The objective of this research is to investigate promising non-neurotoxic insecticides against Asian citrus psyllid (ACP) that could be incorporated into ACP management programs in Florida. We are especially targeting those that have shown promise against insect pests similar to ACP. Such additional tools may not only prove effective against ACP, but also could assist in ACP resistance management programs as needed tools for effective rotation of insecticides. We have been conducting both laboratory and greenhouse studies to determine the efficacy of novaluron against various life-stages of ACP in the past quarter. These investigations are nearing completion. Rimon’ is a commercial blend of novaluron, which is an insect growth regulator used widely in insect management, but is not registered for citrus. The compound was tested on two stages of ACP–fifth instar nymphs and eggs, at three field rates used for similar insect pests, 10, 15 and 20 fl oz/acre. Flush with approximately 15 fifth instar nymphs were dipped into the three preparations of insecticide and mortality was determined 72 hours post-treatment. The three field rates of 10, 15 and 20 fl oz/acre induced 30, 60 and 62% mortality, respectively. For effects on egg hatch, flush with an average of 25 eggs between 0-3 days old were dipped into the insecticide dilutions and the number of live nymphs was determined every 24 hours for eight days. At the 96 hour time point when eggs began to hatch, controls had 1.8-fold more live nymphs over the 10 fl oz/acre treatment and 2.2-fold over 15 and 20 fl oz/acre treatments. Data from this study are currently being analyzed. The last bioassay to be conducted is the evaluation of the viability of eggs from treated females, and it is currently underway. The initial data suggest that the non-neurotoxic insecticide novaluron is effective against the immature stages of ACP in laboratory assays, showing similar effects to other known insect growth regulators against this pest.
The objective of this study is to determine how enhanced nutrition of citrus plants may affect Asian citrus psyllid (ACP) biology. We have initiated this study with complementary field and laboratory experiments. Regarding the field trial, we are still following the Keyplex’ program. The latest two sprays were applied in May and June. We are pursuing the weekly monitoring of the number of psyllids on each tree with tap sampling and flush examination. We plan to continue this survey throughout all of the summer season in addition with a leaf nutrient analysis at the end of the season. We also performed a qPCR analysis 6 months after the first one was conducted, which showed no difference between the trees sprayed with Keyplex and the controls in terms of newly infected trees. From March to May, we consistently observed more psyllids on HLB-infected trees than on uninfected trees. However, during June, when the ACP population strongly increased, this difference was not detected. This may be explained by the higher dispersal rate of psyllids observed in the laboratory from HLB-infected trees than from uninfected trees. We did not find any difference in terms of psyllid density between trees sprayed with Keyplex and the controls. These results from the field are in contrast to those observed in the laboratory, where we observed that psyllids were more attracted to infected plants treated with Keyplex compared with control plants. We initiated an investigation of the pathogen acquisition rate of psyllids from plants treated with various nutrient regimes. We bagged 50 adults on HLB-infected trees. Half of them were treated for one year with Keyplex, and the other half only sprayed with water. After 10 days, ACP adults were removed, and placed on an uninfected citrus plant to allow pathogen multiplication. Subsequently, the adults were submitted to qPCR analysis. So far, there is a significant trend showing that ACP acquired less Candidatus Liberibacter asiaticus (Las) pathogen on the nutritionally supplemented trees than on the controls. This result may be due to a reduction of the presence of the Las pathogen in the trees under Keyplex treatment as compared with the controls. This is still under investigation. We are currently performing the same experiment under field conditions, with adults from our laboratory colony caged on citrus trees, and nymphs from the field directly bagged on the trees. In total, we will analyze, with qPCR, approximately 200 ACP. We also collected volatiles from plants before and after the application of a spray regime, in order to detect a possible eventual change in the volatiles emitted by the plants due to nutrient treatment. But, we have not observed a significant change in the chemical profile emitted by the plants to date.
