The fungal strains used in this study were isolated from the roots of P. notoginseng exhibiting root rot. After isolating and purifying the strains, they were placed on potato dextrose agar (PDA) medium and incubated at a temperature of 28°C for a duration of 7 days. As previously described (Nie et al., 2024), DNA was extracted from the target strain and sequenced. Sequencing results were submitted to GenBank. A comparative analysis using NCBI BLAST revealed that the sequence exhibited 100% similarity to F. proliferatum (MH712158.1). The GenBank accession number is OP430570.1.

Menthone was procured from Shanghai Yuanye Biological Technology Co., Ltd. (CAS. 14073-97-3, purity ≥97%). The effect of menthone on inhibiting the mycelial growth of F. proliferatum was evaluated using the mycelial growth rate method (Zhou et al., 2017). In a sterile environment, menthone was dissolved in a suspension of 20/1,000 Dimethyl sulfoxide (DMSO) and 1/1,000 Tween-80 (2-DMSO-T) and filtered to obtain a sterile filtrate. The menthone solution was diluted to various concentrations using a two-fold dilution method. The prepared menthone was incorporated into the PDA culture medium to create menthone-containing plates. A PDA plate containing only the 2-DMSO-T suspension was used as the negative control. Fungal blocks with a diameter of 5 mm were inoculated onto the central area of PDA plates containing different concentrations of menthone (0 mM, 0.5 mM, 1 mM, 2 mM, 4 mM, 8 mM, and 16 mM). The plates were incubated at 28°C in an incubator at constant temperature for 4 days. Each treatment comprised five replicates. Colony diameters were measured, and the inhibition rate was calculated. A linear regression equation was established using the least squares method, resulting in a toxicity regression equation. The EC50 value was subsequently derived from this toxicity regression equation (Zou et al., 2021). The percentage of mycelial growth inhibition is determined using the following formula. Inhibition rate (%) = (C − B)/(C) × 100, where C is the diameter of the negative control group colony (mm) and B is the diameter of the treatment group colony (mm).

Spore viability was assessed using a fluorescent staining method involving acridine orange and propidium iodide (Manzo-Valencia et al., 2016). The experimental procedure for spore germination was modified from the method described by Tanapichatsakul et al. (2020). Menthone was prepared according to the procedure outlined in section 2.2. Subsequently, the solution was transferred to a sterile EP tube (5 mL) containing 2 mL 1/3 PDA liquid culture medium. The solutions were thoroughly mixed to achieve a menthone concentration of 1 × EC50. An appropriate amount of the spore suspension was then added, with 2-DMSO-T serving as a negative control. Each treatment was performed in triplicates. After incubating the mixture on a shaker at 28°C and 180 rpm in the dark for 24 h, The rate of spore germination is determined using the following formula:

Spore production was assessed as previously described with modification (Zheng et al., 2014). Menthone was added to Bilay’s culture medium at a concentration of 1 × EC50. Subsequently, 2 mL of the spore suspension, at a concentration of 1 × 10⁶ spores/mL, was added to each treatment group. The control group was treated with an equivalent volume of 2-DMSO-T as a solvent control. All samples were incubated in a thermostatic shaker at 28°C and agitated at 180 rpm in darkness for 5 days to facilitate shaking culture. Following incubation, spores were filtered through four layers of sterile cellulose acetate membranes [Sigma-Aldrich (Shanghai) Trading, MO, United States]. The resulting filtrate was thoroughly mixed, and samples were collected for analysis. The number of spores was quantified under a microscope using a hemocytometer. Each treatment included three biological replicates.

