1. Please state project objectives and what work was done this quarter to address them: 1. A repetition of the experiment was conducted in late January through February over a period of five weeks. The protocol established is as follows:a. Selected trees were scouted for flush or pruned to induce flushing.b. Areas of flush were bagged and inoculated with 20 parent ACP.c. Parent ACP were removed from bagged flush after two weeks.d. First generation ACP were counted two weeks after removal of parent ACP.e. The presence of native ACP life stages also documented.2. In March all sentinel trees were sampled and tested using gel electrophoresis and rtPCR to confirm the absence of CTVvv-RNAi per permit requirements. Sentinels were also tested via qPCR for the presence of HLB.3. Trial trees were selected for the next repetition of the experiment. These trees were also tested using gel electrophoresis and rtPCR to confirm the presence of CTVvv-RNAi, and qPCR for the presence of HLB.4. Aphid scouting continues on a biweekly basis. The presence of brown aphid has not been detected. 2. Please state what work is anticipated for next quarter: The project will terminate and a final report will be issued 3. Please state budget status (underspend or overspend, and why): On budget
Objective 1: Compare the effectiveness of biological control in commercial orchards in FL and determine if abiotic factors or management practices influence effectiveness. Use this knowledge to improve the effect of biological control. We have continued to investigate the contribution of natural enemies on population regulation of Asian citrus psyllid (ACP) by measuring psyllid mortality of psyllids in response to natural enemies, where sentinel psyllids were deployed monthly on tree branches with or without screen exclusion cages. During the previous quarter, we have been able to process and analyze abundant data collected during previous quarters. We found that the mortality of uncaged ACP, where natural enemies had access to flush with eggs and nymphs, was higher than of caged ACP at organic sites during all seasons analyzed i.e. Summer, Spring and Fall. These results demonstrated that natural enemies reduced ACP population in organic management groves with no chemical input, which is consistent with previous investigations employing exclusion cages to measure the impact of biological control. As anticipated, psyllid mortality was not different between caged and uncaged sentinel psyllids in conventionally managed groves that received sprays of neurotoxins during the majority of our monitoring period. These results suggest that natural enemies may have contributed to regulation of ACP populations in organic groves more so than under intermittent use of conventional insecticides. We measured populations of ACP under both management regimes and recorded more ACP in areas treated intermittently with conventional insecticides than in organic blocks, suggesting that the level of insecticide input was insufficient to adequately reduce ACP populations. Furthermore, populations of natural enemies densities may have been impacted by the intermittent insecticide applications made in the conventional groves. Natural enemies population were similar in groves sprayed intermittently with conventional insecticides vs. groves managed organically and without use of chemical inputs for ACP. In both management groves, we found six different natural enemies including predators such as ladybeetles (Coccinellidae), syrphid flies (Syrphidae), green lace wings (Chrysopidae), long-legged flies (Dolichopidae), and spiders (Aranae) and parasitic wasp T. radiata (Eulophidae). Among these natural enemies, spiders were the dominant group of predators observed in both management groves. The mortality of ACP in an open cages in the exclusion experiment was higher even though other natural enemies were relatively fewer which may have been caused by abundant spiders. Another dominant natural enemy observed were Dolichopodids, which are predaceous; these especially feed on adult psyllids. The predatory Dolichopodid flies were found in higher numbers after spiders in both management sites. Ladybeetles are an important predator that contributes to mortality of ACP and were the third most frequently observed natural enemy in this study. During our study period, the abundance of ladybeetles was also not affected by the management regime. Tamarixa radiata is one of the key natural enemies of ACP, but we found very few wasps in both organic and conventional groves and occurrence of parasitism was rare. In addition to parasitism, female T. radiata also kill a proportion of psyllid nymphs by host feeding. The low numbers of Tamarixia could be due to indirect effects of predators such as Coccinellidae and Dolichopodidae. In previous work conducted in Central Florida, T. radiata was found to suffer mortality (64-95%) due to predation of parasitized nymphs by ladybeetles. Dolichopodids also compete for resources with Tamarixia and eat them when encountered during their searching behavior. Objective 2. Revise insecticide resistance management for psyllid IPM in new plantings An insecticide resistance management protocol will be developed for young tree protection in Florida citrus that: 1) could be deployed in areas where insecticide resistance is already present and reduce ACP populations and 2) would allow return to normal susceptibility levels for insecticides that have been compromised in effectiveness due to resistance. ACP has developed resistance to neonictinoids in some groves in Florida citrus groves. Therefore, we assessed the mechanism(s) conferring resistance in these ACP populations, compared to the highly susceptible laboratory colony of ACP. Knowing the mechanisms allows development of methods to reverse the problem where it exists and prevent it where it has not yet developed. In the current study, we took three genotypes (groups where individuals within groups are genetically similar, but where the groups differ from one another) of ACP with varying levels of resistance and susceptibility to neonictinoids, and comparatively investigated them. Specifically, we performed a detailed transcriptional analysis comparing all of the ACP genes between resistant and susceptible populations. By figuring out which of these differences are important to development of resistance, we can better understand how the resistant populations changed because of resistance development and therefore what we need to do to reverse it. First, we collected psyllids from these areas, extracted their DNA, and conducted a data analysis called RNA-seq. We identified what are called differentially expressed genes (DEGs) between the resistant populations and our susceptable baseline (‘wild type’) population. These DEGs indicate differences between the resistant and susceptable populations. We found a total of 388 DEGs that distinguish the resistant Lake Alfred population from a known susceptable ACP population and 368 DEGs that distinguish the Wachula resistant psyllids from the susceptible population. After identifying the differences between resistant and susceptable populations (the DEGs), we determinted the molecular function of the DEGs using a Web Gene Ontology Annotation Plot. The Gene Ontology (GO) classifications were used to predict and identify the specific functions for the identified DEGs. In other words, we determined what type(s) of protein differences are produced by the genetic differences between susceptable and resistant ACP. This tells us what the genes do for the insect. Three hundred genes were annotated in this manner for the Lake Alfred population and 290 DEGs were assigned a function for the Wauchula insecticide resistant population. The cellular process, including cell and catalytic activity terms contained 120, 94, and 61 DEGs in Lake Alfred and were dominant in each of the three main categories such as biological process, cellular component, and molecular function. Likewise, 104, 84, and 59 DEGs were assigned to cellular proces, cell, and catalytic activity in the Wauchula population. The functions of some genes involved in metabolic processing, immune system processing, enzyme regulator activity, receptor activity, and catalytic activity were specifically related to expression of insecticide resistance development in ACP. Third, we conducted a procedure called functional enrichment analysis to identify which DEGs were significantly enriched, statistically, in the gene ontology GO using the Kyoto Encyclopedia of Gene and Genomes (KEGG). We did this to visualize the functional involvement of DEGs (differences between resistant and susceptable ACP) in various biological pathways in psyllids. Pairwise comparisons (Lake Alfred vs Wauchula) of the KEGG annotated proteins revealed two proteins absent in Lake Alfred, namely purine-nucleoside phosphorylase, and nicotinamide mononucleotide adenylyltransferase which are critical for the insects ability to metabolize (break down) nicotinates and nicotinamides (like neonicotinoid insecticides). In addition, three proteins that are in the cytochrome P450 family, including glutathione S-transferase and carbonyl reductase 1, were found present in high levels the Lake Alfred populations. In insects, these genes are specifically responsible for metabolism of xenobiotics, including insecticides. For the Wachula population, we identified one glutathione S-transferase that distinguishes this population from normal susceptable ACP populations. Glutathione S-transferases are known specific proteins in insects that break down insecticides, including pyrethroids. To summarize, we identified very specific differences between two populations of ACP in Florida that show significant resistance to several insecticides as compared with wild type susceptable ACP. We know that some of these genes are specific to neonicotinoids, which is congruent with our findings that neonicotinoids are most often the mode of action exhibiting significant resistance, but also found that more general detoxifying enzyme groups are more active in resistant psyllids. These results suggest that our current recommendations for rotation of five modes of action should continue working in Florida as a method for reversing and preventing resistance and that the situation does not appear to have worsened. However, the finding that the majority of the transcripts that we identified in this investigation are novel, and have not been previously implicated in insecticide resistance mechanisms in ACP and other insects, are surprising. Our new results suggests that resistance development to neonicotinoids, such as thiamethoxam, in ACP is more complex than previously described. Our new results implicate the participation of a broad array of novel resistance genes and possible resistance mechanisms working in concert. We continue conducting more detailed quantification of expression of the selected genes we have identified for the first time. These results will be incorporated into our insecticide rotations protocols, and if they improve resistance management, they will be incorporated into our spray recommendations for FL citrus.
Objective: Develop threshold-based models for current use in Florida citrus. The objective of this experiment is to optimize management of Asian citrus psyllid (ACP) by implementing an economic threshold for need-based timing of insecticide applications. We investigated the relationship between insecticide rotation using various modes of action and implementation of specific pest threshold levels for timing of these insecticide applications. Our goal is to determine how these factors influence ACP and insecticide resistance management, as well as their economic viability. First, we monitored adult psyllid populations before and after application for three threshold levels by tap sampling (0.2, 0.5, and 1 adult per tap sample). There are two rotation schemes in replicated plots in an experimental grove in Lake Alfred. The effectiveness was evaluated by weekly counts of ACP adults. Tap samples were taken from 20 trees per replicate and there were 4 replicates for each treatment. Second, we are using an insecticide bioassay to monitor changes in susceptibility to insecticides before and at the end of each rotation modules. The difference in susceptibility before and after each application is being used to determine if susceptibility to a given insecticide is shifting in populations exposed to a given module. For each bioassay, 5 insecticide representing different chemistries and mode of action are being evaluated. For each insecticide, five to seven concentrations were tested that have mortality levels between 1 and 99% based on preliminary studies. Test the effect of concentration for each insecticide, we are using commercially formulated products diluted in distilled water on the day of testing. Before application, we found low to moderate resistance for thiamethoxam (RR = 5.25), imidacloprid (RR = 5.97), cyantraniliprole RR = 1.79) (RR=resistance ratio). Third, we have been collecting samples beginning in March 23, 2020. Leaf samples have also been collected from each plot to determine the presence of Candidatus liberibacter asiaticus in the trees and determine the infection rate. When counts of ACP adults in any plot reach a previously defined threshold level, a spray has been applied within the insecticide rotation developed for that particular treatment. On April 21, 2020, the 0.2 ACP per tap treatment threshold was exceeded, and therefore on May 5, 2020, we applied dimethoate for one rotation scheme and fenpropathrin for the other. After application, the ACP populations in all areas that were treated decreased to low levels. Finally, our goal is to investigate the correlation between the threshold ACP population level required to trigger a management spay within the context of effective insecticide resistance management. For each insecticide application, the insecticide mode of action, we are testing differences in population wide levels of acetylcholinesterase, nicotinic acetylcholine receptors, inhibitors of chitin biosynthesis, glutamate gated chloride channels, allosteric modulators, and voltage gate sodium ion channels. In this manner, we will develop an economical and sustainable management strategy for ACP with insecticides, which is still currently lacking in Florida. Our newly developed methods should have a positive impact on the management of Asian citrus psyllid populations by stabilizing or reducing resistance and increasing their economic viability.
