Performing Department
(N/A)
Non Technical Summary
Florida is the largest citrus producer in the U.S., but the citrus industry relies on the application of medically important antibiotics to control citrus greening disease, which has reduced Florida citrus production by 70%. The impacts of antibiotics on antimicrobial resistance bacteria (ARB) and plant health lacks attention. We hypothesize that antibiotic applications increase frequencies of ARB, compromise plant health, and alter the root-associated microbiome, leading to loss of important ecological bacteria with plant health functions. Overall project goals are to develop foundational knowledge and an applied sustainable approach to mitigate ARB associated with fresh produce, while increasing plant health, and to educate current and future consumers and microbiologists. Our objectives are to: assess the impact of antibiotics on ARB frequencies, plant physiology, metabolites, and microbiome of citrus trees; determine the effects of probiotic supplementation on plant health and ARB; and educate undergraduate students and secondary school teachers on antimicrobial resistance (AMR) across the food chain and strategies for mitigating AMR.
Animal Health Component
80%
Research Effort Categories
Basic
20%
Applied
80%
Developmental
(N/A)
Goals / Objectives
The long-term goal is to develop foundational knowledge and applied tools for deployment of integrated microbiological supplementation as an integrated and sustainable approach to mitigate the negative impacts of antibiotic application. Additionally, we will increase awareness of antibiotic application in agricultural plants by educating undergraduate students and secondary school teachers on antimicrobial use in food crops.This long-term goal will be accomplished by the following objectives:1. Assess the impact of antibiotic application on citrus-associated bacterial community, antibiotic resistant bacteria (ARB), metabolites and physiology of citrus trees relative to untreated trees.2. Determine how supplemented microbial diversity affects antibiotic resistant bacteria associated with citrus and plant health.3. Educate undergraduate students and secondary school teachers on antimicrobial resistance across the food chain, including consequences and strategies for mitigating antimicrobial resistance.
Project Methods
In this first greenhouse experiment, we will track the concentration of each antibiotic in citrus trees over time using 2-year-old citrus trees and soil from a non-antibiotic treated citrus grove where we have previously worked and from which we already have bacteria cultures stored at -80°C. In order to minimize error due to differences in the microbial communities in the soil, all the soils will be collected from an equidistance from citrus trees and mixed before setting up the experiment in the greenhouse. The antibiotic residual contents in roots will be assessed at 1, 3, 6, 11, 20 days post-application (DPA). The goal of this experiment is to determine two timepoints: (T1) the highest concentration of antibiotic and (T2) when the antibiotic concentration is reduced by 80%. These two timepoints will be used in experiment 2.Two-year-old citrus trees will receive antibiotic via spray and drench (i.e., pour over base of tree) using commercial antibiotics labeled for citrus, streptomycin or oxytetracycline, at the concentration of 0.60 g/L. Four replicates for each antibiotic (n=8) will be used. Oxytetracycline and streptomycin analysis will be performed using ACCEL ELISA kit Plexense, Inc., (Davis, CA, USA) and BioVision, Inc. kit (Milpitas, CA, USA), respectively to access the antibiotic from root tissues. A second larger experiment will use T1 and T2, determined in Experiment 1, to measure effects of antibiotic treatment. Antibiotic concentration will be measured at T1 and T2, as described above.Microbiome: At T1 and T2, genomic DNA from roots will be isolated for microbiome analysis (n=10 per treatment). For quantity and purity, DNA from all the samples will be evaluated for quantity by using a Qubit 3.0 Fluorometer (Life technologies InvitrogenTM) and for purity by using a NanoDrop Spectrophotometer.A two-step amplification and dual-barcoding approach will be performed to generate Illumina compatible amplicons. Universal bacterial primers 515F (59-GTGYCAGCMGCCGCGGTAA-39) and 806R (59-GGACTACNVGGGTWTCTAAT-39) will be used to amplify the v4 region of the 16S rRNA gene. The resulting amplicons will sent for sequencing at the Interdisciplinary Center for Biotechnology Research (ICBR) at the University of Florida. Raw read data will be submitted to the