The permeability of the mycelial membrane was evaluated in accordance with the previously described methodology (Liu et al., 2022). A total of 2 g of mycelium was added to a conical flask containing 40 mL of sterile water, followed by menthone to achieve a final concentration equivalent to 1 × EC50. The conductivity was measured at 0, 1, 2, 3, 4, 5, 6, 7, and 8 h and recorded as D1. Concurrently, the conductivity without mycelia for each treatment group was measured and denoted as D0. After boiling the mycelium for 10 min, their conductivity was measured again and recorded as D2. DMSO was used as a negative control, with three biological replicates per treatment group. The formula was employed to calculate the relative conductivity. Relative conductivity = (D1 − D0)/D2 × 100%.

The treatment method for the mycelia was consistent with that described in section 2.5. Samples were collected at time points of 0, 6, 12, 24, 48, and 72 h and subsequently centrifuged at 8,000 rpm for 5 min. The concentration of soluble proteins in the supernatant was determined using the Coomassie Brilliant Blue G-250 staining method (Hatada et al., 2006).

As previously described (Liu et al., 2022), the mycelia of the fungus were collected following treatment with menthone for 24 h, immediately frozen in liquid nitrogen, and stored at −80°C for subsequent experiments. Total RNA extraction was performed following the manufacturer’s guidelines using TRIzol reagent. The quality of the extracted total RNA was assessed with a NanoPhotometer spectrophotometer (IMPLEN, CA, United States), a Qubit 2.0 fluorometer (Life Technologies, CA, United States), and an Agilent 2100 bioanalyzer (Agilent Technologies, CA, United States). A cDNA library was constructed at Novogene Technology Co., Ltd. (Beijing, China) and RNA-seq sequencing was performed. After purifying the cDNA and constructing the library, it was sequenced on an Illumina Novaseq6000 platform. The sequencing data were analyzed using Illumina Casava software (version 1.8) to remove adapter sequences and low-quality reads. Subsequently, the filtered reads were aligned to the reference genome of F. proliferatum (NCBI Taxonomy ID. 1227346) using the Hisat2 (version 2.2.1) software with default parameters.

Gene expression levels were determined as the number of transcript fragments per kilobase of transcript per million fragments (FPKM). The DEseq2 (version 1.22.1) software was used to analyzed DEGs between the two groups, with a screening standard of |log2Fold Change| ≥1 and false discovery rate (FDR) <0.05 (Ling et al., 2024). Finally, the results of the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were visualized using the online platform available at https://cloud.metware.cn.

Histones were prepared according to the instructions provided in the Histone Extraction Kit (BBproExtra^®). The proteins obtained were separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was then incubated with specific antibodies for hybridization. After hybridization, the membrane was blocked with 4% BSA in PBST (PBS-0.1% Tween20) at room temperature for 2 h. The primary antibodies used were anti-H3K27ac (ABconal, A2771, dilution factor is 1:10,000), anti-H3K4me3 (ABconal, A22146, dilution factor is 1:10,000), anti-H3K9ac (ABconal, A21107, dilution factor is 1:1,000), and anti-H3 (ABconal, A2348, dilution factor is 1:10,000). The hybridized membrane was washed three times with PBST for 10 min each before incubation with the primary antibody in PBST containing 4% BSA for an additional 2 h. Subsequently, the membrane was washed six times with PBST for 10 min each. This was followed by incubation with the corresponding secondary antibody, goat anti-rabbit IgG (Abbkine, A23220, dilution factor is 1:1,000), for 2 h. Finally, excess secondary antibody was removed by washing the membrane six times with PBST prior to signal detection in a dark room. The intensity of the bands was quantitatively analyzed using ImageJ software (Perrella et al., 2024).

ChIP experiments were conducted as previously described (Zhou et al., 2023), with modifications. Initially, the hyphae of F. proliferatum were ground into powder using liquid nitrogen. The chromatin was extracted by cross-linking with 1% formaldehyde under vacuum for a duration of 10 min. Following the completion of the cross-linking process, 2.5 M glycine was added and subjected to vacuum for an additional 5 min to effectively terminate the cross-linking reaction. Genomic DNA was fragmented into approximately 500 bp fragments through physical sonication, and ChIP was performed using an anti-H3K27ac antibody (A2771). The sequencing library was constructed following the protocol provided by the Illumina TruSeq^® ChIP Sample Prep Set A. Subsequently, sequencing was conducted on the Illumina HiSeq-PE150 platform, facilitated by Bioacme Biotechnology Co., Ltd.

