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22 protocols using potato dextrose agar (pda)

1

Antimicrobial Potential of Natural Compounds

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Curcumin, dimethylsulfoxid (DMSO), lysogeny broth (LB) agar, phenalenone, and RPMI1640 medium were received from Merck KGaA (Darmstadt, Germany). Potato dextrose agar (PDA) and Mueller–Hinton broth (MHB) were purchased from VWR International (Vienna, Austria). Rose Bengal (RB) was received from TCI Europe (Zwijndrecht, Belgium). Hypericum perforatum extract (ethanol) was prepared from the pharmaceutical drug “Johanniskraut 600 mg forte” (Apomedica, Graz, Austria) by dissolving the filling of the film-coated tablet in ethanol after mechanical removal of the lactose. The 96-well plates (flat bottom) were bought from SARSTEDT (Nümbrecht, Germany).
The U-2001 spectrophotometer for adjusting the McFarland standard was from Hitachi (Chiyoda, Japan). For measurement of the 96-well plates, a Tecan Sunrise Remote Plate Reader (Tecan, Männedorf, Switzerland) was used. The adjustment of pH values was carried out with the pH-meter Mettler Toledo SevenMulti (Mettler-Toledo GmbH, Vienna, Austria).
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2

Antimicrobial Susceptibility Testing Protocol

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Mueller-Hinton Broth (MHB), Muller-Hinton Agar (MHA), streptomycin, ciprofloxacin, ampicillin, tetracycline, metronidazole, myricetin, podophyllotoxin, dimethyl sulfoxide (DMSO ≥ 99.5%), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (≥ 98%) were purchased from Sigma Aldrich (St. Louis, MO, USA). Potato dextrose agar (PDA), methanol, dichloromethane, HPLC-grade acetonitrile, and ethyl acetate were purchased from VWR (Fontenay-sous-Bois, France). Formic acid (FA) was purchased from Merck (Darmstadt, Germany). Water was purified by 0.22 µM membrane filtration and deionization by using a Barnstead Nanopure system from Thermo Scientific (Waltham, MA, USA) or a Milli-Q Plus system (Millipore, Billerica, MA, USA), while the DNeasy UltraClean Microbial kit was purchased from Qiagen (Hilden, Germany) and the GFX PCR DNA and Gel Band Purification Kit were purchased from GE Healthcare (Chicago, IL, USA).
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3

Fungal Strains Cultivation and Analysis

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The S. sclerotiorum strains Ss25 and Ssc51 were kindly provided by the technical institut Terres Inovia (Grignon, France). The S. sclerotiorum strain Ss123 and the B. cinerea strains Bc1, Bc26, Bc82 were provided by the French National Institute for Agriculture, Food and Environment (INRAe, Montfavet, France). Twenty-five μL of a B. cinerea spore suspension (stored at −20°C at 1.3 106 ml−1) were first germinated on Potato Dextrose Agar 39 g L−1 plates (PDA, VWR Chemicals, France). S. sclerotiorum sclerotia were cut in half and the internal face of one piece was placed on 39 g L−1 PDA plates. The pre-cultures were incubated 4 days at 19°C in darkness. Mycelium agar plugs were then excised with a 6 mm diameter cork borer, transferred on fresh PDA plates and incubated for 2 days at 19°C in darkness, until the colonies occupied half of the plate surface. For microscopy observations, mycelium was scraped off the plates with an inoculating loop and inoculated in 2 ml of 12 g L−1 Potato Dextrose Broth (PDB, Sigma-Aldrich, Missouri, United States) in 12-well plates and incubated 24 h at 25°C in darkness, with 60 rpm shaking. To quantify ergosterol content, 5 ml of PDB were inoculated by the scrapping method and incubated in the same conditions for 4 days.
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4

