The objectives of this project are: 1) Improve effectiveness of CLM management strategies with the assessment of the use of pheromone traps for monitoring CLM populations. 2. Monitor susceptibility of CLM field populations exposed to intensive versus modest insecticide use; 3) Relate CLM and canker incidence and severity and 4) Provide recommendations for management of CLM and citrus canker disease. Work on CLM LD50s and LD80s for some commonly used insecticides for management of citrus pests in Florida continued to be calculated for CLM from a susceptible colony. These values are being used to evaluate possible resistance to the tested insecticides in CLM field populations. Since the last report, larvae from the susceptible colony were exposed for 48 h to different doses (0; 0.01; 0.03; 0.1; 0.3; 1; 3; 10; 30; 50; 100, 300, 600 and 1000 ppm) of the following insecticides: Agri-Mek 0.15EC and Delegate WG. These data contributed to the results associated with the following active ingredients (CLM larvae): Actara (thiamethoxam) LD50=2.813 (CL95: 1.07-6.46) ppm, LD80=619.176 (CL95 199.10-3100.06) (n = 681; .2 = 3.81; d.f = 7; Heterogeneity = 0.76); Agri-Mek (abamectin): LD50= 0.259 ppm (CL95: 0.104-0.527), LD80=18.348 ppm (CL95: 7.083-85.113) (n = 1182; .2 = 9.681; d.f = 6; Heterogeneity = 1.613); Cyazypyr (cyantraniliprole): LD50=33.289 ppm (CL95: 15.2-69.1), LD80=8882.7 (CL95: 2905.3-44518) ppm, (n = 954; .2 = 1.749; d.f = 5; Heterogeneity = 0.35); Delegate (spinetoram): LD50=2.589 (CL95: 0.729-6.352), LD80= 95.606 (CL95: 33.676-553.88) ppm (n = 859; .2 = 10.501 d.f = 5; Heterogeneity = 2.100); for Dimethoate (dimethoate) LD50=1.56 ppm (CL95: 0.15-56.31) and LD80=497.45 ppm (CL95: 22.27-0.37E+09) (n = 546; .2 = 13.07; d.f = 6; Heterogeneity = 2.18); and Micromite (diflubenzuron): LD50=88.32 (CL95: 20.69-246.69) LD80=1800.5 ppm (CL95: 522.84-75810.00), (n = 714; .2 = 10.31; d.f = 5; Heterogeneity = 2.06). A novel bioassay technique is currently being developed for Intrepid and will be evaluated in the coming weeks. LD50 and LD80 evaluations for CLM adults continue. Further collections from field populations for laboratory evaluations of insecticides are planned for this summer. A manuscript “Placement Density and Longevity of Pheromone Traps for Monitoring of Citrus Leafminer, Phyllocnistis citrella (Lepidoptera: Gracillariidae)” is being prepared for submission to Economic Entomology.
Issue date for this grant was 13 July 2012. Objectives are: 1. Assess effects of abiotic factors (light quality, photoperiod, air flow, temperature fluctuations) on psyllid movement, 2. Evaluate physiological limits and biotic factors effecting of movement including feeding, egg load, infection status, and population density, 3. Evaluate techniques for tracking psyllid movement in the field for mark recapture studies, 4. Characterize seasonal patterns of ACP distribution and movement at different scales in the field, 5. Develop strategies to protect young trees from colonization by ACP utilizing UV reflection for repellency and insecticide treated trap crops (such as Bergera koenigii) to attract and kill. Objective 1. Bioasssays using a wind tunnel to determine ACP response to different stimuli have shown that ACP movement stops when temperature dips below 67 F, and when there is no light. Wind speeds above 4.25 mph reduce movement greatly, as does humidity over 70%. ACP tend to move with the wind when lighting is even and increased light intensity does not increase ACP movement. Objective 2. Experiments were conducted with a laboratory flight mill in the Stelinski laboratory to determine the effect of applying Fenoxycarb, a juvenile hormone (JH) analogue, on flight behavior of ACP. No significant differences were observed between the controls and the psyllids treated with Fenoxycarb, suggesting that JH may not impact flight capability. Following their recent finding that Clas infection increased flight propensity of ACP, they are now testing the hypothesis that Clas infection may impact other movement patterns, such as male attraction toward female odor(s). In a laboratory olfactometer assay, non-infected ACP males were attracted by non-infected ACP female odors. However, if nymphal development was completed on HLB-infected plants, only infected ACP females were attractive to males. These data will be used to develop a predictive model for spread of the CLas pathogen causing HLB. Objective 3. Mark release trials were not successful and discontinued, but environmental effects on ACP movement was studied by hourly monitoring from sunrise to sunset of sticky cards placed between 3 year old orange trees with a final observation next day at sunrise. Captures increased in the morning with two peaks seen at mid-day followed by a gradual