This project, in general, seeks to determine if there are any observable influences of foliar fertilizers on HLB-affected grapefruit in the Indian River marketing district. To-date, this project has established 3 separate research trials in commercial grapefruit groves and university research farms in St. Lucie county, Florida. Trial #1 is ~25 years old of ‘Flame’ grapefruit on Swingle rootstock. Trial #2 utilizes ~6 year old ‘Ruby Red’ on Sour orange trees. The first 2 trials are located in commercial groves. Trial #3 seeks to determine the effect(s) that foliar fertilizers have on young tree growth and their ability to protract HLB disease symptoms in 1 year old ‘Ray Ruby’ grapefruit on Kuharske rootstock. For trial #1, there are a total of 30 replicated experimental units; for trial #2, there are a total of 50 replicated experimental units; and, for the trial #3, there are a total of 24 replicated experimental units. Foliar fertilizer applications have been started since February 2014. Tree growth data, leaf nutrition data, and CLas titer measurements have been made. Fruit drop monitoring will begin in September 2014 in trials #1 and #2. In prosecution of these efforts, 2-part-time OPS employees have been hired to do our foliar sprays and collect data observations. As of January 2015, the first year of data collection on the commercial grove plots (trials #2 and #3) as well as the young tree trial at the IRREC (trial #1). First-year fruit yields were gathered in the commercial trials (trials #2 and #3) in December 2014. In addition, all of the foliar fertilizer applications were made (once per quarter) to all trials. Currently, data are being analyzed from the first growing season (2014). In 2015, foliar fertilizer treatments, tree growth measurements, and leaf nutrient analyses have commenced as of January and March, 2015, and will continue throughout the year in a manner similar to the calendar of activities last year (2014).
The overall objective of this project is to investigate the feeding deterrence provide by reflective colored kaolin clays on citrus plants from Asian Citrus Psyllid. Previously we reported the development of colored kaolin clays with different dyes, covering the visible spectrum. During this reporting period, experiments have been conducted to determine how the coating with colored clays modifies the reflectance of citrus leaves between 300 -700nm. Reflectance experiments performed using ten modified kaolin clays show a significant improvement in reflective properties as compared to standard kaolin. Of the colored kaolin clays applied, kaolin modified with basic red and violet dyes also altered the reflectance of citrus foliage in the ultraviolet range. In addition, citrus foliage coated with colored kaolin clays, have been categorized using a portable MiniScan spectrophotometer (HunterLab). MiniScan measurements are made based on CIE L*a*b* color space approach. Measurements were made on citrus foliage before and after application of colored coatings. Sites of measurements were marked on the leaves to minimize the error due to variability in the leaves. All colored kaolin clays were sprayed and allowed to completely dry on the leave surface. When comparing the color index measurements before and after coating, each colored clay showed to have a significant change in color strength and showed changes differing in magnitude in L*a*b* scale. For example Kaolin clay colored with violet dye showed significant change in color strength, particularly in b* value, indicating a change in the blue color strength. Further experiments and analysis of change in color index with different coatings are underway. Also experiments to increase the rainfastness of the coatings are underway. To promote rainfastness, clay formulations modified with agricultural grade spreaders and stickers have been prepared with minimal effect on the dyes. In addition, formulations are being prepared with adjuvants that can prevent the run off and enhance the adhesion of clays to the citrus foliage. Additional experiments such as effect of coatings on gas exchange and ACP are planned for coming months.
The overall objective of this research project is to develop a potential non-phytotoxic, environmentally friendly film-forming ACP repellent solution for preventing HLB infection. Earlier we developed a series of Organo-Silica Composite Film (OSCF; ACP repellent) materials using various polymers and cross-linkers. These film materials were screened for temperature safety (heat trapping) and phytotoxicity. Out of which, two best performers, OS-SG 6 and OS-SG 10 were delivered for evaluating ACP deterrent efficacy in field conditions. Field trial results did not show any promising efficacy when compared with control. After receiving feedback from field trial results, we have developed two new OSCF materials (OS-SG-11 and 12) using EPA approved polymers and particulate fillers to increase rainfastness and roughness. and environmentally safe inert ingredients to increase rainfastness. Dynamic Light Scattering (DLS) characterization results revealed formation of large multi-micron composites (~ 1 – 5 microns). Scanning Electron Microscopy (SEM) shows the film with rough surface morphology. UV-Vis and NMR spectroscopy results of OS-SG 11 and 12 show strong interaction among film components. The introduction of filler material improved film rainfastness and roughness. Both OSCF 11 and 12 were non-phytotoxic when tested on ornamental plant Vinca sp (highly susceptible to phytotoxicity). Phytotoxicity experiments were conducted using Panasonic Environmental Test Chamber (Model MLR- 352H) that simulated typical FL summer conditions (85% RH; 35 degree C).
