Knowledge about factors that determine transmission efficiency of Candidatus Liberibacter asiaticus (CLas) by the Asian citrus psyllid (ACP) is important for understanding epidemiology and improving current HLB management strategies. The goal of this project is to investigate how transmission of CLas is influenced by: 1) vector developmental stage, 2) pathogen titer in source plants, 3) plant phenology, 4) inoculation access periods and 5) insecticide treatment. In study 1, we are comparing acquisition efficiency of CLas by different ACP nymphal stadia (1st, 2nd, 3rd, 4th and 5th instars) and adults (1 wk old). After an acquisition access period (AAP) of 48 h on young shoots of infected (source) plants, the insects of each age treatment were first transferred to healthy citrus seedlings for a latent period of 3 wks and then submitted to real-time PCR for detection and quantification of CLas in their bodies. Results of three replications of this experiment using independent CLas-infected plants as sources indicated that nymphs of all development stadia can efficiently acquire the pathogen; adults can also acquire it, but with a lower efficiency. We are still analyzing the data to determine if bacterial multiplication in the vector and transmission efficiency were also influenced by vector age during acquisition. In study 2, we investigated the effect of leaf age (young and asymptomatic x symptomatic mature leaves) in source plants of CLas on acquisition efficiency and vector probing behavior. ACP adults acquired CLas with higher efficiency on fully-expanded young (asymptomatic) leaves (20.4% infective individuals) than on mature leaves (0%). We repeated this experiment with inclusion of a third leaf age treatment (not fully-expanded young leaves), and observed that acquisition efficiency on mature leaves was lower (6.0% infective individuals) than on fully-expanded (25.1%) or not fully-expanded (9.1%) young leaves. By comparing the probing behavior of ACP females on mature x young leaves of infected citrus by the Electrical Penetration Graph (EPG) technique, we noted that phloem ingestion was longer and more frequent on young leaves. On mature leaves, the insects spent most of the time with the stylets in the parenchyma (pathway phase) or non-probing. The higher frequency and longer duration of phloem ingestion appears to explain at least in part the higher acquisition efficiency of CLas when ACP is confined on young asymptomatic leaves. In study 3, we evaluated acquisition efficiency of CLas by ACP adults on infected citrus plants with variable pathogen population. Fifty citrus plants were graft-inoculated with Clas at the same time, and groups of five independent plants were used as source for acquisition assays at 60, 130, 145, 165, 180, 200, 235, and 280 days after inoculations (DAI). Clas was acquired by ACP from both symptomatic and asymptomatic citrus, and a positive linear relationship was observed between pathogen population within the source plant and the percentage of infective psyllid samples. Similarly, the bacteria population within the plant increased over time and became stable at 235 and 280 DAI when HLB symptoms became more evident. In 2010 we will be analyzing the transmission data of this experiment in relation to CLas population in the source plants. The results obtained so far indicate that vector control should be targeted to young shoots of citrus trees, where developing ACP nymphs can efficiently acquire the pathogen and subsequently spread the pathogen as emerging adults. Because ACP can acquire CLas on symptomless plants, vector control should be an important component in HLB management, in addition to eradication of symptomatic trees. Experiments related to studies 4 and 5 will be set up in 2010 and will help to clarify if insecticide treatment can prevent transmission.
Knowledge about factors that determine transmission efficiency of Candidatus Liberibacter asiaticus (CLas) by the Asian citrus psyllid (ACP) is important for understanding epidemiology and improving current HLB management strategies. The goal of this project is to investigate how transmission of CLas is influenced by: 1) vector developmental stage, 2) pathogen titer in source plants, 3) plant phenology, 4) inoculation access periods and 5) insecticide treatment. In study 1, we are comparing acquisition efficiency of CLas by different ACP nymphal stadia (1st, 2nd, 3rd, 4th and 5th instars) and adults (1 wk old). After an acquisition access period (AAP) of 48 h on young shoots of infected (source) plants, the insects of each age treatment were first transferred to healthy citrus seedlings for a latent period of 3 wks and then submitted to real-time PCR for detection and quantification of CLas in their bodies. Results of three replications of this experiment using independent CLas-infected plants as sources indicated that nymphs of all development stadia can efficiently acquire the pathogen; adults can also acquire it, but with a lower efficiency. We are still analyzing the data to determine if bacterial multiplication in the vector and transmission efficiency were also influenced by vector age during acquisition. In study 2, we investigated the effect of leaf age (young and asymptomatic x symptomatic mature leaves) in source plants of CLas on acquisition efficiency and vector probing behavior. ACP adults acquired CLas with higher efficiency on fully-expanded young (asymptomatic) leaves (20.4% infective individuals) than on mature leaves (0%). We repeated this experiment with inclusion of a third leaf age treatment (not fully-expanded young leaves), and observed that acquisition efficiency on mature leaves was lower (6.0% infective individuals) than on fully-expanded (25.1%) or not fully-expanded (9.1%) young leaves. By comparing the probing behavior of ACP females on mature x young leaves of infected citrus by the Electrical Penetration Graph (EPG) technique, we noted that phloem ingestion was longer and more frequent on young leaves. On mature leaves, the insects spent most of the time with the stylets in the parenchyma (pathway phase) or non-probing. The higher frequency and longer duration of phloem ingestion appears to explain at least in part the higher acquisition efficiency of CLas when ACP is confined on young asymptomatic leaves. In study 3, we evaluated acquisition efficiency of CLas by ACP adults on infected citrus plants with variable pathogen population. Fifty citrus plants were graft-inoculated with Clas at the same time, and groups of five independent plants were used as source for acquisition assays at 60, 130, 145, 165, 180, 200, 235, and 280 days after inoculations (DAI). Clas was acquired by ACP from both symptomatic and asymptomatic citrus, and a positive linear relationship was observed between pathogen population within the source plant and the percentage of infective psyllid samples. Similarly, the bacteria population within the plant increased over time and became stable at 235 and 280 DAI when HLB symptoms became more evident. In 2010 we will be analyzing the transmission data of this experiment in relation to CLas population in the source plants. The results obtained so far indicate that vector control should be targeted to young shoots of citrus trees, where developing ACP nymphs can efficiently acquire the pathogen and subsequently spread the pathogen as emerging adults. Because ACP can acquire CLas on symptomless plants, vector control should be an important component in HLB management, in addition to eradication of symptomatic trees. Experiments related to studies 4 and 5 will be set up in 2010 and will help to clarify if insecticide treatment can prevent transmission.
