Management of the Asian citrus psyllid (ACP) currently relies on the use of insecticides; however, aggressive use of insecticides to manage insect vector and greening disease has led to the development of resistance in ACP to various modes of action. Therefore, there is an urgent need to develop and evaluate alternative management programs for ACP and greening disease. The current project proposes to utilize citrus tristeza virus (CTV) engineered to express insecticidal peptides as a novel tool for ACP control. Previously, we have reported the success of two putative insecticidal peptides (A and B) in imparting deleterious effects on the growth and development of ACP. Plants containing peptides A and B have elicited reduced feeding, host selection, fecundity, and longevity of eggs and nymphs of ACP when compared to control plants. ACP surviving on plants containing peptides A and B also exhibited increased developmental time for egg and nymph when compared to control plants. Currently, we are evaluating effects of plants containing peptide C on the growth and development of ACP. We have started quantitative assays to determine the concentration of peptides expressed in samples from test plants are currently being developed. To evaluate possible antifeedant effects of peptide C, we have recently conducted feeding bioassays that measure honeydew production as a surrogate for ACP ingestion. Our preliminary data shows that feeding was significantly reduced on plants containing peptide C when compared to control plants (F=24.22; df: 1, 29; P<0.0001). Additionally, an experiment was conducted to compare the settling behavior of ACP on plants containing peptide C and control plants. Significantly more ACP adults were found on control plants at 48 h (F=9.54; df: 1, 14; P=0.0080) and 72 h (F=13.74; df: 1, 14; P=0.0023) when compared to plants containing peptide C. Future bioassays will be conducted to determine the effects of plants containing peptide C on the growth and development of ACP and another phloem-feeding citrus pest, the brown citrus aphid. In the upcoming months, we will complete experiments evaluating the efficacy of peptide C against the Asian citrus psyllid. Furthermore, we continue to await systemic infection of plants with another novel CTV-vectored peptide. Plants were tested recently for infection with the CTV construct using an enzyme linked immunosorbant (ELISA) assay; however, only two positive plants were found, likely due to the fact that plants were inoculated only one month prior to testing. In addition to evaluating the effect of CTV vectored peptides against ACP and the brown citrus aphid (reported previously), we have recently begun to evaluate the effect of three peptides against the ACP parasitoid Tamarixia radiata. Individuals from a recently-established colony of these insects are currently being evaluated in choice tests and fitness bioassays to determine the effect of peptides on paraitoid host selection and fecundity. Briefly, the effect of peptide plants on T. radiata host selection is evaluated by releasing insects into a T-tube olfactometer. Insects are able to choose between two odor fields located in the arms of the olfactometer. Each arm is connected to an odor source placed in a volatile collection chamber (psyllid-infested plants with CTV-peptide contructs, psyllid-infested plants without CTV constructs, and the respective non-psyllid control plants).
Ultra High Performance Liquid Chromatography – Pesticide Residue Analysis (June 2013 Update) Work continued to compare protocols for analysis of potential insecticide residues in citrus nectar. Early in this quarter, problems arose with the equipment resulting in the inability to obtain a clean baseline. Technical support from the equipment manufacturer was requested and 3 separate visits were made by technicians in attempt to resolve the issues which took nearly 2 months to solve. Multiple issues were identified and resolved one at a time including a bad electronic component that controlled the injector, pump issues requiring replacement, and a clogged rinseate line. Ultimately, issues with the equipment were resolved and method validation for nectar samples continued until low levels of contamination appeared corrupting the baseline. The source of contamination was ultimately identified and resolved. Sample analysis of field collected samples from 2013 began in late June.
