Antifungal Mechanism of 4-(4-Hydroxyphenyl)-2-butanone Against Fusarium solani

Abstract

Fusarium solani, a widely distributed plant pathogenic fungus, poses a significant threat to various crops due to its complex pathogenic mechanisms and being difficult to control. In this study, LC–TOF–MS analysis identified 4-(4-hydroxyphenyl)-2-butanone (RK) as a metabolite detected in Bacillus amyloliquefaciens strain M1, and a commercially available RK standard was subsequently used to evaluate its antifungal activity against F. solani. Antifungal assays demonstrated that RK effectively suppressed fungal growth. Further physiological and biochemical assays confirmed that RK disrupts the cell membrane and mitochondrial function, leading to intracellular reactive oxygen species (ROS) accumulation. This study offers new perspectives on the antifungal mechanism of RK and offers theoretical support for the development of innovative agricultural disease management strategies.

1. Introduction

Root rot is a soil-borne disease which induces infection in many crops and is extremely difficult to control. Fusarium solani is the dominant pathogen and can induce a high incidence of infection [1,2]. Chemical reagents are the main prevention and control measures for the disease. However, chemical reagents may lead to some problems, such as environmental pollution, the breaking of the soil ecological balance, pathogen resistance, and chemical residues. Biological control is environmentally friendly and can minimize pathogen resistance. So, many researchers have applied biocontrol measures, and many microorganisms are reported to antagonize F. solani such as Bacillus [3], Pseudomonas [4], Trichoderma [5], and Streptomyces [6].

Bacillus species are widely reported to have antifungal activity. Their biocontrol activities encompass various mechanisms, including competition, antagonism, the induction of systemic resistance, and the promotion of plant growth [7]. Bacillus amyloliquefaciens (B. amyloliquefaciens) is one of the most important bacteria due to its strong resistance and broad antimicrobial spectrum. Some bioactive substances produced by B. amyloliquefaciens have gradually been identified. Among the bioactive compounds produced by Bacillus species, lipopeptides such as iturin, surfactin, and fengycin are widely recognized for their inhibitory effects against soil-borne fungal pathogens. Additionally, ribosomally synthesized proteins, including bacteriocins, and hydrolytic enzymes, such as chitinases and β-glucanases, play important roles in antagonistic interactions between these biocontrol bacteria and plant pathogens [7,8,9].

According to our previous experiments, B. amyloliquefaciens M1 exhibits significant inhibitory effects against F. solani, Sclerotium rolfsii, and Fusarium oxysporum, especially F. solani [10]. Some peptides and proteins were reported to be related to B. amyloliquefaciens; however, different strains may have different metabolites that suppress pathogens [11]. So, it is necessary to identify the antimicrobial substance produced by M1.

Chalcone derivatives, a class of α, β-unsaturated carbonyl compounds, are well documented for their broad-spectrum antifungal activity. For instance, chalcones bearing thiourea and piperidine moieties showed superior inhibitory effects against multiple plant pathogens, exceeding the efficacy of azoxystrobin and causing disruption of fungal mycelia [12]. Pyrazole-containing chalcone derivatives also displayed strong inhibition of Phomopsis species, with EC₅₀ values surpassing conventional fungicides [13]. Additionally, some chalcones acted synergistically with fluconazole against drug-resistant Candida albicans [14], and 1,3,4-thiadiazole-fused chalcones demonstrated enhanced antifungal effects both in vitro and in vivo [15]. These findings establish chalcone scaffolds as promising templates for antifungal agent design.

4-(4-hydroxyphenyl)-2-butanone (RK) is classified by the U.S. Food and Drug Administration as a safe synthetic flavoring agent when used in small amounts [16]. RK holds significant value in the fields of spices and pharmaceuticals [17]. Structurally, RK can be considered a hydrogenated chalcone-like analogue, retaining the aromatic ketone core but lacking the α, β-unsaturated double bond; however, its antifungal activity against F. solani and potential application as a biological control agent remain unexplored.

In this study, LC–TOF–MS identified RK as an active antifungal constituent produced by strain M1. Combined with physiological and biochemical analyses, we further investigated the antifungal characteristics of RK against F. solani. This work provides a foundation for elucidating the antifungal mechanism of RK and supports its potential development as a biological control agent.

