We completed experiments evaluating the effect of temperature on Las acquisition. Peak acquisition (approximately 50%) occurred at 25oC. Significantly fewer ACP (< 10%) acquired Las below 22.5oC and above 30oC. Experiments to determine the effect of temperature on Las inoculation were also completed and we will begin assessing plants used in these experiments for Las infection later this month. Analysis of endosymbionts from different psyllid populations is underway. Thus far we have amassed a 16S library containing 1000 sequences from psyllid populations collected from seven counties in Florida as well as India, Argentina, Mexico, Texas, and Hawaii. PCR results from these samples indicated that Wolbachia infection differs considerably among psyllid populations within and outside of Florida. In addition, laboratory experiments indicated that there is no apparent effect of temperature on ACP endosymbiont populations. Further experiments are underway to evaluate the role of endosymbionts in Las infection differences observed in Florida psyllids. In experiments to determine the effect of Las on psyllid fitness, we have assessed the fecundity, fertility, development time and adult survival of Las-infected ACP. Results from these experiments suggest that nymphs reared on Las-infected plants develop faster than nymphs reared on healthy plants and that there are no differences in fertility among infected and healthy psyllids. Additional investigations revealed an increase in the fecundity of infected psyllids. Infected psyllids produced nearly 200 eggs per female over a 25 day period, compared to only 100 eggs per healthy female. Vertical transmission of Las by ACP, determined in our previous experiments (Pelz-Stelinski et al. 2010), and increased fecundity of infected psyllids suggest that Las is well-adapted to psyllids. The practical implication of this result is that greater egg production in Las-infected ACP may increase disease spread. Finally, infected adult psyllids exhibited reduced longevity compared to Las-free ACP, suggesting a possible fitness trade-off between reproduction and longevity when infected with Las.
Field data collected mostly in Lake Alfred commercial groves over two growing seasons were used to generate a model and produce recommendations for general optimal field conditions that ensures maximum effectiveness of low volume insecticide applications. Data from the nearby FAWN weather station was used to compare relevant meteorological data from application times against the day 3 (knock-down) and day 28 (residual) percent reduction of Asian citrus psyllid (ACP) nymphs and adults vs. 2 sets of untreated control plots. Applications of test insecticides were made using the Curtis Dyna-Fog’ ‘LV-8’ sprayer at 6 gpa/10 mph in 0.1 acre/3 middles plots of Valencia grove. The volume median diameter of spray droplets was about135 microns. Sprays were made in 4 replicates during conditions of thermal inversions, i.e. ~11 pm onwards and when wind speeds were less than 3-5 mph. Still nights were avoided to ensure label compliance. Products tested were for evaluations of novel active ingredients (AI), existing chemicals requiring LV registration, formulation types as well as experimental investigations into spray adjuvants. Efficacy evaluations were conducted on days 3, 7, 14 and 28 using: a) tap counts, beating randomly selected branches in each plot and enumerating the ACP adults disturbed onto a white sheet, b) yellow sticky traps, deployed 4 per plot and the adults counted on the card, and c) flush rating, 10 flush terminals per plot were collected and the nymphs surveyed in the lab by rankings. All of these data were then analyzed using ANOVA versus the untreated control. Overall the results from the experimental applications were that the LV-8 is an effective method of controlling ACP in a mature citrus grove. The wide scale analysis highlighted the differences in the active ingredients. The insect growth regulators showed lower rates of knockdown, but were effective at the end of the sampling period, whereas the organophosphates showed very effective knockdown, with levels of control tailing off at the end. The newer products that combine two different classes of AI gave good overall control for both periods. Use of the same AI and different classes of adjuvant showed differences in levels of control which prompts further research. Our applications followed the label and best practices defined by the applicators license, so we did not experience suboptimal performance from the products, the use of meteorological data was more akin to fine tuning of best practice. When the micro-meteorological data were incorporated into the rankings, we noticed that the wind speeds during LV applications affected the efficacies. The lowest efficacies were recorded for the higher wind speed; about 3.5 mph. Low humidity during the applications also reduced the efficacy in the long term applications. Conclusions- For the products tested, the LV-8 was an effective application method; products consisting of mixtures of different insecticide classes show good knockdown and longer term control; and that lower, steady wind conditions with higher humidity gave the best control.
