Insecticides are currently the basis of Asian citrus psyllid (ACP) management programs and the number of annual insecticide applications has increased significantly. We continue our investigations of insecticide susceptibility and resistance among field populations of adult and immature Asian citrus psyllid in Florida. Our goals are to monitor resistance and develop effective psyllid management programs that mitigate escalation of resistance development. Five geographically discrete populations of ACP displayed a range of susceptibility levels against twelve tested insecticides. In 2009, one or more field populations of ACP exhibited higher LD50 values to fenpropathrin, imidacloprid, malathion and thiamethoxam compared with the laboratory susceptible population. The highest level of resistance was displayed by the La Belle population with a resistance ratio or RR (LD50 value of field population/LD50 value of laboratory susceptible population) of 38 and 13 to imidacloprid and thiamethoxam, respectively. Three populations displayed a moderate level of resistance to malathion (Ft. Pierce: RR = 5.4, Lake Alfred: RR = 5.0, Groveland: RR = 3.7). The Vero Beach population displayed a moderate level of resistance to fenpropathrin with a RR value of 4.8. In 2010, three diagnostic doses corresponding to 50, 75 and 95 percent mortalities determined from the laboratory susceptible population in 2009 were chosen to assess the susceptibility of field populations in 2010. Susceptibilities of field populations were compared with the laboratory susceptible population as in the previous year. In general, percent mortality of adult ACP from each field population was lower than that of the laboratory susceptible population for all three diagnostic doses tested. This indicates that resistance levels continue to increase from year to year. The susceptibility levels of immature ACP from four field populations to five insecticides were determined. The LC50 value for carbaryl obtained from the laboratory susceptible population was significantly lower than from the highest value obtained from one of the field populations representing a resistance ratio of LC50 [RR50] of 2.88. For chlorpyriphos, the highest LC50 of 8.31 observed from a field population was significantly higher than the laboratory susceptible population (2.58), representing an RR50 of 3.22. The synthetic pyrethroid, fenpropathrin, yielded a range of LC50 values from 0.15-0.57, with the lowest value obtained from the laboratory susceptible and highest from the Groveland population. Two of the field populations tested exhibited significantly lower susceptibility to imidacloprid than the laboratory susceptible population exhibiting RR’s of 3.81 and 2.27, respectively. Two of the populations tested showed significantly lower susceptibility to the microbial insecticide, spinetoram, than laboratory the susceptible population with RR50’s of 2.98 and 5.88. The current results suggest that ACP have developed varying levels of resistance to major insecticides currently registered for its management. Elevated levels of detoxifying enzymes in these populations may be underlying mechanisms of this resistance. Although none of these current resistance levels should result in product failures in the field, continuing elevation of resistance among Florida populations of ACP needs to be stopped. Rotation of insecticides is critical to mitigate this emerging problem.
.-aminobutyric acid (BABA) is known to induce resistance to microbial pathogens, nematodes and insects in several host plant/pest systems. The present study was undertaken to determine if a similar effect of BABA occurred against the Asian citrus psyllid (ACP) in citrus. A 25 mM drench application of BABA significantly reduced the number of eggs/plant as compared with a water control, whereas 200 and 100 mM applications of BABA reduced the numbers of nymphs/plant and adults/plants, respectively. A five mM foliar application of BABA significantly reduced the number of adults but not eggs or nymphs when compared with a water control treatment. BABA induces resistance in plants through a number of physical and biochemical mechanisms. Accumulation of pathogenesis-related (PR) proteins is one of the responses elicited by application of BABA to plants. Expression of systemic acquired resistance (SAR) in plants as a result of BABA application is related to elevated expression levels of the PR-1, PR-2 and PR-5 genes. The objective of this experiment was to determine whether exposure of citrus plants to ACP adult feeding alone or in combination with BABA treatment induces expression of the PR-2 gene. Significant variation in the expression level of the PR-2 gene was observed among treatments. Significantly higher expression of PR-2 was observed in plants exposed to 500 mM of BABA in combination with ACP adult feeding when compared to the control or other treatments tested. In citrus, SAR induction was explained by the high expression of the PR-2 gene, which in turn relates to a higher resistance of citrus against canker (Francis et al., 2009). The results of the present study show that the PR-2 gene was upregulated by more than 150-fold in citrus treated with BABA in combination with ACP adult feeding compared to the control or citrus treated with BABA or ACP feeding alone. These results suggest that PR proteins, or at least one PR protein in citrus, accumulates as a result of the combined effect of BABA and ACP feeding. Our results corroborate findings of another study, where green peach aphid, Myzus persicae, feeding resulted in the activation of defense-related genes, including PR-1 and BGL2 on Arabdopsis (Moran and Thompson, 2001). Lack of elevated PR-2 gene expression in the BABA treatment could have occurred because the concentration of BABA tested may have not been sufficiently high to cause gene expression. Alternatively, the interval between BABA treatment and our assay may have not been long enough to cause PR-2 gene upregulation. Therefore, optimizing the concentration and priming period of BABA is needed to further elucidate the mechanisms imparting induced resistance in citrus after treatment with BABA. Our results show that all three developmental stages of ACP were negatively impacted by BABA through induction of host-plant plant resistance in citrus. Induction of host-plant resistance may be a viable alternative or additional tool for ACP. BABA could potentially be applied as part of a tank-mix with other pesticides, including various plant activators and fungicides. At this stage, however, additional investigations are needed to optimize a cost effective and efficient dosage of BABA for optimal management of ACP under field conditions. In addition, further work is needed to evaluate other commercially available products, such as 2,6-dichloroisonicotinic acid (INA) and benzothiadiazole (BTH), for inducing resistance in citrus against ACP. The effect of BABA and other SAR inducers on other insect pests or diseases of citrus is also needed.
