As reported in the previous update, it was observed that after being held on plants expressing Peptides A, B or C, Asian citrus pysllids harboring Candidatus Liberibacter asiaticus (CLas) at rates between 20-80% appeared to be ‘cleared’ of the bacteria 15 days post-exposure and that F1 progeny were devoid of CLas. To corroborate this finding and to investigate it further, additional plants, control and experimental, were graft-inoculated. Approximately 50% of plants for controls tested positive in ELISA for CTV expression and those expressing peptides are currently being tested. Once full sets of plants are available, experiments will commence. We have concluded feeding inhibition assays previously reported and are currently conducting psyllid development and longevity assays to measure the response of psyllids to peptides A, B, C and D. Although all four peptides appeared to reduce psyllid feeding, peptide D was associated with the greatest significant reduction in psyllid feeding. Quantitative PCR is the method currently used to determine infection rates of CLas within the psyllid and host plant. While qPCR is the most sensitive method to detect CLas, it does have a finite limit at which it can detect any template. The report of false-negatives has wide-sweeping ramifications on the interpretation of the efficiency of D. citri as a vector of CLas, such as in the experiment described above. We optimized the reaction by using corrected forward and reverse primers, invoked the use of a standard curve to determine the dynamic range of the reaction itself, and employed a nested-PCR approach to pre-amplify potential CLas 16S template and used this reaction as a template in qPCR. This last step enabled us to find CLas in samples in which the initial titer was below the dynamic range of the qPCR reaction. We have validated that there are false-negatives being obtained by the old procedure, and we are currently performing experiments to estimate the error rate and to finalize the method to be employed in future experiments.
Our objective has been to determine how Asian Citrus Psyllid (ACP) behavior is affected by Las-infection in response to healthy versus diseased citrus trees. In previous experiments, we have determined that ACP adults initially settle on Las-infected plants as compared with nearby uninfected counterparts. We hypothesized that while the Las-infected plants are initially attractive to ACP, after prolonged feeding, the ACP experiences imbalanced nutrition and choose to seek a better host. In fact, we have proven that infected plants are less preferred by ACP for feeding as compared with uninfected citrus plants. Over the past year, we have conducted numerous settling bioassays to examine host selection behavior of ACP. We conducted binary choice tests between two plant types including: control vs. old infection, control vs. new infection, new infection vs. old infection, old infection without nutritional supplement vs. old infection with nutritional supplement, new infection vs. old infection with nutrient spray, and control vs. old infection with nutrient spray. All plants used in settling experiments were approximately four year old Hamlin sweet oranges. Control plants were uninfected, healthy plants in each case. Infected plants were either newly infected (<5 month since PCR detection) or old infected (>12 months since PCR detection). Nutrient-sprayed plants were old-HLB infected plants. Based on these experiments, we have found that newly infected plants are very attractive to ACP as compared with the other treatments tested. ACP prefer to settle on these plants over controls and old-infected plants. Old infected plants are not as attractive as newly infected plants when compared with controls. ACP initially settle evenly between control and old or nutrient sprayed plants but then choose to move to the infected plant over a seven day period. When ACP are given a choice between newly infected plants and nutrient sprayed plants, the nutrient sprayed plants appear to regain some of their attractiveness and ACP settle more evenly between these two choices of plants. In addition, we have conducted settling experiments between control and newly infected citrus in the presence of high amounts of methyl salicylate (MeSA) released from commercially available dispensers. This experiment was conducted because HLB infection causes citrus trees to release MeSA. Our results indicate that treatment of plants with high levels of methyl salicylate interferes with the ability of ACP to differentiate between infected and healthy citrus. Therefore, use of MeSA may be a promising method to prevent ACP from finding infected citrus trees to acquire the HLB pathogen. In the last quarter we have experiments in which ACP are pre-exposed to methyl salicylate prior to conducting settling assays as described above. Several dosages of MeSA were pipetted onto a cellulose matrix and placed into a heat resistant nylon resin bags, after which adult ACP were placed into the bags for one hour of exposure. After the hour of exposure, the ACP were released into cages containing one healthy and one young infected citrus plant. After 24 hours the number of ACP were counted on each plant. These results were compared to the numbers of untreated ACP settling on healthy versus young-infected plants. Our results indicate that the ability of ACP to choose between uninfected (healthy) versus infected host plants is impaired after pre exposure to high doses of methyl salicylate. These is further evidence that MeSA may be a tool for controlling spread of HLB by impacting the ACP’s ability to locate infected plants to acquire the HLB pathogen.
