Trichoderma guizhouense NJAU 4742 (CGMCC NO.12166, China Microbial Culture Collection Committee General Microbiology Center) was stored in Jiangsu Key Laboratory for Organic Solid Waste Utilization. The strain was grown in potato dextrose agar (PDA) medium (Difco Laboratories, Detroit), and the conidial suspension of NJAU4742 strain was prepared as described by Yang et al. [57 (link)]. The germinated spore was prepared as described by Yedidia et al. [28 (link)], and a final concentration of 107 germinated spores mL−1 was used in the inoculation experiments. The growth of cucumber seedlings (Cucumis sativus L. cv JinChun-No. 4 obtained from Tianjin Cucumber Research Center, China) was undertaken and plant growth medium (PGM) was prepared as previously described with some modifications. The PGM liquid medium were improved with 1/3 Hoagland nutrient solution [8 (link), 58 (link)]. The germinated spores of NJAU4742 strain were added to the PGM liquid medium with 15-day-old seedlings to a final concentration of 105 germinated spores mL−1 under aseptic conditions. The cucumber seedlings were grown under glasshouse conditions (50 rpm, 80% relative humidity, 28 °C and a photoperiod of 14 h light and 10 h dark) for 5 days, after which various parameters including leaf area, shoot fresh weight and relative expression level of TgSWO were detected.
Pda medium
Manufactured by BD
Sourced in United States
The PDA) medium is a laboratory consumable product used in microbiological testing. It serves as a growth medium for the cultivation and enumeration of microorganisms. The core function of the PDA) medium is to provide the necessary nutrients and support for the proliferation of a wide range of microbial species in a controlled laboratory setting.
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Lab products found in correlation
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (3 mentions)
Agar powder, by BD (1 mentions)
Driselase, by Merck Group (1 mentions)
Peg4000, by Merck Group (1 mentions)
Miracloth, by Merck Group (1 mentions)
Ferrous sulfate heptahydrate feso4 7h2o, by Merck Group (1 mentions)
Nanodrop, by Merck Group (1 mentions)
Nanodrop nd 1000 uv, by Thermo Fisher Scientific (1 mentions)
Bc0090 50 t 48s, by Solarbio (1 mentions)
Ampicillin, by Merck Group (1 mentions)
12 protocols using pda medium
1
Trichoderma guizhouense NJAU 4742 Enhances Cucumber Growth
2
Preparation of A. kawachii β-Glucosidase Enzyme
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The A. kawachii enzyme was prepared as previously described [17 (link)]. Briefly, A. kawachii grown in potato dextrose agar (PDA) medium (Difco Laboratories, Detroit, MI, USA) was inoculated into sterilized wheat bran and incubated at 30 °C for 3 days. The resulting mixture was suspended in sodium phosphate buffer (pH 7.0) and incubated for 18 h at 4 °C. The reaction mixture was then centrifuged, and the supernatant was used for fermentation of O. japonicus extracts. The supernatant provided 0.276 U/mL (1 U is defined as the enzyme activity needed to produce 1 mmol of p-nitrobenzene from p-nitrophenyl-β-d -glucopyranoside per min) β-glucosidase activity [17 (link)].
3
Fungal Strain Transformation and Cultivation
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All fungal strains used or constructed in this study are listed in Table 1 . These strains were cultivated on PDA medium (pH 5.6, purchased from BD company) at 25 ℃ for 7 days and used for multiplication of the inoculum. The transformants were selected on PDAS (PDA with 0.8 M D-sorbitol, pH 5.6) supplemented with appropriate antibiotics as needed, such as 100 mg/L of hygromycin B or geneticin, and cultured for 5–7 days at 30 ℃.
