Effect of short-term exposure to the strobilurin fungicide dimoxystrobin: Morphofunctional, behavioural and mitochondrial alterations in Danio rerio embryos and larvae.

Strobilurins, among the most used fungicides worldwide, are considered non-toxic to mammals and birds, but there is growing evidence that these compounds are highly toxic to aquatic species. Dimoxystrobin has been included in the 3rd Watch List of the European Commission, and it has been classified as very toxic to aquatic life. However, previous studies focused on acute toxicity and only two reports are available on its impact on fish, and none on its effects during the early life stages. Here, we evaluated for the first time the effects induced on zebrafish embryos and larvae by two dimoxystrobin sublethal concentrations (6.56 and 13.13 µg/L) falling in the range of predicted environmental concentrations. We demonstrated that short-term exposure to dimoxystrobin may exert adverse effects on multiple targets, inducing severe morphological alterations. Moreover, we showed enhanced mRNA levels of genes related to the mitochondrial respiratory chain and ATP production. Impairment of the swim bladder inflation has also been recorded, which may be related to the observed swimming performance alterations.

Toxicological impact of strobilurin fungicides on human and environmental health: a literature review.

Fungicides are specifically used for controlling fungal infections. Strobilurins, a class of fungicides originating from the mushroom Strobilurus tenacellus, act on the fungal mitochondrial respiratory chain, interrupting the ATP cycle and causing oxidative stress. Although strobilurins are little soluble in water, they have been detected in water samples (such as rainwater and drinking water), indoor dust, and sediments, and they can bioaccumulate in aquatic organisms. Strobilurins are usually absorbed orally and are mainly eliminated via the bile/fecal route and urine, but information about their metabolites is lacking. Strobilurins have low mammalian toxicity; however, they exert severe toxic effects on aquatic organisms. Mitochondrial dysfunction and oxidative stress are the main mechanisms related to the genotoxic damage elicited by toxic compounds, such as strobilurins. These mechanisms alter genes and cause other dysfunctions, including hormonal, cardiac, neurological, and immunological impairment. Despite limitations, we have been able to compile literature information about strobilurins. Many studies have dealt with their toxic effects, but further investigations are needed to clarify their cellular and underlying mechanisms, which will help to find ways to minimize the harmful effects of these compounds.

Low trifloxystrobin-tebuconazole concentrations induce cardiac and developmental toxicity in zebrafish by regulating notch mediated-oxidative stress generation.

Trifloxystrobin-tebuconazole (TFS-TBZ) is a novel, broad-spectrum fungicide that has been frequently detected in both the environment and agricultural products. However, its adverse effects on aquatic organisms remain unknown. In this study, the adverse effects of ecologically relevant TFS-TBZ concentrations (i.e., 75.0, 112.5, and 150.0 µg/L) on the heart and development of zebrafish were investigated. TFS-TBZ was found to substantially hinder development, inhibit growth, and cause significant abnormity at higher concentrations. Moreover, TFS-TBZ caused severe pericardial edema, heart loop failure, cardiac linearization, and ultra-slow heartbeat, implying that TFS-TBZ might induce congenital heart disease. TFS-TBZ inhibited Notch signaling and increased the intracellular generation of reactive oxygen species, resulting in decreased myocardial cell proliferation and increased apoptosis. The use of sodium valproate and Gadofullerene illustrated the relevance of the Notch signaling system and oxidative stress. Finally, TFS-TBZ exposure conveys severe developmental toxicity to the zebrafish heart. The underlying mechanism is regulation notch mediated-oxidative stress generation, implying that TFS-TBZ may be potentially hazardous to aquatic organisms in the environment.

Toxicity of fungicide azoxystrobin to Enchytraeus albidus: Differences between the active ingredient and formulated product.