increase until dusk. Thus far we have not captured any ACP during the overnight period which was expected due to the results of the no light variable in the wind tunnel. This study is a good companion to Objective 1 allowing us to confirm the wind tunnel results in the the field. Objective 4. More ACP were captured on sticky cards in the upper canopy compared to the lower canopy of mature trees in a 2 year study at two locations, Bob Paul (‘Pineapple’ orange) and Tanner Road (‘Valencia’ orange). Sticky cards on the east side of the trees captures more ACP than the west side at both locations. At both locations and also the English grove (‘Valencia’), ACP captures are greatest adjacent to a hammock or wind break on the east and west sides. No differences were seen in trap captures facing insecticide sprayed or unsprayed blocks at Bob Paul. Two-sided clear and yellow sticky cards have been placed in several groves to compare directional responses in an attempt to determine whether ACP adults flying in one direction may turn back and be captured on the opposite side of the yellow card. Objective 5. A paper reporting positive results using UV reflective mulch to repel ACP the first 2 years after planting has been published in the refereed journal Pest Management Science. Subsequent results indicated diminished repellency from the 5 ft strip of metalized mulch after trees exceeded 6 ft in height. In another trial, repellency was demonstrated by the result that fewer ACP were captured on sticky traps set at 1 m in reflective mulch compared to 2 m, where as the opposite was true over white mulch or no mulch (grass) where more ACP were captured more at 1m than 2m.
This is a three-year project. The goal is to determine the optimum combination of chemotherapy, thermotherapy, and nutrient therapy that can registered for use in field citrus and control HLB. The project started in April 1, 2014. The first field citrus grove at Pico farm of USHRL-USDA-ARS was evaluated for ACP and HLB based on past records of ACP and HLB incidence, visual observation and qPCR quantification. A randomized block complete design was carried out to combine nutritional and chemical treatments. The nutritional treatment was applied to three rows (#39, 41 and 43) and the other three rows (#40, 42 and 44) with normal nutrition. The chemical treatment was two antimicrobial compounds, which were loaded into the nano-emulsion formulations of W/O (Water in Oil) for foliar spray or O/W (Oil in Water) for bark application. Blank nano-emulsions without antimicrobial compounds were used for controls. The chemical treatments were applied separately or in combination with the nutrient treatments. All trees involved in this experiment have been confirmed to be Las-positive via a real-time quantitative polymerase chain reaction (qPCR) assay in this quarter. They were also pruned and fertilized. Chemical treatment applications will be made every month when flush is present starting with the spring flush in 2014. The nutrition will be applied as the protocol of Quantum Growth by Ecological Lab. INC. ACP populations will be recorded by stem-tap sampling. Citrus effects of the combined treatments will be investigated, including: a) reduction in fruit drop; b) increased canopy density; c) reduced HLB symptoms and Las titers; d) increased root uptake; e) residues and phytotoxicity. Additional groves, chemical treatments, nutreint treatments, and heat treatments will be added during the next quarter.
In order to speed up their application and registration for use by the citrus industry, this one-year enhancement project immediately tested antimicrobial compounds in the field. The research focused on evaluating five antimicrobial compounds, that were effective against Las in our citrus graft assay test, against HLB in field citrus. The antimicrobials were SDX (a sufonamide), ZS and VA (Chinese antibiotics) and CAR and PCY (natural oils). Positive control was ampicillin and negative control was water. Application methods were foliar-spray, basal bark application and gravity bag infusion. Trees will be evaluated at 2 month intervals. The four month assay, which is still preliminary, showed that the SDX, ZS and VA treatments were more effective in reducing Las titers than water, but not as good as the positive control (AMP). All treatments, except the water control, decreased the Las titer in the new leaves at 4 months after initial treatment. Temporal phytotoxicity was observed in some gravity bag infusion treatments, but all treated trees recovered after four months. After 4 months, trees treated with SDX, ZX and VA still had high titers of HLB bacteria in the older leaves, but less titer in the young leaves. CAR and PCY treatments have not yet been analyzed for Las.
