The movement patterns and dispersal capabilities of Asian citrus psyllid (ACP) required investigation to better understand the spread of huanglongbing (HLB) and to improve management strategies for ACP. Understanding movement patterns and dispersal behaviors of ACP will be essential in creating optimal pest control strategies with the hope of curbing the spread of HLB, and protecting citrus production in Florida. To investigate ACP movement, we adopted an immunomarking technique which utilizes crude food proteins (chicken egg albumin, bovine casein, and soy protein) to track the movement of ACP in Florida citrus. In brief, ACP are marked with proteins in the field and allowed to move naturally. They are re-captured on traps and analyzed for the marking protein. In this way, we can determine exactly: where psyllids came from, how far they moved, and how fast they moved. Initially we found that ACP movement is biased in the direction from abandoned or marginally managed groves into well managed groves. We also found that ACP are capable of moving back and forth between 2 groves separated by 100 yards within 2 days. Furthermore, ACP are capable of invading up to 100 yards into managed grove interiors within 4-7 days. Furthermore, during the spring, we found that ACP move even when there is abundant flush (food/egg laying sites) available. In terms of where psyllids tend to invade most often, we found that invading ACP are most often found within the first 3-4 rows of trees from the plot borders. More recently, we have been studying the seasonality of ACP movement. This research has been conducted by quantifying how many psyllids move on a monthly basis over the course of the entire year. The first year’s results indicate that the majority of psyllid movement occurs during the spring and early summer months, while very little movement occurs September through February. We will need to repeat this experiment for another year to be certain that this pattern is consistent. There may be certain times of the year when frequent insecticide treatment is needed (particularly on plot borders) due to psyllid movement, but there may also be certain other times of the year when fewer treatments are necessary when ACP are not moving. Given that we have proven that unmanaged and abandoned groves serve as a source of ACP infestation, our recent goal has been to determine the status of HLB infection in ACP and citrus trees in paired unmanaged and managed citrus groves separated by 100 yards or less. To accomplish this research, we sampled 7 pairs of adjacent unmanaged and managed groves. Surprisingly, we have documented slightly higher rates of HLB infection in both psyllids and trees in unmanaged groves than managed groves that were separated by 100 yards or less. These results confirm that abandoned and unmanaged groves not only serve as a source of psyllid infestation for adjacent managed groves, but also likely serve as a source of HLB infection. Effective HLB management in Florida citrus will therefore likely require removal of abandoned groves. Also, growers who are next to unmanaged citrus should consider protecting their border areas intensely to prevent immigration of infected psyllids into their groves. In summary, we have found that: 1) Movement of ACP occurs between unmanaged and managed groves; 2) The majority of ACP occur on the border rows of managed groves; 3) ACP adults can move at least 250 yards within 4 days; 4) Movement appears to vary over the course of the season ‘ more movement in summer than fall/winter; 5) The HLB bacterium is present in ACP and trees in unmanaged and nearby managed groves; and 6) infection levels are 2.3 & 1.5 times greater in unmanaged groves compared with nearby managed groves. We have either completed or exceeded our goals for year 1 and look forward to repeating necessary experiments in year 2 and to collecting new data to complete the two year objectives. Some of this research has already been published in a peer reviewed journal (Environmental Entomology, 2009, 38: 1250-1258) and other portions are currently under review.
In Texas, ACP in orchards have been in a state of semi-dormancy since October. ACP from our colonies have been non-responsive in behavioral tests. Behavioral tests will resume once ACP resume being responsive to test stimuli. Tests are planned to compare the responsiveness of ACP to combava petigrain oil, essential oil distilled from the foliage of Citrus hystrix. We have generated a database of plant odors to be analyzed by GC-EAD in Florida when that system becomes operational. If Florida, we are obtaining the first successful recordings from ACP antennae. After weeks of frustration with our Syntech electroantennogram system, we invited Dr. Peter Ockenfels of Syntech, Germany to our lab. He spent 4 days troubleshooting and successfully putting the system back on line. This opens the way to searching for antennally active compounds from host plants and conspecifics. Collections of cuticular hydrocarbons from greenhouse-reared ACP were made and exhaustively characterized by GC/MS. The resulting database and samples have been forwarded to Dr. Fran Webster at Syracuse University for analysis. Dr. Webster is an analytical chemist with expertise in NMR and structure elucidation. To date, we have not seen any obvious difference between hydrocarbon extracts collected from male and female ACP. Approximately 50 compounds have been identified to date. Extracts will be analyzed by GC-EAD to determine if any of these compounds elicit antennal response, followed by behavioral studies.
