Construction of a fusant bacterial strain simultaneously degrading atrazine and acetochlor and its application in soil bioremediation

Abstract

Application of herbicide-degrading bacteria is an effective strategy to remove herbicide in soil. However, the ability of bacteria to degrade a herbicide is often severely limited in the presence of other pesticide. In this study, the atrazine-degrading strain Klebsiella varicola FH-1 and acetochlor-degrading strain Bacillus Aryabhatti LY-4 were used as parent strains to construct the recombinant RH-92 strain through protoplast fusion technology. Compared with the parent strains, RH-92 exhibited enhanced ability to degrade herbicide mixture containing atrazine and acetochlor, exhibiting 63.16 % and 68.48 % higher degradation rates, respectively. RAPD analysis showed that gene rearrangement occurred during protoplast fusion, and the genetic similarity indexes of the fused strain RH-92 and the two parent strains were 0.5853 and 0.4240, respectively. HPLC-MS analysis confirmed that RH-92 shared similar degradation products and pathways with both parent strains but exhibited a novel metabolic pathway for the continuous degradation of CMEPA (degradation product of acetochlor) into MEA through amide bond hydrolysis. The activities of GSH, GST and SOD of RH-92 increased and the level of MDA decreased under the stress of compound herbicides. Strain RH-92 did not show a large number of bacterial apoptosis, and maintained good cell membrane integrity and permeability. The half-lives of atrazine and acetochlor were 4.9 d and 7.6 d when the parent strains FH-1 and LY-4 were applied in unsterilized soil containing herbicide mixture treatment,the application fusant RH-92 strain significantly reduced the half-life to 1.6 and 1.8 d, respectively. Furthermore, 16S rRNA sequencing indicated that RH-92 application effectively restored bacterial taxa with diminished relative abundances under herbicide mixture treatment, ameliorated phytotoxicity in soybean seedlings, and promoted enhanced vegetative growth in the roots and plant height. This study highlighted the application of fusant strains as a bioremediation strategy for combatting atrazine and acetochlor pollution in soil and provided theoretical insights.