Prior to phytohormone extraction, the collected fungal samples (40–200 mg fresh weight, n = 3) were freeze-dried in a Modulyo®D Freeze Dryer (Thermo Scientific, Waltham, USA) for 48 h. The freeze-dried fungal samples and the collected root tissue of co-cultivated seedlings (1 sample = 12–15 seedlings from the same plate; which corresponded to 30–150 mg fresh weight, n = 8) were weighed and homogenized using a Geno/Grinder® (Spex SamplePrep, Stanmore, UK) at 1,100 rounds per min for 1 min. As described in Almeida Trapp et al. (2014 (link)), 1 ml of methanol: water (7:3) containing 20 μg/ml of d4-SA and d5-IAA as well as 10 μg/ml d5-JA and d6-ABA was added to the powdered root and fungal material. After mixing, samples were shaken for 30 min and centrifuged at 16,000 g at 4°C for 5 min. Subsequently the supernatant was transferred into a new tube and concentrated for 4 h at 45°C in an Eppendorf Concentrator plus (Eppendorf AG, Hamburg, Germany). The concentrate was resuspended in 100 μl of 50% methanol with 0.05% formic acid, mixed and centrifuged at 16,000 g at 4°C for 10 min. Afterwards the supernatant was collected and measured on an Agilent 1100 HPLC system (Agilent Technologies, Böblingen, Germany) connected to a LTQ Orbitrap mass spectrometer (Thermo Scientific, Waltham, USA) (Almeida Trapp et al., 2014 (link)).
Free full text: Click here