We have made good research progress this quarter. Laboratory testing of traps: We have conducted several and continue to conduct other laboratory bioassays to determine the behaviors used by psyllids during approach, landing and post landing on key parts of traps. We are characterizing the physical properties of traps including size, angle of surface to substrate, trap grid configurations, relative proportions of trap parts and color relative to their impact on psyllid walking, jumping and flight behavior. These factors clearly affect psyllid behavior in response to host plants, and therefore, will affect trap efficacy and efficiency. These experiments are enabling us to optimize trap configurations and to develop an estimate of the importance and the relative trade-offs in efficacy among trap components. Based on our preliminary lab results we redesigned trap prototypes for field testing and those have been placed in the field. We are now in the process of setting up more technically-rigid psyllid behavior experiments under more controlled laboratory conditions with a Noldus olfactometer and related equipment to get a better handle on how behavioral factors interact and to increase the inferences we may draw from such experiments. Field testing of traps: We are presently conducting replicated field tests of about 10 new and different trap configurations building on our lab and field results as prototypes in a replicated experiment in commercial citrus in the Immokalee area. Results from this field test and the continuing lab results will help us to focus further experiments. We expect to develop an improved trap for Asian citrus psyllids that can be used both in groves and for regulatory purposes in places where citrus plants are sold. This trap would collect the insects and preserve them using methodology that will enable testing for the presence of HLB pathogens at some later date.
Researchers from the Auburn University College of Engineering are constructing a combined empirical and simulation based toolset to ultimately optimize the “how much” (rate of release) and “where” (physical locations within a orange grove) deployment of a citrus psyllid repellent. The current focus is on recording physical measurements of repellent diffusion into air under prescribed ambient conditions and constructing a computational fluid dynamics (CFD) model of the airborne mixing of the repellent within the flowfield generated by a citrus grove. These studies serve as an engineering requirements flow-down, providing the necessary scientific data to shape specific deployment hardware options that balance effects longevity with application cost. Given the recent Citrus Research and Education Center (CREC) laboratory and field studies using the “Splat” (emulsified paraffin wax) approach for dimethyl disulfide (DMDS) application, DMDS is selected as the initial agent for repellent release rate studies. The PIs have traveled to CREC for first-hand discussion and observation of those studies and interfaced with ISCA Technologies, the manufacturer and supplier of “Splat”. Auburn is currently setting up a laboratory facility to measure and study the release rate of DMDS as input for airflow computer simulations. Initial experimental focus is on DMDS in liquid form, coupled with an artificial membrane to provide control over the rate of diffusion into air. It should be noted that the Auburn strategy maintains a broad top-level approach, favoring no one particular deployment method and leaving options open to consider other potential repellant chemical formulations as well. Implementation and evaluation of an initial CFD model is underway. The vegetation canopy is approximated by a porous medium that acts as a volumetric sink on airflow momentum, while insertion of repellent is accomplished by discrete point sources within the hedge row geometry of a typical mature grove. A literature survey supporting the use of CFD modeling applied to vegetation canopy airflow and mixing has provided valuable data and best practices. Of particular interest is the related problem of the effects of tree plantings in urban street canyons for the dispersion of automotive exhaust pollution. The literature reports good correlation between experimental and numerical airflow and dispersion findings and provides parametric information such as average canopy porosity for various tree species.
Researchers from the Auburn University College of Engineering are constructing a combined empirical and simulation based toolset to ultimately optimize the “how much” (rate of release) and “where” (physical locations within a orange grove) deployment of a citrus psyllid repellent. The current focus is on recording physical measurements of repellent diffusion into air under prescribed ambient conditions and constructing a computational fluid dynamics (CFD) model of the airborne mixing of the repellent within the flowfield generated by a citrus grove. These studies serve as an engineering requirements flow-down, providing the necessary scientific data to shape specific deployment hardware options that balance effects longevity with application cost. Given the recent Citrus Research and Education Center (CREC) laboratory and field studies using the “Splat” (emulsified paraffin wax) approach for dimethyl disulfide (DMDS) application, DMDS is selected as the initial agent for repellent release rate studies. The PIs have traveled to CREC for first-hand discussion and observation of those studies and interfaced with ISCA Technologies, the manufacturer and supplier of “Splat”. Auburn is currently setting up a laboratory facility to measure and study the release rate of DMDS as input for airflow computer simulations. Initial experimental focus is on DMDS in liquid form, coupled with an artificial membrane to provide control over the rate of diffusion into air. It should be noted that the Auburn strategy maintains a broad top-level approach, favoring no one particular deployment method and leaving options open to consider other potential repellant chemical formulations as well. Implementation and evaluation of an initial CFD model is underway. The vegetation canopy is approximated by a porous medium that acts as a volumetric sink on airflow momentum, while insertion of repellent is accomplished by discrete point sources within the hedge row geometry of a typical mature grove. A literature survey supporting the use of CFD modeling applied to vegetation canopy airflow and mixing has provided valuable data and best practices. Of particular interest is the related problem of the effects of tree plantings in urban street canyons for the dispersion of automotive exhaust pollution. The literature reports good correlation between experimental and numerical airflow and dispersion findings and provides parametric information such as average canopy porosity for various tree species.
