The cells of W. ichthyophaga were grown to mid-exponential phase at 28°C and 180 rpm in YNB medium supplemented with 3.4 M NaCl (Carlo Erba Reagents). Then 30 mL cell culture was harvested by centrifugation (4600 × g, 10 min, 25°C), and the pellet was washed twice with 30 mL modified OS buffer (50 mM succinic acid, 1 M NaCl, 2.75 M sorbitol, pH 7.5) using the same centrifugation parameters. The pellet was then resuspended in 10 mL modified OS buffer that contained 32 mg cell-wall-degrading enzyme cocktail Caylase (Cayla), and incubated at 25°C and 50 rpm for 1.5 h to disintegrate cellular clumps. Next, the cells were harvested and resuspended in 5 mL YNB containing 2.75 M sorbitol, and then they were 10-fold diluted in YNB containing 2.75 M sorbitol, 100 μg/mL fludioxonil (Sigma Aldrich), and 0.75% low-melting point agarose, and poured onto YNB agar plates that contained different NaCl concentrations (i.e., 2.5, 3.4, 4.3 M) and 100 μg/mL fludioxonil. The plates were incubated at 28°C and their growth was scanned after 7–10 days.
Fludioxonil
Manufactured by Merck Group
Sourced in United States, Germany
Fludioxonil is a fungicide used in agricultural and horticultural applications. It is a broad-spectrum fungicide that inhibits fungal growth by disrupting the synthesis of fungal cell membranes. The active ingredient, fludioxonil, is a synthetic compound that is effective against a variety of fungal pathogens.
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Lab products found in correlation
Iprodione, by Merck Group (4 mentions)
Boscalid, by Merck Group (3 mentions)
Fenhexamid, by Merck Group (3 mentions)
Formic acid, by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Prochloraz, by Merck Group (2 mentions)
Fenarimol, by Merck Group (2 mentions)
Cyprodinil, by Merck Group (2 mentions)
Caffeine, by Merck Group (2 mentions)
Chlorpyrifos, by Merck Group (2 mentions)
Pyraclostrobin, by Merck Group (2 mentions)
Tebuconazole, by Merck Group (2 mentions)
Natamycin, by Merck Group (2 mentions)
Pirimicarb, by Merck Group (2 mentions)
Imazalil, by Merck Group (1 mentions)
Malt extract agar, by Thermo Fisher Scientific (1 mentions)
Fenpropimorph, by Merck Group (1 mentions)
Difenoconazole, by Merck Group (1 mentions)
Dichlofluanid, by Merck Group (1 mentions)
Fenbuconazole, by Merck Group (1 mentions)
15 protocols using fludioxonil
1
Halotolerant Yeast Growth Profiling
2
Fludioxonil Compound Preparation
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Fludioxonil was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Fludioxonil was dissolved in 100% dimethyl sulfoxide (DMSO; Sigma-Aldrich Corp.). The final concentration of DMSO in the cell culture media was 0.1%.
3
Fludioxonil Treatment on Fungal Samples
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After growth on glass cover slips in liquid AMM medium, the medium was discarded and replaced by liquid AMM medium containing 2 µg/mL fludioxonil (Sigma Aldrich). The samples were further incubated at 37 °C for 4 h, fixed and stained as described in Section 2.3.1 .
4
Fungal Strain Cultivation and Fungicide Preparation
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B. cinerea strain B05.10 was obtained from the VTT culture collection (http://culturecollection.vtt.fi , ref. D-071295). To prepare conidial suspension, a mycelial disc of B. cinerea was inoculated onto malt extract agar (Oxoid, Basingstoke, UK) plates and incubated for 2 weeks at 22ºC. The conidia were harvested by flooding the cultures with sterile distilled water or potato dextrose broth (PDB; BD Biosciences, Spark, MD, USA), rubbing the surface with a spreader, and then filtered through four sheets of Miracloth (Chicopee Mills Inc., Milltown, NJ, USA). Schizophyllum commune KUC9080 isolated from a soil of Gurye, Korea was used to construct a microbial culture extract library. The KUC9080 strain was maintained on potato dextrose agar (PDA) plates. Tebuconazole and chlorothalonil were obtained from Kyung Nong Co. Ltd (Seoul, Korea), and boscalid, dichlofluanid, fenhexamid, fludioxonil, iprodione, difenoconazole, fenarimol, fenbuconazole, fenpropimorph, imazalil, and prochloraz in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA).
5
Antifungal Impact on Cochineal Insect
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A group of 15 first instar nymphs of D. opuntiae was fed on a prickly pear pad of Opuntia ficus-indica injected with 5 ml of 20 μg ml−1 antifungal cocktail of Ketoconazol (Sigma), Anfotericine B (Sigma), and Fludioxonil (Sigma). Fleshy leaves were injected weekly for 4 weeks and then female insects were removed. O. ficus-indica leaves without antifungal were similarly infested and used as negative controls. After treatment, a pool of six individuals of each leaf was used to measure differences in dry weight, UA content and uricase activity. Five replicates of this experiment were performed. UA content, uricase activity and dry weight data were compared between controls and treatments using a t-test.