Strains used in this study
| Strains | Characteristics | Reference |
|---|---|---|
| MEFC09 | Wild type, Coleophoma empetri | CGMCC 21058 |
| MEFC09-∆ku80 | MEFC09 derivate, ∆ku80::neo | [34 (link)] |
| MEFC09-F | MEFC09 derivate, PgpdAt-mcfF-TtrpC-hph | This study |
| MEFC09-H | MEFC09 derivate, PgpdAt-mcfH-TtrpC-hph | This study |
| MEFC09-HF | MEFC09-H derivate, PgpdAt-mcfF-TtrpC-neo | This study |
| MEFC09-P | MEFC09 derivate, PgpdAt-mcfP-TtrpC-hph | This study |
| MEFC09-S | MEFC09 derivate, PgpdAt-mcfS-TtrpC-hph | This study |
| MEFC09-∆CEfks1 | MEFC09-∆ku80 derivate, ∆CEfks1::hph | This study |
| MEFC09-∆CEfks2 | MEFC09-∆ku80 derivate, ∆CEfks2::hph | This study |
| MEFC09::CEfks2 | MEFC09 derivate, PgpdAt-CEfks2-Tpgk-hph | This study |
| MEFC09-∆mcfJ | MEFC09-∆ku80 derivate, ∆mcfJ::hph | This study |
| MEFC09-J | MEFC09 derivate, PgpdAt-mcfJ-Tpgk-hph | This study |
| MEFC09-JFH | MEFC09-J derivate, PgpdAt-mcfF-TtrpC-neo, PgpdAt-mcfH-TtrpC-neo | This study |
CGMCC: China General Microbiological Culture Collection Center
4
Fusarium verticillioides Strain Curation
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Fusarium verticillioides strain 7600 (wild type, WT) is deposited in the Fungal Genetics Stock Center, University of Kansas Medical School, Kansas City, KS, USA. The mutant strain ∆fst1 and corresponding complemented stain fst1-comp were previously described by Bluhm et al. [15 (link)]. Cultures were stored long-term in 50% glycerol at −80°C and maintained as working stock on PDA medium (B&D, Sparks, MD).
5
Fusarium pseudograminearum Strains RNA-Seq
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FpgMBV1-containing F. pseudograminearum strain FC136-2A and its isogenic FpgMBV1-free strain FC136-2A-V- were maintained in the laboratory, department of plant protection, Henan Agricultural University, Henan province, China [27 (link)]. For RNA sequencing, these two strains were grown at 25 °C in the dark on potato dextrose agar (PDA) medium (Becton, Dickinson, and Company, Sparks, MD, USA). For HPLC-MS/MS analysis, these strains were cultured in wheat grain media. Typically, 180 g of wheat seeds was soaked in distilled water for 12 h and boiled for 30 min then air-dried and autoclaved at 120 °C for 30 min in triangle flasks to complete the preparation of the wheat grain medium. Three-day-old PDA plugs of FC136-2A or FC136-2A-V- mycelia were cultured in wheat grain media for 30 days in the dark at 25 °C.
6
Quantification of Extracellular Biomolecules and Viable Cells
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We quantified extracellular polysaccharides, proteins, and DNA. A 30-mg sample of the cut towel pieces was suspended in 1 mL 0.1 M NaOH, heated at 100 °C for 3 h, and centrifuged at 13,800 × g for 5 min and the supernatant was collected and used for the subsequent analysis. The extracellular polysaccharides were quantified using the phenol sulphate method23 . The proteins were quantified using the Lowry method24 (link), and the double-stranded DNA was quantified using a Qubit® 3.0 Fluorometer (Thermo Scientific).
The viable cell counts were determined from a 0.5 cm square towel piece. Each sample was mixed with 2.0 mL Lecithin Polysorbate (LP) diluted solution (Fujifilm Wako) and sonicated for 10 min. Then, it was streaked and cultured on SCDA medium (SHIOTANI M.S.) for bacteria and PDA medium (Becton Dickinson) for bacteria and fungi. The number of colonies was counted after culturing at 37 °C for 6 days for SCDA medium and 30 °C for 6 days for PDA medium.
The viable cell counts were determined from a 0.5 cm square towel piece. Each sample was mixed with 2.0 mL Lecithin Polysorbate (LP) diluted solution (Fujifilm Wako) and sonicated for 10 min. Then, it was streaked and cultured on SCDA medium (SHIOTANI M.S.) for bacteria and PDA medium (Becton Dickinson) for bacteria and fungi. The number of colonies was counted after culturing at 37 °C for 6 days for SCDA medium and 30 °C for 6 days for PDA medium.