This project evaluates young tree protection from ACP/HLB using approaches to integrate ground cover, insecticides, and irrigation management. Treatments include 1) soil-applied neonicotinoids interspersed with sprays of a different mode of action on a calendar basis to trees on UV reflective mulch, 2) rotation of insecticide modes of action sprayed twice on each major flush to trees on UV reflective mulch, 3) soil-applied neonicotinoids interspersed with sprays of a different mode of action on a calendar basis to trees on bare ground, 4) rotation of insecticide modes of action sprayed twice on each major flush to trees on bare ground. This experiment is planted at three locations 1) Southwest Florida Research and Education Center (SWFREC), Immokalee, FL, 2) Citrus Research and Education Center (CREC), Lake Alfred, Fl, and 3) Florida Research Center for Agricultural Sustainability, Vero Beach Florida. During this quarter, irrigation set up for the experiment was completed, metalized reflective mulch installed, and 600 Ruby Red grapefruit trees on US 897 rootstock planted at the location in Vero Beach. These trees will receive irrigation at the same rate for a couple of months so that they are well established before they are subjected to deficit irrigation treatment to synchronize flush and start comparing the treatment of soil-applied insecticides rotation with foliar sprays against sprays on synchronized flush. The locations at SWFREC and CREC were already planted and were subjected to treatments of soil and spray applications for ACP control. However, the treatments to control irrigation to synchronize flush were initiated in February this year. In Immokalee, observations were made overtime during this quarter to look for the plant colonization with adult psyllids and plant infestation with eggs and nymphs. For plants on mulch, there was an average of 0.02 adults per plant (n = 984) much less than those on the bare ground averaging 0.08 adults per plant (n = 864). On mulch and bare ground plantings, the percentage of plants observed with adults was 0.6% and 4.7%, respectively. The plants treated with soil-applied neonicotinoids rotation with spray applications averaged 0.04 adults per plant (n = 930) and those treated with spray applications only averaged 0.06 adults per plant (918). There was no difference in the flushes between the mulch and bare ground treatments. There was not much impact of the deficit irrigation treatment to regulate tree flushing probably because the time to measure the impact was not enough for the reported period, or the irrigation schedules need to be adjusted. The percentage of flush infested with psyllid immatures averaged 1% on the mulch, and 10% on the bare ground planted trees. Plant tissue samples (n = 80) were collected and processed from the SWFREC experiment for PCR analysis, and none were positive for HLB. Data on plant growth and incidence of ACP was also recorded at the CREC location. We have also been continuously collecting data on soil moisture, and soil temperature changes by treatments at both locations and soil moisture sensors were installed at the Vero Beach location this quarter.
In this project we are profiling the new scions and rootstocks for their tolerance to HLB by studying the metabolite content by GC-MS, and challenging new varieties with psyllids and HLB.Objective(s) pursued: 1. To understand the mechanism behind the tolerance of different varieties toward HLB. The comparison between the varietal responses will allow us to determine the mechanism of tolerance to CLas. 2. To understand the role of rootstocks in citrus tolerance to HLB. The comparison between rootstock metabolites will allow us to determine the best scion/rootstock combination for tolerating CLas. Progress on Objectives: This quarter we focused in three areas: 1) Marathon Mandarin analyses; 2) CUPS varieties; 3) Lucky biology and plant response.1) For the evaluation of the new mandarin hybrid Marathon, finally we have some cuttings that are growing in the greenhouse. In addition, the source plant is growing very well and we were able to sample many leaves for analysis of volatiles and polar metabolites. These samples have been run on the GCs and need integration.2) The samples from the new varieties we collected from the CUPS which were used for cuttings did not root well. It may be because we did this in the winter. They remain in the mist bed, except for UF 411 an UF 711 did root. The leaves collected for volatiles and metabolites are being prepared now for extraction (they must be diced into 0.1g aliquots). We estimate there will be approximately 150 samples for this phase. 3) For Lucky and its parents Sugar Belle and Nava x Osceola, we began the biology experiment (detailed in the previous report) on 11/30/20 and ended it Jan 30th, which was one month longer than planned because the weather during December was so cold, we did not have good colonization. We ended the experiment by collecting leaf samples from all plants to assess the plant response to ACP, sprayed any remaining insects, and returned them back to their outside cage to recover. The plants did not look well, so we trimmed, repotted and fertilized them. The leaf metabolites from the ACP-exposed plants will be compared to non-infested controls.In addition to these efforts, the seeds from the USDA (US-802, 812, 897, 942, 1283, 1284, 1516) for metabolite profiling and HLB screening were received, planted, and the germination rate was good. It will be some months however before we can do anything with them in terms of sample collection.
