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Collina M. - One of the best experts on this subject based on the ideXlab platform.

  • ROLE OF SINGLE SITE-SPECIFIC ALLELE REPLACEMENT INTO SVHK1 LOCUS IN THE STUDY OF STEMPHYLIUM VESICARIUM Dicarboximide AND PHENYLPYRROLE Fungicides RESISTANCE
    2016
    Co-Authors: Gazzetti K., Brunelli A., Ciriani A., Collina M.
    Abstract:

    Stemphylium vesicarium is the fungal agent of pear Brown Spot and its resistance to Dicarboximide Fungicides has been a known concerning phenomenon since the 1990s. Henceforward, pear orchards have been monitored and field strains have been tested by mycelial growth inhibition assays to understand the sensitivity to Dicarboximide and phenylpyrrole Fungicides. Four phenotype classes were recognized according to in vitro responses to procymidone and iprodione: S (sensitive), S+ (low resistance), R1 (moderate resistance), R2 (high resistance). Cross-resistance to fludioxonil was only detected in R2 phenotype. Previous molecular studies correlated Dicarboximide resistance class with single aminoacid substitutions observed in a two-component histidine kinase (HK1), corresponding to single nucleotide polymorphism (SNPs) in the nucleotidic sequence of SvHK1 gene [1]. The goal of this ongoing study is to define the role of SNPs in SvHK1 sequence on Dicarboximide resistance by the replacement of the S allele with S+, R1 or R2 alleles. A reference sensitive strain was selected through biological and molecular assays and DNA was properly extracted and Fusion PCR technique was used to build the linear disruption vector (KOSvHK1). Fungal protoplast were obtained by enzymatic lysis of cell wall and transformed. KOSvHK1 replacement of SvHK1 gene produced null mutants which were able to grow up on Hygromycin B. Transformants are currently screened for unique, complete and site-specific insertion of KOSvHK1 using PCR-based methods and Southern Blotting assays. Interesting mutants will be transformed with linear complementation vectors, and complemented strains will be tested for the expected acquired resistance level. Assessment of the role of SNP mutations in SvHK1 sequence in S. vesicarium resistant phenotypes to Dicarboximides will allows us to develop a RealTime PCR assay to quickly determine resistant allele-frequency in monitored populations. The results obtained so far will increase the possibility of quantify, prevent and manage the iprodione and fludioxonil resistance risk in the field

  • Role of single site-specific allele replacement into svhk1 locus in the study of Stemphylium vesicarium Dicarboximide and phenylpyrrole Fungicides resistance
    2015
    Co-Authors: Gazzetti K., Brunelli A., Ciriani A., Collina M.
    Abstract:

    Stemphylium vesicarium is the fungal agent of pear Brown Spot and its resistance to Dicarboximide Fungicides has been a known concerning phenomenon since the 1990s. Henceforward, pear orchards have been monitored and field strains have been tested by mycelial growth inhibition assays to understand the sensitivity to Dicarboximide and phenylpyrrole Fungicides. Four phenotype classes were recognized according to in vitro responses to procymidone and iprodione: S (sensitive), S+ (low resistance), R1 (moderate resistance), R2 (high resistance). Cross-resistance to fludioxonil was only detected in R2 phenotype. Previous molecular studies correlated Dicarboximide resistance class with single aminoacid substitutions observed in a two-component histidine kinase (HK1), corresponding to single nucleotide polymorphism (SNPs) in the nucleotidic sequence of SvHK1 gene (Alberoni et al., 2010). The goal of this ongoing study is to define the role of known SNPs in SvHK1 sequence on Dicarboximide resistance by the replacement of the S allele with S+, R1 or R2 alleles. ..........................