Due to the often-excessive usage of fungicides, increasing attention is being paid to their impact on soil and non-target organisms. Risk assessments are usually based on the pure active ingredient and not on the formulated products applied in the environment. The aim of this study was therefore to investigate how azoxystrobin, the best-selling strobilurin fungicide, affects non-target soil organisms Enchytraeus albidus. To investigate the effects of the different types of azoxystrobin, E. albidus was exposed to the pure active ingredient, AZO_AI, and the formulated product, AZO_FP. Survival, reproduction, and molecular biomarkers of E. albidus were determined for different exposure durations (seven and 21 days). AZO_FP (LC50 = 15.3 mga.i./kg) showed a slightly stronger effect on survival than AZO_AI (LC50 = 16.8 mga.i./kg), yet the impact on reproduction was much stronger. Namely, while the tested concentrations of AZO_AI (EC50≥ 8 mga.i./kg) had almost no effect on reproduction, AZO_FP (EC50 = 2.9 mga.i./kg) significantly inhibited reproduction in a dose-dependent manner. Changes in enzyme activities (superoxide dismutase, catalase, glutathione-s-transferase) and malondialdehyde levels in both treatments indicated oxidative stress. Although AZO_FP had a stronger negative effect, the impact depended on the exposure time and the tested concentration. The higher toxicity of AZO_FP was a consequence of increased bioavailability and activity of the active ingredient due to the presence of adjuvants. Overall stronger adverse effects of AZO_FP suggest that the toxicity of azoxystrobin in the agricultural environment on the enchytraeid population may be underestimated. Furthermore, the results of this study highlighted the importance of comparing the toxicity of the active ingredient and the formulated product.

Ecotoxicity evaluation of azoxystrobin on Eisenia fetida in different soils.

Azoxystrobin, a widely used broad-spectrum strobilurin fungicide, may pose a potential threat in agricultural ecosystems. To assess the ecological risk of azoxystrobin in real soil environments, we performed a study on the toxic effects of azoxystrobin on earthworms (Eisenia fetida) in three different natural soils (fluvo-aquic soil, black soil and red clay soil) and an artificial soil. Acute toxicity of azoxystrobin was determined by filter paper test and soil test. Accordingly, exposure concentrations of chronic toxicity were set at 0, 0.1, 1.0 and 2.5 mg kg-1. For chronic toxicity test, reactive oxygen species, activity of antioxidant enzymes (superoxide dismutase, catalase and peroxidase), detoxifying enzyme (glutathione transferase), level of lipid peroxidation (malondialdehyde) and level of oxygen damage of DNA (8-hydroxydeoxyguanosine) in earthworms were determined on the 7th, 14th, 21st, 28th, 42nd and 56th days after treatment. Both acute and chronic toxic results showed azoxystrobin exhibit higher toxicity in natural soil than in artificial soil, indicating that traditional artificial soil testing method underestimate ecotoxicity of azoxystrobin in a real agricultural environment on the earthworm population. Combining with the analysis of soil physicochemical properties, the present experiment provided scientific guidance for rational application of azoxystrobin in agricultural production systems.

Chronic effects of the strobilurin fungicide azoxystrobin in the leaf shredder Gammarus fossarum (Crustacea; Amphipoda) via two effect pathways.

Fungicides pose a risk for crustacean leaf shredders serving as key-stone species for leaf litter breakdown in detritus-based stream ecosystems. However, little is known about the impact of strobilurin fungicides on shredders, even though they are presumed to be the most hazardous fungicide class for aquafauna. Therefore, we assessed the impact of the strobilurin azoxystrobin (AZO) on the survival, energy processing (leaf consumption and feces production), somatic growth (growth rate and molting activity), and energy reserves (neutral lipid fatty and amino acids) of the amphipod crustacean Gammarus fossarum via waterborne exposure and food quality-mediated (through the impact of leaf colonizing aquatic microorganisms) and thus indirect effects using 2 × 2-factorial experiments over 24 days. In a first bioassay with 30 µg AZO/L, waterborne exposure substantially reduced survival, energy processing and affected molting activity of gammarids, while no effects were observed via the dietary pathway. Furthermore, a negative growth rate (indicating a body mass loss in gammarids) was induced by waterborne exposure, which cannot be explained by a loss in neutral lipid fatty and amino acids. These energy reserves were increased indicating a disruption of the energy metabolism in G. fossarum caused by AZO. Contrary to the first bioassay, no waterborne AZO effects were observed during a second experiment with 15 µg AZO/L. However, an altered energy processing was determined in gammarids fed with leaves microbially colonized in the presence of AZO, which was probably caused by fungicide-induced effects on the microbial decomposition efficiency ultimately resulting in a lower food quality. The results of the present study show that diet-related strobilurin effects can occur at concentrations below those inducing waterborne toxicity. However, the latter seems to be more relevant at higher fungicide concentrations.

