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 psyllid feeding. Both protein-based and RNAi strategies were tested by feeding psyllids artificial diets. To support the artificial diet assays, we optimized the diet composition by adding an antimicrobial agent to eliminate fungal growth that is introduced by the psyllids during the assay period. Using this approach we identified suitable buffers and optimal diet pH during the feeding period. In separate experiments, Tryspin Modulating Oostatic factor (TMOF), a mosquito decapeptide hormone, and cysteine protease inhibitor (CPI) from the Asian Citrus psayllids that was identified in our laboratory were added to artificial diets on which psyllids were allowed to feed. TMOF was tested at a concentration of 10 ‘g/’L and CPI was tested at a concentration of 3 ‘g/’L. Since psyllids take in very small amount of the artificial diet, in nl quantities, the amounts of the tested proteins are physiological rather than pharmacological. After 10 days of feeding, 100% mortality was observed in psyllids feeding on diets containing TMOF or CPI, whereas, 40% mortality was found in psyllids feeding on the control diets. The earliest significant effect of added TMOF to the diet was observed at 4 days of feeding. TMOF caused ~15% mortality whereas the mortality of the control group was less than 5%. CPI fed psyllids caused a significant higher mortality than the controls after 7 days of feeding. CPI was tested at lower concentrations that were used for TMOF, because of limited availability of the purified protein. Because of these results we plan to purify more CPI for subsequent testing. In the fourth quarter of the grant period we plan to expand this work and study dose effect and optimal concentrations, as well as, potential for synergistic effects when both TMOF and CPI are fed together to psyllids in the same diet. Continued success of this strategy will allow us to develop transgenic citrus expressing these proteins in the phloem making citrus resistant to adult and nymph psyllids. A third polypeptide was also tested at concentrations similar to TMOF, this polypeptide prevented psyllids from successful eating the artificial diet causing starvation and death. We plan to continue these studies and further characterize the polypeptide and feed it in concert with TMOF and CPI to be able to find out the combined effect on the Asian citrus psyllids. In parallel to these studies, we synthesized dsRNA molecules targeting 11 different psyllids essential genes encoding three different classes of proteins (alpha-tubuliln, V-ATPase, and Cathepsins). The DNA encoding a fragment of each of the 11 genes was synthesized and cloned into appropriate vectors. Primers used for dsRNA synthesis using the Megascript kit from Invitrogen was used to produce double stranded RNA (dsRNA) for RNA inhibition (RNAi) studies. We produced dsRNA for two alpha-tubulins and two V-ATPases. Initial feeding studies with alpha-tubulin dsRNA and V-ATPase dsRNA caused ~60% psyllids mortality as compared to only ~30% mortality for psyllids fed a control diet containing an equal amount of dsRNA not specific to the psyllid. Influence of dsRNA on cognate psyllid transcripts will be tested in psyllids that remain alive at 8 days. In summary we: 1. Prepared dsRNA molecules against alpha-tubulin and V-ATPase and fed them to Asian citrus psyllids showing that they are very effective causing high mortality. 2. Showed that physiological amounts of TMOF, a new polypeptide that was recently discovered in our lab and CPI that were fed to psyllids by an artificial diet caused 100% mortality.
The purpose of this proposal is to identify and develop attractants, both pheromone and host-plant based, 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 to better determine the need for spraying. 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 chemical, potentially a pheromone, that attracts male ACP. Within the past few months, a female-produced sex attractant pheromone was identified in the potato psyllid by researchers in Washington, which is the first example of a psyllid pheromone. 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. Generally, evidence of attraction was stronger in females and in mated individuals of both sexes relative to virgins. 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 were 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. Our ongoing studies are focusing on testing more insecticides with the wax formulation so that more effective psyllid kill can be achieved.
The purpose of this project has been to develop an effective repellent for the Asian citrus psyllid (ACP). Our work was initiated by investigating the volatiles released by guava plants and their effects on ACP behavior. Interplanting citrus with guava, Psidium guajava L., was reported to reduce ACP populations and incidence of HLB. Therefore, we initiated a series of investigations on the response of ACP to citrus volatiles with and without guava leaf volatiles and to synthetic dimethyl disulfide (DMDS), in laboratory studies and in the field. DMDS was recently identified as a metabolite produced in large quantities by wounded guava leaves. 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 response 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 the early spring which show promise. Most recently, we have identified new compounds that are ACP repellents. In general, 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.
