We have completed our first objective to evaluate the effects of nursery practices (watering, citrus variety, potting media) on imidacloprid uptake and retention. The trial was conducted at UCR Agricultural Operations . Two citrus varieties were selected for this trial – Parent Washington navel and Limoneira 8A lemon, both on Carrizo rootstock. The trees were budded in June 2012 and grown in 5′ pots at LREC. Trees were transferred to UCR Agricultural Operations on June 13 2013 and transplanted on June 17 to no. 5 pots. Two potting media were chosen for the study that differed principally in the proportions of sand (10-30%) and redwood (40-60%). The evapotranspiration rate was determined for the two soil mixes and this data was used to select three watering levels for the study. The three levels were defined as ‘replacement watering’ (100-120% ET), ‘overwatering (150-200% ET) and ‘severe overwatering’ (300-400% ET). For the trial, trees were placed in a 12 x 25 grid pattern and randomized according to variety, soil mix and watering level. When the trees had adapted to their new potting media environment, they were treated on July 11, 2013 with 0.33 mls Admire Pro per cu ft potting media. Monitoring for ACP on all trees was also conducted at monthly intervals for the duration of the trial, and imidacloprid residues were determined for all trees by ELISA for 28 weeks post-treatment. There were very clear differences among citrus varieties and watering levels, but somewhat equivocal effects of soil type. Imidacloprid residues were not detectable at 16 weeks after application (especially in lemon at the high watering levels). We are currently preparing to conduct the second objective which will investigate the uptake and persistence of additional neonicotinoid insecticides. The trees (Parent Washington navel on Carrizo rootstock) have already been budded at Lindcove Research and Extension Center.
The objective of this study is to determine how enhanced nutrition of citrus plants may affect Asian citrus psyllid (ACP) biology. We have initiated this study with complementary field and laboratory experiments. We have begun the second year of the field experiments for this investigation. We are repeating the same protocol as last year; however, with this supplemental funding we are able to expand the investigation. This additional experiment is being conducted in a mature Valencia grove and is similar to the one we have been conducting for over a year in a Hamlin reset grove. Nutritional applications will continue to follow the Keplex’ recommendations for this year. At the end of April, we will start monitoring ACP nymph and adult populations, as well as flush development. We have also planned to screen for HLB infection status of the trees in this experiment with qPCR as we have done in the other experiment in Hamlin resets to determine how nutritional treatments may impact tree infection status. This will allow us to compare solid planting resets with mature groves.
We have initiated development of a novel insect behavior-modifying product to control the Asian citrus psyllid (ACP). Decoy is a biodegradable emulsion capable of releasing methyl salicylate (MeSA) at rates sufficient to disrupt ACP behavior and transmission of HLB, while attracting ACP insect predators and parasites to treated plots for three months. The formulation is under development by ISCA Technologies. The repellency of Decoy prototype formulations will be determined in laboratory-based cage studies. We have conducted research on how this pathosystem may affect biological control agents and how this may also be manipulated for control of ACP populations. EXPERIMENT 1: WASP RESPONSE TO VOLATILES FROM INFECTED VERSUS UNINFECTED PLANTS Las-infected plants were more attractive to T. radiata than uninfected counterparts (.2=9.76, d.f.=1, P=0.002). Similarly, uninfected plants that received damage by feeding ACP adults were more attractive to T. radiata than uninfested controls (.2=25.00, d.f.=1, P<0.001). The odors of adult ACP, in the absence of plants and/or plant damage, did not attract T. radiata as compared with blank odor controls (.2=0.03, d.f.=1, P=0.857). Finally, Las-infected plants infested with ACP were not more attractive than uninfected plants also infested with ACP adults (.2=0.12, d.f.=1, P=0.732). EXPERIMENT 2: WASP RESPONSE TO SYNTHETIC ODOR SOURCES MeSA was attractive to T. radiata at the 0.01.g dosage (.2=11.76, d.f.=1, P<0.001), but not at the 0.1.g (.2=0.93, d.f.=1, P=0.336) or 0.001.g (.2=2.79, d.f.