The overall objectives of these studies were to provide predictive knowledge of the time of spring leaf flush and flowering for spring flush ACP control and the time for optimum bee foraging in. Weather, flowering and leaf flush data were collected and used to improve the Citrus Flowering Monitor System. Particular goals were to improve near-bloom temperature responses of the model and add leaf flushing time to the flowering time in the monitor system. The 10 % open flower and petal fall dates as the start and ending times of bee activity were determined. Overall the on-line Citrus Flowering Monitor System was improved to allow easy movement to different FAWN sites for flowering evaluations. Further, it now provides dates of initiation of growth and hours of induction to those dates, plus full bloom dates along with the graphic data. Visible budbreak, spring leaf development and flower bud development stages were monitored for three years. Data were collected in order to determine time of budbreak, leaf feather flush stage as well as the intended 10 % open flowers and 95 % petal fall. The peak flowering period was monitored to test it for the required period for minimal bee toxic ACP sprays. We discussed with growers and beekeepers the spray scheduling feasibility with concern of the grower for adequate ACP control and beekeepers for adequate safe access to citrus flowers for their bees. The active bee period was identified, but scheduling issues exist. About 55 days were required from bud break until full bloom, while 10 to 16 days were required from 10 % open flowers to full bloom and another 8 to 20 days from full bloom to 95 % petal fall, roughly a 24 to 30 day period in each flower bud cohort s development for bee activity. Two examples, normal bloom dates at Lake Alfred in 2015 and 2016 were 2/11 and 3/3 in 2015 (20 days apart) and 3/30 and 4/10 in 2016 (11 days apart). This increased the flowering period 11 or 20 days increasing the bee active bloom period to at least 35 to 50 days. Discussions with the CREC Entomologists suggests that a better psyllid control strategy may be to control (spray for) adult psyllids at budbreak and try to get maximum control into the bloom period (about 55 days until full bloom) and then use bee friendly pesticides for psyllid control until the bloom period is over. With two growers in the Ft. Meade area, using 4 blocks, psyllids were monitored after spraying at budbreak. Spray control of psyllids from one spray at budbreak lasted over 30 days. A second spray then continued control into the bloom period. Monitoring budbreak of the first summer flush also appears to be a good second window for effective psyllid control of the second synchronized flush each year. An aerial spray at spring and first summer budbreak followed by second sprays could provide good protection of the 2 major flushes with just four sprays.
The goal of this project is to develop management strategies which boost natural defense mechanisms to control Huanglongbing (HLB) disease by counteracting salicylic acid (SA) hydroxylase of Ca. Liberibacter asiaticus (Las). This project contains two objectives: 1) Control HLB by optimization of application of SA and its analogs. We are testing the control effect of SA and its analogs, e.g., ASM, Imidacloprid, DL-2-aminobutyric, 2,6-dichloro-isonicotinic acid, and 2,1,3 Benzothiadiazole via trunk injection in field trial. Oxytetracycline is used as a positive control, whereas water was used as a negative control. SA, Acibenzolar-S-methyl (ASM), benzo (1,2,3) thiadiazole-7-cabothionic acid S-methyl ester (BTH), and 2,6-dichloroisonicotinic acid (INA) have also been applied twice onto selected trees by foliar spray in November, 2015 during fall flush, arch 2016 during spring flush, and February 2017 during spring flush. In addition, three field trials for different compounds including SA are being arranged. Materials were applied once onto selected trees by foliar spray in September, 2016 during late summer-fall flush, were applied to selected trees by soil drench in September, 2016 during late summer-fall flush, in early March and June 2017. Trunk injection in August and September, 2016 during summer and late summer-fall flush. Trunk injection of SA showed significant control effect against HLB. The data for trunk injection has been collected and a manuscript has been submitted for publication. HLB disease severity,disease incidence surveys and Las titers were conducted before spray treatment in October, 2015 and at 6 months after the 1st application in April, 2016 and April 2017. To compare the effect of suppressing SA hydroxylase, we also screened multiple SecA inhibitors which suppress the secretion of important virulence factors. Two effective SecA inhibitors are being tested in vitro. We are also investigating the possibility of modifying pathway of citrus to produce more SA in citrus using CRISPR. 2) Control HLB using a combination of SA, SA analogs or SA hydroxylase inhibitors. The SA hydroxylase protein is being expressed in E.coli and purified. Several inhibitors identified using structure based design are being tested for their inhibitory effect against SA hydroxyalse. To further identify SA hydroxylase inhibitors or SA analogs that are not degraded by SA hydroxylase, we have expressed SA hydroxylase in tobacco and Arabidopsis. Overexpression of SA hydroxylase decreased HR induced by Pseudomonas spp, indicating that SA hydroxylase degrades SA. We have qualified SA with HPLC and conducted SAR related genes expression analysis. We have identified multiple SA analogs and are testing whether they can be degraded by SA hydroxylase. One manuscript entitled: ‘Candidatus Liberibacter asiaticus’ Encodes a Functional Salicylic Acid (SA) Hydroxylase That Degrades SA to Suppress Plant Defenses” has been published by MPMI.