The purpose of this investigation has been to develop, evaluate, and optimize biorational management tools for Asian citrus psyllid (ACP) including insect growth regulators and antifeedants. In our first set of laboratory studies with insect growth regulators, we investigated the activity of the insect growth regulators pyriproxyfen (Knack), buprofezin (Applaud) and diflubenzuron (Micromite) on ACP eggs, nymphs and adults to evaluate its potential usefulness as a biorational insecticide for inclusion into an integrated pest management (IPM) strategy 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). A significantly lower percentage of early instar nymphs (first, second and third) survived and emerged into adults at the higher concentrations tested (80-160 ‘g mL-1) compared with late instar nymphs (fourth and fifth). 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 separate 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. Furthermore, ACP adults that fed on citrus leaves treated systemically with lethal and sublethal concentrations of imidacloprid excreted significantly less honeydew (7-94%) compared with controls in a concentration-dependent manner suggesting antifeedant activity of imidacloprid. 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. In our initial investigations, we found that at a 100 ppm dosage applied to citrus plants, pymetrozine inhibits acquisition of Liberibacter by psyllids on Valencia, Clepatra Mandarin, and Persian lime by approximately 50%.
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. Findings on the rate of spread of HLB have been inconsistent, possibly due to variable rates of transmission and latency periods. 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 November. We will need to repeat this experiment for another year. However, if these movement patterns hold up as a real trend for more than one year, then it could have important implications for ACP management decisions. 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 much. 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 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.
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. The first goal of this proposal was to conduct an in depth morphological investigation of the antenna sensilla of this wasp parasitoid, including functional morphological studies, which would reveal the functional details of the discovered sensilla. This first objective was completed and the investigation was published in a peer-reviewed scientific journal (Onagbola, E.O., D.R. Boina, S.L. Herman, and L.L. Stelinski. 2009. Antennal sensilla of Tamarixia radiata (Hymenoptera: Eulophidae), a parasitoid of Diaphorina citri (Hemiptera: Psyllidae). 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. Our analyses revealed Propa-2-one, 1-Butanol and 4,6,8-Trimethyl nonene as female parasitoid-specific volatiles; Dodecane, 4,6-dimethyl, Acetc acid, .-Butyrolactone, and Diphenylamine as male specific volatiles while Decanal and 3-Methyl diphenylalamine were produced by both sexes. 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). Recent field studies deploying synthetic sachets of MeSA in grapes and hops showed that populations of pest species were reduced and populations of natural enemies were increased. We have conducted an initial proof of concept experiment in a small plot setting evaluating the effect of deploying Methyl Salicylate (MeSA) dispensers obtained from AgBio Inc. (Denver, CO) in small plots (35 tree). 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 llady 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. This data need to be confirmed on a large scale and in a wide variety of commercial citrus grove settings. Our goal is to investigate the use of MeSA as a control tool for ACP on a larger scale in a future investigation that has been submitted as a separate new proposal to the CRDF. For other insect pests, MeSA has been shown to not only attract beneficial insects to a source and thus improve biological control, but may have some repellent properties. Field demonstration of the efficacy of MeSA would provide citrus producers an easy, inexpensive, and biorational tool to conserve natural enemies and reduce pesticide use while controlling the ACP vector of citrus greening.
