Imazalil Research Articles

Influence of pH and temperature in a heated flooder application on imazalil residue loading, citrus green mould infection and sporulation inhibition

Green mould (caused by Penicillium digitatum) is the largest contributor to loss due to postharvest decay of citrus fruit. Imazalil (IMZ) in the IMZ sulphate formulation is the most important fungicide in green mould management in citrus packhouses. Studies have highlighted the importance of pH and temperature of IMZ sulphate solutions, as well as the exposure time of fruit to the solution, but were focussed on dip application as this was the most common IMZ application method. The heated flooder (in-line drench or cascade) has widely replaced the use of a dip or bath for IMZ application in citrus packhouses globally; however, studies on its optimal use and efficacy were needed. Variables that were studied included the effects of pH (3, 4, 5 and 6), the temperature of the solution (45, 55 and 65 °C at pH 6), and concentration (250 or 500 mg L−1) in a time of 8 s. Imazalil residue loading models were developed for the effects of pH and temperature. Imazalil residue levels loaded on Satsuma mandarin, lemon, navel and Valencia orange fruit increased as pH, temperature range and concentration were increased, and were generally in the range between 0.4 and 3.0 mg kg−1. The South African Maximum Residue Limit (MRL) of 5.0 mg kg−1 was often exceeded at a pH of 6 at temperatures of 55 and 65 °C. At residue levels below the MRL, IMZ application using the heated flooder offered excellent curative action on 24-hour-old infections, excellent protective control, as well as sporulation inhibition. Sporulation inhibition of P. digitatum from green mould lesions was modelled on residue levels, and 90 % inhibition was obtained at residue levels of 1.84–2.47 mg kg−1.

Imazalil and prochloraz toxicokinetics in fish probed by a physiologically based kinetic (PBK) model.

Azole fungicides are highly suspected endocrine disruptors (EDs) and are frequently detected in surface water. Among them, there are prochloraz (PCZ), a commonly used moleculefor ED studies, and imazalil (IMZ), a highly suspected ED. Little is known about their toxicokinetic (TK) behavior in fish. Hence, research suggested that an improved risk assessment could be achieved by gaining insight into their TK behavior. The aim of this study is to understand and model the TK of both substances in different fish species, irrespective of the scheme of exposure. TK data from the literature were retrieved including different modes of exposure (per os and waterborne). In addition, two experiments on zebrafish exposed to either IMZ or PCZ were performed to address the lack of in vivo TK data. A physiologically based kinetic (PBK) model applied to IMZ and PCZ was developed, capable of modeling different exposure scenarios. The parameters of the PBK model were simultaneously calibrated on datasets reporting internal concentration in several organs in three fish species (original and literature datasets) by Bayesian methods (Monte Carlo Markov Chain). Model predictions were then compared to other experimental data (i.e., excluded from the calibration step) to assess the predictive performance of the model. The results strongly suggest that PCZ and IMZ are actively transported across the gills, resulting in a small fraction being effectively absorbed by the fish. The model's results also confirm that both molecules are extensively metabolized by the liver into mainly glucuronate conjugates. Overall, the model performances were satisfying, predicting internal concentrations in several key organs. On average, 90% of experimental data were predicted within a two-fold range. The PBK model allows the understanding of IMZ and PCZ kinetics profiles by accurately predicting internal concentrations in three different fish species regardless of the exposure scenario. This enables a proper understanding of the mechanism of action of EDs at the molecular initiating event (MIE) by predicting bioaccumulation in target organs, thus linking this MIE to a possible adverse outcome.

Transcriptome Analysis Reveals Potential Regulators of DMI Fungicide Resistance in the Citrus Postharvest Pathogen Penicillium digitatum.