This is the second year of a 3-year project. The overall goal is to efficiently deliver antimicrobial molecules into citrus phloem against HLB bacteria. This quarter’s (From April to July 2014) research continued to evaluate the effect of novel nano-emulsions against HLB bacteria, and optimize nano-emulsions by particle size analysis and evaluation of thermal stability. HLB-affected potted citrus trees treated with W/O and O/W nano-emulsion containing several antimicrobials by foliar spray and basal bark application respectively, were free of HLB bacteria by PCR and recovered from any disease symptoms, after 8 months and 4 months from initial treatment in 2013. Based on pesdo-ternary diagram phase in our pervious study, several O/W and W/O nano-emulsion were selected for optimizing the formulation by analyzing the particle size and evaluating the thermal stability. The particle size of five O/W nano-emulsion formulations ranged from 3.81 nm to 30.30 nm and the formulations were stable at temperatures from -20 ‘C to 45 ‘C. And the particle sizes of 16 W/O nano-emulsions formulations ranged from 2.95 nm to 35.3 nm and they were also stable at temperatures from -20 ‘C to 45 ‘C. Thus, we have developed stable formulations that will effectively deliver antimicrobials to citrus and control HLB under greenhouse conditions. The future work will be focus on: 1) Evaluation of compound loading using the optimized nano-emulsion formulations; 2) Evaluation of effect of the combination of optimized nano-emulsion and ideal penetrants on controlling HLB disease in the field by foliar spray and basal bark application.
The goal of this enhanced project of CRDF#584 is to evaluate effective compounds which were coupled with nano-emulsions and penetrants, against HLB bacteria in the field. It is one year project started on June 30, 2013. HLB-affected trees were pruned and fertilized before treatment. Two experiments were carried out in the field. Experiment I: HLB-affected citrus was treated with two effective compounds which were coupled with nano-emulsion and ideal penetrant, respectively,by foliar spray and bark painting. Experiment II: Four formulations of two effective compounds were applied on HLB-affected trees by gravity bag infusion. In experiment I, the tested compounds were delivered into citrus phloem through leaves by foliar spray and bark by bark painting. The HLB bacteria titer was significantly reduced 2 months after initial treatment. Furthermore, HLB symptoms also were reduced, and the number and height of summer flush was significantly increased under the nano-emulsion treatment when compared to the water control. No phytotoxicity appeared in the treated citrus trees. However, Las bacterial titer increased a little in the treated citrus trees four months after initial treatment. We will continue to test for one year. In experiment II, HLB bacteria titer was significantly reduced by gravity bag infusion with a penicillin solution and summer flush number was much greater, compared with other 3 formulations and water control 4 months after initial treatment. No phytotoxicity was observed in these four formulation treatments. We will continue to test for one year. In summary, effective compounds against HLB bacteria can be delivered into citrus phloem by foliar spray and bark painting or gravity bag infusion. Therefore, combining one or more of these delivery systems with an effective antimicrobial compound and controlling psyllids may be effective strategy to combat the HLB disease in the field. We will continue the field trial for more than one year. ACP populations will be recorded by stem-tap sampling. Citrus effects of the combined treatments will be investigated, including: a) reduction in fruit drop; b) increased canopy density; c) reduced HLB symptoms and Las titer; d) increased root uptake; e) residues and phytotoxicity.
This is the second year of a 3-year project. The overall goal is to evaluate the efficacy of 11 compounds individually and in combination for control of the HLB disease based on reducing titers of HLB bacteria in HLB-affected citrus. Based on our optimized delivery system, during this quarter (April to July, 2014), all 72 treatments of 11 compounds and their combinations were applied by bark-application to greenhouse citrus using nano formulations of the antimicrobial agents. Several compound formulations, such as Amp and ACT+VA, significantly reduced HLB bacteria titer 2 months after the initial treatment. The treated, greenhouse plants recovered normal growth. No phytotoxicity was observed in the treated citrus, except the treatment of STZ and VA. The treatments will be applied each month, and samples will be continue to be analyzed for Las every two months. The effective treatments will be tested in the field.
