Whatman filter no 1

A fluorescent dot-blot assay was used to estimate transglycosylase activity in vitro (Fry, 1997 (link); Chormova et al., 2015 (link)). Whatman No. 1 filters (Whatman, Dassel, Germany) were coated with nine different biologically relevant cell wall polysaccharides [1% (w/v) in dH₂O; see previous paragraph], left to dry and coated with sulphorhodamine-labelled xyloglucan oligosaccharides (XyGO-SRs; ∼5 µM) prepared according to Kosík and Farkaš (2008) (link) from enzymatically digested tamarind xyloglucan (Sulová et al., 1995 (link)). Test papers were loaded with 5 µl of algal enzyme extract and incubated in darkness at ∼20°C between acetate sheets to maintain humidity for 2 h. Papers were washed in ethanol:formic acid:water (1:1:1, v/v/v) for 1.5 h, rinsed twice with dH₂O and dried overnight. Orange fluorescence emitted by bound XyGO-SR was visualized by using a CX-20 work station [excitation 365 nm; Spectronics Corp., Westbury (NY), USA] connected to a Nikon Coolpix 8400 camera (Nikon Corp., Tokyo, Japan). Test papers lacking XyGO-SR or polysaccharide coating, or loaded with heat-inactivated enzyme extracts served as controls.

The fruiting bodies and mycelium of 17 edible and medicinal mushrooms, namely, Agaricus blazei, Dictyophora indusiata, G. lucidum, Hericium erinaceus, Hypsizygus marmoreus, I. obliquus, Lentinus squarrosulus, L. edodes, Lentinus TAFRS007, Lentinus TAFRS011, Lentinus TAFRS014, Morchella esculenta, Cordyceps sinensis, Pleurotus sajor-caju, Pleurotus eryngii, Phellinus igniarius, and Tremella fuciformis were collected from local markets in Thailand. The fruiting bodies or mycelia were cut into small pieces, ground, and lyophilized for 2 days. Then, the dried small pieces of mushrooms were ground into powder and divided into three parts. Each part was extracted with each solvent, distilled water (DW), and 99.5% ethanol and hexane. Ethanol and hexane extractions were carried out by incubating at 37°C in a shaker at 180 rpm overnight, while the DW extraction was performed at 25°C overnight in a shaker at the same rpm. After that, all crude extracts were filtered through Whatman no.1 filters and centrifuged at 7000 rpm, 4°C for 15 min to remove precipitate. The ethanol and hexane solvents were removed using a hot air oven at 80°C, and the DW was removed by lyophilization. The mushroom powder was redissolved in 100% dimethyl sulfoxide (DMSO) to a final concentration of 100 mg/mL. The crude extracts were kept at −20°C until used.

Extractions were carried out in two different media: Methanol (MeOH; HPLC grade) and water (Milli-Q waters). For the methanol extraction, leaves were lyophilized (ModulyoD/23, Thermo Savant; Waltham, MA, USA) and crushed with a grinder. Extraction was performed as follows: 2.5 g of dry material was extracted with 50 mL MeOH in an ultrasonic liquid processor (Qsonica Sonicators; Newton, CT, USA) with a power of 55 W and a frequency of 20 kHz, for 10 min (using 50% power) at room temperature. Extractions were done in triplicate. After sonication, solutions were filtered through Whatman No.1 filters. The solvent was evaporated under reduced pressure in a Hei-Vap Precision rotary evaporator (Heidolf; Schwabach; Germany) at 40 °C. Dried extracts (DE) were stored at −20 °C until analysis.
On the other hand, the extraction with water was carried out in the following way: 2.5 g of fresh leaves (crushed with a grinder) was extracted with 150 mL H₂O at 100 °C in a hot plate (C-MAG HS7, IKA; Staufen, Germany) for 30 min. Extractions were done in triplicate. After that, solutions were filtered through Whatman No.1 filters. Finally, the solvent was evaporated under reduced pressure in a rotary evaporator and the dried extracts were stored at −20 °C until analysis.

Extractions were carried out in methanol (MeOH; HPLC grade). The aerial parts of the plants were lyophilized (ModulyoD/23, Thermo Savant; Waltham, MA, USA) and crushed with a grinder. Extraction was performed as follows: 2.5 g of dry material was extracted with 50 mL MeOH in an ultrasonic liquid processor (Qsonica Sonicators; Newton, CT, USA) with a power of 55 W and a frequency of 20 kHz, for 10 min (using 50% power) at room temperature. Extractions were performed in triplicate. After sonication, solutions were filtered through Whatman No.1 filters. The solvent was evaporated under reduced pressure in a Hei-Vap Precision rotary evaporator (Heidolf; Schwabach; Germany) at 40 °C. Dried extracts (DE) were stored at −20 °C until analysis.

Soil pH was measured using a standard procedure where replicate 10 g aliquots of air-dried soil were suspended in 25 ml freshly-boiled deionised water. Soil organic carbon (SOC) and total N was determined by LECO analysis, subtracting carbonate released by incubation with hydrochloric acid from total C to estimate organic C. To extract soluble C from soil (Ghani et al. 2003 (link)), samples frozen immediately after collection and stored at −80 °C, were shaken in sterile distilled H₂O (sd H₂O - soil: water, 1: 10) at 300 rpm for 30 min, 20 °C. After centrifugation (swing-out rotor, 246×g, 20 min, 20 °C) the supernatant was decanted, soil resuspended and shaken as before for 16 h at 80 °C. The total non-purgeable organic C in the supernatant of this fraction was determined by wet oxidation using a Shimadzu TOC-V WS analyser. A simplified method was used to measure un-decomposed organic detritus (from plants and mesofauna): 5 g soil suspended in 10 ml sdH₂O was shaken for 24 h at 300 rpm, 4 °C. The suspension was centrifuged (swing-out rotor, 50×g, 10 min, 20 °C) and the material remaining in suspension was weighed after collection on pre-weighed Whatman no 1 filters and overnight drying at 80 °C.

To determine the IAA, GA and ACC-deaminase concentrations, the DEMTKz3A0 isolate inoculum (1.0 × 10⁵ conidia per mL) prepared as described before (Section 4.5) was cultivated in 100 mL Erlenmeyer flasks with 20 mL of a modified RB medium supplemented with solutions of glucose (1%) as a carbon source, and the phytohormone precursors tryptophan and methionine (3 mM) sterilized using syringe filters (0.22 μm) at 12, 20, and 28 °C and 60% relative humidity in an Innova 4900 growth chamber (New Brunswick Scientific, Edison, NJ, USA) at 120 rpm for 2, 3, 4, and 5 days. The dry weight of mycelium (mycelium d.w.) was determined after collecting on Whatman no. 1 filters and weighting after drying at 65 °C for 24 h. To determine the ability of the DEMTzZ3A0 strain to synthesize IAA, GA, and deaminase ACC the supernatants of the liquid culture were additionally centrifuged (10,000× g for 10 min).