Carboxin can induce cardiotoxicity in zebrafish embryos.

Carboxin is a heterocyclic systemic fungicide, mainly used to prevent and control grain smut and wheat rust. Although its mammalian toxicity has been reported, its toxicity to acute exposure to aquatic animals is unknown. In our study, we used zebrafish as aquatic organisms to study Carboxin toxicity. Carboxin can cause developmental toxicity and cardiotoxicity in zebrafish embryos. Histopathological staining of cardiac sections reveals structural changes in zebrafish hearts, and fluorescence quantitative PCR results shows the heart developmental genes mRNA expression levels were disrupted significantly. Besides, carboxin can also cause oxidative stress and reactive oxygen species (ROS) accumulation in zebrafish embryos. The accumulation of ROS causes mitochondrial damage, which is where ATP energy is produced. So ATPase activities and gene expression level were measured and significantly decreased after exposure to carboxin. From the confocal images, the number of blood cells in the heart were decreased significantly after carboxin exposure. Besides, Carboxin exposure can inhibit myocardial cell proliferation. These are all causes to the heart failure, eventually leading to embryos death.

Exploring molecular tools for transformation and gene expression in the cultivated edible mushroom Agrocybe aegerita.

Agrocybe aegerita is a cultivated edible mushroom in numerous countries, which also serves as a model basidiomycete to study fruiting body formation. Aiming to create an easily expandable customised molecular toolset for transformation and constitutive gene of interest expression, we first created a homologous dominant marker for transformant selection. Progeny monokaryons of the genome-sequenced dikaryon A. aegerita AAE-3 used here were identified as sensitive to the systemic fungicide carboxin. We cloned the wild-type gene encoding the iron-sulphur protein subunit of succinate dehydrogenase AaeSdi1 including its up- and downstream regions, and introduced a single-point mutation (His237 to Leu) to make it confer carboxin resistance. PEG-mediated transformation of protoplasts derived from either oidia or vegetative monokaryotic mycelium with the resulting carboxin resistance marker (CbxR) plasmid pSDI1E3 yielded carboxin-resistant transformants in both cases. Plasmid DNA linearised within the selection marker resulted in transformants with ectopic multiple insertions of plasmid DNA in a head-to-tail repeat-like fashion. When circular plasmid was used, ectopic single integration into the fungal genome was favoured, but also gene conversion at the homologous locus was seen in 1 out of 11 analysed transformants. Employing CbxR as selection marker, two versions of a reporter gene construct were assembled via Golden Gate cloning which allows easy recombination of its modules. These consisted of an eGFP expression cassette controlled by the native promoter PAaeGPDII and the heterologous terminator Tnos, once with and once without an intron in front of the eGFP start codon. After protoplast transformation with either construct as circular plasmid DNA, GFP fluorescence was detected with either transformants, indicating that expression of eGFP is intron-independent in A. aegerita. This paves the way for functional genetics approaches to A. aegerita, e.g., via constitutive expression of fruiting-related genes.

Design, synthesis, and antifungal activity of novel cinnamon-pyrazole carboxamide derivatives.

Hit, Lead & Candidate Discovery To discover succinate dehydrogenase inhibitors with a novel structure, we introduced cinnamic acid structure to optimize the lead structure 1 and synthesized four series of cinnamon-pyrazole carboxamide derivatives. The bioassay data showed that compounds (E)-N-(1-[4-chlorophenyl]-4-cyano-1H-pyrazol-5-yl)-3-(2-fluorophenyl) acrylamide (5III-d) and (E)-3-(2-chlorophenyl)-N-(1-[4-chlorophenyl]-4-cyano-1H-pyrazol-5-yl) acrylamide (5III-f) showed the significant antifungal activity against three fungi. In addition, 5III-d and 5III-f exhibited the excellent inhibitory effect against succinate dehydrogenase (SDH) enzymes with IC50 values ranging from 19.4 to 28.7 µM. The study demonstrates that the chlorine substituent group is present on both the phenyl and pyrazole rings that have a very good effect on the antifungal effect, and the compounds 5III-d and 5III-f can act as potential SDH inhibitors (SDHI) and throw a sprat for a new generation of SDHI.

