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.
We have initiated two new collaborations in various efforts for the application of the D. citri insect cell lines developed. (1) Dr. W. Hunter, Objective: provide cultured cells for genomic characterization of D. citri. We have scaled-up the growth and culturing of several lines in order to have enough material for extraction and characterization of the genomic DNA derived from the cultured. The requested amount of cells ~0.1-1 g of material. We have gathered approximately 0.2 g of material and will forward the frozen cells to Dr. Hunter’s lab presently. It is anticipated that an additional ~ 0.2 g of cells will be ready within the next month. (2) Dr. Lisa Fontaine-Bodin,CIRAD-BIOS, Montpellier, France, Objective: to use the cell lines in attempts to culture the various European and African Liberibacter strains. In the part quarter, we have sent Dr. Fontaine our cells and they are apparently viable and the cell lines should be established in the lab. It is anticipated that attempts to cultivate the various Liberibacter strains available in Dr. Fontaine’s lab will proceed in the coming year. The laboratory continues in attempts to increase the robustness of the cells lines. A periodicity in the growth has been noted which current efforts are aimed at mitigating. Suspension cells lines appear more uniform, however, contain large clumps and doubling times remain in 2-3 weeks. Attached cells remain heterogeneous with doubling times near 2-4 weeks. Antibiotic free cell lines have also been established and are now in routine growth in the lab.
The purpose of this multi-faceted research project has been to develop or uncover biorational alternatives to conventional pesticides for management of Asian citrus psyllid (ACP). One tactic that we have been recently exploring is the use of systemic induced resistance (SAR) against ACP. .-aminobutyric acid (BABA) is known to induce resistance against several microbial pathogens, nematodes and insects in several host plants. The current investigation was undertaken to determine whether BABA would induce resistance against ACP in citrus under greenhouse conditions. We examined the effect of five different concentrations of BABA applied as a root drench to citrus plants, on the performance of ACP. Observations were made on the number of eggs, nymphs and adults produced per plant. In addition, leaf-dip bioassays were performed using similar concentrations of BABA to rule out the possibility of any direct toxic effect of BABA on early nymphal instar (2nd), later nymphal instar (4th) and adults ACP. The results revealed that BABA-induced resistance in citrus plants can suppress the growth and development of ACP at various growth stages. The total mean (‘ SEM) number of eggs produced per plant was significantly higher in control plants (97.9 ‘ 8.8) than in plants treated with 25 (43.5 ‘ 12.8) and 100 (44.8 ‘ 14.9) mM of BABA. The total mean number of nymphs produced per plant was significantly higher in control plants (74.0 ‘ 7.3) than in plants treated with 25 (47.9 ‘ 10.2), 50 (37.7 ‘ 7.9) and 100 (364.2 ‘ 10.4) mM of BABA. Likewise, the mean number of males and females was significantly higher on control plants (male: 4.7 ‘ 1.3; female: 5.6 ‘ 1.1) than on plants treated with 25 (male: 3.2 ‘ 1.0; female: 2.7 ‘ 0.9), 50 (male: 1.5 ‘ 0.4; female: 1.6 ‘ 0.5) and 100 (male: 1.2 ‘ 0.3; female: 1.4 ‘ 0.7) mM of BABA. The percent mortality of early nymphal instar (2nd), later nymphal instar (4th) and adults of ACP as a result of different BABA concentrations was not significantly different from the percent mortality observed in the control treatment. The above results suggest that the reduced growth and development of ACP on BABA-treated plants is a function of induced resistance in citrus plants, rather than direct toxicity by BABA on various developmental stages of ACP. The results of the current study suggest that BABA has potential as a SAR treatment for management of ACP that may supplement conventional insecticides. However, the effects of BABA under field conditions and its effect on no-target organisms still require further investigation. In addition, since BABA acts by potentiating a normally under-expressed defense pathway; therefore, genetic tools could be used to trigger such pathways by genetic alteration and possible development of transgenic cultivars.
