This represents the final report of an extension of a previously funded CRDF grant. The project has focused on developing a new method of controlling the Asian citrus psyllid by inhibiting its ability to establish a feeding site on citrus. Specifically, to inhibit the formation of feeding structures (salivary sheaths) formed during the mouth-part (stylet) probing process in a way that prevents the insects stylets from reaching the citrus vascular tissue. If successful, this strategy would be a highly targeted control strategy that would not affect other beneficial insects (thus allowing its use as a component of an IPM strategy that employs predetors/parasitoids) and would prevent HLB-associated pathogen transmission that is seen to occur even when systemic broad-spectrum pesticides are used and brief psyllid feeding occurs. During this one-year extension granting period, determination of the chemical composition of the salivary sheaths finalized and verified and this information was used to show that inhibitors of sheath formation did indeed block insect penetration into plant tissues and as a result, prevent the insects stylets from reaching the vascular tissues. Furthermore, knowledge gained from this research allowed identification of multiple inhibitor-classes of compounds, some of which are a class of natural products (Generally Regarded as Safe ‘GRAS’ compounds) that could inhibit sheath biosynthesis. These compounds were selected based on the probability that their structure would be competitive with the substrates used in the sheath polymerizing process. As a result of this work, experiments are being finalized in the greenhouse to show efficacy of topical applications of these compounds in insect control. Furthermore, commercial contacts have been initiated to support commercialization of this control strategy. The specific composition of the sheath and of its inhibitors and the mechanism of action are with-held from public documents at this time to suppport the commercialization process, but this information has been and will be shared confidentially with appropriate members of the CRDF staff. As stated in the most recent quarterly report, the discovery of GRAS compounds that have affect on sheath formation offers the possibility of rapid commercialization if they are shown to be effective in whole plant application trials currently underway in greenhouse and field applications. Some of the tested compounds are available at relatively low costs that would be compatible with commercialization if efficacy as a topically applied compound can be verified. Current research focuses on application in a formulation that provides extended retention in the tissues to which the compounds are applied and efficient translaminar movement so that interaction of inhibitors and salivary sheaths can occur during initial insect probing and throughout the probing process as a method of improving efficacy.
The objective of this enhancement grant to project #570 was to develop novel methods that manipulate psyllid behavior to improve the deployment and efficacy of biological control agents. The research was conducted and is ongoing in Florida and Puerto Rico. The context of the grant is the following: while much excellent science has been conducted and behaviorally-active chemicals (sex pheromone, plant volatiles, etc.) have been identified in the lab, no highly-attractive field lure for the Asian citrus psyllid (ACP), vector of HLB, has proven effective. A method providing higher more-effective levels of attraction suitable for research and management goals is direly needed. Idea: The diversity of available host plants has potential and remains a basic key to meeting this need if it can be novelly and appropriately deployed relative to known ACP behaviors. We sought to address the need by developing the idea. A container system was developed that is economical in cost enough to be practical ~$10.00/unit, and large enough over periods of months to support and manage trees in the 4-6 feet and larger height range. Using the developed system, the “sentinel” trees can be easily transported and placed as needed. They thrive and can be intensively managed – watered and fertilized – in the greenhouse and field as well as paired with other tools that enhance their field attraction (visual cues, courtship vibration calls) to ACP. The test plants attracted ACP in competition with conventional in-ground citrus trees. ACP behavior in response to sentinel trees mirrored within reasonable limits what is observed with ACP response to conventionally-planted trees and will out-compete conventional trees for ACP, if of, or in (as described below), the proper more competitive attraction state. Trees of known preferred citrus cultivars and related species of preferred hosts (e.g., Bergera koenigi and Murraya exotica) attracted more psyllids than less preferred cultivars/species and trees during flushing were most attractive to ACP regardless of cultivar. Sentinel plants were also paired with visual cues to increase the active distance of the attraction to ACP and we tested (in cooperation with R. Mankin, USDA-ARS) and continue to develop use of his electronic “sonicator” that provides ACP vibrational courtship cues to enhance residency time of ACP once they land on the sentinel plants. So far use of the apparatus appears promising but requires some further fine tuning that is ongoing. Parts of this research were executed in Puerto Rico in cooperation with D. Jenkins (USDA-ARS). ACP populations are readily available there in groves available for research without the intensive insecticide application regimes encountered in FL which facilitated this research. In P.R. we tested the sentinel plants as well as 18 trap designs in the field and found a combination of cues and trap characteristics which consistently trapped more psyllids than other trap designs. This novel cylinder trap may be used to survey psyllid populations for pathogens, fungal and viral, (ongoing now under project #570 objective 2) and may be modified to disseminate pathogens in a grove. Relative to our objectives we have demonstrated that sentinel host plants of ACP deployed in the field in portable, self-supporting containers can be used to attract and manipulate ACP in Florida and Puerto Rico. This sentinel plant system can be deployed both in groves, urban citrus and in areas where there is no citrus. In groves either managed or unmanaged, it may be used to manipulate psyllid movement for precision management, e. g., delivery of entomopathogens and enhance or deliver other mortality agents more economically and effectively. Outside of orchards it may be used to identify environmental factors that influence psyllid populations (elevation, shade, etc…).