Green mold, caused by Penicillium digitatum, is the major cause of citrus postharvest decay. Currently, the application of sterol demethylation inhibitor (DMI) fungicide is one of the main control measures to prevent green mold. However, the fungicide-resistance problem in the pathogen P. digitatum is growing. The regulatory mechanism of DMI fungicide resistance in P. digitatum is poorly understood. Here, we first performed transcriptomic analysis of the P. digitatum strain Pdw03 treated with imazalil (IMZ) for 2 and 12 h. A total of 1338 genes were up-regulated and 1635 were down-regulated under IMZ treatment for 2 h compared to control while 1700 were up-regulated and 1661 down-regulated under IMZ treatment for 12 h. The expression of about half of the genes in the ergosterol biosynthesis pathway was affected during IMZ stress. Further analysis identified that 84 of 320 transcription factors (TFs) were differentially expressed at both conditions, making them potential regulators in DMI resistance. To confirm their roles, three differentially expressed TFs were selected to generate disruption mutants using the CRISPR/Cas9 technology. The results showed that two of them had no response to IMZ stress while ∆PdflbC was more sensitive compared with the wild type. However, disruption of PdflbC did not affect the ergosterol content. The defect in IMZ sensitivity of ∆PdflbC was restored by genetic complementation of the mutant with a functional copy of PdflbC. Taken together, our results offer a rich source of information to identify novel regulators in DMI resistance.

Toxic interactions at the physiological and biochemical levels of green algae under stress of mixtures of three azole fungicides

Assessing the interactions between environmental pollutants and these mixtures is of paramount significance in understanding their negative effects on aquatic ecosystems. However, existing research often lacks comprehensive investigations into the physiological and biochemical mechanisms underlying these interactions. This study aimed to reveal the toxic mechanisms of cyproconazole (CYP), imazalil (IMA), and prochloraz (PRO) and corresponding these mixtures on Auxenochlorella pyrenoidosa by analyzing the interactions at physiological and biochemical levels. Higher concentrations of CYP, IMA, and PRO and these mixtures resulted in a reduction in chlorophyll (Chl) content and increased total protein (TP) suppression, and malondialdehyde (MDA) content exhibited a negative correlation with algal growth. The activity of catalase (CAT) and superoxide dismutase (SOD) decreased with increasing azole fungicides and their mixture concentrations, correlating positively with growth inhibition. Azole fungicides induced dose-dependent apoptosis in A. pyrenoidosa, with higher apoptosis rates indicative of greater pollutant toxicity. The results revealed concentration-dependent toxicity effects, with antagonistic interactions at low concentrations and synergistic effects at high concentrations within the CYP-IMA mixtures. These interactions were closely linked to the interactions observed in Chl-a, carotenoid (Car), CAT, and cellular apoptosis. The antagonistic effects of CYP-PRO mixtures on A. pyrenoidosa growth inhibition can be attributed to the antagonism observed in Chl-a, Chl-b, Car, TP, CAT, SOD, and cellular apoptosis. This study emphasized the importance of gaining a comprehensive understanding of the physiological and biochemical interactions within algal cells, which may help understand the potential mechanism of toxic interaction.

Gold nanoparticle-based immunochromatographic assay for rapid detection of imazalil.

Imazalil (IMZ) is a commonly used fungicide for controlling fungus in agriculture, leaving residual IMZ in crops that could be hazardous to human health. In this work, we designed IMZ haptens for mice immunization and prepared sensitive monoclonal antibody (mAb) against IMZ. The subtype of anti-IMZ mAb is IgG2a. It possessed a half inhibition concentration (IC50) of 0.95 ng mL-1 and showed no cross-reactivity against other chemicals in ic-ELISA. Taking advantage of the mAb, we developed a gold nanoparticle-based immunochromatographic assay (GICA) for the rapid detection of IMZ in grapes and tomatoes. The assay gave a visual limit of detection (vLOD) of 25 ng g-1 and cut-off value of 500 ng g-1 in both samples. According to the calibration curves, the calculated LOD were 4.12 ng g-1 and 4.70 ng g-1 in grapes and tomatoes, respectively. The recovery rates of IMZ ranged from 84.7% to 104.4% with variation coefficients (CVs) of 5.7-11.8% in spiked samples, indicating a potent practicability of the GICA. The whole GICA process took 30 min. Therefore, the developed assay can be used for on-site detection and quantitation of IMZ in grape and tomato samples.