NCBI SRA. Read processing and data analysis will be reprocessed using the DADA2 pipeline R package, to obtain the exact sequence variants (ESVs). Community composition will be analyzed using the 'metacoder' R package. ESVs of the bacterial community from antibiotic-treated plants and the control will be compared, as in our previous work.Antibiotic resistant bacteria (ARB): At the time of microbiome analysis, ~100 mg of roots from each plant will be collected and immersed in tubes containing 0.5 mL of 10 mM phosphate buffer, vortexed three times for 5 s each time and serially diluted until 10−4. A volume of 100 μL of each dilution will be spread-plated on nutrient agar medium (3.0 g L−1 meat extract, 3.5 g L−1 meat peptone, 5.0 g L−1 NaCl, 20.0 g L−1 agar) (n=4), which will be amended with streptomycin and oxytetracycline, both at the concentration (50 μg/mL) as performed by other researchers. Plates from each dilution will be incubated at 28°C and colonies counted at 24 hrs and compared to unamended plates. Representative colonies from amended plates will be sequenced for 16S to identify the genus.Plant physiology: Six plants will be randomly selected per treatment, and photosynthetic capacity (mmol CO2 m-2 s-1) and CO2 assimilation rate (mmol CO2 mol air-1) will be evaluated, as we have performed in the past, by using a portable LI-6400XTR infrared gas analyzer (LI-COR Biosciences, Lincoln, NE, USA). Plant physiology parameters will be correlated with abundance of bacterial taxa and ARB using Vegan package.Metabolome: Root tissues will be collected from the same six plants monitored for photosynthetic capacity for metabolic analysis. Briefly, metabolites from frozen and ground roots will be extracted using a single-phase extraction solution (chloroform/methanol/water, 1:2.5:1, v/v/v). Frozen roots will be homogenized and mixed with 1 mL of the extraction solution for 20 min at 4°C, centrifuged for 30 min at 4°C and the supernatant transferred into new tubes from which 200 μL will be taken for further analysis. Metabolite fingerprinting will be performed by FIE-HRMS using a Q Exactive Plus Hybrid Quadrupole Orbitrap Mass Analyser with an Acella UHPLC system (ThermoFisher Scientific, Bremen, Germany). Multivariate analysis will be performed using MetaboAnalyst 4.0 (http://www.metaboanalyst.ca/) and one-way ANOVA (p< 0.05). The multiple comparison and post hoc analysis will be performed using Tukey's HSD. Identification will be based on the MS peaks to pathway algorithm31, including metabolites annotated at KEGG database (https://www.genome.jp/kegg/pathway.html).Objective 2. Determine how supplemented microbial diversity affects antibiotic resistantbacteria associated with citrus and plant health. Hypotheses: (i) supplementation of microbial diversity as a probiotic to citrus will reduce enrichment of antibiotic resistant bacteria (ARB) associated with citrus trees treated with antibiotics and promote plant health. We will use our culture collection obtained from mature citrus trees and composed of approximately 150 bacteria, which we currently have stored at -80°C, to identify potential beneficial taxa (Figure 2). Because of budget constraints and the challenge of growing and maintaining hundreds of citrus trees, we will use common bean (Phaseolus vulgaris), a model plant for screening for probiotic bacteria. A test will be conducted to determine when the antibiotic concentration is reduced by at least 80% of the highest concentration (T2) for common bean plants, similarly as was done for citrus trees.Bacteria to be tested will be delivered via root drench method at T2. The bacterial microbiome of both citrus and common bean is composed of Proteobacteria, Actinobacteria, and Acidobacteria. Moreover, our research has demonstrated the plant probiotic application is not host-specific. For instance, we showed that a strain of Bacillus amyloliquefaciens ALB629, isolated from cacao trees, was able to colonize and mitigate biotic and abiotic stress in common bean plants under controlled and field conditions.The in-vivo screening experiment will use common bean plants at the V4 growth stage, as we have performed in the past6. Up to 150 bacteria from our culture collection will be used by selecting for the screening, targeting those bacteria that belong to the same taxa that were affected by the antibiotic. Antibiotic resistant bacteria (ARB) and plant growth will be assessed at T2 using 4 replicates per treatment. When the top 2 bacterial candidates are found, the experiment will be repeated with citrus trees, and all data types described in Objective 1 will be obtained.