Three new trials in grapefruit have been established in Indian River St. Lucie County, FL. We are establishing foliar nutritional trials similar to what we have done in southwest Florida with oranges using the Boyd cocktail. We are looking specifically at the nitrogen source in combination with individual micro-nutrients. The objective is to determine if there are any observable influences of foliar fertilizers on HLB affected grapefruit in the Indian River marketing district. Trial #1 is ~25 years old of ‘Flame’ on Swingle rootstock with 30 replicated experimental units, and trial #2 of ~6 year old ‘Ruby Red’ on Sour orange trees with 50 replicated experimental units are in commercial groves. Trial #3 at IRREC is to determine the effect(s) that foliar fertilizers have on young tree growth and their ability to ameliorate HLB disease symptoms in 1 year old ‘Ray Ruby’ grapefruit on Kuharske rootstock. Foliar fertilizer applications started in February 2014. Tree growth data, leaf nutrition data, and CLas titer measurements have been made. Fruit drop monitoring will begin in September 2014 in trials #1 and #2. These trials will be carried forward under CRDF project 14-903. One additional year of data in the 5-year nutritional study in a 32 acre commercial grove of ‘Valencia’ in southwest Florida evaluating foliar nutrition has shown yield in HLB infected trees have increased each year and juice quality has been maintained. Economic analysis is showing the treatments with highest yield are the most expensive and yield is not significantly different from treatments with less expensive products that are more productive in dollar returns. The common ingredients in the most cost productive treatments are micro- and macro-nutrients, some with salicylate and phosphite. One additional harvest was acquired in a 13-acre commercial block of ‘Valencia’/Swingle testing effects of: (1) foliar nutrition and (2) chemical control on ACP populations, HLB titer, and yield. Results from the first 4 years yrs have been published. The ACP threshold to trigger insecticide application was lowered in 2014 from 0.2 to 0.1 due to diminished ACP populations in the area. Since the last report, ACP exceeded threshold seven times in nutrition treatments and twice in insecticide treatments according to biweekly monitoring. We applied Mustang @4.3oz/ac on 23 Apr and Exirel @20oz/ac 7 Jul. Insecticide treatments significantly (P < 0.05) reduced ACP adult counts 14 Apr to 26 Jun. HLB samples were taken Nov 2013; no treatment effects were detected for reset trees. Lower (P<0.05) Ct values were detected in insecticide treatments than without insecticide. Treatments with insecticide had higher (P < 0.05) yields. Fruit from nutrition-alone treatments had higher Brix values than untreated controls. No other measures of fruit quality differed among treatments.
