ABSTRACT Three phenotypes were identified among benomyl-resistant strains of Botrytis cinerea in ... more ABSTRACT Three phenotypes were identified among benomyl-resistant strains of Botrytis cinerea in Israel when tested for sensitivity to carbendazim (M BC) and diethofencarb (NPC): Ben(HR)NPC(S) = highly resistant to MBC (50% effective concentration [EC(50)] > 50 mu g/ml) and sensitive to 0.5 mu g/ml NPC; Ben(MR)NPC(R) = moderately resistant to MBC (10 less than or equal to EC(50) < 20 mu g/ml) and resistant to 10 mu g/ml NPC; and Ben(HR)NPC(R) = highly resistant to MBC and resistant to NPC. A 1-kb fragment of the wild-type gene encoding for beta-tubulin (designated benA) in B. cinerea was cloned and sequenced. The deduced partial amino acid sequence of the B. cinerea beta-tubulin showed a high degree of similarity to beta-tubulins of other filamentous fungi. A polymerase chain reaction approach was used to amplify and sequence 992-bp benA fragments from strains representing the three phenotypes. in the eight Ben(R) strains analyzed, three single base-pair mutations were identified and found to correlate with the different phenotypes: codon 198, encoding glutamic acid in the wild type, was changed to an alanine codon in the Ben(HR)NPC(S) phenotype or to a lysine codon in the Ben(HR)NPC(R) phenotype; codon 200, encoding phenylalanine, was changed to a tyrosine codon in the Ben(MR)NPC(R) phenotype. These mutations were similar to those identified in benomyl-resistant field strains of other phytopathogenic fungi.
ABSTRACT A simple and rapid bioassay for the direct determination of carbendazim residues in soil... more ABSTRACT A simple and rapid bioassay for the direct determination of carbendazim residues in soil was developed. Pellets composed of mixtures of soil (200–500 mg) and agar were placed on an agar medium pre–inoculated with the test organism Penicillium digitatum. After cold pre–incubation followed by incubation at 27°C, the size of the inhibition zone was determined. The lowest detectable concentrations of carbendazim and thiabendazole in a sandy soil were 0.25 and 10 μg/g, respectively. The lowest detectable concentration was higher in heavy soils. In a study of carbendazim degradation in soil, chemical analysis and the pellet bioassay technique yielded similar results. This technique requires only small quantities of soil, without the need for soil extraction.
Ten beta-trifluoroalkyl aminovinyl ketone derivatives were synthesized, and their inhibitory effe... more Ten beta-trifluoroalkyl aminovinyl ketone derivatives were synthesized, and their inhibitory effects on several phytopathogenic fungi, an oomycete and plants were assessed. The various compounds were fungitoxic at the 10-100 microM range, with (Z)-3-amino-4,4,4-trifluoro-1-(4-chlorophenyl)but-2-en-1-one exhibiting the highest inhibitory effect on most of the test pathogens. Alternaria alternata and Neurospora crassa were the most tolerant and sensitive fungi to the compounds, respectively. We propose that (Z)-3-amino-4,4,4-trifluoro-1-phenylbut-2-en-1-one is the minimal structural requirement for a beta-trifluoroalkyl aminovinyl ketone fungitoxic derivative.