[FORMATION AND FUNCTIONING OF SYMBIOTIC SYSTEMS AND RHIZOSPHERE MICROBIOCENISIS OF SOYBEAN UNDER VARIOUS FUNGICIDES APPLICATION].

The Relevance. At the recent years in soybean crops the quantity of plant pathogenic fungi has increased. The fungicides of systemic and contact action have been applicated intensively against of them. After introducing into the soil fungicides and/or their deg- radation products can to disrupt the activities of non-target objects - beneficial soil mi- croorganisms, inhibit nodulation process and the nitrogen-fixing activity of diazotrophs. The purpose of the work was to investigate the impact of combined application of fungi- cides with inoculation on the soybean symbiotic system and rhizosphere microorganisms. The Methods. The microbiological and statistical methods, gas chromatography method. The Results. Inoculation of seeds by the highly active Bradyrhizobiumjaponicum UCM B-6035, UCM B-6018 and UCM B-6023 strains the activity of nitrogen-fixing symbiotic systems increased by 1.4-3.4 times in comparison with the variant without of fungicides application and bacterization. Seed treatment by Vitavaks 200 FF fungicide caused a de- crease of'nitrogen-fixing activity of rhizobia industrial strains in symbiosis with soybean by 3-5 times. The seeds inoculation by B. japonicum UCM B-6035 strain promoted to reduce the negative impact of the Maxim Star 025 FS fungicide on the nitrogenase activity of nodulation apparatus. The positive effect of seeds bacterization was observed in the in- crease of the quantity of rhizosphere microorganisms of main ecological trophic groups. In the variant with seed treatment by Maxim Star 025 FS and Kinto duo fungicides was found a decrease in the number of microorganisms of studied groups in comparison with the control variant; the Vitavaks 200 FF fungicide application promoted to improve of these microorganisms development compared with the variant without the fungicides application and bacterization. At the inoculation of rhizobia industrial strains the negative effect of the Maxim Star 025 FS and Kinto duo fungicides to oligoazotrophic and prototrophic micro- organisms was not observed. The Conclusion. The symbiotic system of variant with the combined application of the Kinto duo fungicide with B. japonicum UCM B-6023 strain characterized by the highest nodulation and nitrogen-fixing activity.

Site-directed mutagenesis of the P225, N230 and H272 residues of succinate dehydrogenase subunit B from Botrytis cinerea highlights different roles in enzyme activity and inhibitor binding.

Carboxamide fungicides target succinate dehydrogenase (SDH). Recent field monitoring studies have identified Botrytis cinerea isolates resistant to one or several SDH inhibitors (SDHIs) with amino acid substitutions in the SDH B subunit. We confirmed, by site-directed mutagenesis of the sdhB gene, that each of the mutations identified in field strains conferred resistance to boscalid in B.cinerea, and in some cases cross-resistance to other SDHIs (fluopyram, carboxin). Enzyme inhibition studies showed that the studied modifications (SdhB_P225T/L/F, N230I, H272Y/R/L) affected the inhibition of SDH activity by SDHIs, directly contributing to resistance. Our results confirm the importance of H272, P225 and N230 for carboxamide binding. Modifications of P225 and N230 conferred resistance to the four carboxamides tested (boscalid, fluopyram, carboxin, bixafen). Modifications of H272 had differential effects on the susceptibility of SDH to SDHIs. SdhB(H272L) , affected susceptibility to all SDHIs, SdhB(H272R) conferred resistance to all SDHIs tested except fluopyram, and SdhB(H272Y) conferred fluopyram hypersensitivity. Affinity-binding studies with radiolabelled fluopyram revealed strong correlations among the affinity of SDHIs for SDH, SDH inhibition and in vivo growth inhibition in the wild type. The sdhB(H272Y) mutation did not affect SDH and respiration activities, whereas all the other mutations affected respiration by decreasing SDH activity.

Complete biodegradation of fungicide carboxin and its metabolite aniline by Delftia sp. HFL-1.