  • Field strains of Stemphylium vesicarium with a resistance to Dicarboximide Fungicides correlated with changes in a two-component histidine kinase
    2010
    Co-Authors: Alberoni G., Collina M., Lanen C., Leroux P., Brunelli A.
    Abstract:

    Abstract In Stemphylium vesicarium, four phenotypes were recognized according to their in vitro responses to Dicarboximide Fungicides: S (sensitive), S+ (low resistant to iprodione and procymidone but moderately resistant to vinclozolin), R1 (moderately resistant to iprodione and vinclozolin but highly resistant to procymidone), R2 (highly resistant to all Dicarboximides). Cross-resistance was observed between Dicarboximides and aromatic hydrocarbon Fungicides in all cases while crossresistance to phenylpyrroles was only detected in R2 phenotype. Moreover, no changes were noted in sensitivity to oxidative and osmotic stress inducers. An osmosensing histidine kinase gene, homologous to OS1 from Neurospora crassa, was sequenced from several field isolates of Stemphylium vesicarium. This gene is predicted to encode a 1,329 amino acid protein, comprising a conserved histidine-kinase domain in the C-terminal region and six tandem repeats of about 90 amino acids at the N-terminal end. In S+ and R1 phenotype isolates, a single amino acid substitution was observed in the first amino acid repeat; F267L and L290S respectively. For the R2 isolates, the exchanges T765R or Q777R were located within the histidinekinase domain

Brunelli A. - One of the best experts on this subject based on the ideXlab platform.

  • ROLE OF SINGLE SITE-SPECIFIC ALLELE REPLACEMENT INTO SVHK1 LOCUS IN THE STUDY OF STEMPHYLIUM VESICARIUM Dicarboximide AND PHENYLPYRROLE Fungicides RESISTANCE
    2016
    Co-Authors: Gazzetti K., Brunelli A., Ciriani A., Collina M.
    Abstract:

    Stemphylium vesicarium is the fungal agent of pear Brown Spot and its resistance to Dicarboximide Fungicides has been a known concerning phenomenon since the 1990s. Henceforward, pear orchards have been monitored and field strains have been tested by mycelial growth inhibition assays to understand the sensitivity to Dicarboximide and phenylpyrrole Fungicides. Four phenotype classes were recognized according to in vitro responses to procymidone and iprodione: S (sensitive), S+ (low resistance), R1 (moderate resistance), R2 (high resistance). Cross-resistance to fludioxonil was only detected in R2 phenotype. Previous molecular studies correlated Dicarboximide resistance class with single aminoacid substitutions observed in a two-component histidine kinase (HK1), corresponding to single nucleotide polymorphism (SNPs) in the nucleotidic sequence of SvHK1 gene [1]. The goal of this ongoing study is to define the role of SNPs in SvHK1 sequence on Dicarboximide resistance by the replacement of the S allele with S+, R1 or R2 alleles. A reference sensitive strain was selected through biological and molecular assays and DNA was properly extracted and Fusion PCR technique was used to build the linear disruption vector (KOSvHK1). Fungal protoplast were obtained by enzymatic lysis of cell wall and transformed. KOSvHK1 replacement of SvHK1 gene produced null mutants which were able to grow up on Hygromycin B. Transformants are currently screened for unique, complete and site-specific insertion of KOSvHK1 using PCR-based methods and Southern Blotting assays. Interesting mutants will be transformed with linear complementation vectors, and complemented strains will be tested for the expected acquired resistance level. Assessment of the role of SNP mutations in SvHK1 sequence in S. vesicarium resistant phenotypes to Dicarboximides will allows us to develop a RealTime PCR assay to quickly determine resistant allele-frequency in monitored populations. The results obtained so far will increase the possibility of quantify, prevent and manage the iprodione and fludioxonil resistance risk in the field

  • Role of single site-specific allele replacement into svhk1 locus in the study of Stemphylium vesicarium Dicarboximide and phenylpyrrole Fungicides resistance
    2015
    Co-Authors: Gazzetti K., Brunelli A., Ciriani A., Collina M.
    Abstract:

    Stemphylium vesicarium is the fungal agent of pear Brown Spot and its resistance to Dicarboximide Fungicides has been a known concerning phenomenon since the 1990s. Henceforward, pear orchards have been monitored and field strains have been tested by mycelial growth inhibition assays to understand the sensitivity to Dicarboximide and phenylpyrrole Fungicides. Four phenotype classes were recognized according to in vitro responses to procymidone and iprodione: S (sensitive), S+ (low resistance), R1 (moderate resistance), R2 (high resistance). Cross-resistance to fludioxonil was only detected in R2 phenotype. Previous molecular studies correlated Dicarboximide resistance class with single aminoacid substitutions observed in a two-component histidine kinase (HK1), corresponding to single nucleotide polymorphism (SNPs) in the nucleotidic sequence of SvHK1 gene (Alberoni et al., 2010). The goal of this ongoing study is to define the role of known SNPs in SvHK1 sequence on Dicarboximide resistance by the replacement of the S allele with S+, R1 or R2 alleles. ..........................