Although Mn(2+) is the most abundant substrate of versatile peroxidases (VPs), repression of Pleu... more Although Mn(2+) is the most abundant substrate of versatile peroxidases (VPs), repression of Pleurotus osteatus vp1 expression occurred in Mn(2+) sufficient medium. This seems to be a "biological contradiction". The aim of this study was to explore the mechanism of direct oxidation by VP1 under Mn(2+)-deficient conditions, as it was found to be the predominant enzyme during fungal growth in the presence of synthetic and natural substrates. The native VP1 was purified and characterized using three substrates: Mn(2+), Orange II (OII) and Reactive Black 5 (RB5), each oxidized by a different active site in the enzyme. While the pH optimum for Mn(2+) oxidation is 5, the optimum pH for direct oxidation of both dyes was found to be 3. Indeed, effective in vivo decolorization occurred in media without addition of Mn(2+) only under acidic conditions. We have determined that Mn(2+) inhibits in vitro the direct oxidation of both OII and RB5 while RB5 stabilizes both Mn(2+) and OII oxidation. Furthermore, OII was found to inhibit the oxidation of both Mn(2+) and RB5. In addition, we could demonstrate that VP1 can cleave OII in two different modes. Under Mn(2+)-mediated oxidation conditions, VP1 was able to cleave the azo-bond only in an asymmetric mode while at the optimum conditions for direct oxidation (absence of Mn(2+) at pH 3) both symmetric and asymmetric cleavages. We concluded that the oxidation mechanism of aromatic compounds by VP1 is controlled by Mn(2+) and pH levels both in the growth medium and in the reaction mixture. Versatile peroxidases 1 (VP1) is a member of the ligninolytic heme peroxidase gene family of the white rot fungus Pleurotus ostreatus and plays fundamental role in biodegradation. This enzyme exhibit versatile nature as it can oxidize different substrate under altered environmental conditions. VPs are highly interesting enzymes due to the fact that they contain unique active sites which are responsible for direct oxidation of various aromatic compounds including lignin, in addition to the well-known Mn(2+) binding active site. This study demonstrates the limits of versatility of P. ostreatus VP1 which harbors multiple active sites, exhibiting a broad range of enzymatic activities, but perform differently under distinct conditions. The versatility of P. ostreatus and its enzymes is an advantageous factor in the fungal ability to adapt to changing environments. This trait expands the possibilities for potential utilization of P. ostreatus and other WRF.
The new tetraglycosylceramide neurosporaside (1a) has been isolated from the fungus Neurospora cr... more The new tetraglycosylceramide neurosporaside (1a) has been isolated from the fungus Neurospora crassa. Neurosporaside is a tetraglycosylated glycosphingolipid characterized by a sugar chain unprecedented among natural glycoconjugates. The structure of neurosporaside was elucidated by extensive spectroscopic analysis and microscale degradation analysis, which allowed full structure elucidation using less than 1 mg of compound.
The soil-borne, asexual fungus Fusarium oxysporum f.sp. melonis (FOM) is a causal agent of muskme... more The soil-borne, asexual fungus Fusarium oxysporum f.sp. melonis (FOM) is a causal agent of muskmelon wilt disease. The current study focused on the most virulent race of FOM-race 1,2. The tagged mutant D122, generated by Agrobacterium tumefaciens-mediated transformation, caused the delayed appearance of initial wilt disease symptoms, as well as a 75% reduction in pathogenicity. D122 was impaired in the gene product homologous to the Snt2-like transcription factor of Schizosaccharomyces pombe. Involvement of snt2 in the early stage of FOM pathogenesis and its requirement for host colonization were confirmed by targeted disruption followed by quantitative reverse transcription-polymerase chain reaction analysis of snt2 expression in planta. Δsnt2 mutants of FOM and Neurospora crassa exhibited similar morphological abnormalities, including a reduction in conidia production and biomass accumulation, slower vegetative growth and frequent hyphal septation. In N. crassa, snt-2 is required for sexual development, as Δsnt-2 mutants were unable to produce mature perithecia. Suppressive subtraction hybridization analysis of the D122 mutant versus wild-type isolate detected four genes (idi4, pdc, msf1, eEF1G) that were found previously in association with the target of rapamycin (TOR) kinase pathway. Expression of the autophagy-related idi4 and pdc genes was found to be up-regulated in the Δsnt2 FOM mutant. In N. crassa, disruption of snt-2 also conferred a significant over-expression of idi4.
The gene and cDNA of a novel protein phosphatase were cloned from Neurospora crassa. The pzl-1 ge... more The gene and cDNA of a novel protein phosphatase were cloned from Neurospora crassa. The pzl-1 gene encompasses three introns and is localized to the left arm of chromosome I between cyt-21 and Fsr-12. It encodes a protein of 58.3 kDa containing a Ser/Pro rich N-terminal segment, and a C-terminal domain that is similar to the catalytic subunit of type 1 protein phosphatases. The first 51 amino acid residues, including a potential N-myristoylation site, as well as the C-terminal domain (about 300 residues) have a high level of sequence identity with yeast PPZ phosphatases. However, residues 52–208 do not share high similarity with other proteins. The mRNA of pzl-1 was detected in all phases of asexual development of the filamentous fungus.