The antennae of ACP have relatively few chemoreceptors and it is unclear how or to what extent ACP uses plant odors to orient. We have detected no or very weak antennal responses to the many odor compounds produced by young citrus leaves. We demonstrated that oxidative breakdown products of compounds such as E-‘-ocimene and citral are highly stimulatory to antennal receptors (ms. under review). Degradation of volatile organic compounds (VOCs) is common and results in the release of acetic and formic acids into the atmosphere. In one study, acetic acid comprised 8.4% of VOCs above a citrus grove. Degradation of VOCs can produce acetic and formic acids at rates 200 to 300% higher than the precursor compounds. In our studies, ACP adults probed a substrate impregnated with formic and acetic acids more frequently compared with blank controls and other citrus VOCs. ACP orientation to host plants may involve acetic and formic acid alone or in blends with other compounds. Classically, insect orientation to odors was presumed to occur through peripheral perception and central integration followed by activation of motor neurons. Signal integration can also occur at the level of the sensillum through ephaptic coupling wherein firing of a receptor neuron is modified by that of an adjacent neuron in the absence of synaptic connections. ACP rhinarial plate sensilla on antennae contain three odorant receptor neurons (ORNs) that respond strongly to common citrus volatile agonists. However, acetic acid and possibly formic acid act as inhibitors of ORN responses and may explain the absence of whole antenna responses to blends containing these antagonists. Coutinho-Abreu et al. at the University of California, Riverside demonstrated inhibition by acetic acid in an ORN responsive to 1-hexanol, and proposed an attractant blend containing myrcene, ethyl butyate and p-cymene that has yet to be tested in Florida citrus. Identification of volatile blends with potential behavioral activity will be pursued through gas chromatograph-coupled electroantennography (GC-EAD), single cell recordings and field tests. We will incorporate candidate compounds identified by Coutinho-Abreu et al. Their work and our findings suggest that strategic use of masking odors such as acetic or formic acid and separate attractant blends could be used in the field to modify ACP behavior.
Actigard is commercial SAR inducer which is non-insecticidal and does not have a tree size restriction for use. Collaboration with Syngenta in an EPA-approved Experimental Use Program (EUP) in two east coast grapefruit groves is being repeated again in 2014 to support the labeling of soil applied Actigard integrated with copper sprays for control of canker on bearing grapefruit and sweet orange. Reduction/suppression of fruit infection in citrus trees using foliar applications of soluble copper and novel bactericidal formulations in comparison with film-forming copper formulations: In 2014 trials, fixed copper formulations (e.g. copper hydroxide and copper oxide) sprayed at 2 to 4 lb of product per acre (0.75 to 1.4 lb per acre of metallic copper) are being compared with commercial and experimental soluble copper formulations at much lower metallic rates in grapefruit and Hamlin trials. Improved performance of copper formulations with windbreaks: 6 and 7 yr-old red grapefruit blocks surrounded by a 20-30 ft tall Corymbia torelliana windbreaks are under study. In each block, weather stations are deployed to measure wind gusts in proximity to the windbreak. Cu bioavailability and residual activity of copper formulations: Copper residues on grapefruit copper oxide (Nordox 75G and copper zinc formulations (Nordox 30-30) at the same two rates of metallic copper as in the efficacy trial are being measured during two periods of season when rainfall and fruit expansion rates differ. Firewall (streptomycin): two applications of Firewall allowed by the EPA section 18 label are being testing in the grapefruit trial with and without the penetrant Nanocanopy that is purported to increase uptake and systemic activity of the active ingredient. Efficacy of Firewall + Nanocanopy will be compared to copper treatments as well.
