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.
Report for period ending 9/30/13 Activities of the CHMA program assistant (7/1/13 – 9/30/13): During this reporting period, the CHMA program assistant provided support to the CHMA effort by attending various local CHMA meetings as well as statewide and multi-state meetings. Specifically, Mr. Page attended twelve (12) such meetings which included the following: (8/2) Hardee CHMA meeting, (8/13-8/15) Citrus Expo, (8/22) Sastsuma meeting in Valdosta, GA), (8/29) Packinghouse day, (9/4) Gulf citrus growers meeting in Ft. Myers, (9/10) Highlands grower forum, (9/11) Hardee CHMA meeting, (9/18) FDACS citrus grower/beekeeper meeting at CREC, (9/19-9/20) landscape show in Orlando, (9/23) grower meeting in immokalee, (9/24) Hillbillies Meeting, (9/26) Gulf CHMA meeting in immokalee. In addition to time spent attending meetings, other daily activities performed included continual updating of the CHMA graphs and rankings posted online, helping growers sign up for access to the CHMA mapping service, working our bugs in the mapping program and updating boundaries for new CHMAs (particularly in the gulf region). Other products produced during this time include a new revised CHMA brochure, a new set of pest fact sheets requested by growers at various CHMA meetings, and a brochure and video tutorial explaing how to utilize the CHMA mapping program. Mr. Page also gave updates on the CHMA program to one group of Brazilian visitors, a presentation to a group from Texas Mutual via phone, and also provided information on the Florida CHMA program’s success to a group of satsuma growers from Alabama.
Update for 9/30/13 Previous studies using an electrical penetration graph monitor revealed that not all systemic insecticides provide equal disruption of phloem-related feeding behavior by psyllids which have been demonstrated to be responsible for successful pathogen transmission. During this quarter, laboratory and greenhouse based trials were initiated to further examine the effects of soil-applied systemic insecticides on psyllid mortality, feeding behavior and ultimately measure the rate of pathogen transmission by psyllids on nursery plants treated with each of the insecticides tested. In this study, four soil-applied systemic insecticides were evaluated for effects on psyllid mortality and the rate of HLB infection among the four different treatments as well as an untreated control group of plants (to be assessed via PCR at a later date after a several month holding period). There were 15-20 replicate plants for each treatment tested. Products tested included imidacloprid (Admire Pro 4.6F), thiamethoxam (Platinum 75 WG), clothianidin (Belay 2.13 SC) and cyantraniliprole (Verimark) applied as a soil drench to containerized sweet orange nursery plants. Rates used were based on the recommended per tree field rate for a new reset approximately 3′ or less in height; imidacloprid (0.025 fl oz/tree), thiamethoxam (0.0131 oz/tree) and clothianidin (0.0229 oz/tree). For Verimark, the anticipated label rate for a tree of this size would likely be 15-20 fl oz/A, thus we tested the higher rate of 20 fl oz/A which equates to 0.1428 fl oz / tree. Prior to treating the trees with soil applications, leaf samples were collected for PCR analysis to confirm the HLB status prior to the initiation of the experiment. Next, soil applications were made to the containerized trees. Then trees were then held in a greenhouse for 3 weeks to allow time for systemic movement of the insecticide to all parts of the plant. Three weeks post application, 20 presumably HLB psyllids were caged on each tree and mortality assessed at 24, 48 and 72 hrs. After 72 hrs, all psyllids were removed and placed in alcohol to be later analyzed using PCR to confirm HLB pathogen presence. Plants were then held in the greenhouse for later testing (3-5 months) using PCR to determine if there was a difference in the rate of successful HLB pathogen transmission (inoculation) between treatments. The results obtained thus far assessing psyllid mortality on treated plants showed that there were significant differences in terms of psyllid mortality between soil-applied treatments. At 24 h, more than 90% of the psyllids confined to Admire Pro, Belay and Platinum treated plants were dead compared to 18% dead on the Verimark and 15% dead on the untreated plants. At 48h, 100% of psyllids were dead on Admire Pro, Belay and Platinum treated plants compared to 45% dead on the Verimark and 28% dead on the untreated plants, which at 72h increased to 90% of the psyllids dead for Verimark and 30% dead for the untreated plants. Results of EPG comparison of psyllid feeding between the four treatments tested and PCR results comparing rate of pathogen transmission will be provided in future reports when available.
