We have been investigating levels of insecticide resistance in populations of Asian citrus psyllid (ACP) in Florida. There are three distinct color morphs of ACP. In other words, ACP adults in the field are either gray/brown, blue/green or red/orange, based on their abdominal color. Over the course of an annual season, the proportions of ACP representing a particular color morph change. For example, gray/brown psyllids may be more abundant in the summer than winter, while blue/green ACP may be more abundant in the winter than summer. In insects, variation in body color among individuals of a species is a common phenomenon. These color variations play an important role in mate selection, keeping predators away, body temperature regulation, and habitat selection. Color variations in individuals of a species are possibly the result of phenotypic plasticity, nutritional status, bacterial symbionts, infection status, genetic factors or combinations of two or more factors. We noticed that certain color morphs may be more suspectable to insecticides than others in experiments in which resistance levels of FL ACP populations were being evaluated. Our hypothesis is that factors such as nutritional status and bacterial symbionts dictate the abdominal color of ACP, which in turn influences levels of insecticide resistance in corresponding color morphs of ACP. Previous studies with other insect species have shown that nutritional status and bacterial symbionts influence insecticide susceptibility. Insecticide susceptibility for three insecticides was compared among gray/brown, blue/green and red/orange color morphs of field-collected ACP. ACP from the red/orange morph were significantly more susceptible to fenpropathrin than ACP of the blue/green morph. Imidacloprid caused higher mortality of red/orange than blue/green and gray/brown morphs. Likewise, chlorpyriphos caused significantly higher mortality in red/orange than in blue/green and gray/brown morphs. To confirm the genetic basis of variable susceptibility levels among the three color morphs, relative expression of five CYP4 genes (CYP4C67, CYP4DA1, CYP4C68, CYP4DB1 and CYP4G70) was compared among the color morphs. These genes regulate expression of the specific enzymes that degrade the insecticide imidacloprid (and certain other insecticides/toxins). Expression of all five CYP4 genes was significantly different among the various color morphs tested. CYP4C67 was expressed at significantly higher levels in blue/green than in red/orange and gray/brown morphs. CYP4DA1, CYP4C68, CYP4G70, and CYP4DB1 were expressed at significantly higher levels in blue/green and gray/brown than red/orange morphs. These results suggest that expression levels of five CYP4 genes are highly specific to the color morph of ACP tested. Red/orange ACP color morphs are characterized by lower levels of CYP4 genes, indicating lower levels of cytochrome P450 monooxygenases. Lower levels of CYP4 genes in red/orange morphs suggest that decreased expression of such genes might be responsible for higher susceptibility to the three insecticides tested in ACP. These CYP4 genes may serve as DNA-based screening markers for cytochrome P450-mediated insecticide resistance in field populations of ACP. In addition, these results will be useful for insecticide resistance monitoring, since we know that certain morphs are more susceptible than others. This will allow us to standardize our tests during various points in the season. Finally, during the times when red/orange ACP are most prevalent, ACP populations may be most susceptible to several currently used insecticides.
The goal of this project is to evaluate and find effective botanical repellents for Asian Citrus Psyllid (ACP) management that will be safe to the environment and not harm beneficial insects. The objectives include examination of repellent and toxicological properties of selected botanical chemicals against ACP that are known to be active against other insect species. For behavioral bioassays we are using a customized T-Maze olfactometer. The olfactometer consists of a 30 cm glass tube that is bifurcated into two equal halves with a Teflon strip forming a T-maze. Each half serves as an arm of the olfactometer enabling the ACP to make a choice between two potential odor fields. The olfactometer arms are connected to odor sources placed in solid-phase micro-extraction chambers (ARS, Gainesville, FL) through Teflon’-glass tube connectors. The samples are diluted in appropriate solvent and subsequently pipetted onto a 5 cm Richmond cotton wick, which will serve as the release device. Each chemical is evaluated individually on at least 4 dilutions on a log scale to determine optimum release rate. The control treatment consists of a cotton wick impregnated with solvent only. To evaluate insecticide contact activity of compounds we have secured an automatic microapplicator. Seven serial dilutions for each test chemical are prepared by dissolving the test chemical in acetone or absolute alcohol. Thereafter, 0.4 ‘l of each serial dilution of test chemical or a solvent only (control) treatment is applied from to the dorsal mesothorax of adult psyllids. Treated psyllids are transferred to Petri dishes containing citrus leaf discs placed on a solidified agar beds. Mortality of ACP is scored 48 hr after exposure to treatments. Data are analyzed using standard probit analysis. Toxicity bioassays using treated glass jar techniques will also be carried out using higher dosage for the selected botanicals. In this project we are evaluating 30 plant-produced chemicals for their attractant, repellent, and insecticidal activities against ACP with an intent to identify effective tools for ACP. Preliminary bioassays with some of the botanicals indicated that a ketone compound, gernyl acetone from cardamom attracted psyllids at a 1 ul dosage in a customized T-maze laboratory olfactometer. Geranyl acetone is also a component of general citrus volatiles. Similarly, another ketone, alpha damascene, from rose attracted psyllids at a 10 ul dosage. A sesquiterpenoid, citronellol from lemon grass oil repelled psyllids. However, topical applications of a 0.8 ug dosage per insect of these two chemicals did not show toxic activity against psyllids. Beta-damascone, 9-decen-ol, and geraniol did not influence psyllid behavior up to a 100ul dosage. Behavioral and toxicity bioassays with more botanical repellents/insecticides are in progress.