Total RNA was extracted from the mycelia using TRIzol reagent. First-strand cDNA was synthesized from the total RNA using a reverse transcription kit (TaKaRa). Real-time fluorescence quantitative PCR (RT-qPCR) was conducted on the CFX Manager 3.1 system (Bio-Rad) employing the SYBR Premix ExTaq kit (TaKaRa). In the RT-qPCR and ChIP-qPCR experiments, QTUB or input (unimmunoprecipitated chromatin sample) served as normalization controls. The relative expression levels of genes were analyzed using the 2 − Δ Δ C T method. The primers used for RT-qPCR and ChIP-qPCR are listed in Supplementary Tables S1, S2, respectively.

All experiments conducted in this study were performed in triplicate for each sample, and the data were analyzed using one-way ANOVA with SPSS software version 19.0. Duncan’s multiple range test was used to assess the significance of differences (p < 0.05). All data are presented as mean ± standard deviation. The figures were generated using Adobe Illustrator 2022 and GraphPad Prism 9 software.

To elucidate the antifungal mechanism of menthone against F. proliferatum, we performed transcriptome sequencing analysis of samples treated with menthone using the Illumina NovaSeq 6000 platform. After data cleaning and quality control, high-quality reads were generated for each independent sample, ranging from 21.25 to 31.77 million reads. These high-quality reads were successfully mapped to the reference genome of F. proliferatum, achieving mapping rates exceeding 90%, indicating the high level of reliability of our transcriptomic data (Supplementary Table S3). Principal component analysis (PCA) and correlation analyses demonstrated good biological repeatability within the sample groups, confirming the reliability of our results (Figures 2A,B).

To further explore gene expression patterns, we identified DEGs using criteria of |log2Fold Change| ≥1 and false discovery rate (FDR) <0.05 for screening purposes. Our analysis revealed 3,442 upregulated genes and 3,880 downregulated genes in the menthone-treated group compared with the control group (Figures 2C,D). These findings suggest that menthone modulates the growth and development of F. proliferatum by inhibiting the expression of specific genes. Subsequently, the DEGs following menthone treatment were subjected to Gene Ontology (GO) functional enrichment analyses. The results indicated that upregulated DEGs were predominantly enriched in pathways related to the endoplasmic reticulum, cytoplasm, and lipid biosynthetic and metabolic processes. In contrast, downregulated DEGs were significantly enriched in pathways associated with ribonucleoprotein complex biosynthesis and nucleic acid-binding activities, including RNA- and snoRNA-binding pathways (Figures 3A,B).

To further investigate the biological functions of these DEGs in the growth and development of F. proliferatum, we conducted a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the DEGs, selecting the top 10 most significantly enriched pathways (Figures 3C,D).

DEGs upregulated by menthone treatment were predominantly enriched in oxidative phosphorylation, energy metabolism, steroid biosynthesis, and lipid metabolism. These findings suggest that menthone treatment enables F. proliferatum to mitigate membrane damage caused by menthone by enhancing steroid biosynthesis and lipid metabolism while simultaneously boosting oxidative phosphorylation and energy metabolism pathways to supply cellular energy. Conversely, the downregulated DEGs were primarily enriched in ribosome biogenesis, ribosome biogenesis in eukaryotes, transcription, and spliceosomes. The regulation of ribosomal biogenesis is a critical signal for cell growth and proliferation (Li et al., 2021). We hypothesized that menthone influences ribosome biogenesis in eukaryotes and other metabolic processes, affecting fungal protein synthesis and inhibiting the nutritional growth of F. proliferatum.

