Applications of PGRs to container-grown citrus trees affected the biology and behavior of the Asian citrus psyllid. In greenhouse and growth camber experiments, prohexadione and mefluidide applications to trees reduced ACP fecundity and survivorship, whereas uniconazole affected fecundity and paclobutrazol affected survivorship. These PGRs did not affect adult ACP weight, except when paclobutrazol was applied. Dikegulac appeared to inhibit ACP egg laying, but this was likely due to phytotoxicity. Oviposition was delayed and rate was lower on PGR-treated trees, but chlormequat under greenhouse conditions had an effect. The effect of PGRs on ACP did not appear to be due to growth retardation or specific toxicity of the materials, suggesting a yet undefined effect on plant physiology that altered host quality.
The objective of this study is to determine how enhanced nutrition of citrus plants may affect Asian citrus psyllid (ACP) biology. We have initiated this study with complementary field and laboratory experiments. We started the second year of the field experiments. We are repeating the same protocol as last year, with the difference that this year we will expand and have a second experimental field (given the additional enhancement funding that will be reported on separately). This experiment is being conducted in a solid planting of Hamlin resets that are now approximately three years old. We have already started the application of nutritional spraying, following the Keplex’ recommendations for this year. At the end of April, we will start monitoring ACP nymph and adult populations, as well as flush development. We have also planned a new screening of HLB infection status of trees with qPCR in this reset field for April to observe how infection has changed from last year, depending whether trees were supplemented with nutrients or not. We are currently performing a laboratory experiment where citrus resets are placed in a Las-infected ACP colony (55% of psyllids had been confirmed Las positive). Half of these citrus trees are nutritionally supplemented and half of them are sprayed with water (control). We are following the infection status of these trees over the course of 1 year to observe if the plants treated with nutritional supplement are more or less protected against HLB infection. Interestingly, after 4 months of exposure to infected psyllids only the controls were found to be infected by CLas, whereas all the nutritionally supplemented trees are still negative to CLas. If these results are confirmed, it may show that nutritional supplement may decrease susceptibility of resets to HLB infection. Finally, we are also conducting an experiment where we are comparing the dispersal behavior of ACP that settled on HLB-infected trees, or that settled on HLB-infected trees treated with nutrient supplements. We know that psyllids are first attracted to HLB-infected trees but subsequently disperse from these infected trees. One hypothesis for this subsequent dispersal from HLB-infected trees is that these trees are nutritionally sub-optimal to psyllids. Consequently, we hypothesize that psyllids that settled on HLB-infected trees treated with nutriment spray should disperse less than counterparts that settled on untreated HLB-infected plants. So far, our data appear to confirm this hypothesis as we have fewer psyllids dispersing from HLB-infected trees treated with nutrient supplement sprays than psyllids dispersing from untreated HLB-infected trees.
We are continuing to evaluate the new neonicotinoid, flupyradifurone (active ingredient of SivantoTM , Bayer CropSciences) against the Asian citrus psyllid in various assays in comparison to imidacloprid. We have completed a study to determine the efficacy of this insecticide to reduce feeding on plant tissue at various concentrations and found that flupyradifurone (technical grade) did not perform as well as imidacloprid. At 25 ug/ml treatment, there is no difference in efficacy between the two insecticides. However, at 2.5 ug/ml, imidacloprid continued to deter feeding at the same level as 25 ug/ml, whereas feeding behavior was no different between treatment and controls for flupyradifurone. Thus far, we have found that flupyradifurone does not perform as well as imidacloprid in topical (LD50), contact (LC50) or feeding deterrent assays. Currently, we are conducting assays to compare toxicological efficacy of flupyradifurone and imidacloprid when ingested by the insect. We are also planning experiments to investigate non-lethal effects of the insecticide including settling behavior, fecundity and longevity. We are ready to begin our annual field survey to monitor insecticide resistance in field psyllid populations and will begin those efforts in April. We have begun limited work on development of a vial assay to use in the field to test for insecticide resistance on-site and have completed the base-line susceptibility assays against the six insecticides (carbary, chlorpyrifos, fenpropathrin, flupyradifurone, imidacloprid and thiamethoxam). We hope to evaluate this tool over the course of the summer field survey. This tool would greatly improve the efficiency of insecticide resistance monitoring in the field.