As the number of Citrus growers interested in providing certified organic products increases in the Lower Rio Grande Valley, there is a need to evaluate the efficacy of biopesticides products available for farm use. Many of these biopesticides have previously worked with other horticultural crops throughout the US but remain unproven on South Texas citrus pests and citrus varieties. Previous studies performed at TAMU-Kingsville Citrus Center over the past two years focused on the impacts of various compost and bio-pesticide sources at reducing Asian Citrus Psyllid (ACP) populations. These studies yielded encouraging results on the potential of biopesticides to provide ACP management in organic farms. Objectives: The purpose of this project is to determine the effect of using organic practices such as the use of bio-pesticides and different compost sources (i.e., green waste compost, Sugarcane mill waste compost, and seafood based compost) on the growth parameters of young grapefruit trees and their pest load and compare it to conventionally managed citrus trees in South Texas Data Analysis: A randomized split plot design was used and a ‘Mixed model analysis’ evaluated the effects of the main factors (treatments , controls and pest evaluation) and their interactions on the biopesticides used against pest and or fungal diseases. Correlation analysis will establish relationships between active ingredient levels and densities of each target. Pest survey in the field, pre-treatment sampling and weekly post-treatment sampling and surveys was made to assess pest densities and bio-pesticide control efficacy. Pest population densities and fluctuations will be evaluated throughout the experimental period. Greenhouse and laboratory chamber studies will utilize 1 year-old trees or leaf tissue samples to more accurately determine the mechanism involved in compost or biopesticide source impacts on citrus pest control. Results: Application of bio-pesticides reduced ACP egg, nymph, and adult populations immediately following spray application. Although, the reduction in each case was only temporary, suggesting that routine and continuous spray of bio-pesticides would be essential for ACP management in organic citrus production. Kaolin+Oil treatment had a better trend of reducing ACP populations than the other two programs, but the Kaolin negatively impacted plant growth and photosynthesis and is recommended for only periodic use and not as part of a continual routine spray program. Interestingly, the major finding from this study came from the sugarcane mill waste compost source applied to young citrus trees. A negative trend correlation was found between decreasing ACP populations and increasing calcium (Ca) in citrus leaves. This Ca increase arose from the sugarcane compost source, and was not particularly significant for the other two compost sources. Benefits to Citrus Industry: With an increase in citrus organic farming, growers will benefit from the data as findings may provide safe mechanisms such as appropriate compost source or foliar inorganic sprays containing available Ca to reduce ACP population without the use of bio-pesticides.