Work continues towards estimation of baseline LD50s and LD80s of susceptible CLM some commonly used insecticides. Larvae and adults are 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: Actara 25WG, Agri-Mek 0.15EC, Cyazypyr, Danitol, Delegate WG, Dimethoate, and Micromite 80WGS. Intrepid (methoxyfenozide) was evaluated on leaves immediately transferred to a water source after excision and evaluated after 96 h due to allow effects of this compound’s mode of action (growth regulator) to become apparent. We found this method to be more efficient than caging plants or artificial diets and feel that results are representative of commercial applications. Updated results for CLM larvae: 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= 0.360 ppm (CL95: 0.14-0.80), LD80=34.6 ppm (CL95: 11.7-189.7) (n = 1350; .2 = 13.10; d.f = 9; Heterogeneity = 1.46); 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=2.49 (CL95: 1.00-5.19), LD80= 86.58 (CL95: 36.60-308.13) ppm (n = 964; .2 = 10.08; d.f = 8; Heterogeneity = 1.26); Dimethoate LD50=2.49 ppm (CL95: 1.01-5.19) and LD80=86.58 ppm (CL95: 36.60-308.13) (n = 629; .2 = 10.08; d.f = 8; Heterogeneity = 1.26); 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). For Intrepid, LD50=2.95 (CL95: 0.24-10.30) LD80=64.11 ppm (CL95: 18.36-814.8), (n = 125; .2 = 4.42; d.f = 6; Heterogeneity = 0.736). Updated results for CLM adults: residual contact toxicity of Actara has now been estimated as LD50=16.75 (CL95: 5.00-91.73) LD80=4153 ppm (CL95: 417.4-0.241 E+07), (n = 377; .2 = 0.688; d.f = 3; Heterogeneity = 0.229). Micromite – a further 240 adult CLM were tested at various concentrations up to 1000 ppm since the last report: LD50=476.02 (CL95: 192-1630) LD80=39,483 ppm (CL95: 7988-63,435), (n = 756; .2 = 4.42; d.f = 6; Heterogeneity = 0.736). Delegate (spinetoram) replicates also increased: LD50=24.93 (CL95: 5.37-358), LD80= 742.5 (CL95: 92.76-0.48E+06) ppm (n = 743; .2 = 12.02; d.f = 5; Heterogeneity = 2.4). Agri-Mek LD50=74.51 (CL95: 45.31-165.33), LD80= 364.67 (CL95: 164.59-1974.1) ppm (n = 240; .2 = 4.35; d.f = 3; Heterogeneity = 0.145). Contact toxicity for Intrepid LD50 and LD80 values remain inestimable for CLM adults. Evidence for resistance has not been found in CLM larvae collected from commercial groves.