The overall objective of this research is to develop a push-pull system for the Asian citrus psyllid (ACP) that can complement integrated management systems in young citrus plantings. Towards that goal, visual factors that affect psyllid takeoff into flight and landing are being examined to guide development of an optimal pull component. A vertical flight attraction assay was developed to examine response of ACP resting or in flight towards visual targets that consist of different wavelengths of filtered light. Narrowband width filters (10 nm) were used to allow clear delineation between colors tested. This assay simulates the response of ACP to visual cues that are above them such as when an adult is settled on vegetation and looks up at the sky or when having fallen to the ground looks up and sees its host plant from the ground. ACP response was lowest to red and orange in the visible spectrum. Yellow was more attractive to ACP then orange or red, but less attractive then blue, green, or lime green. Green and lime green were the most attractive to ACP in the visible spectrum. In the ultraviolet spectrum the most attractive wavelength was long UV (405nm) compared to shorter wavelengths arranged in order of decreasing attractiveness 350nm, 380nm, and 365 nm (least attractive). The vertical bioassay differed from the horizontal bioassay, in that ACP responded as strongly to UV as they did to green and lime green. This may be evidence that the ACP orient differently to vertical and horizontal cues. For example, when ACP drop to the ground following a disturbance vertical cues of UV (sky) or green and lime green (leaves) are both used to return to a place of rest (the host plant). Whereas horizontal cues are more typically used when ACP are already situated on a plant and therefore only UV (sky) cues are used to navigate towards increasingly younger and more optimal portions of the host plant (young flush), which are near the edge of the plant (near UV) versus in the center of the plant (away from UV). Ultimately, the UV cues from the horizontal axis may be causing the ACP to move to another plant in search of new host locations. Further studies are on-going with the vertical bioassay to see if there is a difference between 0-1 day old and 4-7 day old ACP in this orientation. Additionally, studies are being undertaken to see if flying ACP will be attracted to different colors then walking/flying ACP by using specially designed walking preventing chambers coated with Fluon. A horizontal walking assay was developed to stimulate walking responses of ACP on vegetation and this assay measured direction of movement as well as speed of movement. Known age studies with ACP in the horizontal bioassay revealed that 1 day old ACP were positively attracted toward green light , whereas 4-7 day old ACP remained relatively neutral to green light. Both 1 day old and 4-7 day old ACP showed more positive attraction towards long UV light. These findings indicate that newly emerging ACP may be more attracted to green, because they need to acquire nutrients from feeding on a plant. Conversely, older ACP may be more interested in movement to other plants in search of mates and oviposition sites. Further studies with the horizontal walking assay will be made between newly emerged unmated female ACP and older gravid females. Examination of the effect of different visual stimuli on flight duration of ACP as measured on flight mills is on-going. Comparisons in the duration of flight and numbers of flight bouts are being made between different colored substrates, different quality of light overhead (colored, with UV, without UV, polarized), contrast between overhead lighting and substrate coloring and presence of visual targets.