To elucidate the antifungal mechanism of menthone, we assessed the changes in histone modification levels using western blotting following menthone treatment. The results indicated that compared with the control group, the acetylation level of H3K27 was significantly reduced in F. proliferatum post-menthone treatment. No significant alterations were observed at other lysine sites of histone H3 (Figure 4; Supplementary Figure S1). This suggests that menthone primarily modulates the histone acetylation of H3K27 in F. proliferatum.

Previous studies have established a close relationship between histone acetylation and gene activation. In this study, H3K27ac levels decreased following menthone treatment, which might have inhibited related gene expression. Therefore, we hypothesized that menthone inhibits the growth of F. proliferatum by suppressing histone acetylation.

To investigate the dynamic changes in H3K27 acetylation in F. proliferatum, we performed a ChIP-seq analysis. The results indicated that the number of genes enriched in high and low acetylation modifications affected by H3K27ac following menthone treatment was 488 and 362, respectively, when compared to the control (Figure 5B). The mapping rates for both ChIP-seq samples exceeded 80%, with approximately 40% of the modified binding sites located near the transcription start site (Figures 5A,D and Supplementary Table S4). Furthermore, H3K27 acetylation peaks were significantly enriched at transcription start sites across different treatment groups (Figure 5C). This finding aligns with the previously reported distribution patterns of histone markers (Papait et al., 2013), validating the authenticity of our sequencing data.

Combining the results from the western blot analysis, we focused on the regulation of genes downregulated by histone modifications. Initially, we conducted GO enrichment analysis of the differentially acetylated peak genes to investigate the biological functions associated with these modified genes. Our findings revealed that these genes were predominantly enriched in tRNA modification, methylation, and processing (Figure 6A). Furthermore, KEGG enrichment analysis indicated that these genes were significantly enriched in cysteine and methionine metabolism and the spliceosome pathway (Figure 6B). These results imply that menthone treatment influences the functionality of the F. proliferatum spliceosome, resulting in disrupted gene expression, ultimately exerting antifungal effects and affecting the normal life processes of the fungus.

To investigate the regulatory mechanism of H3K27ac on gene transcription in F. proliferatum, we conducted a combined analysis of RNA-seq and ChIP-seq data to identify the transcripts associated with H3K27ac peaks following menthone treatment. Our comparative analysis revealed 362 low-acetylation peaks in the menthone-treated group, corresponding to 207 transcripts. Notably, the expression levels of 110 genes were significantly downregulated (Figure 6C). This finding suggests that after menthone treatment, H3K27ac primarily influences the downregulation of F. proliferatum genes, thereby inhibiting their growth and development. KEGG pathway enrichment analysis was performed to elucidate the pathways involved in the co-downregulated genes (n = 110) with low H3K27ac acetylation, indicating a significant enrichment within the ribosome biogenesis pathway (Figure 6D and Supplementary Tables S5, S6).

Several relevant genes were selected for RT-qPCR and ChIP-qPCR to validate the accuracy of the transcriptome and ChIP-seq data. The results indicated (Figure 7) that treatment with menthone significantly downregulated the expression levels of genes involved in ribosome biogenesis (FPRO_06392, FPRO_01260, and FPRO_01372), spliceosome-related genes (FPRO_01894 and FPRO_13101), and tRNA biosynthesis-related genes (FPRO_06284) in F. proliferatum compared to the control group. This finding is consistent with the transcriptome sequencing results, suggesting that the transcriptomic data are reliable.

Furthermore, after menthone treatment, there was a significant decrease in the H3K27ac modification levels of these genes relative to those in the control group. Consequently, reducing H3K27ac levels in the promoter regions of the relevant genes in F. proliferatum inhibited transcriptional activation. Our findings demonstrate that menthone treatment leads to decreased acetylation modifications at H3K27, suppressing gene transcription in F. proliferatum, subsequently affecting its growth and development.