The literature shows that transmission efficiency of Candidatus Liberibacter asiaticus (CLas) by the Asian citrus psyllid (ACP), Diaphorina citri, can be quite variable (1-100%). We are conducting a series of studies to determine the impact of various factors on the efficiency of acquisition of CLas by ACP), such as vector development stadia and duration of acquisition access period (AAP) (study 1), as well as leaf age (study 2), pathogen titer and symptom expression in infected citrus (study 3). We are also investigating how transmission efficiency varies depending on the duration of the inoculation access period (study 4), and if a systemic insecticide can prevent transmission (study 5). We finished study 1 and are close to conclusion of studies 2 and 3. Studies 4 and 5 are being set up and will require another year for completion. In this quarterly report we show results of experiments related to study 1 (see previous reports for partial results of studies 2 and 3). In study 1, we compared acquisition efficiency of CLas by different ACP nymphal stadia (1st, 2nd, 3rd, 4th and 5th instars) and adults (1 wk old). Insects of each age group were allowed a 2-day acquisition access period (AAP) on CLas-infected plantas, then kept on citrus healthy plants for 19 days and tested by real-time (RT) PCR. The experiment was repeated three times with different source plants. Mean acquisition efficiencies by 1st, 2nd, 3rd, 4th and 5th instars, as measured by the proportion of infective psyllids by RT-PCR, were 68.4, 60, 66.6, 84 and 65.3%, respectively. In contrast, only 42.9% individuals were infective when acquisition took place during the adult stage. In a second experiment, we compared CLas acquisition efficiency by 3rd-instar nymphs and adults in relation to duration of the AAP (1.5, 6, 12, 24, 48 or 96 h). Almost 30% of the nymphs and adults were PCR positive for CLas when submitted to the shortest AAP tested, suggesting that the minimum AAP required for acquisition is <1.5 h. Acquisition efficiency by nymphs increased at a logarithmic rate in relation to AAP duration, reaching an average of 91% with 24-h AAP and stabilizing around this level with longer AAPs. In contrast, acquisition efficiency of CLas by adults increased linearly with AAP duration, reaching the maximum level (93%) only after an AAP of 96 h. Overall, the results show that both nymphs and adults can efficiently acquire the pathogen if allowed to feed for 1 and 4 days, respectively, on young shoots of infected plants. In a third experiment, we used the Electrical Penetration Graph (EPG) technique to determine stylet penetration activities and probing periods required for pathogen acquisition and inoculation in citrus. Healthy ACP adults were connected to the EPG system and placed on CLas-infected plants for different probing periods. Probes were artificially terminated by pulling the insects out of the plant after the following stylet penetration periods (treatments; n=30 insects per treatment): I) 20 min in waveform C (pathway phase through epidermis and parenchyma); II) waveform C + 30 s in waveform D (first contact with phloem tissue); III) waveforms C+D + 90 s in waveform E1 (penetration and possibly salivation in the phloem sieve elements); and IV) waveforms C+D+E1+ 1 h in E2 (phloem sap ingestion). Following each probing treatment, the insects were kept on healthy seedlings for 21 days and then tested by RT-PCR for CLas infectivity. We observed infective psyllids (5 out of 30) only when waveform E2 was performed (treat. IV), indicating that CLas acquisition by ACP occurs only after its stylets reach the phloem tissue, and sap ingestion (represented by E2) takes place. We hope to obtain additional replicates per treatment to confirm these results. This knowledge is important to analyze the efficacy of some control tatics (e.g. insecticides, host plant resistance and feeding deterrents) aimed to prevent pathogen acquisition by ACP.
Progress has been made toward developing a GC-EAD method that should be useful for determining which specific chemicals may serve as psyllid attractants. This method records specific impulses from psyllid antennae in response to blends of chemicals allowing precise identification of which specific chemicals the insect antenna is responsive to. Software and hardware-related obstacles to recording antennal signals from the Asian citrus psyllid were overcome during 2010, resulting in the first successful recordings from a psyllid antenna. Citrus volatiles were collected from citrus flush and analyzed by gas chromatograph-coupled electroantennography (GC-EAD). GC-EAD identified several peaks that reliably elicited antennal response. These peaks were subsequently identified by GC-mass spectometry (GC-MS) and will now be confirmed by testing individual compounds in behavioral bioassays. Varying concentrations of ‘pure’ purchased odor compounds will be ‘puffed’ over antennae to generate dose-response curves. In addition to identifying active plant odors, our primary focus will be to use our recording system to search for pheromone compounds that may be present in odors produced by male and/or female psyllids. Currently, we have identified seven compound of particular interest, which are being pursued in behavioral testing. Also, we have been analyzing cuticular extracts of pysllids and have identified several differences between males and females. We are investigating these chemicals that differ between males and females in an effort to identify the specific behaviorally active pheromone components.