  • Field strains of Stemphylium vesicarium with a resistance to Dicarboximide Fungicides correlated with changes in a two-component histidine kinase
    2010
    Co-Authors: Alberoni G., Collina M., Lanen C., Leroux P., Brunelli A.
    Abstract:

    Abstract In Stemphylium vesicarium, four phenotypes were recognized according to their in vitro responses to Dicarboximide Fungicides: S (sensitive), S+ (low resistant to iprodione and procymidone but moderately resistant to vinclozolin), R1 (moderately resistant to iprodione and vinclozolin but highly resistant to procymidone), R2 (highly resistant to all Dicarboximides). Cross-resistance was observed between Dicarboximides and aromatic hydrocarbon Fungicides in all cases while crossresistance to phenylpyrroles was only detected in R2 phenotype. Moreover, no changes were noted in sensitivity to oxidative and osmotic stress inducers. An osmosensing histidine kinase gene, homologous to OS1 from Neurospora crassa, was sequenced from several field isolates of Stemphylium vesicarium. This gene is predicted to encode a 1,329 amino acid protein, comprising a conserved histidine-kinase domain in the C-terminal region and six tandem repeats of about 90 amino acids at the N-terminal end. In S+ and R1 phenotype isolates, a single amino acid substitution was observed in the first amino acid repeat; F267L and L290S respectively. For the R2 isolates, the exchanges T765R or Q777R were located within the histidinekinase domain

Gazzetti K. - One of the best experts on this subject based on the ideXlab platform.

  • ROLE OF SINGLE SITE-SPECIFIC ALLELE REPLACEMENT INTO SVHK1 LOCUS IN THE STUDY OF STEMPHYLIUM VESICARIUM Dicarboximide AND PHENYLPYRROLE Fungicides RESISTANCE
    2016
    Co-Authors: Gazzetti K., Brunelli A., Ciriani A., Collina M.
    Abstract:

    Stemphylium vesicarium is the fungal agent of pear Brown Spot and its resistance to Dicarboximide Fungicides has been a known concerning phenomenon since the 1990s. Henceforward, pear orchards have been monitored and field strains have been tested by mycelial growth inhibition assays to understand the sensitivity to Dicarboximide and phenylpyrrole Fungicides. Four phenotype classes were recognized according to in vitro responses to procymidone and iprodione: S (sensitive), S+ (low resistance), R1 (moderate resistance), R2 (high resistance). Cross-resistance to fludioxonil was only detected in R2 phenotype. Previous molecular studies correlated Dicarboximide resistance class with single aminoacid substitutions observed in a two-component histidine kinase (HK1), corresponding to single nucleotide polymorphism (SNPs) in the nucleotidic sequence of SvHK1 gene [1]. The goal of this ongoing study is to define the role of SNPs in SvHK1 sequence on Dicarboximide resistance by the replacement of the S allele with S+, R1 or R2 alleles. A reference sensitive strain was selected through biological and molecular assays and DNA was properly extracted and Fusion PCR technique was used to build the linear disruption vector (KOSvHK1). Fungal protoplast were obtained by enzymatic lysis of cell wall and transformed. KOSvHK1 replacement of SvHK1 gene produced null mutants which were able to grow up on Hygromycin B. Transformants are currently screened for unique, complete and site-specific insertion of KOSvHK1 using PCR-based methods and Southern Blotting assays. Interesting mutants will be transformed with linear complementation vectors, and complemented strains will be tested for the expected acquired resistance level. Assessment of the role of SNP mutations in SvHK1 sequence in S. vesicarium resistant phenotypes to Dicarboximides will allows us to develop a RealTime PCR assay to quickly determine resistant allele-frequency in monitored populations. The results obtained so far will increase the possibility of quantify, prevent and manage the iprodione and fludioxonil resistance risk in the field

