The goal of this project is to identify insecticidal peptides with efficacy against phloem-feeding citrus pests. Our first objective is to evaluate a range of known insecticidal peptides against the Asian Citrus Psyllid (ACP) in artifical feeding assays. We have procured candidate peptides for screening and are optimizing artificial bioassays for evaluation of their effect on ACP mortality. In a second objective, we have proposed to utilize Citrus tristeza virus (CTV) for delivery of insecticidal peptides into citrus plants for management of the ACP, Brown Citrus Aphid (BCA), and Citrus Leafminer (CLM). Development of CTV as a vector for foreign peptides was previously conducted in a separate project by Dawson et al. Using the CTV expression vector, we have elected to examine four peptides known to have insecticidal activity against phloem-feeding agricultural pests. To facilitate bioassays for screening individual insecticidal peptides, we have recently established laboratory colonies of BCA and CLM. BCA were obtained from a colony currently housed at the Citrus Research and Education Center in the laboratory of Dr. Bill Dawson. The CLM colony was established by collecting insects from citrus groves located in Polk County, Florida. In addition, we have procured plants for use in experiments evaluating CTV-expressed insecticidal peptides. Prior to the inception of the current project, one of the peptides proposed for evaluation, snow drop lectin, Galanthus nivalis agglutinin (GNA), was successfully cloned into the CTV-expression vector and inoculated into citrus plants. At the present time we are conducting experiments to evaluate the efficacy of CTV-vectored GNA against ACP and BCA. These bioassays will evaluate the effect of GNA on insect feeding, survival, development, and fecundity. Concurrently, we are conducting bioassays using synthetic GNA in an artificial feeding system to evaluate the effect of GNA concentration on ACP mortality. In addition to GNA, we have proposed to evaluate three CTV-vectored insecticidal peptides against ACP, BCA, and CLM. Indolicidin, which was previously cloned into the CTV vector, has been inoculated into citrus and should be available for screening in bioassays with the target insects in the next few months. Allium sativum leaf agglutinin (ASAL) and Pinellia ternate agglutinin (PTA) peptide genes have been sequenced and are currently being cloned into CTV-vectors. This process should be completed within two months, after which the CTV-peptide constructs will be bark flap inoculated into citrus. Following successful establishment of CTV vectors into initial citrus hosts, stems from plants exhibiting a high level of CTV will be harvested and graft-inoculated onto subsequent ‘Valencia’ sweet orange plants for use in bioassays. This process is expected to be completed in the next four months. To quantify peptide expression in CTV-inoculated plants, we are developing enzyme-linked immunosorbant (ELISA) assays specific to the peptides under investigation. At the present time, an ELISA assay for GNA is being optimized. Identifying the concentration of expressed peptides should facilitate comparisons between the efficacy observed in artificial bioassays with synthetic peptides and in planta assays with CTV-expressed peptides.
This project is focused on evaluating the impact of psyllid control programs on non target insects and mites. We are conducting replicated experiments at the SWFREC and commercial groves to test effects of insecticides, horticultural oils and nutritionals on psyllids and non-targets. Stem tap sampling, vacuum sampling, yellow sticky card and pheromone traps and shoot and trunk examinations are being used to monitor populations of psyllids and secondary pests such as citrus leafminer, citrus rust mites, citrus red mite, snow scale, Florida red scale, Chaff scale, citrus black and whiteflies, as well as their parasitoids and predators. For this report citrus rust mites (CRM) are compared under different psyllid control programs. The experiment in Collier county compares (1) Nutritional (2) Insecticides (3) Insecticides + Nutritionals and (4) Untreated control. Insecticides currently recommended for psyllid control are used when adults exceed 0.2 per tap sample and nutritional products and their application timing follow a program developed by McKinnon Corporation averaging three sprays per year. Psyllids averaged 0.6 adults per tap sample in the untreated control; significantly more than 0-0.07 per tap sample in the other three treatments which did not differ. Averages of 6 and 4 CRM per lens field were observed in the nutritional and untreated treatments compared to < 1 in the treatments using insecticides indicating that these particular insecticides which included spirotetramat (Movento) were providing control of both psyllids and mites. Each of the two experiments in Hendry county, one in a 35 acre block of 'Early Gold' and the other in a 16 acre block of 'Valencia' include 4 treatments: three spray programs for psyllid, one receiving monthly calendar sprays, two intended to maintain ACP populations below thresholds of 0.2 or 0.7 psyllids per tap sample and an untreated check. Calendar applications are being applied approximately every 1 month and consist of a rotation of insecticides recommended for managing this pest. Sprays in both experiments included carbaryl (Sevin XLR Plus ) at 0.75 gal/ac in April, spinetoram (Delegate WG) at 4.5 oz/ac in May and imidacloprid (Admire Pro) in June at 4.5 oz/acre. No sprays have been applied in threshold treatments because selected population levels have not been reached. No psyllid adults were observed in the tap sampling conducted in the calendar application and 0.01-0.19 per tap sample in the untreated control. CRM averaged 2 per lens field in the untreated 'Early Gold' and increased to 5 per lens field in the calendar application but did not differ between the two treatments in 'Valencia' (average 1.7 per lens field) indicating that calendar application either did not help or increased CRM populations, probably through negative impact on predators particularly phytoseids mites which were significantly lower in the calendar application treatment than in untreated plots. Natural mortality in cohorts of psyllid immatures averaged 82% in the untreated control compared to 69% in calendar application and provided additional evidence of the greater activity of natural enemies in the control. Another trial was conducted at the SWFREC to test some commonly used and experimental insecticides for effects on psyllids and non targets. Delegate, Movento and Danitol all provided significant reduction in psyllid populations compared to the untreated control for more than three weeks. However, CRM populations compared to untreated control were reduced only in Movento and Danitol treatments averaging 2 and 5 per lens field, respectively, compared to an average of 8 per lens field in control and Delegate plots which did not differ.