2. Materials and Methods

2.1. Strain, Materials, and Reagents

The fungal strain F. solani (HNCIMC:35607) and the bacterial strain B. amyloliquefaciens M1 (HNCIMC:26021) were obtained from the Henan Provincial Engineering Laboratory of Preservation and Breeding of Industrial Microbial Strains, Henan University of Technology (Zhengzhou, China). 4-(4-hydroxyphenyl)-2-butanone (RK) was supplied by Aladdin (Shanghai, China). The following assay kits were purchased from Solarbio Technology Co., Ltd. (Beijing, China): Mitochondrial Membrane Potential Assay Kit with JC-1, Reactive Oxygen Species (ROS) Assay Kit, 4′,6-Diamidino-2-phenylindole (DAPI) Assay Kit, Malondialdehyde (MDA) Content Assay Kit, Caspase-3 Activity Assay Kit, Citrate Synthase (CS) Activity Assay Kit, α-Ketoglutarate Dehydrogenase (α-KGDH) Activity Assay Kit, Succinate Dehydrogenase (SDH) Activity Assay Kit, and NAD-Malate Dehydrogenase (NAD-MDH) Activity Assay Kit.

2.2. n-Butanol Extracts of Fermentation Products from Strain M1

B. amyloliquefaciens M1 was initially grown in potato dextrose broth (PDB) at 37 °C with shaking at 200 rpm for 12 h. A 2% (v/v) inoculum was then transferred into fresh PDB and incubated under identical conditions for an additional 48 h to produce 2 L of fermentation broth. The culture was centrifuged at 8000 rpm for 10 min at 4 °C, and the resulting fermentation supernatant was filtered (0.45 μm) and adjusted to pH 2.0 with 5 mol/L HCl, followed by overnight incubation at 4 °C. The precipitate was collected by centrifugation (9000 rpm, 10 min) and extracted with 400 mL n-butanol under ultrasonic assistance. Following centrifugation at 12,000 rpm for 10 min, the n-butanol layer was concentrated using a rotary evaporator and subsequently dissolved in methanol to yield the n-butanol extract [18].

2.3. Preparation of F. solani Spores Suspension

F. solani was initially inoculated onto potato dextrose agar (PDA) plates and incubated at 30 °C for 7 days. Fresh mycelia were collected from the PDA and transferred into carboxymethyl cellulose (CMC) broth, followed by incubation at 30 °C with shaking at 200 rpm for another 7 days. The culture was centrifuged at 8000 rpm for 10 min, and the supernatant was discarded. The resulting pellet was washed with sterile saline and centrifuged again at 8000 rpm for 10 min; this washing step was repeated three times. After discarding the supernatant, the pellet was resuspended in sterile saline to prepare a spore suspension at a concentration of 1 × 10⁸ conidia/mL, which was then stored at 4 °C for later use.

2.4. Antifungal Validation of n-Butanol Extract Against F. solani

Hyphal plugs (8 mm) of F. solani were taken from the edge of freshly activated PDA plates and placed at the center of new PDA plates, which were incubated at 30 °C for 2 days. Antifungal activity was evaluated using the Oxford cup method, with four wells positioned 2 cm from the fungal plug. Each well received 200 μL of M1 sterile filtrate, n-butanol extract (methanol as control). Plates were incubated at 30 °C for 5 days, and the radii of the inhibition zones were measured. The antifungal rate of the n-butanol extract was calculated as follows:

$$\mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}\left(\%\right)={\displaystyle \frac{C-T}{C}}\times 100$$ (1)

where C is the average growth of the control group (mm). T is the average growth of the treatment group (mm).

2.5. Identification of Antifungal Substances from n-Butanol Extract

The concentrated n-butanol extract of M1 was analyzed by LC–TOF–MS at Zhongke e-Test (Qingdao Chengchuang Sci. & Tech. Co., Ltd., Qingdao, China) following Ahmad et al. [19]. Separation was performed on a C18 reversed-phase column using 0.1% formic acid in water (A) and acetonitrile (B) as the mobile phase. MS was operated in positive ESI mode (m/z 150–1500) with a 2.5 kV nebulization voltage, a 350 °C capillary temperature, and nitrogen carrier gas at 3 L/min. Identified compounds were compared with our metabolomics database. A total of five compounds were chosen according to their chemical characteristics and safety profiles, and these were subsequently purchased from commercial sources for further activity verification.

Hyphal plugs (8 mm in diameter) of F. solani were taken from the edge of freshly activated PDA plates and placed at the center of new PDA plates. Two wells were positioned 2 cm from the hyphal plug on each plate, and 200 μL of each test compound was added per well. Five compounds were tested individually, each on a separate plate, with methanol serving as the solvent control. Plates were incubated at 30 °C for 5 days to assess antifungal activity.