  • Role of single site-specific allele replacement into svhk1 locus in the study of Stemphylium vesicarium Dicarboximide and phenylpyrrole Fungicides resistance
    2015
    Co-Authors: Gazzetti K., Brunelli A., Ciriani A., Collina M.
    Abstract:

    Stemphylium vesicarium is the fungal agent of pear Brown Spot and its resistance to Dicarboximide Fungicides has been a known concerning phenomenon since the 1990s. Henceforward, pear orchards have been monitored and field strains have been tested by mycelial growth inhibition assays to understand the sensitivity to Dicarboximide and phenylpyrrole Fungicides. Four phenotype classes were recognized according to in vitro responses to procymidone and iprodione: S (sensitive), S+ (low resistance), R1 (moderate resistance), R2 (high resistance). Cross-resistance to fludioxonil was only detected in R2 phenotype. Previous molecular studies correlated Dicarboximide resistance class with single aminoacid substitutions observed in a two-component histidine kinase (HK1), corresponding to single nucleotide polymorphism (SNPs) in the nucleotidic sequence of SvHK1 gene (Alberoni et al., 2010). The goal of this ongoing study is to define the role of known SNPs in SvHK1 sequence on Dicarboximide resistance by the replacement of the S allele with S+, R1 or R2 alleles. ..........................

Frank David - One of the best experts on this subject based on the ideXlab platform.

  • Stir bar sorptive extraction applied to the determination of Dicarboximide Fungicides in wine
    Journal of Chromatography A, 2001
    Co-Authors: Pat Sandra, Bart Tienpont, Joeri Vercammen, Andreas G. J. Tredoux, Tom Sandra, Frank David
    Abstract:

    Abstract The Dicarboximide Fungicides vinclozolin, iprodione and procymidone were analyzed in white wines using stir bar sorptive extraction (SBSE) in combination with thermal desorption–capillary GC–MS analysis (TD–cGC–MS). The method was optimized using spiked water samples in a concentration range between 0.5 and 100 μg/l. Iprodione was measured as its degradation product 3,5-dichlorophenyl hydantoin. Limits of quantification in the full scan MS mode are 0.5 μg/l for vinclozolin and procymidone and 5 μg/l for iprodione. In the ion monitoring mode, concentrations 100 times lower can be dosed. Because of wine matrix effects on the recoveries, quantification of the target Fungicides in wine had to be carried out by standard addition. For the thermolabile iprodione, the accuracy of SBSE–TD–cGC–MS was verified using SBSE followed by liquid desorption and analysis by liquid chromatography–atmospheric pressure chemical ionization mass spectroscopy. Procymidone and iprodione were detected in wines in concentrations up to 65 μg/l while the highest concentration of vinclozolin detected was smaller than 3 μg/l.

  • Short communication Stir bar sorptive extraction applied to the determination of Dicarboximide Fungicides in wine
    2001
    Co-Authors: Pat Sandra, Bart Tienpont, Joeri Vercammen, Andreas G. J. Tredoux, Tom Sandra, Frank David
    Abstract:

    The Dicarboximide Fungicides vinclozolin, iprodione and procymidone were analyzed in white wines using stir bar sorptive extraction (SBSE) in combination with thermal desorption-capillary GC-MS analysis (TD-cGC-MS). The method was optimized using spiked water samples in a concentration range between 0.5 and 100 mg / l. Iprodione was measured as its degradation product 3,5-dichlorophenyl hydantoin. Limits of quantification in the full scan MS mode are 0.5 mg / l for vinclozolin and procymidone and 5 mg / l for iprodione. In the ion monitoring mode, concentrations 100 times lower can be dosed. Because of wine matrix effects on the recoveries, quantification of the target Fungicides in wine had to be carried out by standard addition. For the thermolabile iprodione, the accuracy of SBSE-TD-cGC-MS was verified using SBSE followed by liquid desorption and analysis by liquid chromatography-atmospheric pressure chemical ionization mass spectroscopy. Procymidone and iprodione were detected in wines in concentrations up to 65 mg / l while the highest concentration of vinclozolin detected was smaller than 3 m g/l . © 2001 Elsevier Science B.V. All rights reserved.

