Entomopathogenic nematodes: Amending soils to increase biological control of insect pests We repeated experiments to test whether EPN community structure can be engineered by managing soil water potential (see June 2014 report for results). Mixtures of Sd, Sx, Hi, and Hz were maintained with weevil larvae in soil low and high water potential. EPNs that recycle in weevils were periodically added to fresh columns with weevil larvae for several ‘generations’ (21 days each). During the first three ‘generations,’ Sd gradually, but consistently increased its presence in the EPN population from 45% to 55% in the dry (6% moisture) soil and remained steady at 22-30% in the wetter (18% moisture) soil. Steinernema sp. (Sx) constituted between 27-41% of EPNs in dry soil and 55-63% in moist soil, whereas Hz represented between 6-23% of EPNs without an effect of soil moisture. It is interesting that Hi did not persist in any treatment, given that Hi is the dominant EPN species in all of the ecoregions where citrus is grown. We reported that Hi is little affected by Catenaria spp., a fungal predator of nematodes, whereas the fungus is lethal to most other EPN species in controlled studies. Similarly, during monthly sampling for two years in 4 citrus groves, detection of Catenaria sp. was found to be inversely related to Sd abundance and positively related to Hi abundance (unpublished). Results such as these suggest the need to increase the complexity of the biota in these experiments to include natural enemies of EPNs in order to recreate selection pressures in microcosms that produce EPN communities comparable to those in the field. A future microcosm experiment will test this hypothesis. We continued comparing the biology of two closely related native species, Steinernema diaprepesi (Sd) and Steinernema sp. (Sx), with respect to survival mechanisms. When subjected to a range of relative humidity (50%-100%), Sx did not survive RH ‘90% whereas, Sd persisted on filter paper disks at 90% and greater RH. Moreover, when the two species were maintained 48 h in aqueous solutions of 30% glycerol, both lost volume with the loss of water but, upon rehydration for 24 h, most Sd became motile whereas all Sx ruptured. Thus, Sd (an inhabitant of well drained, central ridge soils) has superior capacity for osmoregulation and desiccation survival compared to Sx (an inhabitant of poorly drained, flawoods soils). An inferior capacity for osmoregulation may also affect the spatial pattern of Sx which has not been detected in coastal orchards where hypertonic conditions tend to be greater than in the inland flatwoods orchards. Plant parasitic Nematodes: Characterizing a new nematode pest and the prevalence of resistance breaking populations of the citrus nematode. Treated plots two nematicide trials conducted in east coast grapefruit on swingle. We identified a second site on the central ridge with 18-month-old trees heavily infested by dagger nematode (Xiphinema vulgare). Installed irrigation lines adjacent to exiting lines to initiate nematicide trials (oxamyl and an experimental nematicide/fungicide) that will continue for three years.
The project began on May first 2014 with two citrus groves selected for the project. A mature grove owned by English Bothers consists of 20 year-old Hamlin sweet orange trees predominately on Swingle rootstock. Sixteen full row main plots of 40 trees each were selected for irrigation water acidification at one of four target water pH (7.5, 6.0, 5.0, and 4.0). The second experimental site was established in a block of two year old Hamlin sweet orange trees on Swingle rootstock at Orange-Co’s Joshua block. Rows of trees with similar treatment plots were selected randomly with one row split into two main plots of 20 trees each. Initial soil measurements, indicated that soils at both sites were alkaline with average soil pH of 7.43 for English Bothers and 7.05 for Orange-Co. After irrigation water acidification treatments were initiated, soil pH at both sites decreased by October, 2014 to a range of 6.24 to 6.55 with no significant differences for treatment at either site. Soil ph in control blocks with no acidification remained at 7.3 for English Brothers and declined to 6.55 at Orange-Co. These treatments were applied during the rainy season with little irrigation applied at both sites. A total of approximately 39 minutes of irrigation at was recorded for a four month period (July through September) at Orange-Co. Irrigation during these four months were primarily to apply acidifying fertigation. Likewise, no significant differences in tree trunk measurements (17.0to 21.1 Mm), Phytophthora propagules (0.008 to 0.02 propagules/mg soil) ir root density (0.07 to 0.31 mm root/mm soil at 0-15 cm and 0.01 to 0.03 mm/mm at 15 to 30 cm) were found in June, indicating no difference in tree size due or root pathogen density for the acidification treatments. These results indicate that little soil acidification occurred during the summer rainy season because of low irrigation inputs.
