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.
The objective of this project is to evaluate methyl salicylate dispensers to determine whether their deployment in citrus can enhance biological control of Asian citrus psyllid. Locations were selected for the field trials with the commercially available methyl salicylate (MeSA) lure, Predalure (AgBio Inc.; Denver, CO). The first site was an unmanaged grove of ‘Valencia’ oranges near Clermont, FL and plots were established on 4/13/10. Four plots were designated to have 1 lure hung per tree and 4 plots were designated as control plots. The plots were 0.4 ha in area with a 0.4 ha buffer between the plots. Twelve unbaited Pherocon AM yellow sticky traps were placed in all plots in rows 2, 5, 7 and 9 in trees 3, 7 and 12 to determine pretreatment populations of Asian citrus psyllid (ACP) adults and natural enemies. The trees were in late bloom stage. On 4/20/10, MeSA lures were attached to the trees with twist ties at mid-canopy height and sticky traps were replaced. The plots were then checked every 2 weeks. Sampling included replacement of sticky traps, sweep net samples of trees 6 and 11 in rows 2, 7 and 9 of all plots and 5 flush samples/plot, when suitable flush was available. The flush samples were examined in the laboratory and the number of ACP eggs and nymphs were counted. The flush were then placed in a jelly cup with water and a mylar cylinder with a mesh top placed around the flush to capture emerging ACP and parasitoid adults. They were placed in an incubator maintained at 25oC and egg, nymphs and adult ACP as well as any parasitoids were counted at 5-10 day intervals. In the field, MeSA lures were replaced on 6/24/10. The second location was established on 4/22/10 in a minimally managed grove of mixed citrus cultivars in Lake Alfred, FL. In this grove, only 3 replications of MeSA treated plots and controls were possible. Traps were placed as before and lures were hung 1/tree on 4/29/10 (post-bloom) with biweekly sampling as above. Lures were replaced in this grove on 6/30/10. The third site was a heavily managed grove of ‘Hamlin’ oranges in Auburndale, FL at the beginning of fruit set. Replicated plots (4) of MeSA treated and controls were established as above on 5/5/10 with the lures placed in the treated plots on 5/11/10. Biweekly sampling was conducted with traps, sweeps and flush (when available). Lures will be replaced in this grove on 5/15/10. To date, very few ACP and beneficial insects have been collected in sweep samples in any of the 3 groves. Flush has only been available for 3 sampling dates at the Clermont grove and no parasitoids have been found although there was successful adult ACP development from the eggs and nymphs present. Traps are being held in refrigeration until the ACP and beneficial species can be counted. The unusually cold winter most likely reduced overwintering ACP population levels and also had an effect on the flush cycle of the citrus trees, but the most recent samples indicate an increase in ACP populations. An experiment to investigate the effects of MeSA lures on the behavior of ACP is being conducted. When feather flush suitable for oviposition by ACP appeared all but 5 flush were removed and the plants placed in 58 X 58 X 90 cm screen cages. Five plants were designated as treatment and 5 as control. In the cages with treatment plants, 1 MeSA lure was suspended from the top of the cage centrally. The control cages had no lures. All cages were placed under natural conditions outside the laboratory and treatment cages were separated from the control cages by a distance of 50 m. On July 8, 2010, 50 adult ACP from a greenhouse colony were released in each cage and the number of psyllids settling on the plants was observed at 1 hour, 24 hours, 5 days and will be continued at 7 days and 10 days. At 10 days the adult ACP will be removed from the plants and examined to determine the number of males and females. The flush will be examined and the number of ACP eggs and nymphs will be counted.