As of December, 2009, we continue to have over 16 actively growing Asian citrus psyllid derived insect cell lines. Serial dilution of cell lines in order to obtain more homogeneous cell populations are in progress. We have cell lines that are in suspension as well as those that attach to tissue flask substrata. Our cultures remain slow growing and one unanticipated obstacle that has emerged is asymmetric growth patterns for several cell lines, meaning cell population appear to grow slowly, then rapidly, then slowly again. These growth characteristics appear to be independent of media composition. This asymmetric growth is particularly apparent to attached cell lines and several suspension cell lines display robust growth with no asymmetry noted. We currently have cell lines in media without any antibiotics. Overall, we have met many of our 2009 goals which included determining (1) the optimal conditions for passage of the cell lines, (2) the stability of the cell lines and (3) the conditions for storage/preservation of the cell lines (in progress). We have also begun to assess the morphological characteristics of the cell lines and perform preliminary biochemical and genetic analyses on the cells. Since Sept. 2009 we have made our cell lines available to several groups at the Lake Alfred Citrus Research Center. In particular, we have established a collaborations with Dr. Davis in order to attempt to cultivate Liberibacter asiaticus with the cell lines (work in progress). In addition, we have made our cell lines available to Dr. J. Brown (University of Arizona, Dept. of Plant Sciences). Two suspension cell lines appear almost homogeneous in composition and the storage conditions and stability of these lines are being tested. As part of our 2010 goals we plan to begin to use these lines to complement efforts to culture the greening bacterium and to isolate psyllid viruses (proposal under consideration).
In Texas, our studies of the responses of free-flying ACP to host-plant odors was severely hampered by severe heat and drought followed by prolonged rains. The unfavorable weather conditions that existed from July until October precluded us from obtaining ACP that were suitable for behavioral tests. We developed a greenhouse test to quantify the level of ACP movement between potted sour orange trees and traps scented with petitgrain oil. We completed chemical analyses of the odor emitted by the flushing shoots of sour orange, Thai lime (Citrus hystrix), and Mexican lime. These compounds will be assayed by GC-EAD as this method comes on line. A malfunctioning transfer line heater on our GC has been identified as the source of electrical interference that was hampering antennal recording from ACP. A replacement for the unit was loaned by U. of Florida. We are experimenting with different electrodes and methods of preparation. Cornell U. has provided tungsten electrodes and a sharpening system. Tungsten electrodes may allow us to record from individual chemosensory cells and avoid occluding receptor cells that may occur with microcapillary tubes. We are optimistic we will be able to reliably record from ACP antennae in the near future.
Field work- In the past quarter, we have focused heavily on testing of psyllid insecticides as low volume sprays (2 gal/a). This has included the optimization of chemistries that are labeled for the psyllid, such as Danitol, as well as testing of unlabelled compounds. For labeled compounds, we have investigated the effect of product rate and application timing. For unlabelled compounds, we have conducted efficacy testing at standard rates to generate the needed data for label changes. This season’s low volume field trials were conducted with the LV-8 low volume applicator, which is becoming commonly used in the Florida citrus industry. In the past few months, we conducted 9 experiments and tested a total of 43 treatments in Florida citrus groves. Experiments were evaluated by counting psyllids and nymphs. In addition to low volume testing against Asian citrus psyllid, we also evaluated the efficacy of several products applied by low volume against the citrus leafminer. We found that the most effective treatments against psyllid applied by low volume (Pyrethroids) do not impact citrus leafminer populations. However, products known to be effective against psyllid when applied by standard volumes did show some effectiveness against citrus leafminer when applied by low volume. A manuscript on field trials is under preparation and will be completed over the winter months. Laboratory work- In the laboratory, we have been working on characterizing spray droplet size effect on psyllid control. The work involved establishing optimum application parameters for using our monodisperse aerosol generator (VOMAG) with the new signal generator. We have applied ‘Lorsban Advanced’ at six droplet sizes using different nozzle sizes. The spray trials have focused on the mortality (day 3 and day 7) of the egg, nymph as well as the adult psyllid. The other chemistries (Movento, Danitol, Provado and Sevin) will be tested as psyllid infested ‘potted trees’ become available. These trees are being rotated in the infesting area as space becomes available. Trees for the next two replications of Lorsban are currently being infested. A manuscript for the laboratory aspect has been started. It will include all the data for system characterization.
