Antifungal activity and mechanism of the antibiotic fungicide tetramycin against Magnaporthe oryzae

Tetramycin, a polyene macrolide antibiotic known for its excellent antifungal activity, is extensively utilized in agricultural disease control. It has proven highly effective against various crop diseases, including leaf blight, black spot, and root rot. Its broad-spectrum antimicrobial activity, low toxicity, high therapeutic efficiency, and potent fungicidal properties make it a valuable tool in agriculture. This study aimed to investigate the antifungal activity of tetramycin against Magnaporthe oryzae and its underlying mechanism. The results revealed that tetramycin significantly inhibited mycelial growth in a dose-dependent manner, with a median effective concentration (EC₅₀) of 4.41 mg·L^-¹. Further investigations revealed that tetramycin markedly suppressed appressorium formation and conidial germination, reducing spore germination to 0% at high concentrations. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) observations revealed concentration-dependent morphological damage to hyphae, with severe structural alterations including contraction, deformation, fragmentation, extensive vacuolization, and cytoplasmic dilution at elevated concentrations. Cell wall integrity assays indicated no significant effect within the tested concentration range, whereas cell membrane integrity tests demonstrated that tetramycin disrupted the membrane, with the damage escalating as concentration increased. These findings suggest that tetramycin exerts its antifungal effect by compromising cell membrane integrity, leading to severe ultrastructural damage and cellular dysfunction, highlighting its potential for controlling rice blast. Future research should further elucidate its mechanism and conduct field trials to evaluate its practical application, and establish systematic resistance monitoring programs to ensure sustainable use.

1 Introduction

As one of the world’s most important staple crops, rice ranks among the top three in global production output. However, it faces a significant threat from rice blast disease caused by the fungus Magnaporthe oryzae, often referred to as rice’s “cancer.” This disease has been reported in 85 countries worldwide, with the most severe impacts observed in Asia and Africa, leading to annual yield losses that could feed approximately 60 million people (Liu et al., 2022). China, the widespread incidence of rice blast disease also poses immense challenges to agricultural productivity. During outbreaks, yield reductions can range from 10% to 30%, with extreme cases resulting in losses of up to 40% to 50% (Gao et al., 2020). This threatens food security and pressures agricultural economies. Therefore, Understanding rice blast mechanisms and control is crucial for food security and sustainable agriculture.

Rice blast is a disease caused by the fungal pathogen Pyricularia oryzae Cavara, an asexual fungus within the genus Pyricularia, while its sexual form belongs to the Ascomycota, known as Magnaporthe grisea (Hebert) Barr (Couch and Kohn, 2002; Burbano-Figueroa et al., 2009). The complete infection cycle of the rice blast pathogen involves several key steps, including the attachment and germination of conidia, hyphal elongation, the formation of appressoria, differentiation of infection pegs, and the proliferation of invasive mycelium (Wilson and Talbot, 2009). Under favorable conditions, conidia adhere to the leaf surface and secrete a mucilaginous matrix composed of proteins, lipids, and carbohydrates, which facilitates binding to the cuticle (DeZwaan et al., 1999; Liu et al., 2011). The conidium then differentiates and swells at its apex, forming a specialized infection structure: the appressorium (Talbot, 2003; Wilson and Talbot, 2009). As the appressorium matures, internal turgor pressure accumulates, enabling it to withstand up to 8 MPa of mechanical stress (Chung et al., 2022). Notably, the contact region between the appressorium and the leaf surface remains unpigmented, allowing direct penetration of the epidermal layer. The pathogen subsequently forms a penetration peg (de Jong et al., 1997), invades host cells, and establishes lesions. Infectious hyphae emerging from these lesions produce new conidia (Talbot et al., 1993; Khang et al., 2010), thereby completing the infection cycle of Magnaporthe oryzae. Airborne dispersal of these conidia facilitates secondary infections in neighboring rice fields (Talbot, 1995; Fernandez et al., 2014). Symptoms of rice blast vary depending on the infected plant part, classified into seedling blight, leaf blast, neck blast, and panicle blast (Law et al., 2017), with leaf and neck blast being the most prevalent (Greer and Webster, 2001). Neck blast directly causes significant losses in rice panicles, leading to notable impacts on yield and resulting in substantial economic losses in agriculture. Consequently, effective management strategies for rice blast disease remain a critical area of research.