Assays of non-bearing trees soil drenched with the chelated copper formulations Magna-Bon (MB) and Copper Quik (CQ) confirm that copper concentration in leaves of non-sprayed field trees is well above nutritional concentrations. HLB preventative treatments of MB and CQ were applied at 3 month intervals during two growing seasons at 1,2,4,5 or 10 oz/tree in 8 oz of water as soil drenches to visually asymptomatic 1 and 2 year old Valencia trees on Volkamer lemon and Swingle citrumelo, respectively. The solid plantings, located in Sebring and Avon Park, were surrounded by adult trees with 100% incidence of HLB symptoms. After two seasons, leaf samples for determining PCR status of trees in each trial were collected in December 2013 and analysis completed in early 2014. In the 4 year old block of Valencia on Swingle, PCR positive trees across all treatments ranged from approximately 40 to 80% incidence and in the 3 year old block of Valencia on Volkamer lemon ranged from 8-15% incidence. Incidence of PCR positive trees drenched with MB or CQ drenches was not significantly different from untreated trees. Despite termination of the funding in June 2014, soil copper treatments will be continued in the 3 year old block of Valencia on Swingle for one more season. Second year PCR results from a trial at USDA Picos farm in Ft. Pierce treated with MB, CQ and copper or zinc phosphite drench treatments of Hamlin on Swingle trees treated since planting still has a low incidence of HLB positive trees. Drench treaments at the Picos will be continued for another season. In the 6 year old block of Valencia on Swingle in Lake Placid, PCR positive trees with symptoms or pre-symptomatic were were treated with soil drenches of Magna-Bon (MB), Cop-R-Quik (CQ) and an experimental compound (EXP) with well demonstrated systemic activity against citrus canker (caused by Xanthomanas citri subsp. citri) as a soil drench. After two seasons of spring and fall soil drench applications with high and low rates of each of these compounds in 3 replicated blocks, visual tree health ratings on a scale of 1-5 (1=Vigorous, asymptomatic, 2=Slight decline; 3=Moderate decline; 4=Severe decline; 5=Non viable, won’t recover) were higher for treated trees than the untreated check. Tree responses indicate that these treatment were having phytotoxic effects on HLB infected trees rather than achieving reduction in bacterial infection and or reduction in HLB symptom expression.
The objective of this project is to determine overwintering habits of Asian citrus psyllid (ACP), including determining alternative hosts, so as to understand how to improve dormant season control strategies for ACP. During the 2012/2013 winter season, we found high populations of ACP in groves under intermittent management (groves defined as grower use of insecticide treatment between 1 and 5 times per year), based on a field study of 40 groves sampled throughout Florida during January and February of 2013. We have observed a similar trend in field studies conducted during January and February 2014. Over two years of sampling, significantly more ACP were found in intermittently managed groves compared with abandoned, organic, or conventionally managed groves. In addition, we have examined abiotic and biotic data collected from these groves over the same winter months of 2013 and 2014. We found the presence of ACP was affected by the proximity to urban areas, but unaffected by natural and artificial edges such as roads, forests and lakes. The nutrient content of citrus leaves in groves appeared to have a significant effect on ACP abundance, such that psyllids were significantly, positively associated with trees that had high concentrations of phosphate (P) and potassium (K). Experiments to assess the effects of cold and heat acclimation on genes associated with insecticide resistance are completed and data analyses are currently underway.
One goal of this project is to identify the phenotypic effects of Wolbachia infection on ACP, specifically with respect to insect fitness. We determined that manipulation of endosymbionts, including clearing and novel introduction, are only effective when ACP are treated at the nymph stage. Because ACP nymphs are not amenable to microinjection, and cannot survive on the artificial diet used for rearing ACP adults, we have developed an artificial diet that permits rearing of ACP through nymph development. This diet can be used to successful administer antibiotic treatments for manipulation of endosymbionts. We have recently submitted these finding for publication. In previous attempts to remove Wolbachia endosymbionts from ACP, antibiotic applications have resulted in significant mortality in treated insects (P1 generation). We have recently developed a novel antibiotic cocktail, and have successfully used this to rear the first generation of psyllids (F1) from treated parents. Concurrently, we have tried, and discarded, several methods for super-infecting the psyllids with non-native Wolbachia strains (of which we have strain specific primers for) and have decided to pursue microinjection as a method for infecting psyllids. Evaluations of superinfections on Las transmission are underway. An additional objective of this project is to establish germ-line transmformation of ACP to introduce chromosomal changes that will interfere with Las transmission. The major roadblock to successful ACP transformation is the lack of artificial rearing and a thick chorion (egg shell) preventing simple micro-injection. We have made progress in the past four months by improving ACP colony viability and fecundity for optimal egg collections. This includes: 1) testing of