Groundwork to conduct the annual survey to monitor insecticide resistance levels in field populations of Asian citrus psyllid (ACP) has begun. Sites included in the survey will be Fort Pierce, Groveland, La Belle, Lake Alfred, Vero Beach and Winter Haven. Insecticides that will be evaluated are carbaryl, chlorpyrifos, cyantraniliprole, fenpropathrin, flupyradifurone, imidacloprid, spinetoram, sulfoxaflor and thiamethoxam. Two of these insecticides, flupyradifurone and sulfoxaflor, are new chemistries for which baseline susceptibilities will be determined in laboratory strains of ACP. Both insecticides target nicotinic acetylcholine receptors (IRAC Group 4). Resistance levels against imidacloprid, another Group 4 compound, has been detected in past annual surveys. Thus, a sub-study will be included in the annual survey to evaluate potential cross-resistance against these new chemistries in field populations with elevated resistance levels against imidacloprid. Additionally, progress has been made towards procuring resources and developing methodologies to create and maintain an artificially selected resistant laboratory strain of ACP against imidacloprid. This colony will be used to investigate resistance level dynamics, such as time required to reverse resistance to baseline levels of susceptibility. Finally, efforts to elucidate the underlying biochemical mechanism that mediates resistance to the pyrethroid, fenpropathrin, is underway. It is hypothesized that resistance against this compound is caused by amino acid changes in the voltage-gated ion channel, the target site of this insecticide chemistry. Sequencing of the gene coding for the channel is currently being conducted so that comparisons of the channel can be made between the susceptible laboratory strain versus resistant field populations.
The overall goal of this 3-year research project is to efficiently deliver antimicrobial molecules into citrus phloem against HLB bacteria. This quarterly (from April 2013 to July 2013) research continued to evaluate the penetrants based on a lemon cuticle assay. The lemon cuticles were isolated by punching out from lemon leaves and incubating in citrate buffer (pH 4.0), containing 2% (v/v) cellulose and 0.2% pectinase in 0.01 M (50mM) for about 3d. The results indicated that citrus cuticles were more difficult to isolate form HLB-affected leaves than those from the healthy ones. Higher starch and lower Zn concentrations were detected in the HLB-affected citrus, which resulted in the poor isolation of the cuticles from the HLB-affected citrus. Eight penetrants were tested using the isolated cuticles. Compared to the control, seven compounds increased penetration several fold. However, one compound was not effective in promoting penetration. The future work will be focused on the following: 1) Optimizing penetration ability of the chemical compounds through isolated cuticles from different citrus varieties; 2) Evaluating of drug loading capacity using the optimized nanoemulsion formulations; 3) Optimizing the final formulations by combinations of the penetrants and high drug loading capacity formulations.
Based on results from the previous Contest Project (CRDF#400), eleven compounds have been selected by the contest committee as candidates for further research to determine their efficacy for control of HLB based on their ability to substantially reduce the titers of the bacterium Candidatus Liberibacter in our grafted citrus assay, lack of phytotoxicity to citrus and potential for registration. After first evaluating combinations of these molecules using the graft-based chemotherapy method under a separate agreement, this project extension will evaluate these 11 compounds as control agents against the HLB bacterium individually and in combination using infected, container-grown citrus and HLB-affected scions. The objective of this project is to determine an optimum chemical formulation that may be registered for field control of HLB. In this quarter (April to July, 2013), 10 compounds or combinations have been applied to HLB-affected potted-plants coupled with heat treatments (40, 42 and 45 degree). All these treatments will be analyzed in 2 months. The preliminary results showed that heat treatment promoted the growth of new flush from the seriously HLB-affected citrus. No bacterium was detected in the new leaves but was present in the old leaves two months after treatment with compounds coupled with the heat treatments. These same compounds or combinations were also used to treat HLB-affected scions and grafted onto the healthy plants. Because PLA, PDL and BSO were very expensive, only the grafting tests were done. The grafted plants survived well except with Act. All these treatments will be analyzed again in 2 months. These compounds and combinations of compounds will also be tested by bioassay.