Fungicide carboxin was commonly used in the form of seed coating for the prevention of smut, wheat rust and cotton damping-off, leading carboxin and its probable carcinogenic metabolite aniline to directly enter the soil with the seeds, causing residual pollution. In this study, a novel carboxin degrading strain, Delftia sp. HFL-1, was isolated. Strain HFL-1 could use carboxin as the carbon source for growth and completely degrade 50 mg/L carboxin and its metabolite aniline within 24 h. The optimal temperatures and pH for carboxin degrading by strain HFL-1 were 30 to 42 °C and 5 to 9, respectively. Furthermore, the complete mineralization pathway of carboxin by strain HFL-1 was revealed by High Resolution Mass Spectrometer (HRMS). Carboxin was firstly hydrolyzed into aniline and further metabolized into catechol through multiple oxidation processes, and finally converted into 4-hydroxy-2-oxopentanoate, a precursor of the tricarboxylic acid cycle. Genome sequencing revealed the corresponding degradation genes and cluster of carboxin. Among them, amidohydrolase and dioxygenase were key enzymes involved in the degradation of carboxin and aniline. The discovery of transposons indicated that the aniline degradation gene cluster in strain HFL-1 was obtained via horizontal transfer. Furthermore, the degradation genes were cloned and overexpressed. The in vitro test showed that the expressed degrading enzyme could efficiently degrade aniline. This study provides an efficient strain resource for the bioremediation of carboxin and aniline in contaminated soil, and further revealing the molecular mechanism of biodegradation of carboxin and aniline.

Development of a gene-targeting system using CRISPR/Cas9 and utilization of pyrG as a novel selectable marker in Lentinula edodes.

First, we attempted to recombine the Shiitake (Lentinula edodes) pyrG (ura3) gene homologously by introducing a donor vector containing a carboxin resistance gene (lecbxR) flanked by homologous sequences of pyrG into protoplasts of the fungus. However, all the carboxin-resistant transformants only contained ectopic insertions of the exogenous gene and no homologous insertions. Agaricomycetes are generally known for their low efficiency of homologous recombination, and a similar result was shown for L. edodes. We then co-introduced a Cas9 plasmid vector containing a CRISPR/Cas9 expression cassette targeting pyrG and donor plasmid vector. As a result, ∆pyrG strains containing the expected homologous recombination were obtained. However, only two of the seven ∆pyrG strains had the Cas9 sequence; the others did not. Our results suggest that genome editing occurred via the transient expression of the CRISPR/Cas9 cassette in the Cas9 plasmid vector introduced into the fungal cell. Transforming pyrG into a ∆pyrG strain (strain I8) resulted in prototrophic strains with an efficiency of 6.5 strains/experiment.

Cloning and characterization of the gene for the iron-sulfur subunit of succinate dehydrogenase from the violet root rot fungus, Helicobasidium mompa.

The sdhB gene, encoding the iron-sulfur protein (Ip) subunit of succinate dehydrogenase (Sdh, EC 1.3.99.1), has been cloned from the violet root rot fungus, Helicobasidium mompa, and characterized. The promoter region contains a CCAAT box, TATA-like box, and CT-rich region. The gene is interrupted by eight introns and is predicted to encode a polypeptide of 291 amino acid residues. The putative amino acid sequence of the encoded product of sdhB gene from H. mompa shows high homology to the other known sdhB genes and is 79% identical to the Ip subunit of SdhB of Uromyces fabae. Three cysteine-rich clusters associated with the iron-sulfur centers involved in electron transport were particularly well conserved. One of these clusters contains a critical histidine residue implicated in carboxin sensitivity in the basidiomycetes. Only one copy of the gene was present in the genome of H. mompa, and reverse transcription (RT)-PCR analysis of mRNA expression showed that the sdhB gene was transcribed in potato dextrose broth.

Immobilization mechanism of Cd2+/HCrO4-/CrO42- ions and carboxin on montmorillonite modified with Rhizobium leguminosarum bv. trifolii exopolysaccharide.

