Murashige and Skoog medium, 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin, 2-(N-morpholino)ethanesulfonic acid (MES), triphenil-tetrazolium chloride (TTC), xylenol orange, EDTA, succinate, 4-morpholinepropanesulfonic acid (MOPS), Polyvinylpyrrolidone (PVP-40), hydroxylamine, sulphanilamide, α-naphthylamine, ampicillin, NP40, safranin, Salicylhydroxamic acid (SHAM), kalium-cyanide (KCN), linoleic acid, fatty acid free bovine serum albumin (BSA), luminol, p-Coumaric acid, Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), were obtained from Sigma-Aldrich. ProBond Purification System was purchased from Invitrogen. Amicon Ultra 30K Centrifugal Filter Units were purchased from Merck. IPTG was obtained from Duchefa Biochemie, cytochrome c was purchased from Fluka. Primary and secondary antibodies were purchased from Agrisera Antibodies. All other chemicals were of analytical or HPLC grade, and were purchased from Reanal, Hungary. Pierce BCA Protein Assay Kit, GeneJET Plant RNA Purification Kit, and RevertAid First-Strand cDNA Synthesis Kit were obtained from Thermo Scientific; SensiFAST SYBR No-ROX Kit was purchased from Bioline.
Salicylhydroxamic acid
Manufactured by Merck Group
Sourced in United States
Salicylhydroxamic acid is a chemical compound used as a laboratory reagent. It is a crystalline solid that is soluble in organic solvents. Salicylhydroxamic acid is commonly utilized in various analytical and research applications, but a detailed description of its core function cannot be provided while maintaining an unbiased and factual approach.
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Lab products found in correlation
Aminooxyacetic acid, by Merck Group (3 mentions)
Hydroxylamine, by Merck Group (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Sulphanilamide, by Merck Group (1 mentions)
Safranin, by Merck Group (1 mentions)
Kinetin, by Merck Group (1 mentions)
α naphthylamine, by Merck Group (1 mentions)
Carbonyl cyanide p trifluoromethoxyphenylhydrazone, by Merck Group (1 mentions)
Amicon ultra 30k centrifugal filter units, by Merck Group (1 mentions)
Probond purification system, by Thermo Fisher Scientific (1 mentions)
Genejet plant rna purification kit, by Thermo Fisher Scientific (1 mentions)
Np 40, by Merck Group (1 mentions)
Sensifast sybr no rox kit, by Meridian Bioscience (1 mentions)
Fatty acid free bovine serum albumin, by Merck Group (1 mentions)
Luminol, by Merck Group (1 mentions)
Ampicillin, by Merck Group (1 mentions)
Polyvinylpyrrolidone, by Merck Group (1 mentions)
P coumaric acid, by Merck Group (1 mentions)
Linoleic acid, by Merck Group (1 mentions)
9 protocols using salicylhydroxamic acid
1
Plant Cell Culture Optimization
2
Enzymatic Assay Reagent Preparation
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KOD Hot Start DNA Polymerase, 30%(w/w) hydrogen peroxide solution, catechol, pyrogallol, 4-tert-butylcatechol, 4-methylcatechol, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), p-hydroquinone, sodium azide, sodium fluoride, 3-amino-1,2,4-triazole (3TR), potassium cyanide and salicylhydroxamic acid were purchased from Sigma and Merck.
3
Modulating Stress Resistance in Plants
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Salicylhydroxamic acid (SHAM, an inhibitor of the AOX pathway), dimethylthiourea (DMTU, an H2O2 scavenger), DPI (an NADPH oxidase inhibitor), aminooxyacetic acid (AOA, an ethylene biosynthesis inhibitor), and ethylene were purchased from Sigma (StLouis, USA). 1 mM SHAM inhibits the AOX activity, as this concentration is sufficiently low to avoid the possible side effects (Møller et al., 1988 (link)). For BL+SHAM treatment, seedlings were pretreated with 1 mM SHAM, 24 h later were treated with 1 μM BL for another 12 h. Then, these plants were exposed to stress conditions as described earlier. To investigate the role of ROS in the resistance, leaves were pretreated with 100 μM DPI or 5 mM DMTU, then treated with 1 μM BL; 12 h later plants were treated with 10 mM H2O2. Then 1 mM AOA was sprayed to the seedlings for 12 h at room temperature. For the ethylene treatment, one lot seedling was incubated in 3.5 μl l−1 ethylene solution in a closed container at room temperature for 12 h. Then, these plants were exposed to stress conditions as described earlier.