More T. radiata wasps originally imported from Vietnam, China, and Pakistan were produced by colonies established at DPI-Gainesville and Southwest Florida Research and Education Center and released in Florida citrus. That is the major focus of two (number 1 and 3) of the three objectives of this project. From Pakistan, south China, and North Vietnam colonies established at DPI-Gainesville, 26,861, 16,248, and 12,940 wasps were produced and released, respectively, that increased the number of total released wasps to 48,079, 49,495, and 38,544, respectively. Parasitoids from the Pakistani colony were released at the SWFREC experimental grove in Collier County and from south China and N. Vietnam were integrated with biweekly sprays of horticultural oil in replicated trials at two commercial groves, in Glades and Charlotte Counties, respectively, the latter being organic. A total of 1,196 adults of D. citri and 58 adults of T. radiata (4.6% parasitism) emerged from feral nymphs collected in August at SWFREC and reared in the laboratory. No mummified nymphs were recovered on sentinel plants placed at these locations during July or August but 8% of nymphs were mummified among those recovered on sentinel plants placed in a neighboring commercial grove in July. Samples of feral nymphs collected in September from the Glades grove and reared in the laboratory produced 880 adults of D. citri and one adult of T. radiata (0.1% parasitism) from oil-treated plots and 741 adults of D. citri and and 25 adults of T. radiata (3.3% parasitism) from untreated plots. During the same month, nymphs collected from treated and untreated plots in the Charlotte grove produced 103 and 51 adults of D. citri, respectively, but no parasitoids. No mummified nymphs were recovered on sentinel plants placed in July in treated and untreated plots at both locations. The colony at SWFREC of the previously established strain of T. radiata produced 11,855 wasps that increased the previous total to 56,645 produced this year and used to maintain the colony at OrangeCo, conduct laboratory experiments, and release in experimental, organic and conventional groves including the ones mentioned above. These wasps were also released in the treated and untreated plots of an experimental block of a commercial grove in Collier county where treated plots received insecticides and nutritional sprays. The feral nymphs collected in August and reared in the laboratory produced 792 adults of D. citri and three adults of T. radiata (0.4% parasitism) from treated plots, and 764 adults of D. citri and one adult of T. radiata (0.1% parasitism) from untreated plots. In September, 448 and 366 adults psyllids but no parasitoids emerged from nymphs collected from treated and untreated plots, respectively. We are collaborating with California Department of Food and Agriculture, USDA-ARS, USDA-APHIS and Florida Department of Ag & Consumer Services (Division of Plant Industry) to arrange a meeting this November in Florida to discuss mass production and release of T. radiata for biological control of the Asian citrus psyllid. Publications: 1) Mann, R.S., J. A. Qureshi, P. A. Stansly and L.L. Stelinski. 2010. Behavioral responses of Tamarixia radiata (Hymenoptera: Eulophidae) to volatiles emanating from Diaphorina citri Kuwayama (Hemiptera: Psyllidae) and citrus. Journal of Insect Behavior (In press). 2) Barr, N.B., D.G. Hall, A. Weathersbee, R. Nguyen, P. A. Stansly, J. A. Qureshi, and D. Flores. 2009. Comparison of laboratory colonies and field populations of Tamarixia radiata, an ecto-parasitoid of the Asian Citrus Psyllid, using ITS and COI DNA sequences. Journal of Economic Entomology. 102: 2325-2332.
