The B. cinerea strain 05.10 was maintained in our laboratory. Other phytopathogenic strains, including F. oxysporum, F. graminearum, Valsa mali, Phomopsis vaccinii, Pestalotiopsis theae, Rhizoctonia solani, and the oomycete Phytophthora capsici, were also cultured on potato dextrose agar (PDA) at 25°C under dark conditions.

The BcRtn1-GFP, BcIlv2-GFP, BcGFP-SKL, and GFP-BcAtg8 strains were described previously (Wang et al., 2023) and available from the corresponding author’s laboratory. The ΔBcMca1, ΔBcMca2, and ΔBcMca1Mca2 mutants (Wang et al., 2023) were maintained in our laboratory. All these strains were also cultured on potato dextrose agar (PDA) at 25°C under dark conditions.

Fresh tomato leaves were harvested from greenhouse-grown plants, while fresh tomatoes and strawberries were obtained from local markets. Terpinen-4-ol (95% purity; CAS: 20126-76-5) was purchased from Macklin (Shanghai, China). Propidium iodide (PI), 2′,7′-dichlorofluorescein diacetate (DCFH-DA), N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino) phenyl) hexatrienyl) pyridinium dibromide (FM4-64) and annexin V-PE were purchased from Beyotime Biotechnology (Shanghai, China).

Terpinen-4-ol was added to PDA to achieve the desired final concentration of 0, 0.2, 0.4 and 0.8 μl/ml. Mycelial plugs (5 mm in diameter) of the plant pathogens and the B. cinerea mutants ΔBcMca1, ΔBcMca2, and ΔBcMca1Mca2 were inoculated onto PDA plates, which were then incubated at 25°C in the dark for 3 days. The colony diameter was measured, excluding the original plug size. Conidial germination assays were conducted in a 96-well microtiter plate, with 180 μl of spore suspension (4.6 × 10⁴ spores/ml) in each well. The conidial suspensions were treated with different concentrations of terpinen-4-ol (0, 0.2, 0.4 and 0.8 μl/ml). The germination rate of the conidia was then estimated after incubation at 25°C for 6 h. Three independent technical replicates were performed.

Conidia of B05.10 were collected from 7-day-old PDA cultures. Conidial concentration was determined microscopically using a hemocytometer and adjusted to 4.5 × 10⁴ conidia/ml. Detached leaves from 4-week-old tomato plants received 30 μl terpinen-4-ol sprays (0, 0.2, 0.4 or 0.8 μl/ml). After 4 h air-drying, leaves were inoculated with mycelial plugs. The antifungal activity was further assessed using commercially available mature tomatoes and strawberry fruits with artificial equatorial wounds (diameter 1 mm). The wounds were sprayed with 0.4 μl/ml terpinen-4-ol or H₂O for 4 h, then inoculated with either 5-mm mycelial plugs or 20 μl of conidial suspension, and finally incubated in an airtight box. After 3 days, fruits were maintained at room temperature (95% humidity) for a further 3 days before the lesion diameters were measured.

The conidia suspension of B. cinerea was inoculated into 100 ml of yeast extract-peptone-dextrose (YEPD) liquid medium and incubated at 25°C at 120 rpm for 24 h. The B. cinerea mycelia were treated with 0.1 μl/ml of terpinen-4-ol, while a control group remained untreated. Following an additional 4 h of incubation under the same conditions, the mycelia were harvested and stained with PI (20 μg/ml) to assess cell membrane integrity and with DCFH-DA (10 μM) to detect intracellular ROS. Apoptosis was determined by Annexin V-PE assay. Fluorescence was examined using an Olympus fluorescence microscope (Tokyo, Japan). All experiments were performed according to the protocol described in the kit instructions.

To examine whether terpinen-4-ol affects selective or non-selective autophagy, conidia of these strains expressing GFP-tagged markers (BcRtn1-GFP, BcIlv2-GFP, BcGFP-SKL, and GFP-BcAtg8) were cultured in YEPD liquid medium at 25°C for 24 h. The mycelia were then exposed to 0.1 μl/ml of terpinen-4-ol or H₂O for 4 h, as previously described. The samples were stained with FM 4–64, and the fluorescence was examined using an Olympus fluorescence microscope (Tokyo, Japan) (Meng et al., 2025).

