A new antifungal compound from Streptomyces diastatochromogenes

Novel antifungal compounds effective against phytopathogenic fungiwere identified by evaluating an n-butanol extract obtained from the fermentation broth of Streptomyces diastatochromogenes strain No.1628. The extract exhibited had strong antifungal activity against Botrytis cinerea, Fusarium oxysporum, and Rhizoctonia solani, markedly reducing the spore germination rates of F. oxysporum and B. cinerea to 25.65% and 28.23%, respectively, at a concentration of 35 mg/L. In vivo efficacy assays further demonstated that the extract achieved disease control efficiencies of 53.42% and 55.68% against Rhizoctonia rot following irrigation at 10 mg/L for 14 and 21 days, respectively. Subsequent chemical investigation led to the isolation of five antifungal compounds from the n-butanol extract: the novel tetraene macrolide, which was structurally elucidated through spectroscopic analysis as (7E,12Z,13E,15E,17E,19E)-21- ((4-amino-3,5-dihydroxy-6-methyltetrahydro-2 H-pyran-2-yl)oxy -)-12-ethylidene-1,5,6,25-tetrahydroxy-11-methyl-9-oxo-10,27-dioxabi-cyclo[21.3.1] -heptacosa-7,13,15,17,19-pentaene-24-carboxylic acid (compound 1), and four other already known antifungal agents, namely tetrin B (2), tetramycin A (3), toyocamycin (4) and anisomycin (5). Compound 1 exhibited potent inhibitory activity against the hyphal growth of R. solani, F. oxysporum, and B. cinerea, with IC₅₀ values of 0.20, 1.28, and 1.53 μg/mL, respectively. These fundings underscore S. diastatochromogenes as a promising microbial source for the discovery of natural antifungal agents.

Plant diseases caused by phytopathogenic fungi have contributed to major economic losses in agriculture worldwide, and fungal infections destory crops worth millions of dollarsannually [1]. Although chemical fungicides have achieved moderate success in controlling fungal pathogens [2], their persistent and indiscriminate use has been discouraged because of their toxic effects on non-target organisms, environmental toxicity, and role in pathogen resistance [3,4,5,6]. Hence, synthetic chemical control agents are being phased out through regulatory measures or becoming increasingly ineffective [4]. By contrast, microbial secondary metabolites have attracted considerable interest as potential alternatives to synthetic pesticides [7]. For instance, agricultural antibiotics produced by actinomyces have emerged as promising candidate for the development of environmentally friendly and safe integrated crop management [8,9,10,11].

Streptomyces is a major producer of natural antifungal agents, including compounds, such as jinggangmycin and polyoxins. Notably, approximately 70% of antibiotics used in agriculture and horticulture are derived from this genus [12,13,14]. In our previous study, a bacterial strain was isolated from a soil sample collected from Mount Tianmu in Hangzhou, China, and was identified as S. diastatochromogenes (strain No.1628) on the basis of its morphological and physiological characteristics and 16 S rDNA sequence [15]. Subsequent invastigations revealed that the fermentation broth of this strain exhibited potent antifungal activity against a broad spectrum of phytopathogenic fungi.

Novel antifungal compounds with unique chemical structures effective against phytopathogenic fungi were explored by assessing an n-butanol extract from the fermentation broth of S. diastatochromogenes (strain No.1628) was evaluated for its antifungal activity. The inhibitory effect of the extract were compared with those of commercial fungicides, including 50% carbendazim WP, 75% thiophanate-methyl WP, 5% jinggangmycin AS, 80% mancozeb WP and 98% hymexazol WP. In addition, the n-butanol extract was assessed for its ability to inhibit spore germination and its biocontrol efficacy against cucumber pathogens. Bioassay-guided fractionation of the extract led to the isolation of several active compounds, which were structurally characterized using nuclear magnetic resonance (NMR) spectroscopy. Among them, a novel compound with potent antifungal activity was identified and named tetramycin P. The antifungal efficacy of teramycin P was further evaluated against key plant pathogens, including Rhizoctonia solani, Fusarium oxysporum and Botrytis cinerea.

