The objectives of this study are: 1) to develop a series of flexible stochastic models to predict the temporal increase and spatial spread of citrus HLB and canker. They can be used in a number of ways: to predict spread and to analyze the effectiveness of control strategies both in plantations and State-wide. 2) Test various control methods under field conditions to evaluate effects and collect data to parameterize models. SEIDR model. Using Markov-chain Monte Carlo (MCMC) methods, and data from infected areas of South Florida for successive snapshots of the occurrence of symptomatic detected trees in known populations of susceptible trees, we continue to estimate the transmission rates and dispersal kernel for HLB. A working model has been developed and continues to be improved that focuses on the differential effects of host age on epidemiological parameters as well as variability. Both residential and commercial citrus scenarios are being tested with user selectable and changeable via sliding controls. We continue to explore various aspects of Baysian MCMC methods to infer posterior densities on the model parameters. The uncertainty will be incorporated in models to predict spread and to allow for uncertainty in the efficiency and comparison of control methods. We are also considering a second modeling approach known as Approximate Bayesian Computation (ABC) and willcompare model outputs of the ABC versus the MCMC estimates to determine HLB dispersal distances be that we believe may improve control strategies. We have begun to develop a web based version of the model (known as a front end) and will soon go into validation testing. Both residential and commercial citrus scenarios are being tested with user selectable and changeable via sliding controls. We will use the front end to visualize the effect of these various control strategies. This should benefit researchers, growers and regulators to compare the results of thousands of simulations for practical management decision making and/or regulatory intervention/strategy building. Via this model we have begun to examine the effects of various controls such as using insecticide applications or not, removing infected trees or not, and the effect of HLB infection in young versus older trees. Model output confirms that controlling secondary infections by diseased tree removal and insecticide applications plus controlling primary infection from new insect immigrations can reduce disease increase to a manageable 2 to 5% increase per year.
Using an electrical penetration graph monitor, we examined the duration of feeding disruption provided by 5 insecticides commonly used in Florida citrus production. For chlorpyrifos, at 1DAT, while there was no significant difference in probing duration, psyllids on chlorpyrifos performed half of the probing durations when compared to untreated plants. Phloem related feeding behaviors were prevented up to 7 DAT on chlorpyrifos treated plants when chlorpyrifos residues on the leaf surface averaged 27.13 ppm. For both fenpropathrin and imidacloprid-treated plants, there was an immediate reduction 1 DAT in D. citri probe duration, number of probes, non-probing/walking activities, stylet penetration, phloem activities and xylem ingestion when compared to untreated plants. Over time, the duration of those behaviors increased as insecticide residue levels decreased. Fenpropathrin residues provided significant disruption of phloem related feeding behaviors by D. citri up to 21 DAT. Disruption of phloem feeding behaviors for psyllids on imidacloprid-treated plants lasted up to 28 DAT. At this time imidacloprid residue levels were 34.66 ppm, which was 83% lower than the concentration found at 1DAT. Psyllids on spinetoram-treated plants performed probes which were shorter in duration, but disruption of phloem feeding behaviors was not prevented, even at 1 DAT. The decrease in probing duration for psyllids on spinetoram-treated plants was only significant 7 DAT. Psyllids on spirotetramat-treated plants performed normal phloem activities throughout the entire experiment. The results from this study demonstrate that while some insecticides may cause relatively rapid mortality of adult psyllids, there is considerable variability that exists among these products in terms of the duration of feeding disruption provided. While some insecticides provided feeding disruption lasting 3-4 weeks (fenpropathrin and imidacloprid), protection provided by other products was much shorter. Overall, the results of this study can be used to help guide citrus growers in product selection and also determine when additional applications will be necessary.