This research project is directed towards controlling psyllids using biologically-based control strategies that employ the use of RNAi technology against key biological control pathways, peptide hormones and protein inhibitors that, if expressed in transgenic citrus, would enhance plant resistance to psyllids feeding. DIET: Both protein-based and RNAi strategies were tested by feeding psyllids artificial diets. Both protein-based and RNAi strategies were tested using artificial diets on which pysllids were fed. Psyllids in nature, feed on phloem content of citrus and its relatives. Thus, psyllids do not tolerate many alterations to diet composition that is drastically different than the phloem content. Addition of high concentrations of proteins or single stranded and double stranded RNA (ssRNA and dsRNA) reduces psyllids survival. Therefore, we determined the acceptable concentrations of each molecule and cofactor that was added including a suitable buffer to allow continuous feeding and maintenance of a physiological pH that was not detrimental to psyllids. We also identified an antimicrobial agent that was added to the diet and prevented fungal growth but did not harm the psyllids or their associated and obligate symbiotic microflora. Prior to the identification of the antifungal agent, fungal contamination of the diet caused unacceptable high level of psyllids mortality because the fungus is carried by the psyllids and can enter the diet through the psyllids feeding process. Control experiments showed that addition of dsRNA molecules, that did not target psyllids transcripts, at up to ~16 ng/uL improved psyllids performance, but above this concentration, the non-specific dsRNA would reduce psyllids survival. Therefore, comparisons of efficacy of specific psyllids gene targeting dsRNA were done with dsRNA that did not target psyllids genes. PROTEIN: In separate experiments, mosquito peptide hormone, TMOF, and Diaprepes abbreviates (citrus root weevil) cysteine protease inhibitor (CPI) were added to an artificial diet that was fed to psyllids. TMOF and CPI were tested at concentrations of 10 ‘g/’L and 3 ‘g/’L, respectively. After 10 days of feeding, all the psyllids that were fed diets containing either TMOF or CPI died, whereas only 40% mortality was observed in psyllids that fed on the control diets. TMOF caused 15% mortality after 4 days of feeding as compared with less than 5% mortality in the control group. Psyllids that were fed CPI did not show significantly higher mortality than the controls until after 7 days of feeding, because CPI was tested at less than . the concentration that was used for the TMOF because of limited availability. During the second year of the grant’s period more CPI will be synthesized and purified to study dose effect and optimal concentration, as well as, potential for synergistic effects when both proteins are present within the same diet. RNAi: Ten psyllids genes representing three gene families of cathepsins (five genes), vacuolar ATPases (four genes), and tubulin (one gene) were targeted and their dsRNA (16 ng/’L) fed to psyllids using artificial diets. The earliest effects were observed at ~4 days after feeding and feeding continued until day 10. At day 5 after feeding, two cysteine protease dsRNAs targeting cathepsin L and cathepsin B genes had the highest mortality 2-fold higher than controls. At day 10, three vacuolar ATPases and three cathepsins (B, L and F) showed significantly higher mortality than the controls. The current results reflect the use of reduced concentration of dsRNA because some transcripts that are active at higher concentrations in the diet do not show significant activity when reduced to 16 ng/’L. Dose curves and combinatorial experiments are in progress to determine if combinations of these active dsRNA molecules can provide synergistic effects. QRT-PCR experiments are in progress to determine the influence of dsRNA on cognate psyllid transcript levels. Our results show that specific dsRNA and proteins cause psyllids mortlity. Using these observations it is possible now to develop transgenic citrus trees that are resistant to psyllids and express these moieties in their phloem.