Ultra High Performance Liquid Chromatography – Pesticide Residue Analysis (December 2013 Update) The majority of this quarter was spent working with technicians from Thermo to diagnose and repair the LC-MS. Repairs consisted of the Thermo rep systematically replacing parts one at a time until ultimately it appears that the machine is functioning correctly. These repair efforts were slowed by the holidays and waiting for the rep to receive each new part ordered after each succesive “fix” didnt work. We will get back to sample analysis after the new year.
Ultra High Performance Liquid Chromatography – Pesticide Residue Analysis (September 2013 Update) Work continued to compare protocols for analysis of potential insecticide residues in citrus nectar. Analysis of nectar samples collected from early in 2013 continued. More than 100 samples collected were analyzed and the results then compared with laboratories outside UF to compare results. The results from our lab in some cases were comparable to that of outside labs running the same samples, however, over a series of runs, the sensitivity of our analysis decreased such that we lost confidence in the results obtained. After running more samples, the LC-MS ultimately could no longer detect any chemcial standards injected. We are currently awaiting a specialist trouble shooter from Thermo to arrive to assist in diagnosing the problem with the machine.
Report for period ending 3/31/14 Activities of the CHMA program assistant (1/1/14 – 3/31/14): During this reporting period, the CHMA program assistant provided support to the CHMA effort by attending various local CHMA meetings as well as statewide and multi-state meetings. Specifically, Mr. Page attended ten (10) such meetings which included the following: (1/10/14) Volusia CHMA meeting, (1/14/14) Gulf CHMA meeting, (1/15/14) Valdosta CHMA meeting, (1/28/14-1/30/14) Citrus Show in Ft. Pierce, (1/31/14) Hardee CHMA meeting, (2/7/14 – 2/8/14) Alabama fruit and vegetable conference, (2/13/14) East Pasco OJ break, (2/19/14) Hardee CHMA meeting, (3/25/14) Hillbillies, (3/26/14) Dow sponsored CHMA meeting. On 2/20/14, Mr. Page also gave a presentation on the CHMA program for Congressman Tom Rooney who was visiting the area. The presentation informed the congressman of the importance of coordinated psyllid control efforts and the value to the Florida citrus industry. In addition to attending meetings, Mr. Page spent time providing support to the CHMA program by assisting CHMA captains in identifying hotspots of psyllids within their CHMAs, creating seasonal trend graphs for all the CHMAs, and updating the cycle data on the CHMA website. Mr. Page also worked with the computer programmers to rework the CHMA mapping website so that growers no longer must enter a password to view the psyllid count information. This was done to make the website more user friendly.
Entomopathogenic nematodes: Amending soils to increase biological control of insect pests We repeated moisture trials (described in September report) two additional times using both oven dried vs autoclaved soil. We unexpectedly, but consistently, found Catenaria sp. but no other nematophagous fungus survived desiccation in oven dried soil. The series of experiments reported here and in September 2013 demonstrated that S. diaprepesi and H. zealandica survived poorly in saturated soil compared to well-drained soil, whereas only Steinernema sp. survived better in saturated than in well-drained soil. The soil moistures used in these trials did not consistently effect the survival of H. indica. These results from controlled experiments support a recent report (Soil Biology and Biochemistry, 2013, 66:163-174) that the spatial patterns of these four EPN species in Florida citrus orchards were best explained by soil variables related to soil moisture (clay and organic matter content, water holding capacity, depth to groundwater). Comparative proteomic analysis revealed differences in protein expression by S. diaprepesi and Steinernema sp. maintained in well-drained and saturated soil. Detected proteins will be identified by LC-MS-MS to understand mechanisms of habitat adaptation. We found additional evidence for the hypothesis that soil moisture affects EPN community composition by sampling EPNs in 100 field plots from an experiment reported in 2013 (Biological Control 64:26-36). Briefly all four species of EPNs were added to native soil or to sandy soil used to fill tree planting holes in 2007 and again in 2010. The site is poorly drained with groundwater very near the surface and was originally inhabited only sparsely by H. indica. During the 4 year experiment, H. indica and the introduced S. diaprepesi dominated the EPN communities. However samples taken during this December reporting period (3 years after the original experiment terminated) revealed that H. indica and Steinernema sp. are both very abundant in native soil and in sand to the exclusion of S. diaprepesi and H. zealandica. The species specific effect of soil water potential on these Florida EPN natives appears to be the most robust physical variable studied to date for explaining, and potentially manipulating, naturally occurring EPN community structure. Plant parasitic Nematodes: Characterizing a new nematode pest and the prevalence of resistance breaking populations of the citrus nematode. Sampled soil and harvested fruit in 118, 4-tree plots at Ft. Pierce site. Between July and December, the numbers of X. vulgare more than doubled in untreated plots, compared with just a 15% increase in plots treated with oxamyl; however, the difference was not significant (P>0.05). Neither the oxamyl, nor the two experimental compounds reduced numbers of X. vulgare significantly compared to untreated controls. Treatments did not affect fruit yield per tree. However, the incidence of tree loss to citrus greening was especially high (20%) in untreated trees on one of the double-row beds where loss of trees treated with one of the experimental nematicides was just 4.0% (P=0.008). We characterized X. vulgare population densities in 20 sections of a heavily infested grove near St. Cloud and found no relationship between nematodes and either tree condition or root mass density. Soils are being analyzed to determine if there are physical or chemical properties related to the nematode population density or tree quality.