In previous work, we reported that soil-applied neonicotinoid insecticides can significantly disrupt psyllid feeding behavior and thus reduce (if not totally prevent) treated young trees from becoming infected with the HLB bacterium. Since the majority of this work was done in a laboratory setting and we only have anecdotal evidence from the field to support this claim, a multi-year field trial was initiated to validate this claim. At the end of May, a 10-acre block of ‘Valencia’ orange was planted at the Mid-Florida Citrus Foundation Water ConservII grove site in Orange County. Within this planting, 60 24-tree (4 rows x 6 trees) plots were assigned to one of 6 experimental treatments. Each treatment is replicated 10 times across the 10-acre block. The treatments are as follows 1) soil-applied neonicotinoid applications every 6 weeks; 2) monthly foliar-applied broad spectrum insecticide applications only; 3) monthly applications of a selected psyllid deterrent material; 4) soil-applied neonicotinoid applications every 6 weeks plus monthly applications of foliar-applied broad spectrum insecticides; 5) soil-applied neonicotinoid applications every 6 weeks plus monthly applications of the selected psyllid deterrent material and 6) and untreated control plots for comparison. One day after planting, and just prior to application of the first treatments, leaf samples were collected from the two center rows of trees in each plot. These leaves were then analyzed in the laboratory, first for the presence of Las to confirm that our experiment began with HLB pathogen-free trees, and second, to quantify the level of neonicotinoid residues already present within the leaf tissues. Every two weeks following planting, psyllid and leafminer counts will be made in each plot as an assessment of the level of pest control provided by each treatment. On the same day these pest assessments are made, leaf samples will be collected from the two center rows of trees in each plot. These samples will be taken back to the lab for pesticide residue analysis. To determine the rate of HLB infection between treatments, leaf samples will be collected every 3 months from the two center rows of trees in each plot and analyzed for the presence of Las using pcr. Additionally, tree growth measurement will be made every three months to compare for treatment effects on plant growth rate. During the first two months of this particular objective, PCR analysis of all plant material has shown that none of our trees were HLB infected coming out of the nursery. Analysis of leaves collected showed they contained imidacloprid as a result from treatments applied prior to leaving the nursery. Despite the presence of imidacloprid in the leaf tissue, within the first week after planting, leafminer damage was as high as 5% in foliar insecticide and control treatments, and increased to 30-40% approximately 4 weeks after planting. Such results were anticipated since disruption of the root system at the time of planting has been shown to dramatically decrease the duration of control provided. Thus, these results provide further direct evidence for the need to quickly retreat young trees immediately following planting in order to provide maximum protection from both leafminer and asian citrus psyllid. This objective as described above will be continued over the course of the next 4 years to compare the success rate of bringing young trees into production using the different management strategies tested.
Following three freezes in southwest Florida during the 2010-11 winter in which we experienced temperatures of 25 degrees F for several hours resulting in leaf and fruit drop, and some terminal twig damage, spring growth was good and the trees in our trials receiving foliar nutrients rebounded with thick canopies, flowered and set a good crop. Yield from the 2010-11 Valencia crop in our trial gave the same results as the two previous years when comparing the treatments in the Boyd nutritional experiment. The treatments with the three highest yields were from the complete Boyd cocktail, the complete cocktail without SARs, and the complete cocktail without hydrogen peroxide. These treatments all contained the macro- and micro nutrients along with phosphite. Juice quality was affected by some drying in the Valencia fruit resulting from the freezes, and assuming all trees experienced the same amount of damage to the fruit the juice quality results follow. Percentage juice and juice per box was not different among treatments except lowest for the treatment with only SARs + phosphite, which was highest in acid. Acid, Brix, Ratio, and lbs solids/box were highest for treatments with Boyd cocktail, Boyd cocktail w/o SARs, and Boyd cocktail w/o hydrogen peroxide, macro nutrients only, and phosphite only, and lowest with SARs + phosphite. HLB bacteria as detected by real time PCR during the last 3 year shows the incidence at the test site increased from 41% to 79%, and is now at 100%. Furthermore, at the most recent sampling in 2011 regardless of the treatment the ct values were lowest indicating the the bacteria population in the tree was higher than in each of the previous years. We are entering the 4th year of a replicated experiment in a 12-acre experiment commercial block of 8-year-old ‘Valencia’ oranges on ‘Swingle’ to test effects of micro-nutrients + systemic acquired resistance inducers, and Asian citrus psyllid (ACP) chemical control on ACP populations on Can. Libericacter asiaticus (CLas) titer, and plant yield. While we are continuing with the experiment, results from the first 3 years are being written up for publication. Psyllid populations in the insecticide and insecticide+nutritional plots reached the 0.20 threshold on 22Apr, 6May, and the insecticide+nutritional plot reached threshold on 4June. Insecticide and Insecticide+nutritional had significantly (P< 0.05) fewer psyllids than control or nutritional only on dates 8Apr, 22Apr, and 7May. All three treatments were significantly (P < 0.05) less than control on 21May, but no differences were found between treatments on 4Jun. Since our last report we have sprayed one application on the treated plots: Movento MPC at a rate of 16oz/ac + 2gal/ac of 435 horticultural oil. Three years of sticky card data are now being examined to assess movement of adult psyllids among treatment plots. Tap and flush sampling have been modified to include counts of nymphs (all stages) and eggs on flush. In Jan and May, we collected plant samples for HLB detection. Results of the fruit samples sent to the CREC fruit quality laboratory (Apr 11) for analysis of lbs. juice per box, acid, total brix, and ratio have not been received. An initial citrus leafminer damage assessment using a modified Horsfall Barratt scale (22 June) showed significant differences (p < 0.0001) between insecticide + nutritional and insecticide only treatments on the one hand and nutritional on the other with this latter showing most damage.
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.
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