We are beginning the second year of our project. During this year we are focused on evaluating psyllid-derived sequences which can be used as double-stranded RNAs to yield RNA interference-induced negative effects (even death) in recipient psyllids. We are using the tomato/potato psyllid, Bactericerca cockerelli, and its solanaceous plant hosts as an herbaceous plant model system. This herbaceous system is much easier and faster to manipulate than is the perennial citrus system, but we believe our efforts will lead to subsequent application toward the asian citrus psyllid, Diaphorina citri. We have well over 100 candidate sequences identified from last years work, and several thousand more cloned and stored. We are now comparing specific sequences for RNAi activity via three delivery methods: direct injection into the psyllid hemocoel, in vitro acquisition via feeding through parafilm membranes, and by feeding on tomato plants infected with recombinant Tobacco mosaic virus (TMV) constructs containing the candidate sequences. B. cockerelli midgut cDNAs, and those generated last year from the B. cockerelli normalized cDNA library, were engineered for use as templates to generate dsRNAs in vitro (via T7 RNA polymerase transcription) and for insertion into TMV. In vitro-generated dsRNAs were adjusted to specific concentrations and used for subsequent direct delivery experiments while TMV was used to express the sequences in whole plants. The in vitro-generated dsRNAs were tested by feeding psyllids through stretched parafilm membranes containing the candidate dsRNAs in a solution of 15% sucrose. B. cockerelli psyllids readily feed on this solution for up to 7 days, thus allowing adequate time for acquiring the test dsRNAs and assessing potential RNAi effects. We have demonstrated that fluorescent, Cy3-labelled dsRNAs were acquired by membrane feeding. We were able to visualize these in psyllid guts by using fluorescence microscopy after feeding. We also used qPCR to identify acquired dsRNAs and to quantify target mRNAs in psyllids. In some experiments dsRNAs evaluated in initial membrane feeding experiments caused mortality in psyllids. However, we are investigating now the quantitative effects of these treatments to ensure that phenotypic effects, including psyllid mortality, are due to the specific sequence and not due to overly abundant dsRNAs. The dsRNAs also were evaluated via micro-manipulator driven intra-thoracic injection (200 nL/psyllid). Mortality is fairly high, ~50%, due to injection even when injecting buffer controls. Therefore, we are injecting large numbers of insects (at least 15 per treatment) and including buffer controls so as to have sufficient numbers for statistical analysis. We are using injection here only as a comparator, intra-thoracic injection is a standard means to induce RNAi effects in insects so it is a very good positive control for comparison against oral delivery via membrane feeding and TMV-infected plant acquisition. The same psyllid sequences are being evaluated via psyllid feeding on recombinant TMV-infected tomato plants. Our data show that psyllids feeding on these plants acquire the specific test RNAs, as determined by RT-PCR and qRT-PCR. However, this approach is still a little problematic as TMV sometimes loses the inserted test RNA sequence during tomato plant infection, therefore all plants used for feeding experiments are carefully evaluated for inserted sequence retention. We are also assessing how much and what forms of RNAs are acquired during whole plant feeding.
The first year of a 2-year research proposal (FDACS Contract Number 58-1920-9-925 40) was successfully completed when the second year funds were terminated and a no cost extension was granted to complete loose ends. 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 cultured in our laboratory and healthy psyllids as a transmission tool; and, (2) following successful inoculation or loading of the psyllids, we would complete Koch’s postulates. We have been able to cultivate a strain of ‘Candidatus Liberibacter asiaticus’ (Las) Taiwan (B239) in our laboratory using different media components and it has been shown to be Las by PCR and sequencing. We have followed the procedure previous described in our last quarterly report to transmit the bacteria produced in culture media or from infected dodder using stretched parafilm membrane sachets containing 10% sucrose solutions in 1X TE buffer in which the cultured bacteria were suspended. The titer of the bacteria in sucrose was roughly assessed using real time PCR (Ct values ranged from 20 to 38). Seedlings were grown at 25 C with natural daylight supplemented with HID lamps to extend the daylength to 18 hr. No symptoms were observed at 3 months, however, those positive by PCR at 3 months developed typical blotchy mottle symptoms by 6 months. We have repeated membrane inoculations with cultivated bacteria from infected dodder and citrus plants but will not be able to complete the final steps of Koch’s postulates because of lack of funding. A second approach used direct microinjection of the culture fluid into the hemolymph of adult psyllids. Psyllids were immobilized with a low velocity stream of C02 and approximately 0.01’l of bacterial culture was injected into the abdomen of each adult psyllid as described in the last quarterly. After 6 months, we have observed 2 of 7 sweet orange seedlings inoculated with injected bacteria exhibiting HLB symptoms. This has been repeated and we are waiting for results. While this does not serve as additional confirmation of Koch’s postulates for Las, it does provide important insights into the interaction of Las with the vector. We have been continuing to refine our membrane and micro-injection techniques to obtain additional confirmatory data on the ability of D. citri to transmit cultivatable (HLB causing) bacteria obtained from HLB infected sweet orange, dodder, or infectious psyllids.
