From Tano Forest Science Station at University of Miyazaki, white rot-decayed cedar wood samples and a basidiomycetous fruit body were collected from same wood. The decayed xylem was collected to approximately 2 cm depth after removal of the bark. For isolation of cultivable bacteria, wood chips were placed in 10 mL sterile water, vortex mixed and the resulting suspension dilution plated in 0.1 mL aliquots onto R2A agar (peptone 0.5 g/L, yeast extract 0.5 g/L, casamino acids 0.5 g/L, glucose 0.5 g/L, soluble starch 0.5 g/L, K 2 HPO 4 0.3 g/L, MgSO 4 •7H 2 O 0.05 g/L, sodium pyruvate 0.3 g/L, Agar 15 g/L). Available bacterial colonies were isolated after cultures were incubated for 2-7 days at 25 °C. The fungal mycelia were isolated on the agar medium with beech wood powder as the sole carbon source and on the basis of formation of a reddish-colored zone in the culture medium resulting from guaiacol oxidation, type of rot was evaluated. The isolated mycelia were maintained on potato dextrose agar (PDA; Difco Laboratories). Bacterial strains which showed enhancement effect for mycelial growth of isolated fungus were selected preliminary by confrontational assay on PDA according to our previous study [14] . The incubation of mycelium and measurement of mycelial growth were carried out as described "Confrontational assay" section.
Potato dextrose agar pda
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Potato dextrose agar (PDA) is a culture medium used for the growth and isolation of fungi. It consists of potatoes, dextrose, and agar as the main components. PDA provides the necessary nutrients and growth conditions for a wide range of fungal species.
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Potato dextrose broth (pdb), by BD (3 mentions)
Methanol, by Merck Group (3 mentions)
Neutrase 0.8 l enzyme, by Novozymes (2 mentions)
Nylon cell strainer, by BD (1 mentions)
Dm4000m, by Leica (1 mentions)
40 mm nylon cell strainer, by BD (1 mentions)
Hplc grade acetonitrile, by Merck Group (1 mentions)
Polytetrafluoroethylene ptfe syringe filter, by Thermo Fisher Scientific (1 mentions)
Plate count agar, by Thermo Fisher Scientific (1 mentions)
Baird parker agar, by BD (1 mentions)
M17 agar, by Thermo Fisher Scientific (1 mentions)
Violet red bile lactose agar, by Thermo Fisher Scientific (1 mentions)
Brain heart infusion agar, by BD (1 mentions)
Anhydrous sodium sulfate, by Junsei (1 mentions)
Sodium chloride (nacl), by Junsei (1 mentions)
Diazinon, by Chem Service (1 mentions)
Ferulic acid, by Merck Group (1 mentions)
Calcium carbonate, by Merck Group (1 mentions)
Stevioside, by Fujifilm (1 mentions)
Coniferyl alcohol, by Merck Group (1 mentions)
19 protocols using potato dextrose agar pda
1
Isolation and Screening of Bacteria and Fungi from White Rot-Decayed Cedar Wood
2
Soil Fungal Isolation and Cultivation Protocol
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Soil samples were collected in 2014 from agricultural fields at various locations in Gangwon-do (36°54'06.10" N, 127°28'08.20" E), Chungcheongbuk-do (36°46'04.61" N, 127°30'00.52" E), and Gyeonggi-do (37°25'45.60" N, 127°08°14.15° W), Korea. Each soil sample was collected from a depth of approximately from 0~15 cm after removing the surface litter, sealed in a sterile soil sampling polythene bag, air dried, and stored in a plastic bag at 4℃ until use. The fungi were isolated using the conventional dilution technique [6 ] and cultured on potato dextrose agar (PDA; Difco Laboratories, Detroit, MI, USA) supplemented with 100 µg/L chloramphenicol (a bacteriostatic agent) for 5~7 days at 28℃ until fungal colony growth was observed. The pure cultures were maintained on the PDA slants at 4℃ for future use.