Our longterm goal is to identify and then use specific psyllid RNA sequences to induce RNA interference (RNAi) activity resulting in a negative phenotype (even death) in recipient psyllids. We envision that successful application of RNAi towards the Asian citrus psyllid (ACP, D. citri) will negatively impact their ability to colonize and/or reproduce on selected plants and thereby interfere with their ability to transmit C. Liberibacter spp., the causal agents of HLB. This strategy could then be an important component of HLB and/or ACP control. To make rapid progress we are using the tomato psyllid (Bactericerca cockerelli), which colonizes herbaceous plants (tomatoes, tobaccos) and transmits another Liberibacter spp. (C. L. psyllaurous; C. L. solanacearum) to several plant species. This psyllid:Liberibacter systems is very similar to the ACP:L. asiaticus complex associated with HLB. During this year we have established high quality colonies of B. cockerelli and identified three solanaceous plant species (tomato, Nicotiana benthamiana, N. tabacum) for use in our experiments. These plants are good hosts for the virus we will use to deliver interfering RNAs to plants, Tobacco mosaic virus (TMV), and for B. cockerelli and C. L. solanacearum. Furthermore, C. solanacearum induces specific and characteristic symptoms in these plants. We are using all three plant species for our ongoing efforts and by using recombinant TMV we have shown that we can induce production of specific siRNAs corresponding to the recombinant sequence in these plant species. We can also detect the presence of the recombinant RNAs in psyllids after feeding on these plants. We have utilized two complementary approaches to generate psyllid interfering RNAs for our work. The first has been by mining existing GenBank data for potentially useful D. citri sequences, and the second has been by constructing a new, normalized cDNA library for B. cockerelli mRNAs directly in the TMV-based virus delivery system. Both approaches have yielded a number of potential RNAi targets. We analyzed the D. citri EST database and identified 179 RNAs which are predicted to be expressed in D. citri midguts. Based on these analyses we cloned 80 midgut sequences (partial sequences varying in size between 500-1000bp) from D. citri. By BLAST comparative sequence analyses we next cloned 36 D. citri homologs from B. cockerelli. Sequence comparisons showed these to be highly homologous (80 ‘ 90%) to corresponding D.citri sequences. RT-PCR analysis was used to assess RNA presence in psyllids and confirmed our predictions showing that 25 of these were abundant in B. cockerelli gut RNA extracts. We believe that gut RNAs are potentially useful targets, and their knockdown effects can be specifically monitored in gut tissues. Sequence-based analyses identified some, including ion transporter and ATPase sequences, which are ideal candidates for RNA interference. We have developed three approaches for delivering candidate interfering RNAs to psyllids including TMV-based expression, micro-injection of dsRNAs into the psyllid hemocoel, and artificial membrane feeding in vitro. We are presently using all three to determine their abilities to induce RNAi effects in B. cockerelli. RNAi effects are monitored by RNA knockdown (northern hybridization and qRT-PCR) and by phenotype in recipient psyllids. We also generated and are presently analyzing a normalized B. cockerelli cDNA library. The library sequences were cloned directly into our TMV-based plasmid for expression in plants. Nucleotide sequence analysis has allowed us to confirm the quality of the library. Homologs corresponding to NADH dehyrogenase subunit, elongation factor Tu, glutathione peroxidase, peritrophin, alpha-keto reductase etc. have been identified within the library and these are being tested.
The purpose of this project has been to develop an effective repellent for the Asian citrus psyllid (ACP). We recently identified DMDS as a metabolite produced in large quantities by wounded guava leaves, which is a repellent of ACP. Volatiles from guava leaves significantly inhibited attraction of ACP to normally attractive host-plant (citrus) volatiles. A similar level of inhibition was recorded when synthetic DMDS was co-released with volatiles from citrus leaves. In addition, the volatile mixture emanating from a combination of intact citrus and intact guava leaves induced a knock-down effect on adult ACP. Compounds similar to DMDS including dipropyl disulfide, ethyl-1-propyl disulfide, and diethyl disulfide did not affect the behavioral respomse of ACP to attractive citrus host plant volatiles in laboratory olfactometer assays. Our field experiments confirmed the results of our laboratory olfactometer assays. Deployment of synthetic DMDS from polyethylene vials (Alpha Scents) and SPLAT wax dispensers (ISCA technologies) reduced populations of ACP in an unsprayed citrus orchard for up to 3 weeks following deployment. Given that population densities were equivalent among plots prior to the deployment of DMDS treatments, we hypothesize that DMDS repelled adult ACP from treated plots. However, we cannot exclude the possibility that a proportion of the ACP populations in DMDS-treated plots may have been reduced due to direct intoxication. By the fourth week, there was no remaining DMDS in the dispensers, which likely explains why populations were once again equivalent in treated and control plots. Given the volatility of DMDS, one of the main obstacles to the development of a practical DMDS formulation for ACP management will be development of a slow-release device that maintains the chemical above a behaviorally active threshold for long periods. The polyethylene vials and SPLAT dispensers evaluated in these initial proof-of-concept investigations will likely not be economically practical for releasing DMDS for control of ACP in their current form. Both the number of dispensers required per acre (~200) as well as the amount of active ingredient required per three weeks (~3 kg) would likely be economically prohibitive for a hand applied dispenser. Furthermore, the dispensers evaluated in this study resulted in a ~2/3 decrease in field populations of ACP, which would be insufficient for effective control of this pest as a stand alone treatment. Another immediate logistical hurdle for developing DMDS into a practical psyllid management tool is the chemical’s strong and unpleasant odor. This may render field application difficult and potentially limit the use of DMDS depending on fruit harvesting schedules or proximity to urban areas. Ideally, a slow-release dispenser needs be developed that could achieve 150-200 d of behaviorally efficacious release. ACP populations are much more prevalent on crop borders and thus targeted applications of DMDS to those areas may be immediately useful with a dispenser that is not yet optimized. Our current efforts are focussing on further optimizing these dispensers to increase the duration of efficacy. ISCA has recently developed four new formulations of SPLAT dispensers that we will be evaluating in 2010. Most recently, we have identified new compounds that are ACP repellents. Trisulfides (dimethyl trisulfide) inhibited the response of ACP to citrus volatiles more than disulfides (dimethyl disulfide, allyl methyl disulfide, allyl disulfide). Monosulfides did not affect the behavior of ACP adults. A blend of dimethyl trisulfide (DMTS) and dimethyl disulfide (DMDS) in 1:1 ratio showed an additive effect on inhibition of the response of ACP to citrus volatiles and was more effective than DMDS alone in the lab. We met the objectives proposed for year 1 of this project. We hope to continue development of a psyllid repellent in year two by investigating the new formulations developed for sulfur compounds as well as blend of the newly identified chemicals. ISCA technologies (Riverside, CA) is working with CRDF/FCPRAC to register a DMDS-based product for ACP control in Florida.
