Botrytis cinerea (ACCC 37346) was purchased from the Agricultural Culture Collection of China (ACCC). Paeonol was purchased from Aladdin Industrial Corporation (Shanghai, China). Cherry tomato fruit (Solanum lycopersicum ‘Qian xi’) of similar maturity, size, and color and without mechanical damage or disease were purchased from commercial orchards located in Xuzhou, Jiangsu Province, China.

The MIC of paeonol against B. cinerea was determined by sterile 96-well plate assays according to Lin et al. (2021). The absorbance was recorded at 560 nm by UV spectrophotometer (Versa max, Molecular Devices, China). MIC was set as the lowest concentration with no growth in plate assays (Fan et al., 2023). All analyses were performed with three replicates.

The mycelial growth method was based on the previous study of Lin et al. (2019). B. cinerea mycelial disks were positioned at the center of each potato dextrose agar (PDA) plate incorporating varying concentrations of paeonol and were concurrently incubated at 23°C. The diameters of the colonies were measured at 72 h after treatment. The B. cinerea mycelial growth inhibition was quantified using this formula: Inhibition (%) = [control mycelial diameter (mm) − paeonol treated mycelial diameter (mm)] ÷ [control mycelial diameter (mm)] × 100%. The value of EC₅₀ (concentration to inhibit 50% of mycelial growth) was calculated by linear regression of mycelial growth inhibition versus log₁₀ transformation of paeonol concentrations. All analyses were performed with three replicates.

Paeonol was diluted to different concentrations in potato dextrose broth (PDB). The B. cinerea spore suspension was mixed with paeonol-containing PDB and incubated at 23°C for 12 h. Spore germination was determined using a microscope (BX41, Olympus, Japan). The spore germination rate was calculated based on observations of 100 spores. Triple replications were used in all analyses.

Botrytis cinerea mycelia exposed to paeonol at the final concentration of 0, 125, or 250 mg/L at 23°C for 12 h. Treated mycelia were re-suspended in 2.5% glutaraldehyde solution for SEM observation (Teneo VS, FEI Company, United States) (Lin et al., 2022).

Botrytis cinerea spore suspension was exposed to 0, 125, or 250 mg/L paeonol at 23°C for 6 h. After centrifugation, spore cells were stained with propidium iodide (PI) for 15 min. Ten thousand cells were calculated and analyzed using a flow cytometry (FACS Verse, Becton, Dickinson and Company, United States).

To mimic the B. cinerea cell membrane, LUVs were synthesized by mixing phosphatidylcholine (PC)/phosphatidylethanolamine (PE)/phosphatidylinositol (PI)/ergosterol (5:4:1:2) (Cho et al., 2013). The four components were dissolved in chloroform and dried by nitrogen. LUVs-embedded calcein was created by stirring the dried LUVs in dye buffer (70 mM calcein, 10 mM Tris, 150 mM NaCl, and 0.1 mM EDTA). This solution underwent 11 cycles of freezing in liquid nitrogen and passing through polycarbonate filters with a Liposome extruder (Hand extruder, Genizer, United States). Different concentrations of paeonol were applied to calcein-encapsulating LUVs, and calcein release was measured by spectrofluorophotometer (SparkControl, Tecan, Switzerland) at wavelengths (λex = 490 nm, λem = 520 nm). Calcein leakage was calculated as: calcein leakage (%) = [(A_{added paeonol} − A_{without paeonol})/(A_added Triton X-100 − A_{without paeonol})] × 100. All analyses utilized three replications.

Botrytis cinerea mycelia (500 mg) were suspended in PBS and exposed to paeonol at final concentrations of 0, 125, or 250 mg/L at 23°C for 0, 2, 4, 6, or 8 h. The detection of protein, nucleic acid, and ion leakage was conducted utilizing the BCA Protein Assay Kit (Solarbio, Beijing, China), UV spectrophotometer (Versa max, Molecular Devices, China), and ICP-Optical Emission Spectroscopy (Avio 200, PerkinElmer, Waltham, United States), respectively. Each treatment was conducted in triplicate.