The goal of the proposed study is to understand the mechanism of survivor trees. 1. Understanding the role of endophytic microbes from survivor trees. Three healthy and three HLB infected trees were selected for phytobiome analysis from Gapway grove based on the Las QPCR detection results. The microorganisms collected from this experiment were classified as three types: rhizosphere, rhizoplane and endosphere communities. The DNA and RNA samples were sequenced. Around 10 Gb clean reads data was generated per metagenome sample. Totally around 120 billion bp (120 Gb) were pulled together for assembly.The final assembly was composed of 17,676,569 contigs longer than 200 bp, totally 10.8 Gb, the longest contig length was 536,098 bp, average length was 613 bp and the N50 was 651 bp. The statistics indicated the quality of the assembly was good. Multiple known beneficial microorganisms, such as Bradyrhizobium, Lysobacter and Variovorax showed significantly higher relative abundance and activity in rhizoplane microbiome despite of health status. However, several beneficial taxa, including Rhodopseudomonas, Achromobacter, Methylobacterium and Chitinophaga, showed higher relative abundance and activity in healthy rhizoplane microbiome compared with rhizosphere community in healthy trees but not in HLB samples. By performing comparison between healthy and HLB samples, we found several phyla, such as Proteobacteria, Acidobacteria and Bacteroidetes were enriched in healthy root-associated microbiome. HLB altered the rhizoplane microbiome by recruiting more functional features involved in autotrophic life cycle such as carbon fixation, and abandoning the functional genes involved in microbe-host interactions identified above, collectively resulting in downward spiral in rhizoplane microbiome-host interaction. This seems to suggest the manipulation of the root microbiome is necessary. However, the challenge is how to maintain a beneficial microbiome which is under study now. Objective 2. To illustrate whether the endophytic microbes from survivor trees could efficiently manage citrus HLB. As shown in Objective 1, Bradyrhizobium and Burkholderia are the most abundant bacteria that have shown dramatic changes between survivor trees and HLB diseased trees. Members of Burkholderia and Bradyrhizobium have been known to benefit plants. We determined the contribution of Burkholderia to the citrus hosts. We isolated multiple Burkholderia strains. We selected two representative strains A53 (Burkholderia metallica) and A63 (Burkholderia territori) to inoculate citrus plants using the soil drench method. The results demonstrated that the two strains could successfully colonize the root surface and maintain a relative high population even seven months after inoculation. We then conducted a greenhouse study to evaluate the effects of the selected strains on the plant fitness. Salicylic acid (SA)-mediated ISR is an important benefit of beneficial bacteria to the plant host. We determined the expression of three SA mediated ISR marker genes, SAM, PR1 and PR2, of the inoculated trees. Plants treated with strain A53 exhibited a significant upregulation of PR2 gene at 3 dpi compared with negative control plants. A63 induced expression of the SAM gene at 5 dpi and the PR1 gene at 7 dpi. Similarly, Actigard induced the PR1 and SAM gene expression at 5 and 7 dpi. Large scale experiment is ongoing. In addition, we grafted the roots from survivor trees to healthy and HLB diseased trees in greenhouse to check the effect of endophyte changes on the grafted trees. Since endophytes appear to be enriched from the rhizosphere, we also used the soil from the survivor trees to plant both healthy and HLB diseased trees in the greenhouse. We also grafted shoots from survivor trees to further understand the putative mechanisms. The work is ongoing. We are testing the effect of application of isolates on plant defenses and attractiveness to psyllids. Two manuscripts have been accepted for publication.
