Xs105

For UHPLC‐MS/MS analysis, an adapted version of the method described in Buchner et al. (2017) was used. For cellular hormone measurements, 5 mg of lyophilized algal biomass was weighed with an analytical balance (XS 105; Mettler Toledo®). 20 mg of lyophilized agar was used to determine extracellular hormone levels. Algal biomass or agar was placed in 2 mL safe‐lock Eppendorf tubes and extracted in 1.5 mL of ice‐cold acetone/water/acetic acid (80:20:1, v:v:v) after addition of 25 µL stable isotopically labeled internal standard solution (0.5 µM ABA‐d6, 0.5 µM SA‐d4) by shaking (TissueLyser II; Qiagen, Düsseldorf, Germany) at 30 Hz for 5 min using one 5 mm glass bead (pre‐cleaned with methanol) for each Eppendorf tube, followed by centrifugation at 10,000g, 4°C for 12 min. Supernatants were evaporated to dryness using a SpeedVac SPD111 vacuum concentrator (Thermo Fisher Scientific Inc., Waltham, MA, USA) for 1 h to remove the acetone, followed by freezing in liquid nitrogen and lyophilization for 14 h ± 5 min. The lyophilized pellet was resuspended in 75 µL of acetonitrile by vortexing for 10 s and 5 min ultra‐sonication in an ice‐cooled water bath. Then, 75 µL of UPW were added followed by vortexing for 10 s and 5 min ultra‐sonication in an ice‐cooled water bath. The extracts were filtered through 0.2 mm PTFE filters before injection into the UHPLC‐MS/MS system.

For biomasses assay, conidial suspension of the strains was inoculated into PDB medium. The final concentration of conidia in the PDB medium was 1 × 10⁴ conidia/ml, and then the conidia suspension was inoculated at 26°C on a rotary shaker (200 rpm). The biomasses were measured at the 4 dpi. Mycelia were harvested and dried by Vacuum freeze dryer (Martin Christ, Osterode am Harz, Germany) and weighted by Mettler Toledo XS105 (Switzerland). Biomass relative to Vd991 = Biomass (Vd991, ΔVdDrs2, or Com)/Biomass of Vd991. For conidia yield assay, fungal spores were added into PDA (temperature bellow 60°C) to the final concentration of 5 × 10⁴ conidia/ml. Plates were placed at 26^°C for 10 days. Conidia were collected with a puncher and suspended in water with 0.05% Tween 80. Conidial concentration was determined by hemocytometer. All experiments were performed in triplicates with three independent repeats.

The water-absorbing capacity of the prepared hydrogels was evaluated through swelling ratio (S_g/g), according to a previously reported method [30 (link)]. Gravimetrically, 100 mg of dried hydrogels were immersed in Milli-Q water at ambient temperature for 24 h to reach swelling equilibrium. Then, samples were accurately re-weighted, using a Mettler Toledo XS105 precision balance (Mississauga, ON, Canada), in order to calculate their ratios by applying Equation (1) below: $$S_{g/g}=\frac{M_s-M_d}{M_d}$$
where $M_s$ = mass of the swollen hydrogel, and $M_d$ = initial mass of the dry gel. After deswelling the hydrogels at room temperature for 48 h until a constant weight was obtained, the above process was repeated three times under the same conditions, where the samples were allowed to swell again in water for the evaluation of their mechanical behavior through successive swelling–deswelling cycles. The given values are an average of three individual sample readings.

Hindlimb muscle denervation was induced by the unilateral resection of a segment of the sciatic nerve using methods previously described [26 (link)]. Briefly, mice were anesthetized with isoflurane gas (2–3%) and an ~10 mm incision made mid-thigh. Following blunt dissection, the sciatic nerve was visualized and an ~5 mm segment removed. A sham surgery was performed on the contralateral leg. Mice recovered for 28 days prior to muscle collection. After 28 days, mice were fasted overnight, anesthetized with pentobarbital sodium (90–100 mg/kg body weight) for 40 min and euthanized by cervical dislocation. Muscles were excised and weighed to ±0.1 mg using an analytical balance (model XS105, Mettler-Toledo, Columbus, OH, USA), prior to additional experimental perturbations as described below.

Exponential growth of each species was determined to ensure that algal samples taken for subsequent experiments were at the same developmental stage (Gustavs et al. 2009). Algal biomass was harvested every two to three d, transferred to 2 mL Eppendorf tubes, frozen in liquid nitrogen and stored at −20°C prior to lyophilization (Zirbus VaCO₂; Zirbus technology GmbH, Germany) for at least 90 h as described by Bailly and Kranner (2011). Growth curves for each species were constructed using sample dry mass (DM), determined with an analytical balance (XS 105; Mettler Toledo®, Greifensee, Switzerland), to assess the exponential growth phase for each species.