7
Fungal Strain P. chlamydosporia Fermentation
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The deuterium reagents were used from Cambridge Isotope Laboratories, Inc. (USA). The agar powder and PDA medium were used from Becton, Dickinson Co., Ltd. (USA). Yeast extracts and peptones were used from Oxoid, Inc. (UK). Driselase and PEG4000 were used from Sigma-Aldrich Trading Co., Ltd. (China). Miracloth was used from Merck-Calbiochem (Germany). The silica gel for column chromatography was used from Qingdao Haiyang Chemical Co., Ltd. (China). The monosaccharides and silanization reagents (L-cysteine methyl ester hydrochloride and N-trimethylsilylimidazole) were used from Shanghai Macklin Biochemical Technology Co., Ltd. (China). All molecular kits were purchased from Vazyme Biotech Co., Ltd. (China). All organic reagents were purchased from Thermo Fisher Scientific, Inc. (USA). The fungal strain P. chlamydosporia PC-170 was used for the study, and its solid (rice medium) fermentation method was the same as we described before (Qin et al., 2019 (link)).
8
Aspergillus flavus Cultivation and Antioxidant Assays
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A. flavus NRRL3357 was obtained from Sun Yat-sen University (Guangdong, China) and cultivated on potato dextrose agar (PDA) medium (Becton, Dickinson and Company, USA) at 30 °C. The spore suspension was harvested with 0.05 % Triton X-100, and the spore number was counted using a hemocytometer under a microscope. DNA was extracted using a Biospin fungus genomic DNA extraction kit from cultured A. flavus hyphae, and a Nanodrop (Nanodrop ND-1000 UV, Thermo Scientific) was used to measure the purity of DNA using an absorption ratio of 260 to A280, the DNA concentration was measured by Nanodrop and adjusted to a final concentration of 2 × 103 ng/μL. Ferrous sulfate heptahydrate (FeSO4·7H2O) was purchased from Sigma. Chemical kits used for testing activity of CAT (BC0200-50 T/48S), POD (BC0090-50 T/48S) and SOD (BC0170-50 T/24S) were purchased from Solarbio (Beijing Solarbio Science & Technology Co., ltd.). All other chemical agents were analytical grade.
9
Antifungal Activity of EGCG Against A. flavus
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EGCG of 98% purity, was purchased from Waston Technology Co., Ltd. (Shaanxi, China). A. flavus NRRL 3357 was gifted by Prof. Zhumei He of Sun Yat-sen University (Guangdong, China), and cultivated on the potato dextrose agar (PDA) medium (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) at 30°C in dark. Spores were harvested with 0.05% TritonX-80 and their numbers were counted with a hemocytometer.
10
Antifungal Effects of Arabidopsis Transgenic Lines
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Lyophilized leaf powder of each transgenic A. thaliana line was used to study the effect of different GSs on the visible growth of S. sclerotiorum. Anti-fungal activity was essentially assayed as previously described with minor modifications (Kelemu et al., 2004 (link)). S. sclerotiorum was preserved at 4°C and then reactivated in a Petri dish containing potato dextrose agar (PDA) medium (Becton Dickinson, Columbia, MD). The mycelium was inoculated into the center using a 5 mm puncher; the Petri dishes were then incubated at 22°C for 72 h to provide actively growing mycelium for subsequent experiments. New marginal hyphae were excised with a puncher from the Petri dishes. Filter paper disks of 10 mm in diameter were then placed onto the surface of PDA. Each filter-paper disk received 25 mg lyophilized powder and 100 μl ddH2O, along with 100 μl ddH2O only as a control. None of the three filter paper disks was a PDA Petri dish, and each of the three Petri dishes was a biological repeat. The filter-paper disks carrying lyophilized powder and S. sclerotiorum were incubated at 22°C. Fungal growth was assessed by observing visible mycelium growth and sclerotia number per disk after 72 h of incubation. Three technical replicates were performed.
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