The wild-type strain B05.10 and gene-overexpressing strains, including the GFP-BcAtg8, BcRtn1-GFP, BcGFP-SKL, and BcIlv2-GFP strains, were cultivated in YEPD liquid medium at 25°C in a 120-rpm shaker for 24 h. Subsequently, the cultures were treated with terpinen-4-ol or H₂O for an additional 4 h as previously described. Mycelia were then harvested and resuspended in protein extraction buffer. Equal volumes of protein extracts from each strain were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes. Immunoblotting was performed using an anti-GFP antibody (Cat# 32146, Thermo Fisher Scientific) at a dilution of 1:5,000, with an anti-actin antibody (Abcam, Cambridge, MA, USA) serving as a reference.

For the analysis of BcMac1 and BcMac2 gene expression, total RNA was extracted from B. cinerea mycelia under two conditions: B. cinerea treated with either terpinen-4-ol or H₂O, and B. cinerea treated with either terpinen-4-ol or H₂O during subsequent pathogenicity assays. RNA isolation was carried out using the TRIzol method (TaKaRa, Japan) in accordance with the manufacturer’s instructions. The PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa) was employed for reverse transcription of total RNA. TB Green® Premix Ex Taq (TaKaRa) was used to qRT-PCR analyses. Transcript levels were normalized to the expression of the β-actin gene.

All experimental data are presented as the means ± the standard errors. Statistical differences were analyzed using analysis of variance (ANOVA) and followed by Duncan’s multiple range tests in SPSS 21.0 (SPSS Inc.). A value of p < 0.05 was considered statistically significant.

The antifungal efficacy of terpinen-4-ol was evaluated against eight plant pathogens, including fungal and oomycete species, by measuring colony diameter on PDA. Terpinen-4-ol exhibited significant antifungal activity against all eight pathogens, with colony growth inhibited in a concentration-dependent manner (Figure 1A). In the control group, the colony exhibited unrestricted radial expansion, whereas terpinen-4-ol-treated colonies displayed concentration-dependent growth retardation with significantly reduced final diameters. Notably, complete mycelial growth inhibition of B. cinerea and V. mali was achieved at 0.8 μl/ml terpinen-4-ol. From the perspective of antifungal activity, terpinen-4-ol exhibited the strongest inhibitory effect against B. cinerea, with an inhibition rate of 86% at a concentration of 0.4 μl/ml (Figure 1B). Consequently, B. cinerea was selected for further mechanistic studies due to its exceptional sensitivity.

To assess the inhibitory effect on conidial germination, B. cinerea conidial suspensions were prepared and incubated on slide containing terpinen-4-ol at concentrations of 0, 0.05, 0.1, 0.2, 0.4 μl/ml. Conidial germination rates were suppressed by terpinen-4-ol in a concentration-dependent manner, showing significant inhibition at 0.2 μl/ml (Figure 1C). These results demonstrate that terpinen-4-ol possessed an ability to impair both mycelial growth and conidial germination in B. cinerea.

To further investigate the effect of terpinen-4-ol on B. cinerea plasma membrane integrity, the cell membrane integrity was assessed by PI staining. Compared to the control group, terpinen-4-ol-treated mycelia exhibited pronounced red fluorescence (Figure 2A). This result demonstrates that terpinen-4-ol has the capacity to disrupt cell membrane integrity. Additionally, many plant derived compounds strongly induced ROS production, therefore ROS accumulation was monitored using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) staining. The terpinen-4-ol-treated group showed a significant increase in fluorescence intensity, whereas no such increase was observed in the control group (Figure 2B). These findings suggest that terpinen-4-ol exerts antifungal activity against B. cinerea by disrupting plasma membrane integrity and promoting ROS accumulation.