The goal of this project is to determine overwintering habits of Asian citrus psyllid (ACP), including determining alternative hosts, so as to understand how to improve dormant season control strategies for ACP. The dormant season is the ‘weak link’ in the seasonal phenology of ACP and thus the time when populations of psyllids can be affected most, when targeted appropriately. In order to examine ACP population density over winter months during 2012/13 we have sampled 40 citrus groves under differential management (conventional management, intermittent management, organic and abandoned). Our data show that significantly more ACP are found in groves under intermittent management; such groves were so defined as owners used insecticide and fertilizer treatment between 1 and 3 times per year. To further understand ACP distribution within intermittently managed groves, we are currently analyzing additional abiotic and biotic data collected from these 40 groves over the same winter months. Using a geographical information system (GIS), we are examining whether any of the following show correlation to ACP abundance; citrus variety, leaf nutrient analysis, soil type, surrounding landscape, meteorological data, grove layout, and edge effects. In addition, we are examining ACP movement between groves during winter months by conducting analysis of a large data set describing ACP abundance over 2 years in surrounding groves of the 40 we have examined. These data will yield clues in identifying potential ‘winter reservoir groves’ in which targeted management over winter months may reduce the population growth of ACPs in the spring season. Data from the differential vertical sampling realized last winter are currently being analyzed. We observed a difference up to 4’C between the upper and lower canopy height. These differences during winter may explain the differences observed in ACP population densities between seasons, which indicate higher densities at the upper canopy height sampled during this time than at other heights.
The asian citrus psyllids (ACPs) feeding strategy employs the secretion and polymerization of a sheath or tube within the plant tissue during probing with their mouth parts. We have developed a method to purify these sheaths and have been conducting composition studies. These methods include complex GS/MS, LC/MS and NMR studies and during the last quarter we have used this data to develop a deeper knowledge of the composition and cross-linking of these sheaths. These studies have identified the major building blocks of the sheaths and have provided information on how these building blocks are linked together to create an insoluble feeding structure. This information has allowed us to identify new compounds that block the ability of these building blocks to be polymerized into a sheath and different methods of application of these inhibitors are now being tested in plant systems. Also, replicated studies have led to the identification of classes of small peptides that cause increased psyllid mortality when they are added to their diet. These peptides are being characterized further to identify specific peptide sequences with the greatest effect on the psyllid.
This proposal aims to continue improvement to a novel psyllid the 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 and continues to be conducted to increase trap efficiency by exploiting unique vector behaviors in response to traps. Research by others to discover and identify semiochemicals that actively attract or repel ACP is ongoing. While a number of plant volatiles and ACP-produced compounds have shown their presence or activity in laboratory bioassays, only low level capture increases (less than or equal to a 25% increase in trap captures over unbaited traps) and inconsistent results have been manifested in field bioassays. To date psyllid visual is the only behavioral response reliable enough to attract psyllids. Therefore, we have conducted a large number of field and laboratory studies toward obtaining an understanding of ACP trap response behavior by manipulating psyllid behavior around the trap or farther away so that they are moved close enough to the trap to perceive it (i.e., increase trap active distance). This work is continuing. We have also initiated conversations with several private companies to get a trap prototype mass produced.