2.6. Fourier Transform Infrared Spectroscopy Analysis

Samples of 4-(4-hydroxyphenyl)-2-butanone (RK) and the n-butanol extract of M1 fermentation broth were individually mixed with potassium bromide and compressed into pellets approximately 1–2 mm thick. Infrared spectra were recorded using a Thermo Nicolet IS5 spectrometer at a resolution of 4 cm⁻¹ in the range of 400–4000 cm⁻¹.

2.7. Antifungal Validation and Minimal Inhibitory Concentration (MIC) of Antifungal Substance

The spore suspension was diluted 1:1000 in potato dextrose broth (PDB) to achieve a final density of 1 × 10⁵ spores/mL, and the resulting solution was dispensed into the wells of a 96-well plate. A stock solution of 2.5 g RK was prepared in 4 mL methanol to reach the desired concentrations. RK was added to the wells at volumes corresponding to 0, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, and 2.5% (v/v) of the total well volume, resulting in final RK concentrations of 0, 1.5625, 3.125, 4.690, 6.250, 7.813, 9.370, 10.011, 12.500, 14.060, and 15.625 mg/mL. Wells receiving methanol only served as the control. Each concentration was tested in six replicates. The plates were incubated at 28 °C for 48 h, after which fungal growth was quantified by measuring absorbance at 600 nm (A₆₀₀) using a UV–visible spectrophotometer (NanoDrop 2000, Thermo Fisher Scientific, USA) [20]. The MIC was defined as the lowest RK concentration at which no detectable hyphal growth was observed, and the A600 value was comparable to that of the blank medium.

2.8. Hyphal Morphology Observation

Fresh hyphal plugs from PDA plates were transferred into 1 mL of PDB containing RK at 1/2 MIC and MIC, with methanol as the control. Cultures were incubated at 30 °C, 200 rpm for 12 h, and hyphal morphology was examined under a 100× oil immersion objective using a light microscope (Zeiss Axioskop 40, Jena, Germany).

2.9. Spore Germination Inhibition Assay

The F. solani spore suspension was diluted to 1 × 10⁶ spores/mL. RK was added to a total volume of 1 mL, consisting of equal volumes of spore suspension and PDB, to achieve final concentrations of 1/2 MIC and MIC in the system. The mixtures were incubated at 30 °C with shaking at 200 rpm for 12, 24, 36, and 48 h. Spore germination was determined microscopically, with methanol as the solvent control. Each treatment was performed in triplicate [21]. The spore germination rate and inhibition rate were calculated as follows:

$$\mathrm{G}\mathrm{e}\mathrm{r}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}\left(\%\right)={\displaystyle \frac{Number of germinated spores}{Total spores counted}}\times 100$$ (2)

2.10. Time–Kill Curve Assay

The F. solani spore suspensions were adjusted to 1 × 10⁶ spores/mL and treated with RK at final concentrations of 1/2 MIC, MIC, and 2MIC in PDB medium. An equal volume of methanol was used as the solvent control. Cultures were incubated at 30 °C with shaking at 200 rpm. Samples were collected at 0, 3, 6, 12, 24, 36, 48, 60, 72, and 84 h. At each time point, aliquots were serially diluted with sterile PBS and plated onto PDA plates. After incubation at 30 °C for 48 h, colony-forming units (CFU) were counted [22]. The percentage of growth was calculated relative to the control group according to the following formula:

$$\mathrm{G}\mathrm{r}\mathrm{o}\mathrm{w}\mathrm{t}\mathrm{h}\left(\%\right)={\displaystyle \frac{CFU\left(treatment\right)}{CFU\left(control\right)}}\times 100$$ (3)

2.11. Determination of Cell Membrane Integrity, Mitochondrial Membrane Potential (MMP), Intra-Cellular Reactive Oxygen Species (ROS), and Nuclear Damage of F. solani Spores

F. solani spore suspensions were centrifuged and resuspended in phosphate-buffered saline (PBS) containing RK at the MIC, then diluted to a final spore density of 1 × 10⁶ spores/mL. The control group received an equivalent volume of methanol. Both treated and control samples were incubated at 30 °C with shaking at 200 rpm for 24 h, followed by centrifugation to collect the mycelia, which were subsequently washed twice with 10 mM PBS. Negative controls without RK were included. Following the procedure of Xin, Y et al. [23], with minor modifications, mycelial cultures were exposed to 5 μg/mL propidium iodide (PI). According to Luan, P et al. [24], with slight adjustments, the cultures were also treated with 10 μg/mL JC-1, 10 μM DCFH-DA, and 1 mg/mL DAPI for 30 min at 30 °C, then washed and resuspended in PBS. Fluorescence signals were finally observed using a fluorescence microscope (Revolve, Echo Laboratories, San Diego, CA, USA).