Feb 2014 The objectives of this proposal are 1) Determine the base line level of Guignardia citricarpa sensitivity to fungicides registered for disease control in citrus and evaluate new products for efficacy against G. citricarpa in vitro; 2) Conduct and improve implementation of spray trials for efficacy of registered products for citrus and to evaluate novel compounds in the field; 3) Optimize field evaluation of control measures through analysis of the spatiotemporal disease progress utilizing past and current field data of the outbreaks to gain knowledge on the incidence, severity and rate of the epidemic and assess the fungal population to increase the likelihood of successful field research and 4) Evaluate products and treatment conditions for postharvest control of citrus black spot. This quarter we accomplished: Objective 1: All preliminary experiments have been completed for propiconazole, difenoconazole, and tetraconazole. Preliminary experiments with imazalil and fenbuconazole are on-going Objective 2: We’ve taken our first rating in the fungicide trial and will continue until fruit harvest. Too early to say how treatments are going to shake out. We are planning for next season’s trial. CBS lesions are now apparent on fruit at most sites so we’re planning to start some of the survey data next week. Objective 3: Suitable sites to conduct the spatial studies have been located. This study will begin in earnest close to fruit maturity when symptoms become apparent. Objective 4: Since the last progress report, work has continued evaluating the efficacy in vitro of the postharvest citrus fungicides imazalil (Freshgard 700, Active Ingredient: 44.6%) and pyrimethanil (Penbotec 400, Active Ingredient: 37.14%) at concentrations of 0.001, 0.01, 0.1, 1, and 10 mg/l active ingredient in the PDA medium on mycelial growth inhibition of four Guignardia citricarpa isolates. After 14 day, there was a near log-linear increase in mycelial growth inhibition increasing with 100% inhibition at the highest concentration. To evaluate heat effects on G. citricarpa, balls of mycelial masses cultured in liquid media were placed in solution at 55C for 0.5, 1, 2, 3, 4, 5, or 6 minutes, the flasks cooled by placing in an ice-water bath, the mycelia plated on . strength PDA media, and growth measured over 10 days. The control was exposed only to a solution at 25C. No treatment duration killed the G. citricarpa, but 55C exposure for 1 min reduce mycelial growth by half, and inhibition gradually increasing to about 75% after 6 minutes exposure. Neither chitosan at low concentrations (0.5 ‘ 10 mg/L), nor 50, 100, 200, or 300 mg/L salicylic acid (SA) inhibited G. citricarpa mycelia growth, but instead appeared to stimulate its growth slightly (SA) to moderately (chitosan) in the mid to higher concentrations tested. These experiments will be repeated.
We have compared the qualitative and quantitative expression in plants of amiRNAs engineered to target psyllids by directly expressing them from Agrobacterium tumefaciens, by using Tobacco mosaic virus (TMV) and by using a modified Begomovirus expression system (Tomato mottle virus, A component, TAV). Based on the results of Hi-seq illumina deep sequencing, TAV is the best candidate to express artificial microRNAs in plants. We are currently evaluating more samples by deep sequencing and engineering additional constructs to target psyllids. Objective 2 is to evaluate and optimize in planta expression of anti-psyllid interfering RNAs. We have started the leaf-disc feeding assays using our transgenic plants expressing double-stranded RNAs expressed from two different promoters (the 35S promoter for general tissue expression and the AtSuc2 promoter for phloem-specific expression) and the in planta transient expression of specific artificial microRNAs. So far the transient delivery/expression system has not worked for citrus. Thus, we are evaluating additional candidate interfering RNAs by in vitro feeding assays, and evaluating specific interfering RNAs in citrus plants infected with recombinant Citrus tristeza virus (CTV). The latter is in collaboration with Dr. W. O. Dawson. We have also been optimizing the conditions of our on plant feeding tests to have them close to natural feeding environment.