The overall objective of this research is to develop a push-pull system for the Asian citrus psyllid (ACP) that can complement integrated management systems in young citrus plantings. Towards that goal, visual factors that affect psyllid takeoff into flight and landing are being examined to guide development of an optimal pull component. A vertical flight attraction assay was developed to examine response of ACP resting or in flight towards visual targets that consist of different wavelengths of filtered light. Narrowband width filters (10 nm) were used to allow clear delineation between colors tested. This assay simulates the response of ACP to visual cues that are above them such as when an adult is settled on vegetation and looks up at the sky or when having fallen to the ground looks up and sees its host plant from the ground. ACP response was lowest to red and orange in the visible spectrum. Yellow was more attractive to ACP then orange or red, but less attractive then blue, green, or lime green. Green and lime green were the most attractive to ACP in the visible spectrum. In the ultraviolet spectrum the most attractive wavelength was long UV (405nm) compared to shorter wavelengths arranged in order of decreasing attractiveness 350nm, 380nm, and 365 nm (least attractive). The vertical bioassay differed from the horizontal bioassay, in that ACP responded as strongly to UV as they did to green and lime green. This may be evidence that the ACP orient differently to vertical and horizontal cues. For example, when ACP drop to the ground following a disturbance vertical cues of UV (sky) or green and lime green (leaves) are both used to return to a place of rest (the host plant). Whereas horizontal cues are more typically used when ACP are already situated on a plant and therefore only UV (sky) cues are used to navigate towards increasingly younger and more optimal portions of the host plant (young flush), which are near the edge of the plant (near UV) versus in the center of the plant (away from UV). Ultimately, the UV cues from the horizontal axis may be causing the ACP to move to another plant in search of new host locations. Further studies are on-going with the vertical bioassay to see if there is a difference between 0-1 day old and 4-7 day old ACP in this orientation. Additionally, studies are being undertaken to see if flying ACP will be attracted to different colors then walking/flying ACP by using specially designed walking preventing chambers coated with Fluon. A horizontal walking assay was developed to stimulate walking responses of ACP on vegetation and this assay measured direction of movement as well as speed of movement. Known age studies with ACP in the horizontal bioassay revealed that 1 day old ACP were positively attracted toward green light , whereas 4-7 day old ACP remained relatively neutral to green light. Both 1 day old and 4-7 day old ACP showed more positive attraction towards long UV light. These findings indicate that newly emerging ACP may be more attracted to green, because they need to acquire nutrients from feeding on a plant. Conversely, older ACP may be more interested in movement to other plants in search of mates and oviposition sites. Further studies with the horizontal walking assay will be made between newly emerged unmated female ACP and older gravid females. Examination of the effect of different visual stimuli on flight duration of ACP as measured on flight mills is on-going. Comparisons in the duration of flight and numbers of flight bouts are being made between different colored substrates, different quality of light overhead (colored, with UV, without UV, polarized), contrast between overhead lighting and substrate coloring and presence of visual targets.
The overall objective of this research is to develop a push-pull system for the Asian citrus psyllid (ACP) that can complement integrated management systems in young citrus plantings. Towards that goal, visual factors that affect psyllid takeoff into flight and landing are being examined to guide development of an optimal pull component. A vertical flight attraction assay was developed to examine response of ACP resting or in flight towards visual targets that consist of different wavelengths of filtered light. Narrowband width filters (10 nm) were used to allow clear delineation between colors tested. This assay simulates the response of ACP to visual cues that are above them such as when an adult is settled on vegetation and looks up at the sky or when having fallen to the ground looks up and sees its host plant from the ground. ACP response was lowest to red and orange in the visible spectrum. Yellow was more attractive to ACP then orange or red, but less attractive then blue, green, or lime green. Green and lime green were the most attractive to ACP in the visible spectrum. In the ultraviolet spectrum the most attractive wavelength was long UV (405nm) compared to shorter wavelengths arranged in order of decreasing attractiveness 350nm, 380nm, and 365 nm (least attractive). The vertical bioassay differed from the horizontal bioassay, in that ACP responded as strongly to UV as they did to green and lime green. This may be evidence that the ACP orient differently to vertical and horizontal cues. For example, when ACP drop to the ground following a disturbance vertical cues of UV (sky) or green and lime green (leaves) are both used to return to a place of rest (the host plant). Whereas horizontal cues are more typically used when ACP are already situated on a plant and therefore only UV (sky) cues are used to navigate towards increasingly younger and more optimal portions of the host plant (young flush), which are near the edge of the plant (near UV) versus in the center of the plant (away from UV). Ultimately, the UV cues from the horizontal axis may be causing the ACP to move to another plant in search of new host locations. Further studies are on-going with the vertical bioassay to see if there is a difference between 0-1 day old and 4-7 day old ACP in this orientation. Additionally, studies are being undertaken to see if flying ACP will be attracted to different colors then walking/flying ACP by using specially designed walking preventing chambers coated with Fluon. A horizontal walking assay was developed to stimulate walking responses of ACP on vegetation and this assay measured direction of movement as well as speed of movement. Known age studies with ACP in the horizontal bioassay revealed that 1 day old ACP were positively attracted toward green light , whereas 4-7 day old ACP remained relatively neutral to green light. Both 1 day old and 4-7 day old ACP showed more positive attraction towards long UV light. These findings indicate that newly emerging ACP may be more attracted to green, because they need to acquire nutrients from feeding on a plant. Conversely, older ACP may be more interested in movement to other plants in search of mates and oviposition sites. Further studies with the horizontal walking assay will be made between newly emerged unmated female ACP and older gravid females. Examination of the effect of different visual stimuli on flight duration of ACP as measured on flight mills is on-going. Comparisons in the duration of flight and numbers of flight bouts are being made between different colored substrates, different quality of light overhead (colored, with UV, without UV, polarized), contrast between overhead lighting and substrate coloring and presence of visual targets.
