Nitrosoguanidine Mutagenesis Research Articles

Improved Fermentation Yield of Doramectin from Streptomyces avermitilis N72 by Strain Selection and Glucose Supplementation Strategies

Doramectin is a macrolide antiparasitic that is widely used in the treatment of mammalian parasitic diseases. Doramectin is usually produced by Streptomyces avermitilis fermentation using cyclohexanecarboxylic acid (CHC) as a precursor; however, the growth of S. avermitilis is usually inhibited by CHC, resulting in a low fermentation yield of doramectin. In this study, a high-yielding strain XY-62 was obtained using the S. avermitilis mutant strain S. avermitilis N72 as the starting strain, then combined with a CHC tolerance screening strategy using ultraviolet and nitrosoguanidine mutagenesis, and a 96 microtiter plate solid-state fermentation primary sieving and shake flask fermentation rescreening method. Compared with S. avermitilis N72, the doramectin fermentation yield increased by more than 1.3 times, and it was more adaptable to temperature, pH, and CHC concentration of the culture; additionally, the viability of the mycelial growth was enhanced. In addition, further studies on the high-yielding strain XY-62 revealed that the accumulation of doramectin could be further increased by glucose supplementation during the fermentation process, and the yield of doramectin reached 1068 μg/mL by scaling up the culture in 50 L fermenters; this has the potential for industrial production. Therefore, mutagenesis combined with CHC tolerance screening is an effective way to enhance the fermentation production of doramectin by S. avermitilis. Our strategy and findings can help to improve the production of doramectin in industrial strains of S. avermitilis.

Screening of silicon-activating bacteria and the activation mechanism of silicon in electrolytic manganese residue

Electrolytic manganese residue (EMR) is a kind of solid waste with a high silicon content. Most of the silicon in EMR, however, exist in the state of SiO2, which cannot be directly absorbed by plants. Currently, it is very challenge to recover the silicon from EMR. In this study, a preliminary screening of strains with silicon-activating ability was conducted, and four strains were screened out and isolated from the soil around the tailings pond of EMR. Then, single factor experiments were conducted to obtain the optimal growth conditions of the four strains, and the results indicated that the Ochrobactrum sp. T-07 had the best silicon-activating ability from EMR after nitrosoguanidine mutagenesis (Ochrobactrum sp. T-07-B). The available silicon (in terms of SiO2) in the leaching solution was up to 123.88 mg L−1, which was significantly higher than that produced by Bacillus circulans and Paenibacillus mucilaginosus, the two commercial available pure culture strains. Results of direct/indirect contact experiments between Ochrobactrum sp. T-07-B and EMR revealed that bioleaching was promoted under the synergistic effect of bacteria growth on the surface of and metabolism within EMR. The newly isolated strains with silicon-activating effect are different from the existing-known silicate bacteria and may be used for more efficient silicon activation in silicate minerals.

Species identification and mutation breeding of silicon-activating bacteria isolated from electrolytic manganese residue.

A strain of silicon-activating bacteria was isolated from electrolytic manganese residue (EMR); identified as a species of Ochrobactrum by integrated microscopic morphological characteristics, biochemical index determination, and clone analysis (i.e., results of 16S rRNA sequence); and temporarily named as Ochrobactrum sp. T-07 (T-07). The optimal growth conditions of the strain T-07 were obtained as follows: temperature of 30°C, initial pH of 7.0, shaking speed of 180 rev.min-1, and loading volume of 100mL. In order to enhance its activation activity of silicon, T-07 went through the ultraviolet (UV) mutagenesis and nitrosoguanidine (NTG) mutagenesis breeding, and the mutant strain T-07-B with higher activity was obtained. Under the optimal fermentation condition (leaching time of 20days, temperature of 30°C, initial pH of 7, pulp concentration of 5%, shaking speed of 180 rev.min-1, and particle diameter of EMR ≤ 180μm), the available silicon content in the supernatant reached 98.8mgL-1, which was 2.4 times of the original strain T-07. Therefore, T-07 can be used as a good backup in developing biological silicon fertilizer for plants.