6
HNE Impact on Cellular Metabolism and DNA Damage
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The 4-hydroxynonenal (HNE) was provided by Clinisciences (Nanterre, France). Fetal Bovine Serum (FBS) provided by ThermoFisher (Eindhoven, Netherlands). Puromycin, hydromycin and zeocin were provided by Fisher Scientific(Hampton, NH, USA). Epidermal growth factor (EGF), hydrocortisone, insulin, transferrin, sodium selenite (5 nM) and gentamycin sulfate (5 µg/mL), DMSO (dimethylsulfoxyde), procymidone, iprodione, cyprodinil, fludioxonil, lambda-cyhalothrin, methyl methanesulfonate (MMS), Trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP), rotenone, oligomycin and etoposide were provided by Sigma-Aldrich (Saint-Quentin Fallavier, France). Ro 19-8022 was a gift from Hoffman Laroche Ltd. (Basel, Switzerland), and formamidopyrimidine-DNA glycosylase (Fpg) a gift from Serge Boiteux, CNRS, France.
7
Comprehensive Yeast Phenotypic Assays
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Phenotypic plate assays were performed on YNB (without amino acids and ammonium sulfate) media supplemented with 2% glucose and 10 mM ammonium sulfate unless stated otherwise. For UV sensitivity, cells were spotted then exposed for 6 sec to UV light at 48 mJ/cm2 in a UV Stratalinker (Stratagene, USA). For all other assays, the stressor was added to the media immediately prior to pouring at the following concentrations: 2.5 mM caffeine (Sigma, USA), 1 μg/mL cyclohexamide (Sigma, USA), 0.1 mM CuSO4 (Sigma, USA), 0.5 µM Myriocin (Sigma, USA), 1.5 mM Na2MoO4 (Sigma, USA), 5 μg/mL fluconazole (Sigma, USA), 50 μg/mL fludioxonil (Sigma, USA) and 0.01% SDS (Sigma, USA). Melanization assays were performed on solid L-DOPA media supplemented with 10 mM asparagine55 (link). Tenfold serial dilutions were prepared immediately prior to testing, with plates being imaged at 24–96 hr. All assays were performed at both 30 and 37 °C.
8
Analytical Standards for Pesticide Detection
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L-menthol (natural source, food grade, ≥ 99% purity), BHT (food grade, ≥ 99% purity), formic acid, elevated purity grade solvents (acetonitrile, ethanol), as well as analytical standards of azoxystrobin, boscalid, buprofezin, chlorpyrifos, chlorpyrifos-methyl, clofentezine, dodine, fludioxonil, hexythiazox, methoxyfenozide, myclobutanil, penconazole, propiconazole, pyraclostrobin, pyriproxyfen, pyridaben, spirotetramat, tebuconazole and tebufenpyrad were purchased from Sigma Aldrich-Merck S.r.l. (Milan, Italy). A Milli-Q Plus apparatus (Millipore, Bedford, MA, USA.) was used for obtaining ultrapure water.
Weighted amounts of the analytical standards (OhausDV215CD Discovery semi-micro and analytical balance, 81/210 g capacity, 0.01/0.1 mg readability, Ohaus Corporation, Pine Brook, NJ, USA) were dissolved in methanol or toluene (clofentezine and pyraclostrobin) in volumetric flasks, in order to obtain individual stock solutions at a concentration of 1 mg mL−1.The last ones were diluted in methanol for preparing the multi-standard working solutions at 0.02, 0.5, 1.5, 2.5 and 4 ng μL−1 used for the method validation.
Weighted amounts of the analytical standards (OhausDV215CD Discovery semi-micro and analytical balance, 81/210 g capacity, 0.01/0.1 mg readability, Ohaus Corporation, Pine Brook, NJ, USA) were dissolved in methanol or toluene (clofentezine and pyraclostrobin) in volumetric flasks, in order to obtain individual stock solutions at a concentration of 1 mg mL−1.The last ones were diluted in methanol for preparing the multi-standard working solutions at 0.02, 0.5, 1.5, 2.5 and 4 ng μL−1 used for the method validation.
9
Fungicide Sensitivity Assay Protocol
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Assays for chemical sensitivity were performed by transferring conidia/ hyphae using a sterile toothpick onto PDA containing the test compound. The diameter of colony was recorded at 4 to 7 days after incubation at room temperature (~25°C). Changes of radial growth (%) were determined as a cumulative percentage of growth of wild-type and mutant strains cultured on the same plate. Vinclozolin (10 μg/ml), iprodione (10 μg/ml) and fludioxonil (0.1 μg/ml) fungicides (Sigma, St. Louis, MO) were dissolved in 1% DMSO; other chemicals were dissolved in water, filter sterilized, and added to medium. All experiments were repeated at least two times with duplicate of each treatment.
10
Antifungal Evaluation of Natural Compounds
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All chemicals and media, namely polyenes (amphotericin B (AMB), nystatin (NYS), natamycin (NAT)), fludioxonil (FLU), kojic acid (KA), potato dextrose broth (PDB), and potato dextrose agar (PDA), were purchased from Sigma Aldrich Co., St. Louis, MO, USA. Dimethyl sulfoxide (DMSO) was procured from AMRESCO Co., Solon, OH, USA.
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