxy-ethyl) phenol), a biogenic amine neurotransmitter found almost exclusively in invertebrate species. While octopamine occurs in vertebrates, it does not appear to have a major role as compared with norepinephrine. Indeed, appetitive learning is significantly suppressed in insects when octopamine antagonists are administered. It therefore may be possible to introduce a novel form of mating disruption in agricultural settings by disrupting a target species ability to learn. While the potential for such application is distant and would require significant effort to develop a safe and target-specific method of application, pest control options are expanding with better understanding of the target species ecology and behavior.
The overall goal of this project is to improve insecticide resistance management for Florida populations of Asian citrus psyllid (ACP). We are achieving this through investigations of the mechanisms of resistance, monitoring resistance in the field, development of optimized rotation skills, and evaluations of new tools for implementation into these rotation schedules. One of the major obstacles facing Florida citrus growers is a lack of a sufficient number of modes of action for management of to achieve efficacious and cost-effective rotations season-long. Currently, and unfortunately, this sometimes requires application of the same mode of action more than one time per year. We therefore continue to investigate the physiological consequences and effectiveness of alternative modes of action against ACP. We continue to investigate three different rotation modules using dimethoate, adamectin, fenpropathrin, diflubenzuron and imidacloprid. We have there were three rotation models that are producing the best results to minimize development of insecticide resistance and one positive control and one negative control. Each treatment is replicated four times. Before application we use a leaf dip bioassay to determine susceptible levels of ACP populations. We monitor ACP adults, eggs and nymphs weekly and determined when insecticide applications should be made based on a threshold of adults = 2, eggs =5 and nymphs =5 per per average sampling per sample date. This experiment remains currently in progress, but all three rotation models that we have developed appear to be quite promising to maintain the effectiveness of our currently available modes of action for ACP viable. A wide variety of insecticides are used to manage ACP populations within citrus groves in Florida. However, in areas shared by citrus growers and beekeepers the use of insecticides may increase the risks of honeybee (A. mellifera) loss. In addition to developing the most effective insecticide rotation schedules, we have investigated the potential non-target effects of our rotation modules on beneficial insects. The objective of this research was to determine the environmental toxicity of insecticides, spanning five different modes of action used to control ACP, to A. mellifera. The insecticides investigated were imidacloprid, fenpropathrin, dimethoate, spinetoram and diflubenzuron. In laboratory experiments, LD50 values were determined and ranged from 0.10 to 0.53 ng/ l for imidacloprid, fenpropathrin, dimethoate and spinetoram. LD50 values for diflubenzuron were > 1000 ng/ l. Also, a hazard quotient was determined and ranged from 1130.43 to 10893.3 for imidacloprid, fenpropathrin, dimethoate, spinetorama. This quotient was < 0.465 for diflubenzuron. In field experiments, residual activity of fenpropathrin and dimethoate applied to citrus caused significant mortality of A. mellifera 3 and 7 d after application. Spinetoram and imidacloprid were moderately toxic to A. mellifera at the recommended rates for ACP. Diflubenzuron was not toxic to A. mellifera in the field as compared with untreated control plots. Phenoloxidase (PO) activity of A. mellifera was higher than in untreated controls when A. mellifera were exposed to 14 d old residues. The results indicate that diflubenzuron may be safe to apply in citrus when A. meliifera are foraging, while most insecticides used for management of ACP in citrus are likely hazardous under various exposure scenarios.
This project looked at the in vitro sensitivity of Guignardia citricarpa, causal agent of citrus black spot (CBS) for demethylation inhibitor fungicides (DMI; FRAC 3), succinate dehydrogenase inhibitor fungicides (SDHI; FRAC 7), an aniline-pyrimidine (FRAC 9), and a phenylpyrrole (FRAC 12). This allowed for the establishment of baseline sensitivities for those fungicides registered for citrus along with estimating whether it was worth placing them in a year-long field trial. Three DMI fungicides showed promise: difenoconazole, fenbuconazole, and propiconazole. The difenoconazole and fenbuconazole were found to be efficacious in the field trials and were recommended for CBS management. Imazalil is used post-harvest but did not have great in vitro efficacy or an ability to suppress lesion development post-harvest. The three SDHI fungicides were all efficacious in vitro and boscalid was recommended for CBS management based on field trial results. The two newer SDHI fungicides have not been field tested as of this date. The aniline-pyrimidine and the phenylpyrrole were not pursued further based on in vitro results. There was a spatial distribution study of G. citricarpa in the field and it was found that there was a clustered distribution of the disease in groves. There were several post-harvest studies undertaken. One looked at the effect of heated fungicides. It was found that heating the fungicide solution did not improve the efficacy nor did hot water treatments by themselves. Chitosan was also tested as a post-harvest control but did not have any particular effect on lesion development.