The main objective of this series of investigations has been to develop an effective attractant for Tamarixia radiata, the main parasitic wasp attacking Asian citrus psyllid (ACP) in Florida. Development of an effective attractant for this insect will allow for accurate monitoring of this beneficial insect and it will allow us to recruit and establish high populations of this beneficial insect to improve biological control of ACP. This first objective of this research was to describe the antennal capabilities of this insect. This was promptly accomplished and was published in a peer-reviewed scientific journal (Onagbola, E.O., D.R. Boina, S.L. Herman, and L.L. Stelinski. 2009. Annals of the Entomological Society of America. 102: 523-531). Next, we moved onto conducting an in depth analysis of the chemicals produced by both sexes of this parasitoid. We discovered that both male and female ACP parasitoids release several volatile compounds. In laboratory behavioral tests, we found that male parasitoids were attracted to .-Butyrolactone to the same degree as to female parasitoids, indicating that this is likely the sex-attractant pheromone females produce and release to attract males. Subsequently, we developed a polyethylene-tube dispenser for releasing this chemical in collaboration with our industry partner, Alpha Scents. Field trapping tests indicated that in the early summer traps baited with .-Butyrolactone caught more Tamarixia radiata than unbaited traps, but this trend did not hold up in the late summer months. In addition to investigating the chemicals that Tamarixia radiata produces, we conducted an initial investigation with another chemical that is known to attract natural enemies of insect pests and reduce their populations. Methyl salicylate (MeSA) is a known herbivore-induced plant volatile which has been shown to attract natural enemies (e.g. parasitoids, coccinellid beetles, predatory mites) and repel plant pests (e.g. mites, herbivorous beetles, aphids). The treatments compared were plots treated with MeSA versus untreated control plots; all treatments were replicated five times. Two dispensers were deployed per tree in April of 2009 and populations of psyllids and their natural enemies were monitored through September. Our preliminary data indicate that treatment of citrus plots with MeSA increased populations of natural enemies such as lady beetle and fly predators of ACP and well as the ACP parasitoid, Tamarixia radiata. In addition, populations of ACP were lower in MeSA-treated plots compared with untreated controls. Finally, we have described the host searching and mating behavior of this ACP natural enemy. We examined the behavior of T. radiata to in response to volatiles from citrus and ACP or ACP feeding on citrus. We also examined the behavioral response of male and female T. radiata to conspecifics of the opposite sex to determine whether olfactory signals mediate mate location. T. radiata adults exhibited a sexually dimorphic response to volatiles emanating from ACP and citrus. Female T. radiata were attracted to the odors emanating from ACP nymphs. However, female wasps did not respond to odors emanating from ACP adults, ACP honey dew secretions or intact citrus. Odors emanating from ACP-damaged citrus were not attractive to T. radiata but stimulated attraction of wasps to ACP feeding on damaged plants. T. radiata females were not attracted to ACP immatures when they were presented as visual cues. Odors emanating from ACP immatures or adults, their honey dew secretions or citrus plants were not attractive to male T. radiata. Male T. radiata were attracted to the odor of female conspecifics compared with blank controls in the absence of associated citrus host plant volatiles. Collectively, our results provide behavioral evidence that : 1) female T. radiata uses volatiles from ACP nymphs to locate its host and : 2) female T. radiata release a volatile pheromone that attracts males. In summary, we not completed all of the objectives of this research and actually exceeded our goals. Based on this proposal, we will be continuing the development of an MeSA dispenser to recruit T. radiata and improve biological control of ACP in 2010.