Microorganisms

S. diastatochromogenes (strain No. 1628) was isolated from a soil sample collected from Mount Tianmu, Hangzhou, China. The strain has been deposited in the China General Microbiological Culture Collection Center (CGMCC) under the accession number CGMCC 2060 [15]. The phytopathogenic fungi Alternaria solani, B. cinerea, F. oxysporum, R. solani and Colletotrichum lindemuthianum were generously provided by the Institute of Plant Protection and Microbiology, Zhejiang Academy of Agricultural Sciences. All fungal isolates were maintained on potato dextrose agar (PDA) slants at 4 °C.

Culture conditions for S. diastatochromogenes

An 1 mL spore suspension of S. diastatochromogenes (approximately 1.0 ×ばつ 10⁶ conidia/mL) was inoculated into 300 mL Erlenmeyer flasks containing 60 mL of liquid fermentation medium (medium components: soybean meal 40 g/L, bran 10 g/L, soluble starch 20 g/L, FeCl₂ 1 g/L, CaCO₃ 5 g/L, NH₄NO₃ 3 g/L, KHSO₄ 3 g/L). The cultures were incubated on a rotary shaker at 28 °C and 180 rpm for 96 h. Following incubated, the cultures were harvested by centrifugation at 5000 rpm for 15 min. The resulting supernatants were collected and pooled, yielding a total volume of 50 L, which was subsequently used for antifungal activity assay and the isolation of bioactive compounds.

Antifungal activity assay

Antifungal activity was evaluated using the hyphal growth rate assay as described previously [16]. Commercial antifungal agents, including 50% carbendazim WP, 75% thiophanate-methyl WP, 5% jinggangmycin AS, 80% mancozeb WP and 98% hymexazol WP, were applied at their recommended concentrations and served as positive controls. Sterile water was used as the negative control. Each treatment was conducted in triplicate. The diameter of fungal colonies was measured once the mycelial growth in the negative control had completely covered the surface of the Petri dishes. The percentage inhibition of mycelial growth was calculated using the following formula:

, where D_a represents the diameter of growth zone in the control dish, and D_b represents the diameter of growth zone in the experiment dish.

Spore germination assay

A spore suspension of B. cinerea or F. oxysporum was inoculated onto concave glass slides, each containing 50 μL of potato dextrose broth supplemented with varying concentrations of the test samples (i.e., the n-butanol extract from the fermentation broth of S. diastatochromogenes 1628). The slides were incubated at 25 ± 1 °C for 6 h. Following incubation, aliquots were transferred to both chambers of a Neubauer hemocytometer and examined under a light microscope to assess spore germination. A spore was considered germinated when the length of the germ tube was equal to or greater than that of the spore [17]. All experiments were performed in triplicate.

Antifungal activity assay on cucumber

A pot experiment was conducted in a greenhouse to evaluate the biocontrol efficacy of the n-butanol extract against cucumber Rhizoctonia rot. The greenhouse conditions were maintained at an average temperature of 25 °C, relative humidity of 70% and a photoperiod of 16 h light/8 h dark. Cucumber seedlings were transplanted into 14 cm-diameter plastic potsand grown until the fourth leaf stage. At this stage, the seedlings were inoculated with R. solani by root irrigation using a mycelial suspension. After 24 h, the n-butanol extract dissolved in sterile distilled water was applied to each pot. Each treatment consisted of 30 replicates.

