Isolation, characterization, and identification of two methomyl-degrading bacteria from a pesticide-treated crop field in West Bengal, India

Abstract

Two methomyl-degrading bacteria (initially named Disha A and Disha B) were isolated from a pesticide-treated crop field in Baruipur, 24 Parganas (South), West Bengal, India. Both strains could not grow in mineral salt (MS) medium but showed efficient growth in the presence of methomyl. The highest growth was observed in the MS medium containing 0.16% methomyl. When methomyl was supplemented with glucose, no further enhancement of growth was observed, whereas supplementation with yeast extract had a positive effect on growth of both strains, indicating that methomyl could be utilized as the sole source of carbon but not that of nitrogen. In Nutrient Broth and Luria Bertani medium, these strains could tolerate 0.4% methomyl. Optimum pH and temperature for growth of both bacteria in the methomyl-containing MS medium were 7.0 and 30°C, respectively. Protein concentration in the cell-free extracts of bacterial cultures was directly proportional to methomyl concentration in the medium. Disha A was more resistant to the antibiotics amoxicillin and penicillin, as indicated by minimum inhibitory concentration (600 and 500 μg/mL, respectively), which were higher than those obtained for Disha B (350 and 300 μg/mL, respectively). Both Disha A and Disha B were plasmid-bearing, gram-positive, endospore-producing, rod-shaped bacteria. Biochemical studies, 16S rDNA sequencing, and phylogenetic analysis indicated maximum similarity of Disha A to Bacillus cereus ATCC 14579, whereas Disha B showed maximum similarity to Bacillus safensis F0-36b ATCC BAA-1126. The HPLC analysis clearly indicated that B. cereus and B. safensis showed 88.25 and 77.5% of methomyl (Sigma) degradation, respectively within 96 h of growth. This is the first report of Bacillus species that can degrade the carbamate pesticide methomyl and thrive in presence of its high concentrations.