Two Mutations in β-Tubulin 2 Gene Associated with Thiophanate-Methyl Resistance in Colletotrichum cereale Isolates from Creeping Bentgrass in Mississippi and Alabama

Turfgrass anthracnose, caused by Colletotrichum cereale (≡C. graminicola), has become a common disease of creeping bentgrass putting greens during the summer in Mississippi and Alabama over the last 15 years. Thiophanate-methyl is a single-site mode-of-action fungicide applied to control C. cereale. In vitro bioassays were performed to evaluate the sensitivity of 103 isolates to thiophanate-methyl concentrations ranging from 0.039 to 10 μg/ml. Eighty-three isolates were collected from creeping bentgrass in Mississippi and Alabama that had been exposed to thiophanate-methyl. An additional 20 isolates were included from nonexposed turfgrasses. Radial colony growth in amended media was relative to nonamended media for all in vitro bioassays. With thiophanate-methyl at 10 μg/ml, relative growth of exposed isolates ranged from 77.5 to 130.7% with a mean of 99.3% compared with nonexposed, baseline isolates that ranged from 0.0 to 48.7% with a mean of 20.4%. A representative sample of thiophanate-methyl-exposed and nonexposed isolates was used to determine the mechanism of resistance by comparing amino acid sequences of the β-tubulin 2 protein. All of the thiophanate-methyl-exposed isolates that were sequenced had a point mutation resulting in substitutions from glutamic acid to alanine at position 198 or from phenylalanine to tyrosine at position 200 of the β-tubulin 2 protein. These amino acid substitutions in C. cereale isolates from Mississippi and Alabama appear to confer resistance to thiophanate-methyl and differ from those reported previously for this pathogen.

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Occurrence and Molecular Identification of Azoxystrobin-Resistant Colletotrichum cereale Isolates from Golf Course Putting Greens in the Southern United States

Plant Disease ◽

10.1094/pdis-94-6-0751 ◽

2010 ◽

Vol 94 (6) ◽

pp. 751-757 ◽

Cited By ~ 16

Author(s):

Joseph R. Young ◽

Maria Tomaso-Peterson ◽

Lane P. Tredway ◽

Karla de la Cerda

Keyword(s):

United States ◽

Creeping Bentgrass ◽

Amino Acid Sequences ◽

Golf Course ◽

Southern United States ◽

Common Disease ◽

Putting Greens ◽

Qoi Fungicides ◽

Colletotrichum Cereale

Turfgrass anthracnose, caused by Colletotrichum cereale (≡C. graminicola), has become a common disease of creeping bentgrass and annual bluegrass putting greens throughout the southern United States. Strobilurin (QoI) fungicides such as azoxystrobin are single-site mode-of-action fungicides applied to control C. cereale. In vitro bioassays with azoxystrobin at 0.031 and 8 μg/ml incorporated into agar were performed to evaluate the sensitivity of 175 isolates collected from symptomatic turfgrasses in Alabama, Mississippi, North Carolina, Tennessee, and Virginia. Three sensitivity levels were identified among C. cereale isolates. Resistant, intermediately resistant, and sensitive isolates were characterized by percent relative growth based on the controls with means of 81, 23, and 4%, respectively, on media containing azoxystrobin at 8 μg/ml. The molecular mechanism of resistance was determined by comparing amino acid sequences of the cytochrome b protein. Compared with sensitive isolates, C. cereale isolates exhibiting QoI resistance had a G143A substitution, whereas isolates expressing intermediate resistance had a F129L substitution. C. cereale isolates displaying azoxystrobin resistance in vitro were not controlled by QoI fungicides in a field evaluation. The dominance of QoI-resistant C. cereale isolates identified in this study indicates a shift to resistant populations on highly managed golf course putting greens.

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Detection and Characterization of Benzimidazole Resistance in California Populations of Colletotrichum cereale

Plant Disease ◽

10.1094/pdis-92-2-0239 ◽

2008 ◽

Vol 92 (2) ◽

pp. 239-246 ◽

Cited By ~ 31

Author(s):

Francis P. Wong ◽

Karla A. de la Cerda ◽

Rufina Hernandez-Martinez ◽

Sharon L. Midland

Keyword(s):