The objectives of this study are: 1) to develop a series of flexible stochastic models to predict the temporal increase and spatial spread of citrus disease. The models were initially characterized for citrus canker but have subsequently been extended to HLB. They can be used in a number of ways: to predict spread and to analyze the effectiveness of control strategies both in plantations and State-wide. 2) Test various control methods under field conditions to evaluate effects and collect data to parameterize models A SEIDR model (Susceptible, Exposed (latently infected but not yet infectious), Infectious but not yet symptomatic/detected, Detected and infectious and Removed trees) was developed. Using Markov-chain Monte Carlo methods, and extensive data from South Florida for successive snapshots of the occurrence of symptomatic detected trees in known populations of susceptible trees, we have been able to estimate the transmission rates and dispersal kernel for HLB. Current work is focused on the differential effects of host age on epidemiological parameters as well as variability across the plantation. From these it is possible to allow for uncertainty in the parameters as well as variability over time and through space. We used Baysian methods to infer posterior densities on the model parameters. The uncertainty is then incorporated in models to predict spread and to allow for uncertainty in the efficiency and comparison of control methods. A front-end (a web based version of the model) was developed for non-researcher users has now been nearly finalized. Via this web tool, both residential and commercial citrus scenarios can be tested and a wide variety of epidemiological and climate/weather variables have been included and are user selectable and changeable via sliding controls. Various disease control/mitigation parameters can be selected and simulations can been visualized to see the effect of these various control strategies. This web tool runs simulations one at a time and is highly instructive to growers and regulators. It is based on a more formal analytical model that can run thousands of simulations based on the same parameters and make more statistically valid predictions for regulatory intervention strategy building and regulatory/industry decision making. Currently the spread of HLB is being modeled in 10 contiguous blocks in SG, chosen to encompass both young and mature trees, since host age is likely to be an important consideration for HLB. The blocks are also relatively isolated from the surrounding outbreak. The outbreak is noticeably clustered with only a small number of isolated cases. The model continues to be improved upon to capture more and more of the true features of the data and the disease. The model is being extended to estimate spread, should HLB be introduced into new areas such as TX, CA or AZ. Field plots were established at the USDA, ARS Picos Farm to examine the effects of insecticide control and roguing in various combinations, both to evaluate treatment effects and to collect epidemiological data for model development. As with previous trials conducted in south Florida, to date there is no statistical differences among various control strategies. We interpret these results to support the hypothesis that control must be applied on an area-wide basis to see significant benefit from control efforts.
This project was designed to examine the potential disease control of citrus huanglongbing (HLB) by interplanting citrus with guava. In Vietnam guava has been shown to be an effective deterrent to HLB, slowing the disease and keeping plantings alive for up to 15 years that normally succumb in 2-3 year. For all plots and experiments, Guava trees, (Vietnamese white cultivar) were propagated and grown to appropriate size requiring ~1 year. Both nursery and field citrus trees are assayed for HLB every 60 days, and have been assayed multiple times. Psyllid populations are also being monitored continuously every 2 weeks within interplanted plots to document any repulsion of the vector due to guava. Results: Guava vs no guava nurseries: Two nursery sites, a guava protected citrus nursery versus an unprotected nursery, have were established with disease free, PCR-negative citrus trees (2 sweet orange and 1 grapefruit cultivars) in June 2009 and were located in the protected and unprotected plots. The guava trees were grown to appropriate size as indicated in Vietnam prior to outplanintg. To date HLB appears to be progressing more slowly in nursery plots interplanted with guava that non interplanted plots. Citrus/guava interplantings: 3 commercial plantings with multiple replications were established. This required 1 year to grow the guava transplants and a second year to become established in the field before interplanting with citrus per Vietnam protocols. One trial was established in a commercial orchard with collaborators in Southern Gardens Citrus, but two years of freezes and replanting prompted the grower/collaborator to abandon the planting. A second was established in grove in Martin county but the plantation was sold and removed before data could be collected. A third trial planting was established at the USHRL Picos Farm in Fort Pierce. The Picos plot was interplanted with citrus in August 2009. Severe frosts during 2008/2009 and again during 2009/2010 winters affected the USHRL plots and caused a delay in the experiment. To date, several plants have been identified in the USHRL plots after multiple assays. The majority of these positives have occurred as the result of an edge effect. That is, treatment plots closest to an existing HLB+ planting of Valencia were affected. To date, there is no significant difference among the treatments. However this data is insufficient to draw any conclusions. Research was initiated on using a Y-tube olfactometer to investigate guava volatiles as repellents of the psyllid. Initial research involved calibrating the equipment and ensuring there was no bias in the behavior of psyllids under the assay. In an attempt to find a reliable control plant to test guava against, the following have been studied as known attractants: 1) whole lemon plants, (2) C. macrophylla shoots, (3)whole C. macrophylla plants, (4) starved psyllids with C. macrophylla shoots, (5)old psyllids with C. macrophylla shoots, and (6) old psyllids with lemon shoots. The best response was 1 week old psyllids with C. macrophylla shoots, although psyllid attraction to this odor source was weak. Research with white guava (germplasm from Vietnam) as a repellent alone or in combination with citrus will be initiated once the assay using citrus is refined.