2.12. Determination of Ergosterol Content

The method was adapted from Xie et al., with minor modifications [25]. Freshly harvested F. solani mycelia were ground under liquid nitrogen, and approximately 0.1 g of the resulting powder was suspended in 5 mL of KOH-ethanol solution. The mixture was incubated in a water bath at 85 °C for 2 h to saponify the sterols. After cooling, 2 mL of sterile distilled water and 5 mL of n-heptane were added, and the mixture was vortexed for 30 min to extract the sterols. The n-heptane layer was collected, and absorbance was measured over a wavelength range of 230–300 nm using a UV spectrophotometer. Ergosterol content was calculated from the absorbance at 281.5 nm. All assays were performed in triplicate, with methanol as the control, and the results were expressed as a percentage of the dry weight of fungal mycelia.

2.13. Determination of Malondialdehyde (MDA) Content, Caspase-3 Activity, and Tricarboxylic Acid (TCA) Cycle Enzyme Activity

A 2 mL suspension of F. solani spores (1 × 10⁶ spores/mL) was inoculated into 100 mL of PDB medium and incubated at 30 °C with shaking at 200 rpm for 3 days. RK was then added to reach final concentrations corresponding to 1/2 MIC (5.47 mg/mL) and MIC (10.94 mg/mL), while the control received an equivalent volume of methanol. The cultures were further incubated under the same conditions for 48 h. Following incubation, the mycelia were collected by filtration, dried under vacuum, and subsequently ground into powder in liquid nitrogen before storage at −80 °C. MDA and caspase-3 activity contents were measured by using the MDA content assay kit and caspase-3 activity content assay, respectively. The enzyme activities of CS, SDH, α-KGDH, and MDH in the supernatant were measured using the corresponding assay kits.

2.14. Statistical Analysis

All statistical analyses were conducted using GraphPad Prism (version 8.0). Differences among three or more groups were assessed by one-way ANOVA followed by Tukey’s post hoc test. The results were presented as the mean ± standard error of the mean (SEM), and a p-value < 0.05 was considered statistically significant.

3. Results

3.1. Verification of Antifungal Activity of M1

The sterile filtrate from strain M1 inhibited the growth of F. solani, resulting in a 42.36% reduction in colony diameter compared with the control (Figure 1). The colony diameter of F. solani treated with the n-butanol extract was significantly smaller than that of the control group, with an inhibition rate of 71.14%. This extract may contain potential active compounds. Therefore, the n-butanol extract of M1 was selected for further mechanistic studies to explore the molecular basis of its inhibitory effect.

Figure 1.

Verification of antifungal activity of M1. (A) Control—methanol; (B) sterile filtrate of strain M1; (C) n-butanol extract.

3.2. Preliminary Identification of Antifungal Substances in the n-Butanol Extract

LC–TOF–MS analysis was conducted on the n-butanol extract produced by strain M1. Among the metabolites of M1, several antifungal compounds were detected, producing characteristic ion peaks in the mass spectrum. Notably, a distinct peak at 15.506 min corresponds to lauryl diethanolamine (Figure 2). The resulting chromatogram displays several prominent signal peaks, each corresponding to the ion intensity of different compounds at specific retention times. By comparing high-resolution mass spectrometry data (Figure 3) with the NIST database and the literature, 44 secondary metabolites were preliminarily identified (Table S1). Considering factors such as reported bioactivity, physiochemical properties, and safety assessments, five characteristic compounds were selected for in vitro antifungal activity validation. Experimental data show that among the five pure screened compounds, RK exhibited the best potential antifungal activity against F. solani with an inhibition rate of 59.10% (Figure 4). RK was verified by HPLC where the retention time of the standard (8.392 min) showed good consistency with the retention time of the target compound in the fermentation extract (8.812 min), within the ±0.5 min system error range (Figure 5).

Figure 2.

TIC diagram of n-butanol extract from fermentation product of strain M1.

Figure 3.

EIC diagram of n-butanol extract from fermentation product of strain M1.

Figure 4.

Verification of antifungal activity of sample against F. solani. (A) CK; (B) Laurocapram; (C) Saikosaponin a; (D) Di-n-butyl phthalate; (E) Lauryldiethanolamine; (F) RK.

Figure 5.

RK HPLC diagram.

