A highly virulent strain of B. cinerea was isolated from strawberries and cultured at 25°C on potato dextrose agar medium (PDA; containing 1 L of an infusion from potatoes, 20 g/L glucose, and 15 g/L agar) before use in experiments. TTO was purchased from Fuzhou Merlot Lotus Biological Technology Company (Fujian Province, China). Ammonium acetate and ammonium hydroxide were purchased from Sigma Aldrich (St. Louis, MO, USA). Acetonitrile and methanol were purchased from Merck (Germany). Ammonium acetate and acetonitrile were of HPLC-grade. Distilled water was filtered through a Milli-Q system from EMD Millipore Corporation (Billerica, MA, USA). Succinate dehydrogenase (SDH), malic dehydrogenase (MDH), citrate synthase (CS), isocitrate dehydrogenase (ICDH), α-ketoglutarate dehydrogenase (α-KGDH), and hydrogen peroxide (H₂O₂) reagent kits were purchased from Nanjing Jian Cheng Bioengineering Institute (Nanjing, China).

B. cinerea cultures were maintained on PDA at 25°C for 3 days. The spore suspension was harvested by adding 10 mL sterile 0.9% NaCl solution to each petri dish and gently scraping the mycelial surface three times with a sterile L-shaped spreader to free the spores. The spore suspension was adjusted using a hemocytometer to 5 × 10⁶ spores/mL. One milliliter suspension was inoculated into 250 mL flasks containing 150 mL sterile potato dextrose broth (PDB; containing 1 L of an infusion from potatoes, 20 g/L glucose) medium and cultured at 25°C on a rotary shaker at 150 revolutions per minute (rpm) for 3 days. TTO was added to the medium to a final concentration of 5 mL/L, and cultures incubated for 2 h. Mycelia were then collected, rinsed three times with 0.1 M phosphate buffer saline (PBS; pH = 7.4), and immediately frozen in liquid nitrogen. Samples were stored at −80°C. Cultures without TTO were used as a control. Six samples of each group were prepared in parallel for LC-MS/MS analysis and other tests.

Before analysis, control and treated mycelia (~100 mg wet weight) were thawed at 4°C and homogenized. One milliliter of acetonitrile and methanol (1:1, v/v) was added to each sample and mixed by vortexing. Samples were immediately frozen in liquid nitrogen, subjected to ultrasonic grinding for 10 min, stirred for 60 min at −20°C, and then separated by centrifugation at 12,000 × g for 15 min at 4°C. An aliquot of 900 μL supernatant was dried under vacuum, dissolved in 100 μL of acetonitrile and water (1:1, v/v) by vortexing, and centrifuged at 12,000 × g for 15 min at 4°C. The chemical compositions of the samples were analyzed using an UHPLC (1290 Infinity LC, Agilent Technologies) equipped with a quadrupole time-of-flight (AB Sciex TripleTOF 6600) mass spectrometer.

For separation by hydrophilic interaction liquid chromatography (HILIC), samples were analyzed using a 2.1 mm × 100 mm ACQUIY UPLC BEH 1.7 μm column (Waters Corporation, Ireland). In both electron spray ionization (ESI) positive and negative modes, the mobile phase contained (A) 25 mM ammonium acetate and 25 mM ammonium hydroxide in water, and (B) acetonitrile. The elution gradient was: 0–1 min, 85% B; 1–12 min, 85–65% B; 12–12.1 min, 65–40% B; 12.1–15 min, 40% B; 15–15.1 min, 40–85% B; 15.1–20 min, 85% B, with a 5 min re-equilibration period employed.

The ESI source conditions were set as follows: Ion Source Gas1 (Gas1) as 60, Ion Source Gas2 (Gas2) as 60, curtain gas (CUR) as 30, source temperature: 600°C, Ion Spray Voltage Floating (ISVF) ±5,500 V. In the MS only acquisition mode, the instrument was set to acquire over the m/z range 60–1,000 Da, and the accumulation time for the TOF MS scan was set at 0.20 s/spectrum. In the auto MS/MS acquisition mode, the instrument was set to acquire over the m/z range 25–1,000 Da, and the accumulation time for product ion scan was set at 0.05 s/spectrum. The product ion scan was acquired using information dependent acquisition (IDA) with the high sensitivity mode selected. The collision energy (CE) was fixed at 35 V ± 15 eV. The declustering potential (DP) was set to ±60 V.