ACP cage numbers, rotation intervals, response to temperature, and ACP morphotypes (orange/green more fecund than brown color morphotype); 2) testing of plant variety hosts and trimming schedules ‘ most success with lemon and lime versus sweet orange. Plants trimmed in cohorts biweekly maintain high levels of quality flush; 3) determining timing of egg laying ‘ optimal from 0800-1100; and 3) host preference- Swingle hosts yield highest levels of egg lays throughout the day. We have evaluated egg treatment, washes, age, and environment for optimal nymph survival, specifically: 1) Testing of egg treatment, washes, age, and environment for optimal nymph survival -Tested bleach and non-ionic detergent wash treatments to facilitate egg removal from flush and soften chorion for injection. – Found 1 min 0.02% Nonidet P-40 wash with 3x distilled water rinse most effective for 1-3 hr eggs (high mortality with bleach). Optimal incubator environment: 27’C, 14:10 L:D cycle, 70% relative humidity. -Tests for non-injected egg survival after detergent wash removal from flush ‘ Eggs held on held on slide for 3 days (after wash) before transfer had best survival on flush kept on leaf disk ‘ Eggs put on tape (as prepared for injection) have high emergence but nymphs stick and die on tape ‘ Eggs removed from tape after 3 days (1d < emergence) have optimal survival 2) Testing of egg injection on various substrates (flush, agar, glue, double-stick tape) - Washed eggs survive glass-needle micro-injection at a 13% rate ' Nymphal survival should be improved with a goal of 50% - Survival optimal with eggs injected on flush or tape - Egg injections with the pBac[IE1-hr5-DsRed] vector and phsp-pBac helper plasmids will now be initiated on eggs placed in tape and transferred to flush/leaves after 3 days (within 1 day of nymphal emergence)
We have collected five years data in the replicated Hamlin & Valencia trials in southwest Florida evaluating the Boyd foliar cocktail. The yield and juice data shows HLB infected trees have increased yield each year and are healthy, growing well, and maintaining production. Yield for the most productive treatments was about 2-boxes/tree on 8 year-old (7 to 8 foot) trees. The common ingredient in the most productive treatments during the past 5 years has been micro- and macro-nutrients, some also had phosphite (phos acid) and/or salicylate (salicylic acid). Yield has increased in all treatments each year. Juice maturity and quality factors don’t appear to be affected by HLB in trees receiving foliar nutritional sprays. Juice quality among treatments of percent juice/fruit (52.9-58.0%), acid (0.74-0.80), Brix (10.59-11.23), ratio at harvest (13.77-14.96), and pound-solids/box (5.88-6.40) have all been in the normal and acceptable range. The unprecedented fruit drop experienced industry wide was much less. Economic analysis shows treatments with the highest fruit yield are the most expensive but yield is not significantly different from less expensive treatments that are more productive in dollar returns. Three new trials in grapefruit have been established in Indian River St. Lucie County, FL. Trial #1 is ~25 years old of ‘Flame’ on Swingle rootstock with 30 replicated experimental units, and trial #2 of ~6 year old ‘Ruby Red’ on Sour orange trees with 50 replicated experimental units are in commercial groves. Trial #3 at IRREC is to determine the effect(s) that foliar fertilizers have on young tree growth and their ability to ameliorate HLB disease symptoms in 1 year old ‘Ray Ruby’ grapefruit on Kuharske rootstock. Foliar fertilizer applications have been started since February 2014. Tree growth data, leaf nutrition data, and CLas titer measurements have been made. Fruit drop monitoring will begin in September 2014 in trials #1 and #2. In prosecution of these efforts, 2-part-time OPS employees have been hired to do our foliar sprays and collect data observations. We completed the 6th harvest in a commercial block of ‘Valencia’/Swingle testing effects of: (1) foliar nutrition and (2) chemical control on ACP populations, HLB titer, and yield. Results from the first 4 years yrs have been published. The ACP threshold to trigger insecticide application was lowered in 2014 from 0.2 to 0.1 due to diminished ACP populations in the area. Since the last report, ACP exceeded threshold seven times in nutrition treatments and twice in insecticide treatments according to biweekly monitoring. We applied Mustang @4.3oz/ac on 23 Apr and Exirel @20oz/ac 7 Jul. Insecticide treatments significantly (P < 0.05) reduced ACP adult counts 14 Apr to 26 Jun. HLB samples were taken Nov 2013; no treatment effects were detected for reset trees. Lower (P<0.05) Ct values were detected in insecticide treatments than without insecticide. Treatments with insecticide had higher (P < 0.05) yields. Fruit from nutrition-alone treatments had higher Brix values than untreated controls. No other measures of fruit quality differed among treatments. Fluorescent leaf assay to determine if phloem translocation was enhanced by the use of extra nutritional supplements continued. As reported previously dye translocation from samples at SWREC and the Orange Hammock grove in Felda were similar to healthy samples from our greenhouse. In an organic grove operation where good fruit size was produced on HLB qPCR positive trees, some of the trees had good translocation while others did not. Visual assays showed that the leaves from the HLB affected trees had uptake similar to healthy leaves. In all cases visual assays were easier to see differences than readings the fluorescent plate reader, due to leaf extracts having other fluorescent compounds released.