The objective of this project is to optimize attractancy of visual targets and to determine which segments of the ACP population are responding to traps. Knowledge of the factors that enhance psyllid attraction to a target as well as enhance detection by psyllids of the target against the background will result in optimization of the design and placement of a visually targeted trap. Such as optimized trap can enhance surveillance efforts, provide a pull component for localized control of dispersing ACP or serve as the basis for a lethal pesticide-treated visual trap. Within canopies leaves are seen both as reflected light (from a leaf) and transmitted (translucent) light (through a leaf). Most traps focus on the use of reflected light and the role of transmitted light was examined to determine if this could enhance ACP attraction. A landing assay was developed in the laboratory using a high output light source emitting UV light to evaluate attraction of ACP released at the bottom of the chamber and landing on sticky translucent surfaces placed on the top of the chamber. In this assay, the numbers of ACP collected on two sticky surfaces were compared. ACP strongly preferred landing on translucent yellow rather than green translucent substrates. The effect of edges and lines to contrast and enhance detection of the colors was also evaluated with the translucent surfaces. Narrow vertical black lines enhanced attraction by about 20% to dark yellow translucent panels. Similar black lines did not significantly increase attraction to lighter yellow or green sticky translucent panels. Dark leaf silhouettes on green translucent panels only marginally enhanced collections. Collections on grey panels differed significantly with degree of polarization and these studies are on-going with different colors of polarized panels. Large cage choice assays conducted in the laboratory on the attraction of ACP to various sticky targets. Standard commercial sticky traps (yellow or green) which utilize reflected light were about twice as attractive as translucent sticky traps which allowed transmission of yellow or green light. When yellow sticky traps (reflected light) were modified by adding broad green or black vertical stripes to increase the contrast against the yellow, trap collections were reduced by one third. Use of a black border around a yellow sticky trap did not increase or reduce collections of ACP. Overall responses were about 10% greater to solidly colored traps than those with edges or stripes. On-going assays are further examining effects of polarization and background contrast on attraction.