We are in the first year of our project. Our goal is to identify and then use specific psyllid RNA sequences to induce RNA interference (RNAi) activity in recipient psyllids. We propose to express psyllid interfering RNA sequences in plants and induce RNAi when psyllids feed on these plants. We envision that successful application of RNAi towards the Asian citrus psyllid (ACP, D. citri) will confer a negative phenotype (even death) in psyllids, such that psyllids cannot colonize and/or reproduce on selected plants. By controlling the psyllid vector we believe this will complement other efforts to help control HLB. To make rapid progress we are using the tomato psyllid (Bactericerca cockerelli), which colonizes herbaceous plants (tomatoes, tobaccos) and is the vector of another Liberibacter spp. (C. L. psyllaurous). We are using tomatoes (Early Pak 7), and Turkish tobacco plants for evaluating Tobacco mosaic virus (TMV)-driven expression of psyllid genes for inducing RNAi. Both of these plants are easy to use, TMV susceptible and both are readily colonized by B. cockerelli. With Turkish tobacco we obtain more consistent Agrobacterium tumefaciens-based TMV infection than for tomatoes, but both plants, as well as Nicotiana benthamiana, are being used currently, although psyllids do not prefer N. benthamiana. We can induce production of specific siRNAs corresponding to the recombinant sequences in these plants (N. benthamiana and tomato), and we have demonstrated so far that psyllids can acquire TMV-based RNAs from TMV-infected tomato plants. Now we are evaluating acquisition and effects of anti-psyllid RNAs. We have utilized two approaches to obtain psyllid interfering RNAs for our work. The first has been by mining existing GenBank data for potentially useful D. citri sequences, and the second has been by constructing a new, normalized cDNA library for B. cockerelli mRNAs directly in a TMV-based virus delivery system. Both approaches are proceeding very well. We have used the D. citri EST database and have identified 1904 contigs, 179 of which are predicted to be expressed in D. citri midguts. We believe that midgut sequences are very likely good targets for orally-acquired anti-psyllid interfering RNAs or dsRNAs, as the midgut will encounter RNAs via psyllids feeding on plants. We have now successfully cloned 80 midgut genes (partial sequences varying in size between 500-1000bp) from D. citri. BLAST comparative sequence analyses allowed us to design 56 predicted conserved primers for use in cloning D. citri homologs from B. cockerelli. We have cloned 36 putative homologs so far, and 30 have been confirmed by sequence analysis. These are highly homologous (80 ‘ 90%) to corresponding D.citri sequences. So far RT-PCR using RNA prepared from B. cockerelli gut tissues confirmed that 25 were present. Sequence-based analyses identified some, including ion transporter genes, which are ideal candidates for RNA interference, and we are proceeding using TMV-based expression as well as direct micro injection to determine their efficacy against B. cockerelli. We have developed a micro-injection system for psyllids. This allows precise injection of known quantities of dsRNAs into the psyllid hemocoel. This is being used as a known control approach to assist in identifying candidate sequences for RNAi. Sequences that show effects after injection will be immediately tested in plants. We now have a normalized B. cockerelli cDNA library. The library sequences are cloned directly into our TMV-based plasmid for expression in plants. We are now confirming the library quality by nucleotide sequence analysis, and transforming the TMV-based plasmids into A. tumefaciens for use in whole plant anti-psyllid assays. Initial sequencing showed us that the library contains psyllid sequences and thus will be very useful for our effort.