We have completed development of tools for in planta transient (and in the long term, transgenic) expression of dsRNAs and specific artificial micro RNAs (amiRNAs), and expression of siRNAs via using plant viruses. Our intent is to compare specific and non-specific RNAi effects by expressing these various forms of interfering RNAs in plants as such information will be critical to effectively design and even implement RNAi approaches targeting D. citri. So far these experiments have worked very well for N. benthamiana and tobacco plants which can be used to test against B. cockerelli, but we have not have good success in transiently expressing RNAs directly in citrus by using Agrobacterium tumefaciens (but see below for objective 2). However, we are further experimenting with this strategy as it offers great opportunities for rapid and efficient screening of interfering RNAs against D. citri, directly in citrus. We have compared the qualitative and quantitative expression of amiRNAs by directly expressing them from A. tumefaciens, by using Tobacco mosaic virus (TMV) and by using a modified begomovirus expression system (TAV). So far we have generated seven different interfering RNA sequences targeting the B. cockerelli ATPase mRNA. We assessed interfering RNA expression by using RNA hybridization and by using Next generation sequencing of small RNAs. Our data show that by using the TAV system, we can specifically express very high numbers of the exact 22 nucleotide artificial microRNA that we have engineered to target psyllids. Objective 2 is to evaluate and optimize in planta expression of anti-psyllid interfering RNAs. We are on track here with experiments towards B. cockerelli. We have generated transgenic plants using two different promoters (the 35S promoter for general tissue expression and the AtSuc2 promoter for phloem-specific expression). These plants will be characterized this year for expression of interfering RNAs and for efficacy towards B. cockerelli. The results from these experiments will provide critical information for use in objective 3. As noted above, we have not had good success yet with Agrobacterium tumefaciens-directed transient expression of specific interfering RNAs in citrus. However, with our collaborator, Dr. William Dawson of the Univ. of Florida, CREC, we have citrus plants that are infected with different recombinant Citrus tristeza virus (CTV) constructs engineered to express specific D. citri RNAs in citrus plants. The recombinant CTVs are engineered to express RNAs targeting D. citri mRNAs for HSP70, CLIC and/or V-ATPase, alone or in combination. We obtained budwood (under APHIS PPQ and Biotechnology permits) and grafted buds to C. macrophylla plants in the UC Davis CRF. These plants are only one month post grafting, we are pushing buds now and will use these for direct RNAi studies towards the Asian citrus psyllid in the upcoming year. This general approach offers the most rapid way to deploy RNAi strategies for D. citri, and could even be applicable to existing, non-transgenic trees. The CTV being used by us is the same as is being used in Florida for field tests and anti-psyllid peptide approaches. We anticipate that we will use the Florida CTV experimentally (it likely could be used in the field in Florida), and in the long term we will collaborate with Dr. James Ng of UC Riverside to use a mild California CTV for RNAi studies in California.
This is the final report for this project. Exceptional cooperation between the University of Florida, ISCA Technologies Inc. and USDA-ARS was made possible through funding provided by CRDF. ARS and UF researchers identified optimal pheromone blends for attraction and disruption of the citrus leafminer, Phyllocnistis citrella, and for control of the incidence and severity of citrus canker disease. Extensive field trials were carried out in commercial citrus groves thanks to support provided by The Packers of Indian River, TRB Groves and Golden River Fruit Company. Experiments employing small (~1 acre) plots consistently showed trap catch disruption using an off-ratio blend consisting of a single pheromone component that was equal to or better than the ‘natural’ 3:1 blend of two pheromone components. Our results showed that the 3:1 blend was necessary and sufficient for attraction of males (for use in traps) but the triene-only single component dispenser was superior and more cost-effective than dispensers of the blend. Male leafminers are not attracted to the off-ratio blend used in the commercial DCEPT CLM product thereby avoiding attraction of males from outside treated areas. Manufacture of DCEPT CLM is simplified because DCEPT CLM requires only one of the aldehyde pheromone compounds. Identification of optimal blends for attraction and disruption was the result of application of advanced stastistical methods including multivariate analysis and response surface modeling. Reduction of citrus canker incidence in disrupted plots was demonstrated in small plots but is expected to be even greater when large contiguous areas of citrus are treated with pheromone. Experiments conducted under this project are documented in 11 peer-reviewed scientific publications and a twelfth is in preparation. This project will now be carried forward through an agreement with the Commercial Product Deliv ery Committee of the CRDF. 1. Lapointe et al. 2014. J. Econ. Entomol. 107: 718-726. 2. Keathley and Lapointe. 2014. Fla. Entomol. 97: 291-294. 3. Keathley et al. 2014. Fla. Entomol. (in press). 4. Keathley et al. 2013. Fla Entomol. 96: 877-886. 5. Kawahara et al. 2013. Fla Entomol. 96: 1213-1215. 6. Lapointe and Stelinski. 2011. Entomol. Exp. et Appl. 141: 145-153. 7. Stelinski et al. 2010. J. Appl. Entomol. 134: 512-520. 8. Lapointe et al. 2011. J. Econ. Entomol. 104: 540-547. 9. Lapointe et al. 2009. J. Chem. Ecol. 35: 896-903. 10. Lapointe and W. S. Leal. 2007. Fla Entomol. 90: 710-714. 11. Lapointe et al. 2006. Fla Entomol. 89: 274-276.