A new strain of Aspergillus tubingensis for high-activity pectinase production.

Pectinase is a general term for a class of enzymes that decompose pectin. To obtain a fungal strain with high-activity pectinase of potential commercial importance, we screened microorganisms from the soil of vineyards, performed mutation breeding by ultraviolet (UV) and nitrosoguanidine (NTG) mutagenesis, and performed comparisons to commercially available pectinases. We found that the derived pectinase-producing strain Rn14-88A had the highest pectinase activity of 8363.215U/mL, and identified it using internal transcribed spacer sequence analysis as Aspergillus tubingensis. Rn14-88A was the original strain for UV mutagenesis, from which mutant strain R-7-2-4 had the highest pectinase enzyme activity (9198.68U/mL), which was a 9.99% increase compared to that of Rn14-88A. Following NTG mutagenesis of R-7-2-4, mutant strain Y1-3-2-6 had a pectinase enzyme activity of 9843.34U/mL, which reflects a 6.36% increase compared to the pectinase activity of R-7-2-4. Subsequently, another round of NTG mutagenesis was performed on Y1-3-2-6, and the mutagenic strain Y2-6-3-4 exhibited an improved enzyme activity of 21,864.34U/mL, which was 161.44% higher than that of Rn14-88A. Through liquid fermentation experiments of A. tubingensis Y2-6-3-4, it was determined that pectinase activity was the highest at a fermentation time of 20h. Therefore, we conclude that A. tubingensis Y2-6-3-4 has potential for use in commercial production.

Enhanced degradation of perfluorooctanoic acid by a genome shuffling-modified Pseudomonas parafulva YAB-1

ABSTRACTPerfluorooctanoic acid (PFOA) as an emerging persistent organic pollutant is hard to be degraded by conventional methods because of its stable physical and chemical properties. Microbial transformation is an attractive remediation approach to prevent and clean up PFOA contamination. To date, several strains of wild microbes have been reported to have limited capacity to degrade PFOA, selection of superior strains degrading PFOA become urgently necessary. Here, we report the application of genome shuffling to improve the PFOA-degrading bacterium Pseudomonas Parafulva YAB-1. The initial mutant populations of strain YAB1 were generated by nitrosoguanidine and ultraviolet irradiation mutagenesis respectively, resulting in mutants YM-9 and YM-19 with slightly improved PFOA-degrading ability. YM-9 and YM-19 were used as the starting strains for three rounds of recursive protoplast fusion. The positive mutants were screened on inorganic salt medium plates containing different concentrations of PFOA and selected based on their PFOA degradability in shake-flask fermentation test. The best performing recombinant F3-52 was isolated after three rounds of genome shuffling. In batch fermentation, the PFOA degradation rate of mutant F3-52 was up to 58.6%, which was 1.8-fold higher than that of the parent strain YAB1, and 1.6-fold higher than the initial mutants YM-9 and YM-19. Pass-generation test indicated that the heredity character of F3-52 was stable. The results demonstrated that genome shuffling was an efficient method for improving PFOA degradation of Pseudomonas Parafulva YAB1. The bred mutant F3-52 with 58.6% PFOA-degrading rate could be used for the environmental control of PFOA pollutant.

Reduced acetaldehyde production by genome shuffling of an industrial brewing yeast strain

High levels of acetaldehyde produced by yeast during fermentation can be of concern to product quality. A novel approach, based on genome shuffling, was applied to reduce the production of acetaldehyde by industrial brewing strain YS86. Four isolates with different impacts of acetaldehyde concentration were obtained from populations generated by ultraviolet irradiation and nitrosoguanidine mutagenesis. These yeast strains were then subjected to recursive pool-wise protoplast fusion. A strain library that was likely to yield positive colonies was created by fusing the lethal protoplasts obtained from both UV irradiation and heat treatments. After two rounds of genome shuffling, a recombinant YSF2–9 strain produced less acetaldehyde than wild-type strain YS86, by 64.5 and 66.2% in laboratory and pilot plant fermentations, respectively. The shuffled yeast strain YSF2–9 was genetically stable and may have a potential application in brewing industry for managing acetaldehyde in beer. Copyright © 2017 The Institute of Brewing & Distilling

