Modulation of metabolomic profile
Here, ultra-high-performance liquid chromatography quadrupole-time-of-flight high-resolution mass spectrometry (UHPLC-QTOF) and annotation in publicly available databases and large metabolite groups were conducted to obtain wide metabolome coverage. We applied UHPLC-QTOF-based untargeted metabolomic profiling of crude extracts to assess relative differences in the vegetative stage (43 DAT) and reproductive stage (131 DAT) leaf metabolite profiles between inoculated and uninoculated plants. A principal component analysis (PCA) explained 79% of the overall variance. The PCA score plot (Figure 1) showed two main clusters accounting for the vegetative and reproductive stages, respectively. Within each cluster, good separation was achieved between the metabolomic profiles of leaves from inoculated plants and those of leaves harvested from the uninoculated control. This finding was anticipated as the metabolism dramatically shifts as the plant transitions from the vegetative to reproductive phases. This outcome was relevant as PCA provides unsupervised descriptions of relatedness/unrelatedness. We ran a t -test ANOVA (P < 0.01) to identify differentially accumulated metabolites at each plant growth stage. This analysis disclosed > 466 annotated metabolites (Supplementary Tables 2 and 3) that had significantly changed between the vegetative and reproductive stages. Of these, 327 were common to both the first and second sampling points (Figure 2). In contrast, 68 and 71 metabolites differentially accumulated during the vegetative and reproductive stages, respectively (Figure 2). The interactions between microbial inoculants and plants are complex. Nevertheless, metabolomics effectively included the metabolic responses and mechanisms involved in the plant-microbe interactions. As 70% of the metabolites were common to the vegetative and reproductive phenological stages (Figure 2), biostimulant-mediated shifts in the leaf metabolome were recorded for both of them. On the other hand, certain stage-specific responses could be identified as well.
To clarify and visualise the variations between metabolic profiles at the vegetative and reproductive stages, we performed a chemical enrichment analysis using ChemRICH (Figure 3; Tables 4 and 5) and plotted the output by MetaMapp Cytoscape (Figure 4) (Barupal & Fiehn, 2017). Most of the significantly upregulated and downregulated metabolites (fold-change values ≤ 0.5 and ≥ 1.5, respectively; P ≤ 0.01) had a wide range of functions including growth stimulation, antifungal activity, pathogen resistance, energy sources, and secondary signalling cofactors.