Florida citrus production is facing increasing acreage losses due to disease threats arising from pests like the Asian Citrus Psyllid and Citrus Greening Disease (Huanglongbing, HLB). Some citrus growers in Florida have experienced positive results at combating the physiological damage and yield loss commonly associated with HLB by following a good nutrition plan, even in fields with up to 95% of trees confirmed with HLB disease. A foliar nutrient spray solution is one such nutrient solution that has promise at boosting citrus tree vigor to assist in the combating HLB disease. Some growers in Florida have observed varying results when mimicking the Maury Boyd nutrient cocktail mixture in orchards with confirmed HLB present. However, one nutrient currently not listed in the Maury Boyd cocktail provided to many of these growers is the macronutrient Calcium. Initial data from field trials after 12 weeks from these treatments supports finding from preliminary studies, with lower ACP populations ontrees with foliar Ca treatment applied , as compared to other treatments. Objectives: The purpose of this project was to determine the effect of using various micro- and macro-nutrient foliar sprays, in combination with nitrogen and oil spray treatment, on the physiological growth parameters of young grapefruit trees, their pest load, and subsequent impacts on fruit yield and quality. Data Analysis: A randomized split plot design was developed and a ‘Mixed model analysis’ will evaluate the effects of the main factors (treatments , controls and pest evaluation) and their interactions ofthe foliar nutrient sprays used against pests. Correlation analysis will establish relationships between nutrients on mites, leafminer, scale, ACP pest densities and leaf nutrient status. Time is still needed to evaluate findings throughout an entire growing season on all pests, plus to adequately evaluate the impact of the foliar treatments on fruit yield and quality during the harvest season. Funds for this study were only approved in April 2011. Due to the short time span for evaluation, we are only 4 months into a one year assessment to satisfy minimum objectives for this study, thus a request has been made for a continuance of financial support by the Texas Citrus Producers Board to finish the study during the upcoming 2011-2012 year. We have requested and have received continued funding to support this project and pay for the soil and leaf sample analytical procedures with final accomplishments to be finalized by August 2012. Benefits to Citrus Industry: With an increased threat of HLB disease and other insect related diseases facing LRGV citrus growers, we anticipate that this data may allow us to find another aspect of decreasing this threat to the industry without the use of bio-pesticides. Furthermore, yield and fruit quality assessments will provide economic data as the financial incentives for foliar applied nutrient plans for South Texas citrus production.
Research targeting methods to block Asian citrus psyllid feeding as a means to prevent the spread of HLB in citrus was initiated. A technical person was hired to maintain psyllid colonies and these colonies were initiated. A postdoctoral research associate with hired and initiated research. Early work was in developing new methods to study salivary sheath biosynthesis in a way that is compatible with our screening protocol. This work resulted in the identification that the sheaths are primarily of carbohydrate composition with few other substances. Specific proteins were identified and are now being characterized, but abundance of these proteins is so low that they are most-likely not structural components of the sheaths. Our earlier finding of EDTA inhibition of salivary sheath synthesis was incorporated into research showing that EDTA changes secreted protein profile during psyllid feeding. We are currently characterizing the proteins to determine their function. Torrey Pines Institute for Molecular Studies has initiated the synthesis of peptide libraries that will be tested in our psyllid feeding bioassay looking for compounds that will block the feeding process or otherwise affect psyllid behavior.
Screening of a peptide combinatorial library was initiated using structured combinations. Separate peptide combinations that induce either increased psyllid mortality of improved psyllid survival were identified in the first round of screening. Discovery of peptides that improve psyllid survival on an artificial diet may prove to improve laboratory experiments designed to develop new psyllid control strategies. Discovery of a peptide that will block the ability of psyllids to feed on citrus is being pursued in research aimed at developing novel insect control strategies based on either application of the peptides to plants or production of plants that produce this peptide as a resistance mechanism. Furthermore, continued analysis of salivary sheath composition has led to a new hypothesis on how salivary sheaths are synthesized and how their building blocks are recruited for polymerization to form the sheath.