Enhancement of Carotenoids Production from <em>Rhodobacter sphaeroides</em> by Nitrosoguanidine Mutagenesis

To enhance carotenoids production from <em>Rhodobacter sphaeroides</em>, Nitrosoguanidine (NTG) mutagenesis was employed to construct high-carotenoids <em>Rb. Sphaeroides</em> strain in the present study. Experimental results suggested that the NTG concentration and treat duration was 3 mg/mL and 15 min, respectively. One high-carotenoids <em>Rb. Sphaeroides</em> strain was finally obtained and the production of carotenoids was 2 times higher than that of the production from wild type <em>Rb. Sphaeroides</em>. The present study promotes to large scale production of natural carotenoids from <em>Rb. Sphaeroides</em>.

Breeding 3 elite strains of Nannochloropsis oceanica by nitrosoguanidine mutagenesis and robust screening

Nannochloropsis have received extensive attention for biodiesel production in recent years. By integrating random mutation with a robust and cost-efficient screening method, we bred three mutants (LAMB001, LAMB002 and LAMB003) of Nannochloropsis oceanica featuring higher lipid productivity than wild-type N. oceanica. This screening included BODIPY 505/51511BODIPY 505/515: (4,4-difluro-1, 3, 5, 7-tetramethyl-4-bora- 3a,4adiaza-s-indacene). staining and fluorescence microplate reading, during which the optimal concentration (0.225μg/mL) of BODIPY 505/515 was determined for the staining of N. oceanica. Nitrosoguanine was proven to be more potent than ethyl methane sulfonate in mutating lipid-rich strains. The three mutants exhibited increases of 33.54%, 30.93% and 21.44% in lipid productivity over that of wild-type-type Nannochloropsis oceanica. Compared to wild-type N. oceanica, the lipid content of LAMB001, LAMB002 and LAMB003 increased by 17.4%, 23.7% and 29.4%, respectively, with LAMB003 accumulating the most lipid, accounting for 31.34% of dry biomass. The fatty acid profile indicated that C16–C18 of LAMB003 increased by 12.56% relative to that of the wild-type, totaling 66.59% of total fatty acids. Comparisons of the triacylglycerol composition between wild-type N. oceanica and LAMB003 indicated that TAG (18:0/18:1/20:5) and TAG (16:0/16:0/20:5) were absent in LAMB003, which was in accordance with its depressed PUFA22PUFA: polyunsaturated fatty acid. content (29.46%)

Whole Genome Sequencing Identifies a Novel Factor Required for Secretory Granule Maturation in Tetrahymena thermophila.

Unbiased genetic approaches have a unique ability to identify novel genes associated with specific biological pathways. Thanks to next generation sequencing, forward genetic strategies can be expanded to a wider range of model organisms. The formation of secretory granules, called mucocysts, in the ciliate Tetrahymena thermophila relies, in part, on ancestral lysosomal sorting machinery, but is also likely to involve novel factors. In prior work, multiple strains with defects in mucocyst biogenesis were generated by nitrosoguanidine mutagenesis, and characterized using genetic and cell biological approaches, but the genetic lesions themselves were unknown. Here, we show that analyzing one such mutant by whole genome sequencing reveals a novel factor in mucocyst formation. Strain UC620 has both morphological and biochemical defects in mucocyst maturation—a process analogous to dense core granule maturation in animals. Illumina sequencing of a pool of UC620 F2 clones identified a missense mutation in a novel gene called MMA1 (Mucocyst maturation). The defects in UC620 were rescued by expression of a wild-type copy of MMA1, and disrupting MMA1 in an otherwise wild-type strain phenocopies UC620. The product of MMA1, characterized as a CFP-tagged copy, encodes a large soluble cytosolic protein. A small fraction of Mma1p-CFP is pelletable, which may reflect association with endosomes. The gene has no identifiable homologs except in other Tetrahymena species, and therefore represents an evolutionarily recent innovation that is required for granule maturation.