Progress has been made on three primary objectives of the project: 1) Develop a generic epidemiological model that can be used to compare control scenarios and to optimize the probability of controlling and managing high-risk pathogens of agricultural significance. 2) Development and testing of efficient methods of statistical inference to estimate epidemiological parameters from maps of emerging epidemics. 3) Develop user friendly model ‘front ends’ that can be used by researchers and regulatory agencies Most work has been focused on this primary objective and in the underpinning statistical methods that are used to estimate parameters for emerging epidemics. The parameters are then used to inform model predictions of the likely further spread of disease and the effectiveness of different management and control strategies. We now have a set of robust and flexible epidemiological models to analyze and predict the spread of emerging pathogens across a range of scales extending from within-plantation to the landscape and regional scales. The models are being tested using extensively and intensively mapped data for citrus HLB and citrus canker provided. These form unique data sources comprising successive maps of diseased and susceptible hosts over time. The data are used to test the statistical methods for parameter estimation as well as the applicability and flexibility of the epidemiological models to particular host-pathogen systems. The data have the added advantage of enabling us to look, in the case of HLB, at how host age affects the transmission of disease and for canker and HLB at variability across different sites in disease transmission patterns and characteristics. The models are designed to be stochastic and biologically realistic but with relatively few epidemiological parameters to be estimated prior to use. The models are also developed as part of a flexible tool-box so that they can be readily adapted to new disease threats. To date we have completed the following in constructing the models: 1) Development of computationally fast epidemiological models to predict future spread of disease at a range of scales; 2) Coupling of the models with GIS to compute spread and model control over extensive areas and realistic agricultural landscapes and under realistic environmental conditions; 3) Adaptation of the models to incorporate a range of control strategies and to compare the effectiveness of different management and control strategies under uncertainty. The models also allow for incorporation of latent and cryptic periods and for dual sources of transmission, for example distinguishing between tree-to-tree spread within a grove and introductions from outside the area of interest. The MCMC methods have successfully been used to discriminate amongst alternative models (for example between models with different characteristics to describe dispersal, transmission, latent and cryptic periods) and to select the optimal model to describe the spread of HLB within plantations and citrus canker within urban environments. Publications: Cunniffe, N.J., R.O.J.H. Stutt, R.E .DeSimone, T. R. Gottwald, and C.A. Gilligan. 2012. Webidemics: Webbased Interactive Demonstration of Epidemiological Modelling Informing Control Strategies. To be submitted to PLoS One. Gilligan, C. A., F. M. Neri, A.R.. Cook, G. J. Gibson, and T. R. Gottwald. 2012. Bayesian analysis of an emerging epidemic: citrus canker in urban Miami. To be submitted to Journal of the Royal Society Interface.
Ultra High Performance Liquid Chromatography – Pesticide Residue Analysis (June 2012 Update) The purpose of this proposal was to purchase a LC-MS-MS in order to facilitate ongoing CRDF funded research requiring the analysis of pesticide residues in citrus leaf tissue. Funding provided by CRDF payed for a portion of this equipment which was cost-shared by UF-IFAS. Following the funding of this project, we placed an order and received the proposed equipment on June 18, 2012. The facility where the equipment will be housed is being prepared (gas, electric, etc…) so that installation can occur. The installers are scheduled to arrive and setup the equipment next month.
We are continuing to solicit sequences that can be used to prepare RNAi molecules that may silence ASP genes resulting in mortality. As these sequences are selected, we are feeding the resulting RNAi molecules to adult ASP using our feeding system. Results comparing mortality of the these RNAi molecules on psyllids will be reported later after the comparisons are complete.
The objective of this project was to investigate three questions: 1) How long does a leaf needs to be infected by Guignardia citricarpa before ascospore production can be initiated; 2) How does infection and colonization of leaves by Guignardia citricarpa occur and potentially showing how pseudothecia, the sexual spore producing structures, are produced; and 3) what is the interaction between the common twig colonizing pathogen Diaporthe citri and the black spot pathogen Guignardia citricarpa and whether they can co-exist to successfully sporulate on dead twigs. The graduate student, Nan-Yi Wang, whose Ph.D. project this is, continues his studies in Gainesville this term and is making good progress in his classes. He is also conducting research while in Gainesville. We have redesigned the mating gene primers several times but have not found any products with homology to known mating genes. We are evaluating new strategies to approach this program. He has finally had success transforming G. citricarpa with GFP. He found that the fungus is very sensitive to hygromycin, a selectable marker, but was eventually able to transform protoplasts with modifications to his protocol. The isolates are being evaluated to make sure that the growth and other characteristics other than the GFP are similar or the same as wild type The trees have been purchased for the greenhouse experiments but the air conditioner broke down so needed to wait for delivery until the air conditioner in the quarantine house had been repaired.