Disease incidence was assessed on days 14 and 21 following treatment with the n-butanol extract. Plants treated with the commercial fungicide 98% hymexazol (applied at the recommended concentration) served as positive controls, whereas those treated with sterile distilled water served as negative controls. All treatments were conducted in triplicate. Disease severity was evaluated according to a previously described method [18]. The mean disease index and disease control efficiency were calculated using the following formulas:

Isolation and characterization of antifungal compounds

Antifungal compounds were isolated from the n-butanol extract using an in vitro antifungal activity-guided fractionation approach. The initial separation was sequentially performed through liquid-liquid extraction. The fermentation broth was extracted three times with equal volumes of ethyl acetate and n-butanol, respectively. The solvent fractions were concentrated under reduced pressure, yielding 18.25 g of ethyl acetate extract and 30.82 g of n-butanol extract. Both dried extracts were dissolved in water and subjected to antifungal activity assays. As the antifungal activity was primarily enriched in the n-butanol extract, 1 L of its aqueous solution was further fractionated through macroporous resin AB-8 column chromatography (Nankai University Chemical Factory, Tianjin, China) and eluted with a gradient of water and ethanol (from 100:0 to 5:95, v/v). The eluates were monitored through TLC and combined into sixteen fractions (Fraction 1–16). Fractions 6–8 and 11–15, which exhibited antifungal activity against the test pathogens were each subjected to further purification with an AB-8 column with the same water/ethanol gradient elution. Five subfractions (D1-D5) were obtained from fractions 6–8, and seven subfractions (S1-S7) from fractions 11–15. Antifungal activity was detected in subfractions D1 and S4. Subfraction D1 was subsequently purified by silica gel column chromatography (100–200 mesh, Qingdao Marine Chemical Factory, Qingdao, China) using stepwise elution with petroleum ether/ethyl acetate mixtures (100:0, 80:20, 60:40, 40:60, and 20:80, v/v). Antifungal activity assays identified two active compounds, which were designated 1 (103 mg) and 2 115 mg). Subfraction S4 was further purified using a Sephadex LH-20 column (Beijing Greenherbs Science and Technology Development Co., Ltd., China) and eluted with a water/ethanol gradient (100:0 to 10:90, v/v). This process led to the isolation of three active antifungal compounds, designated 3 (201 mg), 4 (187 mg), and 5 (145 mg).

The chemical structures of the active compounds were elucidated through comprehensive spectroscopic analysis. UV spectra were recorded using a UV-1800 spectrophotometer (Shimadzu Corporation, Kyoto, Japan) with a spectral bandwidth of 1.00 nm and a scanning range of 190.00–400.00 nm. IR spectra were obtained using a Bruker ALPHA. Mass spectrometric analysis was conducted on an Agilent 1260–6130 LC-MS system (Agilent Technologies). NMR spectra were acquired using a Bruker Avance 400 MHz NMR spectrometer (Bruker), and the samples were dissolved in deuterated dimethyl sulfoxide and tetramethylsilane as the internal standard. Structural elucidation of the compounds was performed by interpreting the spectral data obtained from UV, IR, MS and NMR analyses and by comparison with previously reported data in the literature.

Statistical analysis

All results were presented as means ± standard deviations. Statistical analyses were conducted using one-way analysis of variance. Differences were considered statistically significant at P < 0.05. All analyses were performed using SPSS software version 15.0 (SPSS Inc., Chicago, IL, USA).

Antifungal activity of the fermentation broth and its solvent extracts

The antifungal activity of the fermentation broth was initially evaluated. The results demonstrated that the broth exhibited strong inhibitory effects against R. solani, (A) solani, F. oxysporum and (B) cirereacinerea. Notably, the growth of R. solani was completely inhibited. Furthermore, the inhibition rates against (A) solani, F. oxysporum, and (B) cinerea all exceeded 88% (Table 1).

