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.

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.

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.

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.

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.

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.

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.

Efficient transformation of Pleurotus eryngii with a safe selective marker mutated from the Pesdi1 gene.

We introduced a site-directed mutation in the sdi1 gene and used it as a selective marker for the polyethylene glycol-mediated transformation of Pleurotus eryngii monokaryon protoplasts. The transformants displayed obvious and stable resistance to the fungicide carboxin indicating that the mutant Pesdi1 gene is an efficient selective marker.

Folate Deficiency Triggered Apoptosis of Synoviocytes: Role of Overproduction of Reactive Oxygen Species Generated via NADPH Oxidase/Mitochondrial Complex II and Calcium Perturbation.

Despite a plethora of literature has documented that osteoarthritis (OA) is veritably associated with oxidative stress-mediated chondrocyte death and matrix degradation, yet the possible involvement of synoviocyte abnormality as causative factor of OA has not been thoroughly investigated. For this reason, we conduct the current studies to insight into how synoviocytes could respond to an episode of folate-deprived (FD) condition. First, when HIG-82 synoviocytes were cultivated under FD condition, a time-dependent growth impediment was observed and the demise of these cells was demonstrated to be apoptotic in nature mediated through FD-evoked overproduction of reactive oxygen species (ROS) and drastically released of cytosolic calcium (Ca2+) concentrations. Next, we uncovered that FD-evoked ROS overproduction could only be strongly suppressed by either mitochondrial complex II inhibitors (TTFA and carboxin) or NADPH oxidase (NOX) inhibitors (AEBSF and apocynin), but not by mitochondrial complex I inhibitor (rotenone) and mitochondrial complex III inhibitor (antimycin A). Interestingly, this selective inhibition of FD-evoked ROS by mitochondrial complex II and NOX inhibitors was found to correlate excellently with the suppression of cytosolic Ca2+ release and reduced the magnitude of the apoptotic TUNEL-positive cells. Taken together, we present the first evidence here that FD-triggered ROS overproduction in synoviocytes is originated from mitochondrial complex II and NOX. Both elevated ROS in tandem with cytosolic Ca2+ overload serve as final arbitrators for apoptotic lethality of synoviocytes cultivated under FD condition. Thus, folate supplementation may be beneficial to patients with OA.

Disruption of spindle checkpoint function in rats following 28 days of repeated administration of renal carcinogens.

We previously reported that 28-day exposure to hepatocarcinogens that facilitate cell proliferation specifically alters the expression of G1/S checkpoint-related genes and proteins, induces aberrant early expression of ubiquitin D (UBD) at the G2 phase, and increases apoptosis in the rat liver, indicating G1/S and spindle checkpoint dysfunction. The present study aimed to determine the time of onset of carcinogen-specific cell-cycle disruption after repeated administration of renal carcinogens for up to 28 days. Rats were orally administered the renal carcinogens nitrofurantoin (NFT), 1-amino-2,4-dibromoantraquinone (ADAQ), and 1,2,3-trichloropropane (TCP) or the non-carcinogenic renal toxicants 1-chloro-2-propanol, triamterene, and carboxin for 3, 7 or 28 days. Both immunohistochemical single-molecule analysis and real-time RT-PCR analysis revealed that carcinogen-specific expression changes were not observed after 28 days of administration. However, the renal carcinogens ADAQ and TCP specifically reduced the number of cells expressing phosphorylated-histone H3 at Ser10 in both UBD(+) cells and proliferating cells, suggestive of insufficient UBD expression at the M phase and early transition of proliferating cells from the M phase, without increasing apoptosis, after 28 days of administration. In contrast, NFT, which has marginal carcinogenic potential, did not induce such cellular responses. These results suggest that it may take 28 days to induce spindle checkpoint dysfunction by renal carcinogens; however, induction of apoptosis may not be essential. Thus, induction of spindle checkpoint dysfunction may be dependent on carcinogenic potential of carcinogen examined, and marginal carcinogens may not exert sufficient responses even after 28 days of administration.

[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.