This proposal aimed to continue improving a novel psyllid trap and to use the trap to gather new information on the behavior, biology, population dynamics and biological control of ACP/Candidatus Liberibacter asiaticus. Lab and field testing was conducted to increase trap efficiency by exploiting unique vector behaviors in response to traps and behaviorally active components. Obj. 1: We continued to conduct field and laboratory studies toward obtaining an understanding of ACP trap response behavior by manipulations of visual cues as well as trap physical components and component orientation. During this final quarter we have successfully tested, winnowed out inferior traps, retested old and modified trap prototype configurations and developed new traps. Results indicate that the cylinder trap is the most efficient trap but other shapes have merit for other functions with ACP. For example, a cone shaped trap was found to attract ACP and also enhanced the ability to capture ACP in ways that indicate the trap could be readily used to protect and dispense fungal spores in the field. No increased responses by ACP with trap additions related to odor cues as reported to be potential attractants in the literature were observed. A sampling regime was initiated to try to delineate the northern most established populations of ACP in Florida. Sampling locations toward this end have been Gainesville, Live Oak, Quincy and Marianna, FL. To date ACP have not been detected at any sampling location north of Gainesville. A subsample of captured ACP (n=150) from Gainesville were analyzed monthly (W. Hunter) for the causal CLas bacteria of greening. Interestingly, to date none of the psyllids analyzed have been infected with CLas. An outbreak of another invasive psyllid species, Acizzi jamatonica, on the mimosa tree, Albizia julibrissin, was found in north Florida and this opportunity was exploited in several ways. ACP traps were tested for sampling of the adult psyllids, psyllid parasitoids were sought via observation and rearing, but none were detected. Adults of the psyllids were also evaluated for pathogen associates (A. Rooney, W. Hunter). The ACP trap worked equally well for monitoring of A. jamatonica. A similar effort was also deployed for the persimmon psyllid, Trioza diospyri. The ACP cylinder trap did not capture this psyllid species, but during sampling, a high percentage of the T. diospyri nymphs were found to be parasitized. Obj. 2: The areawide psyllid sampling to detect and develop new biological controls for use against ACP was pursued around the state. Personnel collected psyllids in “out-of-the -way” places that may hold relic citrus trees and relatively isolated ACP populations. So far several new pathogens have been identified. We have obtained cooperation with other USDA-ARS research personnel with expertise in the identification and rearing of entomopathogenic fungi and they have evaluated samples toward this end from Florida and Puerto Rico. Project Enhancement Grant to develop sentinel plants. A methodology to improve the management and use of sentinel citrus plants that are flushing was tested and developed. Flushing citrus plants in portable containers were highly attractive to ACP and appear to have merit for developing a sentinel plant platform that will provide a number of useful functions in the continued suppression of ACP, especially with initiating fungal epizootics, release of parasitoids, as a monitoring method, etc. This approach appears to have excellent potential as a system to deliver bio-based suppression tactics for ACP in non-commercial citrus venues such as abandoned and organic groves, as well as for urban areas.
The Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae), is naturally infected with Wolbachia (wDi). Recently,we calculated the within-host density of Wolbachia in Florida D. citri populations using a novel quantitative PCR for detection of the Wolbachia outer surface protein gene, wsp. Gene quantities were normalized to the D. citri wingless gene (Wg) to estimate Wolbachia abundance in individual D. citri. Using this method, significant geographic differences in Wolbachia densities were detected among Florida D. citri populations, with higher infection levels occurring in male versus female hosts. More recently, we have determined that Wolbachia densities are low during larval development and increase throughout adulthood. These data were recently published in Environmental Entomology. Three sequence types have been of Wolbachia have been identified in D. citri populations in Florida. These putative Wolbachia strains were characterized by identification of accumulated nucleotide differences in 5 conserved genes using multi locus sequences typing (MLST). Differences between ACP Wolbachia genes were determined by comparing consensus sequences to an MLST database. One sequence type in particular in found in only geographic region of Florida, and as such is a promising candidate for investigating interactions with Las transmission. Consequently, we are working to establish additional isofemale ACP lines from populations of psyllids predominately infected with the low frequency sequence type of Wolbachia through field collections. We are currently evaluating the phenotypic effects of strains (including cytoplasmic incompatibility) in established isofemale psyllid lines. Recent data suggests competition among Wolbachia strains. Evaluations of co-infection with native Wolbachia strains are currently underway to evaluate insect and symbiont fitness. In addition, we have identified an inverse relationship between Wolbachia and the ACP endosymbionts, Ca. Carsonella ruddii and Ca. Profftella armatura. Evidently, densities of these endosymbionts differ geographically, and in relation to Wolbachia strain infection.
Kaolin clays have been shown to be effective in reducing Diaphorina citri feeding on citrus leaves by inhibiting normal behavior. The overall objective of this project is to investigate the feeding deterrence provide by reflective colored kaolin clays on citrus plants from ACP. In the first quarter of this project kaolin clays have been procured from different sources including Surround kaolin clays product. We have also selected dyes, including FDA certified colorants, those approved for agricultural use by the EPA (list ‘180.920), covering a range of visible spectrum for coating clays. Particles size and zeta potential measurements of selected clay materials were performed to characterize the clay particles. Since the selected dyes have different structures and chemical properties, surface modifications of the clay are required for doping with dyes. Surface modification experiments were performed for kaolin clay products from different suppliers. It was observed that some of the clays changed the optical reflectance of the absorbed dyes and while others leached out earthly material during modification treatments which interfered with doping process. Clay that retained the dyes reflectance property as well as remained suspended in solution (making it easier for spraying) were selected for further studies. In the trials conducted, several dyes were successfully were sorbed to kaolin clays. Some examples of dyes used are FD&C Blue 2, FD&C Red 40, Basic Blue 54, Crystal Violet, and Basic Yellow 29, D&C Violet 2 etc. Experiments are being conducted to characterize and scale up the lab procedure to produce color clays for ACP greenhouse bioassays to determine reflective wavelength deterrence efficiency. Tests are also being conducted to further modified kaolin formulations for increasing rain-fastness.
Canker disease on fruit and foliage is under evaluation this month (October) for the following trials. Final results will be reported in the December progress report: 1) Soil-applied Actigard in an EPA-approved Experimental Use Program (EUP) in two east coast grapefruit groves conducted in collaboration with Syngenta. 2) Foliar applications of soluble copper and novel bactericidal formulations in comparison with film-forming copper formulations. Experimental formulations are tested at much lower metallic rates in grapefruit and Hamlin. Evaluation of fruit drop due to canker in Hamlin will be completed in November/December. 3) Evaluation of performance of copper sprays in grapefruit groves with windbreaks: 6 and 7 yr-old red grapefruit blocks surrounded by a 20-30 ft tall Corymbia torelliana windbreaks. 4) Performance of two applications of Firewall (streptomycin) in a 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 Firewall alone. Also evaluating Cu bioavailability and residual activity on grapefruit for copper oxide (Nordox 75G) and copper-zinc formulations (Nordox 30-30) during two periods of summer season (July and August) when rainfall and fruit expansion rates differ.