For the isolation and purification of specific antifungal compounds, the fermentation broth was subjected to solvent extraction using ethyl acetate and n-butanol. The antifungal activity of each extract were then evaluated. Results indicated that the n-butanol extract exhibited considerably stronger antifungal activity than the ethyl acetate extract at equivalent test concentrations. At a concentration of 35 mg/L, the n-butanol extract achieved 100% inhibition of F. oxysporum, R. solani and B. cinerea. Even when the concentration was reduced to 17.5 mg/L, the extract maintained high inhibition rates of 95.86% and 90.26% against R. solani and F. oxysporum, respectively. By contrast, inhibition rates against B. cinerea and A. solani at the same concentration were slightly lower, at 80.52% and 78.86%, respectively. These findings suggest a dose-dependent antifungal effect of the n-butanol extract, with efficacy at 17.5 mg/L comparable to that of commercially available fungicides (Table 1).

Effect of n-butanol extract on fungal spore germination

Fungal spores serve as key propagules for distribution and long-term survival, and their germination is typically tightly regulated by species according to lifestyle and nutritional requirements. In the present study, the inhibitory effect of the n-butanol extract on spore germination of B. cinerea and F. oxysporum was assessed at concentrations of 35, 17.5 and 10 mg/L. The results demonstrated that spore germination was inhibited by the n-butanol extract at all tested concentrations (Table 2). At the highest concentration of (35 mg/L), the extract suppressed the spore germination, resulting in germination rates of only 25.65% for F. oxysporum and 28.23% for B. cinerea. Even at the lowest concentration tested (10 mg/L), the extract retained more than 50% inhibitory activity against both fungal species. These findings indicate a strong and concentration-dependent antifungal effect of the n-butanol extract on spore germination.

Biocontrol activity against cucumber rhizoctonia rot

Cucumber rhizoctonia rot, a severe soil-borne disease caused by R. solani, poses a significant threat to crop productivity. To evaluate the biocontrol potential of the n-butanol extract, a pot experiment was conducted under greenhouse conditions. The results demonstrated that the extract exhibited strong inhibitory effects against rhizoctonia rot (Table 3). Following root irrigation with the n-butanol extract at a low concentration of 10 mg/L, disease control efficiency of the extract was determined as 53.42% and 55.68%% were observed after 14 and 21 days, respectively. Furthermore, the biocontrol efficacy improved withincreasing concentrations of the extract and was significantly higher than that of the positive commercial control. These findings suggest that the n-butanol extract from S. diastatochromogenes effectively suppresses and controls cucumber rhizoctonia rot caused by R. solani.

Structure elucidation of antifungal compounds

The antifungal activity-guided separation of the n-butanol extract led to the isolation of four bioactive compounds: 1, 2, 3, 4, and 5 (Fig. 1). Among them, compound 3 was obtained as a pale-yellow powder and exhibited strong UV absorption maxima at 291, 302, and 320 nm, characteristic of tetraene macrolide compounds (Fig.S1). The structure of 3 was determined by HRMS and NMR spectroscopy. HRESIMS revealed a molecular ion peak at m/z 694.3445 [M + H]⁺, corresponding to a molecular formula of C35H51NO13, indicating eleven degrees of unsaturation (Fig.S2). The spectral data (¹H and ¹³C NMR) is presented in Table 4.

The ¹³C NMR spectrum confirmed the presence of 35 carbon atoms, including two ester carbonyls (δ_C 177.3, 164.7), ten olefinic carbons (δ_C 149.1, 136.3, 135.7, 133.6, 133.0, 131.2, 131.1, 131.0, 128.2, 121.2), ten oxycarbons (δ_C 74.4, 73.0, 72.9, 72.6, 71.6, 69.7, 67.9, 67.8, 65.7, 65.3, 59.2), two semiacetal carbons (δ_C 97.1, 95.5), one azonium carbon (δ_C 56.2) and.