The goal of this project is to determine if infection by Candidatus Liberibacter affects the response of Asian citrus psyllid (ACP) to its citrus host plants to understand a critical component of disease spread. In this project we evaluated if healthy psyllids are attracted more to HLB infected or healthy trees. We have also investigated whether this behavior changes when the ACP vector becomes infected with the pathogen. Behavioral experiments indicated that HLB-infected citrus plants are more attractive to ACP adults than healthy plants in two-choice olfactometer experiments. More ACP were attracted to HLB-infected plants than to healthy plants in open-air cage experiments. However, subsequent dispersal of ACP adults to healthy plants following their initial choice indicated that final setting preference was for healthy rather than diseased plants. We speculated that initial movement of ACP to infected plants and further dispersal to healthy plants may be explained by two hypotheses. 1. Yellow color of HLB diseased plants due to chlorosis and yellowing of shoots may attract the ACP initially, but psyllids move to healthy plants after a test probe. 2. Production of deceptive volatile compounds by the HLB infected plants may attract ACP adults in the field to facilitate the spread of bacteria. Settling experiments with HLB infected and healthy plants in complete dark conditions produced similar results to the ones under light conditions suggesting that initial movement of psyllids to HLB infected plants is not due to yellow color but to some other factors. The head space analysis of HLB infected and healthy citrus plants indicated that HLB infected plants produced significantly more methyl salicylate (MeSA) than healthy plants and less limonene and methyl anthranilate (MA). The complete nutritional analysis of HLB infected and healthy plants showed that HLB plants were deficient in zinc, iron, nitrogen and phosphorus and had excess amount of potassium and boron. Initial bioassays with MeSA showed slight repulsion to psyllids while MA had no effect on psyllids at test dosages. However, MA was toxic to Tamarixia radiata at 10mg or higher dosages in the olfactometer bioassays. More bioassays will be conducted to confirm these finding and also to evaluate the effect of these chemicals at various dosages. In addition, settling preferences of ACP on nutrient deficient plants will be assessed by giving ACP adults a choice between healthy plants and the nutrient deficient plants in the active psyllid season. Procedures are being standardized for inducing nutrient deficiencies in collaboration with soil scientists.
.-aminobutyric acid (BABA) is known to induce resistance against several microbial pathogens, nematodes and insects in several host plants. Laboratory studies were conducted to determine if there was a similar effect of BABA against Asian citrus psyllid (ACP) in citrus. We conducted two studies that examined the effect of varying concentrations of BABA, applied as a root drench to citrus plants, on the performance of ACP. Results from both studies revealed that BABA-induced resistance in citrus plants can suppress the growth and development of ACP by reducing the number of eggs, nymphs and adults produced by females. In addition, leaf-dip bioassays were performed using similar concentrations of BABA and no direct toxic effect of BABA on 2nd and 5th nymphal instars and adults of ACP was found. A study analyzing the head-space volatiles collected from BABA-treated and untreated plants by GC-MS revealed marked differences in their chemical profiles. Production of some volatile chemicals was either greater (amplified) or lesser (inhibited) in BABA-treated plants compared with the profiles from plants without BABA treatment. Identification of these chemicals is continuing. Currently, we are investigating the effect of BABA treatment on the feeding behavior of ACP adults using electrical penetration graph recordings. Studies are also being conducted to investigate to understand the underlying mechanism(s) of BABA-induced resistance by comparing induction of plant defense pathways between BABA-treated and untreated plants. Findings from these studies can provide valuable insight in the potential use of BABA in place of conventional insecticides; although effects of BABA under field conditions and its effect on no-target organisms still needs to be evaluated. In addition, since BABA acts by potentiating a normally underexpressed defense pathway; genetic tools can be used to trigger such pathways by genetic alteration and possible development of transgenic cultivars.
