Petri dish

Each B. bassiana isolate was cultured on 1/4 Sabouraud dextrose agar (SDA, BD Difco, USA) medium for 10 days in darkness at 25°C. The fungal conidia of each B. bassiana isolate was suspended in 0.03% siloxane solution (Silwet, FarmHanong Inc., Nonsan, Korea) at 1 ×10⁷ conidia/ml. A 1.0 ml aliquot of fungal conidial suspension was sprayed on cucumber leaf discs (110 mm diameter) and dried at room temperature for 1 h. Filter paper (No.2 Ø110, ADVANTEC, Tokyo, Japan) moistened with 500 μl distilled water was laid on a 90 mm Petri-dish (SPL Life Sciences, Pocheon, Korea) onto which the sprayed cucumber leaf disc was placed and infested with cotton aphid nymphs (about 52 aphids/leaf disc). All Petri-dishes were sealed and maintained under 26 ± 1°C and 16L:8D conditions, and the numbers of live and dead aphids were observed daily. A 0.03% siloxane solution was used as a negative control. In the first screening step, each treatment had only one replicate; in the following bioassays, three replicates were conducted for each treatment.

The PL1 (control line, K4191) and PL6 (mutant line) seeds were surface-sterilized with 70% ethanol for 1 min and then washed with sterile distilled water. Subsequently, the seeds were placed on moist filter papers in a Petri dish (SPL Life Sciences) until the first leaf of the seedlings appeared. Next, the uniformly germinated seeds were transferred to Incu Tissue culture vessels (SPL Life Sciences) filled with half-strength Hoagland’s culture solution (Sigma-Aldrich, St. Louis, MO, USA). The solutions were replaced daily. The seedlings were grown for 7 days in a well-controlled chamber at 22 °C and 60% humidity, with a photoperiod regime of 16/8 h day/night at 200–300 µmol m⁻²s⁻¹ light. After 7 days of transplanting, the seedlings were subjected to a salt stress treatment of a total volume of 200 mL of the solution containing 150 mM NaCl. Following treatment with 150 mM NaCl, the wheat leaves were collected at 0, 3, 24, and 48 h. Both control and salt-stressed seedlings were collected individually. The samples were immediately frozen in liquid nitrogen and stored at −80 °C until use in further experiments.

To isolate natural H. akashiwo cysts, sediment suspensions were sonicated three times for 10 sec (UT 53N, Sharp, Japan) to break up sediment aggregates and to separate cysts from the sediment particles [6 ]. A 200 μL aliquot of the sediment suspension was diluted with 6 mL filtered seawater and spread on a Petri dish (60 × 15 mm; SPL, Korea). H. akashiwo cysts were isolated using a capillary pipette under an epifluorescence microscope (IX71, Olympus, Japan) equipped with a chlorophyll filter set (450–490 nm band-pass excitation, 520 nm long-pass emission). H. akashiwo cyst identification was conducted following the morphological description of Imai et al. and Kim et al. [6 , 27 ]. For DNA extraction, isolated cysts were transferred to a microfuge tube containing beads and 750 μL lysis buffer from the PowerSoil^® DNA Isolation Kit (MoBio, USA) and maintained at 4°C in the dark until DNA extraction. DNA from these extractions was used to construct the qPCR standard curves.

To test the effect of hydrogen peroxide (H₂O₂) on growth of each strain, we used a previously reported method [28 (link)] with slight modification. Each strain was cultured in XOM2 broth medium at 28 °C with shaking until the population reached 3.5 × 10⁸ CFU/mL. Then, 200 μL of each cell culture was mixed with 20 mL PSA medium (1.0% agar) containing appropriate antibiotics and poured into a Petri-dish (90 × 15 mm, SPL life science, Seoul, South Korea). After solidification, a 6-mm-diameter sterilized Whatman paper disc was placed on the center of the plate, and then 5 μL of H₂O₂ (1 mM and 10 mM) was dropped onto the paper disc. Halo zone formation by H₂O₂ on the PSA plate was monitored, and the diameter was measured after 3 days of incubation at 28 °C.
Survivability of each strain against ROS was tested as described previously [19 (link)]. Each strain was incubated in XOM2 broth medium at 28 °C with shaking to 3.5 × 10⁸ CFU/mL. These cells were transferred to fresh XOM2 containing H₂O₂ (0, 1, or 10 mM) at a density of OD₆₀₀ = 0.2 and then shaken at 200 rpm and 28 °C for 6 h. The cells were spotted after serial dilution onto PSA plates containing appropriate antibiotics to count cell number.

For spontaneous differentiation, the iPSC colonies were fragmented and transferred to a petri dish (SPL Lifesciences, Pochon, Korea) containing an embryoid body (EB) culture medium (DMEM/F12, 10% KnockOut™ Serum Replacement, 1% NEAA, 1X P/S and 0.1 mM β-mercaptoethanol (all from Invitrogen)). The resulting EBs were cultured for 5-10 days in suspension and were transferred onto matrigel-coated slides for 15 days of adherent culture in differentiation medium (DMEM/F12, 1% NEAA, 1X P/S, 0.1 mM β-mercaptoethanol, and 10% FBS (all from Invitrogen)). The cells were immunostained with representative markers of the three germ lineages and were observed under a TE2000U fluorescence microscope (Nikon Corporation, Tokyo, Japan).

For the in vitro examination of pluripotency, both hESCs and iPSCs were mechanically detached from the plate and cultured in a Petri dish (SPL Life Sciences, Pocheon, Korea) by using the embryoid body (EB) medium (DMEM/F12), 10 % Knockout SR, 1 % non-essential amino acid (NEAA), 0.1 mM β-mercapto ethanol, and 1 % P/S (all from Invitrogen) for 15 days. EBs were attached to Matrigel (BD Biosciences)-coated slides, and EBs were immunostained for representative markers (Additional file 1) of the three germ layers.