Genome shuffling of Saccharomyces cerevisiae for enhanced glutathione yield and relative gene expression analysis using fluorescent quantitation reverse transcription polymerase chain reaction

Genome shuffling is an efficient and promising approach for the rapid improvement of microbial phenotypes. In this study, genome shuffling was applied to enhance the yield of glutathione produced by Saccharomyces cerevisiae YS86. Six isolates with subtle improvements in glutathione yield were obtained from populations generated by ultraviolet (UV) irradiation and nitrosoguanidine (NTG) mutagenesis. These yeast strains were then subjected to recursive pool-wise protoplast fusion. A strain library that was likely to yield positive colonies was created by fusing the lethal protoplasts obtained from both UV irradiation and heat treatments. After two rounds of genome shuffling, a high-yield recombinant YSF2-19 strain that exhibited 3.2- and 3.3-fold increases in glutathione production in shake flask and fermenter respectively was obtained. Comparative analysis of synthetase gene expression was conducted between the initial and shuffled strains using FQ (fluorescent quantitation) RT-PCR (reverse transcription polymerase chain reaction). Delta CT (threshold cycle) relative quantitation analysis revealed that glutathione synthetase gene (GSH-I) expression at the transcriptional level in the YSF2-19 strain was 9.9-fold greater than in the initial YS86. The shuffled yeast strain has a potential application in brewing, other food, and pharmaceutical industries. Simultaneously, the analysis of improved phenotypes will provide more valuable data for inverse metabolic engineering.

A Lactobacillus mutant capable of accumulating long-chain polyphosphates that enhance intestinal barrier function.

Inorganic polyphosphate (polyP) was previously identified as a probiotic-derived substance that enhances intestinal barrier function. PolyP-accumulating bacteria are expected to have beneficial effects on the human gastrointestinal tract. In this study, we selected Lactobacillus paracasei JCM 1163 as a strain with the potential to accumulate polyP, because among the probiotic bacteria stored in our laboratory, it had the largest amount of polyP. The chain length of polyP accumulated in L. paracasei JCM 1163 was approximately 700 phosphate (Pi) residues. L. paracasei JCM 1163 accumulated polyP when Pi was added to Pi-starved cells. We further improved the ability of L. paracasei JCM 1163 to accumulate polyP by nitrosoguanidine mutagenesis. The mutant accumulated polyP at a level of 1500nmol/mg protein-approximately 190 times that of the wild-type strain. PolyP extracted from the L. paracasei JCM 1163 significantly suppressed the oxidant-induced intestinal permeability in mouse small intestine. In conclusion, we have succeeded in breeding the polyP-accumulating Lactobacillus mutant that is expected to enhance intestinal barrier function.

Genome Shuffling Enhanced ε-Poly-l-Lysine Production by Improving Glucose Tolerance of Streptomyces graminearus

The productivity of ε-poly-L: -lysine (ε-PL) in currently reported wild-type strains is low. Here we improved glucose tolerance of a Streptomyces graminearus strain LS-B1 by genome shuffling while simultaneously enhancing the ε-PL productivity. The starting population was generated by ultraviolet irradiation and nitrosoguanidine mutagenesis and then subjected for recursive protoplast fusion. The positive colonies from library, created by fusing the inactivated protoplasts were screened on agar plates containing different concentrations of glucose. Characterization of all recombinants and wild-type strain in shake-flask fermentation indicated the compatibility of two phenotypes of glucose tolerance and ε-PL yield enhancement. The best performing recombinant, F3-4, was isolated after three rounds of genome shuffling, whose ε-PL production was about 88% higher than that of the parent strain. In batch fermentation test, the ε-PL concentration was obtained as 2.4 g/L by F3-4 compared with 1.6 g/L of wild type. Fed-batch fermentation by F3-4 was carried out and the ε-PL production accumulated to 13.5 g/L when initial glucose concentration was improved from 50 to 85 g/L. Enzyme activities of hexokinase, pyruvate kinase, and citrate synthase revealed that the glycolytic pathway and tricarboxylic acid circle way in F3-4 were more active than those in wild type, which was a possible reason for enhanced ε-PL production.