A trunk cutting, herbicide spray applicator device was constructed, mounted on a small tractor and tested to kill HLB infected trees. In two tests, 6 cuts were better than 3 and full strength imazapur (Arsenal) worked better than the product diluted 50%. The best treatments killed over 95 % of the canopy in each test, but tree death was relatively slow requiring more than 1 month to show significant tree decline. During the testing process, improvements in the machine provided more reliable cutting and spraying. A third test has been applied for a final evaluation. BASF Chemicals has expressed willingness to obtain a label for their formulation of this herbicide as applied in these tests. Both the University of Florida Office of Technology Transfer and Chemical Containers Inc. were contacted regarding the possibility of patenting this equipment and the treatment procedure. All of this information was presented to the CRDF Committee at a meeting in Lake Alfred, FL. At this time there is no indication that Chemical Containers is interested in commercial development of this equipment. This is probably because the foliar nutrition cocktail mixes appear to be maintaining HLB infected trees in a productive state for several years and fewer growers are scouting and removing infected trees. Additional tree killing chemicals are being tested. The results will be reported in future.
This is the annual report of this project. The outcomes are as follows: 1) A method was developed for evaluating the responses of free-flying ACP to combinations of olfactory and visual cues within the controlled environment of a greenhouse. Tests measured the trapping rate of ACP on scented and unscented greenish-yellow sticky traps (ACP traps, AlphaScents, Inc.). Petitgrain oil, an essential oil distilled from sour orange leaves, was used as a test odorant because it contains substantial amounts of linalyl acetate and linalool, two of the primary volatiles emitted by the foliage of Meyer lemon, a favored ACP host tree in south Texas. ACP were presented with an array of either unscented or scented traps in a no-choice test. The trap array consisted of six rows of traps with two traps per row. Each trap measured 34 cm long x 2.5 cm wide. Scented traps had a rubber septum loaded with 1 mL. petitgrain oil stapled to the front. The traps were positioned upwind of a screened release cage with 1000 ACP collected from a local orchard. To determine the accumulation rate of ACP on the traps, a census of each trap was made at 15-, 30-, and 45 minutes intervals following their release. A total of five replicated tests were conducted for each treatment. Psyllids accumulated more quickly on the scented- versus unscented traps in the initial 30 minute interval following their release from the cage. During this time, the number of ACP caught on the scented traps increased by an average of 52% while on the unscented traps the average increase was 35% (t = 2.3609; P = 0.046). The accumulation rate of psyllids on the scented traps was twice as high as that on the unscented traps between the 30- and 45minute interval (18% for scented, 9% for unscented), but this difference was not statistically significant t = 1.8032; P = 0.71). This test may be useful for screening combinations of olfactory and visual stimuli and for selecting those that show the most promise for further testing in orchards. A drawback of the test is that it requires large numbers of pysllids, many of which fly to the ceiling following their release and do not respond further to the test cues. Pysllids collected from orchards appear to be sensitive to environmental stressors (i.e., drought, heat). A summer long drought in 2009 curtailed tests at the beginning of August because the psyllids ceased to respond to test cues.
