This research project is directed towards controlling psyllids using RNAi technologies, as well as, protease inhibitors and peptide hormones that, if expressed in transgenic citrus, would kill adult and nymph psyllids. We tested this approach by feeding TMOF, a mosquito decapeptide hormone that stops trypsin biosynthesis in adults and larval gut, and a citrus weevil cysteine protease inhibitor (CPI). Each was added separately to an artificial diet on which psyllids were allowed to feed through a membrane. After 10 days of feeding TMOF (10 µg/µL) and CPI (3 µg/µL), all the psyllids that fed on the diet died (100% mortality). In control diets without TMOF or CPI only 40% died. The earliest significant effect on psyllids survival was observed at day 4 after feeding TMOF. The mortality of Psyllids that fed TMOF for 4 days was 15% whereas the mortality of the control group was less than 5%. Psyllids that were fed CPI did not show a significant higher mortality than controls until day 7. However, CPI concentration in the diet is 3.3-fold lower than the concentration that was used for TMOF. Because CPI is produced by genetic engineering of E. coli, its availability is limited, whereas TMOF was commercially synthesized. To continue these studies we are now fermenting large amounts of E. coli in order to purify high amounts of CPI to study the effect of different doses and optimal concentrations of this inhibitor on psyllids. We are cognizant to the possibility that both proteins when fed together may show synergism and will be looking for such phenomenon that was recently discovered in our lab when TMOF and cry toxins were fed together to mosquito larvae. The ultimate purpose of this project is to develop transgenic citrus plants expressing these proteins. Our results show that this will be feasible after dose response curves will be developed to calculate the minimal amounts of the recombinant proteins that are needed to be circulated in the citrus phloem to effectively control adult and nymph psyllids. Currently we are synthesizing double-stranded(ds) RNA targeting 11 different psyllid genes encoding three different classes of essential proteins that control cell division, digestion, water and ion balance in psyllids cells. The DNA that encodes fragments of each of the 11 genes was synthesized and cloned into appropriate vectors. Primers for the dsRNA synthesis using the RNA replicator kit (Finnzyme) were synthesized and used to produce dsRNA. After optimizing the procedure, dsRNA of individual genes will be fed to the psyllids using artificial diet and the effect of the dsRNAs on psyllid-mortality will be monitored for 10 days. At different times during the feeding, psyllids will assayed by qPCR or Northern blot analyses for RNA transcripts that are being targeted by the dsRNAs. To enhance psyllids survival on the artificial diet, we optimized the diet composition by incorporating antimicrobial agent to reduce fungal growth in the diet, and identified suitable buffers to support optimal diet and pH balance during the feeding period. In summary we: 1. Prepared 11 DNA clones that will be converted to dsRNA for feeding experiments 2. Optimized the feeding diets with antimicrobial and fungal agent and balanced the pH. 3. Showed that feeding TMOF or CPI for 10 days causes 100% mortality to psyllids.