The ergosterol content of B. cinerea was measured as reported by Tian et al. (2012). B. cinerea spore suspension was inoculated into PDB containing 0, 62.5, 125, and 250 mg/L of paeonol for 4 days at 23°C. After incubation, mycelia were collected, re-suspended in 3 mL alcoholic KOH (25% w/v), and incubated at 85°C for 4 h. Sterols extraction involved adding 1 mL distilled water and 3 mL n-heptane to each sample. Then, a vigorous vortexing was conducted for 3 min to isolate the heptane layer. Analysis was performed on this layer using a UV spectrophotometer (Versa max, Molecular Devices, China) at 282 and 230 nm. Ergosterol content was calculated as: ergosterol (%) = [(A₂₈₂/290)/mycelia weight] − [(A₂₃₀/518)/mycelia weight]. All analyses were made with three biological replicates.

Botrytis cinerea spores were incubated in PDB (containing 0, 125, or 250 mg/L paeonol) at 23°C and 180 rpm for 4 days, then mycelia were lyophilized and pulverized. Total RNA was extracted with a kit (Sangon Biotech, Shanghai, China) and reverse transcribed with HiScript III All-in-one RT SuperMix Perfect (Vazyme, Nanjing, China). RT-PCR was run with Taq Pro Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) on a Step One System (7,500, Applied Biosystems, United States). The 2^{−△△C(T)} method was used for relative quantification (Livak and Schmittgen, 2001). The Actin gene acted as the internal control for analyzing four ergosterol synthase genes: Erg1, Erg3, Erg10, and Erg13. PCR primer pairs are shown in Table 1. Triple replications were used in all RT-PCR analyses.

Cherry tomato were wounded with a sterile borer (2 mm diameter and 2 mm depth) in each fruit’s equator and then inoculated with 5 μL of B. cinerea spore suspension (2 × 10⁴ spores/mL). About 30 min later, each wound received 5 μL of paeonol solution at 0, 250, 500, 1,000, and 2000 mg/L. Treated fruit were then stored at room temperature and 90% relative humidity. Disease incidence was determined at 72 h after treatment. Each treatment was triplicated with 20 fruits per replicate.

Cherry tomato were immersed in 0 or 2000 mg/L paeonol for 20 min, then stored under 25°C/90% humidity for 15 days. Twenty fruits were sampled at 0, 3, 6, 9, 12, and 15 days to assess physiological and quality indices.

Weight loss was measured based on previously described protocols (Lin et al., 2021). Cherry tomato were weighed at 0, 3, 6, 9, 12, and 15 days. The weight loss percentage was calculated as the difference between the initial weight and the weight recorded on the harvest day.

Total soluble solids (TSS), titratable acidity (TA), and ascorbic acid content were determined according to the method of Lin et al. (2021). The TSS result was reported as a percentage. TA was expressed as the percentage of citric acid. The ascorbic acid content was expressed as mg/Kg. All analyses were performed with three replicates.

Data were analyzed via ANOVA and Duncan’s multiple range with SPSS 19.0. p < 0.05 indicated statistical significance.

The MIC of paeonol against B. cinerea was determined as a means of investigating the effect of paeonol on B. cinerea growth. When paeonol concentration was <125 mg/L, B. cinerea growth was almost unaffected. Furthermore, when paeonol concentration was ≥250 mg/L, B. cinerea growth was completely inhibited (Figure 1A). Thus, the MIC of paeonol against B. cinerea was 250 mg/L.

As demonstrated in Figure 1B, paeonol suppresses the mycelial expansion of B. cinerea in dose-responsive manners. The value of EC₅₀ (concentration to inhibit 50% of the mycelial growth) was 120 mg/L. Mycelial growth exhibits complete suppression when paeonol concentration is elevated to 500 mg/L. Concurrently, spore germination is suppressed demonstrating analogous patterns in the presence of paeonol. These findings validate that paeonol can also effectively inhibit mycelial growth and spore germination of B. cinerea in vitro.