The objective of this 2 year project was to provide direct assistance to growers on the east coast in developing and evaluating their psyllid management programs. Dr. Pasco Avery was hired at the IRREC to provide direct grower support for this program. During the past 2 years, Dr. Avery and the personnel hired to work in his program aided growers on the east coast by helping to assess psyllid population dynamics under differing management regimes implemented in both organic and conventional citrus operations. This helped growers to get a better understanding of what does and does not work in terms of psyllid management. In addition to the direct extension type assistance provided, Dr. Avery also evaluated the use of the fungal pathogen Paecilomyces formosoroseus for control of the Asian citrus psyllid which growers indicated was of interest due to need for additional control measures due to difficulties meeting pesticide MRL requirements for export to foreign markets. Studies were conducted in both and field conditions to better understand what potential this fungal pathogen holds for managing ACP. The following is a list of publications and presentations by Dr. Avery resulting from this work: Avery, P. B., W.B. Hunter, D.D. Hall, M.A. Jackson, C.A. Powell and M.E. Rogers. 2009. Novel delivery of the biocontrol fungi Isaria formosoroseus for managing the Asian citrus psyllid and reducing spread of citrus greening ‘huanglongbing’ disease. Fla. Entomol. 92 (4): 608-618. Avery, P. B., Wekesa, V. W., Hunter, W. B, Hall, D. G., McKenzie, C, L. Osborne, L. S., Powell, C. A. and M. E. Rogers. Antifeedant and lethal effects of the fungi Isaria fumosorosea on the Asian citrus psyllid Diaphorina citri. Biocontrol Science and Technology (submitted). Avery, P. B., Hunter, W. B, Hall, D. G., Jackson, M. A., Powell, C. A. and M. E. Rogers. Potential of a new biopesticide for managing Asian citrus psyllid. Indian River Citrus Seminar ‘ Ft. Pierce, FL, January 29, 2009. Avery, P. B., Hunter, W. B, Hall, D. G., Jackson, M. A., Powell, C. A. and M. E. Rogers. 2009. Broad spectrum potential of the biopesticide, Isaria fumosorosea for managing insect pests of citrus. 42nd Annual Meeting of the Society for Invertebrate Pathology. Avery, P. B., Hunter, W. B, Hall, D. G., Jackson, M. A., Powell, C. A. and M. E. Rogers. 2009. Investigations of the feasibility for managing the Asian citrus psyllid using Isaria fumosorosea. Proceedings of the International Research Conference on Huanglongbing: Reaching Beyond Boundaries ‘ Orlando, FL, December 1-5, 2008 – published in American Phytopathological Society on their Plant Management Network (PMN) website. This grower assistance project ended in spring 2010 with Dr. Avery taking a postdoc position with Dr. Lance Osborne at the UF Apopka REC. Dr. Avery will continue to work under Dr. Osborne on fungal pathogens which will likely include continuing research into the feasibility of managing ACP using fungal pathogens.
This is the final report of the proposal (FDACS Contract Number 58-1920-9-925 [40]). Our research objectives were: (1) Devise and perform alternative methods (microinjection and membrane uptake) to complete Koch’s postulates using a pure culture of bacteria isolated and cultivated in our laboratory and healthy psyllids as a transmission tool; and, (2) following successful inoculation or loading of the psyllids, we will complete Koch’s postulates. Short term cultivation of a strain of ‘Candidatus Liberibacter asiaticus’ (Las) from Taiwan (B239) was accomplished in our laboratory using two different published methods, and determined to be Las by conventional and real-time PCR and sequencing. We prepared stretched parafilm membrane sachets containing sucrose solutions in 1XTE buffer in which the cultivated bacteria were suspended. Sucrose solutions ranged from 5-20%, however, most experiments were conducted with 10% sucrose. The titer of the bacteria in sucrose was roughly assessed using realtime PCR (Ct values ranged from 20 to 38). Membrane sachets were suspended over 15 ml or 50 ml conical tubes within which 15-20 healthy adult psyllids and 5th instar nymphs were allowed to probe the membranes. After 48 – 72 hr acquisition feeding, psyllids were transferred onto sweet orange seedlings and the remaining fluid in the sachets was assayed by real time PCR. Psyllids and nymphs were allowed to feed on the sweet orange seedlings for 14 days after which they were removed, and assayed for Las by real time PCR. From more than 30 membranes attempted, nine test membranes, each containing 250 ‘ls of culture in 0.25 ‘ 0.5 ml sucrose solution, were successful in uptake of bacterial culture by the psyllids. The orange seedlings were observed for symptom development and assayed three months post-inoculation by real time PCR. Although no symptoms were observed at 3 months, Madam Vinous sweet orange seedlings inoculated with infectious culture had Ct values ranging from 19.5 to 43.8. Typical HLB leaf symptoms were observed by six months. A second approach used direct microinjection of the bacterial cultivation fluid into the hemolymph of adult Diaphorina citri. Psyllids were immobilized with a low velocity stream of C02 and approximately 0.01’l of bacterial culture (Ct value of the bacterial suspensions ranged from 32.9 to 38.3) was injected into the abdomen of each adult psyllid. Psyllids were allowed to recover and then placed on young sweet orange seedlings for inoculation feeding for 14 days. Surviving psyllids were captured, assayed by realtime PCR for the presence of Las, and plants were observed for 3 to 6 months for symptom development. Sweet orange plants inoculated in this manner also became infected, and showed symptoms typical of HLB. Our primary objective was to complete Koch’s postulates by transmitting a pure culture of Las using the psyllid vector. Although a stable pure culture was not attained, we did obtain short term cultivation of a fastidious bacterium which reacted to Liberibacter primers/probe in conventional and realtime PCR assays, produced positive infections of healthy sweet orange seedlings, and could be re-isolated. Our research helps to cement the causal relationship of Las to HLB and provides important insights into the nature of the pathogen-vector interaction.