Entomopathogenic nematodes: Amending soils to increase biological control of insect pests EPN communities in the Bartow pH trial were characterized by qPCR in February, Two years after pH was reduced from 7 to 6 or lower by application of elemental sulfur. The composition of EPN communities in untreated and treated plots was similar to that reported in Feb and March 2013. Numbers of S. diaprepesi were nearly threefold higher (NS) and dominated the EPN community (P=0.001; Sd/total EPNs) in sulfur amended compared to non-amended plots. Numbers of H. indica (P=0.001) were sevenfold higher in the non-amended plots. The effects of sulfur were likely independent of EPN infestation by Paenibacillus sp. because the numbers of bacterial spores per plot did not differ between treatments. Although the number of spores per nematode in untreated plots was half that in the sulfur amended plots, the difference was not significant. Effects of EPNs in those plots on sentinel weevils will be evaluated in May. We repeated laboratory assays (reported 12/13) of EPN survival at different water potentials and pH levels. Use of field soil from Bartow experiment produced results consistent with those using soil from the APS experiment. Moisture and pH did not interact for any EPN species. S. diaprepesi and H. zealandica persisted better in dry soil, whereas soil moisture had no significant effect on H. indica or Steinernema sp. in one trial, but saturated soil favored survival of Steinernema sp. in a second trial. Based on the effects of soil moisture and pH on Florida EPN species tested individually, these physical properties appear to be variables that are manageable in orchards in ways that may enhance conservation biological control. We initiated experiments to test whether EPN community structure can be engineered by managing these properties. Mixtures of Sd, Sx, Hi, and Hz were incubated in soil with factorial treatments of low and high pH and low and high water potential. After various times, surviving nematodes were added to columns of soil (maintaining the different soil properties) with weevil larvae. EPNs that recycle in weevils in these columns will be periodically added to fresh columns with weevil larvae for several generations. EPN community composition of each generation in the different soils will be characterized. Field sites will also be identified based on moisture variablilty and soil types to implement field trials that manipulate both moisture and pH. Drafts of two manuscripts were initiated: ‘Soil pH modulates Paenibacillus spore adherance to Steinernema diaprepesi and the longevity of infective juvenile entomopathogenic nematodes’ and ‘Soil water potential and pH structure entomopathogenic nematode communities in microcosms.’ Plant parasitic Nematodes: Characterizing a new nematode pest and the prevalence of resistance breaking populations of the citrus nematode. Treated plots with oxamyl and and two experimental nematicides in Feb-March. Will sample and evaluate effects in May. Analyzed soil texture and fertility in samples from a heavily infested grove near St. Cloud (reported 12/13) and found no relationship with population density of X. vulgare. Established cultures of this species on citrus seedlings in the greenhouse for controlled studies of pathology and comparative root symptomatology. Detected putative resistance breaking biotype of Tylenchulus semipenetrans from orchard on Swingle citrumelo which will be cultured on indicator ‘host range’ rootstocks for comparison to populations recovered from non-Poncirus rootstocks.
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