3.3. FT-IR Spectral Analysis

The FT-IR profile of the RK reference compound displayed distinct absorption signals attributable to its major functional groups, including phenolic O–H stretching at approximately 3370 cm⁻¹, aromatic C–H stretching within 3000–2850 cm⁻¹, conjugated carbonyl stretching near 1690 cm⁻¹, skeletal vibrations of the aromatic ring between 1600 and 1500 cm⁻¹, and out-of-plane bending of para-substituted aromatic C–H around 826 cm⁻¹. In comparison, the n-butanol fraction obtained from B. amyloliquefaciens M1 exhibited a highly comparable spectral pattern, with corresponding bands observed at 3320, 2928, 1657, 1516, and 826 cm⁻¹, respectively (Figure 6), suggesting the presence of compounds structurally analogous to RK. These band assignments were further corroborated using the transmission IR reference spectrum available in SpectraBase [26].

Figure 6.

FT-IR spectral analysis. Blue color: M1: the n-butanol extract of the M1 fermentation product. Red color: RK.

3.4. Effect of RK on F. solani Hyphae

Microscopic examination showed that the untreated F. solani hyphae retained a typical elongated and branched morphology (Figure 7A). Following RK exposure, numerous vacuole-like cavities appeared within the hyphal filaments in a concentration-dependent manner. At 1/2 MIC, only occasional cavities were detected, whereas treatment at the MIC level resulted in a substantial increase in cytoplasmic empty spaces [27].

Figure 7.

Effect of RK on hyphal morphology, spore germination, and time–kill kinetics of F. solani. (A) Effect of RK on F. solani hyphae; Arrows indicate differences in hyphal morphology between the control and RK-treated groups. (B) effect of RK on F. solani spore germination; (C) time–kill kinetics of RK against F. solani. **** p < 0.0001, compared with control.

3.5. Effect of RK on F. solani Spore Germination

As shown in Figure 7B, the spore germination rate in the control group gradually increased over time, reaching nearly 100% at 48 h. In contrast, treatment with RK markedly inhibited spore germination, with rates of 24.99% and 16.21% observed at 1/2 MIC and MIC, respectively, at the same time point. These findings demonstrate that RK suppresses F. solani spore germination in a dose-dependent manner, with higher concentrations exerting stronger inhibitory effects [21].

3.6. Time–Kill Kinetics of RK Against F. solani

The time-dependent antifungal effects of RK on F. solani spores were evaluated using a time–kill assay (Figure 7C). RK exhibited a clear concentration- and time-dependent inhibitory effect. Exposure to 2 MIC completely suppressed spore growth within 36 h, while MIC treatment required 72 h to achieve complete inhibition. At 1/2 MIC, complete inhibition was observed at 84 h. The percentage of growth decreased progressively with increasing RK concentration, demonstrating that RK effectively suppresses spore proliferation in a dose- and time-dependent manner [22].

3.7. The MIC of RK Against F. solani

No reduction in hyphal biomass was observed in the methanol control. In contrast, after treatment with RK, the mycelial growth of F. solani decreased significantly. At RK concentrations ≥ 10.94 mg/mL, no visible hyphal growth, defined as the absence of turbidity in the culture medium upon visual inspection, was observed, indicating complete inhibition (Figure 8). Therefore, the MIC of RK against F. solani was determined to be 10.94 mg/mL.

Figure 8.

The MIC of RK against F. solani. Arrow indicate the concentrations at which no detectable hyphal growth was observed, corresponding to the defined MIC. A600 values were comparable to those of the blank medium.

3.8. Impact of RK on Integrity of F. solani Spore Cell Membranes

Red fluorescence is barely observed in the control group, indicating that most cells are intact and alive. In contrast, F. solani spores treated with RK exhibit red fluorescence under laser scanning confocal microscopy (Figure 9), indicating compromised cell membrane integrity. The cell membrane, a critical protective barrier, maintains selective permeability, facilitates nutrient transport and energy synthesis, and mediates extracellular signaling—functions essential for cellular homeostasis [28].

Figure 9.

Impact of RK on integrity of F. solani spore cell membranes, as revealed by PI (propidium iodide) staining. Red fluorescence indicates spores with compromised cell membranes.

3.9. Impact of RK on MMP Accumulation of F. solani Spores

The green fluorescence of the treatment group is significantly more pronounced compared to the green fluorescence of the blank control group (Figure 10). The results indicate that RK treatment reduced the mitochondrial membrane potential of pathogen spores [29]. Abnormal fluctuations in mitochondrial membrane potential impair ATP synthesis, resulting in excessive ROS accumulation and ultimately triggering programmed cell death [30].

Figure 10.

Impact of RK on MMP accumulation of F. solani spores. Bright field: optical image. Monomer: fluorescent signal of single spores. Aggregates: fluorescent signal of spore clusters. Merge: overlay of bright-field and fluorescent images. Red fluorescence indicates high mitochondrial membrane potential (healthy spores), while green fluorescence indicates decreased mitochondrial membrane potential (compromised spores). Arrows indicate differences between spores in the control and RK-treated groups.

