## 4 Discussion  
The current study comprehensively evaluated the antifungal activity of carvacrol against _P. digitatum_ through \({}^{1}\)H-NMR metabonomics. Following the important metabolites selected by OSC-PLS-DA loading/S-diagram, METPA ([[http://www.metaboanalyst.ca](http://www.metaboanalyst.ca)]([http://www.metaboanalyst.ca](http://www.metaboanalyst.ca))) (Figure 6A) and KEGG ([[http://www.genome.jp/kegg/](http://www.genome.jp/kegg/)]([http://www.genome.jp/kegg/](http://www.genome.jp/kegg/))) were used for path analysis to determine biologically significant metabolic patterns and related pathways (Figure 6B-E). Alanine, aspartate, glutamate, and glutathione metabolisms were imbalanced in the fungal hyphae (Figure 6). These metabolisms were also found in the correlation network of D group at 12 h. A strong positive correlation between aspartate, glutamate, alanine, and glutamine was seen, and a negative correlation between glutathione and lactate was also observed. These metabolic changes revealed \begin{table}
\begin{tabular}{c c c c} \hline \hline \multirow{2}{*}{**No**} & \multirow{2}{*}{**Metabolites**} & \multicolumn{2}{c}{**D/CK**} \\ \cline{3-5}  & & \({}^{a}\) FC & \({}^{b}\)\(\mathbf{p}\) \\ \hline
1 & Isoleucine & 0.04 & \\
2 & Leucine & 0.5 & \({}^{*}\) \\
3 & Valine & \(-\)0.11 & \\
4 & Lactate & \(-\)0.52 & \({}^{*}\) \\
5 & Alanine & \(-\)0.52 & \({}^{**}\) \\
6 & Lysine & 0.25 & \\
7 & Putrescine & \(-\)0.18 & \\
8 & 4-Aminobutyrate & \(-\)0.27 & \\
9 & Acetate & 0.1 & \\
10 & Glutamate & \(-\)0.41 & \\
11 & Acetaminophen & 0.04 & \\
12 & Succinate & \(-\)0.26 & \\
13 & Glutamine & \(-\)0.65 & \({}^{**}\) \\
14 & Glutathione & \(-\)0.3 & \\
15 & 5-6-Dihydroouracil & \(-\)0.3 & \\
16 & Aspartate & \(-\)0.72 & \({}^{**}\) \\
17 & Sarcosine & \({}^{**}\) \\
18 & Phenylalanine & \({}^{**}\) \\
19 & Ethanolamine & \({}^{**}\) \\
20 & Choline & \({}^{**}\) \\
21 & Betaine & 0.06 \\
22 & Argin & \({}^{**}\) \\
23 & Methanol & \({}^{**}\) \\
24 & Glycine & \({}^{**}\) \\
25 & \(\pi\)-Methyllistidine & \({}^{**}\) \\
26 & Uracil & \({}^{**}\) \\
27 & Typtophan & \({}^{**}\) \\
28 & Xanthine & \({}^{**}\) \\
29 & Adenine & \({}^{**}\) \\
30 & Formate & \({}^{**}\) \\ \end{tabular}
\end{table} 
Table 2: Differential expression of metabolites between two groups at 12 h.  
that carvacrol induced the generation of heavy oxidative stress to disturb the energy and amino acid metabolism of _P. digitatum_ (Supplementary Figure S1). A significant decrease in the level of glutamate, glutamine, glycine and precursors of glutathione was observed in the D group. As an ROS scavenger, the level of glutathione also markedly decreased in the D group. Glutathione being endogenously expressed antioxidant enzyme that played an important role in the protection of cells against ROS by quickly eliminating free radicals [24]. Glutathione was consumed in large quantities in fungi against carvacrol-induced ROS. To sustain a certain level of glutathione in fungus, the synthesis of glutathione from glutamate, glutamine and glycine has to be enhanced [25], which will lead to the degradation of glutamate and glycine. In addition, a heavy load of ROS could enhance peroxidation of membrane lipids, leading to damage the cellular membranes [26]. The decreased level of choline and ethanolamine were seen in the D group compared with the CK group, and both are considered as the key players in the stability and integrity of cell membranes [27]. Therefore, any decrease in choline and ethanolamine may be due to excessive utilization for repairing the damaged membranes caused by ROS which are indicators of decreased level of sarcosine in D group [28]. In the current study, the sarcosine level in group D was lower than that in group CK, and was linked with the decrease of choline and glycine levels. These results reflect the disturbance of ROS to the sarcosine pathway caused by carvacrol. The self-protection mechanism of fungus made it consume huge amounts of energy to avoid ROS mediated stress and repair the cell membranes.