Natural ACP populations were sampled in trees to identify alternative hosts in major Florida citrus growing regions (specific to the dormant winter season). We completed a detailed survey of ACP presence in Lake Kissimmee State Park (Polk Country, FL), where we captured ACP consistently throughout a season. In this forest, occurrence of citrus is particularly rare (only three tangerine trees), and no Rutaceae were found in the trapping locations or at nearby (‘500 m) areas to where ACP were captured. In addition to trap capture data, complementary laboratory no-choice feeding bioassays for ACP performed using Red bay, gallberry, and blueberry. These were selected as possible alternative hosts, given their presence in locations where ACP were captured. The results suggest that ACP may have a wider alternative host feeding range than previously thought, which may allow for significant dispersal even through dense forests in Florida up to 2.3 km from large-area plantings of citrus. We have recently completed temperature bioassays evaluating the effects of temperature on the insecticide susceptibility and gene expression of ACP to address the final objective of the proposed project. RNA collected from insects exposed to heat (simulating spring, summer conditions) and cold (simulating winter conditions) have been collected and analyzed using quantitative reverse-transcriptase- (qRT-) PCR. Over two consecutive winters, we examined the abundance of D. citri in 52 groves throughout Florida, in response to four different management regimes defined as: conventional, intermittent, unmanaged, and organic. During both years, the winter abundance of D. citri in groves under intermittent management was greater than in groves under other management regimes. These findings suggest that only aggressive management was able to control D. citri population during winter periods. In contrast, intermittent management was associated with higher D. citri populations. The abundance of D. citri during winter periods was correlated with high levels of potassium and phosphorous in citrus leaves. Geographic information system analysis indicated that D. citri abundance was higher when more than 20% of the surrounding landscape was urbanized. Citrus variety had a significant effect on D. citri abundance; we found almost no psyllids in grapefruit groves in winter seasons, D. citri had higher abundance in sweet orange varieties . Number of rows and grove area significantly affected D. citri distribution; we found fewer psyllids in larger sampled groves. The interaction between row orientation and elevation had a significant positive effect on numbers of D. citri found in groves . While the number of straight edges (shape score) alone had no significant effect on D. citri abundance, the interaction between management regime and grove shape had a significant effect on insect abundance. Management regime affected the shape of groves; specifically abandoned and intermittently managed groves exhibited fewer straight edges due to patches of open space breaking the edge of groves.
Asian Citrus Psyllid (ACP) is the vector of HLB which is primarily responsible for the disease spread. Controlling ACP invasion could potentially minimize the disease spread and crop loss. This project is aiming at developing an organo-silica based composite film (OSCF) barrier material which will serve as an ACP protectant once spray applied. Preliminary ACP trial results conducted on some of the earlier versions of OSCF materials were promising. The OSCF material was applied on 20 citrus plants on every 2-3 weeks and compared against an industrially available compound, ActigardTM (applied every 30 or 60 days interval). Comparable efficacy was observed with an infection rate as low as 68% for OSCF and 55 ‘ 75% for ActigardTM. Both the OSCF and ActigardTM materials reduced infection in comparison to the untreated control group which expressed an infection rate of 85%. In this reporting period, we have further optimized the OSCF materials which involved two different USEPA approved environmentally-friendly polymers and plant micronutrient based ionic cross-linkers. A total of six different version of OSCF materials have been evaluated for phytotoxicity using Vinca sp (an ornamental model plant which is highly susceptible to phytotoxicity) and Persian Lime. As expected, none of these OSCF materials showed any tissue damage. To address any concerns related to heat trapping potential of OSCF film material once spray applied, we have performed a series of studies on Persian Lime. Surround’, a recommended pest deterrent based on Kaolin Clay was used as a standard control. Four of these OSCF materials showed plant temperature comparable to Surround’ standard after application. Further optimization process will involve adjustment of OSCF spray rate. A couple of the most promising OSCF formulations will be delivered for greenhouse testing against ACPs by the end of this year.
