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. Recently, 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. As ACP has become quickly established in Florida (and now other citrus producing states in the US), it is obvious that there is the capacity for substantial dispersal. 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. Most recently, we have been investigating both the seasonality of psyllid movement and the limits of short-range dispersal capabilities. Specifically, we have tracked psyllid movement on a monthly basis to determine when movement is greatest. By determining the annual seasonality of psyllid movement/dispersal, we will be able to better identify periods of greatest need for supplemental border sprays. Thus far, we have measured psyllid movement in the months of June, July, August, and September. Thus far, we have found that the total number of psyllids moving from unmanaged into managed groves during the months of June, July, and August is approximately equal, but decreases in September. In June, the number of psyllids leaving the interior and exterior portions of unmanaged groves is equal and the majority of these psyllids infest the border areas of managed groves. In July, the majority of invading psyllids from unmanaged groves originate from the interior portions of unmanaged groves. Also, in July, the number of psyllids invading the border and interior portions of managed groves is equal. In August, once again, the majority of invading psyllids from unmanaged groves originate from the interior portions of unmanaged groves and mainly infest the border areas of managed groves. As mentioned above, in September this movement appears to decrease drastically. We will continue to monitor psyllid movement monthly until we understand the annual seasonality. This initial portion of the study does indicate that movement patterns of psyllids differ over the course of the season, suggesting that there may be specific times of the year when movement is greatest and thus psyllid control should be intensified. In separate investigations, we have been studying the short-range dispersal capabilities of psyllids. We have found that psyllids are able to move up to 300 yards within the span of 6 days. This movement can occur between groves seperated by a fallow field and directly within continuous grove space. These results are further evidence of the good dispersal capabilities of this insect. Although the psyllid is considered by many to be a weak flyer, it apparently disperses large distances very quickly. It is possible that this movement is aided by wind. Finally, we have also initiated an investigation of the presence of HLB in abandoned groves. Since we know that abandoned groves serve as a source of psyllid infestation into managed groves, we need to determine if these abandoned groves also harbor the HLB pathogen. If so, they would represent a potential source of greening infection into managed citrus. We have surveyed 7 pairs of abandoned and managed groves throughout Florida that are 50-100 yards apart. With respect to tree infection, so far we have found that there is 2.3 fold more infection in abandoned groves than adjacent managed groves. With respect to psyllid infection in June, July, and August, we have found an equal number of HLB-infected psyllids in adjacent managed and unmanaged groves. Our initial results indicate that abandoned citrus groves are a source of HLB infection, harboring inoculum in the form of HLB-infected trees and are a source of infected psyllids.
Sampling – A stratified random sampling procedure was used to assess spatial dispersion of adult ACP according to yellow sticky trap sampling and stem tap sampling procedures, and optimum sample sizes and allocation were determined for both sampling procedures. The intent of the research is to develop sampling guidelines for the purpose of making statistical comparisons among different groups of trees with respect to psyllid population levels. Both trap sampling and tap sampling indicated that adults were aggregated among trees within individual strata and among strata across the block of trees studied. There was little to no value in stratifying and, if the intent of sampling is to reduce between strata variation, less emphasis should be placed on the number of strata than on the number of trees to sample per stratum. Regardless of whether trap sampling or stem-tap sampling is used, sampling should be conducted throughout a block because there can be significant variation across a block of trees in numbers of psyllids. For commercially-acceptable precision levels, preliminary analyses indicated that at a mean density of one psyllid per sample, 30 stem-tap samples or 20 sticky trap samples may be required either per acre or per ten acres. We are in the process of validating these sample sizes. Biological Control – (a) Research indicated that the following pesticides were least compatible with (most toxic to) adult T. radiata based on the toxicity of direct sprays and potential long residual life on leaves: carbaryl, chlorpyrifos, fenpropathrin, and phosmet. Each of the following may be somewhat more compatible because the toxicity of residues of these pesticides was either low at 24 hours or sharply decreased over a few days after application: abamectin, chenopodium oil, fenpyroximate, spirotetramat, sulfur, and 