We are conducting a series of studies to determine the impact of various factors on the efficiency of acquisition of Candidatus Liberibacter asiaticus (CLas) by the Asian citrus psyllid (ACP), Diaphorina citri, such as vector development stadia (study 1) and leaf age, pathogen titer and symptom expression in infected citrus (studies 2 and 3). We are also investigating how transmission efficiency varies depending on the duration of the inoculation access period (study 4), and if a systemic insecticide can prevent transmission (study 5). We have started experiments related to studies 1-4, and the research is progressing as expected in the original plan. In the previous quarterly reports (July/09 and Oct/09), we presented partial results of studies 1 and 2, indicating that nymphs in all development stadia (1st-5th instars) can efficiently acquire the pathogen; adults can also acquire it, but efficient acquisition depends on the availability of young leaves in infected plants, apparently because phloem sap ingestion (and thus pathogen acquisition) by ACP adults is more frequent and last longer on the younger leaves. In this report, we present new and interesting findings about CLas acquisition rate by D. citri adults on infected citrus with variable pathogen population (study 3). Fifty potted sweet orange on Rangpur lime rootstock trees ( 5-month old) were graft-inoculated with Clas at the same time, and groups of five independent plants were used as source for acquisition assays at 60, 130, 145, 165, 180, 200, 235, and 280 days after inoculations (DAI). In the last three periods most of the source plants were symptomatic. In each assay, a group of 50 lab-reared healthy D. citri adults was confined on young shoots of each source plant (inside sleeve cages) for an acquisition access period (AAP) of 7 days, and then kept on healthy citrus seedlings for a latency period of 21 days. Three to 15 psyllid samples (three psyllids per sample) from each source plant were subsequently tested for infectivity using real-time PCR. Clas population in the source plants was quantified right after the AAP by RT-PCR, using DNA extracted from leaves of the shoots used for acquisition. Clas was acquired by D. citri from both symptomatic and asymptomatic citrus, and a positive linear relationship was observed between pathogen population within the source plant and the percentage of infective psyllid samples [y (Ct) = -0.114 x (infectivity rate) +28.36; R2 = 0.33]. Similarly, the bacteria population within the plant increased over time [y (Ct) = -0.045 x (DAI) +28.43; R2 = 0.34) and became stable (t test, P = 0.010) at 235 and 280 DAI when HLB symptoms became more evident. Considering all source plants tested, acquisition efficiency by D. citri ranged from 0 to 100%, with a success ratio of 95% on symptomatic plants (n=17) against 64% on non-symptomatic but infected plants (n=24).
The following is a summary of Dr. Avery’s activities since Ocotber 2009: 1) Packers of Indian River, Ltd. – Ft. Pierce: Monitored psyllids along the border using yellow sticky cards. Data collected was used to produce a distribution graphic for assessing the psyllid population dynamics throughout the year for the grower. Graph showed that the population peaked in March and August. 2) IMG Citrus ‘ Fellsmere: Assessed and compared 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: monitored inside the block with yellow sticky traps, tap sampling, flush sampling, and assessing flush density. Overall, results indicated that there were more psyllids found on orange than grapefruit trees with similar flushing patterns; especially with the tapping samples. There was some fluctuation towards the later part of the year possibly due to aerial sprays conducted either near or on site. 3) Pine Ranch, Inc. ‘ Lorida:Assessed the effectiveness of various chemical sprays for managing psyllids using yellow sticky traps, tap and flush sampling. Generally the population was low, but the numbers of psyllids may be influenced by the abandoned grove near the monitoring site. 4) River Country Citrus, Inc. ‘ Okeechobee: 1.Assessed the effectiveness of various chemical sprays for managing psyllids using yellow sticky traps, tap and flush samples on young trees. Results indicated that very low numbers were present on young (2 yrs old) trees using Nuprid’. 2.Assessed and compared 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. Overall, results indicated that there were more psyllids found on orange than grapefruit trees with similar flushing patterns. 5) Lindsey Groves ‘ Vero Beach: Assessed and compared 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. Overall, results indicated that more psyllids were found on oranges than grapefruit with similar flushing patterns. 6) Premier Citrus ‘ Vero Beach: Assessed and compared 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. Overall, results indicated that there were no differences in psyllid abundance between orange and grapefruit trees with similar flushing patterns. 7) Laboratory and Field Research at IRREC: Assessed persistence and efficacy of PFR 97’ against the adult psyllid in the field on small trees; experiment was replicated 3 times. Overall, results showed fungal spores were viable and infectious against the adult psyllid up to 28 days post-spray. The effects of two formulations of Isaria fumosorosea Wize (blastospores and conidia) on feeding, honeydew deposits, and mortality of adult Diaphorina citri Kuwayama (Hemiptera: Psyllidae) was assessed in bioassay arenas. Psyllids infected by either formulation had reduced feeding as indicated by significantly fewer honeydew deposits [8 and 9 times less after 5 and 7 days, respectively] with mortality reaching 100% in fungal treatments compared to 0% in the controls after 7 days. Blastospores caused a significantly higher mortality than conidia in the first 3 days. The potential of auto dissemination of Pfr 97 by psyllids in the field is still being investigated.
