Agar agar kobe 1

In 2011, 2012, and 2013, we isolated bacteria from CG material sampled in vineyards of Franconia, Germany. The grapevine cultivars consisted of the scion Cabernet Dorsa, Scheurebe, and Müller Thurgau grafted onto the rootstocks 5BB and SO4 (NCBI, BioProject: PRJNA624984). CG material was ground (2 min, 30 Hz) using a ball mill (Retsch, Hannover, Germany), and 300 mg CG powder was suspended in super purified water (RotisolV high-performance liquid chromatography [HPLC] gradient grade; Roth). After incubation for 2 h at 28 °C, the supernatant was used to create serial dilutions at a ratio of 1:10. Agar plates containing yeast extract broth (YEB; 0.5% [wt/vol] tryptone, 0.5% [wt/vol] yeast extract, 0.5% [wt/vol] sucrose, 1.23% [wt/vol] MgSO₄ [AppliChem, Darmstadt, Germany], 1.5% [wt/vol] Agar-Agar Kobe I [Carl Roth, Karlsruhe, Germany]) supplemented with 213 µM cycloheximide (CHX; Sigma-Aldrich, St. Louis, MO, USA) were used for bacterial growth at 28 °C. By growing colonies on rifampicin-containing yeast extract agar (RIF-YEA, 10 µg/mL) plates, spontaneous rifampicin-resistant derivatives were selected for tracking them in their natural environment. Single colonies were subcultured at least 5 times on YEA-CHX-RIF plates and used for de novo shotgun sequencing.

All media and watery solutions were prepared with water processed with an arium^® pro device (Sartorius AG, Göttingen, Germany). If not otherwise stated, E. coli and B. subtilis strains were cultivated in LB medium (10 g/L tryptone, 10 g/L NaCl, and 5 g/L yeast extract) [30 (link)]. LB medium was supplemented with 1.5% (w/v) Agar-Agar Kobe I (Carl Roth GmbH + Co KG, Karlsruhe, Germany) to prepare solid LB agar plates. Overlay agar for plaque assays was prepared via LB medium supplementation with 0.4% (w/v) Biozym LE Agarose (Biozym Scientific GmbH, Hessisch Oldendorf, Germany).

Malt-extract–glucose medium (MEG) contained 10 g L⁻¹ of glucose and 5 g L⁻¹ of malt extract broth (Oxoid, Basingstoke, UK). P. ostreatus culture was maintained at 4 °C on agar plates with MEG and 2% agar–agar Kobe I (Carl-Roth, Karlsruhe, Germany). The inoculum was prepared by placing three agar plugs of the culture (d = 1 cm) into 20 mL of MEG in 250-mL Erlenmeyer flasks. After cultivation (static, 7 days, 28 °C), the inoculum was homogenized by Ultra-Turrax T 25 (IKA, Staufen im Breisgau, Germany), and 5 mL of the homogenate was added to 45 mL of MEG in 500-mL Erlenmeyer flasks. After an additional 7 days of cultivation, the extracellular liquid was separated from the mycelium by a nylon mesh. The liquid was then gradually filtered through a series of glass fibre filters (1.4 and 0.5 µm; Macherey-Nagel, Düren, Germany) and cellulose nitrate membrane filters (0.45 and 0.2 µm; Whatman, GE Healthcare, Chicago, IL, USA) and concentrated approximately 250-fold using ultrafiltration discs (regenerated cellulose, molecular weight cut-off 10 kDa; Merck). The concentrated extracellular enzymes were stored at −20 °C and filtered through a 0.22-µm nylon syringe filter (Membrane Solutions, Auburn, WA, USA) before immediate use.