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. Laboratory Technician, Vincent Ficarrotta has continued to extract DNA from P. citrella samples. An additional 196 samples have been sequenced for COI barcoding to target the 658 bp region of COI gene. We recently switched extraction methods from QIAGEN DNA Blood Tissue Kit to OmniPrep. The dataset was merged with our previously published genetic dataset (Kawahara et al. 2013) generated from this project. The OK Slough had the highest number of samples sequenced, and within these samples, P. citrella was the most common, followed by P. vitegenella. Other species, such as the two new Phyllocnistis species, are lured to traps at a low frequency (’11 specimens/year) in all years sampled thus far. While the numbers show strong bias towards P. citrella, it is clear that in all years, multiple species of Phyllocnistis readily come to CLM lures, following the initial finding of Kawahara et al. (2013). We also generated a new ML tree with additional samples analyzed on UF’s High Performance Computing Cluster (HPC). This tree shows largely similar results as Kawahara et al. (2013), where the majority of samples obtained grouped with already-known specimens of P. citrella. Bootstrap values for clades were generally low for most groups (<50%), which is comparable to prior results based on a single marker (e.g., DePrins and Kawahara [2012]). However, there were some notable results, such as the strong sister group relationship of P. citrella (North American) to P. citrella (Asia). Further, there is strong support for the monophyly of P. citrella (NA and Asia), P. insignis, and Phyllocnistis new sp. 1. Other taxa that were included in the dataset, but not generated from this particular project, also show strong bootstrap support for their monophyly, which will be relevant to future studies on non - P. citrella related species in the genus (e.g., P. hyperpersea (100% BP), P. longipalpus (100% BP), P. perseafolia (100% BP), P. populiella (93% BP), P. saligna (100% BP)). We also see relatively strong genetic distance between each group (>2-4% in many cases), supporting the hypothesis that these clades should be treated as separate species. Finally, 212 sequences of the 658 bp COI barcode region that were recently sequenced were submitted to GenBank (www.ncbi.org) as proposed in the original project proposal. The GenBank numbers are being acquired and verified by NCBI staff. We are awaiting results of review and a decision on “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. Mark-release-recapture work using fluorescent powders has indicated that male moths are capable of flying 140 m in 24 h. We determined that release immediately after application of powders is essential to capturing marked moths. We have replicated traps at the distances over four release dates and are preparing to deploy traps at 280 m and 420 m from the release site. We are also preparing to sample Rb-spiked citrus to evaluate Rb uptake and detection in CLM for mark-recapture applications.
Previously, we have reported that acquisition and transmission of Las are less efficient at temperatures below 22oC and above 27oC, and this effect also occurs when psyllids are pre-exposed to temperature for 2 or 4 days prior to acquisition experiments. Observations of cuticular melanization in psyllids exposed to extreme temperatures suggest that this process may be associated with corresponding reductions in acquisition and transmission efficiency. Seven target genes from five gene families (rhodopsin, bursicon, Yellow C, DcAWD, and multi-copper oxidase) have been identified to evaluate how temperature-mediated gene expression influence Las transmission (objective 1). Quantitative polymerase chain reaction (qPCR) assays were developed for each of the target genes, and the relative expression of each gene among treatments was compared using the 2-..CT method by normalizing to psyllid wg gene expression, followed by normalization to the treatment based on the lowest gene expression. Subsequently, we have identified changes in the relative expression of four genes from this group in association with melanization. Currently, experiments are underway to evaluate temperature mediate changes in expression of these genes, and their correlation with melanization and Las transmission. Whole plant experiments are ongoing, but initial results suggest that that down regulation of at least one gene is associated with reduced acquisition at 16oC compared with 27oC. We will further evaluate the role of this gene in Las acquisition using dsRNA. Establishment, maintenance, and characterization of psyllid isolines from Florida is ongoing (objective 3). We have identified one isoline in particular that exhibits increased egg laying compared with other populations. Currently, we are developing a restriction enzyme-base assay to rapidly screen for Wolbachia strains in these isolines to determine their association with reproductive fitness. An additional component of this project is germ-line transformation of psyllids. Optimization of egg injections is ongoing. Survival of nymphs following egg injections in Petri dishes was low. Successful egg hatches were obtained using an artificial diet as an alternative approach, but was not significantly greater than the petri dishes procedure.