3.10. Impact of RK on ROS Accumulation of F. solani Spores

No green fluorescence signal was detected in the spores of the control group (Figure 11), indicating that their ROS levels were in physiological homeostasis. In contrast, the RK treatment group exhibited a significant fluorescence increase, indicating that the accumulation of ROS was induced by the RK in F. solani spores, which in turn disrupts redox balance, affects cell cycle progression, and triggers programmed cell death [31,32].

Figure 11.

Impact of RK on ROS accumulation of F. solani spores. Bright field: optical image. ROS: fluorescence signal indicating reactive oxygen species. Merge: overlay ofbBright-field and ROS fluorescence images. Fluorescence indicates intracellular ROS levels. Arrows indicate differences between spores in the control and RK-treated groups.

3.11. Effect of RK on Nuclear Damage in F. solani Spores

DAPI is a DNA-specific probe employed to evaluate the integrity of the cell nucleus membrane according to changes in fluorescence intensity and chromatin condensation. Compared with the control group, RK treatment significantly enhanced the nuclear fluorescence intensity in F. solani spores (Figure 12), indicating that RK induces DNA damage in the nucleus of F. solani, leading to cell apoptosis [33].

Figure 12.

Effect of RK on nuclear damage in F. solani. Blue fluorescence indicates nuclear DNA. Arrows indicate differences in nuclear morphology and fluorescence intensity between the control and RK-treated groups.

3.12. Effect of RK on Ergosterol Content of F. solani

Ergosterol is the principal sterol component of fungal cell membranes and plays a critical role in maintaining membrane integrity and fluidity, thereby supporting normal growth and cellular homeostasis [34]. As shown in Figure 13, RK treatment significantly reduced the ergosterol content of F. solani mycelia in a clear dose-dependent manner. The ergosterol content in the control group (CK) was 2.35%, which decreased to 1.19% at 1/2 MIC and further to 0.71% at MIC. Compared with the control, ergosterol levels were reduced by approximately 49.4% and 69.8%, indicating that RK markedly inhibited ergosterol biosynthesis in F. solani.

Figure 13.

Effect of RK on ergosterol content of F. solani. *** p < 0.001, compared with control.

3.13. Effect of RK on MDA and Caspase-3 Activity in F. solani

To further elucidate the regulatory effect of the antioxidant system in mycelia after RK treatment, antioxidant enzyme activity (MDA, caspase-3) is commonly used as a key indicator. MDA is the primary product of lipid peroxidation in cell membranes, and its content directly reflects the extent of membrane peroxidation. The untreated group sustained a minimal amount of MDA, whereas the RK experimental group showed a notable accumulation of MDA (Figure 14A). This indicates that RK enhances the lipid peroxidation cascade, thereby promoting MDA biosynthesis and initiating a vicious cycle of reactive oxygen species metabolism [35]. Moreover, the extent of oxidative damage was positively correlated with RK concentration.

Figure 14.

Effect of MDA and caspase-3 activity in F. solani. (A) Effect of MDA content in F. solani. (B) Effect of RK on caspase-3 activity in F. solani. Enzyme activity is expressed as U/mg prot (prot = protein). **** p < 0.0001, compared with control. ns, not significant.

Caspase-3 activity in the RK treatment group exhibited a significant dose-dependent increase (Figure 14B). Compared with the blank control (0.454 U/mg protein), the 1/2 MIC treatment resulted in an enzyme activity of 0.770 U/mg protein, corresponding to a 69.6% increase, whereas the MIC treatment achieved 1.184 U/mg protein, representing a 161% increase. The activity level in the MIC group was significantly greater than that in the 1/2 MIC group, with the difference between the two groups being statistically significant (p < 0.05). These data confirmed that the activation of the caspase-3 pathway was positively correlated with RK concentration, further validating that RK triggers cell apoptosis through oxidative–metabolic imbalance as a core mechanism [36].

