## 2 Results
### Leaf Metabolome Modulation  
A data matrix considering the ions detected at all the time points was submitted to the MetaboAnalyst R package for statistical analysis. A supervised statistical approach, a partial least squares discriminant analysis (PLS-DA), was applied to the untargeted metabolomics data. The multivariate analysis revealed a separation between the challenged and control samples at all the studied time points (Figure 1), which suggests that a _Plytophthora cinnamomi_ attack influences the _Phimis purpurea_ leaf metabolic profile, enabling discrimination between the control and challenged samples. The combined analysis of both components (C1 and C2) for all the different time points accounts for over 45% of the sample variation. Both the components account for variations of 45.7%, 45.6%, 51.7%, 51.6%, and 44.5% for 6, 12, 24, 48, and 72 hpc, respectively. To understand the biological relevance of these differences, the \(m/z\) values were putatively annotated based on the KEGG and LipidMaps databases (Supplementary Table S3). Bibliographic searches and the KNApSAcK database were used to confirm that the putatively identified metabolites were assigned to the plant kingdom. A total of 56 \(m/z\) values were putatively annotated and presented a significant modulation (\(p\leq 0.05\)). The predicted metabolites from the \(m/z\) peaks, i.e., the observed ions, were putatively assigned to different metabolic classes, with the most predominant being lipids (53.57%), phytochemical compounds (25%), alkaloids (3.57%), phenylpropanoids (3.57%), and carbohydrates (1.79%) (Figure 2). Some metabolites (12.50%) were putatively assigned, although their function is not yet known. A detailed list of the putative metabolites for each of the time points analyzed is presented in Supplementary Table S3. \begin{table}
\begin{tabular}{c c c c c} \hline \multicolumn{5}{c}{**Differentially Abundant Ions**} & \multicolumn{3}{c}{**Putatively Identified Metabolites**} \\ \hline Time Point & Leaf & Roots & Leaf & Roots \\ \hline
6 hpc & 25 & 48 & 11 & 7 \\ \hline
12 hpc & 17 & 40 & 8 & 11 \\ \hline
24 hpc & 39 & 10 & 13 & 0 \\ \hline
48 hpc & 35 & 30 & 11 & 10 \\ \hline
72 hpc & 18 & 19 & 13 & 6 \\ \hline Total & 134 & 147 & 56 & 34 \\ \hline \end{tabular}
\end{table} 
Table 1: Number of differentially accumulated \(m/z\) values and differentially produced putatively identified compounds by LC-MS/MS in leaves and roots of _Phimis purpurea_ at 6, 12, 24, 48, and 72 h post challenge (hpc) with _Plytophthora cinnamomi_. To further evaluate the modulation of the different metabolic classes, a heatmap using all the \(m/z\) values, i.e., the observed ions, putatively annotated from the different time points, was constructed. After 12 h of the _Phlomis purpurea_ being challenged with the _Phytopthora cinnamomi_, a positive modulation of the ions with \(m/z\) values of 579.26, 149.0069, 150.0082, 301.126, 302.13, 191.052, 209.066, and 267.155, mostly associated with lipids and phytochemical compound classes, was observed in the leaves compared to the control samples (Figure 3). From 24 hpc to 72 hpc, an overall decrease in the same masses is observed in the _Phytopthora c  
Figure 1: Partial least squares discriminant analysis (PLS-DA) components 1 and 2 score plots of _Phlomis purpurea_ leaf metabolite profiles at (**A**) 6, (**B**) 12, (**C**) 24, (**D**) 48, and (**E**) 72 hpc with _Phytopthora cinnamomi_. In the score plots, the ellipse represents the Hotelling T2 with 95% confidence interval. The red ellipses represent the controls and the green ellipses the challenged samples. Four biological replicates were performed per analysis. occurred. In the challenged samples, most of the differentially accumulated metabolites presented a negative modulation at 24, 48, and 72 hpc. A metabolite putatively assigned as an alkaloid was up-accumulated at 24 hpc (\(m/z\) value 365.0), two (\(m/z\) values 175.0 and 244.0) were assigned as lipids. From the metabolites up-accumulated at 48 hpc 12.5% could not be identified.