435 spray oil; depending on environmental conditions, imidacloprid and pyridaben might also be somewhat more compatible for the same reasons. The pesticides that consistently appeared to be most compatible with T. radiata were aluminum tris, copper hydroxide, diflubenzuron, fenpyroximate, and kaolin clay. More research is needed on kaolin clay, as rates higher than we tested might be more toxic and residues of this pesticide on leaves can negatively affect insect activity. (b) Releases of the parasitoid Diaphorencyrtus from south China. Over 9,000 wasps have been released in four counties in central and southwest Florida over the last three years (Collier, Hendry, Lake and Orange Co.). About 4,000 were released during 2009. Wasps were recovered in low numbers from commercial and research citrus groves in Immokalee during 2008, but no recoveries have been made during 2009. Studies are being conducted on the biology of the parasitoid.(c) PPQ granted permission to release three new biotypes of Tamarixia from Asia. Seasonal HLB profile in adult psyllids ‘ In this study, psyllids are collected fortnightly from disease trees in a grove. From Feb 2008 through August 2009, a mean of 41% psyllids tested positive, with a minimum of 3% on one sample date and a maximum of 85% on another sample date. In 2008, percentages of psyllids that tested positive during February ‘ May ranged from 11 to 33% while a mean of 50% adults tested positive during June – August. A mean of 71% (maximum 85%) adults tested positive for HLB during September ‘ December. Although during 2009 (through August) there were two sample dates on which only 10% of psyllids tested positive (February and March), in general there has been no definite trend in seasonality. Of interest will be if percentages of infected psyllids increase during the Sep – Dec period as they did in 2008. Seasonality of transmission rates is being investigated and will be reported next time.
The preliminary results showed 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 (Diaphorina citri) until 60 days after application. No transmission results yet. 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 that was applied insecticides (thiamethoxam and imidacloprid), was reduced, in average from 8 to 15 minutes on those treated plants, and 150 minutes on test plants (control). Using foliar application insecticides, until 14 days after application, in plants that was sprayed lambda-cyhalothrin and imidacloprid, adults of ACP probe the citrus trees, but not reach the phloem and died in 24 hours. In the test plant, all ACP tested reached and sucked the phloem sap for 2 hours in a 5 hours record test. The goal is to start others projects in the second semester of the year and all goals and objective of this project will be reached.
Thus far in the examination of the effect of plant nutritional status on Diaphorina citri Kuwayama biology, we focused on the two most important essential nutrients, Nitrogen and Potassium in several combinations. For this purpose ÒValenciaÓ plants, potted individually in plastic containers containing sand as a potting medium were used. Five different fertilization levels were applied to these plants, consisting of high and low nitrogen and potassium levels in all their combinations, plus a nutrition deficient treatment. For each of the plants, psyllid adults from colony reared on sweet orange ÒValenciaÓ plants, were caged in plastic transparent cages to lay eggs. After that, adults were removed and the emerged nymphs remained caged until their adult emergence. After that, the adults were removed and killed under exposure in CO2 and then they were weighed individually in a six digit scale. Results showed that the adult weight of the psyllids fed on plants with Low Nitrogen (N) – High Potassium (K) rates was significantly lower than in High Nitrogen (N) – High Potassium (K), Low Nitrogen (N) – Low Potassium (K) and High N – Low K rates, while there was no significant difference between the deficient plants and all the other treatments. As regards the effect of the host plant on the Asian citrus psyllids fitness, a number of plants were checked about their suitability as host plants for the psyllids. The plant species and varieties were Sour Orange (Citrus aurantium), Sweet Orange (C. sinensis) varieties ÒValenciaÓ and ÒHamlinÓ, Grapefruit (C. paradisi) varieties ÒRio RedÓ and ÒFlameÓ, Cleopatra Mandarin (C. reticulata reshni), ÒSunburstÓ tangerine (C. reticulata x. C. paradisi), ÒFallgloÓ tangerine [Bower citrus hybrid (C. reticulata x C. paradisi) x Temple (C. reticulata x C. sinensis)], C. macrophylla, C. volkameriana, ÒCarrizoÓ citrange (C. sinensis x Poncirus trifoliata), ÒSwingleÓ citrumelo (C. paradisi x P. trifoliata), Curry Leaf Tree (Murraya koenigii) and Orange Jasmine (M. paniculata). For this study, pairs of ACP adults collected from a field wild population and from a population reared on sour orange seedlings were caged on plants with young flushes of the species above to lay eggs. The initial results shown that all the species and varieties above, except Cleopatra mandarin, are suitable as host plants for the Asian citrus psyllids. In Cleopatra mandarin the viability of ACP eggs was very low, because only 4,9% of the laid eggs gave adults. The suitability of the rest of the species and varieties is under experimentation.