Several aliphatic carbons including those assigned to C-4, C-5, C-7, and C-9 (δ_C 47.6, 46.7, 44.6, 39.8, 36.1, 23.4, 18.0, 13.8, 12.3). The spectral assignments were further supported by DEPT, HSQC, HMBC and ¹H-¹H COSY experiments (Table 4; Figs.S3-S9). Notably, HMBC correlations between H-25 and C-1 suggested ester bond formation, while interactions between H-1′ and C-15 indicated that the attachment of an amino sugar moiety at the C-15 position. Within the tetraene macrolide ring, four hydroxyl groups were identified at C-4, C-5, C-7 and C-11. However, owing to the absence of HMBC correlations between H-4, H-5, and H-7 with C9, a dehydration reaction may have occurred between the hydroxyl group at C-9 and the carboxyl group at C-29, resulting in an additional ester bond. On the basis of the combined spectroscopic data, 3 was structurally characterized as (7E,12Z,13E,15E,17E,19E)-21-((4-amino-3,5-dihydroxy-6-methyltetrahydro-2 H-pyran-2-yl)oxy)-12-ethylidene-1,5,6,25-tetrahydroxy-11-methyl-9-oxo-10,27dioxabicyclo[21.3.1]heptacosa-7,13,15,17,19-pentaene-24-carboxylic acid. This compound represents a novel member of the tetraene macrolide antibiotic family. To the best of our knowledge, this is the first report of its isolation from microbial source. Given its structural similarity totetramycin A, the compound was designated tetramycin P [19].

The structural analysis revealed the remaining four active compounds, namely, 1, 2, 4 and 5, are known bioactive compounds. Compound 1 was identified as toyocamycin (C₁₂H₁₃N₅O₄), which is a kind of pyrrolopyrimidine nucleoside antibiotic [20]. Compound 2 was determined to be anisomycin (C₁₄H₁₉NO₄), which is a nitrogen-containing heterocyclic aromatic antibiotic [21]. Compounds 4 and 5 were identified as tetrin B (C₃₄H₅₁NO₁₄) and tetramycin A (C₃₅H₅₃NO₁₃), respectively [22, 23]. All of these compounds were shown to have strong antifungal activities [20,21,22].

Antifungal activity of compound E1 (tetramycin P)

The antifungal activity of tetramycin P was evaluated against five phytopathogenic fungi: R. solani, F. oxysporum, (A) solani, (B) cinerea and (C) lindemuthianum. The IC50 values are summarized in Table 5. Tetramycin P exhibited potent antifungal activity, particularly against R. solani, with an IC50 of 0.20 μg/mL. It demonstrated strong inhibition against B. cinerea and F. oxysporum IC50 values of 1.53 and 1.28 μg/mL, respectively. However, it showed moderate inhibitory effects on A. solani (IC50 = 8.52 μg/mL) and C. lindemuthianum (IC50 = 10.28 μg/mL. These findings suggest that the antifungal efficacy of tetramycin P is fungus dependent. Notably, compared to commercial antifungal agents used as positive controls, tetramycin P displayed strong and broad-spectrum antifungal activity.

The majority of clinically, veterinary, and agriculturally used antibiotics are natural products derived from actinomycetes, particularly members of the genus Streptomyces, which are widely distributed in soil and marine sediments [8, 10]. In this study, S. diastatochromogenes strain No.1628 was isolated from a soil sample collected from Mount Tianmu, which is a renowned nature reserve in Zhejiang Province, China. Previous reports have shown that S. diastatochromogenes can produce antimicrobial metabolites, such as ε-poly-L-lysine (by strain CGMCC 3145) and yokonolide A, which is an inhibitor of auxin signal transduction (by strain B59) [24, 25]. Our findings reveal that strain No.1628 synthesizes three additional classes of antifungal compounds: toyocamycin, which is a pyrrolopyrimidine nucleoside antibiotic; anisomycin, which is a nitrogen-containing heterocyclic aromatic antibiotic; and a group of tetraene macrolide antibiotics, including tetramycin A, tetramycin P, and tetrin B. Although toyocamycin, anisomycin, tetramycin A, and tetrin B are known compounds, this study is the first to isolate them from S. diastatochromogenes [20,21,22, 26]. Notably, tetramycin P, a novel tetraene macrolide antibiotic identified in this study, exhibited strong in vitro antifungal activity against multiple phytopathogens. This newly discovered compound represents a promising lead for the development of novel fungicides and is reported here for the first time as a natural product of microbial origin.