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. We believe that by controlling the psyllid vector this will aid other efforts to control HLB/citrus greening. We don’t know which RNA sequences will prove to be the best for our effort, and we are using both directional cloning of specific sequences and a random cDNA library 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 and/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) in our studies. B. cockerelli readily feeds on and colonizes most tomato cultivars tested by us so far, and on potatoes. It also appears to transmit C. L. psyllaurous to these plants based on our PCR-based detection analyses. We also showed that pJL36 Tobacco mosaic virus-based expression vector can be used to deliver specific RNAs into the plant phloem, and that psyllids can acquire some of these RNAs by feeding on corresponding TMV-infected tomato plants. Moreover, by using recombinant TMV we can induce production of specific siRNAs corresponding to the recombinant sequence in tomatoes. Thus, we will use this approach to induce production of siRNAs corresponding to psyllid genes in plants and to evaluate candidate sequences for RNAi activity against the tomato psyllid. 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 (D.citri), but using B. cockerelli RNA as the template. We cloned these sequences into pGEM-T easy, and the dsRNAs for these sequences were synthesized. We are attempting to use microinjection to deliver these dsRNAs into psyllid nymphs to assess their abilities to induce RNAi effects. Initial efforts showed that the survival of psyllid nymphs after injection is low but acceptable. Further experiments are needed to improve injection and increase psyllid survival. Also, we are attempting to orally deliver dsRNAs to psyllids. We also have cloned eight gene sequences into pJL36 and have infiltrated tomato plants using Agrobacterium tumefaciens. We will place B. cockerelli psyllids on these plants and to determine we can induce specific RNAi effects in recipient psyllids. We have also identified 1904 contig sequences from a D. citri EST database and identified 179 midgut sequences among these. Primers for 80 midgut contigs, which are immediate genes of interest for us, were synthesized and are being used to clone homologous sequences from B. cockerelli. Our hypothesis is that the midgut genes will be important to be targeted by orally-acquired siRNA or dsRNA. We have also taken efforts for an unbiased, high-throughput approach to evaluate and identify additional B. cockerelli target sequences. The normalized cDNA library construction is underway by Bio S&T inc. In this library, the random sequences from B. cockerelli will be directly cloned ino the pJL36 plasmid, and transformed into Agrobacterium tumefaciens GV3101. This will allow us to test them directly by tomato inoculation and psyllid feeding experiments.
This is a cooperative research project between Co-PIs Joseph Morse, Jim Bethke, Frank Byrne, Beth Grafton-Cardwell, and Kris Godfrey. One objective is to coordinate with researchers working on chemical control of ACP in Florida, Texas, Arizona, and elsewhere. Towards that end Grafton-Cardwell and Morse met with Michael Rogers (UF) in San Diego in July to discuss current research and Godfrey and Grafton-Cardwell participated in the Third Citrus Health Research Forum in Denver recently. We are rearing ACP in a contained greenhouse at the Chula Vista Insectary (San Diego County; about 6 miles north of the Mexican border) under permit (#2847) from CDFA. This permit clearly notes experimental protocols and procedures so that the work is done as safely as possible to minimize the chance of ACP escape. At this site, Jim Bethke has initiated ACP tests on various organic and traditional pesticides of interest to California growers. A second location where we are working with ACP is at UC Riverside, working under permit inside the UCR Insectary Facility. Frank Byrne is conducting trials on various neonicotinoid insecticides, Morse is evaluating the baseline susceptibility of CA ACP to various pesticides in comparison to studies done in Florida, and Morse is collaborating with Mark Hoddle in evaluating the impact of registered organic pesticides on both ACP and Tamarixia. Kris Godfrey recently obtained a permit to rear ACP inside UC Davis’ Contained Research Facility and she is presently growing host plant material in order to start a colony this month or next. In collaboration with Jim Bethke, she will focus her efforts on evaluating experimental organic pesticides and microbials. In summary, we continue to expand our ability to conduct ACP research in California. To date, we have been mostly dependent on research done elsewhere but we are beginning to build the infrastructure and capability for conducting trials of specific interest to California here.
Transmission of Candidatus Liberibacter asiaticus by citrus psyllids requires acquisition of the bacteria by the psyllid, passage through the psyllids body, and release of the bacteria in feeding. The causal agent is a fastidious prokaryote that is extremely difficult to grow in quantity in pure culture. We have been attempting to augment a medium acceptable to psyllid feeding on a membrane which contains living bacterial cells. We have been able to demonstrate release of the bacteria into the membrane by feeding psyllids but have not been routinely successful in psyllid acquisition of the cultured bacteria from the membrane sachet. This work is continuing while we are developing a good micro-injection method for introducing the bacterial culture into the psyllid hemolymph. Upon successful establishment of infective psyllids, the plant inoculation process will proceed immediately to obtain biological proof of PCR results.