The goal of this research has been to monitor insecticide susceptibility in Florida populations of Asian citrus psyllid (ACP) and to determine the molecular mechanisms of resistance. We have completed our investigation of the resistance mechanisms to pyrethroids in ACP. The voltage-gated sodium channel (VGSC) is the primary target of pyrethroids, and is prone to mutations which render pyrethroids ineffective through target-site insensitivity. Mutations to the channel have been reported in a wide diversity of arthropod species. We characterized the VGSC of ACP to determine the potential of the channel to develop similar mutations. Pyrethroids are an important component of management rotations for this pest species and their loss would significantly hinder control efforts. The VGSC in ACP shows as much potential for alternative splicing as any other characterized insect, including variants which have been shown to have reduced pyrethroid sensitivity. Furthermore, it shows significant potential to develop target-site insensitivity through kdr mutations, with the greatest potential at kdr position M918. Previous studies demonstrated that ACP has an exquisite enzymatic arsenal to detoxify insecticides resulting in reduced efficacy. The results from this study demonstrate that target-site insensitivity is also a probable basis for insecticide resistance to pyrethroids in this insect species. The VGSC sequence and its molecular characterization should facilitate early elucidation of the underlying cause of an established case of resistance if it should occur. We have evaluated insecticide resistance in field populations of Asian citrus psyllid (ACP) for approximately the past 7 years. This has allowed us to determine the fluctuation of resistance in Florida ACP populations, develop and recommend appropriate rotation schedules, and determine the specific mechanisms mediating resistance in ACP populations. We have already documented reduced susceptibility in regional Florida ACP populations, where prescribed MOAs sometimes are applied up to 12 times/yr to suppress re-infection of trees. By 2011, there was a measurable reduction of insecticide susceptibility among all ACP populations that we examined across the state of Florida. Also, there was a trend for increased resistance from 2009 to 2012. The highest levels of decreased susceptibility found in field and laboratory in Florida ACP varied between 35 to 100 fold. However, at no point was this reduction of susceptibility sufficiently high so as to result in product failure in commercial groves. We estimate that approximately a 100-fold reduction in susceptibility may be associated with product failure based on our research in non-commercial citrus and the laboratory. The coordination of treatments for ACP is meant to enhance insecticide use by preventing rapid re-infestation by psyllids from non-sprayed areas. By the end of 2010, there were 10 CHMAs established. Our 2010 survey was conducted during that inception period of CHMAs. Insecticide resistance was beginning to be a problem at that time. From 2013 to 2014, there has been a progressive drop in resistance observed among ACP populations in Florida, according to our surveys. In fact, based on our sampling capabilities, it appears that since 2013 we have returned to pre-2009 levels of ACP insecticide susceptibility across the state. The number of active CHMAs has risen from the initial 10 to a current 52. We can hope that coordination and effective rotations of insecticides for ACP have contributed to this decline in resistance. However, given that we know that up to 4,000-fold resistance to the very important neonicotinoid insecticides can occur in ACP populations (V zquez-Garc a et al. 2013), we must remain vigilant by rotating MOAs and monitoring resistance.
The main objective of this research was to determine both abiotic and biotic factors that regulate Asian citrus psyllid (ACP) acceptance of plants and pathogen transmission. The goal was to use this information to interfere with the vector’s capability of transmitting pathogen between citrus trees. During the last quarter we have completed our research and data analysis of our wind-break and replanting investigations. We present two orchard experiments where the densities of ACP were compared depending on: (1) the presence or absence of a windbreak and (2) if the groves consisted of a solid set re-planting or an grove with a mixture of mature and reset-replacement trees. (1) Psyllid abundance was measured on the edges of five groves. The factor investigated was the presence or absence of a windbreak. We observed significantly fewer psyllids on the edges of groves with windbreaks as compared to those without windbreaks. We found no significant difference in the number of natural enemies between the edges with or without windbreaks, suggesting that windbreaks do not affect densities of psyllid natural enemies. (2) During two consecutive years, we compared the densities of psyllids on young trees less than 3 years of age in a solid set re-planting versus on resets (trees planted in replacement of dead or huanglongbing-infected trees) present randomly within mature groves. This was conducted in four groves and among three citrus varieties. More psyllids were found in the solid set re-plantings as compared with on the resets within mature groves. To our knowledge, this is the first report to demonstrate that the planting strategy of new trees in groves may impact the populations of a horticultural pest. Overall our data suggest that establishment and conservation of windbreaks might be beneficial to protect groves from ACP. The data also suggest that ACP populations increase more within uniform landscapes of seedling trees as compared with mature groves with randomly interspersed young seedlings. In 4 groves, we sampled the citrus canopy at 3 different heights and at the 4 cardinal directions. We also investigated the potential for alternative habitats for ACP and sampled non-crop vegetation and potential alternative hosts over 2 seasons. We did not find a consistent pattern in the distribution of ACP during winter in citrus, with one exception; canopies facing the southern cardinal direction harbored more ACP than those facing north. We also found that the proportion of psyllids with the green-blue morphotype (the morph with greatest dispersal capability and likely the major driver of spring grove infestations) increased following insecticide application and after cold weather. Finally, we investigated the potential existence of alternative hosts on which ACP could feed and/or reproduce in the absence of young emerging citrus leaves during winter. We did not find evidence of an alternative host during winter for ACP in central Florida.
