6533b872fe1ef96bd12d3669

RESEARCH PRODUCT

Long Term Exposure of Agricultural Soil to Veterinary Antibiotics Changes the Population Structure of Symbiotic Nitrogen-Fixing Bacteria Occupying Nodules of Soybeans (Glycine max)

subject

description

International audience; Antibiotics can be entrained onto agricultural land through the application of animal manures, human biosolids, or recycled wastewater for irrigation. In order to evaluate the impacts on soil microorganisms of exposure to antibiotics, a series of replicated plots were initiated in 1999 at the AAFC research farm in London ON. Every spring a mixture of sulfamethazine, chlortetracycline and tylosin is incorporated directly into the soil to attain concentrations ranging from 0.1 to 10 mg/kg, and these are then seeded with soybeans. In the present study nodules were isolated from soybean plants growing in antibiotic treated and control soils in 2012, after 14 annual treatments. Rabbit polyclonal antisera distinguishing Bradyrhizobium japonicum serogroups 122, 6, 110 and 123 and B. liaoningense serogroup 135 was used for nodule strain typing. A total of 278 bradirhizobia were isolated and confirmed. Genomic DNA from isolates was subjected to RSα fingerprinting, sequencing of the 16S rDNA coding region, and the 16S rDNA- 23S rDNA intergenic spacer (IGS), and MLST analysis using six housekeeping genes. The sensitivity of isolates to the three antibiotics, singly and in combination, was evaluated. Serogroup 135 dominated nodules, with the other serotypes occasionally detected and many nodules being unreactive to the antisera. Based on genomic fingerprinting and MLST analysis the collection of 278 bradyrhizobia was very diverse. The distribution of isolates in RSa fingerprint groups was significantly different in the 3 soils treated with antibiotics compared to control soil, with an increase in proportion of strains belonging to RSa types a, b, c and q and a decrease of strains belonging to RSa types p and s. This result confirms those based on serotyping: strains belonging to B. liaoningense serogroup 135 are more abundant in soils having received antibiotics. Using RSα fingerprinting results, Shannon diversity indexes computed for control and antibiotic treated soils were equal to 1.11, 1.53, 1.43 and 1.3 for control soil, and soils having received low, intermediate and high antibiotic doses, respectively. T-tests revealed that Shannon diversity indexes were significantly different between control soil and soil having received the low (0.1 mg each drug/kg soil) dose of antibiotics (p-value=0.0045). Overall, the present study indicates that long term treatment with environmentally relevant concentrations of the veterinary antibiotics tylosin, chlortetracycline and sulfamethazine alters the composition of Bradyrhizobial populations occupying soybean nodules. The sensitivity of bradyrhizobia to the three antibiotics was not associated with the treatment from which they were recovered, indicating that variation in nodule occupancy was due to an indirect effect rather than direct antibiotic selection.