Three new formulations of SPLAT-DMDS were developed and evaluated. The formulations release the DMDS active ingredient for up to 3 months, which is approximately 3 times longer than previous formualtions. However, results in the field have still been inconsistent. In certain field trials, were have been able to measure reduced ACP populations but in other experiments there appears to be no effect. The most promising results are obtained when the repellent is deployed after the populations of ACP are initially killed off with an insecticide. Subsequently, psyllids appear to re-colonize DMDS-treated plots slower than untreated plots. However, even these experiments have been inconsistent. An experiment was conducted during the spring and summer that tested the effect of an olfactory psyllid repellent under standard psyllid management practices. The experiment has concluded. The formulation tested was the initial SPLAT-ACP Repel. Throughout this period, three applications of the formulation were made (April 29th and May 29th and June 29th). Applications were made to 3-4 acre replicated blocks of citrus and identical adjacent blocks were used as controls. The experiment was arranged as a randomized complete block with four replicates. All blocks received the same standard psyllid management with pesticides. Psyllid populations were monitored once or twice per week March 16th through August 18th. Yellow sticky monitoring traps were used as well as the tap sampling method. Throughout the course of the experiment, psyllid populations were extremely low in the experimental plots (Below 0.25 psyllids per ten traps per block on average). We observed no significant additional reduction of psyllid populations in the plots that received the olfactory repellent as compared with control blocks without the repellent. This result was likely due to the insecticide use practiced in this grove resulting in psyllid population densities that were likely too low to allow measurement of a treatment effect. During the initial part of the season, we observed that the DMDS active ingredient was highly phyto-toxic when the SPLAT formulation was applied to peripheral (pencil-thin) tree branches. However, application to larger branches (3-5 inch diameter or greater) did not cause phyto-toxic symptoms. We have also developed a method to measure DMDS concentrations in the field and we are using this method to determine how much of the DMDS or other repellents occurs when we deploy it from SPLAT or other release devices. Our intent is to determine exactly how much DMDS we need deployed in the field to achive desirable effects, so that a more consistent release device can be developed.
Processing of field samples continues. All of the sweep net samples have been examined for the presence of psyllids, parasitic wasps, coccinellid beetles, ants, spiders and other predators for the 3 grove types (unmanaged, minimally managed and intensively managed.) For this season, there appears to be no increase in the number of beneficial insects in the plots treated with methyl salycilate (MeSA) over that of the control plots in the samples that have been examined. In all groves, psyllid populations remained low throughout the season, even in the unmanaged grove. The overall average number of psyllids per trap was <1 for the intensively managed grove, ~1/trap for the minimally managed grove and up to 1.5/trap for the unmanaged grove for a 2-week trapping interval. Progress continues on the psyllid behavioral studies investigating any attractant or repellent properties of MeSA. Plants for the settling studies are pruned to induce flush and when suitable for psyllids, ethanol solutions with MeSA will be applied to . of the plants. Plants sprayed with ethanol alone will serve as controls. The numbers of psyllids settling on each plant type over time (1 hour, 24 hrs, 3 days, 5 days and 7 days) will be recorded. To determine any effects of MeSA on pysllid survivorship, longevity and fecundity, plants will be sprayed with MeSA and an equal number will be sprayed with ethanol only. Ten male and 10 female adult psyllids will be caged on new flush of each plant. After 7 days, the cages will be removed and the number of surviving adults as well as eggs and nymphs will be counted and compared.