Imidacloprid (Confidor 700 GrDA), 0.35 g AI/plant and thiamethoxam (Actara 250 WG) 0.25 g AI/plant, applied in the nursery tree bags, before planting, was efficient to control ACP until 60 days after application. The time to cause 100% of ACP mortality was between 5 to 7 days after the confinement of adults in treated plants. However, researches using electrical penetration graph (EPG) showed that in plants treated with imidacloprid and thiamethoxam, after the first feeding on phloem, the adults do not do more probing. We carried out the first PCR of the plants in this experiment and the results were negative, no plants have been detected the presence of the bacterium L. Ca asiaticus. No transmission results yet. We finish the second experiment that was performed to determine if the systemic insecticides are effective until 90 days after application and its effect on transmission of the bacteria. In this experiment, the time to reach 100% of mortality ranged from 3 to 7 days for both systemic insecticides tested (imidacloprid and thiamethoxam). The insecticides were effective up to 90 days after application. The results of PCR carried out for the ACP, in some periods, were positive for 100% of the samples, consisting of 10 insects tested, but in the confinement held at 46 days after application, in any sample was detected the presence of the bacteria. No acquisition in this period. In bioassays performed at 75 and 90 days after application, the percentage of positive samples was 50 to 70% and 10 to 40%, respectively. We started the experiment 2, the difference from the experiment 1 is the application of varying doses of the systemic insecticides and confinement of the ACP in plants treated only 7 days after application. To thiamethoxam (Actara 250 WG), the doses tested were: 1, 0.5, 0.1 and 0.05 g/nursery tree and imidacloprid (Provado 200 SC) were: 1.75, 0.9, 0.2 and 0.08 mL/nursery tree. We also started the experiment 3, using different insecticide spraying to determine if they prevent the transmission and for how long. Plants treated with insecticide, the proportion of insects reaching the phloem was similar between plants treated with imidacloprid (0.35 g AI/tree), thiamethoxam (0.25 g AI/tree) and control (untreated plants), being respectively 74, 72 and 76%. The time to perform the first ACP salivation was also similar between treatments, 118.4, 103.2, and 112.6 minutes, respectively. However, the time of phloem ingestion is drastically reduced compared to untreated plants: imidacloprid 6.1, 9.9 and 6.9 min, respectively for 15, 35 and 95 days after application (DAA); thiamethoxam 9.6, 14.5 and 17.5 min, respectively for 15, 35 and 95 DAA; Control 142.0, 80.3 and 129.0 minutes, respectively for 15, 35 and 95 DAA. Apparently, ACP can only distinguish between plants with and without treatment from the moment that start ingesting the phloem sap. In this case, it was observed that after ingestion of sap with insecticide, the ACP removes the stylet from the plant and rarely returns to start a new probe on the same plant. In plants sprayed with mineral oil decreased the percentage of psyllids that can reach the phloem when compared with plants not sprayed, 20 and 70% respectively. However, the few insects that reach the phloem of treated plants carry out long periods of ingestion in this vascular tissue (‘ 1 h). We began the experiment to evaluate the effect of oil on the feeding behavior of ACP and its effect on repellency of the vector. The results showed that up to 21 days after application, mineral oil, 1.5%, shows repellency to adults of D. citri. Using electrical penetration graphs (EPG) techniques, we are studying the probing behavior of ACP in plants that were applied mineral oil. The results showed that the number of insects reaching the phloem is lower. The next step is the study of lower doses of mineral oil.
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