In order to assess TTO affect on the leakage of cell membrane, the absorbance at 260 nm of B. cinerea cell was determined. As described above for fungal culture and sample preparation, TTO was added to PDB medium at a final concentration of 5 mL/L. PDB without TTO served as the control. Treated and control group were centrifuged at 10,000 × g for 10 min at 4°C, and the absorbance of the supernatant was measured immediately at 260 nm with a UV/Vis spectrophotometer (UV-2000, UNICO Instrument Co., Ltd., Shanghai, China). After 2 h incubation with or without TTO, treated and control group were centrifuged and the absorbance at 260 nm of the obtained supernatant were measured. Each treatment was performed in triplicates. The percentage change in A_260nm (Liu et al., 2004) was calculated using the following formula: A260 nm percentage change(%)=[(At−A0)/A0]×100 where A₀ is the mean A_260nm for cultures measured after 0 h of treatment, and A_t is the mean A_260nm for cultures after 2 h of treatment.

Control and treated mycelia were washed with 0.1M PBS (pH = 7.4) three times and then ground in liquid nitrogen. The ground material was suspended in 10 mL 0.05M PBS (pH = 7.2) and centrifuged at 10,000 × g for 10 min at 4°C. Enzyme activities were measured in the supernatant for SDH, MDH, CS, ICDH, and α-KGDH, using commercially available kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) following the manufacturer's instructions. ICDH, MDH, and α-KGDH activities were detected at 340 nm in redox reaction assays. SDH and CS activities were determined at 600 and 412 nm, respectively. All tests were performed in triplicate.

Control and treated mycelia (1 g wet weight) were mixed with 5 mL 0.05 M PBS (pH = 7.0, containing 3% polyvinyl pyrrolidone), subjected to ultrasonic grinding for 15 min, and centrifuged at 10,000 × g for 10 min at 4°C. The supernatant was used to determine H₂O₂ content of the fungal cells using assay kits obtained from Nanjing Jiancheng Institute of Bioengineering (Nanjing, Jiangsu, China) following the manufacturer's instructions. H₂O₂ content was expressed as mmol/g prot. All tests were performed in triplicate.

The raw MS data (.wiff scan files) were converted to MzXML format using ProteoWizard MSConvert and processed using XCMS for feature detection, retention time correction, and alignment. Metabolites were identified by matching high accuracy (<25 ppm) MS/MS data to our standards database.

In the extracted ion features, only variables having more than 50% of the non-zero measurement values in at least one group were retained. For multivariate statistical analysis, the Metabo Analyst (http://www.metaboanalyst.ca) web-based system was used. After Pareto scaling, principal component analysis (PCA) and partial least-squares-discriminant analysis (PLS-DA) were performed. Leave-one-out cross-validation and response permutation testing were used to evaluate the robustness of the model. Metabolites exhibiting significant differences were identified based on the combination of a statistically significant threshold of variable influence on projection (VIP) values obtained from a PLS-DA model and a two-tailed Student's t-test (p-value) on the raw data. Metabolites with VIP-values larger than 1.0 and p < 0.05 were considered significant. All enzyme activity and H₂O₂ content data were analyzed using SAS software (Version 8.2; SAS Institute, Cary, NC, USA). These data were analyzed by one-way analysis of variance (ANOVA). Comparison of means was performed by Duncan's multiple range tests. A value of P < 0.05 was considered statistically significant.

Extracts from TTO-treated and untreated B. cinerea were analyzed using a UHPLC-Q-TOF mass spectrometer in positive and negative ion modes. Multivariate analysis was applied to find potential biomarkers. PCA, an unsupervised pattern recognition method, was performed to examine intrinsic variation in the data set. Samples that have similar metabolomics profiles are clustered together and those that are different are placed further apart in PCA score plots. As shown by the PCA score plots in positive (Figure 1A) and negative (Figure 1B) ion modes, the control group and TTO-treated group clustered separately, which indicates that metabolite levels were altered in the TTO-treated group.