3
Trichoderma Strain Diversity and Cultivation
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Eight Trichoderma strains, representing different genotypes, were included in this study: T. parareesei T6 (air, UK) [55 (link),56 (link)], T. virens T49 (soil, Brazil), T. longibrachiatum T68 (soil, Brazil), T. spirale T75 (solarized soil, Spain), T. koningii T77 (soil, Spain) [6 (link),55 (link)], and T. harzianum T115 (soil, Philippines) [6 (link)], T. hamatum T123 (Marchantia polymorpha rhizoids, Spain), and T. asperellum T140 (strawberry nursery soil, Spain) (references of our collection, CIALE, University of Salamanca, Spain). Strains were routinely grown on potato dextrose agar (PDA, Difco Laboratories, Detroit, MI, USA) at 28 °C in the dark. For long-term storage, the strains were maintained at −80 °C in a 30% glycerol solution. Conidia from 7-day-old PDA plates were harvested, and the conidia concentrations were calculated as previously described [35 (link)].
4
Isolation and Cultivation of Soil Fungi
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Soil samples were collected in March 2016 from crop field soil at various locations in Gyeongnam (35.102124° N, 128.023072° E) and (34.525836° N, 128.275765° E), Gyeongsangnam-do, Korea. Samples were taken from a depth of 10–15 cm, air dried, and stored in plastic bags at 4 °C until use. Fungi were isolated using a conventional dilution technique [21 ]. One gram of each soil sample was suspended in 9 mL of distilled water, and the prepared suspension was vortexed, serially diluted, and cultured on potato dextrose agar (PDA; Difco Laboratories Detroit, MI, USA) plates. The plates were incubated for 7 days at 25 °C, until fungal colony growth was observed. After that, single colonies on these plates were cultured by transferring them onto new PDA plates, supplemented with 100 mg/L chloramphenicol (a bacteriostatic agent) for 5–7 days at 25 °C until the fungal colony was observed. The pure cultures were maintained on PDA slants at 4 °C for future use.
5
Fungal Diversity from Korean Croplands
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Soil sample collection was carried out in 2016 at different crop field locations in Gyeongnam (N 35.2640.46°, E 128.322497° and N 35.092802°, E 128.084769°), Korea. Soil samples were collected at a depth of up to 15 cm by removing crop debris. Each soil sample was air dried and stored in a sterile polythene bag at 4 °C. A conventional soil dilution technique [8 ] was employed to isolate the fungi. Potato dextrose agar (PDA; Difco Laboratories, Detroit, MI, USA) amended with 100 μg L−1 chloramphenicol was used for isolation. The Petri plates, in which diluted soil suspensions were streaked, were incubated at 25 °C for 5 days. The developing colonies were then transferred to fresh PDA medium to obtain pure cultures. The pure isolates were finally transferred to PDA slants and kept at 4 °C until further use.
6
Cultivation and Conidial Harvesting of Scedosporium apiospermum
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Scedosporium apiospermum (strain RKI07_0416) was kindly provided by Dr Bodo Wanke (Evandro Chagas Hospital, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil). The fungus in its mycelial form was cultured in Erlenmeyer flasks containing 200 mL of Sabouraud-dextrose broth (2% glucose, 1% peptone and 0.5% yeast extract) at room temperature (RT), with constant shaking (200 rpm) for 7 days [36 (link)]. Subsequently, the mycelial cells were filtered using filter paper and washed twice with sterile phosphate-buffered saline (PBS; 10 mM NaH2PO4, 10 mM Na2HPO4, 150 mM NaCl, pH 7.2). To obtain conidial cells for the interaction assays, the fungus was grown on potato dextrose agar (PDA; Difco Laboratories, Franklin Lakes, NJ, USA) plates at RT for 7 days. Conidia were then harvested by washing the plate surfaces with PBS and filtering them through a 40 μm nylon cell strainer (BD Falcon, Saffron Walden, UK) to remove hyphal fragments [30 (link)]. The conidial cells were counted using a Neubauer chamber.