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. In this quarter, using Ellman’s biochemical assay, the baseline data for acetylcholinesterase [AChE, the target site for organophosphate (OP) and carbamate insecticides] sensitivity to inhibition by OP and carbamate insecticides have been generated for 5 insecticides that are labeled for psyllid control in Florida citrus along with paraoxon (parathion) which was included as a standard insecticide. The tested insecticides were active forms of chlorpyrifos (chlorpyrifos-oxon), dimethoate (omethoate), malathion (malaoxon), aldicarb (aldicarb sulfoxide) and carbaryl. The toxicity data of a laboratory strain which was established in 2005 and has not been exposed to insecticides were used for comparison. Biochemical assays were conducted on AChE of five psyllid populations collected from one grove each in five counties (Hendry, Indian River, Lake, Polk, and St. Lucie) for determining the baseline AChE sensitivity levels and to compare them with laboratory strain. In general, AChE of all five psyllid populations collected from five Counties showed decreased sensitivity to inhibition to all the tested compounds compared with laboratory strain with few exceptions. The decrease in sensitivity of AChE to inhibition by different insecticides ranged from 1 to 3-fold for Hendry County, 1 to 3-fold for Indian River County, 1 to 3-fold for Lake County, 1 to 2-fold for Polk County and 2 to 4-fold for St. Lucie County. For Hendry County psyllid population, the highest decrease in sensitivity was observed for malaoxon and carbaryl (3-fold). For Indian River County psyllid population, the highest decrease in sensitivity was observed for aldicarb sulfoxide (3-fold) followed by chlorpyrifos-oxon and omethoate (2-fold). For Lake County psyllid population, the highest decrease in sensitivity was observed for carbaryl (3-fold) followed by chlorpyrifos-oxon (2-fold). For Polk County psyllid population, the highest decrease in sensitivity was observed for chlorpyrifos-oxon (2-fold). For St. Lucie County psyllid population, the highest decrease in sensitivity was observed for malaoxon and omethoate (4-fold) followed by chlorpyrifos-oxon (3-fold), paraoxon (3-fold), aldicarb sulfoxide (2-fold) and carbaryl (2-fold). Psyllid population from Polk County is still highly sensitive to most of the OP and carbamate insecticides tested except for chlorpyrifos-oxon when compared to laboratory strain. These data corroborates with insecticide susceptibility data obtained from bioassays and will serve as a baseline for monitoring resistance development at the target site (AChE) for OP and carbamate insecticide in ACP field populations. Further work to screen psyllid populations collected from 4-5 different locations in Florida is under way for determining the baseline sensitivity of AChE and monitoring resistance levels at the target site (AChE). Selection of one field population each for imidacloprid and chlorpyrifos resistance is in progress. Thus far, selection of 7 generations for imidacloprid and 3 generations for chlorpyrifos has been completed. Further selection of future generations and determining the mechanisms of resistance by toxicological and biochemical studies are in progress. These colonies will be used for further studies on determining the resistance and cross-resistance development potential in psyllids and mechanisms of resistance.
As of our last report on October 15, 2009, work has continued on the objectives of this proposal as outlined. However, during the winter months, psyllid populations decline to low levels in the field and also are unreliable for completion of under greenhouse conditions. Thus no new significant results on this project have been produced since the last report. Based on the results of our previous studies, multiple field trials are planned for spring of 2010 to apply the results of greenhouse studies to typical commercial citrus growing conditions. Effects of host plant nutrition on psyllid fitness: In previous experiments we demonstrated that the two most important essentials, Nitrogen and Potassium, have a significant effect on psyllid fitness. Varying levels of high and low Nitrogen and Potassium rates were applied to ‘Valencia’ orange plants grown under greenhouse conditions. Results of these experiments demonstrated that high nitrogen levels resulted in an increase in psyllid weight, an increased level of egg production and a shorter developmental time. Thus, higher nitrogen fertilization above what is needed by citrus plants may lead to an unnecessary increase in psyllid populations due to excessive use of nitrogen fertilizers. Conversely, addition of increased levels of Potassium fertilizer resulted in decreased fitness of psyllid populations. In field trials scheduled for spring of 2010, we will test varying combinations of Nitrogen and Potassium fertilizers under conventional growing conditions to determine the optimal rates of fertilizers to maintain tree health while minimizing psyllid reproduction rates. We will also be comparing these fertilization regimes to those being used by growers as part of an HLB/nutritional management approach to disease management. In our previous work we have also demonstrated that boron (0.25% Borax) applications have a significant negative effect on psyllid populations in the greenhouse as a result of increased adult mortality. Thus, we will also be examining the effects of 0.25% Borax applications on psyllid populations alone and in combination with varying levels of Nitrogen and Potassium fertilizers during filed trials in spring 2010. Effect of host plant species on psyllid fitness: Of the various citrus species tested thus far for effects on psyllid fitness, we have previously reported that Cleopatra mandarin is the most promising prospect for true resistance to Asian citrus psyllid of all the commercially available citrus varieties used today in citrus production. To convince ourselves that our results are real and not due to some unforeseen experimental error, we have conducted multiple trials using new Cleo plants to prove without a doubt that these results are indeed real…its real! Thus, during 2010, two trials are planned to further examine the resistance of Cleo to ACP. First, a series of 40+ genetically distinct Cleo hybrids planted at the UF/CREC will be challenged by caging psyllids on flushing terminals to determine whether any differences in susceptibility to ACP exists. If differences are found, then further investigations will be continued by the plant improvement team to determine whether these traits can be bred into currently available citrus varieties. In our second series of experiments conducted in the CREC teaching/demo grove with varying combinations of scion and rootstock combinations, we will investigate whether scion grown on Cleo has any advantage with regards to psyllid tolerance when compared to the same scion material grown on different rootstocks.
