**3. Results**

#### *3.1. E*ff*ects of Carvacrol on P. Digitatum Mycelial Growth on PDA*

The inhibitory effect of carvacrol on the growth of *P. digitatum* was quite obvious and a significant growth inhibition on PDA medium was seen in a dose-dependent manner (*p* < 0.05) (Figure 1). The increasing carvacrol concentrations had higher mycelial growth inhibition (MGI). As a whole, over one-fifth of the *P. digitatum* mycelial growth was inhibited at 0.0625 mg/mL of carvacrol, but 0.125 mg/mL concentration inhibited more than half (54.84%) of the mycelial growth. The higher carvacrol concentrations (0.25 and 0.5 mg/mL) completely inhibited the mycelial growth of *P. digitatum* (Figure 1).

Based on the observation of *P. digitatum* in mycelial growth on PDA medium with carvacrol treatments at 0, 0.03125, 0.0625, 0.125, 0.25, and 0.5 mg/mL during the incubation period at 25 ◦C, carvacrol treatments at the concentrations of 0.125 mg/mL and 0.25 mg/mL completely inhibited *P. digitatum* in mycelial growth at the 2nd day and 7th days of incubation, respectively. Therefore, the values of MIC and MFC were 0.125 mg/mL and 0.25 mg/mL, respectively (Figure 2).

**Figure 1.** The antifungal efficacy of carvacrol on the in vivo mycelial growth inhibition (MGI) of *P. digitatum* on PDA. Bars indicate the mean ± standard deviation (S.D.) and those labeled with different letters (**<sup>a</sup>**, **b**, **c**, **d**, and **e**) were significantly different according to Duncan's test (*p* < 0.05).

**Figure 2.** The effect of the growth diameter of *P. digitatum* treated with different concentrations of carvacrol (0, 0.03125, 0.0625, 0.125, 0.25, and 0.50 mg/mL) at 2 days and 7 days post-inoculation (dpi).

#### *3.2. E*ff*ects of Carvacrol on Mycelial Weights in PDB*

The mycelial weights of *P. digitatum* in carvacrol treatment and control groups are shown in Table 1. The data showed that mycelial growth biomass was strongly inhibited with increasing the carvacrol concentration. Initially, the wet and dry weights were 3.736 g and 0.393 g/100 mL at the lower carvacrol concentration of 0.0625 mg/mL, respectively. At higher carvacrol concentrations (0.125, 0.25 and 0.50 mg/mL), the effect on mycelial weights was recorded at a significant level (*p* < 0.05) in comparison with the control group.

**Table 1.** The mycelial weights and water-retention rate of *P. digitatum* treated with several concentrations of carvacrol.


Values are mean ± S.E. The data followed by different letters within the column are significantly different according to Duncan's test (*p* < 0.05).

#### *3.3. E*ff*ect of Carvacrol on Water-Retention Rate of P. Digitatum*

Water is the main component in the fungal cell and account for about 90% of mycelial fresh weight and plays an important role in regulating of cell osmotic pressure. The water-retention rate is used as an index of lipid peroxidation that is related to membrane damage leading to cell aging. The lower water-retention index shows higher membrane damages, and vice versa. The effect of carvacrol on the water-retention rate of *P. digitatum* mycelia is shown in Table 1. Different concentrations of carvacrol treatments were used to evaluate the mycelial membrane damage. The water-retention rate of *P. digitatum* mycelia was decreased and treated by carvacrol in a dose-dependent manner. The results demonstrate that the water-retention rate of *P. digitatum* mycelia was more significantly inhibited by the higher concentrations (0.125, 0.25, and 0.50 mg·mL−1) of carvacrol.

#### *3.4. Metabolites Identified in 1H-NMR Spectra*

The representative 500 MHz 1H-NMR spectra of mycelia obtained from the control group and MIC carvacrol administration group are shown in Figure 3. Chemical shifts assignments of metabolites were shown in the Supplementary Material (Supplementary Table S1). Nuclear magnetic resonance (NMR) was allocated by searching publicly-accessible metabolome databases (such as the Human Metabonomics Database and Madison Qingdao Metabonomics Joint Database) based on chemical changes reported in the literature.

**Figure 3.** The typical 500 MHz CPMG 1H-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-Dihydrouracil; 16. Aspartate; 17. Sarcosine; 18. Phenylalanine; 19. Ethanolamine; 20. Choline; 21. Betaine; 22. Arginine; 23. Methanol; 24. Glycine; 25. π-Methylhistidine; 26. Uracil; 27. Tryptophan; 28. Xanthine; 29. Adenine; 30. Formate.

#### *3.5. 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 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.

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, π-Methylhistidine and xanthine, and marked increase of leucine, uracil, tryptophan and adenine were found in the carvacrol dosed group.

**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.


**Table 2.** Differential expression of metabolites between two groups at 12 h.

a FC: Color-coded according to the fold-change value; Color coded according to the log2 (FC), red represents increased

and blue represents decreased concentrations of metabolites. Color bar

b *P* values corrected by Benjamini-Hochberg methods were calculated based on a parametric Student's t-test or a nonparametric Mann-Whitney test (dependent on the conformity to the normal distribution). \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001.

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