We have made good research progress this quarter. Need for trap in regulatory efforts to reduce spread of greening bacteria – 2009 samples. Of all samples from citrus or kumquat about 19% were positive for citrus greening with 21% of discount garden centers and 16 % from miscellaneous retailers. If both nymphs and adults were collected, about 2/3 of the time, nymphs were positive, suggesting infected plants. Field testing of traps: We conducted two field tests of about 25 different trap configurations as prototypes in a replicated experiment in two field locations in southern Florida: Immokalee and Fort Pierce. Results from these two tests helped us to focus further experiments on prototypes with configurations that trap adult psyllids with some degree of efficacy. These results also enabled us to better focus some laboratory experiments to gain a better understanding of psyllid behavior. 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 relative to their impact on psyllid walking, jumping and flight behavior. These experiments will enable us to optimize trap configurations and to develop an estimate of the importance and the relative trade-offs in efficacy among trap components. Once we have a better understanding of how psyllids respond to individual trap physical properties, we can change the configuration of various components in a focused effort to improve overall capture efficacy. 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.
As of September, 2009, we have over 16 actively growing Asian citrus psyllid derived insect cell lines, These cell lines remain heterogeneous in composition and one of our current goals is to adapt and select for more homogenous cell populations. We now routinely grow our cells in media without any antibiotics, a needed step in order to use these cells to attempt to culture the citrus greening agent (see below). In the broadest sense, we have two distinct cell culture types, (a) suspension cells, and (b) cells that attach and spread along the tissue culture flask substrata. These two cell types have different potential uses, e.g. the attached cells may be better suited for culturing of bacteria, whereas the cells in suspension may facilitate isolation of psyllid specific viruses. Images of the cells show elongated diverse cell types in the attached cell cultures, including epithelial-like cells attached to the tissue flask surface and growing in a monolayer from clumps of globular floating cells to round cells growing in small patches. The suspension cell cultures appear more uniform as small and large clumps of round shaped cells. Growth and maintenance of the cell cultures has become routine. We have established collaborations with Drs. Wang and Davis at the Lake Alfred Citrus Research Center in order to attempt to cultivate Liberibacter asiaticus with the cell lines.
USDA test 1. A new block of young, HLB’free citrus (Valencia on Carrizo) was planted on May 1, 2008. 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. None of the trees tested positive during August 2008. During November, a single tree tested positive. This tree, which was removed, was located in one of the Treatment 2 plots. During February 2009, a second tree in the same treatment’2 plot tested positive and was removed. During May 2009, 3 trees tested positive in one of the treatment-3 plots, and 2 trees tested positive in one of the treatment-1 plots. A mean of 0.9, 0.6, and 0.6% of the trees became infected within a year in plots under treatments 1, 2 and 3, respectively. From May 2008 to May 2009, a total of $163, $259, and $93 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. Coincidently, the trees are being grown under an open hydroponic planting system. 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. As of today, we have no results to present for this planting. UF. An experiment is being conducted to evaluate the ability of systemic insecticides to protect a new planting of citrus from psyllids and consequently HLB infection. We planted 160 Hamlins in two rows at 6 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). Evaluations for psyllids were conducted on 3/25, 4/3, 4/30, 6/5, 6/29 and 8/3. No psyllid infestations have been observed. The trees have grown slowly, thus we upped our fertilization program to get the trees more established. Problems have diminished with deer feeding on flush. We have not yet tested any trees for HLB.
