Pendimethalin [ N -(1-ethylpropyl)-2,6-dinitro-3,4-xylidine] is a selective preemergence dinitroaniline herbicide. Several fungi and bacteria have been reported to degrade pendimethalin, but the enzymes or genes involved in this process have not been characterized. Nitroreduction is the initial degradation and detoxification step for pendimethalin. In this study, a pendimethalin nitroreductase (PNR), responsible for the nitroreduction of pendimethalin, was purified from the pendimethalin-degrading strain Bacillus subtilis Y3. Based on a comparison of its mass fingerprints with all of the deduced proteins from the draft genome of strain Y3, a protein annotated as a nitroreductase was identified, and its corresponding encoding gene was termed pnr . PNR was a functional homodimer with a subunit molecular mass of approximately 23 kDa. PNR reduced the C-6 nitro group of the aromatic ring of pendimethalin, yielding 2-nitro-6-amino- N -(1-ethylpropyl)-3,4-xylidine. PNR could also catalyze the nitroreduction of three other major varieties of dinitroaniline herbicides, including butralin, oryzalin, and trifluralin. However, the number of reduced nitro groups was two instead of one, which differed from the nitroreduction of pendimethalin by PNR and which may be due to the symmetry in the chemical structures of the two nitro groups. A detoxification assay revealed that 2-nitro-6-amino- N -(1-ethylpropyl)-3,4-xylidine (PNR-reduced pendimethalin) showed no inhibitory effect on the growth of Saccharomyces cerevisiae BY4741, whereas pendimethalin showed an obvious inhibitory effect on its growth, indicating the detoxification effect of pendimethalin by PNR. Therefore, PNR has potential in pendimethalin detoxification applications. This report describes an enzyme (and corresponding gene) involved in the biodegradation of pendimethalin and dinitroaniline herbicides. IMPORTANCE Pendimethalin [ N -(1-ethylpropyl)-2,6-dinitro-3,4-xylidine] is a widely used selective preemergence dinitroaniline herbicide, and its residue has been frequently detected in the environment. The U.S. Environmental Protection Agency (EPA) has classified pendimethalin as a persistent bioaccumulative toxin. To date, no enzymes or genes involved in pendimethalin biodegradation have been reported. In the present study, the gene pnr , which encodes the nitroreductase PNR, responsible for the nitroreduction of pendimethalin, was cloned from the pendimethalin-degrading strain Bacillus subtilis Y3. PNR could also catalyze the nitroreduction of three other major varieties of dinitroaniline herbicides, including butralin, oryzalin, and trifluralin. The reduction of pendimethalin by PNR might eliminate its toxicity against Saccharomyces cerevisiae BY4741, indicating the application potential of PNR in the detoxification of pendimethalin.

Trifluralin, a widely utilized dinitroaniline herbicide, has emerged as a prevalent environmental contaminant that poses significant risks both to ecosystems and to human health. Microbial degradation represents the primary pathway for preventing trifluralin accumulation in the environment. Although much work has been conducted on the microbial breakdown of trifluralin, numerous challenges persist regarding the identification of efficient degrading strains, the elucidation of the metabolic pathways involved, and the application of bioremediation techniques. In this study, Bacillus sp. TF-1, a strain isolated from a paddy field that can utilize trifluralin as a source of carbon and energy, was applied. Remarkably, it eliminated 86.7% of 100 mg/L trifluralin within 6 h, and 99.7% of trifluralin was eliminated within 48 h. UPLC–MS analysis suggested that trifluralin degradation occurred first through mono-nitroreduction, followed by further nitroreduction and trifluoromethyl oxidation; trifluralin could also be metabolized through complete nitroreduction and N-dealkylation. Furthermore, Bacillus sp. TF-1 effectively mitigated the severe toxicity of trifluralin to sensitive crops. These findings not only expand the repertoire of efficient trifluralin-degrading microorganisms but also increase our understanding of trifluralin biodegradation pathways and highlight the biological importance of employing microbes to eradicate trifluralin residues from the environment.