Scientists and agricultural experts worldwide are actively seeking effective methods for managing rice blast disease. These methods include the development of resistant cultivars, chemical control, and biological control strategies. Breeding disease-resistant varieties is an economical and effective strategy. However, a significant drawback is the rapid accumulation of specific strains of the rice blast pathogen and its high pathogenic variability. This leads to accelerated population turnover, resulting in a notable decrease in resistance of these varieties within 3 to 5 years of large-scale cultivation. In scenarios of large-scale outbreaks, particularly when resistant rice varieties lose their efficacy, the application of chemical pesticides becomes crucial. Chemical control, or pesticide application, can drastically reduce rice losses in a short time. Nonetheless, prolonged reliance on chemical pesticides raises concerns regarding food quality, soil structure, and microbial communities, posing potential risks to human health (Heong et al., 1995; Li et al., 2025). In light of resource, environmental, and ecological safety considerations, alongside the emergence of resistance and variability among rice blast pathogens, biological control is gaining traction as a promising green management strategy. Biological control primarily employs natural microorganisms, fungi, or their metabolites to mitigate disease occurrence, offering a sustainable solution for rice blast management.

Tetramycin is a 26-membered tetraene antibiotic composed of two principal components, tetramycin A and tetramycin B (Dornberger et al., 1971; Sheng et al., 2020). These components differ structurally by a hydroxyl group substitution at the C4 position, with tetramycin B possessing an additional hydroxyl group compared to tetramycin A (Cao et al., 2012; Li et al., 2023). Its conjugated double bond system interacts with ergosterol in fungal cell membranes (Wang et al., 2017), resulting in the formation of hydrophilic ion channels within the plasma membrane. This leads to disrupted membrane permeability and subsequent leakage of intracellular components, ultimately inhibiting fungal growth or causing direct cell death. The biosynthesis of tetramycin is regulated by specific gene clusters in Streptomyces, encoding polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPS) (Gao et al., 2020), which are responsible for assembling the polyene macrolide backbone and modifying the side chain groups. Previous studies have shown tetramycin’s effectiveness against plant pathogens including root rot causative agents (Fusarium oxysporum, Fusarium solani, and Cylindrocarpon destructans) (Gao et al., 2018; Shi et al., 2020; Liu et al., 2025), and its positive effects on Panax notoginseng growth (Boikova et al., 2019). The mechanisms of action are believed to extend beyond membrane disruption, potentially interfering with pathogen metabolism and cell wall integrity. However, there is currently a lack of systematic studies on the specific mechanism by which tetramycin targets the rice blast fungus. Therefore, this study focused on the rice blast fungus as the target pathogen, thoroughly investigating its antifungal activity and mechanisms prior to the practical application of this antibiotic in disease management. More recent investigations have expanded our understanding of tetramycin’s mechanisms beyond direct antimicrobial activity. A 2023 study revealed that tetramycin disrupts both cell wall and cell membrane integrity in Alternaria alternata, causing mycelial distortion, contraction, fragmentation, and severe plasmolysis with reduced intracellular contents (Li et al., 2023). Furthermore, tetramycin B3 was found to inhibit pine wood nematode growth through multiple pathways, including increased reactive oxygen species (ROS) production, lipid accumulation, and significant suppression of detoxification and protective enzyme activities (Sun et al., 2024).

Beyond direct pathogen inhibition, emerging evidence demonstrates that tetramycin can modulate beneficial soil microbiomes. A 2025 study showed that tetramycin reshapes microbial community structure and function to form a rhizosphere barrier, reducing pathogen abundance while increasing beneficial microorganisms, thereby alleviating root rot disease in Panax notoginseng (Liu et al., 2025). Additionally, tetramycin has been shown to enhance plant defense systems when combined with low-dose fungicides, increasing resistance substance content, defensive enzyme activities, and electrophysiological activity in medicinal plants (Sun et al., 2024). The structural basis for tetramycin’s activity has also been characterized at the molecular level, with studies revealing competitive inhibition mechanisms that restore antibiotic efficacy (Yang et al., 2023).

