Disrupting transmission  of Candidatus Liberbacter asiaticus with antimicrobial therapy

Disrupting transmission of Candidatus Liberbacter asiaticus with antimicrobial therapy

Report Date: 11/05/2021
Project: 18-018   Year: 2020
Category: ACP Vector
Author: Kirsten PElz-STelinski
Sponsor: Citrus Research and Development Foundation

1. Please state project objectives and what work was done this quarter to address them: 1. Quantify the effect of citrus antimicrobials on vector fitness.  This objective is to determine if ACP exposed to antibiotic treatments will have reduced lifespans, fecundity, fertility or development time as compared with untreated ACP.Survival and fertility assays were completed previously. Replicate development time and fecundity assays were initiated at during this quarter. 2. Determine the effect of antimicrobials on Las transmission. This objective will determine whether ACP feeding on antibiotic treated infected citrus plants will be less likely to transmit Las. We expect that ACP will be less capable of transmitting Las after feeding on antimicrobials, because 1) trees treated with antimicrobials are more likely to have lower Las titers for acquisition and 2) Las in infected psyllids will be reduced or eliminated when psyllids feed on antimicrobials.This experiment was performed in mature, infected citrus trees located in a research grove located at Lake Alfred, FL to determine whether field applications of foliar antimicrobials were capable of suppressing the acquisition of CLas. Eight-year-old CLas-infected citrus trees received foliar applications (Sept – October 2020) of streptomycin, oxytetracycline, or no treatment (Control).  All trees were treated with monthly insecticide sprays. One day after the application, ten CLas-free insects (5 females and 5 males) per plant from a CLas-negative laboratory colony were caged on young leaves (flush) of treatment and control trees to analyze ACP survival, CLas-acquisition in ACP P1 and F1 progeny, the total trees sampled consisted of 5 individual trees per treatment. Survival of ACP adults was monitored the day after inoculation. After one-week, parental adults were collected in microcentrifuge tubes containing 1 mL of 80% ethanol, ACP adults were collected individually and then stored at -20°C for subsequent CLas detection using real-time PCR.  Egg clutches remained on trees enclosed in mesh sleeves after parental removal. After the nymphs reached the adult stage (approximately 2 weeks after ACP inoculation), adult psyllids were collected for analysis. Objective 3: Determine the effect of antimicrobials on plant response and associated ACP behavior.  Insect choice bioassays were completed. Antibiotic treatments were applied to sweet orange, C. sinensis, trees to determine if they affect ACP host plant selection and acceptance behavior. Six trees (biological replicates) were individually sprayed per treatment. The chemicals evaluated were: FireWall (Streptomycin sulfate), FireLine (Oxytetracycline), and control (adjuvant only). The following treatments were compared with adjuvant only control in paired choice tests: 1) two FirelLne applications, 2) two FireWall applications, 3) one FireWall followed by one FireLine application, 4) one FireLine followed by one FireWall application. In certain treatments, plants that received an initial treatment with FireWall were subsequently sprayed with FireLine and vice versa. In other treatments, plants received two successive applications of FireWall or FireLine (Figures 1 and 2). After treatment applications, all plants were relocated into a growth chamber maintained at 23 ± 3 °C, 60RH, and a 16:8 h (Light: Dark) photoperiod until further bioassays. Psyllid response to treatment versus control (application of adjuvant only) plants was assayed twice. The first assay took place 25 days after the initial treatment applications were made and the second behavioral assay took place another 25 days after the second application of treatments was made (50 days after the experiment was initiated). During bioassays, paired treatment and control plants were placed into sealed bioassay chambers into which ACP adults were released. During assays, 70 D. citri adults were released into a behavioral chamber containing plant pairs for a period of 24 hours. The number of psyllid adults landing and initiating feeding on treated versus control plants was recorded 24 hours after insect release.2. Please state what work is anticipated for next quarter:Obj. 1 Conduct final replicate of development time and fecundity assays and complete data analysis.Obj. 2. Replicate field application of foliar antimicrobials to evaluate supression of CLas acquisition by ACP caged on treated trees. ACP will be collected following acquisition assays. All insects from this replicate and previous replicates, and tree samples, will be processed to determine CLas titers in response to treatments (DNA extraction and PCR) during the final two quarters of the project.Obj. 3. Complete data analysis. 3. Please state budget status (underspend or overspend, and why): We were underspent this quarter due to previouly being unable to spend funds on emplowyees working in the lab due. The funding needed for materials and personnel to perform experiments and process samples is still needed to complete the objectives, but was effected by work delays.  Many purchases were in process when the project ended because samples were still being processed on the final project ended date in November. We have received a no cost extension to complete the experiments, sample processing, and data analysis.       

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