The large-scale validation of citrus leafminer (CLM) disruption with the ISCA DCEPT CLM technology has continued. During this past quarter we have applied our 2015 application of the treatment. The deployment of the treatment was successful. Currently we are monitoring populations of CLM in treated and control plots, as well as, damage incidence. Male moth activity is being monitored with pheromone traps that were deployed after treatments were applied. Damage is being monitored by assessing CLM infestation of new leaves.
This is a continuation of the previous project entitled ‘Screening effective chemical compounds against citrus HLB bacterium Candidatus Liberibacter’ (Project # Contest 400). The aims of this project is to rapidly screen the potential chemical compounds for the control of citrus HLB using a graft-based screening method, then to further evaluate the selected compounds in the greenhouse and field by bark-application, trunk-injection and/or root-uptake. Based on the rank of an expert panel and the action mode of the compound, 102 compounds from the Contest were tested using graft-based screening methods. These compounds were classified into four groups based on Ct value in the inoculated plants, scion infected percentage, Las transmission percentage, and the scion-survival percentage and the scion-growth percentage, i.e. (i) Highly effective (8.9 %); (ii) Effective (24.5 %); (iii) partly effective (21.5 %); (iv) Non-effective (45.0 %). A total of 144 treatments from 24 effective antimicrobials or their combinations, three temperatures (40 ‘C, 42 ‘C and 45’C), and two applications (bark-application and root-uptake) were carried out with three replicates in the greenhouse. Six effective treatments was applied in the field by gravity bag infusion. Some treatments were not effective against citrus HLB in the field because of solubility and/or vascular transport issues. The field trial will continue and merge with our integrated control strategy project(CRDF#910).
This is the first year of a 3-year project. The overall project is focused on determining the optimum combination of chemotherapy, thermotherapy, and nutrient therapy that can be registered for use in field citrus and control HLB. Based on efficiency of eliminating Las bacterium and phytotoxicity of chemical compounds, seven effective antimicrobial compounds (Amp, ZS,Va, Pcy, Carv, SD and Al(OH)3) screened by graft-based assay were applied on HLB-affected citrus trees by gravity bag infusion. The preliminary results showed that the Las bacterium was reduced significantly by Amp and SD treatment at 10 month after initial treatment. The control trees still had high Las bacterial titers. Some compounds, such as Al(OH)3, ZS and Va, were difficult to be dissolve in water, easily blocked the injection tubes, and resulted in less uptake by citrus. So in next quarter, we will try to apply these antimicrobials by foliar spray using water in oil nano formulation. In the last quarter, Pcy and Carv were prepared in nanoemulsion and applied on the HLB-affected citrus. The preliminary results showed more and longer new branches in these treatments. In this quarter (January 2015 to April 2015), we tried to improve the application techniques. Nine hundred ml of antimicrobial solutions were absorbed in 48 hr. In the next quarter, we will continue to apply the chemical compounds to citrus trees by foliar spray or trunk-injection coupled with the thermotherapy, and monitor variation of Las bacterium in the citrus tree. Furthermore, thermotherapy and nutrient therapy will be conducted in the future.