We have continued our laboratory and field studies to further optimize low volume spray application technologies for controlling psyllids in Florida citrus production. In the laboratory, we have tested several fluorescent tracers to develop a reliable methodology for qualitative and quantitative assessment of spray distribution and deposition in low and ultralow volume field applications. We have conducted experiments to quantify droplet spectra generated by low volume applicators. In the field, we have continued to compare various available application technologies as they become available. We have compared high volume dilute spraying of entire tree with low volume mist application. In most recent tests we have evaluated two cold foggers and a mist blower. Thus far, for psyllid control we have found no difference in efficacy between the available technologies. However, we may find differences with some of the more selective chemistries, such as insect growth regulators (IGRs), so we are continuing this research by investigating more pesticide modes of action including IGRs. We have used our custom-made low volume misting machine to conduct experiments with several insecticide chemistries. So far, we have generated efficacy data for several different insecticides. This work along with others, have led to the successful labeling of several products for low volume application in Florida citrus. We have compared the insecticide residues achieved with low volume and standard airblast sprays and have found that the residues from low volume sprayers are lower than those from conventional airblast sprayer. We have also investigated several application parameters for optimizing low volume spraying. While certain insecticides have shown similar efficacies for spraying every row versus every other row or ground speed of 5 mph versus 8-10 mph, some chemistries have shown lower efficacies when spraying every other row or at higher ground speed. In terms of the application volume, we have not found a difference between 2 and 5 gallons per acre. However, one must stay above 2 gallons per acre to remain within the boundaries of current label guidelines. We are also currently investigating whether the rate of insecticide can be reduced with low volume spraying. Preliminary data suggest that this will also depend on the insecticide chemistry. In certain cases, rates can be reduced and efficacy is maintained, while in other cases efficacy declines when reducing the rate, but more testing is needed.
A method for evaluating Asian citrus psyllid (ACP) response to combinations of odor and visual cues in a greenhouse setting was developed. This method allows us to evaluate the responses of free-flying ACP to stimuli combinations in a controlled environment. The greenhouse tests will enable us to efficiently screen combinations of stimuli and select those that show the most promise for testing in orchards. The proof-of-concept test consisted of measuring the trapping rate of ACP on scented and unscented yellow-green sticky traps (ACP traps, AlphaScents, Inc.). Petitgrain oil, an essential oil distilled from the sour orange leaves, was selected for use as a test odorant because it contains substantial amounts of linalyl acetate and linalool, two primary volatiles present in the aroma of Meyer lemon flush. ACP were presented with either unscented or scented traps. The trap array consisted of six rows of traps with two traps per row. Each trap measured 34 cm long x 2.5 cm wide. Scented traps had a rubber septum loaded with petitgrain oil. The traps were positioned upwind of a screened release cage that held 1000 ACP collected from an unsprayed research orchard prior to testing. Once the psyllids were released inside the cage, the lid was opened slightly to allow them to escape. A census of ACP captured by each trap was made at 15-, 30-, and 45 minutes intervals following release. A total of five replicated tests were conducted for each treatment. Psyllids accumulated more quickly on scented traps compared with unscented traps during the interval between the 15 and 30 minute observations. During this interval, the number of ACP caught on scented traps increased by 52 + 5.9% (mean + SEM) while on the unscented traps the average increase was 35 + 3.0%. There was no significant difference in the capture rate of psyllids between the 30 and 45minute observations. During this interval, the number of ACP caught on scented traps increased by an average of 18 + 3.7%. On unscented traps the average increase was 9 + 3.0%. We continued to collect and analyze volatiles emitted by flushing terminal shoots of ACP host plants. We completed analyses of Meyer lemon, Rio Red grapefruit, and orange jasmine, and completed collections of sour orange, kaffir lime, and Mexican lime. Chemical analyses of these volatiles will be completed shortly. We created a six-component mixture modeled on the aroma of orange jasmine flush and showed that it stimulated ACP in screened cages. This formulation will be tested in the greenhouse. Other formulations based on our analyses of host plant flush will be developed and tested in the greenhouse over coming months. Gas chromatogram-coupled electroantennograms (GC-EAD) were conducted on ACP antennae reared on Citrus macrophylla in a greenhouse. ACP antennae responded to a number of plant volatiles and blends. Protocol for distinguishing positive GC-EAD responses above background noise were established. A number of synthetic compounds provided by an industry collaborator were identified by GC-MS after eliciting a positive antennal response. These compounds are being acquired for further study. Behavioral assays will be required to determine if the compounds elicit behavioral responses (attraction or repellence) alone or in mixtures.