ABSTRACT Three phenotypes were identified among benomyl-resistant strains of Botrytis cinerea in ... more ABSTRACT Three phenotypes were identified among benomyl-resistant strains of Botrytis cinerea in Israel when tested for sensitivity to carbendazim (M BC) and diethofencarb (NPC): Ben(HR)NPC(S) = highly resistant to MBC (50% effective concentration [EC(50)] > 50 mu g/ml) and sensitive to 0.5 mu g/ml NPC; Ben(MR)NPC(R) = moderately resistant to MBC (10 less than or equal to EC(50) < 20 mu g/ml) and resistant to 10 mu g/ml NPC; and Ben(HR)NPC(R) = highly resistant to MBC and resistant to NPC. A 1-kb fragment of the wild-type gene encoding for beta-tubulin (designated benA) in B. cinerea was cloned and sequenced. The deduced partial amino acid sequence of the B. cinerea beta-tubulin showed a high degree of similarity to beta-tubulins of other filamentous fungi. A polymerase chain reaction approach was used to amplify and sequence 992-bp benA fragments from strains representing the three phenotypes. in the eight Ben(R) strains analyzed, three single base-pair mutations were identified and found to correlate with the different phenotypes: codon 198, encoding glutamic acid in the wild type, was changed to an alanine codon in the Ben(HR)NPC(S) phenotype or to a lysine codon in the Ben(HR)NPC(R) phenotype; codon 200, encoding phenylalanine, was changed to a tyrosine codon in the Ben(MR)NPC(R) phenotype. These mutations were similar to those identified in benomyl-resistant field strains of other phytopathogenic fungi.
ABSTRACT A simple and rapid bioassay for the direct determination of carbendazim residues in soil... more ABSTRACT A simple and rapid bioassay for the direct determination of carbendazim residues in soil was developed. Pellets composed of mixtures of soil (200–500 mg) and agar were placed on an agar medium pre–inoculated with the test organism Penicillium digitatum. After cold pre–incubation followed by incubation at 27°C, the size of the inhibition zone was determined. The lowest detectable concentrations of carbendazim and thiabendazole in a sandy soil were 0.25 and 10 μg/g, respectively. The lowest detectable concentration was higher in heavy soils. In a study of carbendazim degradation in soil, chemical analysis and the pellet bioassay technique yielded similar results. This technique requires only small quantities of soil, without the need for soil extraction.
Ten beta-trifluoroalkyl aminovinyl ketone derivatives were synthesized, and their inhibitory effe... more Ten beta-trifluoroalkyl aminovinyl ketone derivatives were synthesized, and their inhibitory effects on several phytopathogenic fungi, an oomycete and plants were assessed. The various compounds were fungitoxic at the 10-100 microM range, with (Z)-3-amino-4,4,4-trifluoro-1-(4-chlorophenyl)but-2-en-1-one exhibiting the highest inhibitory effect on most of the test pathogens. Alternaria alternata and Neurospora crassa were the most tolerant and sensitive fungi to the compounds, respectively. We propose that (Z)-3-amino-4,4,4-trifluoro-1-phenylbut-2-en-1-one is the minimal structural requirement for a beta-trifluoroalkyl aminovinyl ketone fungitoxic derivative.