Current management of citrus greening requires preventive control measures targeted to the pathogen (e.g. planting healthy nursery trees, inspection and removal of diseased plants) and to the psyllid vector, Diaphorina citri. In this project we are investigating factors that influence the risks of acquisition or inoculation of the pathogen (Candidatus Liberibacter asiaticus) by D. citri, e.g vector developmental stage, feeding periods, leaf phenology and symptom expression/bacterial population in disease plants, in order to optimize strategies to avoid or reduce disease spread within and between citrus groves. We already learned in this project that bacterial acquisition can occur when the vector feeds on asymptomatic infected plants, although acquisition efficiency is higher on citrus plants with higher bacterial titers, usually symptomatic. We also showed that D. citri nymphs in all development stadia (1st-5th instars) can efficiently acquire the pathogen if allowed to feed for 48 h on young leaves of infected plants (mean acquisition rates ranging from 75-100%); adults can also acquire it, but efficient acquisition depends on the availability of young leaves in infected plants, apparently because phloem sap ingestion (and thus pathogen acquisition) by D. citri adults is more frequent and last longer on the younger leaves. Here we report a further experiment detailing acquisition efficiency of Ca. L. asiaticus by adults and nymphs of D. citri, in which we varied the acquisition access period (AAP). Groups of 30 psyllid adults (1-wk old) or third-instar nymphs were confined on leaves of a young shoot of a symptomatic infected plant, with recently expanded leaves, inside sleeve cages, for AAPs of 1.5, 6, 12, 24, 48 or 96 h. After the AAP, the insects of each group were first transferred to healthy citrus seedlings for a latent period of 15 days at 25C, and then transferred to healthy test seedlings (5 insects/plant) for a 7-day inoculation access period (IAP). After the IAP, total DNA of each insect (sample of 10-20 insects per AAP treatment) was extracted and submitted to nested-PCR with specific primers for Ca. L. asiaticus. The experiment was repeated three times, using different source plants of the pathogen for the AAPs. Partial results showed that around 28% nymphs and adults can acquire the pathogen during an AAP of only 1.5 h. Acquisition efficiency for both nymphs and adults increases linearly with longer AAPs, exceeding 90% after a 96-h AAP. For nymphs, acquisition rates of 38, 55, 91, 88 and 94% were observed when the insects were allowed to feed for 6, 12, 24, 48 and 96 h on the source plants, respectively. Because development time of D. citri nymphs on young citrus shoots ranges between 2-4 wks under field conditions, there is plenty of time for pathogen acquisition if the host plant is infected; in this case, it is likely that most (if not all) emerging adults will be infective. This observation makes imperative to control developing nymphs of D. citri in infected groves. If not controlled, the emerging adults will certainly spread the pathogen to other citrus plants.
This research project is directed towards controlling psyllids using biologically-based control strategies that employ the use of RNAi technology against key biological control pathways, peptide hormones and protein inhibitors that, if expressed in transgenic citrus, would enhance plant resistance to psyllids feeding. DIET: Both protein-based and RNAi strategies were tested by feeding psyllids artificial diets. Both protein-based and RNAi strategies were tested using artificial diets on which pysllids were fed. Psyllids in nature, feed on phloem content of citrus and its relatives. Thus, psyllids do not tolerate many alterations to diet composition that is drastically different than the phloem content. Addition of high concentrations of proteins or single stranded and double stranded RNA (ssRNA and dsRNA) reduces psyllids survival. Therefore, we determined the acceptable concentrations of each molecule and cofactor that was added including a suitable buffer to allow continuous feeding and maintenance of a physiological pH that was not detrimental to psyllids. We also identified an antimicrobial agent that was added to the diet and prevented fungal growth but did not harm the psyllids or their associated and obligate symbiotic microflora. Prior to the identification of the antifungal agent, fungal contamination of the diet caused unacceptable high level of psyllids mortality because the fungus is carried by the psyllids and can enter the diet through the psyllids feeding process. Control experiments showed that addition of dsRNA molecules, that did not target psyllids transcripts, at up to ~16 ng/uL improved psyllids performance, but above this concentration, the non-specific dsRNA would reduce psyllids survival. Therefore, comparisons of efficacy of specific psyllids gene targeting dsRNA were done with dsRNA that did not target psyllids genes. PROTEIN: In separate experiments, mosquito peptide hormone, TMOF, and Diaprepes abbreviates (citrus root weevil) cysteine protease inhibitor (CPI) were added to an artificial diet that was fed to psyllids. TMOF and CPI were tested at concentrations of 10 ‘g/’L and 3 ‘g/’L, respectively. After 10 days of feeding, all the psyllids that were fed diets containing either TMOF or CPI died, whereas only 40% mortality was observed in psyllids that fed on the control diets. TMOF caused 15% mortality after 4 days of feeding as compared with less than 5% mortality in the control group. Psyllids that were fed CPI did not show significantly higher mortality than the controls until after 7 days of feeding, because CPI was tested at less than . the concentration that was used for the TMOF because of limited availability. During the second year of the grant’s period more CPI will be synthesized and purified to study dose effect and optimal concentration, as well as, potential for synergistic effects when both proteins are present within the same diet. RNAi: Ten psyllids genes representing three gene families of cathepsins (five genes), vacuolar ATPases (four genes), and tubulin (one gene) were targeted and their dsRNA (16 ng/’L) fed to psyllids using artificial diets. The earliest effects were