The purpose of this project is to provide direct entomological support to citrus growers on the east coast of Florida, particularly with regards to evaluating current psyllid control programs. Funding provided supports the efforts of Dr. Pasco Avery, located at the IRREC (Ft. Pierce), to meet these needs. The following is a summary of Dr. Avery’s activities since April 2009: 1) Evans Properties – Okeechobee (1,103 acres under field trials)-Bluefield Division :Helped assess the effectiveness of aerial spray program with Malathion 5¨ for managing psyllids using yellow sticky cards. (514 acres).Will evaluate efficacy of PFR 97ª for management of psyllids sprayed on citrus trees to be pushed out. Psyllids will be monitored using yellow sticky traps, tap and flush samples.-Adams Division: Helped assess the effectiveness of aerial spray program with Malathion 5¨ for managing psyllids and Caribbean fruit fly using yellow sticky cards. 2) Packers of Indian River, Ltd. – Ft. Pierce (2, 588 acres); Vero (600 acres); Punta Gorda (440 acres) = 3, 628 acres monitored: Continue monitoring psyllid populations along the border using yellow sticky cards. Produce a graphic distribution for assessing the psyllid population dynamics throughout the year and in the different areas. Use FAWN data to identify possible correlation with weather patterns and abundance of psyllid populations. 3) IMG Citrus – Fellsmere (60 acres under field trials): Will assess the psyllid population abundance on orange and grapefruit to determine if there is a difference between tree types under similar management strategies. This will be assessed using sticky traps, flush samples and flush density throughout 2009. 4) Pine Ranch, Inc. – Lorida (64 acres under field trials): Continue assessing the effectiveness of various chemical sprays for managing psyllids using yellow sticky traps, tap and flush samples. 5) River Country Citrus, Inc. – Okeechobee (83 acres under field trials): Continue assessing the effectiveness of various chemical sprays for managing psyllids using yellow sticky traps, tap and flush samples and flush density throughout the year 2009. 7) St. JohnÕs Management District – Vero Beach and Ft. Pierce (22, 500 acres under field trials): Continue assessing the effectiveness of aerial spray program for managing psyllids using yellow sticky card trap samples. Samples are being assessed from Premier Citrus, Packers of Indian River, Becker Groves and GraveÕs Brothers Company. 8) Orange Avenue GrowerÕs Association – Ft. Pierce (9,800 acres under field trials): Continue assessing the effectiveness of aerial spray programs for managing psyllids using yellow sticky traps. 9) Hammond Citrus and Nurseries Ð Vero Beach: Assessing the effectiveness of various chemical sprays for managing psyllids using yellow sticky traps. 10) Blue Goose Ð Ft. Pierce and Indiantown: Assessing the effectiveness of various chemical sprays for managing psyllids using yellow sticky traps.2. Comparing and assessing the effectiveness of different chemical spray strategies (low, medium and extensive) for managing psyllids. 11) IRREC Ð Ft. Pierce: 1. Evaluating PFR 97ª persistence in the field sprayed on small trees 1, 7, 14 and 21 days post-spray for managing psyllids using dilution plating and counting the number of colony forming units over time. Also percent mortality of adult psyllids after being exposed to leaves collected 1, 7, 14 and 21 days post spray will be assessed.