The overall goal of this 3-year research project is to efficiently deliver antimicrobial molecules into citrus phloem against HLB bacteria. This quarter’s (From January 2015 to April 2015) research was focused on: 1) continuing to evaluate the effect of antimicrobial compounds and their combinations at different concentration against HLB bacteria using nano formulation by bark-application; 2) investigating agronomic performance and HLB bacterial titer of HLB-affected citrus trees treated with nano-formulations by bark painting and gravity bag infusion in the field; 3) comparing efficiency of different emulsions on capturing insects. In this quarter, thirty-two treatments from 14 antimicrobial compounds in combination and alone at various concentrations loaded in oil in water nanoemulsion, continued to be evaluated in the greenhouse. The results indicated that, 6 month after initial treatment, Amp and Pen were the most effective chemical compounds against the Las bacterium. Las bacterium titer was significantly reduced by Act, Act+Va, and Va. No phytotoxicity appeared in citrus trees treated with Act and Act+Va at lower concentrations. Other antimicrobial compounds and combinations (i,e, Pround, Al(OH)3, SDX) were not effective against the Las bacterium. In field trials, Pen and SDX were delivered into citrus phloem by bag gravity infusion and bark-painting. Using bag gravity infusion, Pen can be effectively delivered into citrus phloem against Las bacterium, and Las bacterium titer was kept low (Ct=33.0-38.5) for 12 month. But in HLB-affected citrus trees treated with Pen by bark-painting, Las bacterium was not significantly reduced. In addition, SDX was not effective against the Las bacterium using either application method. Furthermore, agronomic performance (i,e, fruit size, fruit weight, and Brix) of the citrus trees also was investigated in this quarter. The results showed that fruit size and fruit weight under Pen and SDX treatment, were much greater than those in the control. Brix was not significantly different among treatments. In our previous study, water in oil nanoemulsion (Cre-Nano) has been shown to be an ideal delivery system for enhancing effective compounds to penetrate through citrus cuticles. Effects of three nanoemulsion formulations (Cre-Nano, NanoGreen (business product) and Carv-nano) on capturing insects was also evaluated in this quarter. These three nanoemulsion formulations were applied to citrus trees by foliar spray outdoors. Six days after initial treatment, the citrus tree treated with Cre-Nano captured 10 insects, significantly more than those treated with Nanogreen and Carv-nano. In the next quarter, we will complete this project by submitting the final report.
To improve the RNAi strategy for psyllids control, we used artificial micro RNAs (amiRNAs) to target psyllid mRNAs for better specificity and to lower the potential for off-target effects. Previously, we finished making the clones expressing amiRNAs targeting V-ATPase mRNA of Bactericera cockerelli in two viral vectors and a non-viral vector. We used agroinfection-compatible Tobacco mosaic virus (TMV) and Tomato mottle virus (ToMoV-A, TAV) expression vectors, and a virus-free 35S promoter driven binary vector and delivered amiRNAs through transient expression in plants and through artificial diets. To evaluate the quantity and quality of the amiRNAs expressed in plants (Nicotiana benthamiana), we used Hi-Seq small RNA deep sequencing with 3 replicates. The results showed that the TAV vector and the non-viral vector expressed amiRNAs with greater specificity compared to the TMV vector, and the TAV vector expressed the highest amount of amiRNAs in N. benthamiana plants. The 3 replicated trials showed that the expression efficiency is 100% repeatable and reliable. We also confirmed the expression levels of amiRNAs with Northern blot analysis, and the results were in agreement with those from the deep sequencing analyses. We currently are evaluating the functionality and the efficiency of the amiRNAs targeting to the ATPase mRNAs in protoplasts, to confirm the amiRNAs we express are functional and able to target to the target mRNAs. We are also doing feeding tests with different approaches, plant and artificial diet feeding tests. The target mRNA of the psyllids from the feeding tests was evaluated by quantitative PCR (qPCR). Preliminary data show that the levels of target mRNAs from psyllids fed on the amiRNAs were not stable compared to the control psyllids fed on healthy leaves, possibly due to the various quantities of amiRNAs acquired by each psyllid from leaf tissues. We are working on finishing feeding tests for all different amiRNA expression clones in different diets and to evaluate the target mRNA levels by RT-qPCR. We are also developing transgenic plants expressing amiRNAs for further biological analysis.