The purpose of this proposal has been to identify and develop attractants for the Asian citrus psyllid (ACP). The intent is to develop a highly effective attract-and-kill control system for ACP with such attractants, as well as to develop highly effective monitoring traps to effectively evaluate ACP population densities so that pesticide spray schedules can be optimized. Thus far, in collaboration with USDA colleagues, we have confirmed that virgin and mated male ACP are attracted to female ACP. These data suggest that female ACP produce an attractant pheromone that attracts male ACP. Second, we have proven that both male and female ACP are attracted to their host plant volatiles. The host plants tested were: ‘Duncan’ grapefruit, sour orange, ‘Navel’ orange, and Murraya paniculata. Responses varied by plant species and by psyllid sex and mating status. The presence of a visual cue typically enhanced attractiveness of olfactory cues; in no case did unmated individuals show evidence of attraction to host plant odors in the absence of a visual cue. In behavioral assays in the laboratory, we found that .-Butyrolactone is attractive to male ACP, but not to females suggesting that this chemical may be part of the female ACP pheromone blend. In collaboration with an industry partner, (Alpha Scents, West Linn, OR), we obtained custom-made release devices for .-Butyrolactone as well as dispenser for synthetic plant volatiles identified and developed by a USDA collaborator. In our initial field tests, results with .-Butyrolactone have been inconclusive. Although in one trial it appeared that this chemical increased catch of ACP on traps, the results have been inconsistent in follow up trials. We are currently analyzing cuticular extracts of ACP to find further pheromone components because it appears that although .-Butyrolactone may be a component of the pheromone, it is not the only chemical responsible for attracting male ACP. This work is being conducted in collaboration with Stephen Lapointe from USDA-ARS in Fort Pierce. Also, we have evaluated a 5-component blend of synthetic plant volatiles as an ACP attractant based on our work with psyllid attraction to citrus. This blend and its associated dispenser is produced by Alpha Scents. We have shown attraction of ACP to these chemicals in the laboratory, but catch of ACP on traps in the field was not increased by this plant volatile lure in the field. We continue to work on refining this blend and its dosage in an effort to develop an attractive lure for the field. Concurrently with our work towards developing an ACP attractant, we have developed an attract-and-kill formulation for ACP with our industry partner and Co-PI Darek Czokajlo from Alpha Scents. We are working with a gel matrix with UV-protective properties that releases both the attractant and contains a small amount of pesticide. As ACP approach and touch the lure droplet laced with insecticide, they pick up a lethal dose of toxicant and die. We compared formulations containing 6, 14, and 22% imidalcloprid against Asian citrus psyllids in the laboratory. We found the the 14% imidacloprid formulation is superior to the 6% formulation, but that there was no added benefit of the 22% formulation. An optimized attractant is still needed before this formulation could be successfully employed for ACP control and this research is currently in progress. In separate trials working on a different attract-and-kill formulation consisting of an emulsified wax formulation (SPLAT, ISCA Technologies), we compared the insecticides Spinosad, Methoxyfenozide and Tebufenozide against the psyllid. We found that Methoxyfenozide and Tebufenozide are not effective with this formulation and that Spinosad is only marginally effective resulting in about 50% mortality. We have recently established a new technique for collecting the chemicals produced by psyllids in Dr. Lapointe’s lab, which are being analyzed by collaborator, Dr. Webster in New York. We are hopeful that this new technique will allow identification of the ACP attractant pheromone. Dr. Lapointe and his post-doctoral associate, who have considerable expertise in insect chemical ecology, have joined the project and will play a major role in year 2 investigations.
The purpose of this investigation has been to develop, evaluate, and optimize biorational management tools for Asian citrus psyllid (ACP) including insect growth regulators (IGRs), antifeedants, available repellents, and standard insecticides. Initially, we determined the optimal temperatures at which to use currently available insecticides for ACP control. This was published in a peer-reviewed journal (Journal of Economic Entomology, Vol 102, 685-691). Second, we investigated the activity of the IGRs pyriproxyfen (Knack), buprofezin (Applaud) and diflubenzuron (Micromite) on ACP eggs, nymphs and adults to evaluate their potential usefulness as biorational insecticides for inclusion into integrated pest management (IPM) strategies for ACP control. All three chemicals exhibited strong ovicidal and larvicidal activity against ACP eggs and nymphs, respectively, in age- and concentration-dependent manners. Fewer eggs hatched into nymphs at the higher concentrations tested (80-160 ‘g mL-1). Furthermore, all three chemicals exhibited transovarial activity by significantly reducing the fecundity of females and viability of eggs deposited by females that emerged from treated fifth instar nymphs. Topical application of each chemical to adults also significantly reduced fecundity and egg viability. Application of each chemical at 160 ‘g mL-1 resulted in the highest inhibition of egg hatch in younger eggs (0-48 h old) laid before or after treatment and strongest suppression of adult emergence from early instar nymphs compared with other rates tested. Each chemical also markedly reduced female fecundity and egg viability for adults that were exposed either directly or indirectly. Also adults emerging from nymphs treated with pyriproxyfen were deformed and died soon after emergence. The direct (ovicidal and larvicidal) and indirect (transovarial) effects of the IGRs against immature and adult ACP, respectively, suggest that integration of these insecticides as part of an IPM strategy should negatively impact ACP populations over time. In a subsequent investigation, we have been studying the sub-lethal effects of various insecticides. Given the broad use of imidacloprid for management of ACP, particularly on young trees, we investigated it’s possible sub-lethal effects first. Because of the variation in spatial and temporal uptake and systemic distribution of imidacloprid applied to citrus trees and its degradation over time in citrus trees, ACP adults and nymphs are exposed to concentrations that may not cause immediate mortality but rather sublethal effects. Our objective was to determine the effects of sublethal concentrations of imidacloprid on ACP life stages. Feeding by ACP adults and nymphs on plants treated daily with a sublethal concentration (0.1 ‘g mL-1) of imidacloprid significantly decreased adult longevity (8 d), fecundity (33%), and fertility (6%) as well as nymph survival (12%) and developmental rate compared with untreated controls. The magnitude of these negative effects was directly related to exposure duration and concentration. Sublethal concentrations of imidacloprid negatively affect development, reproduction, survival, and longevity of ACP which likely contributes to population reductions over time. Also, reduced feeding by ACP adults on plants treated with sublethal concentrations of imidacloprid may potentially decrease the capacity of ACP to successfully acquire and transmit the HLB causal pathogen. Pymetrozine is a chemical that is known to paralyze the muscles involved in plant probing in plant-sap sucking insects such as aphids and is known to prevent transmission of aphid and whitefly transmitted viruses. Thus, we felt it was an optimal candidate to test if it would prevent transmission of HLB by nymphal and adult ACP. Our results have confirmed that pymetrozine does not prevent transmission of the HLB pathogen by ACP and thus will not be a useful tool for ACP management. During the first year of this investigation, we have exceeded the goals outlined for year 1. We hope to continue in year 2. The positive benefits of IGRs have been recognized by many growers who use them successfully in their annual pest management programs.