We have continued to monitor the long-distance movement of ACP. A protein marking solution made up of 10% chicken egg albumin in water with Silgard added at 2000 ppm was applied to a block of 200 mature sweet orange trees in the central area of a well managed 40 ha grove in Lake Alfred, FL (N 28’07.053′, W 81’44.003′) using an ATV-mounted handgun sprayer run at ‘250psi. Marker spray was applied at the rate of ‘7.5 L/tree. Eight yellow sticky traps were placed within the marked area; two each on the northern, eastern, southern, and western borders. Traps were also placed concentrically, radiating away from the marked area at distances of 100, 300, 400, 500, 650, 1000, 1200, and 2000 meters. At each distance one pair of traps was set up in five separate locations, with an attempt made to get good radial coverage. Some traps placed at distances of 500 m and further from the marked area extended beyond the border of the grove in which the marker protein was applied; into nearby managed and abandoned orange groves. Traps were removed 11 days after application of the marker protein. This experiment was conducted twice, once in June 2010, and again in July 2010. Relative abundance of leaf flush was evaluated on the final day of the experiment in July 2010. A cubic square frame made up of PVC pipe and fittings was randomly placed into the tree canopy, and the number of flush shoots originating from within the square frame was counted. At each trap site, two samples were taken from each of five adjacent trees and the 10 numerical values were averaged to estimate relative flush abundance. Average daily wind direction during the experiment was collected from the Florida Automated Weather Network (FAWN) database with readings from a weathering monitoring station located within the 4 km diameter study area. The degree numbers provided by the database were categorized into eight components (N (338′-22′), NE (23′-67′), E (68′-112′), SE (113′-157′), S (158′-202′), SW (203′-247′), W (248′-292′), NW (293′-337’)). A compass graph was created and superimposed over a satellite map of the study area and used to determine how many days a trap was downwind from the marked area. Grove type was separated into three categories; abandoned (no pesticide applications for at least three years), managed (areas where recommended ACP control protocols are followed), and intensively managed (areas with young trees where insecticides are applied at the maximum allowable rate for ACP control). The results indicate that adult ACP are capable of travelling at least 2000 m within 12 d. Surprisingly, wind direction did not seem to be a major factor during psyllid dispersal, which was influenced more by flush availability and areas of low or no psyllid control (grove type). Flush is a necessary resource for ACP reproduction, and it makes sense that this would affect ACP dispersal. It is not yet understood how the presence of Candidatus Liberibacter asiaticus might affect the biology of ACP. This study has shown that ACP carriers of Ca. Las may be just as fit as their Ca. Las-free cohorts in terms of dispersal abilities. ACP were trapped in both months at all distances except for 1000 m; this is most likely due to the fact that all ten of the traps placed at 1000 m were, due to the constraints of the study area, placed in trees that were either in a state of severe decline or on young trees (< 4 y) in intensively managed areas. Citrus groves that are within 2 km of any other citrus plantings are at risk for ACP infestation and HLB disease introduction from those areas. Further studies should seek to identify if ACP are capable of dispersal greater than the 2 km recorded here. Results from this study should also serve as further evidence of the threat of infestation and infection posed by abandoned groves in proximity to managed citrus as grove type was a significant factor in psyllid dispersal regardless of flush abundance.
We have recently completed a two-year study (2009-2010) to develop baseline studies on levels of insecticide resistance among five geographically discrete populations of ACP across Florida. Briefly, in 2009, one or more field population of ACP exhibited higher LD50 values to fenpropathrin, imidacloprid, malathion and thiamethoxam compared with the laboratory susceptible (LS) population. The highest level of resistance was observed in the La Belle population with a resistance ratio of 38 and 13 to imidacloprid and thiamethoxam, respectively. Three populations exhibited moderate levels of resistance to malathion (Ft. Pierce: RR = 5.4, Lake Alfred: RR = 5.0, Groveland: RR = 3.7). The Vero Beach population had a moderate level of resistance to fenpropathrin with a RR value of 4.8. In 2010, susceptibilities of field populations were compared with the LS population. In general, the percent mortality of adult ACP from each field population was lower than that of the LS population for most of the insecticides tested. In addition, the susceptibility levels of immature ACP from four field populations were determined. Activity of the major classes of insecticide detoxification enzymes was compared for field and laboratory psyllids. Among these enzymes, levels of general esterase, glutathione S-transferase and cytochrome P450 activities were higher in adults in several field-collected populations compared with LS adults. Likewise, among immature ACP, general esterase, glutathione S-transferase and cytochrome P450 activities were significantly higher in certain field populations than LS populations. The current results suggest that ACP have developed varying levels of resistance to major insecticides currently registered for its management. Elevated levels of detoxifying enzymes in these populations may be underlying mechanisms of this resistance. Our results indicate that continuous monitoring of insecticide resistance should be conducted to check for any emerging problem. Insecticide applications in citrus groves against ACP in Florida are conducted under a wide range of temperatures varying from 10-40’C. Therefore, a study was conducted to investigate on the effects of temperature on insecticide susceptibility in uninfected LS, uninfected field-collected, and Candidatus Liberibacter asiaticus (Las) infected adult ACP for five insecticides at labeled rates under laboratory conditions. In addition, the effect of temperature on the activity levels of three detoxifying enzymes was determined for uninfected ACP. All three ACP populations were found to have a significant positive correlation between temperature and percent mortality for chlorpyriphos, imidacloprid, spinetoram and thiamethoxam. The exception was fenpropathrin, where a negative correlation was found. Among the detoxifying enzymes, glutathione S-transferase was the only enzyme found to have significant negative correlation with temperature, whereas cytochrome P450 and general esterase activities were found to have no correlation with temperature. Results from this study suggest that prevailing temperatures under grove conditions at the time of insecticide applications should be considered to decide on an appropriate insecticide.