We have initiated a trial in the Indian River Production Region with grapefruit growers with whom we are establishing foliar nutritional trials similar to what we have done in southwest Florida with oranges using the Boyd cocktail. we are looking specifically at the nitrogen source in combination with individual micro-nutrients. Harvest data the past 5 years from our replicated trial in a 32 acre commercial grove of ‘Valencia’ in southwest Florida evaluating foliar nutrition has shown yield in HLB infected trees have increased each year and juice quality has been maintained. Economic analysis is showing the treatments with highest yield are the most expensive and yield is not significantly different from treatments with less expensive products that are more productive in dollar returns. The common ingredients in the most cost productive treatments are micro- and macro-nutrients, some with salicylate and phosphite. B9: This quarter we completed the 6th harvest in a 13 acre block of ‘Valencia’ on ‘Swingle’ set out in an RCB factorial design with 4 replicates and 4 treatments: insecticide alone, enhanced foliar nutrition along, insecticides + foliar nutrition and untreated control. Both factors significantly affected yields (F = 75.25; df = 1, 9; P < 0.0001 and F = 5.44; df = 1, 9; P = 0.0446 for 'insecticides' and 'nutritionals' respectively). Again, highest yields were obtained in plots receiving both insecticide for ACP plus foliar nutrition (205 ' 7.9 lb of fruit per tree) followed by insecticides only (188 ' 7.5 lb per tree). Yields in plots without insecticide were the least (158 ' 8.3 and 154 ' 9.2 lb per tree in 'nutritional' and 'untreated plots' respectively). Economic analysis will evaluate cost effectiveness. Thresholds: The fourth consecutive year of treatments was completed in two commercial blocks ('Earlygold' and 'Valencia') using a RCB design with 4 reps and 4 ACP management treatments, (1) monthly insecticide sprays, (2) two dormant sprays plus sprays based on a 0.2 ACP adults per stem tap threshold, (3) one dormant spray plus sprays on a 0.7 threshold, and (4) no insecticide sprays. Yields this season were significantly different among treatments (F = 2.53; df = 3, 9; P = 0.0192 and F = 4.77; df = 3, 9; P = 0.0295 for 'Earlygold' and 'Valencia' blocks respectively). Plots receiving calendar sprays (1) had the highest yields followed by treatment (2), and (3) and (4) respectively. No treatment effects on juice quality parameters were found in 'Earlygold' (F = 0.64; df = 3, 25; P = 0.599 and F = 0.64; df = 3, 25; P = 0.599 for brix/acid ratio and lbs solids per box respectively). Yields correlated with ACP numbers assess by biweekly stem tap samples (F = 13.14; df = 2, 14; P = 0.0009; r2 = 0.69 and F = 24.48; d.f. = 2, 14; P = 0.0001 r2 = 0.68, in the 'Earlygold' block and 'Valencia' block respectively). All our results confirm the importance of vector control even when most trees are HLB infected. The correlation between yield losses and ACP densities found in this study is being used in the elaboration of an economic injury level model that permits optimizing the number of insecticides sprays during the growing season based on ACP monitoring. Work continued with the developed fluorescent leaf assay to determine if phloem translocation was enhanced by the use of extra nutritional supplements. We tried this technique using fruit with stems to compare the phloem translocation on normal sized fruit and small fruit. This work was done on fruit from trees that tested positive by qPCR for HLB. We were unable to determine if uptake occurred. Other work consisted in using the fluorescent assay in an organic grove operation where good fruit size was being produced on HLB qPCR positive trees. Visual assays showed that the leaves from the HLB affected trees had uptake similar to healthy leaves.
The objective of this project is to identify the location and movement patterns of Asian citrus psyllids during their winter dormant period. We have previously identified potential overwintering sites based on management practices and surrounding landscape characteristics. Significantly more psyllids were captured in intermittently managed groves during the 2012/2013 and 2013/2014 winter season than in groves under any other management regime. We are currently testing the hypothesis that psyllids move to and ‘shelter’ in intermittently managed groves during winter months, and subsequently disperse in the spring. We have conducted movement studies using capture sticky traps set up at canopy height in concentric buffers surrounding groves to understand movement and directionality of psyllid populations within citrus growing areas. During the winter season of 2013/2014, we evaluated three local citrus area landscapes, each containing three differentially managed grove types; organic, conventional, and intermittent management. Psyllid abundance was evaluated using tap sampling, sticky trap sampling and vacuum insect collection methods. As in the previous year, psyllid abundance was associated with the type grove management conducted at each site. There was no evidence for psyllid movement between groves within our experimental areas. This experiment will continue into the spring season to assess whether movement between groves increases during the spring season. Initial results from this experiment suggest that survival of psyllids in groves is the main factor contributing to population densities in groves under differential management, rather than psyllid movement between groves.