3.14. Effect of RK on TCA Cycle Enzyme Activity in F. solani

Based on the previous results, we further investigated the relationship between mycelial energy metabolism and the inhibition of crucial enzymes involved in the TCA cycle under RK treatment. Compared to the CS activity in the control group (619.103 U/g protein), the RK treatment groups at 1/2 MIC and MIC exhibited significant reductions of 48.7% and 82.6%, respectively (Figure 15A). Additionally, as for MDH, the activity in the control group was 8681.85 U/g protein after RK treatment, and the MDH levels at 1/2 MIC (42.9%) and MIC (78.8%) were markedly lower compared to those in the control group (Figure 15B). Subsequent α-KGDH activity analysis revealed that, compared to the 148.85 U/g protein in the control group, RK treatment at both concentrations resulted in a 40.0% and 70.0% decrease (Figure 15C). Similarly, SDH activity showed a significant reduction of 33.1% and 79.0% under RK treatment at both concentrations (Figure 15D). These results show that RK interfered with energy metabolism by inhibiting the activity of key enzymes whose activity levels are important indicators for assessing mitochondrial function. RK specifically targeted and inhibited rate-limiting enzymes in the TCA cycle, as exemplified by α-KGDH, and key nodes in the electron transport chain, such as SDH. Thus, RK effectively disrupted the cellular energy balance and triggered a cascade of oxidative damage reactions induced by metabolic abnormalities [37].

Figure 15.

Effect of TCA cycle enzyme activity: (A) CS; (B) NAD-MDH; (C) α-KGDH; (D) SDH. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, compared with control. ns, not significant.

4. Discussion

Compared with the M1 fermentation broth, its n-butanol extract exhibited markedly stronger antifungal activity. This observation is consistent with the report by Maung et al. [38], in which the n-butanol extract of Cephalosporium sp. CE100 showed substantial inhibition of Botrytis cinerea mycelial growth, suggesting that the active metabolites are preferentially enriched in the n-butanol fraction. LC–TOF–MS analysis revealed that RK was the predominant antifungal compound in the M1 extract, and this finding was further supported by FT-IR spectra, which closely matched those of the RK standard. RK inhibited the growth of F. solani with a rate of 59.10% at an MIC of 10.94 mg/mL, indicating that RK is a key contributor to the antifungal activity of M1. Furthermore, microscopic examination, spore germination assays, and cell death curve analysis consistently demonstrated that RK significantly affected fungal growth and hyphal structure.

Given that its antifungal mechanism may involve disruption of the fungal cell membrane lipid layer or altered membrane permeability, we subsequently examined the effects of RK on cell membrane integrity, mitochondrial membrane potential, nuclear DNA damage, and TCA cycle-related enzyme activities in F. solani. PI staining results indicated that RK disrupted the cell membrane of F. solani [39], consistent with the findings of Pan et al., who reported that cinnamaldehyde damages the cell membrane of F. solani [40]. Damage to the cell membrane increases membrane permeability, promoting the accumulation of intracellular ROS and other metabolites, thereby triggering oxidative stress. When oxidative stress exceeds the cell’s defense capacity, it leads to lipid peroxidation, protein damage, and DNA lesions, ultimately impairing cellular function and inducing cell death [41].

With RK treatment, the intracellular ROS levels in F. solani significantly increased. According to the study by Sugumaran, M et al., the cytotoxicity of RK can be attributed to the unique structure–activity relationship of its multiple phenolic hydroxyl groups, which enables RK to undergo various redox reactions within the cells. These reactions not only produce corresponding quinone derivatives but also generate many side-chain-dehydrogenated quinone species (cytotoxic quinones). Additionally, a large number of dimers and trimers are formed, all of which can induce the production of ROS, the consumption of cellular thiols, and reactions with cellular macromolecules, including proteins and DNA [42]. This conclusion also validates the reason for the significant increase in ROS content observed in our experimental group following RK treatment. The collapse of the antioxidant system was highly correlated with the increased MDA content (a marker of lipid peroxidation) and DNA damage [43]. Additionally, RK reduced ergosterol content in the fungal membrane, reflecting membrane damage and enhanced oxidative stress, consistent with findings by Wang et al. using a YBG8 fermentation filtrate [44].

JC-1 staining demonstrated a pronounced decrease in mitochondrial membrane potential, indicating that RK treatment impairs mitochondrial function and influences the expression of key genes responsible for maintaining mitochondrial integrity and activity [45]. Mitochondria, enclosed by a double membrane, serve as the cell’s primary energy center, generating ATP through the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. The TCA cycle supplies intermediates for oxidative phosphorylation and is essential for cellular ATP generation. Any dysfunction of mitochondria can negatively impact the growth of pathogenic fungi [37]. The decrease in α-KGDH activity reduces the TCA cycle’s capacity to generate NADH and ATP, thereby leading to TCA cycle dysfunction. Citrate synthase (CS) links nutrient utilization to the production of TCA cycle intermediates and metabolites, thereby helping regulate energy flux and metabolic rate. It catalyzes the condensation of acetyl-CoA and oxaloacetate to form citrate, coenzyme A, and protons. The decreased activity of MDH inhibits the reversible conversion between malate and oxaloacetate. SDH can transfer two electrons to coenzyme Q10, thereby connecting the TCA cycle with oxidative phosphorylation. Similar effects on central metabolism including TCA disturbance have been observed with other antifungal compounds in pathogenic fungi [46]. Cinnamon oil effectively suppressed the growth of Rhizopus nigricans by inhibiting the activities of SDH and MDH [47]. The result is consistent with our experimental findings, indicating that the mitochondrial activity of F. solani was markedly impaired. With increasing concentrations of RK, caspase-3 activity exhibited a dose-dependent increase, indicating the activation of the apoptotic pathway. This may be associated with the leakage of cytochrome C caused by the disruption of mitochondrial membrane integrity [48]. This indicates that RK treatment likely induces apoptosis through mitochondrial dysfunction, activating both apoptotic and autophagic pathways in the cells [49].