The modification of clay minerals by exopolysaccharides (EPSs) may significantly increase their adsorption capacity. Therefore, this study focused on the impact of the EPS synthesised by the soil bacterium Rhizobium leguminosarum bv. trifolii on the adsorptive features of montmorillonite relative to two heavy metal ions (cadmium/Cd(II) and chromium/Cr(VI)) and a pesticide (carboxin). The characterization of montmorillonite was carried out using various methods: X-ray diffraction, X-ray photoelectron spectroscopy, Fourier-transform infrared spectroscopy, scanning electron microscopy. The adsorption/desorption data were modelled using theoretical equations (Langmuir-Freundlich, Redlich-Peterson, etc.). The obtained results showed that EPS promoted the accumulation of heavy metals on the clay mineral and, simultaneously, contributed to a lower degree of their desorption. This resulted from complexation between the biopolymer and the Cd(II)/Cr(VI) ions. After montmorillonite modification with 100 mg/L EPS, the maximum noted growth in Cd(II) adsorption was 26.10%, whereas it was 20.30% for Cr(VI). The adsorbed amounts of Cd(II) and Cr(VI) were then 24.24 and 14.45 mg/g, respectively. In the case of carboxin, the EPS effect on its adsorption/desorption rates was opposite - its adsorption level decreasing by 10.80%, was 0.27 mg/g. Thus, the presence of EPS-producing bacteria could reduce the bioavailability of the heavy metals, but not of the selected pesticide.

Microwave-assisted and continuous flow multistep synthesis of 4-(pyrazol-1-yl)carboxanilides.

A series of 4-(pyrazol-1-yl)carboxanilides active as inhibitors of canonical transient receptor potential channels were synthesized in an efficient three-step protocol using controlled microwave heating. The general synthetic strategy involves condensation of 4-nitrophenylhydrazine with appropriate 1,3-dicarbonyl building blocks, followed by reduction of the nitro group to the amine, which is then amidated with carboxylic acids. Compared to the conventional protocol a dramatic reduction in overall processing time from ~2 days to a few minutes was achieved, accompanied by significantly improved product yields. In addition, the first two steps in the synthetic pathway were also performed under continuous flow conditions providing similar isolated product yields. As an alternative to the three-step protocol, a novel two-step route to the desired 4-(pyrazol-1-yl)carboxanilides was devised involving condensation of 4-bromophenylhydrazine with appropriate 1,3-dicarbonyl building blocks, followed by Pd-catalyzed Buchwald-Hartwig amidation with carboxylic acid amides.

Molecular characterization of boscalid resistance in field isolates of Botrytis cinerea from apple.

Botrytis cinerea isolates obtained from apple orchards were screened for resistance to boscalid. Boscalid-resistant (BosR) isolates were classified into four phenotypes based on the levels of the concentration that inhibited fungal growth by 50% relative to control. Of the 220 isolates tested, 42 were resistant to boscalid, with resistant phenotypes ranging from low to very high resistance. There was cross resistance between boscalid and carboxin. Analysis of partial sequences of the iron-sulfur subunit of succinate dehydrogenase gene in B. cinerea (BcSdhB) from 13 BosR and 9 boscalid-sensitive (BosS) isolates showed that point mutations in BcSdhB leading to amino acid substitutions at the codon position 272 from histidine to either tyrosine (H272Y) or arginine (H272R) were correlated with boscalid resistance. Allele-specific polymerase chain reaction (PCR) analysis of 66 BosR isolates (including 24 additional isolates obtained from decayed apple fruit) showed that 19 carried the point mutation H272Y and 46 had the point mutation H272R, but 1 BosR isolate gave no amplification product. Analysis of the BcSdhB sequence of this isolate revealed a different point mutation at codon 225, resulting in a substitution of proline (P) by phenylalanine (F) (P225F). The results indicated that H272R/Y in BcSdhB were the dominant genotypes of mutants in field BosR isolates from apple. A multiplex allele-specific PCR assay was developed to detect point mutations H272R/Y in a single PCR amplification. Levels of boscalid resistance ranged from low to very high within isolates carrying either the H272R or H272Y mutation, indicating that, among BosR isolates, different BosR phenotypes (levels of resistance) were not associated with particular types of point mutations (H272R versus H272Y) in BcSdhB. Analysis of genetic relationships between 39 BosR and 56 BosS isolates based on three microsatellite markers showed that 39 BosR isolates and 30 BosS isolates were clustered into two groups, and the third group consisted of only BosS isolates, suggesting that the development of resistance to boscalid in B. cinerea likely is not totally random, and resistant populations may come from specific genetic groups.

Five carboxin-resistant mutants exhibited various responses to carboxin and related fungicides.

Five carboxin-resistant mutants from Aspergillus oryzae were characterized by the sensitivities of their mycelial growth and succinate dehydrogenase (SDH) activity to carboxin and three related fungicides. Despite a significant resistance to carboxin, exhibited by all the mutants, their patterns of sensitivity to the other fungicides was distinct. This provides clues to the molecular interaction between SDH and these fungicides.