Creeping Bentgrass ◽

Baseline Sensitivity ◽

Putting Greens ◽

Thiophanate Methyl ◽

Annual Bluegrass ◽

Sensitivity Distribution ◽

Exposed Population ◽

Colletotrichum Cereale

Colletotrichum cereale is the causal agent of turfgrass anthracnose, which has become a serious problem on annual bluegrass (Poa annua) and creeping bentgrass (Agrostis palustris) golf course putting greens. Thiophanate-methyl is a benzimidazole (methyl benzimidazole carbamate [MBC]) fungicide used for the management of anthracnose. In this study, we examined 481 isolates from 10 California populations to determine the presence and frequency of MBC resistance. An in vitro methodology was developed to construct a baseline sensitivity distribution using 60 isolates from an unexposed population (TCGC). The 50% effective dose (ED50) values for the baseline sensitivity distribution for thiophanate-methyl ranged from 0.14 to 2.3 μg/ml with a mean of 0.75 μg/ml. For 60 isolates assayed from an exposed population (AHCC), 57 isolates were not responsive to in vitro concentrations of thiophanate-methyl of up to 30 μg/ml. Isolates nonresponsive to thiophanate-methyl were not responsive to benomyl in vitro. Two isolates nonresponsive in vitro to thiophanate-methyl or benomyl were not controlled in vivo on annual bluegrass plants treated preventively with either fungicide at 11 mg/ml, confirming the results of the in vitro testing. The remaining 361 isolates from eight populations were tested using the single discriminatory dose of thiophanate-methyl at 10 μg/ml. A high proportion (>90%) of isolates from six of the populations were resistant to thiophanate-methyl, indicating the presence of practical resistance at these locations. To determine the molecular mechanism of MBC resistance, the two β-tubulin genes, TUB1 and TUB2, of 12 resistant and 6 sensitive isolates were amplified and sequenced, revealing a glutamic acid to lysine substitution at position 198 of TUB2 that was present in all resistant isolates. This work confirms the presence of MBC resistance in C. cereale populations from California and presents methods and information that can be used to manage resistance to the MBC fungicides and improve anthracnose management programs.

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Triclabendazole-resistant Fasciola hepatica: β-tubulin and response to in vitro treatment with triclabendazole

Parasitology ◽

10.1017/s003118200100124x ◽

2002 ◽

Vol 124 (3) ◽

pp. 325-338 ◽

Cited By ~ 78

Author(s):

M. W. ROBINSON ◽

A. TRUDGETT ◽

E. M. HOEY ◽

I. FAIRWEATHER

Keyword(s):

Amino Acid ◽

Fasciola Hepatica ◽

Amino Acid Sequences ◽

Single Amino Acid ◽

Parasitic Nematodes ◽

Transmission Electron ◽

Primary Amino ◽

Triclabendazole Resistance ◽

Β Tubulin

Resistance in Fasciola hepatica to triclabendazole (‘Fasinex’) has emerged in several countries. Benzimidazole resistance in parasitic nematodes has been linked to a single amino acid substitution (phenylalanine to tyrosine) at position 200 on the β-tubulin molecule. Sequencing of β-tubulin cDNAs from triclabendazole-susceptible and triclabendazole-resistant flukes revealed no amino acid differences between their respective primary amino acid sequences. In order to investigate the mechanism of triclabendazole resistance, triclabendazole-susceptible and triclabendazole-resistant flukes were incubated in vitro with triclabendazole sulphoxide (50 μg/ml). Scanning and transmission electron microscopy revealed extensive damage to the tegument of triclabendazole-susceptible F. hepatica, whereas triclabendazole-resistant flukes showed only localized and relatively minor disruption of the tegument covering the spines. Immunocytochemical studies, using an anti-tubulin antibody, showed that tubulin organization was disrupted in the tegument of triclabendazole-susceptible flukes. No such disruption was evident in triclabendazole-resistant F. hepatica. The significance of these findings is discussed with regard to the mechanism of triclabendazole resistance in F. hepatica.

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Charge influences substrate recognition and self-assembly of hydrophobic FG sequences

10.1101/090159 ◽

2016 ◽

Author(s):

Wesley G. Chen ◽

Jacob Witten ◽

Scott C. Grindy ◽

Niels Holten-Andersen ◽

Katharina Ribbeck

Keyword(s):

Amino Acid ◽

Self Assembly ◽

Amino Acid Sequences ◽

Nuclear Pore ◽

Selective Binding ◽

Charged Amino Acids ◽

Essential Components ◽

Transport Receptors

AbstractThe nuclear pore complex controls the passage of molecules via hydrophobic phenylalanine-glycine (FG) domains on nucleoporins. Such FG-domains consist of repeating units of FxFG, FG, or GLFG sequences, which can be interspersed with highly charged amino acid sequences. Despite the high density of charge exhibited in certain FG-domains, if and how charge influences FG-domain self-assembly and selective binding of nuclear transport receptors is largely unexplored. Studying how individual charged amino acids contribute to nuclear pore selectivity is challenging with modern in vivo and in vitro techniques due to the complexity of nucleoporin sequences. Here, we present a rationally designed approach to deconstruct essential components of nucleoporins down to 14 amino acid sequences. With these nucleoporin-based peptides, we systematically dissect how charge type and placement of charge influences self-assembly and selective binding of FG-containing gels. Specifically, we find that charge type determines which hydrophobic substrates FG sequences recognize while spatial localization of charge tunes hydrophobic self-assembly and receptor selectivity of FG sequences.