Objective 2. Determine the effect of antimicrobials on Las transmission. Hypothesis: ACP will be less capable of transmitting CLas after feeding on antimicrobials because trees treated with antimicrobials are more likely to have lower CLas titers for acquisition. Eight-year old CLas-infected citrus trees have received six foliar applications (May-December) of streptomycin, oxytetracycline (Treatments), or receive no antimicrobials (Control). Ten CLas-free insects per plant from a laboratory colony were caged on young leaves (flush) of treatment and control trees to analyze ACP survival, CLas-acquisition in ACP P1 and F1 progeny, the total trees sampled consisted of 5 individual tree per treatment. In microcentrifuge tubes containing 1 mL of 80% ethanol, ACP adults were collected individually and then stored at -20°C for subsequent CLas detection using real-time PCR. Survival of ACP and CLas-acquisition were replicated twice from June 2019 to March 2020. During the first replicate, ACP P1 adults were collected on the 26th of June. Approximately two weeks later, five to ten ACP adults corresponding to the F1 progeny were collected. The adults collected on the first replicate in June showed higher CT means (> 38.5) and low copy numbers (< 3); indicating that ACP were unable to acquire the pathogen from treated trees. The second (July), the third (September), fourth (October), the fifth (November), the sixth (January), and the seventh (March) replicates were collected using the same conditions previously described. Concurrently, the titer of CLas had been monitored at the same time-points using three leaves per tree to determine the CLas-infection rate. Currently, psyllids collected from late June through March are being processed to analyze the CLas-infection rate. Objective 3: Determine the effect of antimicrobials on plant response and associated ACP behavior. The objective of this experiment is to determine whether antimicrobial treatments applied to citrus plants affect behavior of Asian citrus psyllid that may change plant susceptibility to ACP infestation or pathogen inoculation. Two antimicrobial treatments are being investigated. These are Fireline (oxytetracycline HCL) and Firewall (streptomycin sulfate). Each is being applied to trees at label recommended rates with recommended adjuvants. To date, all treatments have been applied as foliar sprays; however, experiments are in progress. Treatments were applied to two-year-old Citrus sinensis L. Osbeck cv Valencia grafted onto US-812 rootstock. As described in our previous report, experiments comparing all uninfected (treated with antimicrobials versus untreated) versus all infected plants are ongoing using the T-maze olfactometer to determine whether Fireline affected ACP preferences for antimicrobial-treated versus untreated plants. Experiments are still in progress with Firewall to determine whether application will induce an effect on plants that would cause a consequential change in the behavior of the vector to increase or decrease their preference for treated versus untreated trees.
Objective: Develop threshold-based models for current use in Florida citrus. The objective of this experiment is to optimize management of Asian citrus psyllid (ACP) by implementing an economic threshold for need-based timing of insecticide applications. The most recent experiment we are establishing as part of this project also involves insecticide resistance management. We will compare implementation of three economic thresholds by also comparing various insecticide rotation schemes that we have developed based on previous research on managing resistance for ACP. These two factors will be investigated simultaneously. The rotations we will test are comprised of various insecticide modes of action that are currently registered and used for ACP control; these are: acetylcholinesterase inhibitor, acetylcholine receptor agonist, an inhibitor of chitin biosynthesis, chloride channel allosteric modulator, sodium channel modulator and a ryanodine receptor modulator. By rotating these modes of action, we should be able to prevent development of resistance in ACP entirely while implementing an economic threshold. Since the thresholds will determine timing and frequency of applications, it will be important for us to determine how this affects the order of rotating modes of action. Our purpose is to determine the economic threshold level that controls ACP populations most effectively and economically, as well as, which insecticide rotation works best with implementation of such a threshold. Previously, we have established a large-scale experiment at two sites: a commercial site in Frostproof with an estimated size of 80 hectares with 15-25 years old Valencias and Hamlins. There are 8 replicate blocks at this site. We have continued monitoring ACP and flush production at this location since January. The second, newly established site is located in Lake Alfred. Trees at this location consist of 2-3 year old Hamlins. This site was chosen because it has historically shown some of the highest levels of insecticide resistance documented statewide. For example, resistance to neonicotinoid insecticides and associated product failures of imidacloprid and thiamethoxam have been demonstrated at this site. Three economic threshold levels (0.2, 0.5, and 1 ACP per tap sample) will be evaluated for two different insecticide rotation schedules at this site. Each threshold will be evaluated in replicated plots with four replicates per treatment. Ten trees will be selected in each replicate plot to monitor psyllid densities following insecticide applications. Also, these trees will be used for collecting psyllid samples that will be used to determine changes in ACP susceptibility to insecticides in the laboratory. We have already begun collecting adult ACP from these plots to determine the baseline insecticide resistance levels compared to the susceptible laboratory population of ACP using a leaf dip assay. Field populations have been collected, and bioassays are currently being conducted. We use commercial formulations of dimethoate, fenpropathrin, imidacloprid, and cyantraniliprole for this testing. Five to six concentrations of each insecticide are tested and replicated five times. We will begin insecticide applications when adult tap numbers reach the experimental threshold. We will continue to collect samples chosen at random from the central rows of each plot. The plots will be sampled weekly, beginning in late March 2020. The tap sample method will be used to determine the treatment threshold. Ten samples will be taken per plot to determine an everage ACP count. For eggs and nymphs, 10 randomly selected flush samples will be collected per plot, and the number of eggs and nymphs per flush samples will be counted. Leaf samples will be collected from each plot as well to determine the HLB infection rate. When counts of ACP adults in any plot reach a previously defined threshold level, a spray will be applied with the next insecticide in the rotation. We will determine toxicity and dynamic insecticide resistant development. Also, we will collect adult ACP from the rotation sites to determine the relative expression of ten CYP4 and six GST genes compared to the laboratory population. Genes will be selected based on our previous research which has identified specific genes associated with insecticide resistance in ACP. These will severe as diagnostic tools for helping us identify the specific mechanisms conferring resistance. Finally, our goal is to investigate determine the most effective threshold ACP population level required to trigger a management spray within the context of effective insecticide resistance management. In this manner, we will develop an economical and sustainable management strategy for ACP with insecticide, which is still currently lacking in Florida. Our newly developed methods will be having a positive impact on the management of Asian citrus psyllid populations by stabilizing or reducing resistance and focusing on economical viability of spraying.