4
Measuring COX and AOX Respiration
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Determination of COX- and AOX-dependent oxygen consumption was performed using strains cultivated on M2 medium for 2 days and in CM liquid medium for 2 days, as described above. Small pieces of mycelium were subsequently transferred into the high-resolution respirometer (Oxygraph-2k series C and G, Oroboros Instruments, Innsbruck, Austria) and oxygen consumption was measured in liquid CM medium according to the manufacturer’s instructions. Then, 1 mM potassium cyanide (KCN; Fluka, Buchs, Switzerland; 60178) was added to inhibit respiration via COX, and 4 mM salicylhydroxamic acid (SHAM; Sigma-Aldrich, St. Louis, MO, USA; S607) was added to inhibit respiration via AOX. Data were analyzed using the manufacturer’s software DatLab 6.
5
Respirometric Assays Using Inhibitors
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All chemicals for the respirometric assays were of analytical grade. Salicylhydroxamic acid (SHAM), benzhydroxamic acid (BHAM), carbonyl cyanide 3-chlorophenylhydrazone (CCCP), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), 2,4-dinitrophenol (DNP), antimycin A and rotenone were purchased from Sigma-Aldrich and n-propyl gallate (n-PG) from Fluka. Potassium cyanide and sodium azide were supplied from Merck (VWR) and N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) from Roth. Stock solutions were prepared with glass distilled deionized water or absolute ethanol, respectively. Aqueous solutions of potassium azide and sodium cyanide were buffered with 10 mM HEPES-NaOH to pH 7.0. Solutions of SHAM, BHAM and azide were prepared freshly on the day needed and stock solutions with absolute ethanol were handled in glass vials. All stock solutions were kept on ice during measurements and were extra protected from light for storage at -20°C. The anti-foam agent Clerol FBA 5075 (Cognis, Meaux, France) was used for the chemostat medium.
6
Hormonal Regulation of Plant Responses
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The following hormones or hormone donors were used: methyl-jasmonate (mJA; Sigma-Aldrich), ethephon (Eth; Sigma-Aldrich) and sodium 2-hydroxybenzoate (NaSa; Sigma-Aldrich). The following inhibitors of hormone biosynthesis were used: salicylhydroxamic acid (SHAM; Sigma-Aldrich), aminooxyacetic acid (AOA; Sigma-Aldrich) and L-2-aminooxy 3-phenylpropinoic acid (PAL-Inh; Wako-chemicals, Osaka, Japan). mJA and SA inhibitor (PAL-Inh) were dissolved in EtOH before preparing the stock solutions. All chemicals were filter-sterilized and prepared as stocks containing 0.02% Tween 20 (mJA, SHAM, Eth, NaSA, PAL-Inh as 10 mM and AOA as 25 mM). Before the experiments, different concentrations of the chemicals were tested for phytotoxicity. The final selected concentration did not result in any phenotypical changes (data not shown). Hormones and hormone inhibitors were applied to the shoots of 11-d-old plants under sterile conditions in two droplets onto two leaves per plant at the following concentrations: 60 μM mJA, 150 μM SHAM, 400 μM Eth, 15 μM AOA, 500 μM NaSa and 75 μM PAL-Inh. Distilled water containing 0.02% (v/v) Tween 20 was used as a control. After application, dishes were put back into the growing chamber.