Impaired spermatogenesis and associated endocrine effects of azole fungicides in peripubertal Xenopus tropicalis

Early life exposure to endocrine disrupting chemicals (EDCs) has been suggested to adversely affect reproductive health in humans and wildlife. Here, we characterize endocrine and adverse effects on the reproductive system after juvenile exposure to propiconazole (PROP) or imazalil (IMZ), two common azole fungicides with complex endocrine modes of action. Using the frog Xenopus tropicalis, two short-term (2-weeks) studies were conducted. I: Juveniles (2 weeks post metamorphosis (PM)) were exposed to 0, 17 or 178 µg PROP/L. II: Juveniles (6 weeks PM) were exposed to 0, 1, 12 or 154 µg IMZ/L. Histological analysis of the gonads revealed an increase in the number of dark spermatogonial stem cells (SSCs)/testis area, and in the ratio secondary spermatogonia: dark SSCs were increased in all IMZ groups compared to control. Key genes in gametogenesis, retinoic acid and sex steroid pathways were also analysed in the gonads. Testicular levels of 3β-hsd, ddx4 were increased and cyp19 and id4 levels were decreased in the IMZ groups. In PROP exposed males, increased testicular aldh1a2 levels were detected, but no histological effects observed. Although no effects on ovarian histology were detected, ovarian levels of esr1, rsbn1 were increased in PROP groups, and esr1 levels were decreased in IMZ groups. In conclusion, juvenile azole exposure disrupted testicular expression of key genes in retinoic acid (PROP) and sex steroid pathways and in gametogenesis (IMZ). Our results further show that exposure to environmental concentrations of IMZ disrupted spermatogenesis in the juvenile testis, which is a cause for concern as it may lead to impaired fertility. Testicular levels of id4, ddx4 and the id4:ddx4 ratio were associated with the number of dark SSCs and secondary spermatogonia suggesting that they may serve as a molecular markers for disrupted spermatogenesis.

Pre-concentration of pesticides in water using isophorone diamine multiwalled carbon nanotubes-based solid-phase extraction technique and analysis by gas chromatography–mass spectrometry

The existence of pesticides in water at ultra-trace levels necessitates the use of a suitable pre-concentration method for their detection. The objective of this study was to develop an ultra-synthetic adsorbent to extract chlorpyrifos (CPF) and imazalil (IMA) pesticides in water. X-ray diffraction (XRD), thermogravimetric analysis (TGA) and Fourier transform infrared (FTIR) spectroscopy confirm that both oxidised and isophorone diamine multiwalled carbon nanotubes (IPD-MWCNTs) were successfully prepared with an intact structure; which was further confirmed by transmission electron microscopy (TEM) and energy-dispersive spectroscopy (EDS). The Brunauer–Emmet–Teller (BET) showed a high surface area of both oxidised and IPD-MWCNTs, which is linked to the formation of additional active sites. TGA further showed that the nanocomposites were highly stable within the pesticides quantification operating temperature. CPF and IMA were recovered using a low dosage of IPD-MWCNTs adsorbent (0.030 g) and eluted by a combined solvent (ethanol and chloroform (50:50, v/v)). The adsorbent was reusable over seven repeated cycles, with an acceptable percentage relative standard deviation (%RSD) ranging from 3 to 8%. The IPD-MWCNTs adsorption sites are highly stable and cannot be easily fouled, as compared to that of oxidised MWCNTs. Lower limits of detection (LOD) and quantification (LOQ) for CPF (0.026 and 0.078 µg.L−1) and IMA (0.033 and 0.100 µg.L−1) were achieved. Better recoveries for both analytes at low and high concentrations (as well as in real water samples) were obtained by IPD-MWCNTs whereas a conventional adsorbent (i.e. polymeric reverse phase) can only achieve better recoveries at high concentrations.

Hydrophilic interaction liquid chromatography (HILIC) with DAD detection for the determination of relatively non polar fungicides in orange samples

A new, sensitive, rapid, economic and validated high performance liquid chromatography/ diode array detector (HPLC/DAD) method has been developed for the determination of five fungicides namely; Carbendazim (MBC), Thiabendazole (TBZ), Metalaxyl (MET), Imazalil (IMA) and Propiconazole (PCZ). The analytical method was carried on hydrophilic interaction liquid chromatography (HILIC) column using isocratic elution of acetonitrile and water. Prior extraction of orange samples was done using a mixture of diethyl ether and ethyl acetate as an extraction solvent in liquid liquid extraction. The total run time was 15 min which makes it suitable for routine analysis of MBC, TBZ and IMA in oranges. Procedural standard calibration was applied to compensate the matrix effect. In order to measure the lowest concentration of the investigated fungicides below maximum residue limits (MRL), HPLC parameters were optimized. Mean recoveries were obtained ranging from 99.32% to 101.08% with average %RSD < 5.27%. The selected fungicides' residual quantities were successfully measured using the suggested method in various markets and organic orange samples. The investigation of carbendazim's kinetics of degradation, as well as its half-life and predicted preharvest interval (PHI), in real orange samples obtained from orange trees after being treated with the investigated fungicides, is the first to separate relative nonpolar components using HILIC.