Ciriani A. - One of the best experts on this subject based on the ideXlab platform.

  • ROLE OF SINGLE SITE-SPECIFIC ALLELE REPLACEMENT INTO SVHK1 LOCUS IN THE STUDY OF STEMPHYLIUM VESICARIUM Dicarboximide AND PHENYLPYRROLE Fungicides RESISTANCE
    2016
    Co-Authors: Gazzetti K., Brunelli A., Ciriani A., Collina M.
    Abstract:

    Stemphylium vesicarium is the fungal agent of pear Brown Spot and its resistance to Dicarboximide Fungicides has been a known concerning phenomenon since the 1990s. Henceforward, pear orchards have been monitored and field strains have been tested by mycelial growth inhibition assays to understand the sensitivity to Dicarboximide and phenylpyrrole Fungicides. Four phenotype classes were recognized according to in vitro responses to procymidone and iprodione: S (sensitive), S+ (low resistance), R1 (moderate resistance), R2 (high resistance). Cross-resistance to fludioxonil was only detected in R2 phenotype. Previous molecular studies correlated Dicarboximide resistance class with single aminoacid substitutions observed in a two-component histidine kinase (HK1), corresponding to single nucleotide polymorphism (SNPs) in the nucleotidic sequence of SvHK1 gene [1]. The goal of this ongoing study is to define the role of SNPs in SvHK1 sequence on Dicarboximide resistance by the replacement of the S allele with S+, R1 or R2 alleles. A reference sensitive strain was selected through biological and molecular assays and DNA was properly extracted and Fusion PCR technique was used to build the linear disruption vector (KOSvHK1). Fungal protoplast were obtained by enzymatic lysis of cell wall and transformed. KOSvHK1 replacement of SvHK1 gene produced null mutants which were able to grow up on Hygromycin B. Transformants are currently screened for unique, complete and site-specific insertion of KOSvHK1 using PCR-based methods and Southern Blotting assays. Interesting mutants will be transformed with linear complementation vectors, and complemented strains will be tested for the expected acquired resistance level. Assessment of the role of SNP mutations in SvHK1 sequence in S. vesicarium resistant phenotypes to Dicarboximides will allows us to develop a RealTime PCR assay to quickly determine resistant allele-frequency in monitored populations. The results obtained so far will increase the possibility of quantify, prevent and manage the iprodione and fludioxonil resistance risk in the field

  • Role of single site-specific allele replacement into svhk1 locus in the study of Stemphylium vesicarium Dicarboximide and phenylpyrrole Fungicides resistance
    2015
    Co-Authors: Gazzetti K., Brunelli A., Ciriani A., Collina M.
    Abstract:

    Stemphylium vesicarium is the fungal agent of pear Brown Spot and its resistance to Dicarboximide Fungicides has been a known concerning phenomenon since the 1990s. Henceforward, pear orchards have been monitored and field strains have been tested by mycelial growth inhibition assays to understand the sensitivity to Dicarboximide and phenylpyrrole Fungicides. Four phenotype classes were recognized according to in vitro responses to procymidone and iprodione: S (sensitive), S+ (low resistance), R1 (moderate resistance), R2 (high resistance). Cross-resistance to fludioxonil was only detected in R2 phenotype. Previous molecular studies correlated Dicarboximide resistance class with single aminoacid substitutions observed in a two-component histidine kinase (HK1), corresponding to single nucleotide polymorphism (SNPs) in the nucleotidic sequence of SvHK1 gene (Alberoni et al., 2010). The goal of this ongoing study is to define the role of known SNPs in SvHK1 sequence on Dicarboximide resistance by the replacement of the S allele with S+, R1 or R2 alleles. ..........................