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. We carried out the first PCR of the plants in this experiment and the results were negative, no plants have been detected the presence of the bacterium L. Ca asiaticus. 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. In this experiment, the time to reach 100% mortality ranged from 3 to 7 days for both systemic insecticides tested (imidacloprid and thiamethoxam). The insecticides were effective up to 90 days after application. The results of PCR carried out for the ACP, in some periods, were positive for 100% of the samples, consisting of 10 insects tested, but in the confinement held at 46 days after application, in any sample was detected the presence of the bacteria. No acquisition in this period. In bioassays performed at 75 and 90 days after application, the percentage of positive samples was 50 to 70% and 10 to 40%, respectively. We started the experiment 2, the difference from the experiment 1 is the application of varying doses of the systemic insecticides and confinement of the ACP in plants treated only 7 days after application. To thiamethoxam (Actara 250 WG), the doses tested were: 1, 0.5, 0.1 and 0.05 g/nursery tree and imidacloprid (Provado 200 SC) were: 1.75, 0.9, 0.2 and 0.08 mL/nursery tree. 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.
The relationship between the Asian citrus psyllid (ACP) and Candidatus Liberibacter asiaticus (Las) continues to be studied to optimize disease management strategies. In laboratory studies of Las acquisition by ACP, nymphs reared on infected plants were more likely to acquire the pathogen than adults. Acquisition by nymphs ranged from 60-100%, whereas acquisition by adults only reached 40% after 5 weeks of feeding on infected plants. Transmission of Las from parent to offspring (transovarial) occurred at a rate of 2-6% in ACP eggs, nymphs, and adults. Experiments to determine the rate of Las transmission to healthy citrus by ACP were assessed one year after inoculations. Transmission of Las by individual ACP ranged from 4-10%, depending on amount of time Las(+) ACP spent feeding on plants, while groups of 100 or more ACP transmitted the pathogen at a rate of approximately 88%. The proportion of Las(+) adult ACP decreased over time when held on healthy plants. Due to the low rate of acquisition and long time period required for successful transmission by adult ACP, experiments to determine the latent period require for Las replication and transmission by ACP have not produced any Las(+) plants after more than one year of incubation following inoculation. Collectively, these results indicate that healthy adult ACP are poor potential vectors of Las but may, after continuous access to Las followed by oviposition on infected plants, give rise to highly infective nymphs. In subsequent experiments designed to evaluate the fitness of Las(+) ACP reared on infected citrus compared with healthy ACP reared on healthy citrus, Las(+) ACP produced significantly more eggs although adult longevity was reduced. Ongoing studies have thus far indicated no differences in the fertility of infected versus healthy ACP. These results underscore the importance of controlling ACP nymphs to reduce secondary disease spread within a grove. Additionally, oviposition on infective citrus plants, rather than adult acquisition feeding, appears the most important component of the Las transmission cycle. Preventing oviposition on Las-positive plants may provide significant disease control if included as part of an integrated pest management (IPM) strategy. Investigations continue into the seasonality of Las(+) ACP. During 2009, monthly sampling at 5 study sites across the state showed that % Las(+) ACP fluctuated throughout the year, with a season-long average of <1-5% LAS(+) ACP at our central Florida study sites. At our Homestead site where 100% of the trees are Las(+), fluctuations in %Las(+) psyllids were present but the overall % infection rate was much greater (64%). Comparing the results from years 1 and 2 of this study, the months with greatest % Las (+) ACP were not consistent between years. However, we hypothesize that plant flushing patterns, as influenced by climatic conditions, may be a key factor influencing shifts upward in %Las(+) ACP. In 2010, this study will focus specifically on plant-related factors.The effects of insecticides on the feeding behaviors of Las(+) ACP were examined using an electrical penetration graph (EPG) monitor with waveforms produced characterized based on the studies of Bonani et al (2009) and Youn et al (unpublished). For ACP feeding on plants with imidacloprid levels ranging from 7.8-20.7ppm, there was significant reduction in non-probing behaviors (np), stylet penetration (C), phloem salivation (E1), phloem ingestion (E2) and xylem ingestion (G) thus suggesting that pathogen transmission should be significantly reduced on imidacloprid treated plants. However, for aldicarb treated plants, the only statistically significant difference found was an increase in (E2) on aldicarb treated plants. These results suggest that aldicarb applications will not prevent pathogen transmission prior to causing mortality of adult ACP. Plants used in EPG studies are currently being held for subsequent Las analysis to compare actual rate of pathogen transmission between treatments. We are currently determining the level (PPM) of imidacloprid that is required in a plant (degradation studies) to provide effective disruption of psyllid feeding behaviors responsible for pathogen transmission.