Fate, residues and dietary risk assessment of the fungicides epoxiconazole and pyraclostrobin in wheat in twelve different regions, China.

The fungicides epoxiconazole and pyraclostrobin have been widely used to control wheat fusarium head blight. This study was designed to investigate the dissipation behaviors in different climate regions and provide data for the modification of maximum residue limits of the two fungicides. Wheat samples were collected from field sites in twelve different regions, China and analyzed with an HPLC-MS/MS method for simultaneous detection of epoxiconazole and pyraclostrobin in wheat. The average recoveries of epoxiconazole and pyraclostrobin in wheat matrix were 87-112% and 85-102%, respectively, with the relative standard deviations ≤8.1%. The limits of quantification of epoxiconazole and pyraclostrobin in grain and straw were both 0.01 mg/kg. The dissipations of epoxiconazole and pyraclostrobin followed first-order kinetics, with the half-lives of 10.3 days and 7.6 days, respectively. The terminal residues of epoxiconazole and pyraclostrobin in grain were below 0.034 and 0.028 mg/kg, separately, both lower than the maximum residue limits recommended by China. Based on Chinese dietary pattern and terminal residue distributions, the risk quotients of epoxiconazole and pyraclostrobin were 13.9% and 65.9%, respectively, revealing the evaluated wheat exhibited an acceptably low dietary risk to consumers.

Field cross-fostering and in vitro rearing demonstrate negative effects of both larval and adult exposure to a widely used fungicide in honey bees (Apis mellifera).

Pollinators and other insects are experiencing an ongoing worldwide decline. While various environmental stressors have been implicated, including pesticide exposure, the causes of these declines are complex and highly debated. Fungicides may constitute a particularly prevalent threat to pollinator health due to their application on many crops during bloom, and because pollinators such as bees may consume fungicide-tainted pollen or nectar. In a previous study, consumption of pollen containing the fungicide Pristine® at field-relevant concentrations by honey bee colonies increased pollen foraging, caused earlier foraging, lowered worker survival, and reduced colony population size. Because most pollen is consumed by young adults, we hypothesized that Pristine® (25.2% boscalid, 12.8% pyraclostrobin) in pollen exerts its negative effects on honey bee colonies primarily on the adult stage. To rigorously test this hypothesis, we used a cross-fostering experimental design, with bees reared in colonies provided Pristine® incorporated into pollen patties at a supra-field concentration (230 mg/kg), only in the larvae, only in the adult, or both stages. In contrast to our predictions, exposure to Pristine® in either the larval or adult stage reduced survival relative to control bees not exposed to Pristine®, and exposure to the fungicide at both larval and adult stages further reduced survival. Adult exposure caused precocious foraging, while larval exposure increased the tendency to forage for pollen. These results demonstrate that pollen containing Pristine® can induce significant negative effects on both larvae and adults in a hive, though the magnitude of such effects may be smaller at field-realistic doses. To further test the potential negative effects of direct consumption of Pristine® on larvae, we reared them in vitro on food containing Pristine® at a range of concentrations. Consumption of Pristine® reduced survival rates of larvae at all concentrations tested. Larval and adult weights were only reduced at a supra-field concentration. We conclude that consumption of pollen containing Pristine® by field honey bee colonies likely exerts impacts on colony population size and foraging behavior by affecting both larvae and adults.

Embryonic zebrafish response to a commercial formulation of azoxystrobin at environmental concentrations.

Azoxystrobin is a broad-spectrum strobilurin fungicide for use on a wide range of crops available to end-users as formulated products. Due to its extensive application, it has been detected in aquatic ecosystems, raising concerns about its environmental impact, which is still poorly explored. The objective of this work was to study the effects of a commercial formulation of azoxystrobin in the zebrafish embryo model. Sublethal and lethal effects were monitored during the exposure period from 2 h post fertilisation (hpf) to 96 hpf after exposure to azoxystrobin concentrations (1, 10 and 100 µg L-1). The responses of antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR)) as well as detoxifying enzymes (glutathione-s-transferase (GST) and carboxylesterase (CarE)) were evaluated at 96 hpf. Similarly, glutathione levels (reduced (GSH) and oxidised (GSSG) glutathione), neurotransmission (acetylcholinesterase (AChE)) and anaerobic respiration (lactate dehydrogenase (LDH)) -related enzymes were assayed. At 120 hpf, larvae from each group were used for behaviour analysis. Results from this study showed concentration-dependent teratogenic effects, particularly by increasing the number of malformations (yolk and eye), with a higher prevalence at the highest concentration. However, it was found that the lowest concentration induced a high generation of reactive oxygen species (ROS) and increased activity of SOD, GST, and CarE. In addition, GR and GSSG levels were decreased by the lowest concentration, suggesting an adaptive response to oxidative stress, which is also supported by the increased AChE activity and absence of behavioural changes. These findings advance the knowledge of the azoxystrobin developmental and environmental impacts, which may impose ecotoxicological risks to non-target species.