In this project, we are investigating how acquisition efficiency of Candidatus Liberibacter asiaticus by Diaphorina citri varies depending on vector developmental stage (study 1), plant phenology (young leaves x mature leaves) (study 2) and pathogen titer and symptom expression in source plants (study 3). We will also investigate 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 D. citri 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. The experiment is being carried out in a climate-controlled room [25 ± 1oC, 60 ± 20% RH, and 14:10 h (light: dark) photoperiod]. A first trial was set up in May/09; 40 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 extracted and the sample was submitted to real-time quantitative PCR (qPCR). In this first trial, we got an infectivity rate of 50, 75 and 0% when acquisition occurred during the 1st instar, 5th instar and adult stage, respectively, indicating that nymphs acquire the bacterium more efficiently than the adults. A high mortality of 2nd, 3rd and 4th instars during the latent period precluded the analysis of their infectivity. Other two trials of this experiment will be set up in Aug/09 and Sept/09, when we hope to get data for all instars. In the second study, we evaluated the preference of D. citri for different parts of a citrus plant, and 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 D. citri. This insect showed a clear preference for young leaves of the upper portion of the plant (71.3% of the individuals), and main vein on the abaxial leaf surface (87.1%). Groups of healthy lab-reared D. citri adults were then confined separately on a fully-expanded young leaf and on a mature (symptomatic) leaf of source plants of Ca. L. asiaticus. After an 4-day AAP, psyllids from each treatment were kept on healthy plants for 24-day latent period and then tested for infectivity by qPCR. D. citri acquired Ca. L. asiaticus with greater efficiency on young (asymptomatic) leaves (49.5%; n=4) than in mature (symptomatic) leaves (0%; n=4) from infected plants. We plan to repeat this experiment and include a third treatment (not fully-expanded young leaf). To investigate possible reasons for the differences in acquisition efficiency, we analyzed the probing behavior of adult females of D. citri on mature x young leaves of infected citrus plants by the Electrical Penetration Graph (EPG) technique. 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 D. citri is confined on young asymptomatic leaves.
Seasonality of pathogen spread is being investigated by sampling the natural psyllid population in commercial citrus groves through repeated monthly sampling in citrus groves throughout the state. In 2008, our sampling suggested that there were two distinct periods of the year when higher rates of infected psyllids were more likely to be present and thus higher rates of pathogen transmission were likely to occur. The groves sampled in 2008 were sites where HLB management programs were ongoing and included removal of infected trees and application of pesticides for psyllid control. Because the overall rates of infected psyllids at these sites were relatively low, in 2009, we have also included an additional citrus grove study site located in Homestead (south Florida) where 100% of the trees appear to be HLB infected and no insecticides are being applied for psyllid control. To date from our study sites, we have processed 10, 199 psyllids from Lake Alfred, Ft. Meade, Lake Wales, Lake Placid and Arcadia. Since February 2009, we have processed 1,435 psyllids from the Homestead study site. From the data analyzed thus far in 2009, the overall Incidence of HLB+ psyllids at all sites excluding Homestead was 6.8% in January, 1.0% in February, 0.5% in March and 5.8% in April. At the homestead site where all trees are believed to be HLB infected, 100% of the psyllids collected in February tested positive for HLB. In march the incidence of infected psyllids dropped to 15.7% at the Homestead site. Analysis of psyllids collected in April, May and June are not yet completed. This work continues to determine if trends for infected psyllids observed in 2008 hold true in the second year of sampling. In addition to sampling of infected psyllids, a second part of this study was initiated in October 2008. In this study, healthy citrus nursery plants (resets) are being used as indicator plants to determine when pathogen transmission is occurring in the field. Each month, 10 citrus resets are “planted” in each of 3 citrus groves. One of these sites s the Homestead site where the highest rate of HLB+ psyllids has been observed. After 30 days, the plants are dug up and replaced with new healthy plants. The removed plants are then taken back to the CREC and held in secure facilities for later analysis for the presence of the HLB pathogen to determine on a monthly basis, when pathogen transmission os most likely occurring.This is being done to further confirm the results of our psyllid sampling study underway. To date, 230 citrus resets taken back to the CREC are being held for development of HLB symptoms and subsequent testing for the presence of the HLB pathogen.