Despite these advances, there is currently a lack of systematic studies on the specific mechanism by which tetramycin targets the rice blast fungus. Therefore, this study focused on the rice blast fungus as the target pathogen, thoroughly investigating its antifungal activity and mechanisms prior to the practical application of this antibiotic in disease management.

2 Materials and methods

2.1 Pathogen and tetramycin

The wild-type Magnaporthe oryzae strain P131 (causal agent of rice blast) and tetramycin (used for antifungal activity evaluation and mechanistic studies) were provided by the Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine Technology, China.

2.2 Evaluation of the in vitro antifungal activity of tetramycin

The antifungal activity of tetramycin against Magnaporthe oryzae was evaluated using the mycelial growth rate method. In a laminar flow hood, tetramycin was incorporated into PDA medium at final concentrations of 0.12, 0.24, 0.48, 0.96, 1.92, 3.84, 7.68, and 38.40 mg·L^-¹, with sterile water serving as the control (three replicates per treatment). After solidification, 5-mm mycelial plugs obtained from 7-day-old fungal colonies were aseptically transferred to the center of each plate. The plates were incubated at 28°C for 7 days, after which colony diameters were measured using the crosswise method. The inhibition rate (%) of mycelial radial growth was calculated as: [(control diameter - treatment diameter)/(control diameter - 5)]× 100. The half-maximal effective concentration (EC₅₀) was determined through regression analysis.

2.3 Assay of appressorium induction and conidial germination

The wild-type Magnaporthe oryzae strain was revived on OTA medium and cultured at 28°C under light conditions for 5–7 days to induce sporulation. Spores were harvested by adding 1–2 mL of sterile water to the culture plate, then transferred to fresh OTA medium and air-dried. After 48 h of light incubation, the culture was disrupted with sterile water, air-dried again, and sealed with gauze. Following an additional 48 h light incubation at 28°C, spores were eluted with sterile water and filtered through two layers of filter paper. The spore suspension was adjusted to 1×10⁶ spores/mL using a hemocytometer. Tetramycin solutions (7.68 and 76.80 mg·L^-¹) were mixed 1:1 (v/v) with the spore suspension (1×10⁶ spores/mL) in 1.5 mL centrifuge tubes to achieve final concentrations of 3.84 and 38.40 mg·L^-¹, with sterile water as negative control (triplicate experiments). Aliquots (10 μL) were inoculated onto microscope slides placed in humidity chambers, covered with black cloth, and incubated at 28°C for 24 h. Germination rates and appressorium formation were assessed microscopically, with conidia considered germinated when germ tube length exceeded half the spore diameter. Five microscopic fields per treatment were examined for quantification.

2.4 Observing the mycelial morphology and ultrastructure of Magnaporthe oryzae

Magnaporthe oryzae mycelial discs were inoculated into potato dextrose broth (PDB) liquid medium and cultured at 28°C with continuous shaking at 180 rpm. When abundant mycelia appeared in the culture flask, the samples were removed and treated under sterile conditions in a laminar flow hood. Tetramycin solutions were added to achieve final concentrations of 3.84 mg·L^-¹ and 38.40 mg·L^-¹, with sterile water serving as the control. After 12 hours of treatment, the mycelia were transferred into 50 mL sterile centrifuge tubes and centrifuged at low speed to remove the supernatant. A small amount of mycelia was transferred to 2 mL centrifuge tubes, gently washed with phosphate-buffered saline (PBS), centrifuged, and the supernatant was discarded. Glutaraldehyde fixative was added in darkness, and the mycelia were dispersed and suspended in the fixative solution. The samples were fixed at room temperature in darkness for 30 minutes, then transferred to 4°C for overnight storage. Subsequently, the samples were sent to Wuhan Servicebio Technology Co., Ltd. for scanning electron microscopy (SEM) and transmission electron microscopy (TEM) imaging.