Application of catalytic hydrodechlorination for the fast removal of chlorinated azole pesticides in drinking water

Catalytic hydrodechlorination (HDC) is regarded as a promising purifying technology for drinking water treatment. So far, it has proved to be highly effective for the removal of different groups of chlorinated micropollutants including pharmaceuticals, neonicotinoid pesticides, personal care products or chloroacetic acids. The azole pesticides, recently included in the EU Watch Lists (Decisions 2020/1161 and 2022/1307), are a group of micropollutants of particular concern for drinking water given their high toxicity, persistence, and bioaccumulation potential. In this work, the feasibility of HDC for the removal of a representative group of chlorinated azole pesticides tebuconazole (TEB), tetraconazole (TET), prochloraz (PCZ), penconazole (PEN), metconazole (MET) and imazalil (IMZ)) is demonstrated, and their reactivity is compared with that observed for other halogenated micropollutant groups. Notably, all the pesticides investigated in this work (100 μg L−1) were completely dechlorinated within 30 min under ambient conditions using a 1 wt% Pd/Al2O3 catalyst concentration of 0.25 g L−1 and a H2 feeding of 50 mL N min−1. The experimental data were accurately described by a pseudo-first order kinetic equation and rate constant values in the range from 1.08 to 2.60 L gcat−1 min−1 were obtained. These values are quite close to those achieved for the most reactive neonicotinoid pesticides and significantly higher than the obtained for chloroacetic acids and most pharmaceuticals (e.g. diclofenac, sertraline or chlorpromazine). From the identification of the generated reaction intermediates and the final non-chlorinated products, sequential reaction pathways were proposed for each pollutant. Remarkably, despite the high toxicity exhibited by the azole pesticides tested, with LC50 values within the 0.4–7.0 mg L−1 range using A. salina, HDC effluents were non-toxic in all cases. Furthermore, the catalyst showed a remarkable stability upon three consecutive runs. Finally, the versatility of the process was demonstrated in the treatment of real aqueous matrices such as DWTP and tap water, where no significant differences were found either in terms of activity or stability.

Earthworms synergize with indigenous soil functional microorganisms to accelerate the preferential degradation of the highly toxic S-enantiomer of the fungicide imazalil in soil

The roles of soil and earthworm gut microorganisms in the degradation of the chiral fungicide imazalil (IMA) enantiomers were systemically studied in soil-earthworm systems. S-IMA degraded slower than R-IMA in soil without earthworms. After the addition of earthworms, S-IMA degraded faster than R-IMA. Methylibium was the potential degradative bacterium likely related to the preferential degradation of R-IMA in soil. However, the addition of earthworms significantly decreased the relative abundance of Methylibium, especially in R-IMA-treated soil. Meanwhile, a new potential degradative bacterium Aeromonas first appeared in soil-earthworm systems. Compared with enantiomer-treated soil, the relative abundance of indigenous soil bacterium Kaistobacter significantly boomed in enantiomer-treated soil with earthworms. Interestingly, Kaistobacter in the earthworm gut also obviously increased after exposure to enantiomers, particularly in S-IMA-treated soil, which was associated with the significant increase in Kaistobacter in soil. More importantly, the relative abundances of Aeromonas and Kaistobacter in S-IMA-treated soil were obviously higher than those in R-IMA-treated soil after the addition of earthworms. Moreover, these two potential degradative bacteria were also potential bacterial hosts of the biodegradation genes p450 and bph. Collectively, gut microorganisms are important helpers in soil pollution remediation by participating in the preferential degradation of S-IMA mediated by indigenous soil microorganisms.