December 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. Samples collection for inoculum potential continue to be gathered biweekly. Bark is being removed from the twigs and DNA extracted from that. Since determining the best way to extract DNA from the field twigs took longer than expected, processing previously collected twigs continues and qPCR has not been conducted in a systematic manner yet. To get a larger range on temperatures and relative humidities, preliminary studies on the stability of RH measurements for different super saturated salt solutions at a temperature range has been conducted and a statistical analysis to determine whether the variability in RH measurements is excessive for the various salts. The selection will be based on the statistical outcome 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. After incubation in complete darkness to avoid the confounding effects of light, it was found for 5 isolates that 24C was the best temperature for sporulation (P < 0.05) followed by 28C. The repetition of the experiment is not yet completed. 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. The disinfectants have been preliminarily evaluated in the presence of finely ground plant debris (twigs and leaves as would be found on mowers or hedgers). Citrus debris itself had no significant effect on conidia germination but there was a significant effect on the efficacy of the disinfectants. Disinfectant treatment in the presence of citrus tissue debris has a much lower efficacy than determined from previous experiments lacking citrus tissue debris. This loss of efficacy can be attributed to two factors. The first is a reduction in potency due to the presence of tissue debris within the liquid treatment. The second and more profound factor is the availability of disinfectant as a free liquid. When disinfectant is applied at low volumes and entirely or mostly absorbed by the tissue debris, the ai concentration must be raised greater than 200 fold to provide comparable efficacy observed in the presence of free liquid. Confirmatory experiments are being conducted.
Predicting the emergence and arrival of insect pests is paramount for integrated pest management. To achieve this goal, it is important to understand how abiotic factors influence pest dispersal behavior. We investigated the effects of abiotic conditions on flight initiation by the Asian citrus psyllid, Diaphorina citri Kuwayama. We first explored the effect of barometric pressure changes on flight initiation. We used a custom-made barometric chamber and observed the activity of D. citri as measured by the number of psyllids captured on yellow cardboard panels coated with adhesive. We found that psyllid flight initiation changed in response to variations in barometric pressure rather than to differences in stable pressures. D. citri were equally active at 1009 mbar and 1022 mbar. However, D. citri dispersed more as barometric pressure increased, and less when barometric pressure decreased. In a subsequent experiment, we manipulated temperature and relative humidity and observed how D. citri dispersed between citrus plants. Psyllids dispersal increased linearly with temperature. Changes in humidity did not affect dispersal of D. citri. Less than 1% of psyllids dispersed at 15 C, compared to 7.7% at 21 C and 27% at 25 C. The minimal threshold for D. citri to initiate flight is estimated to be 16.5 C. Collectively, our results provide an initial step toward developing predictive models of D. citri movement as influenced by abiotic factors. Densities of an herbivorous pest may be impacted by landscape and orchard architecture. We present two orchard experiments where the densities of the Asian citrus psyllid (Diaphorina citri) were compared depending on: (1) the presence or absence of a windbreak and (2) if the orchards consisted of a solid set re-planting or an orchard with a mixture of mature and reset-replacement trees. (1) Psyllid abundance was measured on the edges of five orchards. The factor investigated was the presence or absence of a windbreak. We observed significantly fewer psyllids on the edges of orchards with windbreaks as compared to those without windbreaks. We found no significant difference in the number of natural enemies between the edges with or without windbreaks, suggesting that windbreaks do not affect densities of psyllid natural enemies. (2) During two consecutive years, we compared the densities of psyllids on young trees less than 3 years of age in a solid set re-planting versus on resets (trees planted in replacement of dead or huanglongbing-infected trees) present randomly within mature orchards. This was conducted in four orchards and among three citrus varieties. More psyllids were found in the solid set re-plantings as compared with on the resets within mature orchards. To our knowledge, this is the first report to demonstrate that the planting strategy of new trees in orchards may impact the populations of a horticultural pest. Overall our data suggest that establishment and conservation of windbreaks might be beneficial to protect orchards from D. citri. The data also suggest that D. citri populations increase more within uniform landscapes of seedling trees as compared with mature orchards with randomly interspersed young seedlings.