An efficient genetic manipulation protocol for Ustilago esculenta.

Ustilago esculenta grows within the flowering stem of the aquatic grass Zizania latifolia, resembling a fungal endophyte. The fungus colonizes Z. latifolia and induces swelling which results in the formation of galls near the base of the plant. Due to their unique flavor and textures these galls are considered as a delicacy in southern China. Efficient genetic manipulation is required to determine the relationship between U. esculenta and Z. latifolia. In this study, we report a protoplast-based transformation system for this unique fungal species. We have explored various factors (enzyme digesting conditions, osmotic pressure stabilizers, vectors and selection agents) that might impact protoplast yield and high frequencies of transformation. A haploid strain (UeT55) of U. esculenta was found to produce higher yields of protoplasts when treating with 15 mg mL(-1) lywallzyme in a sucrose-containing solution at 30°C for 3 h. The transformation frequencies were higher when fungal strain was transformed with a linear plasmid harboring hygromycin or carboxin resistance gene and regenerated on a sucrose-containing medium. A UeICL gene (coding isocitrate lyase) was disrupted and an EGFP (coding enhanced green fluorescent protein) gene was overexpressed successfully in the UeT55 strain using the developed conditions. The genetic manipulation system reported in this study will open up new opportunities for forward and reverse genetics in U. esculenta.

Integrated options for the management of black root rot of strawberry caused by Rhizoctonia solani Kuhn.

An investigation was made to manage strawberry black root rot caused by Rhizoctonia solani (R. solani) through the integration of Trichoderma harzianum (T. harzianum) isolate STA7, mustard oil cake and Provax 200. A series of preliminary experiments were conducted to select a virulent isolate of R. solani, an effective isolate of T. harzianum, a suitable organic amendment, and a suitable fungicide before setting the experiment for integration. The pathogenicity of the selected four isolates of R. solani was evaluated against strawberry and isolate SR1 was selected as the test pathogen due to its highest virulent (95.47% mortality) characteristics. Among the 20 isolates of T. harzianum, isolate STA7 showed maximum inhibition (71.97%) against the test pathogen (R. solani). Among the fungicides, Provax-200 was found to be more effective at lowest concentration (100 ppm) and highly compatible with Trichoderma isolates STA7. In the case of organic amendments, maximum inhibition (59.66%) of R. solani was obtained through mustard oil cake at the highest concentration (3%), which was significantly superior to other amendments. Minimum percentages of diseased roots were obtained with pathogen (R. solani)+Trichoderma+mustard oil cake+Provax-200 treatment, while the highest was observed with healthy seedlings with a pathogen-inoculated soil. In the case of leaf and fruit rot diseases, significantly lowest infected leaves as well as fruit rot were observed with a pathogen+Trichoderma+mustard oil cake+Provax-200 treatment in comparison with the control. A similar trend of high effectiveness was observed by the integration of Trichoderma, fungicide and organic amendments in controlling root rot and fruit diseases of strawberry. Single application of Trichoderma isolate STA7, Provax 200 or mustard oil cake did not show satisfactory performance in terms of disease-free plants, but when they were applied in combination, the number of healthy plants increased significantly. The result of the current study suggests the superiority of our integrated approach to control the sclerotia forming pathogen R. solani compared to the individual treatment either by an antagonist or by a fungicide or by mustard oil cake.

A new direct laser photo-induced fluorescence method coupled on-line with liquid chromatographic separation for the simultaneous determination of anilides pesticides.