Emerging Contaminants Decontamination of WWTP Effluents by BDD Anodic Oxidation: A Way towards Its Regeneration

Electrochemical oxidation using a boron-doped diamond anode (EO-BDD) was tested to remove emerging contaminants commonly present in wastewater treatment plant effluents (WWTPe). The main objective of the work was the regeneration of this water for its possible reuse in high-quality demanding uses. In the first part of the work, we investigated the potential of this technique for removing a group of neonicotinoid pesticides (thiamethoxam (TMX), imidacloprid (ICP), acetamiprid (ACP), and thiacloprid (TCP)) in a WWTP effluent. The influence of operating variables, such as current density, the conductivity of media, supporting electrolyte type (Na2SO4, NaCl or NaNO3), or the natural aqueous matrix on target variables were fully established. Selected target variables were: (1) the percentage of pollutant removal, (2) the kinetics (apparent pseudo-first-order kinetic rate constant), (3) total organic carbon (TOC) removal, and (4) the specific energy consumption (SEC). A response surface methodology (RSM) was applied to model the results for all cases. In the paper’s final part, this technology was tested with a more broad group of common emerging pollutants, including some azole pesticides (such as fluconazole (FLZ), imazalil (IMZ), tebuconazole (TBZ), or penconazole (PNZ)), antibiotics (amoxicillin (AMX), trimethoprim (TMP), and sulfamethoxazole (SMX)), and an antidepressant (desvenlafaxine (DVF)). The results confirm the power of this technology to remove this emerging contamination in WWTP effluents which supposes an interesting way towards its regeneration.

Treatment with Glyphosate Induces Tolerance of Citrus Pathogens to Glyphosate and Fungicides but Not to 1,8-Cineole.

During the postharvest period, citrus fruits are exposed to Penicillium italicum, Penicillium digitatum, and Geotrichum candidum. Pesticides such as imazalil (IMZ), thiabendazole (TBZ), orthophenylphenol (OPP), and guazatine (GUA) are commonly used as antifungals. Glyphosate (GP) is also used in citrus fields to eliminate weed growth. The sensitivity of fungal pathogens of citrus fruit to these pesticides and 1,8-cineole was evaluated, and the effect of GP on the development of cross-resistance to other chemicals was monitored over a period of 3 weeks. IMZ most effectively inhibited the mycelial growth and spore germination of P. digitatum and P. italicum, with minimum inhibitory concentrations (MICs) of 0.01 and 0.05 mg/mL, respectively, followed by 1,8-cineole, GP, and TBZ. 1,8-Cineole and GP more effectively inhibited the mycelial growth and spore germination of G. candidum, with minimum inhibitory concentrations (MICs) of 0.2 and 1.0 mg/mL, respectively, than OPP or GUA. For the spore germination assay, all substances tested showed a total inhibitory effect. Subculturing the fungal strains in culture media containing increasing concentrations of GP induced fungal tolerance to GP as well as to the fungicides. In soil, experiments confirmed that GP induced the tolerance of P. digitatum to TBZ and GP and the tolerance of P. italicum to IMZ, TBZ, and GP. However, no tolerance was recorded against 1,8-cineole. In conclusion, it can be said that 1,8-cineole may be recommended as an alternative to conventional fungicides. In addition, these results indicate that caution should be taken when using GP in citrus fields.

Cross-generational effects of maternal exposure to imazalil on anaerobic components and carnitine absorption associated with OCTN2 expression in mice

Imazalil (IMZ) is a fungicide recommended by the Chinese ministry of agriculture. However, recent study was observed high level of IMZ by dietary exposure in pregnant women. To determine the cross-generational effects, C57BL/6 mice were exposed to IMZ at dietary levels of 0, 0.025‰, and 0.25‰ during the gestation and lactation periods. Then, we assessed the changes in growth phenotypes, carnitine levels, and gut microbiota in F0, F1 or F2 generations. The growth phenotypes of dams didn't observe significant difference, but there were significant changes in the offspring. Plasma samples revealed low levels of free carnitine (C0), long-chain acyl-carnitines and total carnitine. In particular, C0 may be regarded as relatively potential, specific markers by maternal IMZ exposure. Caco2 cell culture and animal experiment confirmed IMZ affected carnitine absorption through the organic cation transporter type-2 (OCTN2) protein encoded by solute carrier family 22A member 5 (SLC22A5) gene in colon. Maternal IMZ exposure also had a greater effect on gut microbiota in offspring, especially anaerobic bacteria, which positively correlated with C0 and acyl-carnitines. These results suggested that maternal IMZ exposure affected carnitine absorption through OCTN2 protein, which led to the decline of anaerobic bacteria and unbalanced intestinal homeostasis.

