Systematic evaluation of chiral fungicide penflufen for the bioactivity improvement and input reduction using alphafold2 models and transcriptome sequencing

Highlights

• Both R-penflufen and S-penflufen were persistent in soil.

• S-penflufen had a stronger binding capacity with the SDH of R. solani.

• There was difference in the bioaccumulation and sub-chronic toxicity to earthworm.

• S-penflufen may be more potential for development.

Abstract

Traditional risk assessment of pesticide concludes at the racemic level, which is often incomprehensive. In this study, systematic studies on environmental stability, bioactivity, and ecotoxicological effects of fungicide penflufen were carried out at the enantiomeric level. The single-enantiomer of penflufen was successfully separated and prepared, and their stability was verified in different environmental matrices. Meanwhile, bioactivity test indicated that S-(+)-penflufen had increased bioactivity with its bioactivities against Rhizoctonia solani, Fusarium oxysporum, and Fusarium moniliforme being factors of 7.8, 1.8, and 4.7, respectively greater than those of R-(-)-penflufen. Molecular docking results showed the strong hydrogen bond interactions with Leu300, enantiomer-specific hydrophobic interactions with Cys299, Arg91, and His93, and the greater binding energy between S-(+)-penflufen and succinate dehydrogenase of Rhizoctonia solani caused the selective bioactivity. Additionally, two enantiomers showed low acute toxicity whereas selective sub-chronic toxicity to earthworms. In sub-chronic toxicity test, the accumulated enantiomers caused abnormalities in intestinal tract structure, enzyme activities, and gene expression of earthworms, especially in the S-(+)-penflufen treatment. The selective interactions between penflufen enantiomers and key proteins were elucidated using molecular docking, which may be the main reason of stereoselective subchronic toxicity. S-(+)-penflufen has high bioactivity and low acute risk, it has great potential for development.

Introduction

Increasing the development and application of novel pesticides has effectively improved agricultural production efficiency (Ogawa et al., 2020). However, the unscientific development and use of pesticides will inevitably cause inestimable harm to food safety, human health, and environmental safety (Huang et al., 2021, Hu et al., 2016). In particular, the effects of pesticides on non-target organisms in soil ecosystems have attracted global attention (Mitchell et al., 2017, Hu et al., 2020). It is noteworthy that reducing the adverse effects of pesticides on the ecological environment and introducing efforts to protect ecosystems have become a national development strategy worldwide. Researching and developing "high efficiency and low risk" pesticides is of great significance to reduce pesticide use and promote "green" development.

Currently, 30 % of the pesticides sold in the market have chiral structures; however, as a result of the limitations of technology and cost, most of them are sold as racemates (Gao et al., 2020). Studies have shown that enantiomers of chiral pesticides have great differences in both the biological activity and environmental behavior of target and non-target organisms, among which high bioactivity and ecotoxicity often exist in different enantiomers (Huang et al., 2012, Wang et al., 2009). For example, Li et al. (2019) found that the S-isomer of fluxametamide had significant biological activity against target pests, whereas the R-isomer was highly toxic to non-target organism honeybees. Therefore, the extensive use of high-risk substances with low activity and high toxicity will undoubtedly increase the economic costs and ecological risks (Zhao et al., 2019). Meanwhile, if the traditional pesticide risk assessment methods only remain at the racemate level, without considering the differences between different enantiomers, the results are often one-sided. Therefore, it is of great significance to explore the environmental behavior differences, biological activity differences in target organisms, and toxicity differences in non-target organisms among chiral pesticide enantiomers for the development of high bioactivity and low toxicity pesticides.

Penflufen (2-[(RS)− 1,3-dimethylbutyl]− 5-fluoro-1,3-dimethylpyrazole-4-carboxanilide) is a class of pyrazolamide fungicides developed by the Bayer Company (EFSA, 2016). This fungicide is a succinate dehydrogenase inhibitor (SDHI), which acts on respiratory chain electron transport complex II and blocks energy metabolism (Di et al., 2021, Zhang et al., 2015). Penflufen has broad bioactivity against many fungal diseases, including potato black scurf, wheat sharp eyespot, rice sheath blight, and root rot in peanut and other similar fungal diseases (EFSA, 2016; Tian et al., 2016a). There are currently 83 patents related to the application of penflufen in agriculture at the declaration or disclosure stage, which demonstrated that it has broad potential applications (Tian et al., 2016a). Notably, Sun et al. (2019) reported that the initial concentrations of penflufen in spinach and Chinese cabbage were greater than 25 mg/kg after spraying with a 22 % penflufen suspension at 300 g a. i. /ha, which is a high residue and may pose a potential risk to the soil environment. However, to the best of our knowledge, few studies regarding the determination method for penflufen enantiomers were reported, which is necessary for monitoring the environmental behavior of penflufen in soil environmental samples. Additionally, no systematic research has been conducted on the toxicological effects, bioactivities, or molecular mechanisms of penflufen in the soil ecological environment at the chiral level.

Analyzing and illuminating the enantioselectivity mechanism is of great significance for efficient utilization and environmental pollution reduction of chiral pesticides. For the target bio-enzyme, the selective biological activity of enantiomers may be derived from the interactions between enantiomers and structurally sensitive biological target-receptors (Abu-Melha et al., 2021). Molecular docking technology can be used to analyze the binding differences between chiral pesticide enantiomers and target proteins and to elucidate the mechanism of enantioselectivity (Abu-Melha et al., 2021, Chen et al., 2019, Tian et al., 2016b). For non-target bio-enzymes, the selective biological toxicity of enantiomers may be induced by enantioselective absorption, transformation, metabolism, accumulation in non-target organisms, or interaction differences between pesticide enantiomers and non-target chiral molecules (Ding et al., 2020). Transcriptome analysis technology is an effective method to characterize differences in the influence of pesticide enantiomers on non-target organisms at the level of genes, proteins, and metabolic pathways (Carrao et al., 2019, Ding et al., 2020, Xiang et al., 2019). Therefore, molecular docking technology and transcriptome analysis technology were used in this study to characterize the enantioselective bioactivity and toxicity mechanisms to target organisms and non-target organisms, respectively.

In this study, a method for the chiral separation, absolute configuration identification, and residual determination of penflufen enantiomers was developed. In addition, the environmental stability of penflufen enantiomers was evaluated. Moreover, the enantioselectivity in bioactivity and toxicity of penflufen enantiomers was confirmed. The purpose of this study was to provide necessary data for risk assessment and rational use of penflufen at the chiral level and to provide a new perspective for the development of potentially high-efficiency and low-risk commercial pesticide products.