A new direct laser photo-induced fluorescence high performance liquid chromatography (DL-PIF-HPLC) method is developed for the simultaneous determination of three anilide pesticides, namely carboxin, monalide and propanil. DL-PIF-HPLC uses a tunable Nd:YAG-OPO laser to obtain fluorescent photoproduct(s) and to simultaneously analyze their fluorescence in a short acquisition time with an intensified CCD camera, which improves the selectivity (by choosing the suitable excitation wavelength), increases the sensitivity (due to the high energy of the laser beam) and reduces the time of analysis, relative to the classical PIF methods. However, one of the main drawbacks of PIF methods is the presence of interferences with other compounds, such as other pesticides from the same group yielding similar fluorescent photoproducts, which reduces their selectivity. The analytical interest of DL-PIF-HPLC to avoid these interferences is demonstrated. The DL-PIF spectra, chromatographic conditions and analytical performances of DL-PIF-HPLC are presented for the simultaneous determination of three anilide pesticides. The calibration curves are linear over one order of magnitude and the limits of detection are in the ng mL(-1) range. The new DL-PIF-HPLC system has the advantage to combine the performances of both techniques, DL-PIF and liquid chromatography, and to improve the analysis selectivity.

Yield loss assessment due to Alternaria blight and its management in linseed.

Field experiments were conducted during 2010-11 and 2011-12 to assess the yield losses due to Alternaria blight disease caused by Alternaria lini and A. linicola in recently released cultivars and their management with the integration of Trichoderma viride, fungicides and plant extract. Disease severity on leaves varied from 41.07% (Parvati) to 65.01% (Chambal) while bud damage per cent ranged between 23.56% (Shekhar) to 46.12% (T-397), respectively in different cultivars. Maximum yield loss of 58.44% was recorded in cultivar Neelum followed by Parvati (55.56%), Meera (55.56%) and Chambal (51.72%), respectively while minimum loss was recorded in Kiran (19.99%) and Jeevan (22.22%). Minimum mean disease severity (19.47%) with maximum disease control (69.74%) was recorded with the treatment: seed treatment (ST) with vitavax power (2 g kg(-1) seed) + 2 foliar sprays (FS) of Saaf (a mixture of carbendazim+mancozeb) 0.2% followed by ST with Trichoderma viride (4g kg(-1) seed) + 2 FS of Saaf (0.2%). Minimum bud damage (13.75%) with maximum control (60.94%) was recorded with treatment of ST with vitavax power+2 FS of propiconazole (0.2%). Maximum mean seed yield (1440 kg ha(-1)) with maximum net return (Rs. 15352/ha) and benefit cost ratio (1:11.04) was obtained with treatment ST with vitavax power + 2 FS of Neem leaf extract followed by treatment ST with vitavax power+2 FS of Saaf (1378 kg ha(-1)).

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.

Mutagenesis and functional studies with succinate dehydrogenase inhibitors in the wheat pathogen Mycosphaerella graminicola.

A range of novel carboxamide fungicides, inhibitors of the succinate dehydrogenase enzyme (SDH, EC 1.3.5.1) is currently being introduced to the crop protection market. The aim of this study was to explore the impact of structurally distinct carboxamides on target site resistance development and to assess possible impact on fitness. We used a UV mutagenesis approach in Mycosphaerella graminicola, a key pathogen of wheat to compare the nature, frequencies and impact of target mutations towards five subclasses of carboxamides. From this screen we identified 27 amino acid substitutions occurring at 18 different positions on the 3 subunits constituting the ubiquinone binding (Qp) site of the enzyme. The nature of substitutions and cross resistance profiles indicated significant differences in the binding interaction to the enzyme across the different inhibitors. Pharmacophore elucidation followed by docking studies in a tridimensional SDH model allowed us to propose rational hypotheses explaining some of the differential behaviors for the first time. Interestingly all the characterized substitutions had a negative impact on enzyme efficiency, however very low levels of enzyme activity appeared to be sufficient for cell survival. In order to explore the impact of mutations on pathogen fitness in vivo and in planta, homologous recombinants were generated for a selection of mutation types. In vivo, in contrast to previous studies performed in yeast and other organisms, SDH mutations did not result in a major increase of reactive oxygen species levels and did not display any significant fitness penalty. However, a number of Qp site mutations affecting enzyme efficiency were shown to have a biological impact in planta.Using the combined approaches described here, we have significantly improved our understanding of possible resistance mechanisms to carboxamides and performed preliminary fitness penalty assessment in an economically important plant pathogen years ahead of possible resistance development in the field.

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.