This is a three-year research project that started in June, 2012. The overall goal of this research is to efficiently deliver antimicrobial molecules into citrus phloem against the HLB bacterium. The quarterly (July~Sep) research focused on designing the W/O (water in oil) and O/W (oil in water) nanoemulsions by evaluating the ideal adjuvant mixtures of solvents (ethanol, acetone, methyl acetate, and ethyl acetate), oils (soy oil, cremorohor, carvacrol, p-cymemen, orchex796) and lipophilic (span80, span85) and hydrophilic surfactants (tween20, tween80). The nanoemulsions were first evaluated for transparency, viscosity, and other physical properties that would make them good candidates for moving molecules into citrus phloem . This evaluation indicated that several O/W nanoemulsion formulations and several W/O nanoemulsion formulations had the desired properties. In addition, seven compounds from the Citrus Research and Education Center, IFAS, UF and one compound, epsilon-L-polyline, from China were tested for their effectiveness against HLB bacterium by our grafted-based chemotherapy method (Zhang et al., 2012). Three effective compounds from the Contest were treated by nano-green (one of the commercial nano-products) and applied to HLB-affected citrus by foliar-spray in the greenhouse. The results will be reported in the future.
The overall objective of this one year project was to rapidly screen and evaluate chemical compounds against citrus HLB using a graft-based screening method (Zhang et al., 2012). A total of 59 chemical compounds were tested based on the ranking of an expert panel and the mode of action of the compounds. HLB-affected lemon scions were soaked in the above solutions overnight in a fume hood and grafted into two-year-old healthy grapefruit rootstocks. First samples for DNA extraction were taken 4 months after inoculation; subsequent samplings were taken at 2 month intervals. Four types of data were collected: Las titer in scions and rootstock based on qPCR Ct values, transmission of the bacteria from the scion to the grapefruit rootstock, survival of the infected/grafted scion and growth of new leaves from the scion. The 59 compounds were placed into four groups based on the above data. (1) Highly effective (Group O): Eight out of 59 compounds were clustered in this group, including Ampicillin, Actidione, Carbenicillin, and Nicotine. All the molecules in this group reduced Las titers to undectable levels (Ct values of more than 38.0) in the inoculated plants (grafted scions and rootstocks). All molecules in this group were antibiotics except Nicotine. (2) Effective (Group I): Twenty-five out of 59 compounds were clustered in this group, including Validoxylamine A, Hygromycin B, Poly-l-arginine, Carvacrol and Gossypol. Some of these compounds, such as Carvacrol, Validoxylamine A and Gossypol were not medical-antibiotics. The effectiveness of group I molecules against HLB bacterium was less than those in Group O, but they could greatly suppress the HLB bacterium with average Ct values of 33.8 and detectable Las in less than 40% of the scions . (3) Partly effective (Group II): Sixteen out of 59 compounds were clustered in this group, including Thujone, Hydroxyurea crystalline, Polymixin B and Neomycin. Compared to the water control, they could partly suppress the HLB bacterium with Ct values of 28.6, and 74.7% scion infection. (4) Non-effective (Group III): Ten out of 59 compounds were classified into this group, including Meso-erythritol, Lincomycin, 2-methyl-4-isothiazolin-3-one and Zlineb. Similar to the water control (CK1 and CK2), the compounds in this group were not effective in suppressing the HLB bacterium with average Ct values of 26.8 and 72.6% scion infection. Three of the 59 compounds were found to be highly phytotoxic to citrus, especially Actidione, Oxytetracycline and M-cresol. The other 56 compounds were not phytotoxic due to more than 80% of scion survival and 40% of scion grown. Based on the this research, two manuscripts will be prepared for publication. The compounds in Group I and Group O may be useful for field control of HLB pending field testing, improved application methods and registration.