Continuous cultivation of D. citri cells in culture has continued for almost 2 years. Although the cells are growing, the major current obstacle is their relative slow growth and heterogeneity. We have attempted to increase the robustness of the cell lines via selective manipulation and transfer of cell patches. Suspension cells are more homogeneous than adherent cells and appear to grow faster. However, several new adherent cell cultures appear to be producing more uniform cell types (fibroblast-looking) that will continue to be selectively cultured with the objective of isolating a relatively homogeneous adherent cell line. These cells also appear to be growing faster than previous cell lines, although additional passage is needed to verify these results. We have also initiated a preliminary screen for isolating D. citri viruses via examination of a cytotoxic response in the cell lines. These experiments are in progress and our current efforts are at optimizing the screening conditions.
Spatial and Temporal Incidence of Ca. Liberibacter in Citrus and Psyllids Detected Using Real Time PCR, December 2010/January 2011. In July 2010, psyllid adults from our HLB negative colony on orange jasmine were caged on new shoots (10-12 adults/shoot/ per tree) that had been caged immediately after trimming and were not exposed to feral psyllid population. On the same tree an additional cage was placed on a previously uncaged shoot that was infested with feral psyllid nymphs. These adults and nymphs remained caged on the shoots for three weeks. Some adults from laboratory reproduced in the cages so when collected there were some nymphs available. Similarly cages with feral populations also had some nymphs and adults at the time of collection. Using PCR, 40 shoots tested HLB negative with average Ct value of 39.8 ‘ 0.07 and 15 shoots tested HLB positive with average Ct value of 27.4 ‘ 0.7. From lab reared adults caged on HLB negative shoots 69 were tested and all were negative. From their nymphal progeny 58 were tested and 1.7% were positive with Ct value 29.8. From the ones caged on HLB positive shoots 51 adults were tested and 12% were positive with average Ct value of 29.6 ‘ 0.7. Among 31 nymphs none were positive. Out of 88 adults that emerged from feral nymphs caged on HLB negative shoots 10% were positive with average Ct value of 28.9 ‘ 0.9. Only 5 nymphs were tested and all were negative. Among 53 adults that developed from feral nymphs caged on HLB positive shoots 9% were positive with average Ct value of 29.6 ‘ 0.9. No nymphs were tested. We are also evaluating the effect of vector management on bacterial titer and fruit yield in the same 12-acre block of a commercial orange grove. The block was divided into 16 plots organized in a randomized block design with eight replicates and four treatments: (1) Nutritional (2) Insecticides (3) Insecticides + Nutritionals and (4) Untreated. Treatments (1) and (4) remained free of insecticides and the vector was allowed to thrive. In treatments (3) and (4), the psyllid population was monitored every 2 weeks and insecticide was sprayed when insect populations surpassed the nominal threshold of 0.5 psyllids per tap sample. To assess the titer values, we selected symptomatic branches on every fifth tree in every row for real-time PCR analysis every four months. Initially CT values and the percentage of PCR positive trees did not differ between treatments. In the last two years, CT values have dropped and most trees eventually tested positive. CT values from insecticide-treated trees became significantly higher in Jan 2010 (25.7 ‘ 0.6), indicating lower bacterial titers, compared with untreated plots (24.0 ‘ 0.1), although no difference between the two treatments in May 2010. Treated trees had significantly higher yields than the untreated trees. These results indicate that mitigation of vector pressure with insecticide decreased alleviated bacterial titer over time and positively affected yield despite the same incidence of HLB. In August 2009, Ct values of psyllids from insecticide treated and untreated trees averaged 38.4’0.9 (0% positive) and 37.5’0.5 (2.4% positive) for adults, 37.8’0.5 (0% positive) and 37.9’0.7 (1.1% positive) for large nymphs and 39.1’0.3 (0%) and 37.6’0.9 (2.5% positive) for small nymphs.