The movement patterns and dispersal capabilities of Asian citrus psyllid (ACP) required investigation to better understand the spread of huanglongbing (HLB) and to improve management strategies for ACP. Understanding movement patterns and dispersal behaviors of ACP will be essential in creating optimal pest control strategies with the hope of curbing the spread of HLB, and protecting citrus production in Florida. To investigate ACP movement, we adopted an immunomarking technique which utilizes crude food proteins (chicken egg albumin, bovine casein, and soy protein) to track the movement of ACP in Florida citrus. In brief, ACP are marked with proteins in the field and allowed to move naturally. They are re-captured on traps and analyzed for the marking protein. In this way, we can determine exactly: where psyllids came from, how far they moved, and how fast they moved. Initially we found that ACP movement is biased in the direction from abandoned or marginally managed groves into well managed groves. We also found that ACP are capable of moving back and forth between 2 groves separated by 100 yards within 2 days. Furthermore, ACP are capable of invading up to 100 yards into managed grove interiors within 4-7 days. Furthermore, during the spring, we found that ACP move even when there is abundant flush (food/egg laying sites) available. In terms of where psyllids tend to invade most often, we found that invading ACP are most often found within the first 3-4 rows of trees from the plot borders. More recently, we have been studying the seasonality of ACP movement. This research has been conducted by quantifying how many psyllids move on a monthly basis over the course of the entire year. The first year’s results indicate that the majority of psyllid movement occurs during the spring and early summer months, while very little movement occurs September through February. We will need to repeat this experiment for another year to be certain that this pattern is consistent. There may be certain times of the year when frequent insecticide treatment is needed (particularly on plot borders) due to psyllid movement, but there may also be certain other times of the year when fewer treatments are necessary when ACP are not moving. Given that we have proven that unmanaged and abandoned groves serve as a source of ACP infestation, our recent goal has been to determine the status of HLB infection in ACP and citrus trees in paired unmanaged and managed citrus groves separated by 100 yards or less. To accomplish this research, we sampled 7 pairs of adjacent unmanaged and managed groves. Surprisingly, we have documented slightly higher rates of HLB infection in both psyllids and trees in unmanaged groves than managed groves that were separated by 100 yards or less. These results confirm that abandoned and unmanaged groves not only serve as a source of psyllid infestation for adjacent managed groves, but also likely serve as a source of HLB infection. Effective HLB management in Florida citrus will therefore likely require removal of abandoned groves. Also, growers who are next to unmanaged citrus should consider protecting their border areas intensely to prevent immigration of infected psyllids into their groves. In summary, we have found that: 1) Movement of ACP occurs between unmanaged and managed groves; 2) The majority of ACP occur on the border rows of managed groves; 3) ACP adults can move at least 250 yards within 4 days; 4) Movement appears to vary over the course of the season ‘ more movement in summer than fall/winter; 5) The HLB bacterium is present in ACP and trees in unmanaged and nearby managed groves; and 6) infection levels are 2.3 & 1.5 times greater in unmanaged groves compared with nearby managed groves. We have either completed or exceeded our goals for year 1 and look forward to repeating necessary experiments in year 2 and to collecting new data to complete the two year objectives. Some of this research has already been published in a peer reviewed journal (Environmental Entomology, 2009, 38: 1250-1258) and other portions are currently under review.
In Texas, ACP in orchards have been in a state of semi-dormancy since October. ACP from our colonies have been non-responsive in behavioral tests. Behavioral tests will resume once ACP resume being responsive to test stimuli. Tests are planned to compare the responsiveness of ACP to combava petigrain oil, essential oil distilled from the foliage of Citrus hystrix. We have generated a database of plant odors to be analyzed by GC-EAD in Florida when that system becomes operational. If Florida, we are obtaining the first successful recordings from ACP antennae. After weeks of frustration with our Syntech electroantennogram system, we invited Dr. Peter Ockenfels of Syntech, Germany to our lab. He spent 4 days troubleshooting and successfully putting the system back on line. This opens the way to searching for antennally active compounds from host plants and conspecifics. Collections of cuticular hydrocarbons from greenhouse-reared ACP were made and exhaustively characterized by GC/MS. The resulting database and samples have been forwarded to Dr. Fran Webster at Syracuse University for analysis. Dr. Webster is an analytical chemist with expertise in NMR and structure elucidation. To date, we have not seen any obvious difference between hydrocarbon extracts collected from male and female ACP. Approximately 50 compounds have been identified to date. Extracts will be analyzed by GC-EAD to determine if any of these compounds elicit antennal response, followed by behavioral studies.
