Metabolic fingerprinting of the ectomycorrhizal roots of an oak tree analysed by 1H NMR

The metabolome of mycorrhizal cork oak (Quercus suber L.) roots colonized by the ectomycorrhizal fungus Pisolithus tinctorius was analysed by High-resolution 1H NMR spectroscopy. Samples (100 ml of N2 root frozen powder) from mycorrhizal and non-mycorrhizal (control) roots were extracted using a semi-polar extraction with a mixture of water/methanol. Extracts from each sample were analysed using a Bruker AVANCE 600 spectrometer equipped with an automatic sample changer and a multinuclear triple resonance (TBI) probe (all from BrukerBiospin, Rheinstetten, Germany) at a field strength of 14.1 T (600.13 MHz 1H frequency). The probe temperature was set to 298.0 K. Following the introduction to the probe, samples were allowed to equilibrate (1 min) prior to the shimming process to ensure good magnetic field homogeneity. All liquid sample handling, automation and acquisition were controlled using TOPSPIN 2.1 software (BrukerBiospin, Rheinstetten, Germany). One-dimensional (1D) 1H-NMR spectra were acquired with suppression of the water residual resonance. The water resonance was pre-saturated using a power level of 55 dB during a relaxation delay of 2 s. Each spectrum was acquired into 32 k data points over a spectral width of 16 ppm as the sum of 128 transients and with an acquisition time of 1.7 s. The total acquisition time was ~8 min per sample. All 1H NMR spectra were phased, base line corrected and referenced to the internal standard (TSP for polar) resonance at δ 0 ppm using the same software TOPSPIN 2.1.