Asian citrus psyllids (ACP) generally rely on olfaction and vision for detection of host cues. Plant volatiles from Allium spp. (Alliaceae) are known to repel several arthropod species. Recently, we examined the effect of garlic chive, (A. tuberosum Rottl.) and wild onion (A. canadense L.) volatiles on behavior of ACP in a laboratory two-port divided T-olfactometer. Citrus leaf volatiles attracted significantly more ACP adults than clean air. Volatiles from crushed garlic chive leaves, garlic chive essential oil, garlic chive plants, wild onion plants and crushed wild onion leaves all repelled ACP adults when compared with clean air, with the first two being significantly more repellent than the others. However, when tested with citrus volatiles only crushed garlic chive leaves and garlic chive essential oil were repellent and crushed wild onions leaves were not. Chemical analysis of the headspace components of crushed garlic chive leaves and garlic chive essential oil by gas chromatography-mass spectrometry revealed that monosulfides, disulfides and trisulfides were the primary sulfur volatiles present. In general, trisulfides (dimethyl trisulfide) inhibited the response of ACP to citrus volatiles more than disulfides (dimethyl disulfide, allyl methyl disulfide, allyl disulfide). Monosulfides did not affect the behaviour of ACP adults. A blend of dimethyl trisulfide and dimethyl disulfide in 1:1 ratio showed an additive effect on inhibition of ACP response to citrus volatiles. The plant volatiles from Allium spp. did not affect the behavior of the ACP ecto-parasitoid Tamarixia radiata (Waterston). Thus Allium spp. or the tri- and di-sulphides could be integrated into management programmes for ACP without affecting natural enemies. The current results provide evidence that volatiles from crushed garlic chive leaves inhibited the response of ACP to its normally attractive host plant volatiles. These volatiles also appeared to have inhibited the psyllid’s normal geotactic and phototactic responses. Furthermore, our results suggest that the sulfur volatiles released by wounded A. tuberosum leaves affected the behavior of ACP. Our current efforts are focusing on formulating these sulfur compounds into controlled release devices for deployment in the field. In field investigations, we continue to evaluate the effect of DMDS on psyllid populations when deployed in the field in the SPLAT release device from ISCA. In one field experiment we eliminated psyllid populations with a standard Danitol spray and subsequently applied DMDS in SPLAT to half the plots, while leaving the other half untreated. We found that it took psyllids significantly longer to re-colonize citrus plots that were first treated with Danitol and then by DMDS than those that were treated with Danitol only. We initiated another large field experiment evaluating DMDS in SPLAT in a commercially managed grove. All plots receive psyllid management sprays, but half also received additional DMDS treatments. Under this low psyllid population density situation, we have observed no additional benefit of adding DMDS. Thus, our results with applying DMDS in the field have been inconsistent to date, with certain experiments showing promise, while others showing little to no effect of the DMDS treatment. We have also discovered that DMDS is highly phyto-toxic. Application of SPLAT containing DMDS was found to kill large tree branches. We are looking into alternate formulations and application procedures to overcome this hurdle. We also have three new SPLAT-DMDS formulations for extended longevity that we are evaluating; however, those tests are ongoing.
The objective of this project is to elucidate mechanisms of resistance in ACP to insecticides and to develop appropriate resistance management strategies. Microorganisms are known to alter insect host physiology, which may benefit or harm the host. Most recently, we determined the effect of Candidatus Liberibacter asiaticus (Las), the bacterium presumably responsible for causing huanglongbing (HLB) disease, on the physiology of its vector, the Asian citrus psyllid (ACP). Specifically, we determined the effects of Las infection on susceptibility of ACP to selected insecticides. Furthermore, total protein content, general esterase, glutathione S-transferase (GST) and cytochrome P450 activities were quantified in Las-infected and uninfected ACP to gain insight into the possible mechanism(s) responsible for altered susceptibility to insecticides due to Las infection. LC50 values were significantly lower for Las-infected than uninfected ACP adults for chlorpyriphos and spinetoram. Furthermore, there was a general trend for lower LC50 values for three other insecticides for Las-infected ACP; however, the differences were not statistically significant. Total protein content (‘g ml-1) was significantly lower in Las-infected (23.5 ‘ 1.3 in head + thorax; 27.7 ‘ 1.9 in abdomen) than uninfected (29.7 ‘ 2.1 in head + thorax; 35.0 ‘ 2.3 in abdomen) ACP. Likewise, mean (‘ SEM) general esterase enzyme activity (nmol