Untargeted exudate analysis
Rood exudate composition was investigated trough an ultra-high-pressure liquid chromatography (UHPLC) coupled to a quadrupole-time-of-flight (QTOF) mass spectrometry as reported by Astolfi et al. (2020) with minor modifications. Briefly, a 1290 series LC system equipped with a binary pump, a JetStream Electrospray source and a G6550 iFunnel QTOF mass spectrometer (Agilent technologies, Santa Clara, CA, USA) working in positive SCAN mode (100–1200 m/z range) was used. The chromatographic separation was achieved by a reverse phase Agilent PFP column (2.0 × 100 mm, 3 μm) using a gradient of water and methanol (from 6% to 94% organic phase in 33 min, flow rate 200 μl min-1). Quality control pooled samples (QCs) were also analyzed in MS/MS mode (data-dependent, 12 precursors per cycle at 1 Hz, 50–1000 m/z, positive polarity, active exclusion after 2 spectra), with collision energies of 10, 20, and 40 eV as previously reported (García-Pérez, Miras-Moreno, Lucini & Gallego 2021).
Compound annotation was carried out according to Astolfi et al.(2020) from raw mass features, using the software Profinder B.07 (from Agilent Technologies, Santa Clara, CA, USA) according to the ‘find-by-formula’ algorithm following mass and retention time alignment. To this aim, the whole isotope pattern (monoisotopic mass, isotopic spacing and isotopic ratio) was used. A custom database produced by combining compounds exported from PlantCyc 9.6 (Plant Metabolic Network, http://www.plantcyc.org; accessed April 2017), Phenol-Explorer 3.6 (http://www.phenol-explorer.eu; accessed April 2017), as well as information on compounds extracted from the literature and that might be present in root exudates was used as a reference for annotation (5-ppm mass accuracy tolerance). The annotation level corresponded to Level 2 of accuracy (putatively annotated compounds) as set out by the COSMOS Metabolomics Standards Initiative (http://cosmos-fp7.eu/msi). Only those compounds identified within 75% of replications within at least one treatment were retained.
The MS-DIAL 4.24 software provided a higher degree of confidence in annotation, based on MS/MS spectra of QCs to confirm features obtained by Profinder B.07. (Tsugawa et al. 2015). To this aim, publicly available built-in MS/MS experimental spectra (Mass Bank of North America) were used.