7
Fungal Cell Wall Composition Analysis
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Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were from Lonza (Verviers, Belgium). YPD medium (yeast extract [10 g/liter], peptone [20 g/liter], and glucose [20 g/liter]), MOPS-buffered YPD medium (YPD medium plus MOPS 165 mM), yeast nitrogen base (YNB) medium, 100× penicillin, and streptomycin were purchased from Sigma (Saint Louis, MO, USA). Dimethyl sulfoxide (DMSO) was from Biomol GmbH (Hamburg, Germany). The inhibitors antimycin A (AA), rotenone, and salicylhydroxamic acid (SHA) were from Sigma, thenoyltrifluoroacetone (TTFA) was from Fluka (Saint Louis, MO, USA), and potassium cyanide (KCN) and H2O2 were from Roth (Karlsruhe, Germany) and Merck (Darmstadt, Germany), respectively. Anti-β-1,3-glucan antibodies were from Biosupplies (Parkville, Australia). MOPS, fluorescein isothiocyanate (FITC), FITC-labeled concanavalin A (FITC-ConA), propidium iodide (PI), and the formalin solution were purchased from Sigma, and stock solutions were prepared: 1 mg/ml in 0.9% NaCl for FITC-ConA, 100 mg/ml in Dimethyl sulfoxide (DMSO) for FITC, and 20 mM for propidium iodide also in DMSO. Trypan blue was from Fluka.
8
Fluorescent Probe Assay for Oxidants
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The molecular probe H 2 DCF-DA was obtained from Biotium (Hayward, CA, USA). 2-(N-morpholino) ethanesulfonic acid (MES), hypotaurine (HT), aminooxy acetic acid (AOA), hydroxylamine (NH 2 OH), potassium pyruvate (C 3 H 3 KO 3 ), ammonia (NH 3 ), ascorbic acid (ASA), catalase (CAT), diphenylene iodonium (DPI), salicylhydroxamic acid (SHAM), L-cysteine, D-cysteine, dimethyl sulfoxide (DMSO), N,Ndimethyl-p-phenylenediamine dihydrochloride and dithiothreitol (DTT) were obtained from Sigma-Aldrich (St Louis, MO, USA). Unless stated otherwise, the remaining chemicals were of the highest analytical grade available from various Chinese suppliers.
9
Screening Fungicide Resistance Mechanisms
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Leaf discs with sporulating colonies at 1mg.L -1 of cyazofamid or ametoctradin with 100mg.L -1 of SHAM (Salicylhydroxamic acid, Sigma-Aldrich, France), as inhibitor of alternative respiration (AOX), were used to isolate resistant strains of P. viticola. Collected populations showing high level of non-specific resistance on fungicide without SHAM were also used to isolate AOX-strains. Thus, monitoring of sensitivity assays using fungicide, alone or with SHAM, was used to select strains developing resistance involving either AOX respiration (non specific resistance) or cytb target mutation mechanism. Indeed, strains showing sporulation on leaf discs with both fungicide and SHAM had specific resistance mechanism, probably inducing cytb target modification. In other hand, strains growing only on fungicide without SHAM involve AOX activity.
To investigate cross-resistance, in vitro sensitivity of these strains to ametoctradin and cyazofamid, applied alone or mixed with SHAM, was measured with increasing concentrations of each fungicide (0.01mg.L -1 to 100mg.L -1 ). Ten discs were analysed for each condition and assays for each isolate were repeated three times per fungicide concentration. After 12 days, individual leaf discs were evaluated for disease incidence and sporulation rate.
To investigate cross-resistance, in vitro sensitivity of these strains to ametoctradin and cyazofamid, applied alone or mixed with SHAM, was measured with increasing concentrations of each fungicide (0.01mg.L -1 to 100mg.L -1 ). Ten discs were analysed for each condition and assays for each isolate were repeated three times per fungicide concentration. After 12 days, individual leaf discs were evaluated for disease incidence and sporulation rate.
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