To investigate whether terpinen-4-ol induces ER-phagy in B. cinerea, strains expressing fluorescent markers were analyzed. In the BcRtn1-GFP (ER marker) strain treated with terpinen-4-ol, GFP fluorescence was localized in the cytoplasm and vacuole, whereas GFP fluorescence in the BcRtn1-GFP strain without terpinen-4-ol treatment mainly localized in the ER (Figure 3A). In contrast, mitochondria-localized BcIlv2-GFP and peroxisome-targeted BcGFP-SKL strains treated with terpinen-4-ol showed no difference from control group (Figures 3B,C). Furthermore, the autophagic flux was analyzed using GFP-BcAtg8. GFP fluorescence was detected in both the cytoplasm and vacuoles of the GFP-BcAtg8 strain treated with terpinen-4-ol, confirming the induction of autophagy (Figure 3D). Next, the process of autophagy was observed through the use of immunoblotting. The results showed that the proportion of free GFP in the terpinen-4-ol-treated mycelia was significantly higher than that of the H₂O-treated mycelia (Figures 3E,F). Collectively, these results indicated that terpinen-4-ol specifically triggered ER-phagy and autophagy in B. cinerea, but did not affect mitophagy and peroxisomal degradation.

Previous studies have shown that terpinen-4-ol induces ROS accumulation, a known trigger of apoptotic cell death. In yeast, the metacaspase Yca1 mediates oxidative stress induced programmed cell death. To investigate the role of metacaspases in terpinen-4-ol-induced apoptosis in B. cinerea, we analyzed the expression levels of BcMca1 and BcMca2 in B05.10 mycelia treated with either terpinen-4-ol or H₂O. Notably, terpinen-4-ol significantly upregulated BcMca1 expression (Supplementary Figure S1). To further confirm this observation, we investigated two single-gene deletion mutants (ΔBcMca1 and ΔBcMca2) as well as a double-deletion mutant (ΔBcMca1Mca2) strains in our further study. The antifungal sensitivity of these mutants was assessed by culturing the wild-type B05.10 strain and mutants on PDA amended with gradient concentrations of terpinen-4-ol. After 4 days, the ΔBcMca1 and ΔBcMca1Mca2 mutants showed decreased sensitivity to terpinen-4-ol compared to that of B05.10. However, ΔBcMca2 displayed no phenotypic divergence from B05.10 (Figures 4A,B). To further evaluate apoptosis, we conducted annexin V-PE and DAPI staining. Notably, POH treatment induced phosphatidylserine exposure on the outer membrane leaflet in both B05.10 and ΔBcMca2 mutants (Figure 4C); whereas no such exposure was detected in either ΔBcMca1 or ΔBcMca1Mca2 mutants. Based on the above results we speculated that terpinen-4-ol activates apoptosis in B. cinerea through the metacaspase BcMca1-dependent pathway.

The potential inhibitory effect of terpinen-4-ol on the pathogenicity of B. cinerea was evaluated using detached tomato leaves, tomato and strawberry fruits. In the detached leaf assay, terpinen-4-ol significantly reduced the pathogenicity of B. cinerea in a dose-dependent manner, with a notable reduction in lesion diameter observed at 2 days post-inoculation (dpi) (Figure 5A). Additionally, the antifungal efficacy of terpinen-4-ol was further evaluated on tomato fruits. After 3 days of storage, mycelial plugs of B. cinerea inoculated onto tomato treated with terpinen-4-ol (0.4 μl/ml) exhibited significantly smaller lesion diameters compared to those untreated controls (Figure 5B). Similarly, conidial suspensions of B. cinerea inoculated onto strawberries treated with terpinen-4-ol (0.4 μl/ml) resulted in significantly reduced lesion diameters after 3 days of storage (Figure 5C). These results demonstrated that terpinen-4-ol effectively inhibited the pathogenicity of B. cinerea on both tomato and strawberry fruits. We further examined the expression level of the BcMac1 and BcMac2 genes in tomato leaves following terpinen-4-ol treatment during the pathogenicity assay. The results revealed that BcMac1 expression was significantly higher than that of BcMac2, indicating that terpinen-4-ol treatment induced the apoptosis in gray mold during host infection.