Feeding behavior of ACP is being studied on P. trifoliata and trifoliate hybrids using an electronic feeding monitor, and choice and no-choice assays to study host selection and probing behavior. 1. Colonization by ACP on certain accessions of P. trifoliata was low in free-choice greenhouse experiments compared with Citrus and hybrids. 2. Electrical penetration graph (EPG) recordings are performed using trifoliate and trifoliate hybrid accessions to study xylem and phloem feeding. EPG waveforms provide information on stylet penetration into leaf tissues including saliva excretion and ingestion from xylem or phloem and hypothesized physical barriers based on electron microscope images. Frequency and duration of probing and feeding from xylem and phloem of trifoliates will be studied. Results show that ACP adults have difficulty in achieving ingestion from phloem of trifoliates. 3. Feeding assays showed that the number of ACP adults feeding on trifoliate leaves and subsequent survival were low compared with hybrids or Citrus. 4. Choice and no-choice assays were designed using ground leaf added to a wax matrix (SPLAT’). Choice assays studied olfactory/gustatory ability of ACP to differentiate between different trifoliates or its hybrids. Salivary sheaths excreted in the SPLAT treatments were stained and counted. ACP produced fewer sheaths on several trifoliates preparations compared with preparations containing citrus or hybrids. No-choice assays are underway.
This project is in furtherance of the commercial release of a new product for control of the citrus leafminer and associated spread of citrus canker disease based on a deployment device for the sex pheromone of the leafminer, DCEPT CLM’ (ISCA Technologies, Inc.). Under an agreement with the Commercial Product Delivery Committee of the CRDF, funds are provided to ISCA to subsidize two years of production of DCEPT CLM sufficient to treat 3,000 acres of citrus, mostly grapefruit, at three locations in St. Lucie and Charlotte counties in April/May of this year. The remaining cost of the product is provided by the growers. Funds are also provide to ARS and University of Florida to support monitoring and analysis of the experiments at the three locations. In addition to monitoring efficacy and longevity, the experiments will provide data on the effect of immigration of gravid female leafminers from outside of the treated areas. The application of DCEPT CLM at one location (Emerald Grove, The Packers of Indian River) is contiguous with untreated citrus, a source of immigrant females. Another site (VPI-5, Golden River Citrus Co.) is isolated and surrounded by natural area and pasture and therefore should receive few immigrants. Efficacy and longevity will be compared between these sites to estimate the magnitude of the effect of immigration. Another variable that is known to affect the ability to establish mating disruption in a grove is the structure of the tree canopy. Declining trees, replants or new plantings allow for increased air movement and presumed loss of pheromone compared with complete mature canopies. This variable will also be considered. Data collected so far indicate that trap catch disruption at the VPI-5 site is excellent; trap catch disruption at Emerald is good but may be compromised by low canopy vigor in some locations. Leaf samples will be analyzed to determine actual mining damage at all locations. Finally, the effect of intentional skip rows is being studied at the TRB site. Data to date indicate that trap catch disruption in skip rows is significantly lower than adjacent rows treated with DCEPT CLM. We hypothesize that this is related to the release dynamics from the solid pheromone dispenser compared to previous experience with SPLAT. The dissipation of pheromone from the DCEPT dispenser is being followed by extracting and analyzing DCEPT devices deployed in the field for varying periods of time. A linear decline in pheromone content has been seen over 12 weeks. This will be followed for up to one year to document pheromone evolution.