In the first experiment that was conducted to know the efficiency of systemic insecticides to control the Asian Citrus Psyllid (ACP), Diaphorina citri, and its effect on transmission of the bacteria Candidatus Liberibacter asiaticus, indicated that imidacloprid (Confidor 700 GrDA), 0.35 g AI/plant and thiamethoxam (Actara 250 WG) 0.25 g AI/plant, applied in the nursery tree bags, before planting, was efficient to control ACP until 60 days after application. The time to cause 100% of ACP mortality was between 5 to 7 days after the confinement of adults in treated plants. However, researches using electrical penetration graph (EPG) showed that in plants treated with imidacloprid and thiamethoxam, after the first feeding on phloem, the adults do not do more probing. No transmission results yet. We started the second experiment to determine if the systemic insecticides are effective until 90 days after application and its effect on transmission of the bacteria. Using electrical penetration graphs (EPG) techniques, we are studying the probing behavior of ACP. In the preliminary results we observed that the time feeding on phloem, in plants which were applied the systemic insecticides imidacloprid, was 7.58 and 6.95 minutes to 35 and 95 days after application, respectively. When applied thiamethoxam the results were 13.28, 15.41, and 17.50 minutes, respectively to 20, 35 and 95 days after application. In the control, the average time feeding on phloem was 129.79 minutes. For both insecticides, all specimens died in two days after those times of phloem sap ingestion. After first probing and ingestion of phloem sap, the insect stopped feeding and no more probing was observed. In foliar application, were tested imidacloprid (Provado 200 SC) and lambda-cyhalothrin (Karate 50 SC). Until 14 days after application, adults of ACP probe the citrus trees, but did not reach the phloem and died in 24 hours. In the test plant, approximately 80% of the ACP adults reached and sucked on phloem for 2 hours, in average, in a 5-hour record test.
This project’s goal is to determine factors influencing transmission efficiency of Candidatus Liberibacter asiaticus by the Asian citrus psyllid (ACP), Diaphorina citri, which have implications on current HLB management strategies, particularly rouging and vector control. We are currently studying how acquisition efficiency varies depending on vector developmental stage (study 1), citrus phenology (young leaves x mature leaves) (study 2) and pathogen titer and symptom expression in source plants (study 3). We will also study the time period required for psyllid adults to inoculate the pathogen in healthy citrus (study 4) and if a systemic insecticide can affect this process (study 5). So far we have started experiments related to studies 1-4, and have partial results for studies 1 and 2, which are described below. The project is progressing as planned in the original proposal. In study 1, we are comparing different ACP nymphal stadia (1st, 2nd, 3rd, 4th and 5th instars) and adults (1 wk old) with respect to acquisition efficiency of Ca. L. Liberibacter on citrus. Around 10-20 psyllid adults and nymphs of each instar were confined on distinct leaves of a young shoot of a symptomatic infected plant, with recently expanded leaves, inside leaf cages. After an acquisition access period (AAP) of 48 h, the insects of each age treatment were first transferred to healthy citrus seedlings for a latent period of 15 days, and then transferred to healthy test seedlings (5 insects/plant) for a 7-day inoculation access period (IAP). After the IAP, total DNA of each insect was extracted and the sample was submitted to real-time PCR. In a first trial of this experiment (set up in May/09), the results suggested that nymphs acquire the bacterium more efficiently than the adults. Two recent trials set up in July/09 and Sept/09 confirmed this trend; mean psyllid infectivity rates of 100, 79, 80, 75, 83 and 60% were obtained when acquisition occurred during the 1st, 2nd, 3rd, 4th, 5th instars and adults, respectively. We are still waiting the data of a fourth trial (Oct/09) to run a statistical analysis, but these partial results indicate that the pathogen is efficiently acquired by all nymphal instars of ACP. In study 2, we observed the effect of leaf age (young and asymptomatic x symptomatic mature leaves) in source plants infected with Ca. L. asiaticus on acquisition efficiency and probing behavior of ACP adults. In a first experiment, groups of healthy adults were confined separately on a fully-expanded young leaf and on a mature (symptomatic) leaf of source plants of Ca. L. asiaticus. After a 4-day AAP, psyllids from each treatment were kept on healthy plants for 24-day latent period and then tested for infectivity by qPCR. ACP adults acquired Ca. L. asiaticus with higher efficiency on young (asymptomatic) (20.4% infective individuals) than on mature (0%) leaves from infected plants. We now 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, as reported previously. By analyzing the probing behavior of adult females of ACP on mature x young (fully-expanded) leaves of infected citrus by the Electrical Penetration Graph (EPG) technique, we noted that phloem ingestion was longer and observed more often on young leaves. Within 5 h, around 50% individuals on young leaves started sustained phloem ingestion (E2), whereas less than 15% individuals on mature leaves did so. 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 Ca. L. asiaticus when ACP is confined on young asymptomatic leaves.