For this enhancement study, we conducted experiments to assess ACP gene expression in response to simulate cool temperature challenges to determine the impact of cool temperatures, and expression of melanization genes, on ACP vector competence. We have recently completed inoculation and transmission assays with ACP pre-exposed to a range of temperatures typically experienced by this pest under field conditions. Following transmission assays, qRT-PCR was used to assess changes in the expression of heat shock and melanization protein genes associated with immune defense and thermal protection. Preliminary data analysis suggested that pre-exposure to cool temperatures resulted in up-regulation of several target genes and reduced the vector capacity of ACP. Both acquisition and inoculation of Las by ACP were reduced following exposure to cool temperatures. These data support our hypothesis that the prevailing climatic conditions during Florida winters may have a reductive effect on Las transmission. Cool temperatures, ranging from 15-22’C, resulted in the production of dark-colored ACP adults. Altered coloration was correlated with up-regulation of melanization genes. Insect development slowed following exposure to cooler temperatures, as expected; however, there were no discernible impacts on insect longevity or reproductive output. Due to the short-term nature of this project, it was not possible to complete assays to assess the susceptibility of ACP to challenge with entomopathogenic fungi following exposure to cool temperatures, although we expect that susceptibility to fungi will be altered as a result of the changes in the expression of melanization genes. Similar studies of insect immunity indicate that individuals with a strong melanization responses are more likely to clear microbial infections. These findings suggest potential novel gene targets for disrupting ACP vector competence that could be exploited as part of an integrated HLB management program. Furthermore, these targets may be harnessed to improve the efficacy of entomopathogenic fungi as biological control agents for ACP.
The goal of this project was to enhance our understanding of resistance mechanisms for Asian citrus psyllid (ACP). The entire voltage-gated sodium channel of ACP was cloned and mapped back to the genomic sequence from the genome database. What was discovered is that ACP does not have predisposition to develop the typical L1014F mutation associated with pyrethroid resistance that is observed in many other insect species, at least without multiple changes to the nucleotide sequence. However, it does have the propensity for other changes that are associated with as high, and in some cases, higher, pyrethroid resistance (M918T and L925I). These two changes have been observed in other hemipteran species. Taken together, these results suggests that with sufficient pyrethroid pressure, target-site changes are likely to be observed. In addition, we found significant potential for alternative splicing, another mechanism that has been associated with pyrethroid resistance in field populations of insects. In our studies, we identified two isoforms in nymphs, demonstrating that alternative splicing occurs in this insect within this channel, again underscoring the importance for judicious use of pyrethroids. These results indicate that target site resistance for pyrethroids is a distict possibility and judicious rotation is very important to keep these tools useful.