To further identify ion peaks that could discriminate between the groups, a supervised pattern recognition method (PLS-DA) was suitable for the selection of biomarkers in similar metabolic profiling data (Schneider et al., 2009). In the PLS-DA score plots for both the positive (Figure 1C) and negative (Figure 1D) ion modes, metabolic differences made it possible to resolve the control and TTO-treated groups into distinct clusters. The R² and Q² parameters are used to evaluate the quality of the PLS-DA model. The R² and Q² were 0.99 and 0.97 in positive ion mode, respectively, and R² and Q² were 0.99 and 0.96 in negative ion mode, indicating that the model had high predictability and reliability.

To find the endogenous metabolites that contributed most to the separation of the control and TTO-treated samples, VIP scores were determined after PLS-DA. VIP measures the contribution made by each variable to the PLS-DA model. Ions were considered as potential biomarkers if they satisfied the criteria VIP-value > 1 and p-value < 0.05. Based on this algorithm, 91 metabolite ions were identified as primary contributors to the separation between control and TTO-treated groups. The data are summarized in Table 1, which also classifies the metabolites as either upregulated (8 metabolites) or downregulated (83 metabolites).

Table 1 shows the metabolic changes in fungal cells after treatment with TTO. The metabolites identified include products and intermediates generated by the TCA cycle, fatty acids, amino acids, and carbohydrates. The results suggest that B. cinerea adapts to environmental changes by regulating metabolic pathways. To provide an intuitive representation of the discriminatory power of the selected biomarkers in untreated and TTO-treated B. cinerea, a visual hierarchical clustering analysis (HCA, Figure 2) was performed. Each rectangle in the heat map represents one metabolite and is colored based on a normalized scale from −2 (low) to 2 (high). Differences between control and treated groups are the result of metabolite alterations after treatment with TTO. In positive mode (Figure 2A), seven metabolites [glycyl-L-leucine, L-carnosine, imidazoleacetic acid, L-methionine, N-(omega)-hydroxyarginine, guanosine, and deoxyadenosine] were upregulated in the TTO-treated sample, accounting for 10.3% of metabolites in positive mode. Other metabolites such as L-arginine, D-glucose 6-phosphate, phosphatidylcholine, alpha-linolenic acid, D-mannitol, and L-glutamate were downregulated in the TTO-treated sample, accounting for 89.7% of metabolites in positive mode. In negative mode (Figure 2B), glycyl-L-leucine, 2-oxoadipic acid, and guanosine were upregulated in the TTO-treated sample, accounting for 7.3% of metabolites in negative mode, while other metabolites such as isocitrate, L-malic acid, succinate, L-alanine, and trehalose were downregulated in the TTO-treated sample, accounting for 92.7% of metabolites in negative mode.

Isocitrate, L-malic acid, succinate, and α-ketoglutarate are all involved in the TCA cycle. The contents of these compounds were all markedly decreased by 81.2, 84.1, 94.9, and 91.9%, respectively, in TTO-treated B. cinerea compared to the untreated control (Figure 2). Phosphatidylcholine and alpha-linolenic acid are related to the fatty acid. The levels of phosphatidylcholine and alpha-linolenic acid compared with control downregulated 68.3% and 83.3% respectively in TTO-treated cells (Figure 3). As illustrated in Table 1, the detected L-carnosine and imidazoleacetic acid in TTO-treated groups compared with control was distinctly increased to 8.54- and 3.44-fold, respectively.

As shown in Table 2, the activities of MDH, SDH, CS, ICDH and α-KGDH in untreated cells was 54.87 U/g, 23.75 U/g, 366.24 U/g, 647.50 U/g and 355.55 U/g, respectively. After treatment with TTO at 5 mL/L for 2 h, the activities for these enzymes decreased about 79.3, 78.9, 83.3, 66.0, and 91.7%, respectively. It indicated that all of these key enzymes involved in TCA cycle were significantly reduced in B. cinerea treated with TTO.

Meanwhile, H₂O₂ content increased from 43.65 mmol/g prot to 120.95 mmol/g prot because of TTO treatment (Table 2). Absorbance at 260 nm increased by 14% after TTO treatment for 2 h, while the control group increased by only 1% (Figure 4). It was suggested that TTO led to the accumulation of H₂O₂ and the leakage of cell membrane.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.