We have initiated this investigation with two laboratory experiments. The aim of the first experiment is to understand how communication between plants may affect psyllid host acceptance. Recent studies show that plants in the vicinity of herbivore-infested plants release inducted volatiles in the absence of damage. This indicates that plants communicate and that a pest-damaged plant causes its nearby undamaged neighbor to release these volatiles, which act as ‘SOS’ signals that attract natural enemies. This plant communication allows plants to ‘anticipate’ the arrival of herbivores (pests) and increase efficiency of induced plant defenses. The Las-pathogen (Candidatus Liberibacter asiaticus) induces the same response as Asian citrus psyllid (ACP) feeding. Consequently, we hypothesize that uninfected plants located nearby Las-infected plants may be ‘fooled’ and also release volatiles that attract vectors. This system may increase the spread of the pathogen that causes HLB disease. To test this hypothesis, replicates of two ‘Valencia’ plants were placed into glass domes, and sampled during three days to collect the volatiles from either a Las-infected plants versus the volatiles from uninfected citrus plants (control). Subsequently, these two plant treatments were placed in a cage for a choice test, and 50 ACP were released into each cage replicate. The number of ACP on each plant was recorded during 3 days after initiation of the experiment. Early evidence suggests that there may be plant to plant communication taking place, signaling infection by Las. The goal of the second experiment was to test the effect of drought stress on recruitment of natural enemies of ACP and the expression of Las-induced plant volatiles. ‘Swingle’ plants were submitted either to a control treatment, where water content was maintained at 90% per pot water capacity, or to a drought stress treatment where plants were deprived of water until the leaves began wilting and then were subsequently watered. Using laboratory olfactometer experiments, we tested if plants under drought stress recruit natural enemies differently as compared with non-stressed controls. We first demonstrated that well-watered plants infested with 50 ACP were more attractive to the parasitoid, Tamarixia radiata, than a well-watered plant without psyllids. This experiment confirmed that a citrus plant infested by psyllids releases volatiles that attract natural enemies. However, under drought stress, plants infested with 50 ACP were not more attractive than an uninfested drought stressed plant. This experiment demonstrated that under drought stress, herbivore-inducted volatiles are decreasing to such an extent that recruitment of natural enemies is impacted. Our next objective is to test if the attractiveness of HLB-infected plants is lowered by submitting them to a drought stress treatment. Finally, regarding the field portion of this project, we selected plots where an entire grove or grove portion has been replanted (2 plots) and where only a fraction of total trees (less than 50%) has been replanted within a setting of otherwise mature and bearing trees (2 plots). Collection of biotic and abiotic data, as well as, conducting ACP monitoring will be implemented this summer. Our objective is to determine the biotic and abiotic factors that impact grove colonization by psylids in this field study.
Our objective for this project has been to evaluate botanical compounds as repellents for Asian citrus psyllid (ACP) with the purpose of developing possible repellent formulations for use in the field. In previous quarters we reported on evaluation of five botanical oils as repellents for ACP. In particular, we have found that fir oil is an effective repellent of ACP. Based on this finding, we began working with the commercial pest management industry to begin developing dispensers for practical application in the field. Several types of dispensers and formulations were designed and so far one was developed for field testing. Specifically, in the last quarter a fir oil high-release device was formulated by Alpha Scents, Inc and we obtained a sufficient number of these for field evaluation. The test was conducted in replicated bocks of sweet orange resets in central Florida. Our goal was to conduct the trial within resets to evaluate the potential of this repellent device for young tree protection. Trees were treated with one dispenser per tree at a rate that was based on previous laboratory tests. Out intent was to treat plots with a rate that was effective in previous laboratory experiments. Identical nearby untreated plots served as controls. There were five replicates or treatment and controls. Treatments were applied to plots at random. Plots treated with the devices, as well as controls, were monitored weekly and the number of adult psyllids per tree was measured by tap counts. In addition, we counted the number of ACP nymphs and the number of active leafminer mines on flush when present. The results of this initial experiment suggest that the amount of fir oil released by these initially designed dispensers did not affect the host acceptance behavior of ACP or citrus leafminer sufficiently. Populations of both pests were not deterred sufficiently from infesting treated plants for practical effectiveness of the dispensers that were tested. In a separate test, we evaluated a commercial prototype product that incorporates several of the plant botanical compounds we evaluated previously into an oil for direct application to the crop. In this case, we did observe significant reduction of flush infestation by ACP as compared with control plots. We are pursuing development of products that incorporate these active ingredients into formulations that cover the leaf surface. Finally, we continue to work with ISCA Technologies on prototype product development. We are planning further evaluation of the various active ingredients we and others have discovered for incorporation in ISCA’s SPLAT technology. A field trial of this latest formulation is planned in Florida in the near future.
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.
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