## 3 Results
### Multivariate Analysis of 1H-NMR Spectral Data  
To evaluate the antifungal activity of carvacrol on _P. digitatum_, the OSC-PLS-DA model was constructed and all NMR data obtained from the control group (CK) and carvacrol administration group (D) at 4, 8 and 12 h were analyzed. In Figure 4B-D, each point manifested a sample, and each clustering represented a corresponding metabolic pattern in different groups. Figure 4B shows that the two groups were not well separated at 4 h. However, with the passage of time, the control group and the drug group were separated further and further, and the CK and D groups were the furthest away in the scores plot at 12 h (Figure 4D). This result shows that the metabolomic changes in D group increased from 4 h to 12 h. At 12 h, the metabolic spectrum of group D changed fundamentally, reflecting the rapid response of the strain to carvacrol. The trajectory plot (Figure 4A) also exhibited a good separation between the CK and D groups, showing an apparent time-dependent antifungal activity of carvacrol on _P. digitatum_.  
Figure 3: The typical 500 MHz CPMG \({}^{1}\)H-NMR spectra for 2 groups. Keys: 1. Isoleucine; 2. Leucine; 3. Valine; 4. Lactate; 5. Alanine; 6. Lysine; 7. Putrescine; 8. 4-Aminobutyrate; 9. Acetate; 10. Glutamate; 11. Acetaminophen; 12. Succinate; 13. Glutamine; 14. Glutathione; 15. 5,6-Dihydroouracil; 16. Aspartate; 17. Sarcosine; 18. Phenylalanine; 19. Ethanolamine; 20. Choline; 21. Betaine; 22. Arginine; 23. Methanol; 24. Glycine; 25. \(\pi\)-Methylhistidine; 26. Uracil; 27. Tryptonham; 28. Xanthine; 29. Adenine; 30. Formate.  
In the 12 h S-plot (Figure 5B), different shapes and colors of the dots show different metabolites. The contribution of these metabolites to the group is related to their distance to the center; variables farther away from the center contribute more to the group separation. On the basis of the correlation coefficient, the 12-hours loading plots (Figure 5C,D) are coded with cold and warm tones, and the correlation increases gradually from blue to red. Significant decrease of lactate, alanine, glutamate, glutamine, glutathione, aspartate, sarcosine, phenylalanine, ethanolamine, choline, arginine, methanol, glycine, \(\pi\)-Methylhistidine and xanthine, and marked increase of leucine, uracil, tryptophan and adenine were found in the carvacrol dosed group.  
Figure 4: (**A**): Score trajectory of the OSC-PLS-DA analysis in the CK group and D group at 4, 8, 12 h. (**B–D**): Scores plots of CK and D groups at 4 h, 8 h, and 12 h, respectively.  
Figure 5: OSC-PLS-DA analysis of NMR data at 12 h. (**A**) The OSC-PLS-DA Score plot. (**B**) S-plot. (**C**,D) Color-coded loadings plots. The use of color bars, where red and blue represent metabolites, is statistically significant or insignificant in facilitating the separation of groups. In the CK group, the peak of the positive and negative states showed that the decrease and increase of metabolites were correlated with the score plot.  
The results of the normality test of metabolites are shown in Table 2. The folding changes (FC) of metabolites and their associated P values were calculated and corrected in color tables. Collapse change values are color-coded after log conversion. Cell units were filled with red or blue to indicate the increase or decrease respectively of metabolites in the carvacrol-treated group compared with the control group.