**4. Discussion**

The current study comprehensively evaluated the antifungal activity of carvacrol against *P. digitatum* through 1H-NMR metabonomics. Following the important metabolites selected by OSC-PLS-DA loading/S-diagram, METPA (http://www.metaboanalyst.ca) (Figure 6A) and 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

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. In order to produce more ATPs, the aerobic respiration in fungal hypha was enhanced while the glycolysis pathway was checked to a grea<sup>t</sup> extent. Such reduction in glycolysis was supported by the decreased level of lactate in D group. In addition, a marked decrease in phenylalanine, alanine, aspartate, and arginine levels was observed in D group. The glycogenic amino acids underwent degradation [29] and reflected an enhancement in the gluconeogenesis pathway that compensates the deficiency of glucose. The results further suggested that carvacrol helps produce energy and amino acid metabolic disorders in *P. digitatum*.

A significant increase in the transcriptional initiators e.g., uracil and adenine were found in the D group compared with the CK group (Table 2) [30]. The increased level of uracil and adenine revealed that carvacrol caused RNA damage to *P. digitatum*.

In this pioneering study, the antifungal activity of carvacrol against *P. digitatum* was studied by using the metabolomics approach based on 1H-NMR. Carvacrol can cause an abnormal metabolic state of *P. digitatum* by interfering with di fferent metabolic pathways such as energy and amino acid metabolisms. Furthermore, carvacrol could also induce damage to the RNA molecules and ROS production. Metabolomics provide a powerful and feasible tool for evaluating antifungal activity and exploring its potential mechanism.

**Figure 6.** (**A**) In this study, metabolic analysis was used to analyze the pathway topology related to antifungal activity. The term "log p" is the conversion of the original p value calculated from enrichment analysis, and "impact" is the path impact value calculated from the path topology analysis. The bubble area is proportional to the e ffect of each path, and the color indicates the importance from the highest red to the lowest white. (**B**) Alanine, aspartate, glutamate metabolism; ( **C**) glutathione metabolism.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2076-3417/9/11/2240/s1.

**Author Contributions:** Conceptualization, C.W. and J.C.; methodology, C.W. and C.C.; formal analysis, C.W. and C.C.; investigation, Y.S.; W.Q. and C.C.; data curation, C.C.; writing—original draft preparation, C.W.; Y.S.; W.Q. and C.C.; writing—review and editing, C.W. and M.F.N.; supervision, C.W.; project administration, C.W. and C.C.; funding acquisition, J.C.

**Funding:** This research was funded by National Natural Science Foundation of China (NO.31760598) and Advantage Innovation Team Project of Jiangxi Province (NO.20181BCB24005).

**Conflicts of Interest:** The authors declare no conflict of interest.