Using low dose fungicide by combining with intermittent ozone treatment to reduce fungicide residue, microbial load and quality losses in orange fruit during long term storage

In the present study, the effects of intermittent ozone (IO3) application combined with low dose fungicide and high-dose dissolved ozone in water on storage life and quality of orange cv. Washington Navel were investigated. Harvested orange fruit were subjected to four different treatments: immersion in ozonated (4 mg L−1) water (OW) and only distilled water (Control) for 10 min, treatment with fungicide solutions containing 500 mg L−1 (half dose) and 1000 mg L−1 (common dose) imazalil (IMZ) for 60 s, respectively. Ozone and 500 mg L−1 IMZ treated fruit were kept in cold room under IO3 gas exposure (2.14 mg m−3) for 30 min at one-week intervals during cold storage. Other two groups (Control and 1000 mg L−1 IMZ) were stored at the same conditions without ozone exposure. Treated fruit were stored at 5 °C for 150 d. As a result, while the OW + IO3 treatment was more effective in reducing microbial load, 500 mg L−1 IMZ + IO3 was the best treatment for inhibiting decay rate of orange fruit during cold storage. Although the 1000 mg L−1 IMZ treatment provided a significant reduction in the initial microbial load, it could not maintain its effectiveness at the end of cold storage when compared to OW + IO3 treatment. The fungicide residue in 500 mg L−1 IMZ + IO3 treated fruit was significantly lower than that of common dose (1000 mg L−1 IMZ), as expected. The combined use of low dose IMZ with IO3 gas exposure also gave positive or at least similar results for quality losses compared to common dose during storage. Ozone and fungicide treatments significantly decreased the weight loss and respiration rate, and maintained the visual quality of orange compared to control. Collectively, these results suggest that 500 mg L−1 IMZ + IO3 and OW + IO3 treatments are potential techniques for maintaining the fruit quality of Washington Navel oranges during long term cold storage.

Postharvest application of imazalil sulphate using a heated flooder for control of citrus green mould

High volume aqueous applications of imazalil (IMZ) are commonly used in major citrus producing industries for control of green mould, caused by Penicillium digitatum. The flooder application is a more novel means of application where the heated fungicide solution is applied in high volume and low pressure through weirs creating a waterfall over a set of rotating brushes. A five-weir flooder is compared to the conventional dip tank application in terms of residue loading, curative and protective control as well as sporulation inhibition. Imazalil sulphate was applied with a flooder (250 mg L-1 at pH ≈ 5 for 8 s) or a dip tank (500 mg L-1 at pH ≈ 3 for 60 s) on various citrus types at solution temperatures of 25, 35 or 52 °C. All treatments deposited IMZ residues in the fruit of ≈ 1.00 mg kg-1, except residues of ≈ 2.00 mg kg-1 after treatment at 52 °C. Flooder application gave similar curative control and better protective control compared to the dip application. Sporulation control by IMZ was significantly improved when applied with the flooder at 52 °C, compared to the dip treatment. Imazalil sulphate application at 250 and 500 mg L-1 was optimised at temperatures from 25° to 60°C. Regression models describing residue loading in a pH 5, 250 mg.L-1 solution applied by flooder at 26.9 or 47.4 °C would deposit residues of 1 and 2 mg.kg-1, respectively. Regression models describing residue loading in a pH 3, 500 mg.L-1 solution applied by flooder at 38.0 or 63.20 °C would deposit residues of 1 and 2 mg.kg-1, respectively. Curative and protective control was generally good (> 80%), but consistently better after treatment with 500 mg L-1. The influence of number of flooder weirs (1−5) and IMZ solution temperature range of 25–55 °C was determined. A single weir loaded an imazalil residue between 1.00 and 1.50 mg kg-1 regardless of solution temperature. Residue loading was markedly increased at 45 °C with three weirs and 2 mg kg-1 was only exceeded at 55 °C with 3–5 weirs. Curative green mould control (≈90%) and sporulation inhibition (>50%) were more consistent and better when imazalil was applied at 45 °C or higher using three or more weirs. The heated flooder application is an effective alternative to the fungicide dip tank.