The goals of this project were to 1) identify toxins derived from Bacillus thuringiensis (Bt) with toxicity against Asian citrus psyllid (ACP), 2) isolate a peptide that binds to the gut of ACP, and 3) modify the selected Bt toxin with the gut binding peptide to provide an artificial anchor for the toxin resulting in enhanced toxicity. We screened toxin mixtures derived from 35 strains of Bt with diverse insect toxicities and toxin profiles for toxicity against ACP. The bioassay protocol was optimized to avoid toxin precipitation, but control mortality was high in some of the bioassays. Of the 35 strains screened, statistical analysis could only be conducted on data generated for 18 strains. Of these, toxin mixtures derived from a total of six Bt strains showed toxicity to adult ACP at 500 g/mL of proteolytically activated toxin. Individual toxins from one of these strains were identified by LC-MS/MS, cloned, expressed, and tested against adult ACP. Of four individual toxins tested, one showed significant toxicity against adult ACP at 500 ug / ml. Given the smaller size of nymphs and large volumes of phloem ingested, it is expected that toxin efficacy will be greater against nymphs than against adult ACP. ACP mortality was associated with severe disruption of the midgut epithelium for the individual toxin and toxin mixture. The microvilli that line the gut epithelium were disorganized or lost, consistent with the mode of action of Bt-derived toxins. Toxicity also correlated with a drop in honeydew secretion indicative of reduced feeding which is also consistent with Bt toxin effects. We screened a phage display library to identify short amino acid peptide sequences that bind to the gut epithelium of ACP. This screen resulted in identification of four candidate peptides. Of these, two were shown to bind to the ACP gut when fused to the reporter enzyme, mCherry but only one, peptide 15, was found to bind specifically to the gut in competition assays. Peptide 15 was shown to bind to a 50 kDa gut protein. Having identified specific insertion sites in the ACP-active Bt toxin, we constructed a set of modified toxins by addition to- or replacement of- amino acid sequences at four different sites in the toxin, with peptide 15. Some of the modified toxins did not express well using standard expression in E. coli. Expression of these modified toxins using different expression strategies is now underway. Once the expression protocol has been optimized, modified toxins will be purified and tested against ACP. It is expected that peptide 15 will provide a peptide anchor for increased toxin binding to the ACP gut epithelium, resulting in enhanced toxicity against ACP.
Obj. 4. The purpose of this objective is to determine whether prior pathogen or dsRNA exposure inhibits Las acquisition by psyllids. We investigated if D. citri exhibits immune priming and produces a different response to secondary infections and the specificity of that protection. To force D. citri to consume bacteria, they were held on an artificial feeding sachet. The artificial feeding sachet was constructed from a petri dish (35 mm x 10 mm) with the bottom removed and covered with thinly-stretched Parafilm (Bemis NC, Neenah, WI). A and two pieces of thinly stretched Parafilm (Bemis NC, Neenah, WI) with a filter paper disc (2.6 cm dia) with 300 l of diet solution was placed on the Parafilm and covered with an additional Parafilm layer (Russell and Pelz-Stelinski, 2015). The diet solution consisted of 17% sucrose in deionized, distilled water, 30 l/ml of neon green food coloring (McCormick & Company, Inc., Sparks, MD). Total bacteria concentration in diet was 1e7 cells/ml. Diet solutions were placed in a dry heat block at 95 C for 15 min, shaken after 7 min to kill bacteria, and stored at -20 C until used. Diet solutions were plated on nutrient agar plates and incubated at 37 C for 24 to ensure bacteria were not viable. For the duration of the trials, feeding sachet were placed in clear, acrylic 85 mm x 70mm x 30 mm boxes and held in an environmentally-controlled chamber (description of incubator) at 16:8 hr light:dark cycle, 27 2 C, and 60-65% RH. Between 15-25 adult, unmated, sex-separated D. citri were primed by placing them on feeding sachets for 4 days. Surviving D. citri were moved to a second sachet containing 1e6 cells/mL live S. marcescens where they remained until all D. citri were dead. Mortality was recorded once daily. Transgenerational immune priming bioassays were conducted with psyllids placed on artificial diets containing heat inactivated E. coli, M. luteus, or no bacteria. To force D. citri to consume bacteria, they were held on an artificial feeding sachet. The artificial feeding sachet was constructed from a petri dish (35 mm x 10 mm) with the bottom removed and covered with thinly-stretched Parafilm (Bemis NC, Neenah, WI). A and two pieces of thinly stretched Parafilm (Bemis NC, Neenah, WI) with a filter paper disc (2.6 cm dia) with 300 l of diet solution was placed on the Parafilm and covered with an additional Parafilm layer (Russell and Pelz-Stelinski, 2015). The diet solution consisted of 17% sucrose in deionized, distilled water, 30 l/ml of neon green food coloring (McCormick & Company, Inc., Sparks, MD). Total bacteria concentration in diet was 1e7 cells/ml. Diet solutions were placed in a dry heat block at 95 C for 15 min, shaken after 7 min to kill bacteria, and stored at -20 C until used. Diet solutions were plated on nutrient agar plates and incubated at 37 C for 24 to ensure bacteria were not viable. For the duration of the trials, feeding sachet were placed in clear, acrylic 85 mm x 70mm x 30 mm boxes