The following progress has been made toward Objective 2 of this proposal: -Objective 2: An insecticide resistance management protocol will be developed for young tree protection in Florida citrus that: 1) could be deployed in areas where insecticide resistance is already present and reduce ACP populations and 2) would allow return to normal susceptibility levels for insecticides that have been compromised in effectiveness due to resistance. Thiamethoxam (Platinum 75 SG) has been one of the most overused neonicotinoids for Asian citrus psyllid (ACP) management. Among insecticides currently in use for Asian citrus psyllid, resistance is greatest for neonicotinoids, like thiamethoxam. First, we investigated the possible mechanisms involved in thiamethoxam resistance and the stability of thiamethoxam resistance in the laboratory by establishing populations of field collected resistant ACP. Second, we investgated how to reverse a resistant population back to susceptibility in the field by implementing rotation schedules. Investigations were conducted in areas that were previously treated with consecutive neonicotinoid applications and where the resistance ratio (RR) to thiamethoxam reached between 1266.20 and 1395.00 fold compared with a susceptable population. Finally, we developed a protocol that can be implemented in the field to reverse resistance to thiamethoxam in areas where it is found. First, RNA was extracted using a total RNeasy mini prep kit from adult ACP collected from specific areas where resistance to thiamethoxam was present at two locations: Lake Alfred and Wauchula, FL. A laboratory susceptible population was used as a control for comparison. For each population, ten tubes containing ten ACP adults were used for RNA extractions. The quality of RNA from each sample was assessed on a Nano Drop 2000 Spectrophotometer using A260/A280 ratios, at approximately 2.0. Thereafter, 500 ng of total RNA from the two insecticide resistant populations and the laboratory susceptible population were used for cDNA synthesis. Relative expression of three CYP4, three GST, and one EST gene(s) in each population was compared by qPCR using SYBR Green Power Up PCR master mix in an ABI 7500 Real Time PCR system. The production of the gene specific product and absence of primer dimers was verified by 1% agarose electrophoresis in Tris-acetate EDTA buffer with gel red. qPCR was performed in a 15 µL reaction volume containing 7.50 µL of SYBR green PCR master mix, 1 µl of cDNA, 0.45 µL of each forward and reverse primers and 5.60 µL of nuclease free water. Amplification cycles consisted of an initial denaturing step at 95°C for 10 min, followed by 40 cycles of 95°C for the 30s, 60°C for 30s and 72°C for 30s, and final melting curve step. Three biological replicates were performed for each gene. Actin was used as a reference gene, and to normalize changes in specific gene expression to the ACP laboratory colony. Transcription levels of three CYP, three GST, and one EST genes that are potentially involved in metabolic resistance to insecticides were compared between the laboratory and field selected strains. Second, recovery to susceptibility was determined for two ACP populations where thiamethoxam resistance was present by quantifying the LC50 value of every generation after constant selection for neonicotinoid resistance was ceased. The treatments (rotational schemes) were: dimethoate followed by cyantraniliprole, fenpropathrin, and diflubenzuron (Rotation A) and fenpropathrin followed by dimethoate, cyantraniliprole, and imidacloprid (Rotation B). The third treatment consisted of thiamethoxam followed by clothianidin, thiamethoxam, and imidacloprid (no mode of action rotation). After week 13, the order in rotation A was thiamethoxam, spinetoram, fenpropathrin and abamectin+thiamethoxam. In rotation B, the order was diflubenzuron, dimethoate, abamectin+thiamethoxam, fenpropathrin, and spinetoram. The third area where only neonicotinoids had been applied, received a recovery order consisting of: fenpropathrin, dimethoate, spinetoram, cyantraniliprole, and diflubenzuron. Insecticides were diluted in water at maximal label rates and applied by air-blast sprayer delivering approximately of 100 g of carrier/acre. Adult ACP were collected in the field for the Lake Alfred and Wauchula sites and placed into 60 × 60 × 90 cm insect-proof cages on four citrus plants and maintained in a greenhouse under the rearing conditions. The initial population in each cage contained at least 400 adult ACP from each no rotation treatment and location. In the field, we modified the rotation schedule to cease neonicotinoid applications in plots that indicated high levels of neonicotinoid resistance. After each application, the toxicity of thiamethoxam to ACP was determined to assess how levels of resistance changed over time. The resistance ratios decreased from 1266.20 to 21. 57 and from 1395.00 to 28.71 after four applications of the recovery mode of action rotation order at Lake Alfred and Wauchula, respectively. Under laboratory conditions, the resistance ratios decreased from 1266. 20 to 28.86 and 1395 to 36.71 for Lake Alfred and Wauchula populations, respectively, after five generations of no insecticide exposure. The qPCR analysis showed that expression of CYP4C67 was significantly increased in both resistant populations relative to the laboratory susceptible population. We also showed that resistance in ACP to thiamethoxam declined significantly in the absence of selection pressure under laboratory conditions and rotation application in field. Both laboratory and field investigations indicated susceptibility to thiamethoxam fully recovered after five generations. Three main mechanisms of insecticide resistance typical among insects are: metabolic detoxification, target site modification, and reduced penetration/increased excretion. Previous studies reported five cytochrome P450s (CYP4C67, CYP4DA1, CYP4C68, CYP4DB1, and CYP4G70) analyzed in this investigation that are known to be upregulated after ACP obtain sub-lethal dosages of imidacloprid. In addition, CYP4 genes have been previously implicated in ACP resistance to imidacloprid. There are several cases where up-regulation of one or several detoxification enzyme genes has been attributed to neonicotinoid insecticide resistance or insecticide detoxification among insects. In the present investigation, populations of ACP