This project has 5 objectives: (1) evaluate efficiency of potential ACP control techniques in cooperation with growers, (2) develop efficient monitoring methods for ACP, (3) accelerate testing of new chemistries and techniques for ACP management, (4) evaluate the economic component of the comprehensive program, and (5) provide an information bridge between researchers, growers, and industry. The positive response from growers to the extension program funded by this project is illustrated by the adoption of practices such as area wide dormant sprays and ACP monitoring using tap samples. Virtually all commercial groves in the region are participating in cooperative area-wide dormant sprays. More than 70,000 acres were sprayed at least once by air and most of the rest by ground during the past two dormant seasons in the SW Florida region according to data provided by aerial applicators and surveys conducted by Hendry County Extension. More acreage is being sprayed by ground with LV equipment this dormant season, with aerial applications accounting for 27,250 acres to date. Field counts provided by growers and an FDACS-DPI ACP sampling team trained under this project verified effectiveness of the program, and populations have declined steadily over the entire region during the last 3 years, even in untreated plots. The area wide program in SW Florida, now called the ‘Gulf “CHMA” (www.crec.ifas.ufl.edu/extension/chmas/gulf_chma.htm) is conducted in cooperation with Gulf Citrus Growers Association, FDOACS-DPI-CHRP and Hendry County Cooperative Extension (Stansly et al., Citrus Industry, 2009-2010). Its success has stimulated efforts in other citrus growing regions of the state with the help of 10 presentations this quarter to citrus association boards and members (Indian River, Peace River, Gulf Coast) professional societies and other groups. Another successful extension effort is seen in adoption of the “stem tap” sample and other recommended techniques for monitoring ACP populations. Sixty-three percent of growers surveyed in 2010 reported monitoring ACP more than 12 times a year (75% using the tap method) compared to 37% in 2009. An extension (EDIS) document describing ACP sampling techniques with the code ENY857/IN867 is in press. Our present management plan is based on four principles: (1) preemptive insecticidal sprays during winter targeting adult ACP to reduce the population reproducing on spring flush; (2) a rapid and reliable psyllid monitoring system to guide timing of insecticidal control during the growing season; (3) conservation and augmentation of psyllid natural enemies, and (4) testing of insecticides and application technology including low-volume sprayers and timing. More than 90% of surveyed growers in SW Florida responded as either satisfied or very satisfied with their HLB/psyllid management programs. Field and laboratory experiments conducted in 2010 include: (1) A 120 acre trial comparing a standard dormant spray (Mustang), and application of spirotetramat directly to spring flush accompanied or not with applications of aldicarb before or after spring flush. All treatments successfully maintained populations lower than the control until May with treatments that included spirotetramat being effective until July, (3-7) Five field trials evaluating ACP suppression with insecticides, 3 foliar and 2 drench, submitted for publication in Arthropod Management Tests (www.entsoc.org) and placed on our website www.imok.ufl.edu/entomology. Three additional trials of systemic insecticides are being conducted in a newly planted 5 acre block of “Hamlin” orange on ‘US802’ at our Center. Included is a new active ingredient, cyazypyr (cyantraniliprole), from Dupont, that showed excellent potential in lab bioassays and could be an important addition to the arsenal of soil-applied systemic insecticides, presently limited with the loss of Temik to the neonicotinoids imidacloprid and thiamethoxam.