The objectives of this project are establishing baseline toxicity data of various insecticides to field populations of the Asian citrus psyllid (ACP). Determining the resistance and cross-resistance development potential in field populations of the ACP and determining the mechanisms of insecticide resistance as part of insecticide resistance management program. Using a topical bioassay method, the baseline susceptibility data (LD50) have been generated for 14 insecticides that are labeled or in the process of labeling (chlorpyrifos, dimethoate, malathion, aldicarb, carbaryl, abamectin, bifenthrin, cypermethrin, fenpropathrin, lambda-cyhalothrin, acetamiprid, imidacloprid, thiamethoxam and spinetoram) for psyllid control in Florida citrus. The toxicity data of a laboratory strain which was established in 2005 and has not been exposed to insecticides were used for comparison. Bioassays were conducted on five psyllid populations collected from one grove each in five counties (Hendry, Indian River, Lake, Polk, St. Lucie) for determining the baseline susceptibility levels and to compare them with laboratory strain. In general, all five psyllid populations collected from five Counties showed decreased susceptibility to all the tested compounds compared with laboratory strain with few exceptions. The decrease in susceptibility to different insecticides was ranged from 1 to 34-fold (Hendry County); 1 to 18-fold (Indian River County); 1 to 14-fold (Lake County); 1 to 12-fold (Polk County); and 1 to 14-fold (St. Lucie County). For Hendry County psyllid population, the highest decrease in susceptibility was observed for imidacloprid (34-fold) followed by thiamethoxam (10-fold). For Indian River County psyllid population, the highest decrease in susceptibility was observed for chlorpyrifos (18-fold) followed by imidacloprid (10-fold). For Lake County psyllid population, the highest decrease in susceptibility was observed for imidacloprid (14-fold) followed by thiamethoxam (12-fold). For Polk County psyllid population, the highest decrease in susceptibility was observed for chlorpyrifos (12-fold) followed by imidacloprid (7-fold). For St. Lucie County psyllid population, the highest decrease in susceptibility was observed for chlorpyrifos (14-fold) followed by imidacloprid (9-fold). Psyllid populations from five Counties are still highly susceptible to most of the synthetic pyrethroids tested when compared to laboratory strain. Further work to screen psyllid populations collected from 4-5 different locations in Florida is under way for determining the baseline toxicity and monitoring resistance levels. In another study, two field colonies have been established in a greenhouse and are being subjected to imidacloprid or chlorpyrifos selection pressure in every generation for developing resistant colonies. This is because field collected psyllid populations from five Counties showed decreased susceptibility to imidacloprid and chlorpyrifos. These colonies will be used for further studies on determining the resistance and cross-resistance development potential in psyllids and mechanisms of resistance. Thus far selection of five successive generations has been completed for imidacloprid and a resistance level of up to 150-fold has been observed. Further selection of future generations and determining the mechanisms of resistance by toxicological and biochemical studies are under progress. The information from these studies should help monitor the onset and progress of resistance in psyllids to different insecticides which in turn will enable us to take remedial measures as part of insecticide resistance management program.