This proposal aims to continue improvement to a novel psyllid the 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 and continues to be conducted to increase trap efficiency by exploiting unique vector behaviors in response to traps. Research by others to discover and identify semiochemicals that actively attract or repel ACP is ongoing. While a number of plant volatiles and ACP-produced compounds have shown their presence or activity in laboratory bioassays, only low level capture increases (less than or equal to a 25% increase in trap captures over unbaited traps) and inconsistent results have been manifested in field bioassays. To date psyllid visual is the only behavioral response reliable enough to attract psyllids. Therefore, we have conducted a large number of field and laboratory studies toward obtaining an understanding of ACP trap response behavior by manipulating psyllid behavior around the trap or farther away so that they are moved close enough to the trap to perceive it (i.e., increase trap active distance). We have a number of positive results from our bioassays with which to attempt to increase trap efficiency but have yet to reach a level of trap capture rate which is a satisfactory representation of ACP populations in the vicinity of the trap. This work is continuing and we are conducting a series of experiments to further exploit the success from the addition of a LED light source to the trap. These include tests of a number of available narrowband bulbs on psyllid attraction. We have also in this quarter begun developing the procedures and components of the final prototype trap with a third party distributor to enable mass production of the traps in preparation for the field sampling objectives. This areawide intensive sampling will begin in late first or early second quarter of 2013 and should provide a unique data set from which to detect and determine the natural infection rate of adult vectors by known and novel entomopathogens. The genome sampled will provide a benchmark for future research as appropriate and remain available for continued use in posterity.
This proposal aims to continue improvement to 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 and continues to be conducted to increase trap efficiency by exploiting unique vector behaviors in response to traps and behaviorally active components. Research by others to discover and identify semiochemicals that actively attract or repel ACP is ongoing. While a number of plant volatiles and ACP-produced compounds have shown their presence or activity in laboratory bioassays, only low level capture increases (less than or equal to a 25% increase in trap captures over unbaited traps) and inconsistent results have been manifested in field bioassays to date. To date psyllid visual is the only behavioral response reliable enough to attract psyllids. Therefore, we continue to conduct field and laboratory studies toward obtaining an understanding of ACP trap response behavior by manipulating psyllid behavior around the trap or farther away so that they are moved close enough to the trap to perceive it (i.e., increase trap active distance). We have a number of positive results from our bioassays with which to attempt to increase trap efficiency but have yet to reach a level of trap capture rate which is a satisfactory representation of ACP populations in the vicinity of the trap. This work is continuing and we are conducting a series of experiments to further exploit these ideas. During this quarter we have cooperated with several USDA-ARS personnel to develop, test and incorporate other trap features that affect psyllid visual response but also involve other sensory modalities including sound productions during psyllid courtship. This work is ongoing and more experiments will be conducted in the next quarter. This quarter we continued developing the procedures to manufacture components of the final prototype trap with a third party distributor to enable mass production of the traps in preparation for the field sampling objectives. We have developed and tested a number of different prototype trap components including ones made with 3-D printing to reduce the cost of the enterprise. In preparation for the areawide psyllid sampling objective, preliminary field tests were conducted with the trap prototype under South Florida conditions to determine how traps endure more intense field conditions of heat and moisture. This areawide intensive sampling will begin in soon.
June 30, 2013 Update: Activities of the CHMA program assistant (Brandon Page) March 31 – June 30, 2013: During this reporting period, the CHMA assistant provided support to the CHMA effort by attending various local CHMA meetings as well as statewide meetings. Specifically, Mr. Page attended 9 meetings where he either gave an oral presentation, setup CHMA displays such as posters, or participated in group discussions. Venues included Hardee Co CHMA meeting (2x), Florida CItrus Growers Institute, Gulf CHMA meeting, All Florida Ag Show, Volusia CHMA meeting, Immokalee growers meeting, and the Florida State Horticultural Society meeting. Time was also spent out of the office giving a presentation on the CHMA program to the Extension Dean of IFAS. In addition to attending meetings, daily updates were made to the CHMA website based on communication with CHMA grower leaders. Time was also spent signing growers up for the mapping program that went live online at the end of December 2012. Grove visits were also made to followup on scouting reports for some CHMAs that appeared to not follow the trends observed by growers in the field.