The goal of this project is to develop potential repellent formulations for Asian citrus psyllid (ACP) that originate from guava (and related) plant volatiles. We have made progress in identifying volatiles that repel ACP in the laboratory, but the hurdle has been how to effectively deliver these volatiles in the field so as to reduce ACP populations. The main obstacles have been: 1) that these repellent chemicals are highly volatile in nature and it is challenging to maintain sufficiently high concentrations in the field so as to affect psyllid population densities, and 2) that these chemicals are quite noxious and foul smelling. The most promising formulation to release DMDS and related psyllid repellent chemicals that we have evaluated to date is the flowable and wax-based SPLAT formulation. This is a proprietary and established release device for insect behavior modifying chemicals and is manufactured and distributed by ISCA Technologies in California. We have seen mixed results with DMDS released from SPLAT. Although some tests in the field verified laboratory tests, showing reduced psyllid population densities, other tests have shown no discernible effect of the treatment. Our release rate analyses with the initial SPLAT formulation indicated that the DMDS active ingredient was released rapidly from this formulation (approximately 80-95% loss within 5-10 days). More advanced slow release formulations were developed. Initial field testing with these formulations showed promising results–psyllid populations were reduced beyond three weeks as compared with control plots. However, a follow-up experiment in the early spring of 2011 under low psyllid population densities again resulted in no effect of the SPLAT-DMDS treatment with the advanced slow-release formulation that was determined as the best one based on 2010 results. It is possible that this test failed because of low psyllid population densities during the timing of the experiment; however, it has become evident that we need to be able to directly quantify DMDS airborne concentration levels in the field in order to directly establish how much DMDS is needed to affect psyllid population densities. Also, this information will allow us to understand how environmental factors influence how much DMDS is present in the air surrounding citrus trees treated with the SPLAT-DMDS formulation. Our objective is to use this DMDS-monitoring technique and the information it provides to figure out why the treatment appears to work during certain applications, but fails during others. Therefore, we recently developed a purge and trap procedure to directly quantify levels of DMDS in the field following application of the SPLAT-DMDS formulation. The purge and trap analytical procedures for the analysis of DMDS in air from the four compass points surrounding a test tree has been optimized. The newer, slow-release DMDS concentration formula was evaluated. Depending on wind direction, initial DMDS concentrations ranged from 0.4 to 1.5 ng/L. Maximum concentrations were observed 3-4 days after application. These maximum concentrations ranged from 1.6 to 2.6 ng/L. Concentrations decayed exponentially during the next 29 days. Final concentrations after 29 days ranged from 0.18 to 0.62 ng/L. We will be correlating how concentration of DMDS relates to behavioral activity on psyllids with the objective of figuring out how much DMDS is needed in the field to affect psyllids and what factors potentially affect concentration changes in the field. We hope that this will help us understand why the emerging technology appears to be effective in some experiments, while showing inconsistent results in others.