=1, P=0.094) dosages as compared with the responses to solvent negative controls (Figure 2). Also, T. radiata were not attracted by either .-ocimene or D-limonene treatments, at dosages known to be attractive to ACP (.2=0.04, d.f.=1, P=0.835; .2=0.03, d.f.=1, P=0.853; respectively), as compared with the solvent negative controls (Figure 2). EXPERIMENT 3: EFFECT OF LAS INFECTION AND MESA LURE ON PARASITIZATION OF D. CITRI BY T. RADIATA The number of nymphs that were not found during the experiment on Las-infected versus uninfected (control) trees was not different (2.50'0.29 and 2.75'0.48, respectively). Similarly, the number of nymphs that were not found between MeSA-treated versus control trees was not different (3.00'0.40 and 3.25'0.25, respectively). Significantly more ACP nymphs were parasitized on Las-infected plants than on paired uninfected control plants in direct choice assays (Estimate= -2.37, SE= 0.43, z= -5.48, P<0.001, Figure 3a). In addition, treatment of uninfected citrus plants with a synthetic MeSA lure significantly increased parasitization of ACP nymphs by T. radiata as compared with otherwise identical uninfected plants that were not treated in this manner (Estimate= -2.50, SE= 0.42, z= -5.91, P<0.001, Figure 3b). We found that the specialist parasitoid of D. citri, Tamarixia radiata, was attracted more toward Las-infected than uninfected plants. We demonstrated that this attractiveness was due to the release of methyl salicylate. Parasitization of D. citri nymphs on Las-infected plants was higher than on uninfected controls. Also, parasitization was higher on uninfected plants baited with methyl salicylate than on non-baited controls.
We have completed the molecular cloning and characterization of the voltage-gated sodium ion channel in Asian citrus psyllid (ACP), and have designed an assay to investigate the gene frequency of allelic variations associated with pyrethroid resistance noted in other insect species as described in the January 2014 report. Validation of the assay is underway and is poised to begin screening insects from different locations across Florida concurrent with the annual field survey to monitor insecticide resistance. To better understand potential for pyrethroid resistance in ACP, we are pressuring colony insects with fenpropathrin and now several generations into a pressurized colony. In addition, we found that psyllids raised on Indian curry leaf vs. orange jasmine show a significant difference in response to fenpropathrin at the LD50 level, and are using this model to investigate the underlying biochemistry and/or molecular biology that contributes to this differential response. These results will allow us to better understand how insecticide resistance develops to pyrethroid insecticides in ACP, so we may better understand how to prevent it. This is important because pyrethroids are one of the most important insecticide modes of action currently being used for ACP management.
The objective of this study is to evaluate non-neurotoxic insecticides against Asian citrus psyllid (ACP), and provide growers with insecticides with varying mode of action for management of ACP. We are investigating those non-neurotoxic insecticides those have shown promise against insect pests similar to ACP. These insecticides with varying modes of action that differ from neurotoxic insecticides may not only prove effective against ACP, but also may assist in ACP resistance management. Diofenolan was evaluated for its ability to inhibit the egg hatch of 49-96 hrs aged eggs. Percent egg hatch inhibition of 7, 18, 31, 42, 65 and 91 % was observed when eggs were treated with concentrations of 0, 20, 40, 80, 160 and 360 ‘g/ml, respectively. To determine the effects of diofenolan on survival of various developmental stages of ACP, we treated first instar nymphs with concentrations ranging between 0-360 ‘g/ml of diofenolan. We observed 95, 69, 38, 27, 23, 8 percent survival of ACP into the second instar for 0, 20, 40, 80, 160, 320 ‘g/ml concentrations, respectively. Currently, we are investigating the effect of diofenolan on the development of third and fifth instars nymphs. We will also investigate the effect of diofenolan on fertility and fecundity of ACP females. Following completion of evaluations of diofenolan (JH analog), we will initiate evaluation of tebufenozide and methoxyfenozide (ecdysteriod agonists). The results of our research suggest that non-neurotoxic insecticides are effective in disrupting growth of the immature stages of Asian citrus psyllid and are possible useful tools as part of an integrated program.