Using a topical bioassay method, the baseline susceptibility data (LD50) have been generated for all of the insecticides commonly used (chlorpyrifos, dimethoate, malathion, aldicarb, carbaryl, abamectin, bifenthrin, cypermethrin, fenpropathrin, lambda-cyhalothrin, acetamiprid, imidacloprid, and thiamethoxam) for psyllid control in Florida citrus. For laboratory strain, bioassays were conducted using psyllids from a greenhouse colony established in 2005, which has not been exposed to insecticides. Bioassays were also conducted on three psyllid populations collected from one grove each in three counties (Polk, Lake and St. Lucie) for determining the baseline susceptibility levels and to compare them with laboratory population. The three psyllid populations from three Counties showed decreased susceptibility to all the tested compounds except lambda-cyhalothrin and acetamiprid compared with laboratory population. The decrease in susceptibility to different insecticides was ranged from 1.2 to 14.2-fold (Lake County); 1.2 to 13.5-fold (St. Lucie County); and 1.0 to 12.0-fold (Polk County). For Lake County psyllid population, the highest decrease in susceptibility was with imidacloprid (14.2-fold) followed by thiamethoxam (11.8-fold). For St. Lucie County psyllid population, the highest decrease in susceptibility was with chlorpyrifos (13.5-fold) followed by imidacloprid (8.9-fold). For Polk County psyllid population, the highest decrease in susceptibility was with chlorpyrifos (12.0-fold) followed by imidacloprid (6.5-fold). Psyllid populations from three Counties are still highly susceptible to lambda-cyhalothrin and acetamiprid compared with laboratory population tested. Further work to screen psyllid populations collected from 6-7 locations (covering the entire state of Florida) are under way for determining the baseline toxicity and monitoring resistance levels. In another study, a field colony has been established in a greenhouse and is being subjected to imidacloprid selection pressure in every generation for developing a resistant colony for imidacloprid. This colony will be used for further studies on determining the resistance and cross-resistance development potential in psyllids and mechanisms of resistance. The information from these help monitor the onset and progress of resistance in psyllids to different insecticides which in turn will enable us to take remedial measures and to develop integrated resistance management programs.
This proposal is new and the funding allocated to the subcontractors has been delayed within the institution. The cooperators have spent the time getting the research organized, developing trap prototypes, identifying locations for conducting field work and in depth discussions as to how to proceed. We are preparing to begin the field work very soon.