This is a three-year research project that started in June 1, 2012. The overall goal of this research is to efficiently deliver antimicrobial molecules into citrus phloem against the HLB bacterium. The objectives focused on will be : 1) Designing nanoemulsions by evaluating the ideal adjuvant mixtures of solvents, oils, lipophilic and hydrophilic surfactants based on the physicochemical characterization of the compounds; 2) Evaluating penetrants to promote uptake, translocation and diffusion of molecules into phloem based on leaf and bark surface characteristics; 3) Developing an efficient transdermal/translaminar delivery process and ideal formulations of compounds for foliar-spray or basal bark applications. Recent studies from Contest (CRDF#400) indicated that some non-medical antibiotics were effective in eliminating or suppressing the HLB bacterium using the graft-based screening method (Zhang et al., 2012). The major limitation to applying these compounds in the field will be to rapidly and efficiently deliver these compounds to the citrus phloem where the HLB bacterium resides. We therefore propose to screen and assess the components of nanoemulsions to improve the efficiency of delivery of the compounds into citrus in the first quarter research of this project. The overall experimental plan has been developed. The physicochemical characteristics of these effective compounds were evaluated and tested, such as solubility. The chemicals were purchased and nanoemulsions were prepared with water in oil (W/O) for lipophilic compounds and oil in water (O/W) for hydrophilic compounds. The HLB affected citrus to be used for this research was identified in the greenhouse and in the field. Some effective compounds combined with one kind of commercial nanoemulsion product (Nano-green) were used to treat HLB-affected citrus by foliar-spray and soil soaking in the greenhouse in June 2012. One OPS student will be hired and will be available in August, 2012.
Having shown that oral uptake of ~300 bp dsRNA fragments matching the coding region to either psyllid Vacuolar ATPase or cathepsin can induce mortality in the Asian citrus psyllid, comparisons were made to determine the optimal dsRNA size. Psyllids were fed either the ~300 bp dsRNAs directly or after processing to siRNAs with the Dicer enzyme. Results showed that the 300 bp dsRNAs induced greater mortality and that observed with processed siRNAs. Furthermore, non-linear dose dependent toxicity of the ~300 bp dsRNAs suggesting complex interactions that have not yet been characterized with respect to dsRNA induced toxicity in insects.
The project was initiated during this quarter. Initiation involved Hiring of personnel and adaptation of facilities to support project operations including increasing plant production to support psyllid colonies and increasing psyllid rearing. Experimental protocols were also optimized for large scale screening to be conducted during this time. Meetings were held with Innocentive review panel to chose the dsRNAs that will be used in the screening process. Initial testing of dsRNAs were performed and are now being replicated with 3 dsRNA targets.
Florida growers have reported that supplementary foliar nutrient applications maintain productivity of HLB-infected trees. However, efficacy and sustainability of the nutritional approach for HLB disease management has not been validated. The main cause of visible HLB symptoms, yield reduction, and tree decline appears to be disruption of phloem tissue, which blocks the flow of photosynthate and nutrients from source to sink tissue. If supplemental nutrition is a sustainable approach, it is expected that foliar nutrients will reduce or eliminate damage and plugging in citrus phloem tissue caused by the bacterium and possibly reduce spread or replication of the bacterium in infected trees. A greenhouse study is underway to evaluate a mixture of foliar nutrients representative of that used by Florida growers for HLB management. Infected and non-infected Hamlin trees under different combinations of nutritional treatments are being monitored for bacterial titer in phloem tissue and development of disease symptoms, including phloem cell morphological changes: plugging, necrosis, and starch accumulation. Initial results show no difference in the infection rate or bacterial populations in leaf midribs. Multiple microscopy techniques including TEM, light, and fluorescence microscopy with callose-specific dyes are being used to monitor phloem plugging and necrosis as the infection progresses. A complimentary field trial is evaluating bacterial titer, yield, and tree health in a south Florida Hamlin grove with a mixture of healthy, asymptomatic (PCR+), and HLB symptomatic trees.