Our objective for this project has been to evaluate botanical compounds as repellents for Asian citrus psyllid (ACP) with the purpose of developing possible repellent formulations for use in the field. Current investigations are focusing on a longer time course of effectiveness of these extracts and subsequent field testing. We have continued to analyze behaviorally active botanical oils by GC-MS to determine possible constituents that may contribute to behavioral activity in the associated laboratory and field bioassays and for development of synthetic odor blends as tools for ACP management. The components were categorized according to their relative percent area on GC-MS chromatograms have been determined. In recent evaluations, we detected nine different compounds in crushed citrus flush. The two most abundant compounds were sabinine and ocimene, followed by limonene, .-pinene, and ‘-3-carene. Low amounts of .-pinene, terpinene, phellandrene, and terpinolene were also detected. Camphor oil headspace contained a large amount of cineole and lesser amounts of limonene, .-pinene, and cymene. Sabinene, .-pinene, terpinene, phellandrene, camphene, and myrcene were also detected in camphor oil headspace. Citronella oil contained large quantities of limonene, lower amounts of citronellal, linalool, geraniol, and isopulegol. Litsea also contained large amounts of limonene and .-pinene and contained lower amounts of: sabinene, .-pinene, terpinene, cineole, camphene, myrcene, linalool, citral, and santene. Fir oil contained large amounts of .- and .- pinene, ‘-3-carene, and camphene, and lesser amounts of limonene, terpinolene, cymene, myrcene, santene, tricyclene, and bornyl-acetate. Surprisingly, clove oil did not share any major constituents with young citrus leaves (flush) utilized by ACP for egg laying and development. Clove oil was comprised of caryophyllene and eugenol, acetic acid, humulene, and furfural. GC-MS analysis was the basis for creation of two synthetic clove odor blends: a .-caryophyllene and eugenol mixture, representing the major components, and a mixture of .-caryophyllene and eugenol, acetic acid, and humulene. Settling Bioassays. In no-choice settling experiments, neither the low nor high fir oil treatment deterred ACP from settling on plants as compared with the negative control. Thereafter, ACP were presented with a choice between control plants and fir oil ‘treated plants with a single dose of fir oil released from polyethylene vials. In this case, the ACP disproportionately settled on control plants as compared with treated plants. Fir needle oil repels ACP females in laboratory assays. ACP were deterred from settling on plants treated with fir oil. Based on these results, the efficacy of fir oil as a repellent in the field may be affected by the availability of surrounding untreated citrus hosts as well as by physiological deprivation of ACP from their feeding and egg laying sources. Gravid female ACP deprived of an egg laying source for a certain period due to deployment of repellents may eventually lay eggs on plants treated with the repellent because of increased physiological need to deposit eggs as described by the rolling fulcrum model (Miller & Strickler, 1984). In other words, female ACP may eventually begin to deposit eggs on sub-optimal resources, such as those treated with a repellent, if deprived sufficiently long. In this scenario, citrus grown by homeowners among agricultural landscapes may serve as a refuge for ACP in areas where repellents, such as fir oil, are implemented for management. The proximity of backyard, or otherwise unmanaged citrus, may therefore impact the efficacy of repellents as also observed with insecticides.
The objective of this research is to evaluate non-neurotoxic insecticides against Asian citrus psyllid (ACP), and provide growers with additional tools for management of ACP. We are investigating those non-neurotoxic insecticides that have shown promise against insect pests similar to ACP. These additional tools may not only prove effective against ACP, but also could assist in ACP resistance management programs as needed tools for effective rotation with current insecticides. In the past quarter, we have been further evaluating methoprene in the laboratory. Methoprene is a juvenile hormone (JH) analog that acts as an insect growth regulator. We evaluated effects of methoprene on fertility and fecundity of Asian citrus psyllid. Reduced fertility of females was observed when they emerged from fifth instar nymphs that were treated with methoprene at 128 and 256 ‘g/ml concentrations. When female adults were topically treated with 128 and 256 ‘g/ml concentrations of methoprene, reduced fertility of ACP was observed for a week following treatment. Methoprene treatments, at 128 and 256 ‘g/ml concentrations, had no effect on the fecundity of females emerging from treated fifth instars or topically treated females. We also investigated feeding behavior of ACP adults on plant surfaces treated with methoprene concentrations ranging from 0-320 ‘g /ml. There was reduced feeding of ACP adults when placed on surfaces treated with methoprene at 160 and 320 ‘g/ml concentrations.