Design, synthesis and antifungal evaluation of novel mandelic acid derivatives containing a 1,3,4-oxadiazothioether moiety.

A series of novel mandelic acid derivatives containing a 1,3,4-oxadiazothioether moiety were designed and synthesized. Bioassay results showed that some target compounds exhibited certain antifungal activity against six kinds of pathogenic fungi in vitro. Among the compounds, the EC50 values of T41 against Gibberella saubinetii, Verticillium dahlia and Sclerotinia sclerotiorum were 31.0, 27.0 and 32.1 µg/ml, respectively, and the EC50 value of T14 against S. sclerotiorum was 14.7 µg/ml. The antifungal activity against the resistant fungus S. sclerotiorum indicated that this series of target compounds may have the similar action modes or sites as the commercialized succinate dehydrogenase inhibitor carboxin. A morphological study with fluorescence microscope demonstrated that T41 can significantly destroy the membrane integrity of G. saubinetii.

Structure of Escherichia coli succinate:quinone oxidoreductase with an occupied and empty quinone-binding site.

Three new structures of Escherichia coli succinate-quinone oxidoreductase (SQR) have been solved. One with the specific quinone-binding site (Q-site) inhibitor carboxin present has been solved at 2.4 A resolution and reveals how carboxin inhibits the Q-site. The other new structures are with the Q-site inhibitor pentachlorophenol and with an empty Q-site. These structures reveal important details unresolved in earlier structures. Comparison of the new SQR structures shows how subtle rearrangements of the quinone-binding site accommodate the different inhibitors. The position of conserved water molecules near the quinone binding pocket leads to a reassessment of possible water-mediated proton uptake networks that complete reduction of ubiquinone. The dicarboxylate-binding site in the soluble domain of SQR is highly similar to that seen in high resolution structures of avian SQR (PDB 2H88) and soluble flavocytochrome c (PDB 1QJD) showing mechanistically significant structural features conserved across prokaryotic and eukaryotic SQRs.

Agrobacterium-mediated transformation of arthroconidia obtained from the edible mushroom Hypsizygus marmoreus.

Using the carboxin resistance gene from Pleurotus eryngii as a selective marker, we introduced foreign DNA into the arthroconidia of Hypsizygus marmoreus through Agrobacterium-mediated transformation. The function of the exogenous GUS (ß-glucuronidase) gene driven by the CaMV35S promoter was detected in the transformants.

The dominant Hc.Sdh (R) carboxin-resistance gene of the ectomycorrhizal fungus Hebeloma cylindrosporum as a selectable marker for transformation.

In an attempt to get a marker gene suitable for genetical transformation of the ectomycorrhizal fungus Hebeloma cylindrosporum, the gene Hc.Sdh (R) that confers carboxin-resistance was isolated from a UV mutant of this fungus. It encodes a mutant allele of the Fe-S subunit of the succinate dehydrogenase gene that carries a single amino acid substitution known to confer carboxin-resistance. This gene was successfully used as the selective marker to transform, via Agrobacterium tumefaciens, monokaryotic and dikaryotic strains of H. cylindrosporum. We also successfully transformed hygromycin-resistant insertional mutants. Transformation yielded mitotically stable carboxin-resistant mycelia. This procedure produced transformants, the growth of which was not affected by 2 microg l(-1) carboxin, whereas wild-type strains were unable to grow in the presence of 0.1 microg l(-1) of this fungicide. This makes the carboxin-resistance cassette much more discriminating than the hygromycin-resistance one. PCR amplification and Southern blot hybridisation indicated that more than 90% of the tested carboxin-resistant mycelia contained the Hc.Sdh (R) cassette, usually as a single copy. The AGL-1 strain of A. tumefaciens was a much less efficient donor than LBA 1126; the former yielded ca. 0-30% transformation frequency, depending on fungal strain and resistance cassette used, whereas the latter yielded ca. 60-95%.

Investigating dominant selection markers for Coprinopsis cinerea: a carboxin resistance system and re-evaluation of hygromycin and phleomycin resistance vectors.