1: Localization of Liberibacter asiaticus (Las) in internal organs of Asian citrus psyllid (ACP). In order to study the cellular interactions of Las in its psyllid vector, the following four methodologies have been used for localization of this bacterium in dissected organs and hemolymph smears of ACP and in leaf sections and extracts of HLB-diseased plants: A. Immunofluorescence confocal laser scanning microscopy. Two polyclonal and several monoclonal antibodies prepared against Las membrane proteins have been tested at various dilutions (1/20 to 1/400) and incubation times (3-27 hrs). Some of the monoclonal antibodies produced positive fluorescent labeling in the phloem area in sections of HLB- infected citrus leaves and in hemolymph smears of psyllids collected from HLB-infected trees, but not in whole-mount insect organs. These results are encouraging but appear to indicate that such antibodies may be slow to penetrate whole insect organs. Efforts are now underway to resolve this issue with various permeabilization procedures or by immunolabeling of paraffin sections rather than whole organs. B. Fluorescence In situ hybridization (FISH). The oligonucleotide primer tested so far is based on the following sequences (20 bases) of Las, tagged with Alexa Fluor 488: TCGAGCGCGTATGCAATACG. Several FISH protocols have been tested on dissected organs of ACP and on leaf sections and extracts from HLB-diseased plants. Green fluorescence, indicating Las, was detected in the filter chamber and midgut of field-collected ACP, but not in healthy controls. It was also detected in leaf sections and plant extracts from HLB-diseased plants but not in those from healthy plants. We are continuing to refine the FISH procedure and to test two other recently-designed Las primers . C. Quantitative RT-PCR of dissected insect organs. We tested two different RT-PCR procedures for detection of Las in dissected salivary glands, alimentary canals and other parts of individual ACP adults. In two successive experiments, using DNeasy Kit for extraction of DNA, Las was detected in 17-33% of the salivary glands, 27-47% of the alimentary canals, and in 47-53%of the rest of the body of psyllids collected from HLB-infected trees in Fort Pierce, FL. during November and December 2009. To our knowledge, this is the first direct demonstration of Las (using PCR) in the alimentary canal and salivary glands of ACP. D. Transmission electron microscopy (TEM) We are currently using a combination of TEM and RT-PCR, comparing the ultrastructure, including the bacterial fauna, of psyllid adults that have never been exposed to infected plants with those collected from HLB-infected trees and tested positive for Las with RT-PCR. Fixation and embedding of samples have been done, while thin-sectioning and TEM observations are underway, providing the base for TEM-immunolabeling studies. 2: Clarification of various acquisition and transmission parameters between ACP and Las. [A] Limited success has thus far been achieved using an artificial diet-in vitro feeding method for Las detection in live ACP. Further improvements in this technique are continuing. [B] Among adults field collected from Las-infected trees, averages of 37% females and 38% males have tested positive by PCR. No differences in infectivity among the three color morphs of adult ACP have been noted. [C] To assess the incubation period of Las in young citrus with respect to testing PCR-positive, groups of field-collected adult ACP were caged on individual citrus plants for 5 days and then assayed individually for Las. Plants that had been infested by Las-infected ACP were then PCR assayed weekly. Among trees infested by at least one infected ACP, 10%, 25%, and 50% tested positive within 4, 6 and 31 weeks, respectively. No relationship was found between the number of infected ACP per plant and the incubation period of Las. [D] There was no evidence of sexual transmission of Las between males and females of ACP. [E] In an acquisition study, greater percentages of females acquired Las than males.