We have made good research progress this quarter. Laboratory testing of traps: We have conducted several and continue to conduct other laboratory bioassays to determine the behaviors used by psyllids during approach, landing and post landing on key parts of traps. We are characterizing the physical properties of traps including size, angle of surface to substrate, trap grid configurations, relative proportions of trap parts and color relative to their impact on psyllid walking, jumping and flight behavior. These factors clearly affect psyllid behavior in response to host plants, and therefore, will affect trap efficacy and efficiency. These experiments are enabling us to optimize trap configurations and to develop an estimate of the importance and the relative trade-offs in efficacy among trap components. Based on our preliminary lab results we redesigned trap prototypes for field testing and those have been placed in the field. We are now in the process of setting up more technically-rigid psyllid behavior experiments under more controlled laboratory conditions with a Noldus olfactometer and related equipment to get a better handle on how behavioral factors interact and to increase the inferences we may draw from such experiments. Field testing of traps: We are presently conducting replicated field tests of about 10 new and different trap configurations building on our lab and field results as prototypes in a replicated experiment in commercial citrus in the Immokalee area. Results from this field test and the continuing lab results will help us to focus further experiments. We expect to develop an improved trap for Asian citrus psyllids that can be used both in groves and for regulatory purposes in places where citrus plants are sold. This trap would collect the insects and preserve them using methodology that will enable testing for the presence of HLB pathogens at some later date.
Researchers from the Auburn University College of Engineering are constructing a combined empirical and simulation based toolset to ultimately optimize the “how much” (rate of release) and “where” (physical locations within a orange grove) deployment of a citrus psyllid repellent. The current focus is on recording physical measurements of repellent diffusion into air under prescribed ambient conditions and constructing a computational fluid dynamics (CFD) model of the airborne mixing of the repellent within the flowfield generated by a citrus grove. These studies serve as an engineering requirements flow-down, providing the necessary scientific data to shape specific deployment hardware options that balance effects longevity with application cost. Given the recent Citrus Research and Education Center (CREC) laboratory and field studies using the “Splat” (emulsified paraffin wax) approach for dimethyl disulfide (DMDS) application, DMDS is selected as the initial agent for repellent release rate studies. The PIs have traveled to CREC for first-hand discussion and observation of those studies and interfaced with ISCA Technologies, the manufacturer and supplier of “Splat”. Auburn is currently setting up a laboratory facility to measure and study the release rate of DMDS as input for airflow computer simulations. Initial experimental focus is on DMDS in liquid form, coupled with an artificial membrane to provide control over the rate of diffusion into air. It should be noted that the Auburn strategy maintains a broad top-level approach, favoring no one particular deployment method and leaving options open to consider other potential repellant chemical formulations as well. Implementation and evaluation of an initial CFD model is underway. The vegetation canopy is approximated by a porous medium that acts as a volumetric sink on airflow momentum, while insertion of repellent is accomplished by discrete point sources within the hedge row geometry of a typical mature grove. A literature survey supporting the use of CFD modeling applied to vegetation canopy airflow and mixing has provided valuable data and best practices. Of particular interest is the related problem of the effects of tree plantings in urban street canyons for the dispersion of automotive exhaust pollution. The literature reports good correlation between experimental and numerical airflow and dispersion findings and provides parametric information such as average canopy porosity for various tree species.
Researchers from the Auburn University College of Engineering are constructing a combined empirical and simulation based toolset to ultimately optimize the “how much” (rate of release) and “where” (physical locations within a orange grove) deployment of a citrus psyllid repellent. The current focus is on recording physical measurements of repellent diffusion into air under prescribed ambient conditions and constructing a computational fluid dynamics (CFD) model of the airborne mixing of the repellent within the flowfield generated by a citrus grove. These studies serve as an engineering requirements flow-down, providing the necessary scientific data to shape specific deployment hardware options that balance effects longevity with application cost. Given the recent Citrus Research and Education Center (CREC) laboratory and field studies using the “Splat” (emulsified paraffin wax) approach for dimethyl disulfide (DMDS) application, DMDS is selected as the initial agent for repellent release rate studies. The PIs have traveled to CREC for first-hand discussion and observation of those studies and interfaced with ISCA Technologies, the manufacturer and supplier of “Splat”. Auburn is currently setting up a laboratory facility to measure and study the release rate of DMDS as input for airflow computer simulations. Initial experimental focus is on DMDS in liquid form, coupled with an artificial membrane to provide control over the rate of diffusion into air. It should be noted that the Auburn strategy maintains a broad top-level approach, favoring no one particular deployment method and leaving options open to consider other potential repellant chemical formulations as well. Implementation and evaluation of an initial CFD model is underway. The vegetation canopy is approximated by a porous medium that acts as a volumetric sink on airflow momentum, while insertion of repellent is accomplished by discrete point sources within the hedge row geometry of a typical mature grove. A literature survey supporting the use of CFD modeling applied to vegetation canopy airflow and mixing has provided valuable data and best practices. Of particular interest is the related problem of the effects of tree plantings in urban street canyons for the dispersion of automotive exhaust pollution. The literature reports good correlation between experimental and numerical airflow and dispersion findings and provides parametric information such as average canopy porosity for various tree species.