The SEM images showed the impact of paeonol on the morphology and structure of B. cinerea (Figure 2). Control group mycelia were smooth, regular, and plump, but 125 mg/L paeonol induced slight irregularities and collapse, while 250 mg/L paeonol led to severe irregularities and collapse.

Propidium iodide (PI) was used to measure cell membrane integrity as it can only enter cells that have damaged plasma membranes and are dying or already dead. The results of staining B. cinerea cells with PI are shown in Figure 3A. Paeonol treatment at different concentrations causes varying degrees of damage to B. cinerea, escalating with increasing paeonol concentration. In comparison with the control, cell damage increased from 28.9 to 51.3% when B. cinerea cells were exposed to 125 and 250 mg/L paeonol, respectively.

In order to confirm the ability of paeonol to disrupt fungal plasma membranes, the percentage of calcein leakage from LUVs was determined (Figure 3B). LUVs-embedded calcein was promptly liberated in a paeonol concentration-dependent manner. A concentration of 500 mg/L paeonol could instigate a leakage rate of 93.5%.

To further confirm the integrity of the cell membrane was disrupted, leakage of cytoplasm, including nucleic acids, proteins, potassium ions, and calcium ions, was measured. As shown in Figures 3C–F, nucleic acids, protein, K⁺ and Ca²⁺ of the supernatant increased in a time- and dose-dependent manner when B. cinerea mycelial was exposed to paeonol.

The effects of paeonol on ergosterol content in the plasma membrane of B. cinerea are illustrated in Figure 4A. The ergosterol content of B. cinerea was remarkably suppressed by varying concentrations of paeonol. Following incubation of B. cinerea in 0.5 MIC or 1 MIC paeonol, ergosterol content was reduced by 24.7 and 48.2%, respectively, compared to the control.

Ergosterol biosynthesis is tightly regulated by Erg genes (Song et al., 2022). To gain insight into paeonol’s role in regulating ergosterol synthesis, expression levels of Erg1, Erg3, Erg10, and Erg13 were analyzed (Figure 4B). Paeonol significantly suppressed the transcript levels of Erg1, Erg3, and Erg10 genes.

Considering that paeonol showed effective antifungal effects against B. cinerea in vitro, cherry tomato fruit was treated with paeonol during the infection assay. The incidence of gray mold decreased slightly when paeonol concentration increased from 0 to 500 mg/L at 72 h after the inoculation of the pathogen (Figure 5), with no significant difference between them (p > 0.05). However, disease incidence decreased significantly when paeonol concentration was increased to 2,000 mg/L (p < 0.05) as gray mold was completely inhibited.

Figure 6 illustrates the effects of paeonol treatment on fruit weight loss, TSS, TA, and ascorbic acid in cherry tomato. Figure 6A showed that the weight loss of the control fruit and paeonol-treated fruit all increased with the extension of the storage period. The paeonol-treated fruit’s weight loss was significantly lower than the control group (p < 0.05) from the ninth to the fifteenth day.

TSS content of control fruit increased on the third day, then declined until the fifteenth day. TSS content of the paeonol-treated group increased until the sixth day, then decreased until the ninth day. A significant difference (p < 0.05) was observed between the paeonol treated-tomato and the control group from the sixth to the fifteenth day.

As displayed in Figure 6C, TA content decreased in control and paeonol treatments during the whole storage period, but no significant differences were discovered in the two treatments of cherry tomato.

The ascorbic acid content in the control group rapidly declined during the whole storage time, while the paeonol-treated group exhibited a minor decrease (Figure 6D). Further comparison found that the ascorbic acid content in paeonol-treated group was observably higher than those in the control group.