2.5 Evaluation of cell wall integrity in hyphae and conidia

Hyphae from 7-day-old Magnaporthe oryzae cultures grown on OTA medium under light conditions were aseptically scraped and treated with tetramycin (3.84 or 38.40 mg·L^-¹) or sterile water (control) for 3 h at 28°C in darkness. Similarly, spore suspen-sions (1×10⁶ spores/mL) were prepared and exposed to identical treatments. Following centrifugation (8,000 × g, 4°C, 5 min), pellets were stained with 0.01 mg·mL^-¹ calcofluor white (CFW) in darkness, and fluorescence intensity changes were quantified using fluorescence microscopy.

2.6 Assessment of cell membrane integrity in hyphae and conidia

Hyphae from 7-day-old Magnaporthe oryzae cultures grown on OTA medium under light conditions were aseptically harvested and treated with tetramycin (3.84 or 38.40 mg·L^-¹) or sterile water (control) for 3 h at 28°C in darkness. Similarly, spore suspensions (1×10⁶ spores·mL^-¹) were prepared and exposed to identical treatments. After centrifugation (8,000 × g, 4°C, 5 min), pellets were stained with 0.04 μg·mL^-¹ propidium iodide (PI) in darkness at room temperature for 30 min, followed by fluorescence microscopy analysis to quantify fluorescence intensity changes.

2.7 Statistical analysis

All data in this study were analyzed using SPSS software. The EC₅₀ value was calculated by regression analysis using the inhibition rate against the log₁₀ value of the tetramycin concentration. Statistical differences between treatments were evaluated using one-way analysis of variance (ANOVA) followed by Tukey’s test at p = 0.05. Means followed by the same letter were not significantly different. For appressorium formation and conidial germination assays, germination rates and appressorium formation rates were assessed microscopically. Conidia were considered germinated when germ tube length exceeded half the spore diameter. Five microscopic fields per treatment were examined for quantification, and each treatment was performed with at least three replicates. For cell wall and cell membrane integrity assays, fluorescence intensity was quantified and compared among treatments using fluorescence microscopy by examining at least five microscopic fields per treatment. Each treatment was performed with at least three replicates, and representative results are presented.

3 Results

3.1 Inhibitory effects of tetramycin on mycelial growth of Magnaporthe oryzae

Tetramycin significantly inhibited mycelial growth of Magnaporthe oryzae in a dose-dependent manner (Figures 1A, B). Tetramycin demonstrated concentration-dependent inhibition, achieving 56.52% suppression of mycelial growth at 3.84 mg·L^-¹ and complete inhibition at 7.68 mg·L^-¹. The toxicity regression equation was determined as y = 0.9143x + 4.4105, yielding an EC₅₀ value of 4.41 mg·L^-¹ (Figure 1C). Comparative analysis revealed that tetramycin exhibited superior antifungal potency against M. oryzae compared to several other agricultural antibiotics. The EC₅₀ value of tetramycin (4.41 mg·L^-¹) was notably lower than that of AMEP412 protein (41 mg·L^-¹) and Antifungalmycin 702 (37 mg·L^-¹), demonstrating approximately 9-fold and 8-fold higher efficacy, respectively (Gohbara et al., 2013; Wang et al., 2024). However, the strobilurin fungicide coumoxystrobin displayed markedly stronger activity with an EC₅₀ of 0.0163 mg·L^-¹ (Xin et al., 2020), approximately 270-fold more potent than tetramycin (Figure 1D). These comparative data highlight that tetramycin possesses moderate to strong antifungal activity within the spectrum of biocontrol agents tested against rice blast pathogen, positioning it as a promising candidate for integrated disease management strategies.