Citrus greening disease, also called Hu’ngl’ngb’ng (HLB) is a severe threat to the growth and sustainability of the FL citrus industry. HLB infection is caused by C. liberibacter spp transported by the Asian Citrus Psyllid (ACP) during feeding between infected and non-infected trees. The objective of this research is to develop and optimize a promising film-forming ACP repellent material for preventing HLB infection. The film-forming material has been named hereafter organo-silica based composite film (OSCF). A series of OSCF materials has been synthesized using a variety of polymers, silanes and crosslinkers, as proposed. Preliminary phytotoxicity (plant tissue damage) studies has been conducted using Vinca sp. and calamondin lime (purchased from the local Home Depot). Phytotoxicity studies indicated that the first series of OSCF materials are safe to plants. In our next report, we will include OSCF material characterization data and results obtained from rainfastness studies.
Citrus leafminer (CLM) is a major pest of citrus, causing direct damage and increased incidence/intensity of citrus canker. Pheromone traps are valuabe for monitoring CLM but need further evaluation to optimize use for management. The objectives of the proposed project include: (1) assess species composition of trap catches, (2) optimize spatial and temporal distribution of pheromone traps. Trapping of wild populations of Phyllocnistis citrella, congeners and other similar moths continued. Species composition in trap captures also continued to be evaluated by analysis for Cytochrome Oxidase 1 (CO1) sequence generation incorporating an anchored hybrid enrichment probe set from Illumina HiSeq data with the Lemmon Lab at FSU. Each sample is associated with locality, collection date, type of storage (dry or ethanol), and trap number. Samples collected in 2013-2014 and received in March 2014 are being processed. Photographs of the specimens will help elucidate morphological characters in order to provide more accurate identifications. David Plotkin’s work on photographing moths through a microscope and generating COI sequence data in the Kawahara Lab as a part-time assistant continued. Almost two years of CLM monitoring for a study to evaluate trap densities and lure efficacy decay under field conditions demonstrated that densities of 1 trap per 2 ha (5 acres) were sufficient to obtain reliable moth counts. Lure efficacy of the two pheromone brands tested declined by 25% after 3-5 weeks and 50% by 6-10 weeks during the spring through fall growing season in Florida. Therefore, large correction factors need to be applied to trap catch readings if traps are replaced at 8-13 weeks. Results obtained will help growers to optimize CLM monitoring programs. A manuscript submitted to the Journal of Economic Entomology retunred with useful comments from reviewers and editor that will allow us to make improvement to the article for publication and dissemination of these results. Mark-recapture work continued. A release of 1500 CLM adults in a naturalized and therefore low-citrus region of Southwest Florida (Picayune Strand) was conducted using the fluorescent powder marking technique. No released moths were recovered in the three week duration of this trial. Pyridine marking has also been attempted without captures of released moths. Efforts to improve the marking technique and study design continue.
Citrus greening is a devastating disease of citrus that has cost the Florida citrus industry over $3.64 billion. ‘Push-pull’ strategies because of their multi-component approach are often synergistic and have been successful in controlling insect vectors. The overall long-term objective of this research was to develop a push-pull system for the Asian citrus psyllid (ACP) that can complement integrated management systems in young citrus plantings. To achieve this goal, studies were conducted to optimize the attraction of psyllids to visual targets utilizing previously discovered wavelengths of light that were most attractive to ACP. ACP showed strong preferences to two choice visual targets. ACP were most attracted to unfiltered light that possessed UV. Polarized white light was found to be slightly more attractive then unpolarized white light. ACP were as attracted to yellow targets compared to green targets as to white light with UV. However, excluding UV from yellow targets caused a marked decline in ACP attraction to yellow compared to green. Contrast patterns using a black mark increased attraction of both yellow and green hues compared to the same hue that was unmarked. The results of the vertical assays provide a better understanding as to the various levels of attraction of ACP to different visual targets. An unexpected finding was that ACP were more responsive to light following a 12 hour decline in pressure. Elements of these findings were incorporated into cage assays containing two types of sticky traps. Standard yellow and green sticky traps were more attractive then translucent traps of the same wavelength. Black stripes on the green sticky traps did not enhance attractiveness towards ACP, while black stripes and black borders on yellow sticky traps did increase the attractiveness towards ACP by 10%. Polarization did not affect trap catch on clear or colored traps significantly on sticky traps, except for yellow translucent traps, which showed improve catches with polarizing filters present. Lastly, both green and yellow sticky traps were more attractive in the vertical rather than the horizontal plane. Effects of adding UV reflectance to yellow and green sticky traps with and without contrast patterns are in development to be tested in cage assays. Results from these studies represent crucial steps in the development and optimization of traps that can be incorporated into a push-pull management system. Furthermore, these results improve efficiency of passive traps to better serve stakeholders by providing a more accurate count of ACP in the field for management purposes.