The objective of this project was to evaluate methyl salicylate dispensers to determine whether their deployment in citrus can enhance biological control of Asian citrus psyllid (ACP). Locations selected for the field trials with the commercially available methyl salicylate (MeSA) lure, Predalure (AgBio Inc.; Denver, CO) were unmanaged grove plots (‘Valencia’ oranges, Clermont, FL), minimally managed grove plots (mixed citrus cultivars, Lake Alfred, FL) and heavily managed grove plots (‘Hamlin’ oranges, Auburndale, FL). The plots were 0.4 ha in area. Treated plots had 1 MeSA lure attached per tree and control plots were untreated. Lures were replaced at 8 week intervals and data were collected season-long. Adult ACP and natural enemies were monitored every 2 weeks with Pherocon AM yellow sticky traps and by sweep net samples. Collection of field data for the methyl salicylate (MeSA) study was concluded on 10/5/2010. In all groves, psyllid populations remained low throughout the season. The overall average number of psyllids per trap was <1 for the intensively managed grove, ~1/trap for the minimally managed grove and up to 1.5/trap for the unmanaged grove. No significant differences were recorded between psyllid numbers or in the number of beneficial insects captured between plots treated with MeSA and control plots. These results were different from our initial investigation in 2009, which indicated that natural enemies were higher in MeSA-treated plots. However, in that year, psyllid populations in that grove were much higher than in the groves studied in 2010. In 2009 an average of > 10 psyllid adults were captured per week in control plots whereas ~2 psyllids were captured/week in MeSA-treated plots. Also in 2009 we saw an increase in natural enemies, particularly coccinellid beetles, in MeSA-treated plots as compared with control plots. We hypothesize that the low psyllid numbers overall in 2010 could account for the lack of effect of MeSA treatment. If natural enemies were initially attracted to the plots, the lack of psyllid prey may have caused them to leave the plots in search of resources elsewhere. Annual variation in attraction of natural enemies to MeSA has been observed in other crops (Rodriquez-Saona et al. 2011. Biological Control. In Press. doi:10.1016/j.biocontrol.2011.06.017). It is also possible that insecticide use or the unusually cold winter affected our findings in 2010. In laboratory bioassays, MeSA was repellent at a high concentration (100 ug) but attractive at a low concentration (0.01 ug) to adult ACP in an olfactometer. In settling experiments on plants, however, more ACP were recorded on MeSA-treated plants than on control plants for the 2 concentrations tested (0.001 and 0.01 M), perhaps indicating that the sprayed concentration was not high enough to observe repellency. In conclusion, MeSA treatment in citrus had mixed results with respect to attraction of natural enemies and reducing psyllid populations in citrus in Florida. MeSA may have an effect in situations when psyllid populations are high at attracting natural enemies and thereby increasing the effect of biological control by earlier and enhanced recruitment. MeSA appears to be a psyllid attractant at low dosages and a repellent at high dosages.
We proposed to identify and assess gene sequences for their negative effects on sap-sucking Hemipteran insects via RNAi using both in vitro and in planta dsRNA feeding assays. To date, we have cloned sequences at least 400 bp in length from nine homologous D. citri and M. persicae transcripts. In addition, we have carried out artificial feeding assays on M. persicae using dsRNA derived from the salivary gland-specific Coo2, midgut-specific glutathione-S-transferase S1 (GSTS1) and constitutively expressed S4e ribosomal protein from M. persicae, as well as a control derived from green fluorescent protein (GFP) sequence. In a side project, we have cloned and characterized two novel IP-free SUS1 promoter alleles (CsSUS1-1 and 2) from Citrus sinensis cv. valencia and have found them to drive phloem-specific expression in both Arabidopsis and tobacco when fused to a reporter gene. Since recent evidence suggests that RNAi in sap-sucking insects may operate more effectively in planta than in vitro, we evaluated the RNAi strategy in planta for its effects against our model insect, M. persicae (objective 2). In this objective, Gateway-based vectors were used to express the selected insect dsRNA either constitutively (35S promoter) or in a phloem-specific manner. We successfully generated Gateway vectors that will result in constitutive (35S promoter) or phloem-specific (CsSUS1 promoter) expression, respectively, of M. persicae-specific Coo2, GSTS1 and S4e dsRNA, as well as a control derived from GFP. Our results suggest that the M. persicae-specific dsRNA expressed in planta has a negative effect on both the lifespan of the insects and the number of offspring generated. In the fall of 2010, we began working on objective 3: to transform citrus with RNAi-inducing transgenes against D. citri. Previously, we conducted 3′ rapid amplification of cDNA from vacuolar ATP synthase subunit G, S4e, and .-tubulin transcripts from D. citri. We have now inserted sequences of the aforementioned transcripts into Gateway-based vectors downstream of both the constitutive 35S and our novel phloem-specific citrus CsSUS1 promoters. To date, we are in the process of transforming and regenerating citrus with the D. citri-specific gateway vectors for evaluation and use by the Florida citrus industry. Initial attempts to transform and regenerate Citrus sinensis ‘Valencia’ and ‘Hamlin’ containing reporter gene constructs were successful. Currently we have completed transformation of citrus callous tissue using gateway vectors with the vacuolar ATP synthase subunit G or S4e transcripts inserted downstream of a phloem-specific citrus CsSUS1 promoter. To date, we have generated several lines, and we are in the process of growing them up for testing in Florida. At the same time, we are preparing to generate additional lines of the aforementioned constructs and with the other transcript/promoter combinations from the second objective. In summary, we have cloned a number of transcripts from both D. citri and our model organism, M. persicae, and have analyzed a subset of derived dsRNAs to test their effect on M. persicae using in vitro assays (objective 1). We have also cloned and characterized several novel phloem-specific promoters from C. sinensis, and have evaluated their expression patterns. We have also created new Gateway-derived vectors bearing a native citrus phloem-specific promoter, for use in RNAi in our M. persicae model and evaluated them in planta (objective 2). Finally, we’ve now generated similar vectors specifically designed against D. citri (objective 3), and are generating transgenic lines expressing D. citri dsRNA for evaluation in Florida.