Figure 1

Figure 1. Antifungal activity of tetramycin against Magnaporthe oryzae. (A) Inhibition rate of mycelial growth by tetramycin. (B) Colony morphology of M. oryzae on PDA plates containing different tetramycin concentrations. Data represent mean ± SE of three replicates. Different letters indicate significant differences (p < 0.05). The same convention applies to subsequent figures. (C) Antifungal activity of tetramycin against Magnaporthe oryzae. Toxicity regression curve of tetramycin inhibiting the mycelial growth of M. oryzae. The probit of inhibition rate was plotted against the logarithm of tetramycin concentration (mg·L^-¹). The regression equation is y = 0.9143x + 4.4150, with a coefficient of determination (R²) of 0.9076. The calculated EC₅₀ value is 4.41 mg·L^-¹. (D) Comparative analysis of the antifungal activity of different agents against Magnaporthe oryzae. The median effective concentration (EC₅₀) values of AMEP412, Antifungalmycin 702, Tetramycin, and Coumoxystrobin against M. oryzae. Different lowercase letters above the bars indicate significant differences among treatments (p < 0.05).

3.2 Inhibitory effects of tetramycin on appressorium formation and conidial germination in Magnaporthe oryzae

Tetramycin treatment virtually abolished appressorium formation (0% incidence), compared to a 32.01% formation rate in control hyphae (Figures 2B, C). Conidial germination exhibited dose-dependent inhibition, with rates declining from 98.96% (control) to 95.83% and 0% at 3.84 and 38.40 mg·L^-¹ tetramycin, respectively (Figures 2A, C).

Figure 2

Figure 2. Conidial morphology after 24 hours of treatment with varying concentrations of tetramycin. (A) Conidial germination rate; (B) Appressorium formation rate; (C) Conidial morphology after 24 h of treatment with tetramycin at different concentrations. The red arrow in “a “ indicates the appressorium. a: tetramycin concentrations are 0 mg·L^-1; b: tetramycin concentrations are 3.84 mg·L^-1; c: tetramycin concentrations are 38.40 mg·L^-1. Scale bar = 62.3 μm. Different letters indicate significant differences (p < 0.05). “CK” denotes the negative control.

3.3 Damage of tetramycin on the morphology and ultrastructure of Magnaporthe oryzae hyphae

Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) observations revealed concentration-dependent morphological damage to Magnaporthe oryzae hyphae following tetramycin treatment (Figure 3). Control hyphae exhibited normal cylindrical structures with smooth surfaces, intact intracellular organization, clearly defined cell membranes, and uniformly distributed cytoplasm. Following treatment with 3.84 mg·L^-¹ tetramycin, hyphae began to show subtle morphological alterations, including irregular surface depressions and shrinkage, accompanied by abnormal intracellular changes characterized by uneven cytoplasmic density and localized vacuolization. When tetramycin concentration increased to 38.40 mg·L^-¹, hyphae underwent significant morphological changes, displaying severe contraction and deformation with pronounced surface depressions and irregular shapes, while some hyphae exhibited fragmentation or distortion. Concurrently, intracellular structures were severely compromised, showing extensive vacuolization, marked cytoplasmic dilution, loss of membrane integrity, and evident leakage of cellular contents.

Figure 3

Figure 3. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) observations of Magnaporthe oryzae mycelial morphology and ultrastructure after tetramycin treatment. Upper panel: SEM images showing hyphal surface morphology. Lower panel: TEM images showing internal cellular ultrastructure. (a) tetramycin concentration 0 mg·L^-¹ (control); (b) tetramycin concentration 3.84 mg·L^-¹; (c) tetramycin concentration 38.40 mg·L^-¹. Scale bar for SEM = 10 μm; Scale bar for TEM = 1 μm.

3.4 Effects of tetramycin on the integrity of the cell wall in Magnaporthe oryzae

Mycelium with intact cell walls exhibited bright blue fluorescence under fluorescence microscopy, while mycelium with compromised cell walls displayed diminished or absent fluorescence intensity. Both the control and tetramycin-treated groups of Magnaporthe oryzae mycelium and conidia showed comparable fluorescence intensities (Figures 4A, B), indicating that tetramycin did not significantly affect cell wall integrity within the tested concentration range. This suggests that tetramycin may not disrupt the deposition or structural integrity of key cell wall components, such as chitin.