exhibiting high neonicotinoid resistance were associated with upregulated expression of CYP4C67, implicating the gene product in neonicotinoid resistance in ACP. We compared the expression of CYP, GST and EST genes between known neonicotinoid resistant and susceptible ACP. Similar to the trend in CYP4 expression, GST and EST genes generally exhibited overexpression in insecticide resistant ACP as compared to susceptible counterparts, although these differences were not always statistically significant. Our findings suggest that populations of ACP that exhibited resistance to neonicotinoids in Florida also showed evidence of overexpression of genes implicated in metabolic detoxification. Further research is necessary to determine whether these differences among field populations of ACP have become genetically fixed. Our current focus is on developing an RNA-seq based database to further understand the mechanism(s) underlying thiamethoxam resistance in field selected populations of ACP. Our results revealed that ACP populations develop high levels of resistance to thiamethoxam under continuous selection by label rate applications in cultivated citrus. A high level of resistance occurred following only 3-4 consecutive neonicotinoid sprays and within five egg to adult generations and was associated with subsequent product failure. We also showed that resistance in ACP to thiamethoxam declined significantly in the absence of selection pressure under laboratory conditions and when modes of action rotation was implemented under field conditions. Recovery to a susceptable state under rotation in the field was more rapid than under no selection in the laboratory population. These results suggest that thiamethoxam resistance is likely unstable under the field conditions. Collectively, our results indicate that rotation of thiamethoxam with insecticides from other chemical classes, including cyantraniliprole, fenpropathrin, dimethoate, spinetoram and diflubenzuron effectively mitigates neonicotinoid resistance in areas where ACP are managed with insecticides.
This project evaluates ACP control using various combinations of conventional and organic insecticides and biological control agents. Four Integrated Pest Management (IPM) programs include 1) conventional and organic insecticides plus biological control 2) organic insecticides and Horticultural Mineral Oil (HMO) plus biological control, 3) conventional insecticides plus biological control, and 4) HMO plus biological control. Program 5 is based on biological control only. Populations of Asian citrus psyllid and predators were monitored using tap sampling and suction sampling methods. Tap sampling was conducted twice a month for a total of 6 times and 2,160 tap samples to detect ACP and predators. ACP populations were very low across all programs averaging below 0.1 adults per tap sample. Shoot infestation averaged 2-4% across all programs from a sample of 300-400 shoots available from each program. A total of 24,000 Tamarixia radiata were released across all programs. A cohort study to evaluate contribution of natural mortality factors in suppression of psyllid population in all programs was conducted in November and data being analyzed. Lacewings and spiders were common predators. Ceraeochrysa cubana was dominant species, 76-82% of all lacewings collected from any program. Considering that psyllids averaged below treatment threshold of 0.1 adult per tap sample across all programs, no sprays were conducted except dormant season spray. The dormant sprays which do not require a treatment threshold were applied in Programs 1-4 in December. Imidan was sprayed in programs 1 and 3, Pyganic + 435 oil in program 2 and 435 oil only in program 4.
This project evaluates young tree protection from ACP/HLB using approaches to integrate ground cover, insecticides, and irrigation management under four treatments at three locations. Treatments include 1) soil applied neonicotinoids interspersed with sprays of different mode of action on a calendar basis to trees on UV reflective mulch, 2) rotation of insecticide modes of action sprayed twice on each major flush to trees on UV reflective mulch, 3) soil applied neonicotinoids interspersed with sprays of different mode of action on a calendar basis to trees on bare ground, 4) rotation of insecticide modes of action sprayed twice on each major flush to trees on bare ground. Designated soil and spray applications of insecticide treatments were made to the trees planted on mulch and bare ground at the Gulf and Ridge locations in Immokalee (SWFREC) and Lake Alfred (CREC), respectively. In Immokalee, 1148 trees observations each on mulch and bare ground trees were made overtime during this quarter to look for the plant colonization with adult psyllids and plant infestation with eggs and nymphs. Only 2 trees on mulch were observed to contain adult psyllids compared to 7 on the bare ground, averaging 0.002 and 0.006 adults per plant, respectively. Only one plant on mulch was infested with 2 eggs but no nymphs (0.09% infestation rate) compared with 5 plants on bare ground infested with both eggs and nymphs (0.4% infestation rate). In Lake Alfred, 432 trees observations each on mulch and bare ground trees were made during this quarter to look for the plant colonization with adult psyllids and infestation with eggs and nymphs. Adult psyllids were observed on 3 trees on mulch, averaging 0.02 adults per plant, and on 23 trees on the bare ground, averaging 0.2 adults per plant. Thirteen plants on mulch were observed with eggs and nymphs, infestation rate of 3% compared with 93 plants on bare ground, infestation rate of 22%. Soil moisture sensors were installed in plots with and without the reflective mulch at CREC and SWFREC this quarter. Data will be collected continuously during the duration of the project to document water savings and applicability of the irrigation schedules and rates in a commercial setting. Irrigation installation is underway in Vero Beach, at a grower site and tree planting will be completed in the next quarter. Deficit irrigation treatments to regulate tree flushing will be imposed in Spring 2020, to compare flush sprays against rotation of soil and calendar spray applications. Trees were young and planted late, therefore, allowed to establish through spring 2020 before subjecting to reduced irrigation to synchronize flush and evaluate spray applications.