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In vitro and In Silico Approach For Characterization of Antimicrobial Peptide From Probiotics Against Staphylococcus Aureus and Escherichia Coli

10.21203/rs.3.rs-366314/v2 ◽

2021 ◽

Author(s):

Amrutha Bindu ◽

Lakshmi Devi

Keyword(s):

Amino Acid ◽

Antimicrobial Peptide ◽

Lactobacillus Plantarum ◽

Exchange Chromatography ◽

Amino Acid Sequences ◽

Proteinase K ◽

Kinetic Assay ◽

Wide Range

Abstract The focus of present study was to characterize antimicrobial peptide produced by probiotic cultures, Enterococcus durans DB-1aa (MCC4243), Lactobacillus plantarum Cu2-PM7 (MCC4246) and Lactobacillus fermentum Cu3-PM8 (MCC4233) against Staphylococus aureus and E. coli. The growth kinetic assay revealed 24 h of incubation to be optimum for bacteriocin production. The partially purified compound after ion-exchange chromatography was found to be thermoresistant and stable under wide range of pH. The compound was sensitive to proteinase-K, but resistant to trypsin, a-amylase and lipase. The apparent molecular weight of bacteriocin from MCC4243 and MCC4246 was found to be 3.5 KDa. Translated partial amino acid sequence of plnA gene in MCC4246 displayed 48 amino acid sequences showing 100% similarity with plantaricin A of Lactobacillus plantarum (WP_0036419). The sequence revealed 7 β sheets, 6 α sheets, 6 predicted coils and 9 predicted turns. The functions on cytoplasm show 10.82 isoelectric point and 48.6% hydrophobicity. The molecular approach of using Geneious Prime software and protein prediction data base for characterization of bacteriocin is novel and predicts “KSSAYSLQMGATAIKQVKKLFKKWGW” as peptide responsible for antimicrobial activity. The study provides information about broad spectrum bacteriocin in native probiotic culture and paves a way towards its application in functional foods as biopreservative agents.

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Differentiating closely affiliated Dehalococcoides lineages by a novel genetic marker identified via computational pangenome analysis

Applied and Environmental Microbiology ◽

10.1128/aem.02181-21 ◽

2021 ◽

Author(s):

Siyan Zhao ◽

Chen Zhang ◽

Matthew J. Rogers ◽

Xuejie Zhao ◽

Jianzhong He

Keyword(s):

Amino Acid ◽

Microbial Communities ◽

Genetic Marker ◽

Genetic Markers ◽

Unknown Function ◽

Computational Approach ◽

Amino Acid Sequences ◽

Reductive Dehalogenation ◽

Wide Range

As a group, Dehalococcoides dehalogenate a wide range of organohalide pollutants but the range of organohalide compounds that can be utilized for reductive dehalogenation differs among the Dehalococcoides strains. Dehalococcoides lineages cannot be reliably disambiguated in mixed communities using typical phylogenetic markers, which often confounds bioremediation efforts. Here, we describe a computational approach to identify Dehalococcoides genetic markers with improved discriminatory resolution. Screening core genes from the Dehalococcoides pangenome for degree of similarity and frequency of 100% identity found a candidate genetic marker encoding a bacterial neuraminidase repeat (BNR)-containing protein of unknown function. This gene exhibits the fewest completely identical amino acid sequences and among the lowest average amino acid sequence identity in the core pangenome. Primers targeting BNR could effectively discriminate between 40 available BNR sequences ( in silico ) and 10 different Dehalococcoides isolates ( in vitro ). Amplicon sequencing of BNR fragments generated from 22 subsurface soil samples revealed a total of 109 amplicon sequence variants, suggesting a high diversity of Dehalococcoides distributed in environment. Therefore, the BNR gene can serve as an alternative genetic marker to differentiate strains of Dehalococcoides in complicated microbial communities. Importance The challenge of discriminating between phylogenetically similar but functionally distinct bacterial lineages is particularly relevant to the development of technologies seeking to exploit the metabolic or physiological characteristics of specific members of bacterial genera. A computational approach was developed to expedite screening of potential genetic markers among phylogenetically affiliated bacteria. Using this approach, a gene encoding a bacterial neuraminidase repeat (BNR)-containing protein of unknown function was selected and evaluated as a genetic marker to differentiate strains of Dehalococcoides , an environmentally relevant genus of bacteria whose members can transform and detoxify a range of halogenated organic solvents and persistent organic pollutants, in complex microbial communities to demonstrate the validity of the approach. Moreover, many apparently phylogenetically distinct, currently uncharacterized Dehalococcoides were detected in environmental samples derived from contaminated sites.