We are attempting to identify and then deliver to psyllids, RNAs capable of inducing RNA interference (RNAi) activity in recipient psyllids. Our goal is to use RNAi to confer a negative phenotype (even death) in psyllids, such that they cannot colonize and/or reproduce on selected plants. By controlling the psyllid vector we believe this aid other efforts to control HLB. We don’t know which RNA sequences will prove to be the best for our effort, and we are taking three different approaches to identify effective sequences. In order to test and identify effective interfering RNAs, we are attempting to develop an efficient, high throughput screening approach that can be used with random or specific potential interfering RNA sequences. We are using the tomato psyllid (Bactericerca cockerelli), which colonizes herbaceous plants and is the vector of another Liberibacter spp. (C. L. psyllaurous), which is closely related to C. L. asiaticus, the causal agent of HLB. This system, using herbaceous plant hosts as opposed to using citrus directly, offers the opportunity to rapidly make progress that will be applicable to the citrus psyllid:HLB complex. We have evaluated a number of tomato and potato cultivars, as well as Nicotiana benthamiana, as potential host plants for B. cockerelli and for our expression vehicle of choice, Tobacco mosaic virus (TMV). B. cockerelli readily feeds on and colonizes most tomato cultivars tested and on potatoes. It also appears to transmit C. L. psyllaurous to these plants based on our PCR-based detection analyses. B. cockerelli does not colonize N. benthamiana plants, but PCR-based analyses suggest that it does transmit C. L. psyllaurous to N. benthamiana plants. The latter will prove to be useful later on in additional studies. To identify candidate tomato cultivars, we agro-inoculated plants of several cultivars with TMV engineered to express the green fluorescent protein (GFP) (plasmid pJL24). The tomato cultivars Bush Ace, Early Pak 7 and Giant Pink Belgium showed good GFP expression, as determined by exposure to UV light. Systemic infections were slow to develop, taking ~25 days for GFP expression to be visible in the upper, young leaves. We then placed B. cockerelli psyllids on these plants and allowed them to feed for three days. Psyllids were removed and total nucleic acids were extracted. We then used RT-PCR to screen psyllids for the presence of GFP RNAs and detected specific GFP product only in psyllids that fed on the GFP-expressing, but not healthy plants. The above experiments show that TMV can be used to deliver specific RNAs into the plant phloem, and that psyllids can acquire some of these RNAs. We will use this approach to evaluate candidate sequences for RNAi activity against the tomato psyllid. We do not yet know which forms of RNAs are acquired by the psyllids, but experiments are underway to determine this. We have demonstrated that 21 ‘ 23 nucleotide GFP-specific siRNAs are produced in the TMV-GFP infected plants, supporting another aspect of our hypothesis. This is, that by using recombinant TMV we can induce production of specific siRNAs corresponding to the recombinant sequence in plants. Thus, we will use this same approach to induce production of siRNAs corresponding to psyllid genes in plants. We have already cloned sequences for eight psyllid genes. These are highly conserved insect gene sequences such as for actin, and we will use these in our initial experiments. Our initial sequences were obtained using primer sequences based on the asian citrus psyllid, but using B. cockerelli RNA as the template. This is encouraging and suggests that the two psyllid species will have useful similarities for our research.
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 determined that virgin and mated male ACP colonized citrus plants that were currently or had been previously colonized by virgin or mated female ACP in greater numbers than control plants without females. However, males or females did not accumulate more on plants colonized by conspecifics of the same sex compared with uninfested plants and females showed no preference for plants pre-infested with males compared with uninfested controls. In complementary Y-tube behavioral assays in the laboratory, virgin and mated males chose arms with odor sources from mated females compared with blank controls in the absence of associated citrus host plant volatiles. In both behavioral assays mated female ACP appeared more attractive compared with virgin females. Collectively, our results provide behavioral evidence for a female-produced volatile sex attractant pheromone in ACP. Subsequently, we determined that male ACP, irrespective of abdominal color, exhibited stronger evidence of attraction to crushed blue/green females than to crushed gray/brown females. Gray/brown individuals of both sexes showed an increase in body mass 5Ð6 d after transfer to a new citrus seedling, suggesting that abdominal color (which is closely related to body mass) may be influenced at least in part by plant quality. Next, we examined the behavioral responses of mated and unmated ACP of both sexes to odors from host plants in laboratory tests, with and without visual cues in collaboration with USDA colleagues. The host plants tested were: ÔDuncanÕ grapefruit, sour orange, ÔNavelÕ 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. Antennal responses to citrus volatiles were confirmed by electroantennogram. The results suggest that ACP uses olfactory and visual cues in orientation to host plants, and suggest the possibility of using plant volatiles in monitoring and management of this pest. Subsequently, we analyzed the chemicals produced by psyllids using gas chromatography and mass spectrometry. We discovered a total of 85 compounds including 8 male and 13 female ACP-specific volatile chemicals with both sexes having 40 volatile compounds in common. Interestingly, we discovered that both ACP and its parasitoid produce .-Butyrolactone. 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 incleased catch of ACP on traps, the results were inconsistent in follow up trials. However, simultaneously releasing .-Butyrolactone and synthetic citrus volatiles did increase catch of ACP on traps compared to unbaited traps. We continue testing the chemicals that have been identified thus far in field trapping tests in an effort to optimize release rates and blends. Also, work is ongoing on identification of further possible attractants with chemical and behavioral testing of ACP.