observed at ~4 days after feeding and feeding continued until day 10. . We have identified two of the best target genes to date as being a Cathepsin (CF2) and a Vacuolar ATPase (Vatpase-3). When specific dsRNA molecules to each are fed separately to adult psyllids we see a doubling in psyllid mortality over control non-specific dsRNA molecules at 6 to 7 days with 48 ng/uL of dsRNA in the diet. However, when they are supplied together we see similar mortality rates at 1/10th the concentration. At the lowest concentrations tested (3 ng/uL) we see mortality that appears to be non-sequence specific. However,sequences targeting CF2 and Vatpase-3 show a much more rapid increase in mortality as concentrations exceed 6 ng/uL. These results suggest that antagonistic effects of ingestion of low concentrations of any dsRNA may provide limited benefit and that by combining this effect with psyllid specific dsRNAs significant control can be realized. Based on these results, the production of transgenic plants expressing a chimeric CF2/Vatpase-3 dsRNA producing gene is being initiated.
Laboratory-based investigations In the laboratory, we have been evaluating the effect of droplet size on psyllid mortality using a controlled droplet applicator (CDA) and a droplet counting device (DC-III). Our first objective is to determine the optimum droplet size for psyllid control. Our second objective is to identify appropriate spray adjuvants that achieve the optimal droplet size when mixed with formulated insecticides. The DC-III was successfully used to screen candidate spray adjuvants for their effect on droplet size production from the CDA. Of the adjuvants that were evaluated, two of these significantly increased droplet size. This increase in droplet size is part of the ongoing effort to ensure low volume applications meet EPA label requirements, i.e. droplets of 90-‘m or greater. These products were then used in the truck mounted low volume applicator to assess their effect on the droplet size produced in the field when applied with a commercially available insecticide labeled for ACP control in citrus by low volume. We found that organosilicone adjuvants were optimal for making droplet size uniform and conforming to label guidelines when mixed with the pesticide Danitol. The CDA has also been used to apply insecticide treatments to ACP on potted citrus plants. To date two out of the five selected chemicals have been applied in a standard toxicity test. The other three chemicals have been screened for suitability in the CDA and further applications are scheduled for mid August. Our preliminary results indicate that effectiveness of these pesticides increases as droplet size is decreased; however, an optimum is reached at approximately 100 micron sized droplets. We anticipate having this portion of the laboratory investigations completed in September. Field-based investigations Several field investigations of low volume sprays are underway. We are conducting efficacy tests of 12 conventional pesticide treatments and their effect of populations of ACP, citrus leafminer (CLM), and their biological control agents. In these investigations, we are comparing efficacy between conventional airblast and low volume sprayers. A separate trial is underway investigating three insect growth regulators is the field. This work mirrors the doses that have proved effective against ACP in laboratory toxicity assays.
We have continued our research on the movement behavior and seasonal dispersal of Asian citrus psyllid. Our main objective is to improve psyllid management by gaining a better understanding of the psyllid’s dispersal behavior and capabilities. To investigate the potential impact of abandoned citrus on nearby managed citrus, we used an in situ immunomarking technique, in combination with ELISA to quantify the movement of ACP from abandoned citrus plots into nearby managed plots. Pairs of abandoned and managed citrus plots were chosen that were separated by a distance of 100 meters. Two crude food proteins were used to mark abandoned citrus plots (bovine casein on the edge row, and chicken egg albumin 150 meters to the interior). Yellow sticky traps were placed within the marked areas of the abandoned plots, as well as between groves and on the edge rows and 150 m to the interior of the managed plots. Traps were collected 5 days after application of the protein markers, and captured ACP were subjected to an ELISA to determine the presence of a protein mark. This study has been conducted monthly for 14 months. We found significantly more ACP, and correspondingly a higher number of ACP moving from abandoned into managed plots during June, July and August 2009 than at any other time during the experiment. Populations are much lower this summer than last. In July of 2009, we trapped 674 adult ACP; of those, 42% were found to have moved from abandoned plots into managed plots. In July of 2010, we trapped only four ACP; all of which were found to have moved from abandoned plots into managed plots. In addition to quantifying ACP movement, we used PCR analysis on all ACP that had moved from abandoned into managed citrus over the course of the study to determine whether they were carrying Ca. Las. Through ELISA and PCR we confirmed that HLB-infected psyllids are moving from abandoned citrus into nearby managed citrus. To evaluate the dispersal range of ACP, we used the in situ immunomarking technique, spraying chicken egg albumin on 200 citrus trees in the central area of a managed grove. Yellow sticky traps were placed within the marked area, and concentrically at distances of 100, 300, 400, 500, 650, 1000, 1200, and 2000 meters away from the marked area. This experiment was expansive and trap distances extended well beyond the border of the grove in which the marker protein was applied; into other managed groves, as well as some abandoned groves. Traps were removed 11 days after application of the marker protein, and captured ACP were subjected to an ELISA to determine the presence of the marker. A total of 179 adult ACP were captured, and 19% carried the protein mark. Marked ACP were found on traps within the marked area, and at each distance except for 1000 meters. Our results indicate that ACP can move at least 2000 m within 11 days.
