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The present study was aimed at assessing the impact of azoxystrobin-a fungicide commonly used in plant protection against pathogens (Amistar 250 SC)-on the soil microbiota and enzymes, as well as plant growth and development. The laboratory experiment was conducted in three analytical terms (30, 60, and 90 days) on sandy clay (pH-7.0). Azoxystrobin was applied to soil in doses of 0.00 (C), 0.110 (F) and 32.92 (P) mg kg -1 d.m. of soil. Its 0.110 mg kg -1 dose stimulated the proliferation of organotrophic bacteria and actinobacteria but inhibited that of fungi. It also contributed to an increase in the colony development index (CD) and a decrease in the ecophysiological diversity index (EP) of all analyzed groups of microorganisms. Azoxystrobin applied at 32.92 mg kg -1 reduced the number and EP of microorganisms and increased their CD. PP952051.1 Bacillus mycoides strain (P), PP952052.1 Prestia megaterium strain (P) bacteria, as well as PP952052.1 Kreatinophyton terreum isolate (P) fungi were identified in the soil contaminated with azoxystrobin, all of which may exhibit resistance to its effects. The azoxystrobin dose of 0.110 mg kg -1 stimulated the activity of all enzymes, whereas its 32.92 mg kg -1 dose inhibited activities of dehydrogenases, alkaline phosphatase, acid phosphatase, and urease and stimulated the activity of catalase. The analyzed fungicide added to the soil at both 0.110 and 32.92 mg kg -1 doses inhibited seed germination and elongation of shoots of Lepidium sativum L., Sinapsis alba L., and Sorgum saccharatum L.

Triazoles are compounds with various biological activities, including fungicidal action. They became popular through cholinesterase studies after the successful synthesis of the dual binding femtomolar triazole inhibitor of acetylcholinesterase (AChE, EC 3.1.1.7) by Sharpless et al. via in situ click chemistry. Here, we evaluate the anticholinesterase effect of the first isopropanol triazole fungicide mefentrifluconazole (Ravystar ® ), developed to overcome fungus resistance in plant disease management. Mefentrifluconazole is commercially available individually or in a binary fungicidal mixture, i.e., with pyraclostrobin (Ravycare ® ). Pyraclostrobin is a carbamate that contains a pyrazole ring. Carbamates are known inhibitors of cholinesterases and the carbamate rivastigmine is already in use for the treatment of Alzheimer's disease. We tested the type and potency of anticholinesterase activity of mefentrifluconazole and pyraclostrobin. Mefentrifluconazole reversibly inhibited human AChE and BChE with a seven-fold higher potency toward AChE ( K i = 101 ± 19 μM). Pyraclostrobin (50 μM) inhibited AChE and BChE progressively with rate constants of (t 1/2 = 2.1 min; k i = 6.6 × 10 3 M -1 min -1 ) and (t 1/2 = 1.5 min; k i = 9.2 × 10 3 M -1 min -1 ), respectively. A molecular docking study indicated key interactions between the tested fungicides and residues of the lipophilic active site of AChE and BChE. Additionally, the physicochemical properties of the tested fungicides were compared to values for CNS-active drugs to estimate the blood-brain barrier permeability. Our results can be applied in the design of new molecules with a lesser impact on humans and the environment.

This study aimed to evaluate how the combined presence of the synthetic fungicide azoxystrobin (AZ) and the biosurfactant-producing Bacillus sp. Kol B3 influences the growth of the phytopathogenic fungus Fusarium sambucinum IM 6525. The results showed a noticeable increase in antifungal effectiveness when biotic and abiotic agents were combined. This effect manifested across diverse parameters, including fungal growth inhibition, changes in hyphae morphology, fungal membrane permeability and levels of intracellular reactive oxygen species (ROS). In response to the presence of Fusarium and AZ in the culture, the bacteria changed the proportions of biosurfactants (surfactin and iturin) produced. The presence of both AZ and/or Fusarium resulted in an increase in iturin biosynthesis. Only in 72 h old bacterial-fungal co-culture a 20% removal of AZ was noted. In the fungal cultures (with and without the addition of the bacteria), the presence of an AZ metabolite named azoxystrobin free acid was detected in the 48th and 72nd hours of the process. The possible involvement of increased iturin and ROS content in antifungal activity of Bacillus sp. and AZ when used together are also discussed. Biosurfactants were analyzed by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Microscopy techniques and biochemical assays were also used.