Evaluation of hematological parameters, oxidative stress and DNA damage in the cichlid Australoheros facetus exposed to the fungicide azoxystrobin.

Azoxystrobin (AZX) is a broad-spectrum systemic fungicide massively used worldwide. Its mode of action consists in the inhibition of mitochondrial respiration decreasing the synthesis of ATP and leading to oxidative stress in the target fungus. However, whether this effect occurs in non target organisms has been scarcely studied. The objectives of this work were (1) to evaluate biomarkers of oxidative stress, hematological, physiological and of genotoxicity in the native cichlid fish Australoheros facetus exposed to environmentally relevant concentrations of AZX and (2) to compare these biomarkers in different developmental stages using juvenile and adult fish (n = 6) exposed during 48 h. The exposure concentrations were 0 (negative control, C (-)), 0.05, 0.5, 5 and 50 µg/L AZX of the commercial formulation AMISTAR®. Blood was drawn to evaluate hematology, and DNA damage through the comet assay (CA) and micronucleus test (MN). Genotoxicity was observed by mean of both biomarkers in juvenile and adult fish at 50 µg/L AZX. Samples of liver and gills were used to determine antioxidant enzymes activity, hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents. In juvenile fish inhibition of superoxide dismutase (SOD) was observed in liver at 0.05, 5 and 50 µg/L AZX and in gills at 5 and 50 µg/L AZX. Glutathione- S- transferases (GST) activity increased in gills at all AZX concentrations tested. In adult fish, increase of hepatic catalase (CAT) activity at 0.5 and 50 µg/L AZX and MDA content at 50 µg/L AZX were observed. In gills only H2O2 content showed changes at 50 µg/L AZX. The sensitivity showed by gills constitutes the first report about AZX toxicity in this organ. All these negative effects were observed in the range of realistic AZX concentrations, which warns of the possible consequences that it may have on the health of aquatic biota. Differences between juvenile and adult fish demonstrate the relevance of considering the developmental stage on the evaluation of biomarkers.

Evaluation of the acute toxic effect of azoxystrobin on non-target crayfish (Astacus leptodactylus Eschscholtz, 1823) by using oxidative stress enzymes, ATPases and cholinesterase as biomarkers.

Azoxystrobin is a broad-spectrum fungicide used worldwide. Since azoxystrobin spreads to large areas, its toxic effects on non-target organisms have aroused interest. In this study, the acute toxicity (96 h) of azoxystrobin on the crayfish (Astacus leptodactylus) was examined by using various biomarkers. The 96 h-LC50 dose (1656 mg L-) and its three sub-doses (828, 414, 207 mg L-1) were applied to crayfish. Superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities were increased significantly compared to the control in hepatopancreas, gill and muscle tissues. The activities of acetylcholinesterase (AChE) and glutathione S-transferase (GST) increased, and glutathione reductase (GR) activity decreased significantly in hepatopancreas. Level of reduced glutathione (GSH) decreased significantly. The content of malondialdehyde (MDA) increased in a dose-dependent manner in all azoxystrobin treatments with the exception of the lowest dose (207 mg L-1)treatment. ATPases (Na+/K+ -ATPase, Mg2+ -ATPase, Ca2+ -ATPase, total ATPase) were significantly inhibited in gill and muscle tissues. The results of the present study indicate that azoxystrobin induces oxidative stress, and has adverse effects on activities of AChE and ATPases in crayfish.

N-acetylcysteine reduced the immunotoxicity effects induced in vitro by azoxystrobin and iprodione fungicides in mice.