The objective of this project is to investigate three questions: 1) whether HLB symptoms or boron/zinc deficiencies alone affect how ACP responds to citrus; 2) whether feeding patterns by adults, length and location of feeding, are altered by HLB infection or boron/zinc deficiencies; and 3) whether different strains of Ca. Liberibacter asiaticus (Las) differentially affect the response of ACP to citrus. In other pathogen/host/vectors systems, such as that with Ca. Phytoplasma mali and Cacopsylla picta (the apple psyllid), the pathogen manipulates the plant host metabolism so that diseased plants become more attractive to the psyllid vector, thereby spreading the pathogen more rapidly than if no plant host manipulation occurred. Since nutrient deficiencies are often associated with HLB in citrus, we wished to confirm that the reported attraction of Diaphorina citri to HLB symptomatic plants over uninfected plants was due to changes in host metabolism by the pathogen rather than physiological changes due to poor nutrition. The production of greenhouse-grown citrus trees with nutrient deficiency symptoms for both zinc and boron continues. Early deficiency symptoms have begun to develop for boron and nutrient analysis shows low borderline deficient boron levels in the leaf. Zinc deficiency has yet to develop. Nutrient analysis show that zinc levels are falling, but not yet low. The high mobility of zinc within the plant allows redistribution of existing zinc to new flush, delaying the development of deficiency and the associated symptoms. Multiplication of our stocks of HLB infected citrus is also underway to provide a constant source of symptomatic tissue for experimentation once strong nutrient deficiency symptoms have developed. As soon as strong deficiency symptoms develop, then psyllid testing for objective 1 and 2 will commence and is expected to move rapidly. We are also interested to determine if strains of Las will have any effect on the attractiveness of trees to D. citri. It has been reported that Las strains have varying levels of virulence and symptomatology (Tsai et al. 2008). We have analyzed DNA samples from HLB positive trees from Polk and Highlands counties as well as the ‘Smoak Grove’ CREC greenhouse strain by PCR and sequencing. Three putative strains of Ca. Liberibacter asiaticus (Las) were found with 5 (CREC greenhouse isolate), 13, and 15 tandem repeats of DNA in the LAPGP locus described by Chen et al. 2010 and have identified sources of budwood. Cloning and sequencing of loci including the b-operon, OMP (outer membrane protein) gene and phage DNA polymerase to support the differentiation of the three strains is complete (Bastianel et al. 2005; Lin et al. 2008; Okuda et al. 2005; Tomimura et al. 2009). Results from sequence analysis clearly defines two strains based on conserved mutations in the b-operon sequence, matching strains from Japan and Vietnam and a strain from Vietnam for the northern and southern Florida strains, respectively.
We have concluded the low volume spray trials testing insecticides in different chemical classes for control of the Asian citrus psyllid. The insecticides evaluated included insect growth regulators, organophophates, pyrethroids, neonicotinoids and one microbial insecticides and were applied at the recommended label rate or an experimental rate with a spray volume of ~5 gal/acre. Ambient weather conditions were recorded during applications and up to 28 days following applications. The relative humidity (68%-95%), temperature (19.5-38.2 deg. C), mean wind speed at application (0.9 -5.1 mph; 0.4-2.3 m/sec), total solar radiation (203.5-250.8 Watts/sq. m), rainfall up to day 3 (0.02″-2.31″; 0.5 mm-58.7 mm) and total rainfall at 28 days (5.0″-11.4″; 127.5 mm-289.6 mm) were measured. From the relative humidity data the delta T statistic was calculated. The value of delta T allows for the estimation of the evaporation time of an insecticide droplet and may have a significant impact for low volume applications where the droplet sizes are much smaller than high volume applications. Ideally, the value of delta T should be between 2-8. Values lower than 2 and between 8-10 indicate marginal spraying conditions. Values higher than 10 indicate conditions unsuitable for spraying. The delta T value at application in these studies ranged from 1.0-1.8. Efficacy of each application was assessed by sampling populations of both adults and nymphs in treated plots compared with untreated control plots on days: 3, 7, 14, 21, and 28 in a commercial citrus grove. Unbaited yellow sticky cards and tap counts onto white cards from multiple branches were used to determine the numbers of adults. Populations of nymphs were estimated by examination of individual flush terminals using a ranking scale. Regression analysis was used to determine if there was a relationship between the meteorological conditions measured during the trials and observed mortality of the psyllid nymphs and adults. The efficacy data for day three was compared to the above canopy wind speed at application. There was a significant positive correlation, indicating that the most effective applications of residual insecticides (organophosphates and pyrethroids) occurred at lower wind speeds but not for insecticides from other classes. No correlation with the value of delta T and efficacy was shown for all insecticides in all classes tested. However, applications were made during conditions within the recommended ranges for delta T. The maximum daily temperature for the first three days was positively correlated with increasing efficacy for the residual insecticides tested but not for other classes. The cumulative solar radiation to day 21 post-application was negatively correlated with efficacy for the residual insecticides. There was no correlation between cumulative rainfall to day three and efficacy for all insecticides tested.