Fungicides are widely used in agriculture for crop protection. Succinate dehydrogenase inhibitors (SDHIs) and strobilurins inhibit mitochondria electron transport chain (ETC) in fungi, by blocking complex II and complex III, respectively. Questions regarding their selectivity of action for fungi have been raised in the literature, and we previously showed that boscalid and bixafen (SDHIs) alter the mitochondrial function of human hepatocytes. Here, we analyzed the impact of the exposure of human hepatocytes to pyraclostrobin, a fungicide belonging to the class of strobilurins. Using electron paramagnetic resonance (EPR), we observed a decrease in oxygen consumption rate (OCR) and an increase in mitochondrial superoxide levels after 24 h exposure to 0.5 µM concentration. As a consequence, the content in ATP amount in the cells was reduced, the ratio reduced/oxidized glutathione was decreased, and a decrease in cell viability was observed using three different assays (PrestoBlue, crystal violet, and annexin V assays). In addition, as SDHIs and strobilurins are commonly associated in commercial preparations, we evaluated a potential "cocktail" toxic effect. We selected low concentrations of boscalid (0.5 µM) and pyraclostrobin (0.25 µM) that did not induce a mitochondrial dysfunction in liver cells when used separately. In sharp contrast, when both compounds were used in combination at the same concentration, we observed a decrease in OCR, an increase in mitochondrial superoxide production, a decrease in the ratio reduced/oxidized glutathione, and a decrease in cell viability in three different assays.

The fulfilment of the European "Farm to Fork" strategy requires a drastic reduction in the use of "at risk" synthetic pesticides; this exposes vulnerable agricultural sectors-among which is the European risiculture-to the lack of efficient means for the management of devastating diseases, thus endangering food security. Therefore, novel scaffolds need to be identified for the synthesis of new and more environmentally friendly fungicides. In the present work, we employed our previously developed 3D model of P. oryzae cytochrome bc1 (cyt bc1 ) complex to perform a high-throughput virtual screening of two commercially available compound libraries. Three chemotypes were selected, from which a small collection of differently substituted analogues was designed and synthesized. The compounds were tested as inhibitors of the cyt bc1 enzyme function and the mycelium growth of both strobilurin-sensitive (WT) and -resistant (RES) P. oryzae strains. This pipeline has permitted the identification of thirteen compounds active against the RES cyt bc1 and five compounds that inhibited the WT cyt bc1 function while inhibiting the fungal mycelia only minimally. Serendipitously, among the studied compounds we identified a new chemotype that is able to efficiently inhibit the mycelium growth of WT and RES strains by ca. 60%, without inhibiting the cyt bc1 enzymatic function, suggesting a different mechanism of action.

Fungicides often cause genotoxic stress and neurodevelopmental disorders such as autism (ASD). Fungicide-azoxystrobin (AZOX) showed acute and chronic toxicity to various organisms, and remained a concern for ill effects in developing neurons. We evaluated the neurotoxicity of AZOX in developing mouse brains, and observed prenatal exposure to AZOX reduced neuronal viability, neurite outgrowth, and cortical migration process in developing brains. The 50% inhibitory concentration (IC50) of AZOX for acute (24 h) and chronic (7 days) exposures were 30 and 10 μM, respectively. Loss in viability was due to the accumulation of reactive oxygen species (ROS), and inhibited neurite outgrowth was due to the deactivation of mTORC1 kinase activity. Pretreatment with ROS scavenger- N-acetylcysteine (NAC) reserved the viability loss and forced activation of mTORC1 kinase revived the neurite outgrowth in AZOX treated neurons. Intra-amniotic injection of AZOX coupled with in utero electroporation of GFP-labelled plasmid in E15.5 mouse was performed and 20 mg/kg AZOX inhibited radial neuronal migration. Moreover, the accumulation of mitochondria was significantly reduced in AZOX treated primary neurons, indicative of mitochondrial deactivation and induction of apoptosis, which was quantified by Bcl2/Bax ratio and caspase 3 cleavage assay. This study elucidated the neurotoxicity of AZOX and explained the possible cure from it.

Long-term use of a single fungicide increases the resistance risk and causes adverse effects on natural ecosystems. Controlled release formulations of dual fungicides with different modes of action can afford a new dimension for addressing the current issues. Based on adjustable aperture and superhigh surface area, metal-organic frameworks (MOFs) are ideal candidates as pesticide release carriers. This study used Al 3+ as the metal node and 2-aminoterephthalic acid as the organic chain to prepare aluminum-based metal-organic framework material (NH 2 -Al-MIL-101) with "cauliflower-like" structure and high surface area of 2359.0 m 2 /g. Fungicides of azoxystrobin (AZOX) and diniconazole (Dini) were simultaneously encapsulated into NH 2 -Al-MIL-101 with the loading content of 6.71% and 29.72%, respectively. Dual fungicide delivery system of AZOX@Dini@NH 2 -Al-MIL-101 demonstrated sustained and pH responsive release profiles. When the maximum cumulative release rate of AZOX and Dini both reached about 90%, the release time was 46 and 136 h, respectively. Furthermore, EC 50 values as well as the percentage of inhibition revealed that AZOX@Dini@NH 2 -Al-MIL-101 had enhanced germicidal efficacy against rice sheath blight ( Rhizoctonia solani ), evidenced by the synergistic ratio of 1.83. The present study demonstrates a potential application prospect in sustainable plant protection through co-delivery fungicides with MOFs as a platform.