The goal of this project was to determine if infection by Candidatus Liberibacter affects the response of Asian citrus psyllid (ACP) to its citrus host plants to understand if the causal pathogen of HLB affects the plant so as to promote its own spread. Initially, we evaluated if uninfected psyllids are attracted more to HLB-infected versus healthy trees. Also, we determined whether this behavior changes when the ACP vector is itself a carrier of the pathogen. We conducted a series of behavioral experiments to investigate whether HLB-infected citrus plants are differentially attractive to ACP as compared with healthy citrus plants. Our results proved that HLB-infected citrus plants released volatile chemicals that were more attractive to ACP adults than healthy plants. Also, more ACP were attracted to HLB-infected plants than to healthy plants in open-air cage experiments. However, subsequent dispersal of ACP adults to healthy plants following their initial choice indicated that final settling preference was for healthy rather than diseased plants. This behavior also occurred under complete darkness, indicating that volatile chemicals were mediating this response more than visual cues such as yellowing of leaves on diseased plants. Initial movement of ACP to infected plants and further dispersal to healthy plants was likely explained by production of deceptive volatile compounds by HLB-infected plants to attract ACP adults so as to facilitate the spread of bacteria. However, we hypothesized that the subsequent movement to new plants could be due to poor nutritional status or a nutritional imbalance in HLB-infected plants. Our next line of research was to identify the specific mechanisms that mediated psyllid response. If we could find the specific chemical cues that mediate greater psyllid attraction to infected versus health plants, we could potentially: 1) make use of such attractants to improve psyllid monitoring, and 2) turns off genes that result in the pathways that lead to production of these chemical psyllid attractants in infected plants. In preliminary experiments in this project, we found evidence of specific psyllid chemical attractants produced by infected plants and potential specific nutritional imbalances that cause psyllids to leave infected plants and finally settle on healthy plants. Elucidating and confirming these specific mechanisms has been the topic of the follow-up project to this one (project 439), which is ongoing.
We have successfully completed our major objective of establishing a continuous tissue culture cell line derived from Diaphorina citri. Several of these cultures have been in continuous cultivation for over two years. We continue to maintain these cells and to attempt to increase the robustness of the cell lines via selective manipulation and transfer of cell patches. As noted in previous updates, suspension cells are more homogeneous than adherent cells and appear to grow faster. A number of more homogeneous looking adherent cell lines appear to be emerging. We have disseminated the cell lines to several laboratories both nationally and internationally. Frozen cell suspensions have also been made for storage of the cell lines.
The objective of this project was to investigate three questions: 1) what is the seasonal pattern of Ca. Liberibacter asiaticus (Las) prevalence in leaf tissue on a grove scale; 2) what are the flushing patterns of citrus and do the flushing patterns affect the prevalence of Las in Diaphorina citri or citrus leaves; and 3) what is the prevalence of Diaphorina citri carrying Las on a grove scale and how does it compare the results from the citrus trees in the same grove. In 2008 and 2009 Ebert and Rogers demonstrated that the prevalence of Las in the Asian citrus psyllid (ACP) varied seasonally but the pattern between seasons was not consistent. It was suggested that perhaps the reason for the differences between the years related to the flushing patterns of citrus and the prevalence of the bacterium in the leaves where ACPs are feeding. This project aims to determine if there is a relationship between the frequency of disease on branches and ACPs. Three sweet orange groves have been sampled from June 2010 for HLB prevalence in branches and ACP. Citrus phenology measurements began in October 2010. In each grove, two hundred trees were selected from 10 consecutive rows. One leaf/tree was randomly picked every fortnight. For qPCR detection of Las, the midribs of five random leaves were pooled to obtain 40 samples/date. An estimated Las prevalence in the branches was generated from the pools with PooledInfRate v3. Pooling needed to be reduced in the groves with high HLB prevalence as the the season progressed. Simultaneously, ACPs were collected from the same location to compare the seasonal prevalence of Las in citrus branches and ACPs. There were 2 peaks of Las prevalence in the moderately infected grove at approx. 