This is a cooperative research project between Co-PIs Joseph Morse, Jim Bethke, Frank Byrne, Beth Grafton-Cardwell, and Kris Godfrey. One objective is to coordinate with researchers working on chemical control of ACP in Florida, Texas, Arizona, and elsewhere. Towards that end, Grafton-Cardwell and Morse attended the CHRP meeting in Denver 15-17 October 2013; in addition, there have been substantial emails, phone calls, and interaction at meetings with ACP researchers from FL and TX over the past 6 months. Jim Bethke is rearing ACP in a contained greenhouse at the Chula Vista Insectary (San Diego County; about 6 miles north of the Mexican border) under permit from CDFA. Since March 2013, Jim and staff have run 19 screening trials (14 focusing on organic products) and a Citrograph article will appear in the next issue summarizing all work on organic pesticides. A second location where we are working with ACP is at UC Riverside, working under permit inside the UCR Insectary Facility. Frank Byrne is conducting trials on various neonicotinoid insecticides (7 trials with early instars, 3 with late instars since 3-13) and Morse is evaluating the baseline susceptibility of CA ACP to various pesticides (27 trials since 3-13) in comparison with studies done in Florida. Three ACP field trials have been run since 3-13 evaluating organic products useful in control of ACP; also 7 local field trials (6 organic) in which leaves are treated in the field, allowed to weather naturally, and bioassays are done with adult ACP to determine how long residues continue to kill adults. Kris Godfrey is rearing ACP under permit at a 3rd location, i.e. inside UC Davis’ Contained Research Facility. She has run trials evaluating new pesticides such as Pest Out and Grandevo and has a third trial with Venerate planned for later this year. Beth Grafton-Cardwell has updated online ACP pest management guidelines. She serves as a focal point for communicating with ongoing ACP management programs in the SJV, Ventura, and elsewhere. Over the past year, she has given 15 talks to various organizations on ACP-HLB topics and Morse has given 8. Both Grafton-Cardwell and Morse serve as co-chairs of CDFA’s recently convened Science Advisory Panel dealing with ACP and HLB. The SAP held a meeting in Ontario 3-4 December 2013 and their report was submitted to CDFA 16 January 2014. In summary, we continue to expand our ability to conduct ACP research in California with a clear focus on management of HLB (rather than ACP).
A “Bugphone” microcontroller device that detects Asian citrus psyllid male vibrational calls and plays back female replies was tested for its ability to attract males to a sticky cone attached to a small tree. In these studies, 20-30% of tested males have responded to the Bugphone, but for various reasons, only a small percentage of the males that approached the cones were captured. Alternative trap shapes and devices are being tested to increase the percentages of males captured. In addition, Dr. Eisenstadt and several of his electrical engineering students at the University of Florida are interested in improving operation of the Bugphone by reducing its energy usage. We are at the beginning stages of modifying the system to enable operation for a month or more without battery replacement. In a new phase of this study, we have purchased outdoor speakers that can be operated with small amplifier systems to produce signals interfering with male and female ACP duetting calls field environments. Initial studies of the capability of the systems to disrupt mating have begun.