and held in an environmentally-controlled chamber (description of incubator) at 16:8 hr light:dark cycle, 27 2 C, and 60-65% RH. Between 15-25 adult, unmated, sex-separated D. citri were primed by placing them on feeding sachets for 4 days. Data presented here are preliminary, as additional replicates were collected during February and March 2017. These insects are currently being processed and analyzed via QPCR. Offspring of female D. citri that were fed a diet containing M. luteus (n = 17, 2.60E7 1.35E7) or E. coli (n = 18, 2.09E6 8.40E5) had higher CLas titers than offspring of non-primed females (n = 8, 1.22E5 6.20E4). The Ct of plant material was correlated with the titer of CLas in D. citri (b = -0.140, F1,41 = 4.115, p = 0.050, R2 = 0.09). Previously, we demonstrated that the D. citri immune response is induced due to recognition of Gram-positive bacteria, such as M. luteus. Given that M. luteus protected D. citri from S. marcescens, and CLas is also a Gram-negative bacterium, it was expected that CLas titers would be lower in offspring of M. luteus primed adults. In fact, the opposite was observed. Females fed a diet containing M. luteus prior to mating had higher CLas titers and produced offspring that acquired CLas at a much higher rate than those from control or E. coli fed females. This suggests that immune priming with M. luteus infection may facilitate CLas infection.
This project has been reported under ‘Epidemiology and Cultural Control’, but it seems more appropriate to be under ‘Vector Management’, see the category above. 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 a first wave flowering full bloom date of February 11 to 20 depending on the location within the Florida citrus production regions, but actually occurred a day earlier. A second wave of flowering was projected to occur from March 8 to 11 depending on location, but this wave was a week early. Warmer than normal weather caused the advanced development and initiation of earlier full bloom. Data collection was started with bud break estimated to occur in early January. It appeared that bud break may be more reliably estimated from initiation of differentiation than from the estimation of full bloom date. Some preliminary plotting showed that in several years mean temperature the first 2 or 3 weeks after initiation of growth was important to determining how many weeks were required to reach full bloom. Dr. L. Stelinski 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 properties near Fort Meade were evaluated from early January until present. At one property, 2 blocks were sprayed January 3rd and at the other location two blocks were sprayed January 17th. No adult psyllids were detected by tap counts in 30 days where the spray was applied at budbreak in early January. However adults psyllids were detected at the location where the spray was applied later even though this was a shorter control time. The idea of spraying at budbreak but before feather flush appears to have some merit. We had information for growers on full bloom and 10 % open flowers but did not work out a way to make this information available to bee keepers. We need to develop an extension plan for bee keeper information. We had some success in an associated project in reducing off-season winter bloom by applying gibberellic acid monthly in the fall. We will monitor these trials to see if yields were improved. It did appear however that fewer flowers were present in the first flowering wave also. We will monitor the five locations that growers sprayed and one we sprayed to determine yield of the next crop.
We have continued to investigate movement of Asian Citrus Psyllid (ACP) in response to abiotic and biotic factors. Currently, we are investigating the flight duration as it relates to humidity and influence of wind velocity on movement in the laboratory and field. A flight mill was used to measure the flight of ACP under differing humidity and temperature treatments. We have thus far tested increasing humidity with increasing temperature in the following three treatments: 70 F/relative humidity (RH) 60%, 77 F/ RH 60%, 82 F/ RH 75%. Increasing temperature and humidity caused psyllids to fly longer distances. Psyllids flew the longest distances in the 82 F/ RH 75% treatment but initiated flight 2x more in the 77 F/ RH 60% treatment. This preliminary result suggests that ACP may be flying longer distances on hotter, more humid days but making more frequent short flights on cooler, lower humidity days. We are currently expanding this data set with additional temperature and RH treatments. We are investigating the influence of wind on ACP dispersal in the laboratory and field. In the laboratory, we are using a wind tunnel set up. ACP are allowed to settle on a young citrus plant for 24 hours before exposed to a wind treatment for 24 hours. To track their dispersal, a sticky trap is set up behind the plant and to the surrounding cage at 1, 2, 3, and 24 hours of wind exposure. We have tested the following wind velocities: 1.8 meters per second, 1.5 m/s, 1 m/s, and 0.38 m/s. At higher wind velocities, ACP does not move and the most movement occurs in no wind controls. The results from this study currently indicate that ACP is dispersing most at 0.38 m/s. In the field, we are assessing ACP movement using a wind vane fitted with sticky traps alongside an anemometer. The wind vane shifts direction to align with the wind. We have been preliminarily field testing this apparatus with great success. Currently, in low density winter ACP populations, it appears that the ACP are moving with the wind and moving more at wind velocities under 1m/s.