June 2013 update: As part of this grant, we are investigating the use of kaolin clay as a supplemental tool to protect both young and mature trees from becoming colonized by Asian citrus psyllid. The fact that kaolin can deter insects from colonizing plants has been previously demonstrated by other researchers which have shown reduction (not elimination) of certain pests, including psyllids, on kaolin treated plants. Our goal of the ongoing investigations described below is to determine the feasiblity of kaolin to deter psyllids from colonizing citrus trees, thus serving as a repellent that could be used in conjunction with pesticide sprays which we have previously reported as providing very short residual activity. In laboratory trials, choice tests were conducted by releasing psyllids into arenas with plants treated with different rates of kaolin applied. Three of the four rates evaluated demonstrated a repellent effect with psyllid choosing the untreated over kaolin treated plants. The lowest rate evaluated did not deter psyllids from landing and feeding on treated plants. In young tree field trials we previously reported on, we have shown reductions in psyllid populations on young trees treated with kaolin as well as a subsequent decrease in HLB infection rate that appeared to not increase until the rainy season when it is more difficult to keep leaves coated with the kaolin residues. In order to determine how rapdily rain can degrade the repellent effects of kaolin, laboratory trials were conducted to quantify the dislodgeable kaolin residues based on different rates of material applied. Through this study, we were able to predict dislodgeable residue levels that should deter psyllid feeding across a range of concentrations tested. Based on the results obtained from our laboratory studies, we intiated three field trials this year (two of which are still ongoing) to examine the efficacy of kaolin applied to mature citrus in order to first determine the efficacy provided by different rates of product applied and secondly determine how long such residues will last when exposed to rainfall. THe results from this work will be presented in future reports.
As a more sustainable and environmentally friendly method of psyllid control, we evaluated a RNA interference (RNAi) approach based on psyllid oral uptake of dsRNA molecules that target specific psyllid genes. This approach is based on the finding that cellular uptake of dsRNAs, that match the sequence of essential genes, results in down regulation of those genes and can lead to cell/organism death. These dsRNA molecules were introduced into the psyllids through feeding on citrus engineered to express the dsRNA using a Citrus tristeza virus as a paratransgenesis vector. Increased toxicity was observed when adult psyllids were fed on citrus producing dsRNA targeting two gut protease genes. No increased psyllid toxicity was observed in psyllids fed on citrus producing green fluorescent protein (GFP) dsRNA. These results suggest that RNAi-based control may be a viable alternative to current pesticide use for control of psyllids and all phloem feeding pests.
Xambr’ Paran’ Brazil Plots: To quantify the effect of windbreaks, copper sprays, and insecticide sprays individual and combined effects on citrus canker management. In 2010 replicated field plots were established. I was soon evident that the screens used as windbreaks were not strong enough to provide the required effect. Thus in addition to the screens, we established natural windbreaks using Casuarina. Casuarina trees that are currently ~6 m high. There are a few canker-affected plants in the experimental area that will be pruned every 3 weeks. We forecast that in September/October 2013, trees will have developed enough canopy to allow inoculation and to start the experiment. During May, 2013 inspection, 25 plants showed HLB like symptoms. St Lucie Co. windbreak studies: Data from the windbreak system at the first location (5-y old Ray Ruby grapefruit, Estes Orchard, 11 acres, surrounded by 25-ft Corymbia torelliana) demonstrated a windbreak effect on canker incidence on fruit in August 2012. The weather stations (deployed in east-west and north-south directions) measured the effect of proximity to the windbreak on wind speed expressed as the number of wind gusts ’11 mph. The relationship with incidence of fruit canker lesions was explored. In the period May -August 2012, the greater frequency of wind gusts ‘ 11 mph, the higher the canker fruit incidence. The number of wind gusts ‘ 11 mph increased with distance from the windbreak whether measured within the north to south rows or across the rows from east to west, resulting in a relationship between wind and disease incidence on fruit (R2=0.70). In August, the highest incidence of fruit disease (3.5%) was in the center of the 11 acre bock and the lowest incidence (0.4%) was on the end of the eastern most row nearest to the windbreak. Programmable leaf wetness controller (PLWC): During the previous quarter we developed a complex sensor that closest emulates a leaf. This pin sensor, is a flat surface, made