RK is an aromatic compound initially discovered in raspberries. As research on RK has deepened over the years, its application range has expanded to include both food and household fragrances [50], and it is also used as an insect attractant in agriculture [51]. As a result, the production of RK has transitioned from natural sources to large-scale industrial manufacturing, significantly improving both its yield and cost-effectiveness [52]. RK is unstable under light exposure [53]. According to the study by McPherson, P.A.C et al., RK is unstable under light exposure and shows greater stability under acidic and neutral pH conditions [54]. Moreover, a study by Sung Eun Lee et al. on 4-(4-hydroxy-3-nitrophenyl)-2-butanone, similar in structure to RK, explores its use against fungal contamination [55]. The compound, applied as a solvent treatment, showed volatility, prompting the recommendation of stabilization methods. We believe that RK could benefit from techniques like emulsion, microencapsulation, or nanoemulsion to improve its stability and effectiveness in biocontrol applications.

5. Conclusions

RK significantly inhibited the growth of F. solani, leading to changes in hyphal morphology, and reduced spore germination. Physiological and biochemical experiments showed that RK disrupted the integrity of F. solani spore cell membranes, damaged nuclear structures, impaired mitochondrial function, and led to excessive ROS accumulation. Meanwhile, RK affected the activity of enzymes associated with the TCA cycle, ultimately inhibiting the growth of F. solani. The experimental results confirmed that RK possesses potent antimicrobial activity, laying the foundation for the future development of environmentally friendly biocontrol strategies. But the tests have not been confirmed in vivo, so it has not been tested whether RK has negative effects on plants (phytotoxicity) when applied in real conditions. Considering the relatively high MIC value of RK, from an economic feasibility perspective, future research could focus on reducing the MIC by controlling certain environmental and extraction conditions. Additionally, combining RK with other biocontrol agents for integrated pest management in agriculture could effectively reduce the associated economic burden.

Abbreviations

The following abbreviations are used in this manuscript:

MIC	Minimum inhibitory concentration
ROS	Reactive oxygen species
LC–TOF–MS	Liquid Chromatography–Time of Flight–Mass Spectrometry
FPKM	Fragments Per Kilobase of exon model per Million mapped fragments
MMP	Mitochondrial membrane potential
PI	Propidium iodide
DCFH-DA	2′,7′-dichlorofluorescin diacetate
DAPI	4′,6-diamidino-2-phenylindole
MDA	Malondialdehyde
TCA	Tricarboxylic acid
CS	Citrate synthase
SDH	Succinate dehydrogenase
α-KGDH	α-ketoglutarate dehydrogenase
MDH	Malate dehydrogenase

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/microorganisms14020322/s1, Table S1: Components of substances identified by LC–TOF–MS.

Author Contributions

Conceptualization, N.L.; methodology, L.Z., Y.C. (Yifan Chen), Y.C. (Yan Chen), W.G., H.W., Z.X. and X.C.; software, W.G.; validation, L.Z., Y.C. (Yifan Chen), Y.C. (Yan Chen), W.G., H.W., Z.X. and X.C.; formal analysis, Y.C. (Yifan Chen); investigation, Y.C. (Yifan Chen) and L.Z.; resources, L.Z.; data curation, Y.C. (Yifan Chen); writing—original draft preparation, Y.C. (Yifan Chen); writing—review and editing, X.W., A.L., Y.H. and Y.C. (Yifan Chen); visualization, N.L.; supervision, N.L.; project administration, N.L.; funding acquisition, N.L. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This research was funded by Major Science and Technology Projects in Henan Province (No. 231100110300) and the Open Project Program of the National Engineering Research Center of Wheat and Corn Further Processing, Henan University of Technology (No. NL2023004).