The objective of this study was to determine differential detoxification enzyme levels among different abdominal color morphs (orange/yellow, blue/green and gray/brown) of Asian citrus psyllid (ACP). Glutathione S-transferase, cytochrome P450 and esterase activity were measured. First, we used a topical bioassay to determine the insecticide susceptibility to adults of three color morphs. The insecticides chosen were from four different modes of action. Three color morphs of adult ACP were collected in the field from Lake Alfred, FL. Tested insecticides were of analytical grade and included bifenthrin (99.8%), dimethoate (99.8%), flupyradifurone (99.5%) and aldicarb, (99.7%). The LD50 value and 95% fiducial limits for aldicarb (carbamate) were1.52 ng/ l (0.86-2.74) for orange/yellow; 2.06 ng/ l (1.17-3.71) for blue/green and 2.29 ng/ l (0.52-12.37) for gray/brown ACP. The LD50 value and 95% fiducial limits for diamethoate (organophosphate) were 0.32 ng/ l (0.18-0.56) for orange/yellow; 0.50 ng/ l (0.28-0.91) for blue/green and 0.62 ng/ l (0.35-1.12) for gray/brown ACP. For bifenthrin (pyrthroid), the LD50 value and 95% fiducial limits were 0.10 ng/ l (0.06-0.18) for orange/yellow; 0.13 ng/ l (0.07-0.24) for blue/green and 0.13 ng/ l (0.07-0.23) gray/brown ACP. The LD50 value and 95% fiducial limits for flupyradifurone (butenolid) were 3.79 ng/ l (2.03-7.52) for orange/yellow, 4.78 ng/ l (0.92-46.9) for blue/green and 6.16 ng/ l (3.30-12.50) for gray/brown ACP. The susceptibility to aldicarb, dimethoate and flupyradifurone was significantly higher for the orange/yellow morph as compared to blue/green and gray/brown morhphs. Secondly, we quantitatively measured the detoxification enzyme activity levels of orange/yellow, blue/green and gray/brown color morphs to determine difference between the physiological states of these three color morphs. Cytochrome P450 activity was quantified and expressed in terms of general oxidase level. A heme peroxidation method was used to indirectly determine the P450 activity using substrate of 3,3′,5,5′-tetramethylbenzidine (TMBZ). General esterase activity was measured using 4-nitrophenyl acetate (pNPA) as a substrate. Glutathione S-transferase activity were conducted using CDNB (1-chloro-2.4-dinitrobenzene) (CDNB) as the substrate. GST activity was significantly lower in orange/yellow color (299.70 1.24 mol/min/mg protein) than gray/brown (350.86 1.19 mol/min/mg protein) and blue/green (412.25 1.37 mol/min/mg protein) ACP adults. Likewise, mean cytochrome P450 activity was significantly lower in gray/brown (0.152 0.006) and blue/green (0.149 0.005) equivalent units (EU) cytochrome P450/mg protein than orange/yellow (0.179 0.008) equivalent units (EU) cytochrome P450/mg protein. Mean esterase activity was significantly higher in blue/green (416.72 5.12 mol/min/mg protein) and gray/brown (154.25 5.46 mole/min/mg protein) than orange/yellow (282.56 2.93 mol/min/mg protein) ACP. Results indicated the GST and esterase activity may be correlated with insecticide susceptibility levels. The study shows that activity levels of three important detoxifying enzymes in ACP are potentially different depending on the color morph and may influence insecticide efficacy depending on differential abundance in the field. However, further investigations are needed to compare expression levels of associated genes between difference color morphs. Importantly, insecticide resistance does not appear to be shifting populations of ACP toward more resistant color morphs based on our observations thus far. Work continues on the field investigation comparing different insecticide rotational schemes. We completed the latest rotation sprays in October 5, 2016 but were unable to collect sufficient numbers of psyllids to assess for insecticide resistance. Currently, we are rearing insects collected from the different plots to obtain sufficient numbers for testing in the laboratory. These laboratory cultures established from our field experiments will allow us to determine which rotation schedules tested are best to minimize the chance of resistance development in the field. We expect to have more results at the end of next quarter to begin answering this question.
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 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.
(863) 956-8817
(863) 956-5894
700 Experiment Station Road
Lake Alfred, FL 33850
Email Us