The purpose of this investigation has been to develop, evaluate, and optimize biorational management tools for Asian citrus psyllid (ACP) including insect growth regulators (IGRs) and antifeedants. In the second series of laboratory studies with insect growth regulators, we investigated the activity of both Buprofezin (Applaud) and Diflubenzuron (Micromite) on ACP eggs, nymphs and adults to evaluate their potential usefulness as biorational insecticides for inclusion into an integrated pest management (IPM) strategy for ACP control. Both chemicals exhibited strong ovicidal and larvicidal activity against ACP eggs and nymphs, respectively, in age- and concentration-dependent manners. Fewer eggs hatched into nymphs at the higher concentrations tested (80-160 µg mL-1). A significantly lower percentage of early instar nymphs (first, second and third) survived and emerged into adults at the higher concentrations tested (80-160 µg mL-1) compared with late instar nymphs (fourth and fifth). Furthermore, both chemicals exhibited transovarial activity by significantly reducing the fecundity of females and viability of eggs deposited by females that emerged from treated fifth instar nymphs. Topical application of each chemical to adults also significantly reduced fecundity and egg viability. Application of each chemical at 160 µg mL-1 resulted in the highest inhibition of egg hatch in younger eggs (0-48 h old) laid before or after treatment and strongest suppression of adult emergence from early instar nymphs compared with other rates tested. Both Buprofezin and Diflubenzuron 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 Buprofezin and Diflubenzuron against immature and adult ACP, respectively, suggest that integration of these insecticides as part of an IPM strategy should negatively impact ACP populations over time. Current studies are underway to determine the effects of field aged residues. Also, further field scale testing is underway to determine how to best incorporate each chemical into an integrated management program for ACP. Depending on dosage, both of these insect growth regulators reduced or eliminated psyllid egg hatch and reduced or eliminated nymph to adult survival (the effect was greater when younger nymphs were treated). Furthermore, adult female psyllids exposed to both of these IGRs produced fewer eggs and viability of eggs from treated females was reduced. In a separate investigation, we have studied a psyllid antifeedant, named Pymetrozine. If psyllid feeding can be prevented or greatly reduced, perhaps HLB transmission can be reduced or prevented. Pymetrozine is a chemical that is known to paralyze the muscles involved in plant probing in plant-sap sucking insects such as aphids. Pymetrozine is also known to prevent transmission of aphid and whitefly transmitted viruses. In our initial investigations, we found that at a 100 ppm dosage applied to citrus plants, Pymetrozine inhibits acquisition of Liberibacter by psyllids on Valencia, Clepatra Mandarin, and Persian lime by approximately 50%. However, acquisition is not completed prevented and occurs in approximately 20% of psyllids. Our ongoing research is focussing on optimizing the dosage of Pymetrozine to see if we can further reduce acquisition of Liberibacter by psyllids. Also, our initial investigation has shown that only 25% of psyllid nymphs feeding on Pymetrozine-treated plants survive. This is because the majority of psyllids feeding on Pymetrozine-treated plants are starved and do not develop into adults. Thus this chemical both reduces acquisition of the greening pathogen and kills psyllid nymphs by starvation. Our next steps in addition to optimizing dosage are to determine if pymetrozine affects transmission of HLB and to investigate the effects of this chemical applied to citrus in the field on both psyllid mortality and transmission of HLB.
The goal of these 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. 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. In previous studies, we found that .-Butyrolactone serves as an attractant for Tamarixia radiata wasps in laboratory experiments. We have conducted initial field trapping tests, which have been inconsistent. In the early summer, traps baited with .-Butyrolactone appeared to catch more Tamarixia radiata than unbaited traps, but this trend did not hold up in the late summer months. Current research is aimed at determining whether more consistent captures of Tamarixia radiata can be achieved by optimizing the dosage of the attractant. Also, we are investigating whether these inconsistent captures are due to the seasonal phenology of the wasps in terms of population densities, natural behavior, and commercial citrus management practices. We have also investigated a second chemical, Methyl Salicylate (MeSA) and associated dispenser from a second collaborator (AgBio, Corporation). This chemical is known to recruit beneficial insects and improve biological control. We have conducted our initial field testing of MeSA to determine whether we can improve biological control of ACP. There is abundant research showing that Methyl Salicilate (MeSA) is an effective chemical for simultaneously repelling pests and attracting natural enemies. Deployment of MeSA from controlled release devices has been shown to keep pests including aphids, thrips, and mites below economically damaging levels. In addition, this chemical has been shown to attract predatory mites, predatory bugs, coccinellids, lacewings, parasitic wasps and flies, and predatory flies. This chemical is readily synthesized at low cost. The company AgBio, the US sales rep of Chem-Tica, (who produces and distributes MeSA dispensers to researchers) was contacted and we received MeSA dispensers from them. In our initial investigation, we confirmed that deployment of MeSA reduces ACP populations and increases populations of psyllid natural enemies in small scale, non-commercial plots. Currently, we are identifying and cataloging the types of natural enemies that were recruited by deployment of MeSA as many more types of natural enemies were recruited by this chemical in addition to Tamarixia radiata. Our future goal is to optimize MeSA dispensers for larger scale use and to determine if similar results can be achieved in commercially managed groves. We plan on continuing work with AgBio to determine if MeSA dispensers will improve biological control of the psyllid. If large scale field trials prove effective, then this company will be in position to commercialize the product for use in Florida citrus.