Our purpose is to develop RNA interference in the tomato/potato psyllid (Bactericerca cockerelli) as an effective model for application towards the Asian citrus psyllid, Diaphorina citri. Our hope is that by inducing effective RNAi effects in recipient psyllids, we can help to control psyllid-vectored plant pathogens with the ultimate target being Ca. Liberibacter asiaticus, the causal agent of citrus greening. We originally generated a B. cockerelli normalized cDNA library and have continued to work with this library to identify candidate sequences for RNAi studies. So far less than 100 targets have been sequenced, but this is ongoing . We have used several B. cockerelli sequences for RNAi studies and cloned the D. citri actin, ATPase, Hsp70 and CLIC, D. citri homologs. We have used two primary approaches to evaluate dsRNAs for RNAi activity. Direct intrathoracic injection of dsRNAs into the psyllid hemocoel is done using 200 nl of 100ng/ul dsRNA. This is used as a known control standard to compare against oral delivery of the same dsRNAs. Increased mortality was seen for actin dsRNA when compared with the control GFP dsRNA 3 days after injection. Injection of an ATPase specific dsRNA also consistently suppressed the endogenous ATPase mRNA expression by 30% in independent experiments. We have developed and used an artificial diet system for screening candidate dsRNAs for B .cockerelli . Psyllid acquisition of the dsRNAs was confirmed by end point and quantitative RT-PCR, and by Cy3-labelled dsRNA visualization. We have screened 18 sequences so far by this method, and have confirmed 4 of them as promising candidates for RNA interference. We also cloned C002 and aquaporin (aqp1) homolog genes from B. cockerelli, and are evaluating these sequences now. We see increased mortality of B. cockerelli in a dsRNA concentration-dependent manner for some of our selected candidate sequences. In addition, consistent and specific down-regulation of endogenous target actin and ATPase mRNAs also was shown by quantitative real time PCR.. We also confirmed RNAi activity after a 3 day oral acquisition by using RNA hybridization to detect siRNAs in psyllids. These data clearly demonstrate RNAi activity in psyllids. We are making transgenic plants now to test candidate sequences that have shown promise via in vitro feeding assays. The candidate sequences were cloned in the plant-expression gateway vector pCB2004 and are ready for production of transgenic tomatoes. We are also attempting to rapidly evaluate RNAi effects in plants. We have used a Tobacco mosaic virus (TMV)-based plant expression system to express B. cockerelli sequences in plants, and psyllids are then fed on these plants and monitored for RNAi effects. So far we have seen decreased survival of B. cockerelli for 2 of 6 sequences after 10 days feeding on recombinant TMV-infected plants. However, TMV systemic expression of insert sequences requires approximately 3-4 weeks and resulting infections are non-uniform within tomato plants. These factors likely contribute to the inconsistency seen so far in our experiments. We are working to improve this system and will have additional data by the next reporting period.
This project includes experiments being conducted by USDA-ARS and University of Florida on protecting newly planted citrus trees from Asian citrus psyllid and huanglongbing. This progress report reviews research conducted by University of Florida in Southwest Florida. The original experiment to compare a granular slow release imidacloprid product (Suscon) to the standard liquid formulation was discontinued in spring 2010 due to a poor stand (deer damage) and lack of psyllids during the interval following applications. Three other studies evaluating soil applied products for psyllid control were initiated in spring/summer 2010. The first study included 11 different treatments and an untreated control on 2 year old Hamlin trees and was designed to evaluate the difference between a single application of Admire Pro, Suscon (slow release) Platinum or Cyazapyr versus a split application of these products or the inclusion of Temik and Movento (foliar) as a rotation partner with the Admire Pro (imidacloprid). Initial applications were conducted 25,26 ‘ Feb with the follow up applications on 19,20 ‘ May. Psyllid eggs and small nymphs were found in low numbers on very few flushes on some sample dates and significant differences were found only on 29-Apr when untreated plots contained 8.1 nymphs per flush which was significantly greater than other treatments which did not differ from each other. Nymphs were only found on the low rate of Cyazapyr and two different rates of platinum. Differences in leafminer activity was noted with the split application of Platinum and admire providing greater control of leafminers than the corresponding single application but this trend did not reveal itself with Cyazapyr as the different treatments were not significantly different from each other. Neither Movento or Temik had a significant effect on leafminer incidence. The second study compares three rates of cyazapyr against Admire Pro and Platinum applied on 19,20 Jul to newly planted Hamlin trees. Evaluations were conducted 10,23 ‘ Sep. and no psyllids were found. However, all products have been 100% effective for leafminer control. Leaf samples are being analyzed for residues of all insecticides. Another study is designed to last 3 years with applications of Cyazpyr, Platinum and Admire Pro every three month in rotation with each other. The objective is to assess the ability of these treatments to delay or prevent the onset of HLB in an area of high incidence. Initial treatments were made 29-Jul on newly planted Hamlin trees and evaluations conducted 2,22-Sep with no psyllids being observed although all treatments have been 100% effective in controlling leafminer. Leaf samples are being analyzed for residues of all insecticides.
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