Citrus leafminer (CLM) is a major pest of citrus, causing direct damage and increased incidence/intensity of citrus canker. Pheromone traps are valuable 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. Species composition in trap captures continues 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. The probe set is designed to capture a tiled, 250 bp region of the 658 bp CO1 ‘barcode region’. Blast-searches against the comprehensive BOLD sequence library has allowed a faster, bulk-sequencing approach to the identification of leafminers. CO1 genes continue to be sequenced from samples to contribute to a maximum likelihood phylogenetic analysis. Sampling from Silver Strand B9 block, Okaloacoochee Slough State Forest, and Charleston Grove continues; several months’ samples are currently being prepared to ship to FSU for analysis. Photographs are taken of specimens prior to extraction, so that new species collected in OK Slough can be distinguished from P. citrella by external morphological characters, also continues. Photographs of the specimens will help elucidate these characters in order to provide more accurate identifications. Vince Ficarrotta presented some of this work at the Lepidopterists’ Society Annual meeting in Park City, UT in June 2014. Vince’s assistant, David Plotkin, now a graduate student, continued work photographing moths through a microscope and generating COI sequence data. David will be presenting some of the leaf-miner work at the upcoming ESA meeting in Portland Oregon. Recently, an undergraduate senior student, Emma Roulette, was added to the project to focus further on dissecting specimens of the two unknown species of Phyllocnistis that are attracted to lures. She plans to dissect and illustrate the genitalia and compare them with type specimens in the National Museum of Natural History to verify their identities. This portion of the project was initially conducted by Q. Jia in 2013, but we are continuing this work and examining additional specimens. A manuscript describing trapping efficiencies was submitted to the Journal of Economic Entomology. We are currently incorporating reviewer comments. Work towards marking techniques continues. Due to inconsistencies with surface-applied powders and diet-incorporated dyes, we will evaluate marking CLM with Rb as a dietary supplement. We are currently evaluating application techniques for this substance including work towards an artificial diet. Once a calibration curve is established, releases will be made and captures compared to background Rb concentrations to establish recapture rates and spatial features of CLM populations.
We have completed our annual survey for the insecticidal response of field populations of the Asian citrus psyllid (ACP) to a panel of insecticides with various modes of action. In years 2009 through 2012, we began to see a drop in susceptibility to all major insecticide classes in a number of ACP populations across central and southern Florida. In the surveys conducted in 2013 and 2014, we found a reversal in the LD50 response of all field populations to levels that were not significantly different from the reference laboratory strain. However, most population dose-response curves were significantly different from the laboratory strain and between one another in both years, suggesting that there is divergence in insecticide response between populations that may have a genetic basis. These results indicate that certain populations of ACP may be primed for resistance development if insecticide use does not continue to be managed strategically and conservatively. We will continue to monitor the insecticidal response in ACP closely. This year we added two additional sites in the survey for select insecticides, and plan to add additional ones next year as well to further our understanding of the natural and induced response of ACP to insecticidal exposure. Overall, it appears that rotations have been effective in decreasing the incidence of insecticide resistance in populations of ACP.