0.50 mid-October 2010 and April 2011, dipping to 0.20 in February 2011. Las prevalence in ACP had a similar pattern but the main peak reached 0.75 in December. In the highly infected grove, there has been a steady increase of Las prevalence in branches since July 2010 until March 2011 where a plateau of 0.75 was reached. There was the same general trend observed with ACP although the prevalence levels were 0.15 to 0.20 higher, reaching 0.90 in March 2011, but there was a large decline in ACP prevalence to 0.60 in May 2011. In the low HLB intensity grove, no Las has been detected in since the initiation of sampling in July 2010. However, very low prevalence has been detected in branches. Prevalence has hovered below 0.02 until April 2011 when it reached 0.06. The phenology data was collected at all sites since November 2010. Flush was highly synchronous in 2011 likely because of the extended cold temperatures this winter and the drought conditions. As would be expected with the very dry conditions, there is currently little new flush on the trees. Presentations at Conferences: Parkunan V., Wang N.-Y., Ebert T.A., Rogers M.E. and Dewdney M.M. 2011. Seasonal prevalence of citrus Huanglongbing (Candidatus Liberibacter asiaticus) in a central Florida sweet orange grove. at the 2nd International Research Conference on Huanglongbing January 10-14, Orlando FL. V. Parkunan, T. Ebert, N. Wang, M. Rogers, M. Dewdney. 2011. Seasonal variation of Candidatus Liberibacter asiaticus in citrus branches and in vector, Diaphorina citri, in Central Florida sweet orange groves. Phytopath 101:S138.
Using a food protein-based marking system, we have investigated the movement patterns of the Asian citrus psyllid (ACP)in citrus groves in Florida. We have documented the seasonal activity of movement between abandoned and managed groves; movement between managed groves; and investigated within grove movement. We have also shown that psyllids infected with Candidatus liberibacter asiaticus (Las) move from abandoned groves into managed groves, confirming that abandoned groves can serve as a reservoir of both ACP and citrus greening inoculum. Based on studies conducted from June 2009-Sept. 2010, the seasonal movement of psyllids between abandoned and managed groves can be characterized as primarily local or within grove movement from January-March, dispersal (between groves) from April-September and little or no movement from October-December when ACP populations are at lowest. A study to determine movement of ACP between managed groves and the effects of fallow fields on ACP movement showed that ACP dispersal was not inhibited by fallow fields and ACP dispersed a distance of 300 m in one day crossing an unplanted area to the next grove. A study of long-distance movement determined that ACP are capable of moving at least 2000 m in 12 days. The major factor correlated with ACP movement was the abundance of citrus leaf flush, a resource necessary for ACP for egg laying. Wind direction was not significantly correlated with ACP dispersal, suggesting that ACP may be capable of substantial directed flight. We also confirmed that ACP dispersing from abandoned groves moved the Candidatus liberibacter asiaticus (Las) pathogen into commercial groves. This movement occurred at distances of 100-150 meters. PCR analysis of protein-marked psyllids in this larger study showed that 0.02-0.2 % of captured dispersing psyllids carried the Las pathogen. Infected psyllids were found to move at least 2 km (1.25 miles) from the protein-sprayed area indicating that groves within a minimum of this distance from trees with citrus greening are at risk of infection. We also investigated within grove movement of ACP. A 305 m x 230 m study area was established. The perimeter trees (total 200 trees) of this grove were sprayed with a 20% egg albumin solution and an area in the center of the plot (122 m x 91 m; 192 trees total) was sprayed with a 10% milk solution. Twenty yellow sticky traps (5/side) were placed in the egg-sprayed area, 20 traps in the milk-sprayed area and 20 traps in the unsprayed interior of the plots and removed from the field after 4 days. ACP were removed from the traps and subjected to ELISA analysis to determine if the psyllids had been sprayed with either milk or egg solutions. A total of 48 psyllids were captured on the 60 traps. Of these, only 5 were marked with protein. Three of the 5 were marked with egg protein and captured in the area where egg protein was sprayed. No marked psyllids were captured in the milk-sprayed area. In the unsprayed interior area, 1 psyllid was marked with milk protein and 1 with egg protein. This experiment is currently being repeated. Funding from the CRDF has supported the research of one M.S. degree candidate (expected graduation August 2011) and has resulted in 2 journal publications: Tiwari et al. 2011. Incidence of Candidatus Liberibacter asiaticus infection in abandoned citrus occurring in proximity to commercially managed groves. J. Econ. Entomol. 103:1972-1978. Lewis-Rosenblum et al. 2011. Seasonal short-range movement patterns and long-range dispersal of the Asian citrus psyllid, Diaphorina citri (Kuwayama) (Hemiptera: Psyllidae) in Florida citrus. In Preparation.