The objective is to evaluate soil-applied neo-nicotinoids and other SAR inducers on HLB disease progress in newly planted citrus trees subjected to psyllid-mediated infection or graft-inoculation. One yr-old Hamlin trees were planted in May 2009 and treated as follows: 1) untreated check (UTC), 2) foliar insecticide to control psyllids, 3) soil-applied imidacloprid/thiamethoxam (IMID/THIA) to induce SAR, 4) soil-applied IMID/THIA plus foliar insecticides, 5) graft-inoculated UTC, 6) graft-inoculated with IMID/THIA. There were 50 trees per treatment (5 blocks of 10 trees). In 2009, the effect of SAR inducers on HLB infection progress was inconclusive perhaps attributable to the interaction of IMID/THIA with psyllid control which may have an uncontrolled effect on psyllid transmission. In 2010, the SAR inducer acibenzolar-S-methyl (ASM, Actigard 50WP) which does not control psyllids was substituted in treatments 3, 4 and 6. At 24 months after treatments began, 105 trees were PCR+ (35%) in the trial. Higher number of PCR+ trees occurred in the UTC (20), the UTC with graft inoculation (22), and the IMID/THIA/ASM with graft-inoculation (28). A lower number of PCR+ trees occurred in the treatments with SAR inducers (11), foliar insecticides (12), and foliar insecticide plus SAR inducers (12). Two years after treatments were initiated, the effect of SAR on HLB disease progress has been minimal, which indicates a lack of promise for SAR inducers in HLB management.
The objective is to evaluate soil-applied neo-nicotinoids and other SAR inducers on HLB disease progress in newly planted citrus trees subjected to psyllid-mediated infection or graft-inoculation. One yr-old Hamlin trees were planted in May 2009 and treated as follows: 1) non-treated check (UTC), 2) foliar insecticide to control psyllids, 3) soil-applied imidacloprid/thiamethoxam (IMID/THIA) to induce SAR, 4) soil-applied IMID/THIA plus foliar insecticides, 5) graft-inoculated UTC, 6) graft-inoculated with IMID/THIA. There were 50 trees per treatment (5 blocks of 10 trees). In 2009, the effect of SAR inducers on HLB infection progress was inconclusive perhaps attributable to the interaction of IMID/THIA with psyllid control which may have an uncontrolled effect on psyllid transmission. In 2010, the SAR inducer acibenzolar-S-methyl (ASM, Actigard 50WP) which does not control psyllids was substituted in treatments 3, 4 and 6. At 17 months after treatments began, 65 trees were PCR+ (22%) in the trial. Higher number of PCR+ occurred in the UTC (14), the UTC with graft inoculation (13), and the IMID/THIA/ASM with graft-inoculation (18). Lower number of PCR+ trees occurred without graft inoculation in treatments with SAR inducers (6), foliar insecticides (8), and foliar insecticide plus SAR inducers (6). At this time, the effect of SAR on HLB disease progress is minimal, which indicates a lack of promise for use of SAR inducers in HLB management.
Objective 1: On Young fruiting Hamlin trees, elevation of imidacloprid (IMID) to 2X the rate per season to compensate for the higher tree volume produced canker control effects on leaves and fruit that matched those of 6 applications of copper sprays (the recommended program for canker control on young fruiting trees). This control is attributed to SAR-mediated reduction in canker incidence on the foliage not to control of the citrus leafminer interaction. Trunk application of IMID produced canker control on leaves and fruit that was equivalent to that of the IMID soil application. The advantage of trunk application is that IMID is not reaching the soil which alleviates the risk of leaching in the soil profile. Objective 2 Integration of soil applied IMID with foliar applications of copper sprays for control of canker. IMID applied once at the beginning of the season followed by 11 CH sprays gave the best control in the 2010 trials. This confirms that SAR and copper could be integrated for augmentation of canker control for young fruiting trees. Objective 3 is to evaluate of the complementary use of acibenzolar S- methyl (ASM), thiamethoxam (THIA )and IMID soil applications to increase and/or extend canker control in 2-yr-old grapefruit trees. In 2010, ASM at different frequency of soil application with THIA and CH sprays at 21 day interval in 2 yr-old Ray Ruby grapefruit and 1-yr-old Vernia sweet orange was highly effective for suppression of foliar canker compared to the non-treated check in each trial.
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