Understanding the interactions between the Asian citrus psyllid (ACP) Diaphorina citri and Candidatus Liberibacter asiaticus (Las), is key in identifying potential controls and management of citrus greening. Recently we found that exposure and pre-exposure to different temperature alters ACP acquisition and inoculation of Las. Thus, variable climatic conditions in Florida are likely to influence ACP transmission of Las. In addition, in cool temperatures ACPs exhibit altered coloration indicating up-regulation of melanization genes. This response is a typical insect response to fungal pathogens. In order to investigate the effect of melanization further, we are conducting fitness assays on melanized ACPs to determine how this change in morphology affects developments, longevity, and fecundity. We are also currently conducting qRT-PCR assays to quantify regulation of a number of heat shock proteins and melanization genes such as phenoloxidases and genes within the rhodopsin pathway. It is possible that expression of these genes may also play a role in ACP-Las interactions within the insects, and to this end we are currently conducting transmission assays on melanized ACP nymphs and adults. Experiments to assess ACP gene expression in response to simultaneous challenges of cool temperature and entomopathogenic fungi are underway. We expected these additional data will 1) identify potential targets for disrupting ACP vector competence, and 2) yield a better understanding of the potential for use of entomopathogenic fungi as an overwinter biocontrol agent of ACPs.
Based on results from the previous Contest Project (CRDF#400), eleven compounds have been screened by the contest committee as candidates for further research to determine their efficacy for control of HLB based on their ability to substantially reduce the titers of the bacterium Candidatus Liberibacter in our grafted citrus assay, lack of phytotoxicity to citrus and potential for registration. After first evaluating combinations of these molecules using the graft-based chemotherapy method under a separate agreement, this project extension will evaluate these 11 compounds as control agents for the HLB bacterium individually and in combination using infected, container-grown citrus and HLB-affected scions. The objective of this project is to determine an optimum chemical formulation that may be registered for field control of HLB. In this quarter (Jan. 2014 to March, 2014),all graft-based tests have been done in 16 treatments of 11 compounds alone and in combination. Some treatments were effective in reducing the Las bacterium to undetectable levels by qPCR. However, some treatments had serious phytotoxicity to citrus, such as ACT and its combinations. We continued the third-round test of the potted-plant experiment including 72 treatments of 11 compounds and their combinations. Based on our delivery research, we changed the application from root-soaking to bark-application using nano formulation of the antimicrobial agents. No bacterium was detected in the new leaves but was present in the old leaves two months after treatment with some of the compounds. Six months after treatment, several treatments were effective in reducing the Las bacterium both in the new and the old leaves, especially SDX, AMP. The research is ongoing.
Ultra High Performance Liquid Chromatography – Pesticide Residue Analysis (Final Report) The purpose of this proposal was to purchase a LC-MS-MS in order to facilitate ongoing CRDF funded research requiring the analysis of pesticide residues in citrus leaf tissue. Funding provided by CRDF paid for a portion of this equipment which was cost-shared by UF-IFAS. The LC-MS-MS was delivered at setup in the Rogers’ lab at the Citrus Research & Education Center. Despite lengthy delays in getting the equipment to function properly due to installation of faulty parts, the LC-MS-MS is functional and is being used to meet the goals of other CRDF funded research projects. During the course of our method development period, we split research samples being analyzed for independent verification with the research labs of Bayer, Syngenta and Valent. Such interaction with these companies help use refine and perfect our methods being used for pesticide residue analysis. To date, the LC-MS-MS has been used to quantify parent compounds and metabolites of imidacloprid, thiamethoxam and clothianidin in citrus nectar collected as part of CRDF funded projects. The goal of these nectar analyses is to support pesticide label changes needed to help protect young trees from becoming infected with HLB. Our results from this project have played an important role in a larger group effort which has resulted in changes in the Admire Pro label, for which a 24C SLN was granted to increase the annual rate of product applied from 0.5 lbs a.i. per acre to 1.0 lbs a.i. per acre, and Belay Insecticide which now has a bearing label permitting use for trees in the 5-9′ size class. This equipment will continue to be used in similar efforts and additional studies looking at maximizing the effectiveness of currently used pesticides for psyllid control.