Update for 3/31/14 During this quarter, Enhancement Funds were used for work that continued on a field trial examining the timing of soil applied insecticides on young trees with the goal of determining the potential for insecticide translocation of product to floral nectar. Monthly applications continued through February which was the time when bloom began. During the bloom period, nectar was sampled on three dates. Sampling was conducted by hand picking 100+ blooms per plot and taking back to the laboratory where a capillary tube was used to collect nectar from each flower. Nectar collected from all flowers from each plot was pooled in one capillary tube and then placed into the freezer for analysis using LC-MS-MS at a later date. Enhancement Funds were also used to supplement work on a greenhouse trial initiated in the fall where four soil-applied systemic insecticides (imidacloprid, thiamethoxam, clothianidin and cyazypyr) were applied to potted citrus and challenged with psyllids containing the Las bacterium. The purpose was to provide direct evidence of the ability of these insecticides to prevent pathogen transmission via disruption of phloem feeding by psyllids. To date, we have collected psyllid mortality data from this experiment and have recently completed collecting and grinding all leaf material (allowing 6 mo for pathogen latency in plants) from the 100 plants used in this study. The DNA extractions from processed leaf samples followed by PCR to confirm successful pathogen inoculation will be conducted prior to the next reporting cycle.
Materials have been secured, plans have been made, and the project has been initiated.
Abandoned citrus groves in US citrus-producing regions are potential sources for both Asian citrus psyllid (ACP), and Candidatus Liberibacter asiaticus (CLas), the bacterium that causes Huanglongbing (HLB). In Florida and Texas where grower-driven area-wide management programs are underway in commercial groves, no psyllid control is being implemented in abandoned citrus. Since ACP adults are highly mobile, they can disperse from abandoned to productive citrus groves. If not controlled, these psyllids will stymie the effectiveness of area-wide management programs aimed at containing the spread of HLB in commercial citrus. ACP is susceptible to a native entomopathogenic fungus, Isaria fumosorosea (Ifr). For our project, we are developing a novel autodissemination system that will inoculate ACP with Ifr and use these infected psyllids to instigate epizootics and rapidly reduce ACP populations in abandoned groves. To accomplish our project goals, we had previously located five pairs of adjacent managed groves and abandoned groves in the Rio Grande Valley. These pairs of managed and abandoned groves consisted of orange or grapefruit trees and the abandoned groves had not been managed for at least 2 years but still retained a large number of live trees. When we revisited these five pairs of groves during January of 2014, all of these sites were no longer suitable for our field trials because either the abandoned groves had been removed or the previously managed groves were now also abandoned. As a result, we had to resurvey the citrus producing areas of Hildago and Cameron counties for suitable field sites. Fortunately, during February and March of 2014, we located another eight pairs of managed groves and abandoned groves that met our requirements. These groves will be used for field trials during the spring and summer of 2014 that will evaluate: (1) Impact of Ifr autodisseminators on ACP populations in abandoned and managed citrus trees, and (2) Effect of time and exposure on Ifr autodisseminators. Before the start of our field trials, it was essential to evaluate potential field sites and refine project goals, protocols, and time lines with our USDA-ARS collaborators. During February 19-21 of 2014, Drs. Christopher Dunlap (USDA-ARS, Peoria, IL), Mark Jackson (USDA-ARS, Peoria, IL), and Joseph Patt (USDA-ARS, Fort Pierce, FL) met with us at the Texas A&M Citrus Center in Weslaco. For this meeting, we provided our collaborators with a summary of the studies completed to date, discussed the studies that were planned for 2014, and took them to potential field sites. Three important outcomes of our meeting were: (1) the design of our autodisseminator was modified to not only enhance it’s ability to attract and retain ACP adults but also improve it’s protection of the Ifr formulation from degradation due to exposure to UV light, rainfall, and other environmental factors, (2) the Ifr formulation will be modified by Dr. Jackson to not only enhance its infectivity of ACP adults under field conditions but also facilitate its application onto the parts of the autodisseminator that the ACP adults will be attracted to, and (3) it was decided that key components of our autodisseminator will be pre-fabricated by AlphaScent Inc., a US supplier of insect monitoring systems, to facilitate the assembly of the large numbers of autodisseminators required for our field trials.