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In vitro biosynthesis and processing of composite common precursors containing amino acid sequences identified immunologically as neurophysin I/oxytocin and as neurophysin II/arginine vassopressin

FEBS Letters ◽

10.1016/0014-5793(80)80381-4 ◽

1980 ◽

Vol 121 (2) ◽

pp. 358-362 ◽

Cited By ~ 27

Author(s):

Hartwig Schmale ◽

Dietmar Richter

Keyword(s):

Amino Acid ◽

Amino Acid Sequences ◽

In Vitro Biosynthesis ◽

Neurophysin Ii

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Effects of peptide hormone structure on H+ secretion by Necturus gastric mucosa

American Journal of Physiology-Legacy Content ◽

10.1152/ajplegacy.1976.231.2.573 ◽

1976 ◽

Vol 231 (2) ◽

pp. 573-578 ◽

Cited By ~ 2

Author(s):

JM Berkowitz ◽

M Praissman ◽

ME LeFevre

Keyword(s):

Amino Acid ◽

Gastric Mucosa ◽

Peptide Hormone ◽

Amino Acid Sequences ◽

Gastrointestinal Hormone ◽

Gastric Tissue ◽

The Common ◽

Subsequent Addition ◽

Lower Molar

The actions of human synthetic gastrin I(G), the C-terminal tetrapeptide of gastrin (T), and the C-terminal octapeptide of cholecystokinin (OP) on acid secretion and transepithelial potential difference (PD) of the isolated Necturus gastric mucosa were determined. All three peptides induced H+ secretion, but the maximum H+ output was less with OP than with G or T. G and OP produced their maximum H+ output at lower molar concentrations than T. G- and OP-stimulated secretion was long sustained, but T-stimulated secretion rapidly returned to basal levels. T- and G-stimulated secretion was partially inhibited by the addition of OP. Evidence is presented that T rapidly disappears from solutions exposed to gastric mucosa, whereas G does not. Washing sensitized the mucosa to subsequent addition of T. The results suggest that the action of the common C-terminal tetrapeptide of G, T, and OP is modified by the preceding amino acid sequences, and that T, the smallest of the three peptides, is rapidly degraded by gastric tissue in vitro. The implications of the work for the study of gastrointestinal hormone structure-function relationships in isolated tissue preparations are discussed.

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Identification of Mimotope Peptides Which Bind to the Mycotoxin Deoxynivalenol-Specific Monoclonal Antibody

Applied and Environmental Microbiology ◽

10.1128/aem.65.8.3279-3286.1999 ◽

1999 ◽

Vol 65 (8) ◽

pp. 3279-3286 ◽

Cited By ~ 45

Author(s):

Qiaoping Yuan ◽

James J. Pestka ◽

Brandon M. Hespenheide ◽

Leslie A. Kuhn ◽

John E. Linz ◽

...

Keyword(s):

Monoclonal Antibody ◽

Alkaline Phosphatase ◽

Protein Synthesis ◽

Amino Acid ◽

Synthetic Peptide ◽

Amino Acid Sequences ◽

In Vitro Translation ◽

Enzyme Linked Immunosorbent Assay ◽

Translation System

ABSTRACT Monoclonal antibody 6F5 (mAb 6F5), which recognizes the mycotoxin deoxynivalenol (DON) (vomitoxin), was used to select for peptides that mimic the mycotoxin by employing a library of filamentous phages that have random 7-mer peptides on their surfaces. Two phage clones selected from the random peptide phage-displayed library coded for the amino acid sequences SWGPFPF and SWGPLPF. These clones were designated DONPEP.2 and DONPEP.12, respectively. The results of a competitive enzyme-linked immunosorbent assay (ELISA) suggested that the two phage displayed peptides bound to mAb 6F5 specifically at the DON binding site. The amino acid sequence of DONPEP.2 plus a structurally flexible linker at the C terminus (SWGPFPFGGGSC) was synthesized and tested to determine its ability to bind to mAb 6F5. This synthetic peptide (designated peptide C430) and DON competed with each other for mAb 6F5 binding. When translationally fused with bacterial alkaline phosphatase, DONPEP.2 bound specifically to mAb 6F5, while the fusion protein retained alkaline phosphatase activity. The potential of using DONPEP.2 as an immunochemical reagent in a DON immunoassay was evaluated with a DON-spiked wheat extract. When peptide C430 was conjugated to bovine serum albumin, it elicited antibody specific to peptide C430 but not to DON in both mice and rabbits. In an in vitro translation system containing rabbit reticulocyte lysate, synthetic peptide C430 did not inhibit protein synthesis but did show antagonism toward DON-induced protein synthesis inhibition. These data suggest that the peptides selected in this study bind to mAb 6F5 and that peptide C430 binds to ribosomes at the same sites as DON.

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