USDA test 1. A new block of young, HLB’free citrus (Valencia on Carrizo) was planted on May 1, 2008 in a grove with substantial levels of HLB. Three psyllid control treatments (programs) are being compared in this planting: 1) a monoculture of citrus receiving monthly insecticide applications; 2) citrus interplanted with orange jasmine with a relaxed insecticide program for the citrus and orange jasmine not treated with insecticides; and 3) citrus interplanted with orange jasmine with a relaxed insecticide program for the citrus and regular applications of imidacloprid to orange jasmine. For plots with jasmine, a jasmine plant was planted between each citrus tree along some rows in each plot. Treatment 2 is being studied because psyllids may be strongly attracted to jasmine and killed, reducing numbers of psyllids that go to citrus. Treatment 3 is being studied because psyllids may be strongly attracted to jasmine thus reducing numbers of psyllids that go to citrus, and population levels natural enemies of the psyllid may be enhanced by having jasmine in the vicinity of citrus. None of the trees tested HLB positive just before planting in May 2008. By one year later, a mean of 0.9, 0.6, and 0.6% of the trees had been identified as infected under treatments 1, 2 and 3, respectively. During August 2009, a mean of 9.8, 3.9, and 4.2% of the trees had been identified as infected under treatments 1, 2 and 3, respectively. The rapid increase in incidence of HLB in the plots between May and August 2009 was attributed to increases in psyllid infestations during June and July, particularly in one Treatment 1 plot that was adjacent to older citrus. During 2009, a total of $148, $268, and $118 per acre worth of insecticides (materials only) was applied to treatments 1, 2 and 3. The large cost of treatment 2 was due to two applications of Bayer Feed and Protect to jasmine. USDA test 2. A new block of young, HLB-free citrus (Valencia on Carrizo) was planted during December 2008. Three treatments (psyllid control programs) are being compared in this planting: 1) citrus receiving monthly insecticide applications; 2) citrus under a relaxed insecticide program; and 3) citrus treated once every three weeks with spray oil. There are three replications of each treatment. None of the trees tested positive prior to planting. All of the trees were treated just after planting with imidacloprid. A hard freeze on Jan 22 damaged almost every tree, and a number of the trees died. These were replanted during February. All trees were treated with imidacloprid during February, April and May giving time for the trees to recover. The experiment was to officially began in June, but five trees tested positive in June. We removed these and replaced them with healthy trees during early August and resampled every tree, confirming that no tree tested positive. As of today, we have no results to present for this planting. UF test. An experiment was initiated to evaluate the ability of systemic insecticides to protect a new planting of citrus from psyllids and consequently HLB infection. We planted 112 Hamlins in two rows at 15 ft spacing on 03 Mar 2009. Half of each row was considered a replicate and divided into two main plots, treated and untreated. Treated plots were split into two subplots on 13 March, one receiving a liquid formulation of imidacloprid (Nuprid 2f @ 32 oz/acre ‘ 0.5 lbs a.i./acre) and the other receiving a solid formulation of the same (Suscon 13 @ 10 lbs/acre ‘ 0.5 lbs a.i./acre). On 11-Sep 2009, leaf samples were obtained from all 112 Hamlin trees and were sent to the HLB laboratory at the IFAS ‘ SWFREC facility in Immokalee Florida. Results from this testing showed no significant treatment effects (Chi square = 3.61, P = 0.182). At that time, 10 of the 56 Control or untreated trees tested positive for HLB whereas 10 of the 28 trees treated with the granular slow release imidacloprid were positive and 6 of the 28 trees treated with the liquid formulation of imidacloprid were positive for HLB.
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. Trap sampling and tap sampling indicated that adults were aggregated among trees, within individual strata and among strata across the block of trees studied. 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. Validation sampling indicated that, for commercially-acceptable precision levels at means of 1 or more adult psyllids per sample, 30 stem-tap samples or 28 sticky trap samples are required in a block of trees up to ten acres in size. Larger numbers of samples were required at lower densities. Biological Control – (a) Release of three new biotypes of Tamarixia from Asia were initiated in Florida. It is too early to gauge establishment. (b) The insect pathogen Hirsutella citriformis may sometimes provide significant levels of adult psyllid mortality. Adult psyllid cadavers mycosed by this fungus were most common in a group of mature trees during the fall and winter from October through March. Near absence of cadavers mycosed by the fungus during May ‘ June may be due to environmental conditions unfavorable to the fungus. In collaboration with the University of Florida, a method of culturing the fungus on media was developed. We are in the process of verifying pathogenicity of the culture. A study is planned to evaluate a number of citrus chemicals for toxicity to the fungus. (c) 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 continued on the biology of the parasitoid. Seasonal HLB profile in adult psyllids ‘ In this study, psyllids are collected fortnightly from diseased trees in a grove. Over all sample dates, an average of 91.7 (SEM 5.9) % of the trees tested HLB positive (mean CT 23.9, SEM 0.3) in the sample area. From Feb 2008 through Nov 2009, a mean of 33% (SEM 1.8) psyllids tested positive, with a minimum of 3% on one sample date and a maximum of 85% on another sample date. An average CT value of 25.2 (SEM 0.2) was recorded for infected psyllids over the entire study period. No definite trends in seasonality of HLB in adult psyllids have yet been identified over the course of this nearly two-year study with respect to percentages of psyllids infected or titers of the bacterium based on RT-PCR CT values. Beginning June 2008, once a month psyllids from the infected trees were caged individually on citrus seedlings for 7 days. After the 7 day period, the psyllids were tested for HLB and seedlings infested by an infected psyllid were assayed 6 months later for the pathogen. Over all study dates, an average of 57% psyllids tested HLB positive; an average of 25 (SEM 9.7) % infected psyllids transmitted the disease, with means per sample date as low as 0% and as high as 75%. It is too early to determine if there are any seasonal trends in transmission rates. The study was expanded during fall 2009 to include identifying the sex and body color of psyllids in the individual transmission experiments.
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