Monascus Cultivation on Rice Substrate

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Monascus sp. DBM 4361, isolated from a non-sterile dried red fermented rice sample, was maintained on Potato-Dextrose agar (VWR Chemicals) slants at 4°C. The strain was deposited at the Department of Biochemistry and Microbiology (DBM), University of Chemistry and Technology Prague.
An amount of rice (Giana, Thailand) (150 g) was washed with hot water, then boiled for 1 min. The rice was evenly divided into three autoclavable plastic bags, which were closed with a metal ring and a cotton plug. The use of plastic bags instead of glass vessels enabled manual separation of rice kernels after sterilization without opening of the bags. The cultivation was inspired by soy koji preparation (Lotong and Suwanarit, 1983 (link)). The bags were placed in a beaker sealed with aluminum foil and sterilized at 121°C for 20 min. Sterilization was repeated after 24 h to eliminate contamination by spore-forming bacteria. Spores from the Monascus culture (mixture of ascospores and conidia, because the strain formed both asexual and sexual spores, see Figure 1) were transferred to sterile water using a sterile loop. The sterile rice in the bags was inoculated with 5 mL of the spore suspension. Cultivation of the fungus on rice was performed for 10 days at 30°C. The rice was mixed by hand daily.
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5

Preparation of Physiological Solution

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Physiological solution (0.9% NaCl) was acquired from Applichem Panreac (Darmstadt, Germany), and potato dextrose agar (PDA) and potato dextrose broth (PDB) were purchased from VWR (VWR International, Radnor, Pennsylvania, USA).
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6

Isolating and Characterizing Soil Fungi

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Approximately 0.5–1 g of soil from the AWI site was plated on potato-dextrose agar (VWR, Radnor, PA) and incubated at ~20°C until growth was visible (~2–5 days). This short incubation time was chosen in order to avoid plate overgrowth. Soils were also diluted 10-800 fold and plated on Rose Bengal agar (BD, Franklin Lakes, NJ). Phenotypically distinct colonies were transferred to fresh potato-dextrose agar plates until pure cultures were isolated. Pure cultures were confirmed by DNA sequencing and using NCBI’s nucleotide BLAST tool [21 ]. Spore suspensions were obtained by rinsing mature cultures with basal medium (containing 5 g (NH4)2SO4/L, 2.5 g MgSO4·7H2O/L, 20 mg CaCl2·2H2O/L, 1.0g KH2PO4/L, 1.01 mg MnSO4·H2O/L, 0.24 mg Na2MoO4·2H2O/L, 0.10 mg NiCl2·6H2O/L, 0.17 mg CuCl2·2H2O/L, 1.36 mg FeSO4·7H2O/L, 0.24 mg CoCl2·6H2O/L and 0.58 mg ZnSO4·7H2O/L) and centrifuging at speeds greater than 10,000xg in microfuge tubes (Eppendorf, Hamburg, Germany) [22 (link), 23 (link)].
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7

Monascus Fungus Cultivation on Sterilized Rice

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Monascus sp. DBM 4361, isolated from a nonsterile dried red fermented rice sample, was maintained on Potato-Dextrose agar (VWR Chemicals) slants at 4°C. The strain was deposited at the Department of Biochemistry and Microbiology (DBM), University of Chemistry and Technology Prague. An amount of rice (Giana, Thailand) (150 g) was washed with hot water, then boiled for 1 minute. The rice was evenly divided into three autoclavable plastic bags, which were closed with a metal ring and a cotton plug. The bags were placed in a beaker sealed with aluminium foil and sterilized at 121 °C for 20 minutes. Sterilization was repeated after 24 hours to eliminate contamination by spore-forming bacteria. Spores from the Monascus culture (mixture of ascospores and conidia, because the strain formed both asexual and sexual spores, see Fig. 1) were transferred to sterile water using a sterile loop. The sterile rice in the bags was inoculated with 5 mL of the spore suspension. Cultivation of the fungus on rice was performed for 10 days at 30 °C. The rice was mixed by hand daily.
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8