Azoxystrobin (AZO) and Iprodione (IPR) fungicides are extensively used worldwide, and therefore, contaminate all environmental compartments. The toxicity and the mechanisms by which they affected immune cells are complex and remain unknown. This study investigated the impact of AZO and IPR on the in vitro function of mice peritoneal macrophages including lysosomal enzyme activity and tumor necrosis factor (TNF)α and nitric oxide (NO) production in response to lipopolysaccharide (LPS) stimulation, the proliferation of mice splenocytes stimulated by concanavalin (Con)A and LPS, and the production of the Th1cytokine interferon-gamma (IFNγ) and the Th2 cytokine interleukin (IL)-4 and IL-10 by ConA-activated splenocytes. This is the first report indicating that AZO and IPR fungicides dose-dependently inhibited mice macrophage lysosomal enzyme activity and LPS-stimulated production of TNFα and NO. Mitogen-induced proliferation of mice splenocytes was also suppressed by AZO and IPR in a dose-dependent manner. More pronounced impact was observed on ConA-induced response. The production of IFNγ by ConA-stimulated splenocytes was dose-dependently inhibited; however, the production of IL-4 and IL-10 increased in the same conditions. These results suggested that AZO and IPR polarized Th1/Th2 cytokine balance towards Th2 response. Overall, marked immunosuppressive effects were observed for AZO. The immunomodulatory effects caused by AZO and IPR were partially reversed by the pharmacological antioxidant N-acetylcysteine (NAC), suggesting that both fungicides exerted their actions through, at least in part, oxidative stress-dependent mechanism. Collectively, our data showed that AZO and IPR fungicides exerted potent immunomodulatory effects in vitro with eventually strong consequences on immune response and immunologically based diseases.

Azoxystrobin Impairs Neuronal Migration and Induces ROS Dependent Apoptosis in Cortical Neurons.

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.

Determination of pyraclostrobin dynamic residual distribution in tilapia tissues by UPLC-MS/MS under acute toxicity conditions.

As a lipophilic fungicide, pyraclostrobin is highly toxic to aquatic organisms, especially to fish. In recent years, research has mainly focused on the pyraclostrobin residue in fish tissues under chronic toxicity, but less is known about its distribution in fish tissues under acute toxicity conditions. In this study, the distribution of pyraclostrobin in fish tissues (blood, liver, muscle and gill) was determined by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The purification effects of different purification materials [1) mixtures of PSA, C18 and MgSO4; 2) QuEChERS-PC; and 3) Oasis HLB SPE] were compared for the detection of pyraclostrobin in fish tissues. Finally, the quick and easy clean-up tool of the Oasis HLB SPE procedure was selected. Under optimum conditions, the linearities had a good relationship (determination coefficient R2 > 0.999). The mean recoveries of the analyte for all tested concentrations ranged from 86.94% to 108.81% with RSDs of 0.7%-4.9%. The pyraclostrobin residue amount was much different in fish tissues. Furthermore, the pyraclostrobin residue in different fish tissues increased initially and then decreased gradually. The concentrations in each tissue were initially ranked before 120 min in the following order: gill > liver > blood > muscle. These phenomena may be attributed to the stress response of fish under acute poisoning. This is the first study to document the distribution of pyraclostrobin in fish tissues under acute toxicity conditions, and it provides reference for the management of agrochemicals in terms of aquatic ecological risks.

Fungicide pyraclostrobin affects midgut morphophysiology and reduces survival of Brazilian native stingless bee Melipona scutellaris.

Native stingless bees are key pollinators of native flora and important for many crops. However, the loss of natural fragments and exposure to pesticides can hinder the development of colonies and represent a high risk for them. Nevertheless, most studies are conducted with honeybees and there are not many studies on native species, especially in relation to the effects of fungicides on them. Therefore, the objective of this paper is to evaluate the effects of sublethal concentrations of pyraclostrobin, on Melipona scutellaris forager workers. These Brazilian native stingless bees were submitted to continuous oral exposure to three concentrations of pyraclostrobin in sirup: 0.125 ng a.i./µL (P1), 0.025 ng a.i./µL (P2), and 0.005 ng a.i./µL (P3). Histopathological and histochemical parameters of midgut, as well as survival rate were evaluated. All concentrations of fungicide showed an increase in the midgut lesion index and morphological signs of cell death, such as cytoplasmic vacuolizations, presence of atypical nuclei or pyknotic nuclei. Histochemical analyzes revealed a decreased marking of polysaccharides and neutral glycoconjugates both in the villi and in peritrophic membrane in all exposed-groups in relation to control-groups. P1 and P2 groups presented a reduction in total protein marking in digestive cells in relation to control groups. As a consequence of alteration in the midgut, all groups exposed to fungicide showed a reduced survival rate. These findings demonstrate that sublethal concentrations of pyraclostrobin can lead to significant adverse effects in stingless bees. These effects on social native bees indicate the need for reassessment of the safety of fungicides to bees.

