The aim of the present investigation was to determine the active ingredients in Amaranthus tricolor L. leaves and develop a biological pesticide. Organic solvent extraction, column chromatography, liquid chromatography, ODS-C18 reverse elution, Sephadex LH-20 gel filtration, H spectrum, and C spectrum were used to isolate the pure product for an assessment of the agricultural activity and bacteriostatic mechanisms. The results showed that the activity of the crude extract following carbon powder filtration was 1.63-fold that of the non-filtered extract. Further isolation was performed to obtain two pure products, namely, hydroxybenzoic acid (HBA) and benzo[b]furan-2-carboxaldehyde (BFC), and their molecular formulas and molecular weights were C₇H₆O₃ and 138.12, and C₉H₆O₂ and 146.12, respectively. Our study is the first to determine that HBA has bacteriostatic activity (MIC 125 μg/mL) and is also the first to isolate BFC from A. tricolor. The ultrastructure observation results showed that HBA caused the bacteria to become shriveled, distorted, and deformed, as well as exhibit uneven surfaces. After HBA treatment, 70 differentially expressed metabolites were detected in the bacteria, of which 9 were downregulated and 61 were upregulated. The differentially expressed metabolites were mainly strigolactones, organic acids and derivatives, fatty acids, benzene and substituted benzene derivatives, amino acids and associated metabolites, and alcohols and amines. Among all of the downregulated differentially expressed metabolites, MEDP1280 was the most critical, as it participates in many physiological and biochemical processes. The enrichment analysis showed that the differentially expressed metabolites mainly participate in tyrosine metabolism, biosynthesis of amino acids, cysteine and methionine metabolism, and arginine and proline metabolism. Additionally, HBA was found to disrupt cell membrane permeability and integrity, causing the leakage of substances and apoptosis. The physiological and biochemical test results showed that HBA could increase the pyruvate levels in bacteria but could decrease the activities of respiratory enzymes (malate dehydrogenase (MDH) and NADH oxidase) and antioxidant enzymes (superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX)). Inverse molecular docking was used to study the binding between HBA and respiratory and antioxidant enzymes. The results showed that HBA could bind to MDH, NADH oxidase, SOD, and GSH-PX, suggesting that these enzymes may be the effector targets of HBA. Conclusion: The optimal active ingredient in A. tricolor that can inhibit Acidovorax avenae subsp. citrulli was identified as HBA. HBA mainly disrupts the cell membrane, damages the metabolic system, and inhibits respiration and antioxidant enzyme activity to control bacterial growth. These results provide a reference for the further development of biological pesticides.

To explore the molecular mechanisms of the antifungal compound phenazine-1-carboxamide (PCN) inhibits Rhizoctonia solani and discover potential targets of action, we performed an integrated analysis of transcriptome and metabolome in R. solani mycelium by whether PCN treating or not. A total of 511 differentially expressed genes (DEGs) were identified between the PCN treatment and control groups. The fluorescence-based quantitative PCR (qPCR) got the accordant results of the gene expression trends for ten randomly selected DEGs. The Gene Ontology (GO) enrichment analysis revealed that fatty acid metabolic process, fatty acid oxidation, and lipid oxidation were among the most enriched in the biological process category, while integral component of membrane, plasma membrane, and extracellular region were among the most enriched in the cellular component category and oxidoreductase activity, cofactor binding, and coenzyme binding were among the most enriched in the molecular function category. KEGG enrichment analysis revealed the most prominently enriched metabolic pathways included ATP-binding cassette (ABC) transporters, nitrogen metabolism, aminobenzoate degradation. The DEGs related functions of cellular structures, cell membrane functions, cellular nutrition, vacuole-mitochondrion membrane contact site and ATPase activity, pH, anti-oxidation, were downregulated. A total of 466 differential metabolites were found between the PCN treatment and control groups after PCN treatment. KEGG enrichment found purine, arachidonic acid, and phenylpropanoid biosynthesis pathways were mainly affected. Further results proved PCN decreased the mycelial biomass and protein content of R. solani, and superoxide dismutase (SOD) activity reduced while peroxidase (POD) and cytochrome P450 activities increased. The molecule docking indicted that NADPH nitrite reductase, ATP-binding cassette transporter, alpha/beta hydrolase family domain-containing protein, and NADPH–cytochrome P450 reductase maybe the particular target of PCN. In conclusion, the mechanisms via which PCN inhibits R. solani AG1IA may be related to cell wall damage, cell membrane impairment, intracellular nutrient imbalance, disturbed antioxidant system, and altered intracellular pH, which laid foundation for the further new compound designing to improve antifungal efficacy.

To explore the molecular mechanism through which the novel fungicide N-(naphthalen-1-yl) phenazine-1-carboxamide (NNPCN) inhibits Rhizoctonia solani, we clarified the target and mode of action, explored lead compounds, and developed novel fungicides. Methods: Growth observation, scanning electron microscopy, transmission electron microscopy, transcriptome sequencing technology, quantitative real-time PCR (qRT-PCR), physiological and biochemical determination, and reverse molecular docking technology were used to study the effects of this compound on the microscopic morphology of R. solani. The differentially expressed genes (DEGs), functions, and metabolic pathways were analyzed. The genes displaying significant differences were randomly selected for qRT-PCR verification and confirmed by physiological and biochemical determination to construct their binding mode with key targets. The results showed that the mycelium treated with NNPCN produced a red secretion and exhibited progressive creeping growth. Under a scanning electron microscope, hyphal swelling, uneven thickness, fractures, deformities, and hyphal surface warts increased. Under a transmission electron microscope, the cell wall was separated, the subcellular organelles were disintegrated, and the septum disappeared. Furthermore, there were 6838 DEGs under NNPCN treatment, including 291 significant DEGs, of which 143 were upregulated and 148 downregulated. Ten DEGs were randomly selected for qRT-PCR verification, and the gene expression trend was consistent with the transcriptome sequencing results. Gene Ontology enrichment analysis showed that the DEGs were significantly enriched in cell wall glucan decomposition and metabolism, cell membrane synthesis, metabolism, composition, organic hydroxyl compounds, oxidoreductase activity, and transition metal ion binding. Metabolic pathway enrichment analysis showed that there were 16 significant metabolic pathways, such as steroid biosynthesis and ABC transporters. Further study found that genes, such as the glycosyl hydrolase family 10 domain-containing protein, which is related to glucan catabolic process function as tied to the cell wall, were downregulated. Lipid oxidation, modification, and other genes related to the cell membrane were also downregulated. Secondly, genes related to lipid modification, lipid metabolism processes, integral components of the membrane, and other ABC transporters were downregulated. Fatty-acid oxidation and carbohydrate metabolic processes, which are related to antioxidant and metabolic functions, displayed significant differences in their target genes. Nitrite reductase [NADH] activity and mitochondrial organization gene expression were downregulated. These results revealed that target genes may involved in the cell wall, cell membrane, antioxidant and metabolism, nitrogen metabolism, and mitochondria. The results of the physiological and biochemical tests showed that NNPCN decreased the β-1,3-glucanase, malondialdehyde, and ATPase activities and nucleic acid leakage but increased the activity of nitrate reductase. The results of the reverse molecular docking showed that NNPCN could freely bind to target proteins such as β-1,3-glucanase, ABC transporter, and NADPH nitrate reductase, whereby NNPCN could bind to glucanase via van der Waals and electrostatic forces and to ABC transporter and NADPH nitrate reductase via hydrogen bonding. Conclusion: The mechanism via which NNPCN inhibits R. solani may be related to the cell wall structure, cell membrane damage, antioxidant activity, and metabolism.