This project is focused on 1) refinement of sampling methods, 2) testing the influence of adult density and shoot infestation on precision of estimated means and distribution of population within blocks, and 3) evaluation of methods for assessing psyllid density, shoot density, and infestation rates and their integration into a user friendly system accessible to consultants and managers. We have made significant progress on all three objectives 1) Comparisons of stem tap sampling, sticky traps and sweep nets in commercial citrus were published in a peer reviewed EDIS document. Data indicated that although all methods detect adult psyllids, the stem tap method is most rapid, works under either dry or wet conditions, and has proven to be reliable and consistent. A little more than 100 tap samples would be necessary to detect with confidence 15 psyllids with 75% precision, a reasonable threshold during the growing season when trees are producing new growth. We recommend 100 samples per block divided into 10 stops, five along the perimeter where psyllids tend to congregate, and five inside the block. This scheme could lead to a decision to spray only the block perimeter if necessary. Ten tap samples should be taken at each stop, one per tree. Then 10 young shoots, each containing ‘feather flush’ should be examined with a hand lens to determine if they are infested with psyllids. The search is terminated if 10 young shoots cannot be found after examining 20 trees. We are now comparing stem tap sampling with vacuum sampling in commercial citrus. The vacuum sampler is made from a leaf blower and collects many psyllids even at low populations. A single strike captured an average of about twice what a single tap sample would catch and the two samples were significantly correlated. 2) A three year study comparing the stem tap sampling and sticky traps under different levels of psyllid infestation in commercial citrus was completed. These and other data collected on comparisons of stem tap and sweep net sampling are being analyzed using regression and bootstrapping procedures. We are using precision levels of 0.25 and 0.01 SEM:mean in order to determine the number of samples required using these methods for routine monitoring and analytical modeling respectively. 3) Methods to monitor psyllids using tap sampling method and flush examination along with data sheets to record data have been posted at our website: swfrec.ifas.ufl.edu/entlab. A pest scouting workshop and citrus field day in collaboration with Hendry county extension were conducted during this quarter and appropriate sampling methods promoted to 125 participants. So far, more than 4000 tap sampling kits have been distributed to clientele through workshops conducted at SWFREC and IFAS extension. This method has been widely adopted by the citrus industry. More than 75% growers in SW Florida value tap sampling in their decision to manage psyllids along with flush examination. The success of the tap sample, is also illustrated by its adoption by APHIS and DPI CHRP to monitor 6,000 blocks of citrus in Florida every 3 weeks. Tap sampling method was also used to evaluate several experimental and recommended insecticides at SWFREC and large scale studies in commercial citrus designed to control psyllids using insecticides, horticultural oils and nutritionals. Arevalo, A. H, J. A. Qureshi and P. A. Stansly. 2011. Sampling Asian citrus psyllid (ACP) in Florida citrus groves. EDIS, http://edis.ifas.ufl.edu/in867.
The results analyzed from the 2010 field season indicate that application of sachets of methyl salicylate (MeSA) in the field plots did not significantly increase population densities of natural enemies of Asian citrus psyllid (ACP) compared with untreated plots in the large scale experiment that was conducted in groves with various levels of ACP management. In addition, psyllid populations were not different between MeSA-treated plots and untreated plots. In all of the grove types tested (unmanaged, minimally managed and intensively managed), psyllid populations were low (< 1.5 psyllids/trap/14 days). These results were different from our initial investigation in 2009, which indicated that natural enemies were higher in MeSA-treated plots. However, in that year, psyllid populations in that grove were much higher than in the groves studied in 2010. In 2009 an average of > 10 psyllid adults were captured per week in control plots whereas ~2 psyllids were captured/week in MeSA-treated plots. Also in 2009 we saw an increase in natural enemies, particularly coccinellid beetles, in MeSA-treated plots as compared with control plots. We hypothesize that the low psyllid numbers overall in 2010 could account for the lack of effect of MeSA treatment. If natural enemies were initially attracted to the plots, the lack of psyllid prey may have caused them to leave the plots in search of resources elsewhere. One recent study in strawberries indicated no increase or limited increase in natural enemies in MeSA-treated plots and no significant decrease in pest populations (Lee, J. C. 2010. Environ. Entomol. 39:653-660). It is also possible that insecticide use affected our findings in 2010. Laboratory bioassays to test the effect of MeSA on the behavior of the Asian citrus psyllid adults were conducted. In a two-choice laboratory olfactometer, adult psyllids were repelled by MeSA at the dosage tested as compared with a clean air control. We are starting studies to see if foliar applications of MeSA on citrus plants reduces the number of psyllids settling on plants compared with untreated plants. In conclusion, MeSA treatment in citrus had mixed results with respect to attraction of natural enemies and reducing psyllid populations in citrus in Florida. MeSA may have an effect in situations where psyllid populations were sufficiently high at the onset of the tests. The repellent effect observed may help reduce psyllid populations and needs further study. If MeSA reduces psyllid establishment on plants and inhibits feeding, this effect may reduce the inoculation of Candidatus liberibacter asiaticus and the incidence of citrus greening disease.
For this project, we have been investigating whether female Asian citrus psyllid (ACP) attract males with a pheromone. Laboratory olfactometer and field studies were conducted to examine the behavioral responses of male and female ACP to their cuticular extracts with a goal of identifying a female-produced sex pheromone. In laboratory experiments, more male ACP were attracted to the odor of 1, 5, or 10 ACP female cuticular extract equivalent units than clean air suggesting that female ACP produce pheromone to attract males. The results were confirmed in field studies in which clear or yellow traps baited with 10 female cuticular extract equivalent units attracted more males than the clear traps baited with male cuticular extract or unbaited traps. Furthermore, clear traps baited with 5 or 10 male or female cuticular extract equivalent units attracted more ACP than unbaited (control) traps. However, catch of psyllids on yellow sticky traps was not increased by baiting them with the psyllid cuticular extract. Collectively these investigations indicate that the extract from female ACP adult cuticle attracted male ACP in laboratory and field bioassays suggesting that female ACP produces a pheromone to attract males. Field bioassays with whole body male and female ACP cuticular extracts suggested that a constituent of the ACP cuticular hydrocarbons serves an attractant. In order to determine the specific chemicals responsible for the behavioral effects observed with psyllid cuticular extracts, chemicals analyses of these extracts were performed to determine whether differences in profiles exist between male and female extracts. Males and females were analyzed separately. There were unique chemicals associated with male and female cuticular hydrocarbon extracts. A greater amount of Dodecanoic acid was specifically identified from the female cuticular extract. In laboratory experiments, synthetic dodecanoic acid attracted significantly more male ACP than clean air. However, field experiments with dodecanoic acid have not yet generated conclusive results for its attractiveness to ACP males. In collaboration with chemists, we have modified our chemical analysis techniques for more precise identification of chemicals of male and female cuticular extracts using 10X traps. Preliminary results with modified techniques yielded more precise differences in chemical profiles between male and female psyllids. More bioassays and field experiments are in progress with newly identified chemicals.