This project is in furtherance of the commercial release of a new product for control of the citrus leafminer and associated spread of citrus canker disease based on a deployment device for the sex pheromone of the leafminer, DCEPT CLM’ (ISCA Technologies, Inc.). Under an agreement with the Commercial Product Delivery Committee of the CRDF, funds are provided to ISCA to subsidize two years of production of DCEPT CLM sufficient to treat 3,000 acres of citrus, mostly grapefruit, at three locations in St. Lucie and Charlotte counties in April/May of this year. The remaining cost of the product is provided by the growers. Funds are also provide to ARS and University of Florida to support monitoring and analysis of the experiments at the three locations. In addition to monitoring efficacy and longevity, the experiments will provide data on the effect of immigration of gravid female leafminers from outside of the treated areas. The application of DCEPT CLM at one location (Emerald Grove, The Packers of Indian River) is contiguous with untreated citrus, a source of immigrant females. Another site (VPI-5, Golden River Citrus Co.) is isolated and surrounded by natural area and pasture and therefore should receive few immigrants. Efficacy and longevity will be compared between these sites to estimate the magnitude of the effect of immigration. Another variable that is known to affect the ability to establish mating disruption in a grove is the structure of the tree canopy. Declining trees, replants or new plantings allow for increased air movement and presumed loss of pheromone compared with complete mature canopies. This variable will also be considered.
Update for 3/31/14 During this quarter, work continued on a field trial examining the timing of soil applied insecticides on young trees with the goal of determining the potential for insecticide translocation of product to floral nectar. Monthly applications continued through February which was the time when bloom began. During the bloom period, nectar was sampled on three dates. Sampling was conducted by hand picking 100+ blooms per plot and taking back to the laboratory where a capillary tube was used to collect nectar from each flower. Nectar collected from all flowers from each plot was pooled in one capillary tube and then placed into the freezer for analysis using LC-MS-MS at a later date. Work also continued on a greenhouse trial initiated in the fall where four soil-applied systemic insecticides (imidacloprid, thiamethoxam, clothianidin and cyazypyr) were applied to potted citrus and challenged with psyllids containing the Las bacterium. The purpose was to provide direct evidence of the ability of these insecticides to prevent pathogen transmission via disruption of phloem feeding by psyllids. To date, we have collected psyllid mortality data from this experiment and have recently completed collecting and grinding all leaf material (allowing 6 mo for pathogen latency in plants) from the 100 plants used in this study. The DNA extractions from processed leaf samples followed by PCR to confirm successful pathogen inoculation will be conducted prior to the next reporting cycle.
Update for 3/31/14 During this quarter, work continued on a field trial examining the timing of soil applied insecticides on young trees with the goal of determining the potential for insecticide translocation of product to floral nectar. Monthly applications continued through February which was the time when bloom began. During the bloom period, nectar was sampled on three dates. Sampling was conducted by hand picking 100+ blooms per plot and taking back to the laboratory where a capillary tube was used to collect nectar from each flower. Nectar collected from all flowers from each plot was pooled in one capillary tube and then placed into the freezer for analysis using LC-MS-MS at a later date. In addition to the experimental trial described above, we also sampled commercial citrus groves around the state to determine whether if growers follow the currently recommended young tree care program utilizing soil applied neonicotinoids, would there be detectable levels of such products in floral nectar of young tree plantings. During the bloom period of 2014, blocks of young trees were sampled from both central and the east coast of Florida. Special attention was made to survey primarily those trees in the 5-9′ size class. Blooms collected from each commercial grove were taken back to the lab and processed as described above. For the large majority of samples analyzed, residues levels were at or below the level of detection. In cases where residues were detected, they were below the level of concern where applications were made more than 6 weeks prior to bloom. Work also continued on a greenhouse trial initiated in the fall where four soil-applied systemic insecticides (imidacloprid, thiamethoxam, clothianidin and cyazypyr) were applied to potted citrus and challenged with psyllids containing the Las bacterium. The purpose was to provide direct evidence of the ability of these insecticides to prevent pathogen transmission via disruption of phloem feeding by psyllids. To date, we have collected psyllid mortality data from this experiment and have recently completed collecting and grinding all leaf material (allowing 6 mo for pathogen latency in plants) from the 100 plants used in this study. The DNA extractions from processed leaf samples followed by PCR to confirm successful pathogen inoculation will be conducted prior to the next reporting cycle.
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