7
Fungal Pathogens and Biological Control
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The fungal pathogens used in this study were V. longisporum (C. Stark) (Karapapa et al., 1997 (link)) strain ELV25 and V. dahliae Kleb. strains ELV22, V004 and V024. The strains ELV22 and ELV25 from the collection of the Institute of Environmental Biotechnology (Graz University of Technology), were described by Messner et al. (1996) (link). The mild-virulent strain V004 was classified as non-defoliating pathotype (Blanco-López et al., 1989 (link)), and the high-virulent strain V024 was classified as defoliating pathotype (Varo et al., 2016b (link)). Both were obtained from the fungal collection of the Agronomy Dpt. of the University of Córdoba. The non-pathogenic F. oxysporum strain FO12, also from the fungal collection of the Agronomy Dpt. of the University of Córdoba, was applied as BCA. Single-spore cultures of all isolates were prepared prior to use by means of the serial dilution method and maintained on potato dextrose agar (PDA; Difco® Laboratories, MD, United States) slants at 4°C. 7-day-old single spore cultures incubated on PDA at room temperature were used as an inoculum source.
8
Fungal Biofilm Cultivation Protocol
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Colletotrichum musae D128, Fusarium dimerum F30 and Fusarium oxysporum D221 (Department of Food, Environmental and Nutritional Sciences, University of Milan collection) were used as model systems for fungal biofilm. C. musae and F. oxysporum were grown on Potato Dextrose Agar (PDA, Difco Laboratories, USA) while F. dimerum was cultured on Czapek Agar (CA, Sigma Aldrich, USA). The fungi were maintained at 21 °C for 15 days until conidia collection. The conidia were collected in water and filtered through a double layered sterile gauze according to Kunova et al. (2016) , and the concentration determined by conidia counting using a light microscope (Leica DM4000 M, Leica Microsystems, Germany) and Thoma counting chamber.
9
Culturing and Isolation of Scedosporium Fungi
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Scedosporium apiospermum (strain RKI07_0416) was kindly provided by Dr. Bodo Wanke (Evandro Chagas Hospital, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil), and S. minutisporum (strain FMR 4072), S. aurantiacum (strain FMR8630) and L. prolificans (strain FMR 3569) were kindly given by Dr. Josep Guarro (Microbiology Unit, Medical School and Institute of Advanced Studies, Reus, Spain). Fungal cells were maintained in Sabouraud (2% glucose, 1% peptone and 0.5% yeast extract) liquid culture medium for 7 days at room temperature with orbital shaking (200 rpm). Conidia were obtained by growing each fungus at room temperature in Petri dishes containing potato dextrose agar (PDA; Difco Laboratories, USA). Subsequently, conidial cells were obtained by washing the plate surfaces with phosphate-buffered saline (PBS; 10 mM NaH2PO4,10 mM Na2HPO4, 150 mM NaCl, pH 7.2) followed by filtration through a 40-mm nylon cell strainer (BD Falcon, EUA) in order to remove hyphal fragments (Mello et al., 2016 (link)). The conidial cells were counted in a Neubauer chamber.
10
Conidia Generation and Antifungal Assays for Penicillium Species
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For generation of conidia, fungi were cultured on Potato Dextrose Agar (PDA; Difco-BD Diagnostics, Sparks, MD, USA) plates for 5 (P. chrysogenum strains)–7 (P. digitatum, P. italicum and P. expansum strains) days at 25 °C. For antifungal assays, P. digitatum CECT 20796 (PHI26) [32 (link)], P. italicum CECT 20909 (PHI1) and P. expansum CECT 20906 (CMP-1) [33 (link)] strains were used. Representative images of fungal growth on PDA plates are shown in Figure 1 a.
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