The objective of this project was to quantify the relative effect of copper (Cu), windbreak (Wb) and leafminer control (Lc) on the spatial and temporal progress of Asiatic citrus canker (ACC) under conducive conditions for epidemics and crop loss. The experiment was set up in a 10 ha plot planted with Valencia sweet orange grafted on Rangpur lime located in the municipality of Xambre, Paran , Brazil. The different treatments were the combination of up to three control measures (Cu, Wb, Lc) or none. The presence or absence of windbreak represented a plot. The presence or absence of copper sprays and leafminer control represented a subplot. Each subplot was composed of 112 trees. Each of the eight treatments had three replicates. Cu treated plots were sprayed with Kocide (35% metallic copper) at 1 kg metallic copper/ha every 21 days. Lc was performed with application of abamectin at 150 ml/ha every 21 days. Casuarina was used as a natural Wb around the plots. Disease evaluations started in December 2013 and included percentage of trees, leaves and fruits with ACC symptoms, and fruit yield. In the second season, the assessment of fruit drop was included. The present CRDF funding covered the period of November 2015 to October 2016, which corresponds to the second entire season assessed in the trial. The results observed during this season (2015-2016) followed the same trend of the previous one. Although the incidence of trees with citrus canker reached 100% for all treatments, the progression rate was lower in plots under complete management than trees with no management. The incidence of diseased trees in non-managed plots reached 100% in March 2015, 16 months after epidemics started, whereas, in managed plots this incidence was observed only a year later. Peaks of leaves with ACC symptoms in the last season for plots with complete management and no management were 2 and 42%, respectively. Fruit drop per tree due to canker was 109 and 18 for unmanaged and managed trees, respectively. These drops represented 37 and 5% of the crop loads, respectively. At harvest, incidence of fruit with ACC was 8 and 70% for managed trees and non-managed trees, respectively. Finally, average yield of trees treated with Cu+Wb+Lc was 84 kg, 83% higher than in trees under no management, which produced 46 kg. All measures reduced disease losses, but the combination of Cu and Wb provided the greatest disease control. Lc did not significantly contribute to reduce ACC incidence and disease impact on fruit loss.
June 2016 The objectives of this proposal are 1) To determine the temperature and relative humidity optima for Guignardia citricarpa pycnidiospore infection and production on citrus twigs, leaf litter, and fruit; 2) To determine the relative potential of Guignardia citricarpa to form pycnidiospores on citrus twigs, leaf litter, and fruit; 3) To determine whether Guignardia citricarpa can survive and reproduce on citrus debris on grove equipment. Experiments to confirm initial relative humidity findings continue. After the inconclusive results of the second experiment, we started a third experiment. We used fresh cultures to ensure better pycnidia performance. The results were gathered at 5 weeks post-inoculation but again the results were inconclusive because a mistake was made in the incubation conditions by a new employee. A site has been found to conduct field experiments of inoculum potential and preliminary work is continuing. We have improved our method of collection of conidia from twigs. Collection continues at two week intervals. Methods to improve sampling for DNA extraction with the quantity of twigs are being examined. Experiments were started to look at the effect of temperature on the level of sporulation of P. citricarpa. It can be quite difficult to get consistent sporulation even under controlled conditions. The temperatures that are being tested 15, 20, 24, 28, 32, and 36C. Data has not been collected yet. Work on the effect of FDACS recommended disinfectants (200 ppm bleach or 2000 ppm quaternary ammonium) on conidia germination was conducted. Effective concentrations to inhibit either 50% or 90% of conidia germination for 2 quat products, Canker Solve and C-Quat, and bleach. were found to be well below 5 ppm for all products. Bleach was about ten times more effective but is not as stable as quat. New experiments are being planned to look at the effect of plant debris on the efficacy of these products to have a more ‘real’ world test of these products.
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