The goal of this project was to determine if infection by Candidatus Liberibacter asiaticus (Las) affects the response of Asian citrus psyllid (ACP) to its citrus host plants. In a two-choice laboratory olfactometer experiment, more ACP were attracted to volatile chambers containing Las-infected citrus plants than chambers containing uninfected citrus plants. In settling experiments, Las-infected plants were more attractive to ACP adults than uninfected plants initially; however, psyllids subsequently dispersed to uninfected plants as their final settling point rather than infected plants. Settling experiments with Las-infected and uninfected plants under complete darkness produced similar results to experiments conducted under light conditions. Both Las-infected and uninfected psyllids exhibited similar behavioral responses to Las-infected and uninfected plants in olfactometer and settling assays. Volatiles were collected from citrus plants that were either uninfected (control) or infected with Las. The volatile profiles of these plants were analyzed using a machine-learning algorithm called ‘Random Forests’. This is a data-mining technique which allowed for the selection of the most appropriate predictor volatiles of observed ACP behavior and provided probabilities for classification of each plant as infected or uninfected based on volatiles alone. This analysis effectively divided the sampled plants into two groups, supporting a volatile basis for discrimination between infected and uninfected plants by Asian citrus psyllid. Methyl salicylate and methyl anthranilate were selected as the major predictor variables for the classification of the plant infection treatments. There was significantly more methyl salicylate (MS) and less methyl anthranilate (MA) in Las-infected plants than uninfected plants. Methyl salicylate was found to repel psyllids in laboratory olfactometer experiments at a 100 mg dosage. There was no effect of MA on the behavior of psyllids. In addition to investigating ACP response to plant odors and whether it changes due to Las infection, we investigated the hypothesis that infection may influence plant nutritional status and that this may affect psyllid host choice behavior. Nutritional analysis revealed that HLB-infected plants in our experiments were deficient in zinc, iron, nitrogen and phosphorus and produced more potassium and boron than uninfected plants. Experiments investigating how nutritional status of plants due to infection affects psyllid behavior are underway as part of our ongoing and future studies. Collectively, our results indicate that ACP host seeking behavior is modified by changes plant volatile release due to infection with Las, which alters headspace volatile release and nutrient profiles of infected plants and compared with uninfected controls.
Insecticides are currently the basis of Asian citrus psyllid (ACP) management programs and the number of annual insecticide applications has increased significantly. A two-year study (2009-2010) was conducted to develop baseline studies on levels of insecticide resistance among five geographically discrete populations of ACP across Florida. In 2009, one or more field population of ACP were less susceptable to fenpropathrin, imidacloprid, malathion and thiamethoxam compared to a laboratory reared susceptible population. In 2010, susceptibilities of field populations were compared with the laboratory susceptible population. In general, the susceptibility of ACP from field collected populations was lower than that of the laboratory susceptible population for most of the insecticides tested. In addition, immature ACP from four field populations were determined to have decreased susceptibility levels to the majority of insecticides tested. Increased levels of insecticide resistance among field populations was positively correlated with increased levels of three detoxifying enzymes: general esterases, glutathione S-transferases and cytochrome P450 monooxygenases. In order to better understand the genetic mechanism underlying increased levels of cytochrome P450 monooxygenases in insecticide resistant populations, we investigated the expression levels of family 4 cytochrome P450 (CYP4) genes in adult ACP when exposed to varying levels of imidacloprid. Five novel CYP4 genes (CYP4C67, CYP4DA1, CYP4C68, CYP4DB1 and CYP4G70) were identified from adult ACP. Expression of all five CYP4 genes was induced by exposure of ACP to imidacloprid suggesting their involvement in metabolism of this toxin. In addition, four of the five CYP4 genes were expressed at significantly higher levels in uninfected than Las-infected male ACP, whereas only one was expressed at significantly higher levels in uninfected than Las-infected females. These results suggest that levels of cytochrome P450 monooxygenases in ACP may be linked to expression levels of these CYP4 genes. Previously, we reported that Candidatus Liberibacter asiaticus (Las)-infected ACP are characterized by lower levels of cytochrome P450 monooxygenases than uninfected counterparts. In addition, expression of some of these CYP4 genes appears to be sex-specific. Higher expression of the five CYP4 genes occurred in nymphs than adults, which is congruent with previous results indicating higher levels of cytochrome P450 monooxygenases in nymphs than adults. These five CYP4 genes may be promising candidates for RNA-interference to silence over-expression of genes associated with insecticide resistance in ACP. These newly identified genes may also serve as DNA-based screening markers for cytochrome P450-mediated insecticide resistance in field populations of ACP.