The objective of this research was to evaluate non-neurotoxic insecticides against Asian citrus psyllid (ACP), in order to identify additional tools for management of ACP in Florida. We investigated several non-neurotoxic insecticides that were known to be effective against insect pests similar to ACP. These additional tools may not only prove effective against ACP, but also could assist in ACP resistance management programs as needed tools for effective rotation with current insecticides. Methoprene is a juvenile hormone (JH) analog that acts as a growth regulator. We evaluated this compound for its ability to inhibit ACP egg hatch of eggs of various ages. We treated 0-48 hrs and 49-96 hrs aged eggs with six different concentrations of methoprene ranging from 0-320 ‘g/ml. For 0-48 hrs aged eggs, we observed egg hatch inhibitions of 14, 24, 31, 41, 65 and 84 percent for concentrations of 0, 10, 20, 40, 80 and 160 ‘g/ml, respectively. Similarly, the concentrations of 0, 10, 20, 40, 80, 160 and 320 ‘g/ml led to 13, 23, 29, 34, 39, 62 and 95 percent egg hatch inhibitions of 49-96 hrs aged eggs. These results show that treatment with methoprene successfully inhibited ACP egg hatch. We investigated the effects of cyantraniliprole against ACP. The contact toxicity of cyantraniliprole was 297 fold higher against ACP than its primary parasitoid, Tamarixia radiata. ACP settled and fed less on cyantraniliprole-treated plants than controls at concentrations as low as 0.025 and 0.125 ‘g AI mL-1, respectively. ACP egg production, first instar emergence and adult emergence were significantly reduced on plants treated with 0.25, 0.02 and 0.25 ‘g AI mL-1 of cyantraniliprole, respectively, when compared with control plants. Sub-lethal effects of cyantraniliprole were observed by comparing ACP settling behavior on treated vs. control plants. During the first 48 h of the experiment, there was no clear trend; however, at 72 h fewer adults settled on plants treated at the 0.025 ‘g AI mL-1 rate than on control plants. We investigated diofenolan to determine inhibition of egg hatch. Percent egg hatch inhibition of 7, 18, 31, 42, 65 and 91 % was observed when eggs were treated with concentrations of 0, 20, 40, 80, 160 and 360 ‘g/ml, respectively. We investigated the effects of diofenolan (Juvenile hormone analog) on survival of various developmental stages of ACP. We treated first instar nymphs with concentrations ranging between 0-360 ‘g/ml of diofenolan. We observed 95, 69, 38, 27, 23, 8 percent survival of ACP into the second instar for 0, 20, 40, 80, 160, 320 ‘g/ml concentrations, respectively. We investigated the effect of diofenolan on the development of third and fifth instars nymphs. We found that this compound significantly reduced development of these nymphal stages. We also investigated the effect of diofenolan on fertility and fecundity of ACP females. We found that this compound significantly reduced both fertility and fecundity. There were no significant differences in the population of adult ACP after treatment with Confirm 2F and Intrepid 2F as compared with the untreated control, whereas the Provado positive control (imidaclopried) significantly reduced the adult population as compared with the control. Intrepid 2 F caused the highest reduction of nymphs followed by Provado, and then Confirm as compared with the control. Therefore, these insect growth regulators do appear to have field activity against ACP immature stages. In general summary, the insect growth regulators tested were effective at suppressing ACP immature stage development. Also, novel chemistries, such as cyantraniliprole, were effective against ACP.
The main objective of this research is to determine both abiotic and biotic factors that regulate Asian citrus psyllid (ACP) acceptance of plants and pathogen transmission. The goal is to use this information to interfere with the vector’s capability of transmitting pathogen between citrus trees. Since April, we examined the effect of wind breaks on psyllid population density. We have investigated five different groves. In these groves, we sampled the edge that was facing the wind break, and the opposite edge that was not facing the wind break. We also sampled middle rows as a type of control. In four out of the five groves, psyllid abundance was significantly lower on the edge row that was facing the wind break compared with rows without a wind break. This difference was up to 87% in some groves. One hypothesis that may explain this difference was that windbreaks may act as a habitat reservoir for natural enemies. To test this hypothesis, we recorded the number of psyllid predators found on edges. We did not find a difference in the number of ladybeetles, Chrysopidae or spiders on the border edge facing a windbreak versus edges without windbreaks. We plan to record the level of parasitism for ACP nymphs on the edges of these different treatments to test if the parasitic biological control agent Tamarixia radiata might be more present in edges facing wind breaks as compared with edge without windbreaks. Other hypotheses that we are considering are that wind breaks provide shade and therefore reduce ACP numbers or prevent ACP from colonizing edge rows as compared with rows without wind breaks. We started the second year of our field experiment investigating the effect of grove architecture (solid reset plantings versus mature groves with resets) on psyllid population densities. Last year, we observed that reset trees in an environment consisting of a mixture of mature and reset trees contained fewer ACP and had lower HLB infection levels than reset trees planted in a grove solely composed of reset trees. To confirm these results, we will conduct this study in three different groves, each divided into two plots consisting of a reset planting and the other of mature trees containing resets sporadically placed in between mature trees. Trees will consist of ‘Hamlin’, ‘Valencia’, and Grapefruit.