We have been investigating the application of synthetic sex pheromones to disrupt mating of the citrus leafminer (CLM) as a possible effective and environmentally friendly alternative to pesticide applications. We have conducted large-scale field experiments combined with data modeling to optimize the use of this technology for the purpose of practical application. Optimizing mating disruption through examination of multiple interrelated variables was a method used for possible wider adoption of this technique in Florida citrus, especially since this particular pheromone active ingredient is quite expensive to produce. Simulations and field experiments were designed to produce response surfaces to investigate data. We varied the distribution and number of pheromone dispensers in plots. Moth catch data in traps were used to understand the efficacy of our treatments. We used commercially available dispensers for mating disruption of CLM obtained from ISCA Tech. For modeling, Monte Carlo simulations were used for a spatially explicit agent-based model. These resulted in nonlinear disruption profiles with increasing point source density. Field trials conducted in citrus infested by CLM varied the amount of pheromone applied at each point source and point source density using attractive and non-attractive disruption blends. Trap catch disruption in the field resulted in nonlinear disruption profiles similar to those observed with simulations. Response surfaces showed an interaction between the amount of pheromone applied and the number of point sources for the attractive blend, but not for the non-attractive blend. Disruption surfaces were combined with cost curves to optimize trap catch disruption under real world cost constraints. Our bottom line results were as follows. Our data indicate that the more complex and more expensive 3:1 blend of two pheromone components was not more economically effective than the less expensive single component blend. Also, our data suggest that both formulations actually cause disruption by the same noncompetitive mechanisms. Therefore, we have definitively shown that the less expensive single component dispenser is the most effective for practical adoption for use to manage CLM in Florida using a combination of filed trials and data modeling.
Densities of an herbivorous pest may be impacted by landscape and orchard architecture. We have been conducting two experiments where the densities of the Asian citrus psyllid (ACP) were compared depending on: (1) the presence or absence of a windbreak and (2) if the groves consisted of a solid set of new plantings or groves with a mixture of mature and reset-replacement trees. (1) Psyllid abundance was measured on the edges of five groves. We have finished analyzing these data in the previous quarter. The factor investigated was the presence or absence of a windbreak. For the five groves, we observed significantly fewer psyllids on the edges of groves with windbreaks as compared to those without windbreaks. We found no significant difference in the number of natural enemies between the edges with or without windbreaks, suggesting that windbreaks do not affect densities of psyllid natural enemies. (2) During two consecutive years, we compared the densities of psyllids on young trees less than 3 years of age in a solid set re-planting versus on resets (trees planted in replacement of dead or huanglongbing-infected trees) present randomly within mature groves. This was conducted in four groves and among three citrus varieties. These data have now been analyzed. More psyllids were found in the solid set new plantings as compared with on the resets within mature groves. Overall our data suggest that establishment and conservation of windbreaks might be beneficial to protect groves from ACP. The data also suggest that ACP populations increase more within uniform landscapes of seedling trees as compared with mature groves with randomly interspersed young seedlings. We consistently found that young trees replanted as solid sets contain higher populations of ACP than seedling trees replanted as resets within otherwise mature orchards. These data may indicate that replacing individual infected trees with uninfected seedlings in a mature grove structure may be a more effective method to prevent infestation by ACP (and associated HLB re-infection) than replacing entire blocks of orchards. Changes in microclimate may also reduce the population of ACP on young citrus resets compared with young plantings in the solid sets configuration. We found that canopy temperature was greater, on average, in young trees present within solid sets as compared with resets re-planted within mature orchards. This increase in temperature was particularly evident in the morning (up to 4′ C) and is probably correlated to light exposure at this time of the day in Florida. Sun exposure is likely to have a strong impact on ACP colonization of plants and dramatically differs among trees depending of their size and the depth of the canopy. Presence of mature trees surrounding young trees likely reduces wind and sun exposure on young citrus trees analogously to a windbreak. Additionally, the concentration of young trees in solid sets, with high production of flush season-long is likely to attract ACP at a higher rate than within mature orchards containing randomly interspersed resets. It has been demonstrated that the proportion of ACP immigrating into a grove was correlated to the flush production of citrus trees. Overall our two experiments tend to demonstrate that microclimate and grove complexity are likely to influence ACP population densities. In the context of integrated pest management for ACP and associated HLB, our results advocate for conservation of windbreaks and hedgerows and support the implementation of new ones.
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