Famoxadone-cymoxanil induced cardiotoxicity in zebrafish embryos.

Famoxadone-cymoxanil is a new protective and therapeutic fungicide, but little research has been done on it or its toxicity in aquatic organisms. In this study, we used zebrafish to investigate the cardiotoxicity of famoxadone-cymoxanil and the potential mechanisms involved. Zebrafish embryos were exposed to different concentrations of famoxadone-cymoxanil until 72 h post-fertilization (hpf), then changes of heart morphology in zebrafish embryos were observed. We also detected the levels of oxidative stress, myocardial-cell proliferation and apoptosis, ATPase activity, and the expression of genes related to the cardiac development and calcium-signaling pathway. After famoxadone-cymoxanil exposure, pericardial edema, cardiac linearization, and reductions in the heart rate and cardiac output positively correlated with concentration. Although myocardial-cell apoptosis was not detected, proliferation of the cells was severely reduced and ATPase activity significantly decreased, resulting in a severe deficiency in heart function. In addition, indicators of oxidative stress changed significantly after exposure of the embryos to the fungicide. To better understand the possible molecular mechanisms of cardiovascular toxicity in zebrafish, we studied the transcriptional levels of cardiac development, calcium-signaling pathways, and genes associated with myocardial contractility. The mRNA expression levels of key genes in heart development were significantly down-regulated, while the expression of genes related to the calcium-signaling pathway (ATPase [atp2a1], cardiac troponin C [tnnc1a], and calcium channel [cacna1a]) was significantly inhibited. Expression of klf2a, a major endocardial flow-responsive gene, was also significantly inhibited. Mechanistically, famoxadone-cymoxanil toxicity might be due to the downregulation of genes associated with the calcium-signaling pathway and cardiac muscle contraction. Our results found that famoxadone-cymoxanil exposure causes cardiac developmental toxicity and severe energy deficiency in zebrafish.

Respiratory Toxicity of Azoxystrobin, Pyraclostrobin and Coumoxystrobin on Chlorella vulgaris.

Azoxystrobin (AZ), pyraclostrobin (PYR) and coumoxystrobin (COU) exert negative impacts on Chlorella vulgaris. Thus, in this study, C. vulgaris was used to assess the respiratory toxicity of AZ, PYR and COU by determining the acute toxicity, complex III activity and ATP viability. The 96 h-EC50 values of AZ, PYR and COU for C. vulgaris were 1.85, 2.21 and 1.62 mg/L, respectively. AZ, PYR and COU exerted significant effects on complex III activity and ATP viability after exposure to 0.71, 1.01 and 1.08 mg/L of the fungicides. The binding potentials of AZ, PYR and COU toward ubiquinone were - 10.44, - 9.31 and - 12.98 kcal/mol, respectively, which had adverse effects on amino acids. These results provided new insight into the potential acute respiratory toxicity mechanisms of these strobilurin fungicides in algae.

Acute toxicity of the fungicide azoxystrobin on the diatom Phaeodactylum tricornutum.

Azoxystrobin (AZ) is an effective broad-spectrum fungicide. Due to its extensive application, AZ is detectable in aquatic ecosystems and thus influences aquatic organisms. In this study, the acute toxicity (96 h) of AZ at concentrations of 1.0 mg/L and 5.0 mg/L on the diatom Phaeodactylum tricornutum were examined. At the tested concentrations, AZ significantly inhibited P. tricornutum growth and destroyed its cellular structure. Furthermore, the mechanisms of AZ-induced toxicity on P. tricornutum changed as the exposure time extended. Forty-eight hours after exposure, AZ inhibited P. tricornutum growth primarily via inducing oxidative stress, which increased the activity of two main antioxidant enzymes, superoxide dismutase and peroxidase, and inhibited energy metabolism. However, after 96 h of treatment, the decline in the photosynthetic capacity of P. tricornutum demonstrated that the photosystem was the main AZ target. The pigment content and expression levels of genes related to photosynthetic electron transfer reactions were also significantly decreased. The present study describes AZ toxicity in P. tricornutum and is very valuable for assessing the environmental risk of AZ.