This research seeks to determine whether young trees infected with CLas and displaying typical HLB symptoms can be brought to maturity and produce an economically viable yield. This will be achieved by managing a 58 acre grove of 3-year-old ‘Valencia’ / Kuharske Carrizo trees using a combination of three different foliar and three different ground applied nutritional programs. Factorial AxB treatments consist of A) ground-applied: 1) Liquid/dry+Ca (BHG standard), 2) Liquid+Ca, 3) Liquid/dry-Ca B) foliar-applied: 1) BHG standard-Ca, 2) BHG standard+Ca, 3) “Prescription”(+Ca). The prescription treatment was designed to be dynamic, customized for optimization, with feedback based on frequent leaf tissue analyses, visual symptoms, and the growth of the tree canopies and yield. There are six replications of treatments, with two being pure replications. The grove still continues to look better, despite the nearly 100% HLB incidence. Foliation of canopies is dense, and leaf color going into Summer season was a healthy green. Tree canopy sizes were measured in June 2014, and analyzed. Selected data is summarized below: Table 1. Tree canopy volume (cu.ft/tree) FOLIAR FERT STD-Ca STD+Ca Prescrip SOIL FERT Liq/Dry 397 315 429 Liq 350 303 423 Liq/Dry-Ca 384 312 313 The routine leaf tissue analyses for this project are behind schedule because we were prevented from submitting samples to our regular analytical lab due to restrictions on the movement of plant materials and soil. These rules are being enforced by DPI as part of the citrus quarantine for Florida. Once the necessary permitting is in place, we will resume leaf nutrient reporting.
The objective of the current project is the identification of a Bacillus thuringiensis (Bt) crystal toxin with basal toxicity against Asian Citrus Psyllid (ACP) and enhancement of this toxin by addition of an ACP gut binding peptide. A phage display library will be screened to identify peptides that bind to the gut of the ACP. Addition of the gut binding peptide to the Bt toxin will increase toxin binding and associated toxicity against the ACP. During the reporting period, the proteolytic profile of 15 Bt isolates provided by Dr. Michael Blackburn, USDA Maryland was characterized at Iowa State University. Briefly, Bt toxins were solubilized using sodium carbonate pH 10.5, 10mM DTT, lysozyme 200 micrograms per milliliter for 3 to 16 hours at 37’C, according to the solubility of each strain. The samples were dialyzed for 21 hours with three buffer exchanges against 50 mM Tris-Cl pH 8.5. Ten micrograms of Bt toxins were incubated with bovine trypsin at a final concentration of 10% of the toxin concentration at 37 ‘C for 1 or 2 hours for determining the proteolytic profile. Finally a 1 hour activation of a high protein concentration sample was performed to obtain sufficient toxin for toxicity assays. Removal of trypsin was carried out using benzamidine sepharose incubating the toxin sample at room temperature for 30 minutes with removal of the sepharose by centrifugation. The samples were verified by SDS-PAGE and the protein concentration was quantified by Bradford assay. Samples were shipped to USDA ARS Florida for testing by Dr. David Hall . ACP bioassays of five of the Bt isolates have been conducted at USDA ARS Florida. However, the psyllid mortality rates exposed to diet alone confounded conclusions from these assays. A series of experiments were conducted to address the high mortality in the negative control treatment. The psyllid survival rates have now improved, apparently because of seasonal effects on psyllid acceptance of the artificial diet, with low survival in the winter months. Additional challenges associated with the psyllid bioassays were 1) the amounts of Bt isolates provided only allowed for 4 replications of each dose; Additional replications may minimize the impact of variation between replicates. 2) The diet typically includes a yellow-green food coloring dye, but this dye precipitated when the Bt samples were added. The dye is now omitted from the diet and something green placed behind the diet sachet to promote feeding. 3) Bt toxins activated at Iowa State University are supplied in buffer (Tris-HCl pH 8.8). Assays are underway with this buffer to test for toxicity against psyllids. An appropriate amount of buffer will be added to control diet for future assays.
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