Figure 4

Figure 4. Analysis of cell wall integrity in Magnaporthe oryzae. (A) CFW staining of hyphae after 3 h treatment with tetramycin. Scale bar = 20.8 μm. (B) CFW staining of conidia after 3 h treatment with tetramycin. Scale bar = 62.3 μm. a: tetramycin concentrations are 0 mg·L^-1; b: tetramycin concentrations are 3.84 mg·L^-1; c: tetramycin concentrations are 38.40 mg·L^-1. Lowercase letters designate the different tetramycin concentrations.

3.5 Effects of tetramycin on hyphal plasma membrane integrity in Magnaporthe oryzae

Hyphae with intact plasma membranes excluded propidium iodide (PI) staining, whereas membrane-compromised hyphae exhibited red fluorescence. Both control and treated hyphae displayed PI penetration (Figure 5), indicating membrane permeability to the nucleic acid-binding dye. While control and low-concentration treatment groups showed limited fluorescence (suggesting minor membrane perturbations or basal cell mortality), high-concentration tetramycin treatments (38.40 mg·L^-¹) induced extensive PI staining, demonstrating severe membrane disruption and enhanced dye influx.

Figure 5

Figure 5. Analysis of hyphal and conidial cell wall integrity after 3 hours of tetracycline treatment using CFW staining. Scale bar = 62.3 μm. (a) tetramycin concentrations are 0 mg·L^-1; (b) tetramycin concentrations are 3.84 mg·L^-1; (c) tetramycin concentrations are 38.40 mg·L^-1.

3.6 Effects of tetramycin on conidial plasma membrane integrity in Magnaporthe oryzae

After 30 minutes of PI staining in the dark, fluorescence microscopy revealed that the conidia of Magnaporthe oryzae treated with tetramycin exhibited red fluorescence signals, whereas the untreated control conidia showed no red fluorescence (Figure 6B). In the control group, the proportion of PI-stained conidia was only 0.48%, which significantly increased with higher concentrations of tetramycin. When the tetramycin concentration reached 38.40 mg·L^-1, the proportion of PI-positive conidia rose dramatically to 34.80% (Figure 6A). These results indicate that tetramycin disrupts the integrity of the cell membrane in conidia of Magnaporthe oryzae.

Figure 6

Figure 6. Analysis of conidial plasma membrane integrity by PI staining after 3 h tetramycin treatment. (A) Percentage of PI-stained conidia. (B) Microscopic evaluation of membrane integrity (scale bar = 62.3 μm). Data represent mean ± SE of three replicates. Different letters indicate significant differences (p < 0.05). a: tetramycin concentrations are 0 mg·L-1; b: tetramycin concentrations are 3.84 mg·L-1; c: tetramycin concentrations are 38.40 mg·L-1. “CK” denotes the negative control.

3.7 Potential resistance development and monitoring strategies

Although tetramycin demonstrates significant antifungal activity, Magnaporthe oryzae has exhibited the capacity to develop resistance to various commonly used fungicides. Compared to other antifungal agents, resistance development to polyene antibiotics is typically rare, primarily attributed to their mechanism of targeting ergosterol, a critical membrane component (Carolus et al., 2020). However, prolonged and exclusive application of a single fungicide may still lead to the emergence of resistant mutants, which often carry fitness costs but may maintain competitive advantages under continuous fungicide selection pressure (Silva et al., 2022).

To ensure the sustainable application of tetramycin, establishment of a systematic resistance monitoring program is recommended: (a) regularly collect field isolates to monitor baseline shifts in tetramycin sensitivity; (b) track resistance-associated gene mutations through molecular markers; (c) implement fungicide rotation strategies, integrating tetramycin with agents possessing different modes of action to delay resistance development (Castroagudín et al., 2015; Lucas et al., 2015). These proactive measures can maximize the effective lifespan of tetramycin and provide safeguards for sustainable agricultural production.