Optimization of β-carotene production by a mutant of the lactose-positive yeast Rhodotorula acheniorum from whey ultrafiltrate

The present work investigated on carotenogenesis with high β-carotene content by a new isolated high-activity strain-producer Rhodotorula acheniorum mutant MRN in cheese whey ultrafiltrate. After a serial of UV, ethymethanesurfonate (EMS), and nitrosoguanidine (NTG) mutagenesis, a mutant named MRN of the red lactose-positive yeast strain R. acheniorum was obtained. Then, the effects of different growth medium factors on carotenoid production by this mutant at batch-scale level were identified and optimized by means of response surface methodology (RSM) in order to achieve high-level production of β-carotene. The optimum conditions required to achieve the highest level of β-carotene (262.12±1.01 mg/L) were determined as follows: whey ultrafiltrate (WU) lactose concentration 55 g/L, pH 5.85, ammonium sulfate concentration 3.5 g/L, temperature 23°C, and aeration rate 1.56 vvm. The medium optimization resulted in a 6.45-fold increase in volumetric production (262.12±1.01 mg/L) and a 4.62-fold increase in the cellular accumulation (10.69±0.19 mg/g) of β-carotene.

Signature gene expression profile of triclosan-resistant Escherichia coli

To gain further insight into the defence mechanisms against triclosan in a mutant derived from an Escherichia coli strain carrying the triclosan-resistant target enzyme, FabI(G93V). An E. coli imp4231 FabI(G93V) strain was constructed by replacing intact fabI with a linear DNA cassette, fabI(G93V)-CmR, that contains a single mutation, GGT to GTT, at codon 93 of fabI(G93V) and a chloramphenicol resistance gene (CmR) as a marker for the mutant allele by a Red-mediated recombination system. Using this E. coli imp4231 FabI(G93V) strain, nitrosoguanidine (NTG) mutagenesis was performed to generate E. coli IFNs [imp4231 FabI(G93V) treated with NTG] displaying higher MICs of triclosan than its parent strain. The genes overexpressed in E. coli IFN4 were identified by DNA microarray analysis. An E. coli imp4231 FabI(G93V) strain displays approximately 400-fold increased MICs of triclosan (MIC approximately 8 mg/L) compared with the parent strain (MIC approximately 0.02 mg/L). Furthermore, E. coli IFN4 has the highest MIC of triclosan (MIC approximately 80 mg/L). DNA microarray analysis of E. coli IFN4 shows that many genes involved in the biosynthesis of membrane proteins, including transporters, reductases/dehydrogenases and stress response regulators, were highly expressed in the mutant. These results strongly indicate that E. coli IFN cells might protect themselves from triclosan by activating various defence mechanisms, such as (i) changing efflux activities; (ii) capturing the triclosan; and (iii) increasing the expression of important regulators or metabolic enzymes.

Assignment of avian reovirus temperature-sensitive mutant recombination groups E, F, and G to genome segments

Avian reoviruses (ARV) are less well understood than their mammalian counterparts. ARV are ubiquitous in commercial poultry and frequently isolated from acutely infected chickens. We previously described isolation of ARV temperature-sensitive (ts) mutants after nitrosoguanidine mutagenesis of wild-type ARV138, their assignment to 7 recombination groups (A–G), and genetic mapping of mutants in groups A–D to specific gene segments. For this study, wild-type serotype ARV176 was crossed with ts mutants tsE158 (Group E), tsF206 (Group F), or tsG247 (Group G) and reassortant progenies analyzed. Reassortant temperature-sensitivities were determined by efficiency of plating at permissive and non-permissive temperatures. Mapping results indicated tsE158, tsF206, and tsG247 mapped to the L1, S4, and L3 genes, respectively, which encode the λA core shell, σNS non-structural, and λC core spike proteins, respectively. Specific amino acid substitutions in each mutant were determined and locations of structural protein alterations were placed within the 3-dimensional structure of homologous mammalian reovirus proteins. Mapping recombination groups E–G marks completion of gene assignments for all seven ts mutant groups previously generated.