Successful area-wide management of the Asian citrus psyllid (ACP) and Huanglongbing (HLB) requires effective control of the psyllid in abandoned citrus groves. Since ACP adults are highly mobile, they can disperse from abandoned to productive citrus groves and spread HLB. ACP is susceptible to Isaria fumosorosea (Ifr), a native entomopathogenic fungi. ‘Autodissemination’ is insect dispersal of a pathogen to members of its own population. Our project goals are to develop and field test an ‘autodissemination’ system for inoculating ACP with Ifr and use these infected psyllids to instigate epizootics that will rapidly reduce ACP populations in abandoned citrus groves. During the summer (June-July) of 2014, we collaborated with Paramount Citrus to conduct field trials in Hildago County with three of their managed groves and three adjacent abandoned groves. The first site was a managed Rio Red Grapefruit grove next to an abandoned Rio Red Grapefruit grove. The second site was a managed Valencia Orange grove next to an abandoned Rio Red Grapefruit grove. The third site was a managed Rio Red Grapefruit grove next to an abandoned Valencia Orange grove. At each site, we evaluated the impact of Ifr autodisseminators on ACP movement and infestation in plots of abandoned citrus trees and managed citrus trees. For treatment plots, we hung pairs of autodisseminators coated with Ifr spore formulation and equipped with citrus-blend lures in trees on the edge rows of the abandoned groves. The Ifr formulation was provided by Dr. Mark Jackson (USDA-ARS, Peoria, IL) and based on a south Texas isolate (Ifr 3581). For control plots, we hung autodisseminators with only citrus-blend lures. To monitor ACP movement and populations, we released up to 1200 ACP adults marked with fluorescent powder on abandoned trees every week for four consecutive weeks and hung ACP sticky traps on the managed trees directly across from the abandoned trees. We used different fluorescent colors to distinguish psyllids released in treatment plots from psyllids released in control plots. Every seven days during the first to fourth weeks of the trials, we replaced dispensers and inspected ACP traps. During the fifth week, we also ‘tap sampled’ managed trees and abandoned trees. Marked psyllids released within treatment plots were not recovered on either managed trees or abandoned trees. Marked psyllids released within control plots were recovered on abandoned trees that the psyllids were not released on; however, only unmarked psyllids were captured by ACP traps on managed trees. We are repeating our trials in the three Paramount Citrus groves during the fall and winter (October to December) of 2014. To improve recovery of marked psyllids, we will tap-sample trees during each week of these trials and / or increase ACP trap densities. To facilitate future scalability and commercial production, the autodisseminators used for our summer field trials were assembled from durable plastic components fabricated by AlphaScent Inc. To reduce the cost for end users, AlphaScent designed and provided us with cardboard versions of key autodisseminator components. During August and September of 2014, we field-tested the cardboard version and plastic version of our autodisseminator and found that the cardboard version was adversely affected by heavy rain. As a result, we will use only the plastic version for our fall and winter trials. By the second week of October 2014, we expect to receive sufficient autodisseminator components from AlphaScent for our fall and winter trials.
Work continues towards calculation of LD50 and LD80s for some commonly used insecticides for management of citrus pests in Florida for a susceptible CLM colony. These values are being compared to wild populations to assess for possible resistance to the tested insecticides. Larvae and adults from the susceptible colony were exposed for 48 h to different doses (0; 0.01; 0.03; 0.1; 0.3; 1; 3; 10; 30; 50; 100, 300, 600 and 1000 ppm) of the following insecticides: Actara 25WG, Agri-Mek 0.15EC, Cyazypyr, Danitol, Delegate WG, Dimethoate, Intrepid 2F and Micromite 80WGS. The following baselines for active specific ingredients against CLM larvae have been completed: Actara (thiamethoxam) LD50=4.18 (CL95: 1.86-8.51) ppm, LD80=524.12 (CL95 209.70-1827) (n = 1047; .2 = 9.54; d.f = 10; Heterogeneity = 0.95); Agri-Mek (abamectin): LD50= 0.314 ppm (CL95: 0.13-0.69), LD80=16.68 ppm (CL95: 6.14-78.56) (n = 