Tetraene macrolides are well recognized for their potent antifungal properties. Their primary advantages over other antifungal agents lie in their fungicidal activity and the remarkably low incidence of resistance among target pathogens [22, 23, 26]. A known inhibitor of protein synthesis, anisomycin is the major active ingredient in Agricultural Antibiotic 120, which is a widely used biopesticide in China [27]. Toyocamycin inhibits rRNA maturation and specifically disrupts auxin signaling via the SCF–TIR1 pathway [28, 29]. Given the presence of these active compounds, the n-butanol extract exhibited broad-spectrum antifungal activity against a variety of phytopathogenic fungi. This study demonstrates that S. diastatochromogenes (strain No.1628) is a promising source of bioactive antifungal metabolites, and its potent antifungal properties hold significant potential for future development in agricultural biocontrol applications.

The data and materials supporting the findings of this study are available from the corresponding author upon reasonable request. All relevant data, including NMR spectra, HRESIMS results, and other analytical data used for the structural elucidation of the compounds, are provided in the supplementary material accompanying this manuscript. Additionally, the raw data from antifungal activity assays, spore germination tests, and biocontrol experiments are available upon request. The strain Streptomyces diastatochromogenes (No.1628) used in this study has been deposited in the China General Microbiological Culture Collection (CGMCC) under the accession number CGMCC 2060.

• (2025年08月25日 追記)

  25 August 2025

  A Correction to this paper has been published: https://doi.org/10.1186/s12896-025-01028-7

  (追記ここまで)

PDA:

Potato dextrose agar

TMS:

Tetramethylsilane

FB:

Fermentation broth

EAE:

Ethyl acetate extract

BE:

n-butanol extract

C:

Carbendazim

TM:

Thiophanate-methyl

M:

Mancozeb

J:

Jinggangmycin

H:

Hymexazol

S. diastatochromogenes:

Streptomyces diastatochromogenes

A. solani :

Alternaria solani

B. cinerea :

Botrytis cinerea

F. oxysporum :

Fusarium oxysporum

R. solani :

Rhizoctonia solani

C. lindemuthianum :

Colletotrichum lindemuthianum

This work was supported by "Pioneer" and" Leading Goose" R&D Program of Zhejiang (Grant No. 2022C02047, 2023C02030).

1. Ming-Yun Wang and Ke-Qi Ye contributed equally to this work. Author order was determined both alphabetically and in order of increasing seniority.

Authors and Affiliations

1. Key Laboratory of Microbiological Metrology, Measurement & Bio-product Quality Security, State Administration for Market Regulation, College of Life Science, China Jiliang University, Hangzhou, 310018, China

   Ming-Yun Wang, Ke-Qi Ye, Dan-Ting Li, Xiao-Ping Yu & Xu-Ping Shentu

2. Liaoning Wkioc Bioengineering Co., Ltd, Zhaoyang, 122000, China

   Chun-Li Guo

3. Hailir Pesticides and Chemicals Group Co., Ltd, Qingdao, China

   Jia-Cheng Ge

Contributions

Ming-Yun Wang and Ke-Qi Ye, Formal analysis, Investigation, Methodology, Writing - review and editing; Chun-Li Guo and Jia-Cheng Ge, Investigation; Dan T. Li, Writing - review and editing; Xiao-Ping Yu and Xu-Ping Shentu, Conceptualization, Supervision, Funding acquisition, Writing - review and editing. All authors contributed to manuscript drafting, reviewed the final version and approved its submission.

Corresponding author

Correspondence to Xu-Ping Shentu.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

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The original version of this article was revised: the authors identified an error in Fig. 1.

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