Genome shuffling enhanced l-lactic acid production by improving glucose tolerance of Lactobacillus rhamnosus

Genome shuffling is a powerful strategy for rapid engineering of microbial strains for desirable industrial phenotypes. Here we applied the genome shuffling to improve the glucose tolerance of Lactobacillus rhamnosus ATCC 11443 while simultaneously enhancing the l-lactic acid production. The starting population was generated by ultraviolet irradiation and nitrosoguanidine mutagenesis and then subjected for the recursive protoplast fusion. The positive colonies from library created by fusing the inactivated protoplasts were more likely to be screened on plates containing different concentrations of high glucose and 2% CaCO 3. Characterization of all mutants and wild-type strain in the shake flask indicated the compatibility of two optimal phenotypes of glucose tolerance and lactic acid enhancement. The lactic acid production, cell growth and glucose consumption of the best performing strain from the second round genome shuffled populations were 71.4%, 44.9% and 62.2% higher than those of the wild type at the initial glucose concentration of 150 g/l in the 16 l bioreactor. Furthermore, the higher lactic acid concentrations were obtained when the initial glucose concentrations increased to 160 and 200 g/l in batch fermentation.

Regulation of Arsenate Resistance in Desulfovibrio desulfuricans G20 by an arsRBCC Operon and an arsC Gene

Desulfovibrio desulfuricans G20 grows and reduces 20 mM arsenate to arsenite in lactate-sulfate media. Sequence analysis and experimental data show that D. desulfuricans G20 has one copy of arsC and a complete arsRBCC operon in different locations within the genome. Two mutants of strain G20 with defects in arsenate resistance were generated by nitrosoguanidine mutagenesis. The arsRBCC operons were intact in both mutant strains, but each mutant had one point mutation in the single arsC gene. Mutants transformed with either the arsC1 gene or the arsRBCC operon displayed wild-type arsenate resistance, indicating that the two arsC genes were equivalently functional in the sulfate reducer. The arsC1 gene and arsRBCC operon were also cloned into Escherichia coli DH5alpha independently, with either DNA fragment conferring increased arsenate resistance. The recombinant arsRBCC operon allowed growth at up to 50 mM arsenate in LB broth. Quantitative PCR analysis of mRNA products showed that the single arsC1 was constitutively expressed, whereas the operon was under the control of the arsR repressor protein. We suggest a model for arsenate detoxification in which the product of the single arsC1 is first used to reduce arsenate. The arsenite formed is then available to induce the arsRBCC operon for more rapid arsenate detoxification.

Genome-shuffling improved acid tolerance and l-lactic acid volumetric productivity in Lactobacillus rhamnosus

Genome shuffling is an efficient approach for the rapid improvement of industrially important microbial phenotypes. Here we improved the acid tolerance and volumetric productivity of an industrial strain Lactobacillus rhamnosus ATCC 11443 by genome shuffling. Five strains with subtle improvements in pH tolerance and volumetric productivity were obtained from the populations generated by ultraviolet irradiation and nitrosoguanidine mutagenesis, and then they were subjected for recursive protoplast fusion. A library that was more likely to yield positive colonies was created by fusing the lethal protoplasts obtained from both ultraviolet irradiation and heat treatments. After three rounds of genome shuffling, four strains that could grow at pH 3.6 were obtained. We observed 3.1- and 2.6-fold increases in lactic acid production and cell growth of the best performing at pH 3.8, respectively. The maximum volumetric productivity was 5.77+/-0.05 g/lh when fermented with 10% glucose under neutralizing condition with CaCO(3), which was 26.5+/-1.5% higher than the wild type.