Imazalil and its metabolite imazalil-M caused developmental toxicity in zebrafish (Danio rerio) embryos via cell apoptosis mediated by metabolic disorders

Imazalil (IMZ) is a highly effective fungicide employed in crop production. It has been consistently detected in aquatic environments. The main environmental metabolite of IMZ is imazalil-M (IMZ-M). Limited studies have focused on the toxicity of IMZ and IMZ-M in aquatic organisms. This study systematically evaluated the developmental toxicity of IMZ and IMZ-M on zebrafish (Danio rerio) embryos and explored the potential mechanisms involved. The results showed that IMZ and IMZ-M caused developmental toxicity, characterized by decreased heart rate, hatching inhibition, and pericardial cyst in zebrafish embryos. Subsequently, acridine orange (AO) staining revealed cell apoptosis in the area around the heart regions of zebrafish larvae. Besides, the expression levels of apoptosis-related genes also varied significantly. Furthermore, 1H NMR-based metabolomics analysis showed that IMZ and IMZ-M exposure could induce metabolic profiles disorder in zebrafish larvae. Importantly, zebrafish exposure to IMZ and IMZ-M significantly affected the metabolism of branched - chain amino acids, energy, and ketone bodies, which are related to cell apoptosis. Overall, the toxicity of IMZ and IMZ-M in zebrafish embryos and larvae was characterized, suggesting a theoretical basis for the potential environmental risks of IMZ and its metabolite IMZ-M on non-target organisms.

Transcriptome sequencing reveals an inhibitory mechanism of Penicillium digitatum by sodium dehydroacetate on citrus fruit

Sodium dehydroacetate (SD) has been widely used in various fields due to its high antimicrobial activities. However, the inhibitory mechanisms of SD against fungi, especially on transcriptional level, are still unclear. In this study, the inhibitory property of SD against Penicillium digitatum (imazalil (IMZ)-sensitive P. digitatum (Pds01) and IMZ-resistant P. digitatum (Pdw03) strains) was investigated. Here, we found that SD treatment had a distinct inhibitory effect on Pdw03 strain in vitro and mitigated the disease prevalence of inoculated citrus. Morphology and microstructure observation showed that SD caused remarkable changes in the hyphae of Pds01 and Pdw03. Transcriptomic analysis displayed that SD treatment blocked the expression of genes related to the ribosome, genetic information processing and energy metabolism, while promoting the genes expression related to cell walls degradation, membrane lipid metabolism and biosynthesis of sphingolipid. Among them, the ribosomal pathway was most prominent, and key genes in this pathway (RPSA, RPS9, RPL7A and RPL5) were inhibited by SD treatment in both strains. Moreover, we found that the expression of 12 MFS (major facilitator superfamily) transporters, 6 ABC (ATP-binding cassette transporter) transporters, and 9 glutathione metabolism encoding genes related to SD detoxification were increased. The results of fourier transform infrared spectroscopy (FT-IR) fingerprints further confirmed that SD caused remarkable changes in cell membrane lipids, nucleic acids, proteins and cell wall polysaccharides in both strains. These findings indicated that the interference with ribosome, genetic information processing, cell membrane metabolism and energy metabolism might be closely involved in the antifungal mechanism of SD against P. digitatum. Our results may provide novel insights into the antifungal molecular mechanism of SD against P. digitatum and deepen our understanding of the regulatory network of SD.

Isolation, characterization and industrial application of a Cladosporium herbarum fungal strain able to degrade the fungicide imazalil

Imazalil (IMZ) is an imidazole fungicide commonly used by fruit-packaging plants (FPPs) to control fungal infections during storage. Its application leads to the production of pesticide-contaminated wastewaters, which, according to the European Commission, need to be treated on site. Considering the lack of efficient treatment methods, biodepuration systems inoculated with tailored-made inocula specialized on the removal of such persistent fungicides appear as an appropriate solution. However, nothing is known about the biodegradation of IMZ. We aimed to isolate and characterize microorganisms able to degrade the recalcitrant fungicide IMZ and eventually to test their removal efficiency under near practical bioengineering conditions. Enrichment cultures from a soil receiving regular discharges of effluents from a FPP, led to the isolation of a Cladosporium herbarum strain, which showed no pathogenicity on fruits, a trait essential for its biotechnological exploitation in FPPs. The fungus was able to degrade up to 100 mg L−1 of IMZ. However, its degrading capacity and growth was reduced at increasing IMZ concentrations in a dose-dependent manner, suggesting the involvement of a detoxification rather than an energy-gain mechanism in the dissipation of IMZ. The isolate could tolerate and gradually degrade the fungicides fludioxonil (FLD) and thiabendazole (TBZ), also used in FPPs and expected to coincide alongside IMZ in FPP effluents. The capacity of the isolate to remove IMZ in a practical context was evaluated in a benchtop immobilized-cell bioreactor fed with artificial IMZ-contaminated wastewater (200 mg L−1). The fungal strain established in the reactor, completely dominated the fungal community and effectively removed >96% of IMZ. The bioreactor also supported a diverse bacterial community composed of Sphingomonadales, Burkholderiales and Pseudomonadales. Our study reports the isolation of the first IMZ-degrading microorganism with high efficiency to remove IMZ from agro-industrial effluents under bioengineering conditions.