from wax. Contact wires are then connected together in a pattern, so that a drop of water will bridge a ‘+’ to a ‘-‘, creating a connection. The more drops of water, the more connections; the more connections, the more current flow. Results from tests of the new leaf wetness ‘pin sensor’ show that this arrangement is way more sensitive to water amount than any previously tested sensor. After some tweaking of the gain circuit on the DC-AC-DC board, we were able to show that with each successive drop of water, the voltage increased until there was a saturation of water. This is what we have been looking for as this will produce a scalar approximation of leaf wetness as opposed to the current Boolean systems. The drying time is a bit faster than a citrus leaf. To correct this we will add layer of silk to the top of the pin sensor. Previous work with the silks has shown that it dries in a similar amount of time to a leaf. In theory, this thin layer of silk will hold water to the sensor for a more realistic time. Project publications: (In addition to the 7 previously reported publications relative to this project, the following was recently submitted as well) Bock, C. H., Gottwald, T. R. and Graham, J. H. 2013. A comparison of the bioassay test and culture to detect Xanthomonas citri subsp. citri . Plant Pathology 62: xx-xx.
During this quarter, particular attention was given to the three primary objectives of the project: 1) Develop a generic epidemiological model that can be used to compare control scenarios and to optimize the probability of controlling and managing high-risk pathogens of agricultural significance. 2) Development and testing of efficient methods of statistical inference to estimate epidemiological parameters from maps of emerging epidemics. 3) Develop user friendly model ‘front ends’ that can be used by researchers and regulatory agencies. Following an extensive period of testing methods to parameterize stochastic models for HLB and citrus canker, and the development of flexible models for spread of epidemics through heterogeneous landscapes, we have focused on (i) the practical use of the models to compare control scenarios at the landscape scale; (ii) to allow for uncertainties in the location of hosts at different scales in the landscape and in a policy makers knowledge of parameter estimates (iii) to incorporate meteorological dynamics in the models, particularly to allow for real weather in dispersal events. One of the principal challenges ‘ also addressed in the development of user-friendly models – is to be able to run the models sufficiently fast to enable the user to compare a range of ‘what-if’ control scenarios with different levels of uncertainty, allowing rapid presentation of results within real time. Substantial progress has been made through the development and testing of efficient computational methods (to allow finely-resolved computation of multi-county and state-wide spread of disease/ Two approaches have been used, the familiar individual-tree–based models and meta-population models. We have also analyzed the effectiveness of greatly increasing the speed of execution of stochastic, spatially-extended model using dedicated multi-core computers available to the Epidemiology and Modeling Group. The additional computing power has also allowed the incorporation of meteorological dispersal models into the epidemiological toolbox in objective (1) above. These models can be used to track intermediate and long-distance movement of vectors: providing measures of primary introduction of inoculum. Turning to detailed models within plantations, analysis of the effects of host planting age on the transmission of HLB has been completed. We have also completed the analysis of how to allow for removal or treatment of infected trees in a region in which the dispersal and transmission are being estimated for an emerging epidemic. We are continuing to test the risk-based method to show where disease is most likely to spread in order to optimize control that involves removal of more susceptible hosts around key infected sites. Recent work on citrus canker has examined weather-driven variability in the transmission rate of infection by extracting signals from Markov chain Monte Carlo estimates of parameters averaged over different time-scales of one to several months and correlating these with weather variables. The method suggests that it will be possible to use Florida data to estimate the spread of disease under different environmental conditions, for example those typical of Texas or California and to compare the risk of spread under different runs of months/years of favorable or unfavorable weather conditions. Building on a model for HLB within individual plants, we have compared the relative importance of pathogen transmission within the vascular system of the tree and transmission between leaves via psyllid vectors. The model is being used to compare the efficacy of roguing, application of insecticide and the use of nutritional products and thermotherapy for disease control and mitigation.
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