Identification of alkane monoxygenase genes inAcinetobacter venetianus VE-C3 and analysis of mutants impaired in diesel fuel degradation

Cells ofAcinetobacter venetianus strain VE-C3 are able to degrade diesel fuel oil by a complex mechanism requiring the formation of cell aggregates and their further adhesion to fuel oil drops. In this work the biodegradation process inA. venetianus was studied by a combination of genetic, molecular and physiological methods. PCR amplification, sequencing and Southern blot analysis ofalkM andrubA genes coding for the alkane hydroxylase and rubredoxin were carried out. Then, 22 Alk− mutants impaired in diesel fuel degradation were obtained by nitrosoguanidine mutagenesis and characterised by i) growth on alkanes as sole carbon and energy sources, ii) modification of cell electrophoretic properties, and iii) analysis of plasmid content. Data obtained revealed that the genetic determinants for alkane degradation are located on both the chromosome and the two plasmids harboured by VE-C3 strain (pAV1 and pAV2, 11 Kbp and 15 kbp, respectively). This organization of genes coding for alkane monoxygenase complex seems to be similar to the arrangement found in Acinetobacter sp. strains ADP1 and M1, where genes are scattered through the chromosome but, as a novelty, that some genes involved in hydrocarbon degradation are plasmid borne also.

Characterization of Laboratory Mutants of Botrytis cinerea Resistant to QoI Fungicides

Mutants of Botrytis cinerea with moderate and high resistance to pyraclostrobin, a Qo inhibitor of mitochondrial electron transport at the cytochrome bc1 complex, were isolated at a high mutation frequency, after nitrosoguanidine mutagenesis and selection on medium containing pyraclostrobin and salicylhydroxamate (SHAM), a specific inhibitor of cyanide-resistant (alternative) respiration. Oxygen uptake in whole cells was strongly inhibited in the wild-type strain by pyraclostrobin and SHAM, but not in the mutant isolates. Cross-resistance studies with other Qo and Qi inhibitors (QoIs and QiIs) of cytochrome bc1 complex of mitochondrial respiration showed that the mutation(s) for resistance to pyraclostrobin also reduced the sensitivity of mutant strains to other QoIs as azoxystrobin, fluoxastrobin, trifloxystrobin and picoxystrobin, but not to famoxadone and to the QiIs cyazofamid and antimycin-A. An increased sensitivity of pyraclostrobin-resistant strains to the carboxamide boscalid, an inhibitor of complex II, and to the anilinopyrimidine cyprodinil, a methionine biosynthesis inhibitor, was observed. Moreover, no effect of pyraclostrobin resistance mutation(s) on fungitoxicity of the hydroxyanilide fenhexamid, the phenylpyrrole fludioxonil, the benzimidazole benomyl, and to the phenylpyridinamine fluazinam, which affect other cellular pathways, was observed. Study of fitness parameters in the wild-type and pyraclostrobin-resistant mutants of B. cinerea showed that most mutants had a significant reduction in the sporulation, conidial germination and sclerotia production. Experiments on the stability of the pyraclostrobin-resistant phenotype showed a reduction of resistance, mainly in moderate resistant strains, when the mutants were grown on inhibitor-free medium. However, a rapid recovery of the resistance level was observed after the mutants were returned to a selective medium. Studies on the competitive ability of mutant isolates against the wild-type parent strain, by applications of a mixed conidial population, showed that, in vitro, all mutants were less competitive than the wild-type strain. However, the competitive ability of high resistant mutants was higher than the moderate ones. Pathogenicity tests on cucumber seedlings showed that all mutant strains tested exhibited an infection ability similar with the wild-type parent strain. Preventive applications of the commercial product of F-500 25EC (pyraclostrobin) were effective against lesion development on cotyledons by the wild-type, but ineffective, even at high concentrations, against disease caused by the pyraclostrobin-resistant isolates. Boscalid (F-510 50WG) was found equally effective against the disease caused by the wild-type or pyraclostrobin-resistant mutants. This is the first report indicating the appearance of B. cinerea strains resistant to QoI fungicides by the biochemical mechanism of site modification and the risk for field resistance.