The goal of this project is to evaluate existing and known insect repellents to determine if they can be useful tools for Asian Citrus Psyllid (ACP) management. Also, we are focusing our investigations on chemicals that would otherwise be safe to the environment and not harm beneficial insects. The objectives include examination of repellent and toxicological properties of selected botanical chemicals against ACP. We will plan to evaluate 30 plant produced chemicals that are known insect repellents for their repellent and insecticidal activities against ACP with an intent of identifying effective tools for ACP management. We have selected and purchased the candidate compounds and standardized a protocol for evaluation of their effects on ACP behavior. For behavioral bioassays, we are using a customized T-Maze laboratory olfactometer. The olfactometer consists of a 30 cm glass tube that is bifurcated into two equal halves with a Teflon strip forming a T-maze. Each half serves as an arm of the olfactometer enabling the ACP to make a choice between two potential odor fields. The olfactometer arms are connected to odor sources placed in solid-phase micro-extraction chambers (ARS, Gainesville, FL) through Teflon-glass tube connectors. The samples are diluted in appropriate solvent and subsequently pipetted onto a 5 cm Richmond cotton wick, which serves as the release device. Each chemical is evaluated individually and tested with at least 4 dilutions (dosages) on a log scale to determine optimum release rate. The control treatment consists of a cotton wick impregnated with solvent only. The olfactometer is housed within a temperature-controlled room and positioned vertically under a fluorescent 900 lux light bulb fixed in a fiber board box for uniform light diffusion. This position takes advantage of the negative geotactic (inclination to move up) and positive phototactic (attraction to light) response of ACP. Preliminary experiments with some tested chemicals indicated that citronellol, a sesquiterpenoid from lemon grass oil, repelled psyllids in the laboratory experiments. Behavioral bioassays with more botanical chemicals are in progress. To evaluate insecticide contact activity of these chemicals, we have obtained an automatic microapplicator that is being used to apply known and minute quantities of tested chemicals onto psyllids. Seven serial dilutions for each test chemical are prepared by dissolving the test chemical in acetone or absolute alcohol. Thereafter, 0.4 ‘l of each serial dilution of test chemical or a solvent only (control) treatment is applied to the dorsal mesothorax of adult psyllids. Treated psyllids are transferred to Petri dishes containing citrus leaf discs placed on a solidified agar beds. Mortality of ACP is scored 48 hr after exposure to treatments. Data are analyzed using standard probit analysis. Currently, these toxicological investigations are underway.
Leaf residue toxicity studies. The objective of this experiment has been to determine the residual efficacy of low volume and high volume insecticide applications on citrus leaves. Fenopropathrin (16 oz/acre), phosmet (1.5 lb/acre) and imidacloprid (15 oz/acre) were applied to citrus either as a low volume (LV) at 12 gpa or a high volume (HV at 200 gpa) application. For the fenpropathrin and imidicloprid treatments, 2% V/V of 435 crop oil was added and for the phosmet treatment LI-700 was added to adjust the pH of the solution to the recommended range. At 4 and 8 days post-application, leaves were removed from the plots and a leaf disc mortality bioassay was performed in Petri dishes. Approximately 15 adult Asian citrus psyllids were placed in the dishes with leaf discs of either the treated leaves or untreated control leaves. Adult mortality was assessed at 24 h and 48 h post exposure. Four days after treatments were applied, the 24 h exposure mortality for HV treatments was 100% for fenpropathrin, 84% for phosmet and 61% for imidicloprid. The only treatment with any mortality for LV applications was phosmet at 2.2%. After 48 hrs of exposure, all the HV treatments had >90% mortality. Mortality in all the LV treatments was <10%. Leaves removed from the field at day 8 caused much lower mortality in HV applications than at day 4. After 24 h of exposure exposure the mortality for fenpropathrin was 79%, phosmet was 14% and imidicloprid was 16%. Interestingly, the LV mortality at day 8 was somewhat higher than at day 4 for phosmet (14%) and imidicloprid (18%). After 48 h of exposure, mortality was higher for both HV and LV treatments. Border row treatment. In these experiments our goal is to compare the reduction in psyllid populations when treating all rows of citrus grove plots versus border row only treatment. All treatments are made to plots where psyllid populations were low initially because of a previous comprehensive treatment. In a 44 acre commercial grove, plots have been sprayed with 16 oz/acre of fenpropathrin with 2% V/V 435 crop oil added as a LV application of 12 gpa. In 3 of the plots, every tree was sprayed and in another 3 plots, only the outer 2 rows of trees were sprayed. Three plots were left untreated as control plots. We have completed the first trial of this experiment and are analyzing the results. A second replication is planned for early July, a third in August. Canopy investigation. This experiment is designed to investigate the distribution of adult psyllids within the tree canopy with the goal of optimizing insecticide spray applications. Sampling of the mid and upper parts of the canopy of mature trees for psyllid adults has been initiated. To date, three fold more adult psyllid have been found in the upper canopy than at mid-canopy level.
