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.

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.

[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.

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)).

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.

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.

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.

Transformation of Mucor circinelloides with autoreplicative vectors containing homologous and heterologous ARS elements and the dominant Cbx(r) carboxine-resistance gene.

Mucor circinelloides transformants prototrophic to leucine and resistant to carboxine (Leu(+) Cbx(r)) have been obtained by treatment of protoplasts with plasmid constructs containing homologous leuA gene and adjacent autonomously replicating sequences (ARS) element combined with the Cbx(r)(carboxine-resistance) gene of Ustilago maydis and ARS sequences from this basidiomycete (plasmid pGG37) or from the 2 mu plasmid of Saccharomyces cerevisiae (plasmid pGG43). The presence in the same plasmid molecule of the M. circinelloides leuA gene and adjacent ARS element together with heterologous ARS elements produced an increase in the transformation frequency of about 65-120%. The presence of autoreplicating plasmid molecules in the transformants was demonstrated by mitotic stability experiments, by Southern analysis, and by the rescue of plasmids from transformed bacterial cells.

Controlling food-contaminating fungi by targeting their antioxidative stress-response system with natural phenolic compounds.

The antioxidative stress-response system is essential to fungi for tolerating exposure to phenolic compounds. We show how this system can be targeted to improve fungal control by using compounds that inhibit the fungal mitochondrial respiratory chain. Targeting mitochondrial superoxide dismutase with selected phenolic acid derivatives (e.g., vanillyl acetone) resulted in a 100- to 1,000-fold greater sensitivity to strobilurin or carboxin fungicides. This synergism is significantly greater with strobilurin than with carboxin, suggesting that complex III of the mitochondrial respiratory chain is a better target than complex II for fungal control, using phenolics. These results show certain natural compounds are effective synergists to commercial fungicides and can be used for improving control of food-contaminating pathogens. These results suggest that the use of such compounds for fungal control can reduce environmental and health risks associated with commercial fungicides, lower cost for control, and the probability for development of resistance.

Molecular breeding of white rot fungus Pleurotus ostreatus by homologous expression of its versatile peroxidase MnP2.

Using a DNA-mediated transformation technique, a molecular breeding approach to isolate Pleurotus ostreatus strains with enhanced productivity of its versatile peroxidase MnP2 was conducted. A recombinant mnp2 construct under the control of P. ostreatus sdi1 expression signals was introduced into the wild-type P. ostreatus strain by cotransformation with a carboxin-resistant marker plasmid. A total of 32 transformants containing the recombinant mnp2 sequence were isolated in a screening with specific amplification by PCR. Productivity of MnP2 in the recombinants was evaluated by the decolorization ability of Poly R-478 on agar plates in the absence of Mn2+. Recombinant P. ostreatus strains with elevated manganese peroxidase (MnP) productivity were successfully isolated. One of the recombinants, TM2-10, was demonstrated to secrete recombinant MnP2 predominantly on a synthetic medium containing 15 mM ammonium oxalate, which was confirmed by reverse transcription PCR (RT-PCR) and isozyme profile analysis using anion-exchange chromatography. The benzo[a]pyrene-removing activity by fungal treatment was also analyzed using the isolated recombinant strains.

Flutolanil and carboxin resistance in Coprinus cinereus conferred by a mutation in the cytochrome b560 subunit of succinate dehydrogenase complex (Complex II).

A gene that confers resistance to the systemic fungicide flutolanil was isolated from a mutant strain of the basidiomycete Coprinus cinereus. The flutolanil resistance gene was mapped to a chromosome of approximately 3.2 Mb, and a chromosome-specific cosmid library was constructed. Two cosmid clones that were able to transform a wild-type, flutolanil-sensitive, strain of C. cinereus to resistance were isolated from the library. Analysis of a subclone containing the resistance gene revealed the presence of the sdhC gene, which encodes the cytochrome b560 subunit of the succinate dehydrogenase (SDH) complex (Complex II) in the mitochondrial membrane. Comparison between the sdhC gene of a wild-type strain and that of a mutant strain revealed a single point mutation, which results in the replacement of Asn by Lys at position 80. Measurements of succinate-cytochrome c reductase activity in the transformants with mutant sdhC gene(s) suggest that flutolanil resistance of the fungus is caused by a decrease in the affinity of the SDH complex for flutolanil. This sdhC mutation also conferred cross-resistance against another systemic fungicide, carboxin, an anilide that is structurally related to flutolanil. In other organisms carboxin resistance mutations have been found in the genes sdhB and sdhD, but this is the first demonstration that a mutation in sdhC can also confer resistance. The mutant gene cloned in this work can be utilized as a dominant selectable marker in gene manipulation experiments in C. cinereus.