Effects of host plant nutritional status on Diaphorina citri Kuwayama fitness: In recent experiments we focused on the two most important essentials, Nitrogen and Potassium in several combinations. ‘Valencia’ orange plants were potted individually in containers using sand. Five different fertilization levels were applied consisting of high and low nitrogen and potassium levels in all their combinations. We also included a nutrition deficient treatment. Psyllid fitness was checked in each of these treatments. Results shown that the adult weight of the psyllids fed on deficient plants was significantly lower than in all the other treatments. Additionally, the average number of eggs per female laid by psyllids reared on deficient plants was significantly lower than in all the other treatments. The effect of boron on psyllid fitness was tested. Sour orange seedlings were sprayed with 275ppm boron (0.25% Borax). Two days after treatment, 10 psyllids were caged in each seedling for a week afterwhich time mortality was assessed. IN these experiments, there was a significant effect of boron on psyllid survival with higher rates of psyllid mortality occurring in the boron treated plants. Effect of host plant species on psyllid fitness: Of the various citrus species tested thus far for effects on psyllid fitness, we have shown that Cleopatra mandarin is not a suitable host for the Asian citrus psyllid. Specifically, the survivorship of psyllid immature stages on Cleopatra mandarin did not exceed 5%, whereas survivorship on sour orange was 62.21%. Furthermore, significantly fewer eggs found to be laid on Cleopatra mandarin than sour orange. The potential for induced host preference of the psyllids was evaluated. Psyllids reared on host plants of two different genera (Murraya and Citrus) were transferred to different Citrus species and check for survivorship. Specifically, psyllids form colonies reared on Murraya koenigii (curry leaf tree) and Citrus aurantium (sour orange) were caged in Sour Orange ‘Valencia’ Sweet Orange, ‘Flame’ Grape Fruit ‘Sunburst’ tangerine and Volkameriana lemon. Results shown that no significant difference occurred in the survivorship of psyllids that were previously reared on the two hosts. Effect of the Plant Growth Regulators (PGR’s) on psyllids fitness: citrus seedlings were sprayed with 6 different PGR’s (Embark, Sumagic, Atrimmec, Profile, Cycocel). Psyllid fecundity and survivorship on those plants was studied. The initial results shown that in all PGRs tested, except Cycocel, psyllids laid fewer eggs per female compared to the untreated plants. Regarding the survivorship, significant differences occurred only between Cycocel and Embark, which was higher than in Profile.
Activities (2009) of Pasco B. Avery, postdoctoral associate who is supported by these funds to provide assistance to citrus growers on the east coast. 1) Packers of Indian River, Ltd. – Ft. Pierce : Continue monitoring psyllids along the border using yellow sticky cards. Data collected is being used to produce a distribution graphic for assessing the psyllid population dynamics throughout the year for the grower. Populations peaked in March and August. 2) IMG Citrus ‘ Fellsmere: Assessing and comparing the abundance of psyllids in orange and grapefruit trees managed by the same chemical spray program in two 10 acre blocks (1 block for each fruit tree type) using the following techniques: monitoring inside the block with yellow sticky traps, tap sampling, flush sampling, and assessing flush density. Results thus far indicate that there appear to be more psyllids found on orange than grapefruit trees with similar flushing patterns; but have only assessed for 2 months since August. 3) Pine Ranch, Inc. ‘ Lorida: Continue assessing the effectiveness of various chemical sprays for managing psyllids using yellow sticky traps, tap and flush samples. Generally the population is low, but the numbers of psyllids may be influenced by the abandoned grove near the monitoring site. 4) River Country Citrus, Inc. ‘ Okeechobee: Continue assessing the effectiveness of various chemical sprays for managing psyllids using yellow sticky traps, tap and flush samples on young trees. Very low numbers present on young (2 yrs old) trees using imidacloprid. Also assessing and comparing the abundance of psyllids in orange and grapefruit trees managed by the same oil spray program in two 10 acre blocks (1 block for each fruit tree type) using the following techniques: monitoring inside the block with yellow sticky traps, tap sampling, flush sampling, and assessing flush density. In general, results indicate that there appear to be more psyllids found on orange than grapefruit trees with similar flushing patterns; but have only assessed for 2 months since August. 5) Lindsey Groves ‘ Vero Beach: Assessing and comparing the abundance of psyllids in orange and grapefruit trees managed by the same organic approved program in two 10 acre blocks (1 block for each fruit tree type) using the following techniques: monitoring inside the block with yellow sticky traps, tap sampling, flush sampling, and assessing flush density. In general, results indicate that there appear to be more psyllids found on oranges than grapefruit with similar flushing patterns; but have only assessed for 2 months since August. 6) Premier Citrus ‘ Vero Beach: Assessing and comparing the abundance of psyllids in orange and grapefruit trees managed by the same chemical spray program in two 10 acre blocks (1 block for each fruit tree type) using the following techniques: monitoring inside the block with yellow sticky traps, tap sampling, flush sampling, and assessing flush density. In general, results indicate that there appear to be no differences in psyllid abundance between orange and grapefruit trees with similar flushing patterns; but have only assessed for 2 months since August.