We are in the first year of our project. Our goal is to identify and then use specific psyllid RNA sequences to induce RNA interference (RNAi) activity in recipient psyllids. We propose to express psyllid interfering RNA sequences in plants and induce RNAi when psyllids feed on these plants. We envision that successful application of RNAi towards the Asian citrus psyllid (ACP, D. citri) will confer a negative phenotype (even death) in psyllids, such that psyllids cannot colonize and/or reproduce on selected plants. By controlling the psyllid vector we believe this will complement other efforts to help control HLB. To make rapid progress we are using the tomato psyllid (Bactericerca cockerelli), which colonizes herbaceous plants (tomatoes, tobaccos) and is the vector of another Liberibacter spp. (C. L. psyllaurous). We are using tomatoes (Early Pak 7), and Turkish tobacco plants for evaluating Tobacco mosaic virus (TMV)-driven expression of psyllid genes for inducing RNAi. Both of these plants are easy to use, TMV susceptible and both are readily colonized by B. cockerelli. With Turkish tobacco we obtain more consistent Agrobacterium tumefaciens-based TMV infection than for tomatoes, but both plants, as well as Nicotiana benthamiana, are being used currently, although psyllids do not prefer N. benthamiana. We can induce production of specific siRNAs corresponding to the recombinant sequences in these plants (N. benthamiana and tomato), and we have demonstrated so far that psyllids can acquire TMV-based RNAs from TMV-infected tomato plants. Now we are evaluating acquisition and effects of anti-psyllid RNAs. We have utilized two approaches to obtain psyllid interfering RNAs for our work. The first has been by mining existing GenBank data for potentially useful D. citri sequences, and the second has been by constructing a new, normalized cDNA library for B. cockerelli mRNAs directly in a TMV-based virus delivery system. Both approaches are proceeding very well. We have used the D. citri EST database and have identified 1904 contigs, 179 of which are predicted to be expressed in D. citri midguts. We believe that midgut sequences are very likely good targets for orally-acquired anti-psyllid interfering RNAs or dsRNAs, as the midgut will encounter RNAs via psyllids feeding on plants. We have now successfully cloned 80 midgut genes (partial sequences varying in size between 500-1000bp) from D. citri. BLAST comparative sequence analyses allowed us to design 56 predicted conserved primers for use in cloning D. citri homologs from B. cockerelli. We have cloned 36 putative homologs so far, and 30 have been confirmed by sequence analysis. These are highly homologous (80 ‘ 90%) to corresponding D.citri sequences. So far RT-PCR using RNA prepared from B. cockerelli gut tissues confirmed that 25 were present. Sequence-based analyses identified some, including ion transporter genes, which are ideal candidates for RNA interference, and we are proceeding using TMV-based expression as well as direct micro injection to determine their efficacy against B. cockerelli. We have developed a micro-injection system for psyllids. This allows precise injection of known quantities of dsRNAs into the psyllid hemocoel. This is being used as a known control approach to assist in identifying candidate sequences for RNAi. Sequences that show effects after injection will be immediately tested in plants. We now have a normalized B. cockerelli cDNA library. The library sequences are cloned directly into our TMV-based plasmid for expression in plants. We are now confirming the library quality by nucleotide sequence analysis, and transforming the TMV-based plasmids into A. tumefaciens for use in whole plant anti-psyllid assays. Initial sequencing showed us that the library contains psyllid sequences and thus will be very useful for our effort.
We are conducting a series of studies to determine the impact of various factors on the efficiency of acquisition of Candidatus Liberibacter asiaticus (CLas) by the Asian citrus psyllid (ACP), Diaphorina citri, such as vector development stadia (study 1) and leaf age, pathogen titer and symptom expression in infected citrus (studies 2 and 3). We are also investigating how transmission efficiency varies depending on the duration of the inoculation access period (study 4), and if a systemic insecticide can prevent transmission (study 5). We have started experiments related to studies 1-4, and the research is progressing as expected in the original plan. In the previous quarterly reports (July/09 and Oct/09), we presented partial results of studies 1 and 2, indicating that nymphs in all development stadia (1st-5th instars) can efficiently acquire the pathogen; adults can also acquire it, but efficient acquisition depends on the availability of young leaves in infected plants, apparently because phloem sap ingestion (and thus pathogen acquisition) by ACP adults is more frequent and last longer on the younger leaves. In this report, we present new and interesting findings about CLas acquisition rate by D. citri adults on infected citrus with variable pathogen population (study 3). Fifty potted sweet orange on Rangpur lime rootstock trees ( 5-month old) were graft-inoculated with Clas at the same time, and groups of five independent plants were used as source for acquisition assays at 60, 130, 145, 165, 180, 200, 235, and 280 days after inoculations (DAI). In the last three periods most of the source plants were symptomatic. In each assay, a group of 50 lab-reared healthy D. citri adults was confined on young shoots of each source plant (inside sleeve cages) for an acquisition access period (AAP) of 7 days, and then kept on healthy citrus seedlings for a latency period of 21 days. Three to 15 psyllid samples (three psyllids per sample) from each source plant were subsequently tested for infectivity using real-time PCR. Clas population in the source plants was quantified right after the AAP by RT-PCR, using DNA extracted from leaves of the shoots used for acquisition. Clas was acquired by D. citri from both symptomatic and asymptomatic citrus, and a positive linear relationship was observed between pathogen population within the source plant and the percentage of infective psyllid samples [y (Ct) = -0.114 x (infectivity rate) +28.36; R2 = 0.33]. Similarly, the bacteria population within the plant increased over time [y (Ct) = -0.045 x (DAI) +28.43; R2 = 0.34) and became stable (t test, P = 0.010) at 235 and 280 DAI when HLB symptoms became more evident. Considering all source plants tested, acquisition efficiency by D. citri ranged from 0 to 100%, with a success ratio of 95% on symptomatic plants (n=17) against 64% on non-symptomatic but infected plants (n=24).
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