The goal of the present project is to establish economic thresholds under different juice price scenarios that optimize returns on investment when a nutrient/SAR package is being applied in groves with moderate to high incidence of HLB. Two 3-year field experiments were initiated in two commercial orange blocks in Hendry County (southwest Florida). One of the groves is planted with ‘Earlygold’ oranges and the other with ‘Valencia’ oranges. Average HLB incidence estimated in both groves based on PCR analysis of a random sample of 160 trees is 98% in ‘Earlygold’ and 76% in ‘Valencia’. Experimental design is randomized complete block with 4 replicates and 4 treatments: (1) No insecticide, (2) Calendar applications, in order to drive vector populations close to 0, (3) nominal threshold of 0.2 psyllids per tap, and (4) nominal threshold of 0.7 psyllids per tap. Calendar applications are being applied approximately every 1 month and consist of a rotation of insecticides recommended for managing this pest. Treatment (2) has been sprayed in both blocks with carbaryl (Sevin XLR Plus ) at 0.75 gal/ac in April, spinetoram (Delegate WG) at 4.5 oz/ac in May and imidacloprid (Admire Pro) in June at 4.5 oz/acre. No sprays have been applied for treatments (3) and (4), during the last 3 months because nominal threshold populations have not been reached. Adult psyllid populations are being monitored every two weeks by tap sampling. Flushing patterns and flush infestation are being estimated by assessing the number of new shoots per tree and evaluating the proportion infested with psyllids. Effects of treatments on natural enemy populations are being evaluated by counting beneficials in tap samples, suction samples, flush observations. Additionally, exclusion studies on ACP infested flushes are being conducted in treatments (1) and (2) to evaluate the potential negative incidence of the sprays on the ACP beneficials. A branch with emerging flush is caged with a small number of adult psyllids for 3 days. Resulting eggs and 1st instar nymphs are counted and the cages replaced on half the branches. When the nymphs have reached 5th instar or are ready to emerge as adults the brances are removed and all surviving nymphs are counted. The difference in survival between caged and uncaged branches is taken as the effect of predation on the psyllid cohort. The first such study run in June indicated ACP mortality of 82.8 ‘ 4.9 % in treatment (1) and 69.1 ‘ 9.0 % in treatment (2). The greater reduction in treatment (1) may indicate greater activity of natural enemies in the absence of insecticide applications, and provide a better picture of the faunal changes that have occurred in response to treatments. Secondary pests and their natural enemies are also being studied during the growing season in the four threshold treatments. Citrus leafminer (CLM) densities are being monitored using pheromone traps. A trend toward lower CLM activity in treatment (2) has been observed although differences are not yet significant (P = 0.1024; df = 3,15; F = 2.577 for the ‘Earlygold’ block and P = 0.1018; df = 3,15; F = 2.638 for the ‘Valencia’ block). In contrast, citrus red mites showed significantly higher populations in treatment (2) compared to other treatments in the ‘Valencia’ block (P = 0.0003; df = 3,1599; F = 6.424), in concert with significantly lower populations of predatory mites (phytoseids) (P = 0.0009; df = 3,1599; F = 5.600). These results would indicate that the calendar applications had a negative effect on phytoseids, and therefore their prey, citrus red mites, increased their numbers. Further studies in the following months on these and other secondary pests will help us to elucidate the costs incurred by spraying due to disruption of biological control.