min-1 mg-1 protein) was significantly lower in Las-infected (111.6 ‘ 4.5 in head + thorax; 109.5 ‘ 3.7 in abdomen) than uninfected (135.9 ‘ 7.5 in head + thorax; 206.1 ‘ 23.7 in abdomen) ACP. GST activity (‘mol/min/mg protein) was found to be significantly lower in Las-infected ACP (468.23 ‘ 26.87) when compared to the corresponding uninfected adults (757.63 ‘ 59.46). Likewise, mean P450 activity (EU of cytochrome P450/mg of protein) was significantly higher among the uninfected (0.49 ‘ 0.05) than in Las-infected (0.23 ‘ 0.02) ACP. Susceptibility of ACP to selected insecticides from five major chemistries was greater in Las-infected than uninfected ACP. The lower total protein content, and reduced general esterase, GST and P450 activities in Las-infected than uninfected ACP may partly explain the observed higher insecticide susceptibility of Las-infected ACP. Therefore, the results of our study indicate that Las infection may be detrimental to ACP suggesting a non-symbiotic relationship. Higher mortality of Las-infected than uninfected ACP suggests that Las-infected psyllids may be selected against under commercial ACP management practices relying on insecticides. Selection against Las-infected ACP may limit spread of HLB. This hypothesis is consistent with the notion that insecticide resistance contributes to the spread of vector-borne disease. Our subsequent investigations should help elucidate the mechanisms of altered host physiology with respect to insecticide resistance management programs for ACP. In other concurrent investigations, we have recently determined baseline toxicity of various insecticides to late nymphal instar (4th) ACP to determine if resistance levels differ between ACP adults and nymphs. Baseline data were collected using an ACP culture maintained at a CREC greenhouse for 5 commonly used insecticides. LC50 (mg ai/l) values of the susceptible greenhouse population were compared with those of field collected populations from 2 sites. LC50 of carbaryl (50.71) and spinetoram (3.88) was significantly higher for the population collected from one of the field sites when compared to the greenhouse population (carbaryl: 17.59; spinetoram: 0.66). Immature psyllids collected from two field sites were significantly less susceptible to imidacloprid (with LC50 values of 0.84 and 0.50) than the susceptible lab population (0.22). Our results show that immature psyllid nymphs exhibit resistance levels similar to those previously documented for adults.
The goal of this project is to determine if infection by Candidatus Liberibacter affects the response of Asian citrus psyllid (ACP) to its citrus host plants to understand a critical component of disease spread. In this project we evaluated if healthy psyllids are attracted more to HLB infected or healthy trees. Also, we determined whether this behavior changes when the ACP vector becomes infected with 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. We also examined if psyllids known to be infected with the pathogen behaved differently from uninfected controls in response to both healthy and HLB-infected plants. Our preliminary results indicate that HLB-infected citrus plants are more attractive to ACP adults than healthy plants in two-choice olfactometer experiments. 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. Initial movement of ACP to infected plants and further dispersal to healthy plants may be explained by production of deceptive volatile compounds by HLB-infected plants to attract ACP adults in the field to facilitate the spread of bacteria as occurs with apple phytoplasma Candidatus Phytoplasma mali, responsible for apple proliferation disease. Ca.P. mali hijacks the apple trees to produce specific chemical that attracts the plant-sap sucking psyllid vector to infected trees. This is a major factor for facilitating disease spread in apple. Alternatively, the yellow color of HLB-diseased plants due to chlorosis and yellowing of shoots may attract the ACP initially but psyllids move to healthy plants after sampling the phloem of diseased trees. The movement to new plants could be due to poor nutritional status of HLB infected plants. It is known that ACP adults are attracted to yellow color; therefore, initial attraction of ACP adults to diseased plants may be due to chlorosis of leaves caused by HLB. Also HLB-infected plants are deficient in zinc, iron, manganese, calcium, sulfur and/or boron and hence the subsequent movement of psyllids to healthy plants could be due the poor host suitability of HLB-infected plants. To answer these questions, we are analyzing the head space volatiles produced by HLB infected plants versus healthy plants to evaluate if the differential response of ACP to HLB infected and healthy citrus plants is due to differential chemical production. In addition we are continuing to conduct more experiments in light and dark conditions to evaluate the role of visual cues in the differential response of ACP to HLB infected and healthy citrus plants.
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