Combined effects of maternal exposure to fungicides on behavioral development in F1‐generation mice: 3. Fixed‐dose study of imazalil

Few published studies are reported for the neurobehavioral toxicity of combined exposure to fungicides in mammals. This study was aimed to re-evaluate the reproductive and neurobehavioral effects of maternal exposure to combined imazalil (IMZ) and thiabendazole (TBZ) with fixed-dose of IMZ in mice. IMZ/TBZ was given in the diet to provide levels of 0%/0% (control), 0.006%/0.006% (IMZ/TBZ), 0.006%/0.018%, and 0.006%/0.054% during the gestation and lactation periods. Selected reproductive and neurobehavioral parameters were measured in the F1 generation. No adverse effect of IMZ/TBZ was observed in litter size, litter weight, or sex ratio at birth. Concerning behavioral developmental parameters, the time required of olfactory orientation was accelerated significantly in higher-dose groups in female offspring on postnatal day 14 in a dose-related manner. Exploratory behavior examination indicated that the frequency of mice with urination increased in a significantly dose-related manner in male offspring. After weaning, any variables of exploratory behavior indicated no significant effects in both sexes of adult mice in the F1 generation. In spontaneous behavior, the longitudinal pattern of total distance was not parallel (different pattern) among the control and IMZ/TBZ treatment groups in adult males. The results from these series of combined exposure studies of IMZ/TBZ suggest that the main effects on exploratory and spontaneous behavior of adult mice in the F1 generation after finishing chemical administration have been caused by the IMZ concentration of maternal exposure, with TBZ's effects being complementary.

Natural Biostimulants Elicit Plant Immune System in an Integrated Management Strategy of the Postharvest Green Mold of Orange Fruits Incited by Penicillium digitatum.

This study was aimed at testing the integrated use of a natural biostimulant based on seaweed (Ascophyllum nodosum) and plant (alfalfa and sugarcane) extracts and reduced dosages of the conventional synthetic fungicide Imazalil (IMZ) to manage postharvest rots of orange fruits. The following aspects were investigated: (i) the effectiveness of postharvest treatment with natural biostimulant alone or in mixture with IMZ at a reduced dose against green mold caused by Penicillium digitatum; (ii) the differential expression of defense genes in orange fruits treated with the natural biostimulant both alone and in combination with a reduced dose of IMZ; (iii) the persistence of the inhibitory activity of both biostimulant and the mixture biostimulant/IMZ against green mold; and (iv) the residue level of fungicide in citrus peel when applied alone or in combination with the biostimulant. Treatments with the chemical plant resistance-inducer potassium phosphite, alone or with a reduced dose of IMZ, were included for comparison. The mixture of natural biostimulant and IMZ at a low dose consistently reduced the incidence and severity of fruit green mold and induced a significant increase of the expression level of β-1,3-glucanase-, peroxidase (PEROX)-, and phenylalanine ammonia-lyase (PAL)-encoding genes in fruit peel, suggesting that the natural biostimulant elicits a long-lasting resistance of citrus fruits to infections by P. digitatum. Interestingly, the residual concentration of IMZ in fruits treated with the biostimulant/fungicide mixture was significantly lower than that of IMZ in fruits treated only with the fungicide at the same dose and by far below the threshold values set by the European Union. This study laid the foundations for (i) conceiving a practical and more eco-friendly alternative to the conventional postharvest management of green mold of citrus fruits, based almost exclusively on the use of synthetic fungicide IMZ, alone or mixed with potassium phosphite and (ii) providing a better insight into the mechanisms of disease resistance induction by biostimulants.