Fig 1.
Detection by LC-MS of iPR and IAA exuded by R. irregularis spores.
(A) Structure and fragmentation pattern of iPR. (B) UPLC-MRM-MS chromatograms of iPR in positive mode. Blue lines represent the signals obtained for iPR external standard (100 nM). Red lines represent the signals obtained with GSE produced by 10,000 R. irregularis spores. Plain lines are for m/z transition 336 > 204. Dashed lines are for m/z transition 336 > 136. (C) LC-HRMS extracted ion chromatogram (XIC) for m/z = 336.1666 (+/-5ppm). The blue line represents the signal obtained for iPR external standard (300 nM). The red line represents the signal obtained with GSE produced by 250,000 R. irregularis spores. (D) Structure and fragmentation pattern of IAA. (E) UPLC-MRM-MS chromatograms of IAA in positive mode. Blue lines represent the signals obtained for IAA external standard (100 nM). Red lines represent the signals obtained with GSE produced by 10,000 R. irregularis spores. Plain lines are for m/z transition 176 > 131. Dashed lines are for m/z transition 176 > 77. (F) LC-HRMS XIC for m/ z = 176.0705 (+/-5ppm). The blue line represents the signal obtained for IAA external standard (300 nM). The red line represents the signal obtained with GSE produced by 250,000 R. irregularis spores. Signal intensities are displayed in counts per second (cps).
Fig 2.
GA4 detection in multiple reactions monitoring (MRM) mode.
(A) Structure and fragmentation pattern of GA4. (B) UPLC-MRM-MS chromatogram of GA4 in negative mode. Top (blue lines): External standard (30 nM) of GA4. Middle (red lines): pre-purified SPE fraction from 250,000 ground spores of R. irregularis. Bottom (purple lines): pre-purified SPE fraction from 250,000 ground spores of R. irregularis spiked with GA4 standard to a final concentration of 30 nM. Plain lines are for m/z transition 331 > 213. Dashed lines are for m/z transition 331 > 257. Signal intensities are displayed in counts per second (cps).
Fig 3.
Ethylene production by R. irregularis in response to different treatments.
20,000 spores were germinated for three days in the dark, in the presence or absence of 10 mM methionine (Met) or 1 μM α-keto γ-methylthio butyric acid (KMBA). Tubes were then sealed with a gas-tight stopper and exposed to light or darkness for 24h. One mL of the headspace gas was then analysed by gas chromatography. Different letters indicate different statistical groups (pairwise Kruskal-Wallis test with FDR correction, P < 0.05).