4 Discussion

Tetramycin offers dual advantages: it directly inhibits M. oryzae growth and germination while enhancing rice defense systems. For instance, tetramycin significantly reduces the formation rate of attachment cells and the penetrating ability of infection structures, effectively interrupting the infection process. Additionally, tetramycin has shown growth-promoting effects in cucumber (Tian et al., 2025). In inoculation experiments, we observed both protective and therapeutic effects of tetramycin, which reduced the incidence of rice blast disease. Furthermore, the combination of tetramycin with other fungicides can enhance efficacy, such as leaf spot and viral diseases (Zhang et al., 2022). Compared to chemical pesticides, tetramycin exhibits good environmental compatibility, rapid degradation, and low residual risk, aligning with the development needs of sustainable agriculture. Our findings align with previous studies on tetramycin’s antifungal mechanisms. Similar to Alternaria alternata, where tetramycin disrupted membrane integrity causing plasmolysis (Li et al., 2023), our study revealed membrane disruption as the primary mechanism in M. oryzae. However, unlike A. alternata, cell wall integrity remained intact, suggesting pathogen-specific selectivity. The EC₅₀ values against M. oryzae (4.41 mg·L^-¹) and Colletotrichum scovillei (5.2 mg·L^-¹) were comparable (Gao et al., 2018). Notably, our study uniquely demonstrates complete inhibition of appressorium formation—a critical infection structure specific to M. oryzae pathogenesis, distinguishing it from tetramycin’s effects on Fusarium species.

This study represents the first systematic investigation of tetramycin against M. oryzae, addressing a critical gap in rice blast management. While tetramycin has been studied against soil-borne pathogens and post-harvest diseases, its application to rice blast remained unexplored. Our research demonstrates potent inhibition (EC₅₀ = 4.41 mg·L_-¹) and provides the first evidence of complete appressorium abolishment (0% vs 32% in controls), directly disrupting the pathogen’s unique penetration strategy. Ultrastructural analysis revealed concentration-dependent membrane disruption without cell wall compromise—a mechanism profile distinct from other pathosystems, establishing tetramycin as a promising novel agent for rice blast management.

5 Conclusions

This study systematically evaluated the antifungal activity and mode of action of tetramycin against Magnaporthe oryzae. Tetramycin exhibited significant dose-dependent inhibition of mycelial growth with an EC₅₀ of 4.41 mg·L^-¹, demonstrating potent suppression of fungal expansion at low concentrations. Furthermore, tetramycin substantially impaired both appressorium formation (a critical infection structure) and conidial germination, thereby disrupting pathogenic penetration and dissemination. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) observations revealed concentration-dependent morphological damage to hyphae, with severe structural alterations including contraction, deformation, fragmentation, extensive vacuolization, and cytoplasmic dilution at high concentrations. PI staining revealed concentration-dependent plasma membrane disruption, with severe integrity loss observed at 38.40 mg·L^-¹. As the primary barrier for cellular homeostasis, membrane compromise would critically impair vital physiological processes in the pathogen, ultimately leading to fungal growth inhibition or cell death.

Data availability statement

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

Author contributions

HYJ: Investigation, Writing – original draft, Writing – review & editing. HJ: Software, Writing – original draft. DL: Methodology, Writing – review & editing. YX: Conceptualization, Writing – original draft. KS: Conceptualization, Writing – original draft. KP: Methodology, Writing – original draft. XY: Conceptualization, Resources, Writing – review & editing. XS: Data curation, Resources, Writing – original draft, Writing – review & editing.

Funding

The author(s) declared that financial support was received for this work and/or its publication. This work was supported by the "Pioneer" and "Leading Goose" R&D Program of Zhejiang (2022C02047, 2023C02030), the Scientific Research Fund of Zhejiang Provincial Education Department (Y202456387), and the Basic Scientific Research Business Expenses Project of China Jiliang University (2024YW89, 2024YW110).

Conflict of interest

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.

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The author(s) declare that no Generative AI was used in the creation of this manuscript.

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