.-aminobutyric acid (BABA) is known to induce resistance against several microbial pathogens, nematodes and insects in several plant species. Several laboratory studies were conducted to determine if a similar effect of BABA occurs against Asian citrus psyllid (ACP) in citrus. Previously, drench applications of BABA to citrus plant roots were found to suppress the growth and development of ACP by reducing the number of eggs, nymphs and adults produced per plant. Head-space volatiles collected from BABA-treated and untreated plants were analyzed using GC-MS. Differences in chemical profiles between BABA-treated and control plants were observed. Identification of these chemicals is underway. To further optimize the use of BABA for ACP management, another set of greenhouse experiments was conducted to investigate the effects of a reduced rate of BABA alone and in combination with a reduced rate of imidacloprid, made as foliar applications. The treatments were evaluated by tracking the growth and development of psyllids. BABA (5mM) alone caused 30.7%, 17.7%, 69.5% and 56.1% reductions in the numbers of eggs, nymphs, and adults per plant, respectively, when compared to control plants. The combination of BABA (2.5 mM) and imidacloprid (312.5 ppm) caused 90.7%, 82.9%, 76.6% and 65% reductions in the numbers of eggs, nymphs, and adults per plant, respectively, when compared to control plants. Likewise, in a follow-up experiment, BABA (5mM) alone caused 15.2%, 45.3%, 59.8% and 56.6% reductions in the numbers of eggs, nymphs, and adults per plant, respectively, when compared to control plants. The combination of BABA (2.5 mM) and imidacloprid (312.5 ppm) resulted in 99.1%, 99.7%, 100.0% and 100.0% reductions in the numbers of eggs, nymphs, and adults per plant, respectively, when compared to control plants. BABA alone or in combination with imidacloprid may be a useful method for managing ACP. In this project, we also investigated the activity of Pyriproxyfen, Buprofezin and Diflubenzuron (Micromite) on ACP eggs, nymphs, and adults to evaluate their potential usefulness as biorational insecticides as part of an integrated pest management (IPM) strategy for ACP control. All three chemicals exhibited strong ovicidal and larvicidal activity against ACP eggs and nymphs, respectively, in age- and concentration-dependent manners. Fewer eggs hatched into nymphs at the higher concentrations tested (80-160 ‘g mL-1). A significantly lower percentage of early instar nymphs (first, second and third) survived and emerged into adults at the higher concentrations tested (80-160 ‘g mL-1) compared with late instar nymphs (fourth and fifth). Furthermore, the chemicals exhibited transovarial activity by significantly reducing the fecundity of females and viability of eggs deposited by females that emerged from treated fifth instar nymphs. Topical application of each chemical to adults also significantly reduced female fecundity and egg viability. Application of each chemical at 160 ‘g mL-1 results in the highest inhibition of egg hatch in younger eggs (0-48 h old) laid before or after treatment and the strongest suppression of adult emergence from early instar nymphs compared with other rates tested. They also markedly reduced female fecundity and egg viability for adults that were exposed either directly or indirectly. The direct (ovicidal and larvicidal) and indirect (transovarial) effects of these insect growth regulators against immature and adult ACP, respectively, suggest that integration of this insecticide as part of an IPM strategy should negatively impact ACP populations over time.