Acquisition of pathogen: For assessment of adult acquisition, groups of 10-50 healthy adult ACP were confined in mesh enclosures on psyllid-free branches of infected citrus plants for acquisition access periods (AAPs) ranging from 1 to 52 days. To test nymphal acquisition of Las, flush containing first instar nymphs from our healthy psyllid colony was excised and placed on mature leaves of Las-infected citrus plants. Branches containing nymphs were enclosed in mesh bags to prevent dispersal of nymphs. Adults from eight replicate groups were collected upon emergence and preserved for Las-detection as described above. The acquisition of the HLB pathogen by adult ACP, determined by real-time PCR, ranged from 0% (< 1 week of acquisition feeding) to 38.8% (> 5 weeks of feeding). When ACP were reared on infected plants for acquisition as nymphs, the percentage of positive psyllids rose to 62.2%. Transovarial transmission: Sexually mature, Las-infected D. citri obtained from a laboratory colony were caged on uninfected sweet orange seedlings for oviposition. Eggs laid by single females were collected in pools of 20-30 eggs or transferred to healthy ACP host plants. Nymphs arising from the eggs of single, Las-positive females were collected as 1st-2nd instars in pools of 20-30 nymphs. Of the pools tested, 2.1% of eggs and 6.8% of nymphs were positive for Las. In addition, 2.4% of adults emerging from the eggs of infected females also tested positive for Las. Cumulatively, these results suggest that bacteria are transmitted to offspring at a low rate. Transmission of Las by individual ACP: Psyllids from an infected colony were held individually on healthy sweet orange seedlings and allowed to feed for inoculation access periods (IAPs) of 1, 4, 7, 15, and 28 d. Plants used in transmissions were held in an insect-proof greenhouse and tested bimonthly via real-time PCR for the presence of Las. To date, Las detection in plants ranged from 0% (28d IAP) to 10% 4 day IAP). Results of experiments to determine the rate of transmission by healthy ACP will be included in the next report pending plant testing. In determining the effects of host plants on the acquisition and transmission of Ca. Liberibacter asiaticus (Ca. Las) two hosts were studied this quarter. Chinese box orange (Severinia buxifolia) previously listed as a host for the Asian psyllid and a host of HLB was utilized as well as rough lemon (C. jambhii). Healthy Asian citrus psyllids readily fed on Las infected box oranges and rough lemons and after the appropriate acquisition periods were PCR tested. Between 30-40% of the psyllids from the both hosts were PCR positive for Ca. Las. To determine transmission rates from Chinese orange boxwood psyllids reared on infected plants were transferred in groups to Valencia orange seedlings. Infection results are pending. HLB infected rough lemon stems were PCR assayed at various locations to determine if symptomatic or asymptomatic differed in bacterial concentrations. Psyllid acquisition experiments from rough lemon are in progress. Seasonality of HLB infected psyllids: During 2009, psyllid collections continued at the same 5 locations sampled during 2008. During 2009, a noticeable increase in the overall percentage of HLB infected psyllids was found. While there was still a trend for periodic increases and decreases in the percentage of HLB+ psyllids, the periods when infection rates were highest occurred in January, April and July. While the average psyllid infection rate was less than 5% across all study sites, at one of these locations (Lake Alfred) the percentage of HLB+ psyllids was above 15% on each of these three months. Data collected thus far in 2009 from a 6th new location (Homestead) have shown that there does appear to be fluctuations in the number of HLB+ psyllids even where 100% of the citrus host plants are HLB+. However, the numbers of infected psyllids is much greater ranging from 20-100% based on data analyzed thus far.
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