Impact of 1-octen-3-ol on ECM Fungal Growth

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To evaluate the effect of the 1-octen-3-ol dose on the development of the mycelium of the three ECM fungi (Terfezia leptoderma, Lactarius deliciosus and Boletus reticulatus), we tested three doses per plate: 0 µg (CT); 0.17 µg (VOC_low); and 1280 µg (VOC_high). For that, we used PDA (VWR) with pH 5.5 as the culture medium (without antibiotics addition). On the border of the Petri dishes (9 cm diameter) with the PDA medium, we added 10 µL of 1-octen-3-ol (VOC) in different doses.
The solution of 1-octen-3-ol at 1 µM was prepared using 1-octen-3-ol 98% (Alfa Aesar CAS 3391-86-4) diluted in Chloroform 99% (Merck CAS 67-66-3) and sterilized by filtration (0.2 μm, 47 mm membrane filter, Minisart® NML, Sartorius, Gottingen, Germany). The mycelial plugs with 0.25 cm2 of T. leptoderma, L. deliciosus and B. reticulatus were transferred from 1-month pure cultures (in PDA) and placed in the centre of the Petri dishes of each culture medium. Each treatment was replicated 10 times (N = 10 Petri dishes).
The ECM fungal cultures were placed in a growth chamber at 23 °C ± 1 °C in the dark. Colonies’ growths were measured every 7 days for 63 days by measuring mycelia radial growth at the bottom of the Petri dish.
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9

Fungal Morphology and Colony Characteristics

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The morphology and colony characteristics of the isolates were examined on three different media: 2% (w/v) MEA (VWR, Debrecen, Hungary), 3% (w/v) oatmeal agar (OA, Merck, Darmstadt, Germany), and potato dextrose agar (PDA, VWR, Debrecen, Hungary), and incubated for seven days in the dark at 25 °C [2 (link),7 (link)]. Microscopic studies were performed using light—(Zeiss Primostar, Carl Zeiss, Suzhou, China), stereo—(Zeiss Stemi 305, Carl Zeiss, Suzhou, China), and scanning electron microscopes. SEM samples were prepared by stabilization in 0.1 M phosphate buffer (pH 7.3) containing 2.5% glutaraldehyde (12 h, 4 °C). The samples were then dehydrated with ethanol-water, gradually increasing the volume ratio of ethanol (50% v/v, 60% v/v, 70% v/v, 80% v/v, 90% v/v, 95% v/v, 100% v/v). Finally, the samples were dried (3 h, 30 °C) and coated with gold for microscopic examination. Electron microscopy images were taken with a 10 kV accelerating voltage Hitachi S-4700 Type II FE-SEM microscope, observing secondary electrons with magnitudes of 150×, 250×, 600×, and 2000× [68 (link)].
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10

Antifungal Activity Evaluation of Talaromyces Extracts

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Antifungal activity was tested against three Aspergillus species associated with stonebrood disease (A. flavus ATCC9170, A. fumigatus ATCC10894, and A. niger ATCC10549) from Fraunhofer strain collection (STO20519-STO29521) using CLSI M51-A parameters [72 (link)]. The Aspergillus strains were cultivated on PDA (VWR International, Radnor, PA, USA) for 7 days at 25 °C. The spores were washed by 5–6 mL of 0.05% Tween-80 (Sigma-Aldrich, St. Louis, MO, USA) and filtered through three layers of miracloth to remove the hyphal structures. The inoculum was adjusted to OD530 = 0.14–0.46 based on spore morphology [73 (link)]. The inoculum was spread evenly over the surface of Mueller–Hinton agar (Carl Roth, Karlsruhe, Germany) using a sterile cotton swab. The depth of the medium was consistent in each Petri dish (4 mm). The extracts from the liquid T. purpureogenus strains cultures (Figure 1) were diluted with methanol to 10 mg/mL, and 25 µL of the crude extract was applied to 6 mm cellulose disks and left in the laminar flow cabinet for 30 min to dry. Methanol was used as a control. The disks were placed on the inoculated media and pressed down with sterile forceps. The zones of inhibition were evaluated in triplicate after incubation at 35 °C for 24 h.
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