Issue date for this grant was 13 July 2012. Objectives are: 1. Assess effects of abiotic factors (light quality, photoperiod, air flow, temperature fluctuations) on psyllid movement, 2. Evaluate physiological limits and biotic factors effecting of movement including feeding, egg load, infection status, and population density, 3. Evaluate techniques for tracking psyllid movement in the field for mark recapture studies, 4. Characterize seasonal patterns of ACP distribution and movement at different scales in the field, 5. Develop strategies to protect young trees from colonization by ACP utilizing UV reflection for repellency and insecticide treated trap crops (such as Bergera koenigii) to attract and kill. Objective 1. Bioasssays using a wind tunnel to determine ACP response to different stimuli have shown thus far a plateau effect in light intensity and air temperature. Movement increases incrementally with these two parameters to a point and then levels off. Also, ACP tend to move up wind as wind speed increases within the range of the fan. Objective 2. Dr. Stelinski’s lab ahs continued to investigate the movement capability of ACP using laboratory flight mills as a tool to quantify duration and distance of movement using a technique that was recently published as part of this investigation (Martini, X., A. Hoyte, and L.L. Stelinski. 2014. Abdominal color of the Asian citrus psyllid (Hemiptera: Liviidae) is associated with flight capabilities. Annals of the Entomological Society of America. 107: 842-847.). they are trying to understand the factors that may influence flight capability/capacity/or motivation in the Asian citrus psyllid (ACP). Currently, they are testing the hypothesis that application of a juvenile hormone analogue, fenoxycarb, may increase flight activity of ACP. One to four day old ACP were chilled for 3 min at -20 C’ and then 0.2 .L of a solution of fenoxycarb at 2.5 .g/.L in acetone was applied onto the abdomens of treated psyllids with a micro-injector. 0.2 .L of acetone only was applied onto psyllids used as negative controls. The psyllids were then maintained in Petri dishes with citrus leaf discs for 3 days. After 3 days, psyllids were tested on the flight mill to determine the effect of treatment on flight capability. The results collected to date indicate an increase of ACP flight activity following treatment with fenoxycarb. If the results are confirmed in subsequent replicates, we will perform biochemical bioassays to measure the titer of juvenile hormone in the green-blue morphotype of ACP (this morphotype is capable of long distance flights as documented in the above mentioned publication) versus in the gray-brown morphotype of ACP (this morphotype performs short flights). Our goal is to understand the underlying physiological mechanisms that allow a certain proportion of ACP to move long distances, while other proportions of the population do not appear to disperse. Objective 3. Mark release trials have been put on hiatus due to extremely low recapture rates. Objective 4. Yellow sticky traps set at three canopy heights continued with mixed results after 1784 captures. More ACP captures in the upper canopy in the older Charleston grove and a more even distribution at the younger and more uniform Bob Paul grove that still slightly favor the higher sticky card locations. However, the east side of the trees captures more than the west side at both locations. At both locations and also the English grove, ACP captures are greatest adjacent to a hammock or wind break. At Bob Paul, traps set up to assess ACP movement between insecticide sprayed and unsprayed blocks with low and high populations of ACP respectively indicate no significant differences with 153 captures to date. Objective 5. A paper reporting positive results using UV reflective mulch to repel ACP the first 2 years after planting has been published in the refereed journal Pest Management Science. Subsequent results indicate that the 5 ft strip of metalized mulch is not effective on trees more than 5 ft tall. Trials to determine the impact of reflective mulch on the height of ACP flight were resumed with warmer spring weather during which ACP movement increases and final results are pending.
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