Objective: Develop threshold-based models for current use in Florida citrus.
This objective is in the process of being established. We began scouting for appropriate field sites in Janaury of 2020. During this time, we secured adequate field sites to establish experiments and mapped specific plots for subsequent trials. In 2020, we initiated monitoring of ACP population levels at these locations in preparation for the investigation. We have also aquired all necessary materials to initiate the research and have finished developing necesarry protocols for monitoring. There are a total of four replicates established in the experiment for each treatment threshold that will be investigated. In addition to monitoring for ACP, we have protocols for monitoring plant health, tree productivity, and pathogen levels.
Objective 1: Compare the effectiveness of biological control in commercial orchards in FL and determine if abiotic factors or management practices influence effectiveness. Use this knowledge to improve the effect of biological control.
The goal of this research is to better understand the contribution of natural enemies on ACP population regulation among citrus groves characterized by differing management inputs in Florida. Two ACP management practices were compared: i) Organic and ii) Low input (2-4 annual sprays) conventional insecticide treatment. Trees were 10-12 year old sweet orange ‘Valencia’. The study was conducted in 2019 form March to June (Spring) and July to September (Summer). We released two pairs of ACP sentinels with and without exclusion cages to measure mortality of ACP cuased by natural enemies. The number of living and dead ACP were counted for three weeks after deployment. We also monitored abundance of ACP and natural enemy abundance in all groves. Natural enemies were recorded during 2-minute visual inspections. Sixteen trees were sampled at each site per sampling date. Tap samples were used to monitor the abundance of ACP. Numbers of ACP adults were monitored weekly by sampling twenty trees per replicate block per week. Mortality of uncaged sentinel ACP was higher than that of psyllids within exclusion cages indicating that natural enemies were killing ACP. Mortality of uncaged ACP was significantly higher than caged ACP during summer in organically managed groves, but not so in conventionally treated groves. Among the natural enemies collected, spiders were most prevalent followed by Dolichopodid flies and coccinellids (lady beetles). Adult ACP populations were significantly higher at sites that were intermittently sprayed with conventional insecticides than in organically managed groves throughout the season. Populations of natural enemies were similar in groves sprayed intermittently with conventional insecticides vs. groves managed organically and without use of toxicants for ACP. However, populations of ACP adults were higher in conventionally treated than organic groves. These results suggest that the level of insecticide input was insufficient to reduce ACP populations, but may have negatively impacted the effect of biological control even though we could not document this directly by survey of natural enemy densities. Our results also suggest that ACP populations can be regulated more effectively by the action of natural enemies than by intermittent spraying under conditions of endemic HLB where curtailing spread of disease is not intended.
Objective 2. Revise insecticide resistance management for psyllid IPM in new plantings
In order to effectively implement resitance management, we have to understand the mechanisms causing it. We therefore initiated a global transcriptome-based analysis of Asian citrus psyllid (ACP) involved in the neonicotinoid resistance using bioinformatics techniques coupled with high throughput RNA-sequencing. First, we conducted insecticide toxicity bioassays on ACP collected from two field populations where neonicotinoid insecticides were used considerably. We used the leaf dipping bioassay technique to determine the level of existing resistance to thiamethoxam at these two sites. We determined that the populations of ACP were 1,394 and 1,266 times less sensitive to thiamethoxam at these two locations as compared with our laboratory susceptable control population. Therefore, we collected adult ACP samples from these to locations for analysis and also from the laboratory susceptible population as a comparison. These sample were immediately frozen and stored at -80°C. Then the total RNA was extracted from these samples using Trizol according to the manufacture’s protocol. RNA samples of acceptable quality were used to construt non-strand-specfic sequening libraries with the TruSeq RNA sample prep kit. These libraries were sequenced using the PE150 mode on an Illumina HiSeq 3000 platform at GENEWIZ. We are in the process of analyzing and interpreting these data. We believe that this transcriptomic profiles will contribute to a comprehensive understanding of the mechnisms of neonicotinoid resistance in ACP. In the future, we will analyze data by mapping to the ACP referrence genome and compare differential gene and transcription factor expression between susceptable and resistant ACP populations. To date, our results indicate that increased cytochrome P450 metabolic detoxification is the mechanism responsible for ACP resistance to neonicotonoids. However, some of our recent findings also suggest that target site insensitivity should be re-investigated; it is possible that over 12 years of selection pressure (continued insecticide application), the mechanism by which ACP are developing resistance could have changed. Understanding this possible shift will allow us to continue to develop the best possible rotation schedules for mitigating resistance.
Objective 2. Determine the effect of antimicrobials on Las transmission.
Hypothesis: ACP will be less capable of transmitting CLas after feeding on antimicrobials because trees treated with antimicrobials are more likely to have lower CLas titers for acquisition.