We have continued this investigation by conducting a second year of replication. Specifically, we sampled Asian citrus psyllid (ACP) populations during the winter season at various heights within tress and with respect to the cardinal direction of trees within Florida citrus groves. We sampled two locations and each one was divided into two plots: one oriented north-south and the other east-west. For each plot, we sampled the citrus canopy at three different heights within trees and the four cardinal orientations within the grove using a custom-made, high powered insect vacuum sampler to collect ACP. Our goal was to develop a two dimensional picture of ACP distribution within citrus groves during winter. Hobo data loggers (which collect environmental data such as temperature) were placed at these various locations to correlate temperature and relative humidity with ACP distribution within groves. The data are currently still being analyzed. However, preliminarily, it appears that ACP populations were high within the groves sampled. We collected 2 psyllids per sample with a maximum sample with 33 psyllids per sample during our winter collections from individual portions of trees. We compared our results with those obtained and posted by personnel from USDA-APHIS that sampled the same plots for the CHMA project and who used the tap sampling method. While tap sampling conducted by USDA-APHIS did not find psyllids in these particular areas during February and March, we collected over 100 psyllids in a single day using the vacuum method in these areas.
One objective of this project is to determine how cold and heat acclimation and temperature-mediated gene expression influence Las transmission by Asian citrus psyllids (ACP). We previously reported that climatic conditions appear to influence the likelihood of Las transmission. Moreover, at some temperatures ACPs exhibit altered coloration indicating up-regulation of melanization genes. We continue to identify additional cryoptotective gene targets for quantification using qRT-PCR. Currently, qRT-PCR assays to quantify several heat shock protein and melanization genes expressed by psyllids under varying temperatures are ongoing. Understanding how cold and heat acclimation, and underlying temperature-mediated gene expression, influence transmission of Las should provide novel targets for managing transmission by ACP populations. We continue to develop isofemale lines of Wolbachia and Wolbachia-free psyllids derived from 10 distinct geographic locations in Florida. Currently, replicate isofemale lines from seven distinct regions of the state have been established. Additional collections will be conducted during the next month to establish additional psyllid lines. Phenotypic screening of isofemale lines is expected to begin in the next quarter. Recently, personnel have been hired by Handler to optimize rearing and egg collection protocols needed to support for germ-line transformation of ACP from these isofemale lines.
The objective of the current project is the identification of a Bacillus thuringiensis (Bt) crystal toxin with basal toxicity against Asian Citrus Psyllid (ACP) and enhancement of this toxin by addition of an ACP gut binding peptide. A phage display library will be screened to identify peptides that bind to the gut of the ACP. Addition of the gut binding peptide to the Bt toxin will increase toxin binding and associated toxicity against the ACP. During the current reporting period partially purified Bt toxins from selected Bt strains provided by Dr. Michael Blackburn, USDA, Maryland were trypsin activated. Proteolytic activation of Bt toxins was carried out at Iowa State University. Briefly, Bt toxins were solubilized using sodium carbonate pH 10.5 + 10mM DTT for three to 16 h at 37’C and dialyzed against 50 mM Tris-Cl pH 8.5. Bt toxins were then incubated with bovine trypsin at a final concentration of 10% of the toxin concentration at 37 ‘C for 1 h. Removal of trypsin was carried out using benzamidine sepharose. The samples were boiled in denaturing SDS sample buffer for 5 min, separated on 10% (wt/vol) SDS/PAGE and stained with Coomassie blue. The SDS-PAGE profiles of trypsin-treated Bt toxins indicated the presence of two different toxin groups with different molecular mass protein bands in the protoxin samples. Samples in the first protoxin group consisted of one toxin band of ~39 kDa, while samples in the second protoxin group consisted of two protein bands of ~40 and ~38 kDa. In contrast, all Bt strains had a single toxin band of ~35 kDa in the trypsin activated state. Samples of 1.8 to 2.3 milligrams of five trypsin-activated Bt toxins were sent to Dr. David G. Hall, USDA-ARS for ACP membrane feeding assays for toxicity analysis.
(863) 956-8817
(863) 956-5894
700 Experiment Station Road
Lake Alfred, FL 33850
Email Us