The purpose of this investigation is 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 pyriproxyfen, a juvenile hormone mimic, 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. Pyriproxyfen exhibited strong ovicidal and larvicidal activity against ACP eggs and nymphs, respectively, in age- and concentration-dependent manners. Irrespective of egg age and timing of treatment, a significantly lower percentage of eggs (5-29%) hatched into nymphs at the higher concentrations tested (64 and 128 µg mL-1). A significantly lower percentage of early instar nymphs (first, second and third) survived and emerged into adults (0-36%) at the three higher concentrations tested (16, 32, and 64 µg mL-1) compared with late instar nymphs (fourth and fifth) (25-74%). However, 15-20% of those adults that emerged from late instar nymphs exhibited morphological abnormalities. Furthermore, pyriproxyfen 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 pyriproxyfen to adults at 100 µg mL-1 also significantly reduced fecundity and egg viability. Application of pyriproxyfen at 64 µg mL-1 results 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. Pyriproxyfen also markedly reduced female fecundity and egg viability for adults that were exposed either directly or indirectly. The direct (ovicidal and larvicidal) and indirect (transovarial) effects of pyriproxyfen against immature and adult ACP, respectively, suggest that integration of this insecticide as part of an IPM strategy should negatively impact ACP populations over time. Future studies are needed to determine the effects of field aged residues. Also, further field scale testing is needed to determine how to best incorporate pyriproxyfen into an integrated management program for ACP. We are now moving onto detailed investigations of other insect growth regulators including buprofezin, and diflubenzuron. 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. The objective of this laboratory study 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 µg 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. Ongoing investigations include the effects of feeding inhibitors on HLB transmission.
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. As originally proposed, transmission electron microscopy (TEM) studies of T. radiataÕs antennal sensilla were required to guide further electrophysiological investigations of this ACP parasitoid, which would allow identification of chemical attractants. This first objective has been completed and the investigation has been 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). Specifically, we examined the external and functional morphology of the antennal sensilla of adult male and female T. radiata using scanning (SEM) and transmission (TEM) electron microscopy, respectively, to gain insights into the behavioral ecology of this parasitoid. The antennae of male and female T. radiata were composed of a long scapula-shaped scape with a basal radicula, a barrel-shaped pedicel and a long flagellum with a basal ring-like annulus. Five morphologically distinct sensilla including two aporous sensilla trichoidea (AST-1 and AST-2), one multiporous sensilla trichoidea (MST), one multiporous placoid sensilla (MPS), and one aporous basiconic capitate peg sensilla (BCPS) were identified on the antennae of both sexes. Male antennae consisted of four funicular flagellomeres and possessed a greater number of olfactory MST than female antennae suggesting their possible function in perception of mate-related volatile cues. Female antennae were characterized by three funicular flagellomeres and a greater number of MPS than male antennae suggesting their possible function in the perception of host-related volatile cues. Thus male antennae likely function to detect female-produced pheromones, while female antennae function to detect host volatiles used in finding ACP for parasitization. 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 pheromones females produce and release to attract males. Electrophysiological recordings from male parasitoid antennae confirmed that males are capable of detecting this chemical. Subsequently, we set out to develop a dispenser for releasing this chemical in the field for both monitoring of parasitoid populations and for recruiting parasitoids into groves to increase their population densities and improve biological control of ACP. We partnered with an industry collaborator (Alpha Scents, West Linn, OR) to develop an appropriate dispenser for releasing .-Butyrolactone. We have developed a polyethylene-tube dispenser for releasing this chemical. We also obtained a second chemical (Methyl Salicylate) and associated dispenser from a second collaborator (AgBio, Corporation). This chemical is known to recruit beneficial insects and improve biological control. We are currently field testing both products to determine whether we can improve biological control of ACP.
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