1249; .2 = 13.17; d.f = 8; Heterogeneity = 1.65); Cyazypyr (cyantraniliprole): LD50=43.36 ppm (CL95: 15.80-131.55), LD80=49,413 (CL95: 0.19E+07) ppm, (n = 1,196; .2 = 7.64; d.f = 8; Heterogeneity = 0.96); Danitol (zeta-cypermethrin): LD50=381.78 (CL95: 86.33-11,771), LD80= 37, 220 (CL95: 31.70-53761.1) ppm (n = 561; .2 = 5.29; d.f = 9; Heterogeneity = 0.59); Delegate (spinetoram): LD50=2.67 (CL95: 0.87-6.42), LD80= 93.75 (CL95: 33.89-489.58) ppm (n = 938; .2 = 10.69; d.f = 7; Heterogeneity = 1.53); Dimethoate (dimethoate) LD50=1.56 ppm (CL95: 0.15-56.31) and LD80=497.45 ppm (CL95: 22.27-0.37E+09) (n = 546; .2 = 13.07; d.f = 6; Heterogeneity = 2.18); and Micromite (diflubenzuron): LD50=74.30 (CL95: 13.63-285.15), LD80=5,723 ppm (CL95: 994-0.129E+07), (n = 816; .2 = 19.92; d.f = 9; Heterogeneity = 2.21). Because methoxyfenozide (Intrepid) is an insect growth regulator with apparently relatively low contact efficacy, its bioassay requires time to allow CLM larvae to develop through at least one instar. We are currently experimenting with modifications of the established bioassay to allow this time. LD50 and LD80 values for CLM adults with tested compounds are also currently being estimated. Preliminary results for Delegate (spinetoram) indicate LD50=2.84 (CL95: 0.77-73.74), LD80= 183.52 (CL95: 16.47-0.44E+07) ppm (n = 430; .2 = 6.30; d.f = 4; Heterogeneity = 1.58). Intrepid and Micromite LD50 and LD80 values are thus far inestimable (n= 703, n=381) as no treatment-associated mortalities have differed significantly from controls. This work continues. Since the last report, no incidents of putative resistance in field populations have been identified. However, we will continue to sample and test populations from commercial groves. A revision of the manuscript “Placement Density and Longevity of Pheromone Traps for Monitoring of Citrus Leafminer, Phyllocnistis citrella (Lepidoptera: Gracillariidae)” by P. Vanaclocha, M. M. Jones, C. Monz’ and P. A. Stansly is beng prepared for submission to the Journal of Economic Entomology
The overall goal of this three-year research project is to efficiently deliver antimicrobial molecules into citrus phloem against HLB bacteria. This quarter’s (from June to October 2014) research was focused on evaluating the effectiveness of 14 compounds in combinations and alone at various concentration against HLB bacterium. These compounds were formulated in O/W nanoemulsion for bark application. In addition, the enhanced-delivery field trial was also continued on evaluating efficiencies of these nano-formulations for combating HLB bacterium by bark application and gravity bag infusion. The results from greenhouse experiments showed that several treatments can suppress HLB bacterium with no phytotoxicity at 2 months after initial treatments, including Act, Va, Act+Va, penicillin and Zs. Eleven compounds have been treated by bark application in the potted plants, including Carv, Pcy, Pcy+Carv, Al(OH)3, Amp, KLS, SD, Proud, Act+Sdx, qui, and qui+Sdx. The qPCR analysis will be in the next quarter. In field trials, Pen and SDX in nanoemulsions can be effectively delivered into citrus phloem through bark application. After 2 months from initial treatment, HLB bacterial titer was reduced significantly, and no phytotoxicity appeared in these treated citrus trees. However, the bacterial titer increased a little after 6 months from initial treatment. Meanwhile, in gravity bag infusion field trials, the HLB bacterial titer was still lower after 6 months with Pen solution treatment , compared with other three treatments (combination of Pen and insecticide, SDX, and combination of SDX and insecticide). Due to the water insolubility of the insecticide, the compounds coupled with insecticide could not be effectively delivered into citrus phloem by gravity bag infusion. Therefore, the combination of compounds and insecticide treatment was replaced with the compounds alone. Insecticide was foliar- sprayed alone if necessary. In the future, all effective compounds in combination and alone against HLB bacterial will continue to be evaluated using a O/W nano-delivery system in the field. In the field trial, effectiveness of the compounds (Pen and SDX) against HLB bacterium by bark application and gravity bag infusion will be evaluated further, and agronomic performance of HLB-affected citrus treated with Pen and SDX will be investigated in next quarter.
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