Although Mn(2+) is the most abundant substrate of versatile peroxidases (VPs), repression of Pleu... more Although Mn(2+) is the most abundant substrate of versatile peroxidases (VPs), repression of Pleurotus osteatus vp1 expression occurred in Mn(2+) sufficient medium. This seems to be a "biological contradiction". The aim of this study was to explore the mechanism of direct oxidation by VP1 under Mn(2+)-deficient conditions, as it was found to be the predominant enzyme during fungal growth in the presence of synthetic and natural substrates. The native VP1 was purified and characterized using three substrates: Mn(2+), Orange II (OII) and Reactive Black 5 (RB5), each oxidized by a different active site in the enzyme. While the pH optimum for Mn(2+) oxidation is 5, the optimum pH for direct oxidation of both dyes was found to be 3. Indeed, effective in vivo decolorization occurred in media without addition of Mn(2+) only under acidic conditions. We have determined that Mn(2+) inhibits in vitro the direct oxidation of both OII and RB5 while RB5 stabilizes both Mn(2+) and OII oxidation. Furthermore, OII was found to inhibit the oxidation of both Mn(2+) and RB5. In addition, we could demonstrate that VP1 can cleave OII in two different modes. Under Mn(2+)-mediated oxidation conditions, VP1 was able to cleave the azo-bond only in an asymmetric mode while at the optimum conditions for direct oxidation (absence of Mn(2+) at pH 3) both symmetric and asymmetric cleavages. We concluded that the oxidation mechanism of aromatic compounds by VP1 is controlled by Mn(2+) and pH levels both in the growth medium and in the reaction mixture. Versatile peroxidases 1 (VP1) is a member of the ligninolytic heme peroxidase gene family of the white rot fungus Pleurotus ostreatus and plays fundamental role in biodegradation. This enzyme exhibit versatile nature as it can oxidize different substrate under altered environmental conditions. VPs are highly interesting enzymes due to the fact that they contain unique active sites which are responsible for direct oxidation of various aromatic compounds including lignin, in addition to the well-known Mn(2+) binding active site. This study demonstrates the limits of versatility of P. ostreatus VP1 which harbors multiple active sites, exhibiting a broad range of enzymatic activities, but perform differently under distinct conditions. The versatility of P. ostreatus and its enzymes is an advantageous factor in the fungal ability to adapt to changing environments. This trait expands the possibilities for potential utilization of P. ostreatus and other WRF.
The new tetraglycosylceramide neurosporaside (1a) has been isolated from the fungus Neurospora cr... more The new tetraglycosylceramide neurosporaside (1a) has been isolated from the fungus Neurospora crassa. Neurosporaside is a tetraglycosylated glycosphingolipid characterized by a sugar chain unprecedented among natural glycoconjugates. The structure of neurosporaside was elucidated by extensive spectroscopic analysis and microscale degradation analysis, which allowed full structure elucidation using less than 1 mg of compound.
The soil-borne, asexual fungus Fusarium oxysporum f.sp. melonis (FOM) is a causal agent of muskme... more The soil-borne, asexual fungus Fusarium oxysporum f.sp. melonis (FOM) is a causal agent of muskmelon wilt disease. The current study focused on the most virulent race of FOM-race 1,2. The tagged mutant D122, generated by Agrobacterium tumefaciens-mediated transformation, caused the delayed appearance of initial wilt disease symptoms, as well as a 75% reduction in pathogenicity. D122 was impaired in the gene product homologous to the Snt2-like transcription factor of Schizosaccharomyces pombe. Involvement of snt2 in the early stage of FOM pathogenesis and its requirement for host colonization were confirmed by targeted disruption followed by quantitative reverse transcription-polymerase chain reaction analysis of snt2 expression in planta. Δsnt2 mutants of FOM and Neurospora crassa exhibited similar morphological abnormalities, including a reduction in conidia production and biomass accumulation, slower vegetative growth and frequent hyphal septation. In N. crassa, snt-2 is required for sexual development, as Δsnt-2 mutants were unable to produce mature perithecia. Suppressive subtraction hybridization analysis of the D122 mutant versus wild-type isolate detected four genes (idi4, pdc, msf1, eEF1G) that were found previously in association with the target of rapamycin (TOR) kinase pathway. Expression of the autophagy-related idi4 and pdc genes was found to be up-regulated in the Δsnt2 FOM mutant. In N. crassa, disruption of snt-2 also conferred a significant over-expression of idi4.
The gene and cDNA of a novel protein phosphatase were cloned from Neurospora crassa. The pzl-1 ge... more The gene and cDNA of a novel protein phosphatase were cloned from Neurospora crassa. The pzl-1 gene encompasses three introns and is localized to the left arm of chromosome I between cyt-21 and Fsr-12. It encodes a protein of 58.3 kDa containing a Ser/Pro rich N-terminal segment, and a C-terminal domain that is similar to the catalytic subunit of type 1 protein phosphatases. The first 51 amino acid residues, including a potential N-myristoylation site, as well as the C-terminal domain (about 300 residues) have a high level of sequence identity with yeast PPZ phosphatases. However, residues 52–208 do not share high similarity with other proteins. The mRNA of pzl-1 was detected in all phases of asexual development of the filamentous fungus.
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