Benzimidazole- and QoI-resistance in Corynespora cassiicola populations from greenhouse-cultivated cucumber: An emerging problem in China.

Target leaf spot caused by Corynespora cassiicola is an economically important foliar disease on cucumber. In recent years, this disease has caused a serious problem on greenhouse-cultivated cucumber in China. In this study, to explore the characteristics and possible causes of heavy occurrence of the disease, we monitored the resistance of C. cassiicola strains from different provinces of China to benzimidazole and quinone outside inhibitor (QoI) fungicides. The results from sequence comparison of target genes ß-tubulin and Cytb of 619C. cassiicola strains indicate that resistance frequency to benzimidazoles and QoIs is up to 100%. Furtherly, molecular resistance mechanism of C. cassiicola to benzimidazoles and QoIs was analysed. One single mutation E198A and three double mutations E198A&M163I, E198A&F167Y and E198A&F200S were observed in target gene ß-tubulin, which confers resistance to benzimidazoles. To our knowledge, this is the first report that double mutations of ß-tubulin confer resistance to benzimidazoles in filamentous fungi. Compared with single mutation E198A, three double mutations significantly decreased sensitivity to benzimidazoles. Moreover, significant difference of sensitivity to benzimidazoles was observed among three double mutations. These mutation genotypes of ß-tubulin have different geographical distribution and the mutation E198A&M163I is prevalent, occupying for 63.94%. In addition, strong cross resistance patterns between carbendazim, benomyl and thiabendazole were observed in C. cassiicola strains conferring different ß-tubulin mutations. For QoI resistance, the only mutation G143A of Cytb was detected in tested 619C. cassiicola strains. Strong positive cross resistance was observed when comparing the EC50 values of sensitive and resistant strains of C. cassiicola for six intrinsically different QoIs such as azoxystrobin, fluoxastrobin, pyraclostrobin, fenaminstrobin, picoxystrobin and coumoxystrobin. Taken together, all the results not only provide novel insights into understanding resistance mechanism to benzimidazoles and QoIs in filamentous fungi, but also provide some important references for resistance management of target leaf spot on cucumber.

In vitro toxicity of fungicides with different modes of action to alfalfa anthracnose fungus, Colletotrichum destructivum.

Sensitivity of 24 isolates of Colletotrichum destructivum O'Gara, collected from alfalfa plants in Serbia, to eight selected fungicides, was investigated in this study. Molecular identification and pathogenicity test of isolates tested were also performed. Fungicide sensitivity was evaluated in vitro, using mycelial growth assay method. All isolates exhibited significant pathogenicity, causing necrosis at the alfalfa seedling root tips two days after inoculation. Using the primer pair GSF1-SR1 and by comparing the amplified fragments of the tested isolates with the marker (M), the presence of the amplicon of the expected size of about 900 bp was determined for all isolates. The isolates tested in this study showed different sensitivity towards fungicides in vitro. Mycelial growth was highly inhibited by QoI (quinone outside inhibitors) fungicide pyraclostrobin (mean EC50=0.39 µg mL-1) and by DMI (demethylation-inhibiting) fungicide tebuconazole (mean EC50=0.61 µg mL-1), followed by azoxystrobin (mean EC50=2.83 µg mL-1) and flutriafol (mean EC50=2.11 µg mL-1). Multi-site fungicide chlorothalonil and MBC (methyl benzimidazole carbamate) fungicide thiophanate-methyl evinced moderate inhibition with mean EC50=35.31 and 62.83 µg mL-1, respectively. Thirteen isolates were sensitive to SDHI (succinate dehydrogenase inhibitors) fungicide boscalid and fluxapyroxad, (mean EC50=0.49 and 0.19 µg mL-1, respectively), while the rest of isolates were highly resistant.