Under Objective 1 and 2 the following was submitted and published in Plant Disease: Soil applications of inducers of systemic acquired resistance (SAR), imidacloprid, thiamethoxam or acibenzolar-S-methyl, at various rates and application frequencies were evaluated for control of citrus canker caused by Xanthomonas citri subsp. citri in a field trial of 3- and 4-year-old ‘Ray Ruby’ grapefruit trees in southeastern Florida. Reduction of foliar incidence of canker produced by one, two or four soil applications of imidacloprid, thiamethoxam, and acibenzolar-S-methyl was compared with 11 foliar sprays of copper hydroxide and streptomycin applied at 21-day intervals. In 2008 and 2009 crop seasons, canker incidence on each set of vegetative flushes was assessed as the percentage of the total leaves with lesions. By the end of the 2008 season, despite above average rainfall and a tropical storm event, all treatments significantly reduced foliar incidence of citrus canker on the combined Spring-Summer-Fall flushes. Sprays of copper hydroxide and streptomycin were effective for reducing canker incidence on shoot flushes produced throughout the season compared to the untreated control, whereas soil applied SAR inducers reduced foliar disease depending on rate, frequency and timing of application. Except for the treatment of four applications of acibenzolar-S-methyl at 0.2 g a.i. per tree or two applications of imidacloprid, SAR inducers were ineffective for reducing foliar disease on the flushes that were present during the tropical storm. In 2009, all treatments significantly reduced the incidence of foliar canker on the combined Spring-Summer-Fall flushes but not all treatments of Spring-Summer flushes with SAR inducers were effective compared to the untreated control. Hence, depending on rate, frequency and timing of application, soil-applied SAR inducers reduced incidence of canker on foliar flushes of young grapefruit trees under epidemic conditions. Under Objective 2 Integration of soil applied SARs with foliar applications of copper sprays for control of canker, we are testing the efficacy of the inducers for control of citrus canker on young fruiting Red grapefruit and Hamlin orange trees. We previously demonstrated that trunk application of imidacloprid is effective for canker control on fruit as 21 day copper sprays. To keep the chemicals out of the soil, we are spraying the SAR inducers on the trunk. These trials involve testing of non-registered acibenzolar-S-methyl and the insecticides imidacloprid, thiamethoxam at elevated rates. Hence, fruit from the treated trees must be dropped and destroyed (called ‘crop destruct’ of nonregistered chemical treatments). Under Objective 3, In 2011 the complementary use of ASM, THIA and IMID soil applications to increase and/or extend canker control in 3-yr-old grapefruit and 2-yr-old Vernia orange trees. The highest incidence of disease trees and/or leaves is in the unreated check in each trial compared with a very low incidence of canker in the integrated SAR treatments. A field trial with soil applied neonicotinoids in Parana, Brazil was evaluated. IMID (Confidor) as a soil drench and IMID (Winner) applied to trunk gave comparable in disease control activity on 2-yr old Valencia orange trees, as well as, the other neonicotinoids tested, THIA and Clothianidin. Clothianidin is now registered for use on non-bearing citrus in Florida, hence all of neonicotinoids registered for non-bearing citrus in Florida have been shown to have SAR-inducing activity against canker as well.
Four CTV-vectored insecticidal peptides have been proposed for evaluation against the Asian Citrus Psyllid (ACP) ACP, Brown Citrus Aphid (BCA), and Citrus Leafminer (CLM). Since our last report, we have successfully cloned all of the sequenced genes from the proposed peptides into CTV vectors. We have recently completed bark flap inoculations of the final two CTV-peptide constructs into Nicotiana benthamiana plants to obtain sufficient material for graft-inoculation into citrus, a process that is expected to take two months. The peptide, indolicidin, which was previously cloned into the CTV vector and amplified in Nicotiana, has been successfully inoculated into five citrus plants. Currently, these plants are being used in bioassays (e.g. host preference, feeding, survival, development, and fecundity) with ACP and BCA. Similar bioassays with CTV-vectored snow drop lectin (GNA) are also ongoing, which we expect to complete in the next three months. In addition, begun evaluating efficacy of CTV-vectored GNA against CLM; however, due to low production of insects from our laboratory colony, we expect that these bioassays will need to be completed next spring/summer when CLM can be obtained from the field. Artificial feeding bioassays continue to be conducted to evaluate the efficacy of synthetic peptides, outlined in this proposal, against ACP. Two artificial feeding methods for exposing ACP to synthetic GNA have been evaluated since the last proposal: leaf uptake and feeding sachets. The first method, wherein ACP were placed on single, detached leaves held in a solution containing GNA, proved to be unsuccessful. Feeding sachets have subsequently been used for artificial feeding assays, with preliminary results suggesting that GNA reduces ACP survival at high concentrations. Development of enzyme-linked immunosorbant (ELISA) assays specific to the peptides under investigation are ongoing. Identifying the concentration of expressed peptides should facilitate comparisons between the efficacy observed in artificial bioassays with synthetic peptides and in planta assays with CTV-expressed peptides. Although we are able to successfully quantify CTV expression with ELISA, we have not been successful in accurately quantifying peptide expression in in CTV-GNA plants with this method.
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