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. Most recently, we have moved forward with developing an attract-and-kill formulation for ACP with our industry partner and Co-PI Darek Czokajlo. In the past decade, a gel matrix with UV-protective properties was developed as an attracticide for lepidopteran pests. This formulation was registered in Switzerland under the trade name Sirene and subsequently as LastCall in the U.S.A., Europe, and South Africa. The original target pest of this formulation was the codling moth in apples; however, LastCall has been adopted for multiple other pests since then. One of the main goals of this proposal is to develop effective attract-and-kill for ACP by adopting the controlled release gel, termed MalEx, which is the currently licensed name for a formulation similar to LastCall. There is a large precedent for effective attracticides targeting insect pests of agricultural crops, which combine the use of a very low dose of both synthetic attractant (plant volatile or sex pheromone) and insecticide. Such formulations are typically applied as small droplets, which release pheromone at a rate highly attractive to males. Responsive males are thought to follow the plumes from attracticide droplets and obtain a lethal dose of toxicant upon final contact with the source of attractant. Thus, the MaleEx formulation is well suited for this purpose. In our initial research, the MaleEx formulation was loaded with permethrin as the insecticide. However, we learned that it would be difficult to register the formulation for use in Florida citrus with this active ingredient. Thus, our recent investigations have focussed on developing a formulation that is laced with the neonicotinoid insecticide imidacloprid instead of permethrin. We compared formulations containing 6, 14, and 22% imidalcloprid against Asian citrus psyllids in the laboratory. We found the the 14% imidacloprid formulation is superior to the 6% formulation, but that there was no added benefit of the 22% formulation. Thus, we have optimized the dosage of toxicant in the MalEx attract-and-kill formulation for the psyllid using imidacloprid as the insecticide. In seperate trials working on a different attract-and-kill formulation consisting of an emulsified wax formulation (SPLAT, ISCA Technologies), we compared the insecticides Spinosad, Methoxyfenozide and Tebufenozide against the psyllid. We found that Methoxyfenozide and Tebufenozide are not effective with this formulation and that Spinosad is only marginally effective resulting in about 50% mortality. Our ongoing studies are focusing on testing more insecticides with the wax formulation so that more effective psyllid kill can be achieved. We have continued our work on developing a psyllid attractant in collaboration with both USDA colleagues and our industry partner, Alpha Scents. Most recently, we have conducted four field trapping experiments of synthetic citrus plant volatiles that have been shown to attract psyllids in the laboratory. Our tests to date have been inconsistent. In some cases, the proprietary blend of plant volatiles increase captures of psyllids by 2-3 fold, but in other cases there is no apparent increase in psyllid capture as compared with blank control traps. We are continuing our work on optimizing blends and dosages of currently identified chemicals. We also continue our efforts at identifying further, perhaps more potent attractant chemicals. We are also continuing our research on optimizing trap placement for best psyllid monitoring within groves. This also includes further research on optimizing trap color and design.
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.
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