Ultra low-volume and Aerial Application of Insecticides and Horticultural Mineral to Control Asian Citrus Psyllid in Commercial Orchards. Low volume (LV) aerial and ground sprays have become an important method of application during the dormant season and at other times in SW Florida. We continue our evaluations of LV application of 435 horticultural mineral oil (HMO) which has shown promising results the last 3 years. This report documents updated results from the fifth trial begun on February 2011 in a 10.9 acre plot of ‘Valencia’ orange in Lee County that compares the efficacy of low volume spray spray of 435 horticultural spray oil (HMO) with the grower standard and an untreated control. Seven sprays of 435 oil (23Mar, 12Apr, 21Apr, 28Apr, 5May, 19May, 2June, 14June) have been completed since the last report. A grower standard of 6 oz of Actara/ac + 2gal HMO/ac was applied in 10 gpa spray mix on 2 May. On 14 June, 10fl oz/ac of abamectin 0.15 (Agri-mek) plus 2 gal HMO oil was applied in a 10-gal mix. Thus far mean psyllid populations have been below the 0.2 threshold for grower standard between 12 Apr and 20 June, whereas the oil treatment has been above threshold for 26 Apr, 9 May, 24 May, 6 June, and 20 June. There were significant differences between grower standard and oil treatments on 9 May (p = 0.06), 24 May (p = 0.04), with grower standard having less psyllids present (0.08 +/- 0.06SEM, 9 May; 0.07 +/- 0.06SEM 24May) than oil (0.25 +/- 0.06SEM 9 May; 0.25 +/- 0.06SEM). However, during June tap sampling, these significant differences disappeared (p > 0.05), and the mean psyllid counts for grower standard were 0.12 +/- 0.08SEM, 0.10 +/- 0.10SEM, and for oil (0.23 +/- 0/8SEM, 0.33 +/- 0.10SEM) on 6 June, 20 June, respectively. We added citrus leafminer assessments to this grove to determine if treatments are affective at reducing leafminer numbers. We placed pheromone traps in the grove on 24 May and have conducted one damage assessment of leaves using a modified Horsfall Barratt scale. There were significant differences between the grower standard and oil (p = 0.03) with the grower standard having less leafmining damage (0.50 +/- 0.05SEM) than the oil treated plot (0.68 +/- 0.06). All the spraying with broad-spectrum insecticides appears to be increasing incidence of various secondary pests including scale insects, mites and citrus leafminer (CLM). Furthermore, canker is an increasing problem throughout the area which has increased concern about CLM. CLM control requires precise application timing on emerging flush to be effective so there is considerable interest in aerial application. Therefore, we are evaluating aerial application of two products widely used for leafminer control, the insect growth regulator methoxyfenozide, and spinetoram, an analog of spinosyn A which is derived from actinomycete bacteria. Both products are selective with low vertebrate toxicity. The replicated trial in being conducted on an 80 acre block divided into 12 large plots in a replicated complete block design with 4 treatments and 3 replications. Pheromone traps are being used to monitor CLM populations within all plots before and after the applications which are planned for 7 July when summer flush will be optimal for maximum control.
In California, citrus nursery stock must be treated with both a systemic and foliar pesticide in order to move it within areas quarantined for the Asian citrus psyllid (ACP). The purpose of these treatments is to prevent ACP from establishing in nurseries and being moved around California when the plants are sold. Imidacloprid is one of the systemic insecticide options available to nurserymen and is the focus of our research at UC Riverside. However, there are other systemic neonicotinoids recommended by the CDFA that growers are permitted to use on their citrus before shipments leave the nursery. These insecticides include Safari (dinotefuran) and Platinum (thiamethoxam), and this report summarizes our data on these compounds. We are evaluating Platinum and Safari uptake and persistence in 2 citrus varieties, ‘Kinnow’ mandarins (1 year old) and ‘Tango’ mandarins (3 years old). Admire Pro (imidacloprid) was also included in the trial. Both varieties are budded on ‘Carrizo’ rootstock. Trees were treated with the current label rate for Platinum and Safari, and the 0.33 ml/cu ft potting media rate of Admire Pro. Within 1 week of treatment with Admire Pro, imidacloprid levels were above the 200 ppb threshold established by Mamoudou Setamou as the dose needed to kill ACP nymphs. We use this concentration to evaluate the efficacy of our treatments. Mean concentrations in both the Kinnows and Tangos were at 1800 ppb. At week 2, the concentrations had increased further. The levels in the Kinnows and Tangos were 6,000 ppb and 6,550 ppb, respectively. Clearly, the trees were well protected within 1 week of treatment. As yet, there are no data available to guide us on how much thiamethoxam (Platinum) or dinotefuran (Safari) we need to have in the trees to protect them. However, at the label rates we tested, the concentrations were in the ppm range within 1 week of treatment, suggesting that the trees would also be protected from ACP. Thiamethoxam concentrations in the Kinnows and Tangos were 1,600 ppb and 2,400 ppb, respectively, at one week, while concentrations of dinotefuran in the Kinnows and Tangos were 12,000 ppb and 8,100 ppb, respectively, at one week. The substantially higher concentrations of thiamethoxam and dinotefuran compared to imidacloprid are not surprising given their higher water solubilities. Thiamethoxam (4 g/l) is about 8-fold more water soluble than imidacloprid (0.5 g/l) and dinotefuran (40 g/l) is 80-fold more water soluble than imidacloprid. The higher water solubility will permit a more rapid uptake of insecticide into the trees; however, it could also result in a less persistent product due to more rapid depletion of soil reservoirs of insecticide. We will monitor the concentrations in these trees for 6 months to determine the longevity of residues within the trees. This information will guide future uses of these products on nursery citrus. We are continuing to monitor imidacloprid applications in a commercial nursery. By doing this, it allows us to evaluate the efficacy of treatments in citrus growing under normal production conditions, as opposed to the more controlled environment of a laboratory or greenhouse trial. We generally measure lower levels of imidacloprid in commercial citrus, although the treatments do reach the 200 ppb threshold concentration. The more frequent appearance of flush at the commercail sites results in lower titers of imidacloprid because the new tissue grows at a rate that is faster than the rate of movement of imidacloprid. This result reaffirms the importance of the foliar treatments for nursery stock that is due to leave the faciity. The foliar treatments will protect the citrus until the imidacloprid establishes at threshold concentrations.
(863) 956-8817
(863) 956-5894
700 Experiment Station Road
Lake Alfred, FL 33850
Email Us