The purpose of this proposal is to identify and develop pheromone based attractants for the Asian citrus psyllid (ACP) in order to develop effective monitoring traps to evaluate ACP population densities and better determine the need for spraying. Behavioral bioassays in the laboratory confirmed that virgin and mated male ACP adults are attracted to female ACP in olfactometers. These data suggest that female ACP produce an attractant for male ACP. Most recently, we analyzed whole cuticular extracts of male and female ACP in behavioral olfactometer experiments and in field trials. The cuticular extract from female ACP adults attracted male ACP in laboratory bioassays. In no case did male ACP cuticular extract attract female ACP in the laboratory. Additionally, male and female ACP were not attracted or repelled by same sex ACP individuals in laboratory biaoassays. Field trials with male and female ACP cuticular extracts at various dosages using yellow and clear sticky traps indicated that cuticular extracts of both female and male ACP were attractive to feral psyllids as compared with blank untreated traps in the field up to 3 days. However, more males than females were attracted to the extracts of females. Traps with female cuticular extracts attracted significantly more male than female ACP adults. Additionally, traps loaded with male or female cuticular extract attracted more ACP than control traps with no extract. All traps attracted roughly equivalent numbers of ACP adults when the traps were left in field for 15 days or more. The clear traps generated more conclusive results than the yellow traps although yellow traps attracted more total ACP adults than the clear traps. These results indicated a possibility of female produced attractant, but also suggest the possibility of an aggregation pheromone. Our results in the field were different from those observed in the lab given that it appeared in the field that male extracts were also attractive; however, those extracts were not attractive in laboratory assays. It is possible that females captured on traps, due to attraction to the yellow color, contributed to subsequent attraction of males via a chemical attractant, which may obscure the field data compared with the lab data. More field testing is needed. Chemical analysis of female and male ACP cuticluar extracts with GC-MS indicated that female are characterized by certain chemicals that were not present in male cuticular extracts including isomers of lactones and decanoic acids. Also, there were certain chemicals that were present in higher relative amounts in female cuticular extracts than in those of males. We also analyzed male and female honey dew secretions. GC-MS of honey dew secretions indicated that there were several chemicals that were common between honey dew secretions of both sexes and those of male and cuticular extracts. Behavioral bioassays with chemicals found exclusively in female cuticular extracts indicated that male ACP were attracted to dodocenoic acid in laboratory bioassays. Our previous laboratory experiments with ACP yielded similar results with . butyralactone; however ACP were not attracted to this chemical in the field. We continue to evaluate chemicals that are exclusively found in female cuticular extracts to determine if they attract males to refine the blend and its dosage in an effort to develop an attractive lure for the field. Observations of ACP indicate that adults of both sexes oscillate their abdomen dorso-ventrally on both host plants and within olfactometers prior to mating. We examined the external morphology of the sensilla present on the subgenital plates and genital regions of ACP adults with scanning electron microscopy (SEM) to gain insight into the their function with respect to communication. We continue to determine the putative functions of the identified sensilla using transmission electron microscopy (TEM).
April & May, 2010 ‘ Continued servicing the traps and accumulating information. Compiled the data from the McPhail trap servicing records, DPI’s Caribbean Fruit Fly records (including all CFF spray dates, fly catches and trapping reports) and CHRP records showing Asian Citrus Psyllid catches and worked to determine how best to format data to be submitted to Dr. Steve Rogers for comparison. June, 2010 ‘ Until June 16, the traps were serviced and data gathered for the project. The traps were removed on June 16 (two weeks early) due to personnel being reassigned to the Medfly project.
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