Dominant selectable markers are beneficial for transformation of many fungi, particularly those model species where repeated transformations may be required. A carboxin resistance allele of the Coprinopsis cinerea sdi1 gene, encoding the iron-sulphur protein subunit of succinate dehydrogenase, was developed by introducing a suitable point mutation in the histidine block responsible for binding of the associated iron ion. This modified gene was used successfully to confer carboxin resistance upon transformation of C. cinerea protoplasts. Plasmids previously used to establish hygromycin transformation systems of several basidiomycete species, such as pAN7-1 and phph004, failed to give rise to hygromycin-resistant transformants of C. cinerea, whilst pPHT1 was successful. Sequencing of these constructs showed that the hygromycin resistance gene in pAN7-1 and phph004 had been modified removing the codons encoding two lysine residues following the N-terminal methionine. Replacement of the deleted 6 bp (AAA AAG) in the truncated hph gene led to generation of hygromycin-resistant transformants indicating the importance of these two codons for expression in C. cinerea. Phleomycin-resistant (ble) transformants were also obtained, but only with the intron-containing construct pblei004, showing that an intron is necessary to obtain phleomycin-resistant C. cinerea. This contrasts with hygromycin-resistance, where introns are not required for expression, emphasising the variability in importance of these elements.

Transformation of an oleaginous zygomycete Mortierella alpina 1S-4 with the carboxin resistance gene conferred by mutation of the iron-sulfur subunit of succinate dehydrogenase.

The sdhB gene encoding an iron-sulfur (Ip) subunit of succinate dehydrogenase (SDH, EC 1.3.99.1) complex was cloned from Mortierella alpina 1S-4. The deduced amino acid sequence of SdhB from M. alpina 1S-4 showed high similarity to those of SdhB from other organisms. The mutated sdhB (CBXB) gene encodes a modified SdhB with an amino-acid substitution (a highly conserved histidine residue within the third cysteine-rich cluster of SdhB replaced by a leucine residue) and is known to confer carboxin resistance. We succeeded in transforming M. alpina 1S-4 by using the CBXB gene as a selectable marker gene and expressing the heterologous uidA gene encoding beta-glucuronidase of Escherichia coli. Moreover, transformation efficiency was up to 40-50 transformants per 4.0 x 10(8) spores. This carboxin-transformation system, characterized by marginal background growth and mitotic stability in M. alpina 1S-4, is considered to be widely useful for the wild strain, M. alpina 1S-4, and various derivative mutants without laborious preparation of auxotrophic mutants as a host strain.

Identification of three mutant loci conferring carboxin-resistance and development of a novel transformation system in Aspergillus oryzae.

Mutants exhibiting resistance to the fungicide, carboxin, were isolated from Aspergillus oryzae, and the mutations in the three gene loci, which encode succinate dehydrogenase (SDH) B, C, and D subunits, were identified to be independently responsible for the resistance. A structural model of the SDH revealed the different mechanisms that confer carboxin-resistance in different mutations. The mutant AosdhB gene (AosdhB(cxr)) was further examined for possible use as a transformant selection marker. After transformation with AosdhB(cxr), carboxin-resistant colonies appeared within 4 days of culture, and all of the examined colonies carried the transgene. Insertion analyses revealed that the AosdhB(cxr) gene was integrated into AosdhB locus via homologous recombination at high efficiency. Furthermore, AosdhB(cxr) functioned as a successful selection marker in a transformation experiment in Aspergillus parasiticus, suggesting that this transformation system can be used for Aspergillus species.

Carboxin and its major metabolites residues in peanuts: Levels, dietary intake and chronic intake risk assessment.

We developed an ultra-performance liquid chromatography-tandem mass spectral method to determine the fungicide carboxin and its metabolites, oxycarboxin and carboxin sulfoxide in peanut samples. The method was used to detect the concentration of the analytes in the samples from fields and markets. The total residue quantities in peanut kernels were used to evaluate the chronic dietary risk of total carboxin upon peanut consumption. The estimated dietary intake of carboxin from peanuts whose seeds had been treated with carboxin at the recommended dose was between 0.020% and 0.344% of acceptable daily intake and the risk was found to be negligible. The chronic dietary risk assessment from markets and commercial field samples for various groups of humans indicated that the group with the greatest degree of exposure was 45 to 75-year-old women who lived in rural areas. However, their acceptable daily intake percentage was 0.006%, meaning that their health risk was extremely small.