Table 3 lists the strains used in this study. Escherichia coli DH10β was used as an intermediate host for cloning purposes. Bacillus subtilis and E. coli cells were routinely grown in LB-Broth (Luria/Miller) for molecular biology (tryptone 10 g l⁻¹, yeast extract 5 g l⁻¹, NaCl 10 g l⁻¹, and pH 7.0; Carl Roth, Karlsruhe, Germany) at 37°C with agitation. 1.5% (w/v) agar (Agar-Agar Kobe I, Carl Roth, Karlsruhe, Germany) was added to prepare the corresponding solid media. Strains were stored at −80°C in their corresponding growth media containing 20% (v/v) glycerol (Carl Roth, Karlsruhe, Germany). Ampicillin (Carl Roth, Karlsruhe, Germany) 100 μg ml⁻¹ was used for selection of E. coli, when required. Chloramphenicol (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany), 5 μg ml⁻¹ was added to select B. subtilis biosensors, where appropriate.

Beetroot (Beta vulgaris subsp. vulgaris, variety “Pablo”, grown in the greenhouse at Agroscope-Wädenswil (CH)) was harvested, the soil was removed, and small parts of the roots were resuspended in phosphate buffer (15 mM K₂HPO₄ and 9 mM KH₂PO₄, pH 7). After 10-fold serial dilution (up to 10⁻³), 100 µL of the respective dilution steps were plated on tryptic soy broth (TSB, Oxoid/Thermo Fisher Scientific, Waltham, MA, USA) agar plates (15 g/L Agar-Agar, Kobe I (Carl Roth, Karlsruhe, Germany)). Plates were incubated at 26 °C for 24 h; single colonies were picked and subcultured to ensure pure cultures. Bacterial isolates were identified at the genus level with MALDI-TOF by smearing bacterial cell material from a colony on agar plate onto a MALDI targe for the identification of specific biomarker peptides [30 (link)] and 16 S rRNA gene sequencing. Forty-four bacterial isolates (Supplementary Table S1) were then tested for their antagonistic potential against M. incognita.

Single colonies (sphere diameter: 5 mm) of A. niger were transferred to 500 µL of treatment solutions containing different concentrations of PS diluted in DBPS. Three controls were used for each experiment: C−/− (without a PS and illumination), PS control (with PS in the highest concentration used in the experiment and no light) and light control (illumination with the samples but without a PS). The samples were then incubated for 20 min in the dark at room temperature with constant shaking (400 rpm). Afterward, the samples, except for C−/− and PS control, were illuminated with an LED array (435 nm, 15.8 J cm⁻²) from below under constant shaking (400 rpm). The C−/− and the PS control were incubated under constant shaking in the dark for one hour using the same shaker as the samples. The colonies were then transferred to an SB agar plate containing 1.5% Agar-Agar Kobe I (for microbiology, Carl Roth GmbH + CoKg) and incubated for three days at 37 °C (adapted from [43 (link)]).
Growth inhibition was investigated by imaging the cultures at different time points and measuring the colony area using the image processing and analyzing software ImageJ 1.48v (National Institute of Health, Bethesda, MD, USA [44 ]). Each experiment was reproduced nine times.

Table 1 lists the strains used in this study. B. subtilis cells were routinely grown in Luria-Bertani (LB-Medium (Luria/Miller), Carl Roth, Karlsruhe, Germany) medium at 37 °C with agitation. 1.5% (w/v) agar (Agar-Agar Kobe I, Carl Roth, Karlsruhe, Germany) was added to prepare the corresponding solid media. Strains were stored at −80 °C in their corresponding growth media containing 20% (v/v) glycerol (Carl Roth, Karlsruhe, Germany). Chloramphenicol (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) 5 µg mL⁻¹, kanamycin (Carl Roth, Karlsruhe, Germany) 10 µg mL⁻¹, tetracycline hydrochloride (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) 12.5 µg mL⁻¹, erythromycin (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) 1 µg mL⁻¹ and lincomycin (Alfa Aesar™ Lincomycin hydrochloride, Fisher Scientific, Kandel, Germany) 25 µg mL⁻¹ were added to B. subtilis when required.