Eight-year old CLas-infected citrus trees have received six foliar applications (May-December) of streptomycin, oxytetracycline (Treatments), or receive no antimicrobials (Control). Ten CLas-free insects per plant from a laboratory colony were caged on young leaves (flush) of treatment and control trees to analyze ACP survival, CLas-acquisition in ACP P1 and F1 progeny, the total trees sampled consisted of 5 individual tree per group. Survival of ACP and CLas-acquisition were replicated twice from June to November. During the first replicate, ACP P1 adults were collected on the 26th of June. Approximately two weeks later, five to ten ACP adults corresponding to the F1 progeny were collected. The second (July), the third (September), fourth (October), and the fifth (November) replicates were collected using the same conditions previously described. In microcentrifuge tubes containing 1 mL of 80% ethanol, ACP adults were collected individually and then stored at -20°C for subsequent CLas detection using real-time PCR. Concurrently, the titer of CLas had been monitored at the same time-points using three leaves per tree to determine the CLas-infection rate. Currently, psyllids collected from June through November are being processed to analyze the CLas-infection rate.
Currently, psyllids collected from June through November are being processed to analyze the CLas-infection rate.
Objective 3: Determine the effect of antimicrobials on plant response and associated ACP behavior.
The objective of this experiment is to determine whether antimicrobial treatments applied to citrus plants affect behavior of Asian citrus psyllid that may change plant susceptibility to ACP infestation or pathogen inoculation. Two antimicrobial treatments are being investigated. These are Fireline (oxytetracycline HCL) and Firewall (streptomycin sulfate). Each is being applied to trees at label recommended rates with recommended adjuvants. To date, all treatments have been applied as foliar sprays; however, experiments are in progress and other methods of treatment application will be explored. Treatments are being applied to two year old Citrus sinensis L. Osbeck cv Valencia grafted onto US-812 rootstock. Separate experiments are underway comparing all uninfected (treated with antimicrobials versus untreated) versus all infected plants. In the first experiment, we compared response of ACP to the odors of treated and control plants 4, 6, and 8 weeks after treatment with Fireline. C. sinensis plants were placed in glass chambers with air throughput delivered into a psyllid two-choice (T-maze) behavioral assay. In this manner, ACP were tested to determine their response to treated versus control plants using either all uninfected or all infected plants. ACP response was evaluated with the T-maze olfactometer to determine whether Fireline affected ACP preferences for antimicrobial-treated versus untreated plants. There was no difference in behavioral response of ACP to plants treated with Fireline versus untreated controls 4, 6, and 8 weeks after treatment. These results were consistent when both uninfected and CLas-infected treatment and control plants were compared. Experiments are still in progress with Firewall. Thus far, it does not appear that application of antimicrobial treatments (Fireline) to citrus should induce an effect on plants that would cause a consequential change in the behavior of the vector to increase or decrease their preference for treated versus untreated trees, based on the odors released by trees.
During this 4th quarter of the Project we have continued our work as predicted in the Chronogram.
Objective 1: We continued monitoring tree trunk diameter (rootstock and scion) and canopy areas. So far, no differences were found in trunk diameter, but leaf and canopy areas are bigger in IP-covered trees. All IPC-covered trees are still HLB-negative. After replacing the old 4-ft IPCs with new 8-ft covers, donated by The Tree Defender, Inc, canopy area expanded by branch unfolding. We have documented this by photography and also by leaf are index measurements. Objective 2. We have already planted most of the 700 trees of SugarBelle, Tango and Early Pride mandarins. After performing initial measurements of the tree parameters (trunk diameter, and leaf sampling, for CLas, chlorophyll and sugar analysis), we will continue regularly with these analysis.
Objectives 3 and 4. We are continuing monitoring fruit development inside the IPCs and comparing this with our CUPS planting. We are going to assay this winter deficit irrigation to induce blooming in both IPC and CUPS. We have set up an irrigation system that will allow to perform these studies.
Outreach, Professional Presentations and Extension Activities for this quarter : – A CUPS Day.“CUPS, mini-CUPS and other strategies to manage HLB”. Talk on “Individual Protective Covers” . SWFREC, to be delivered on Dec 17. 45 people registered. -International invited seminar at IVIA, Valencia, Spain. “Living with HLB. The new reality of Florida Citriculture”. -Industry Magazine Article: “Individual Protective Covers for Psyllid Exclusion and HLB Disease Prevention in Young Trees”. Citrus Industry, October 2019.
This main objective of this project is to manage ACP using various combinations of conventional and organic insecticides and biological control agents. Four Integrated Pest Management (IPM) programs were established. These included 1) conventional and organic insecticides plus biological control 2) organic insecticides and Horticultural Mineral Oil (HMO) plus biological control, 3) conventional insecticides plus biological control, and 4) HMO plus biological control. Program 5 relied only on biological control. Between July-September, there were six sampling events in which 2,160 tap samples were conducted to detect ACP and predators and 2,267 shoots examined for infestation with ACP immatures. ACP populations were very low across all programs averaging below treatment threshold of 0.1 adults per tap sample. Shoot infestation rate averaged 7%. No spray applications were made in any program. Psyllid adults averaged 0.05 per tap sample in the program 5 compared to 0.01 adults per tap sample across programs 1-4, showing a significant reduction of 80, which persisted from previous applications as no new sprays were conducted due to low populations. Shoot infestation averaged 18% in program 5, and 2-10% across programs 1-4. Lacewings, spiders, and ants averaged 0.05, 0.006, and 0.32, respectively, in program 5, and 0.05, 0.002, and 0.19, respectively, across programs 1-4. A total of 12,000 Tamarixia radiata were released across all programs. Nymphs were not available to evaluate parasitism.
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