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Article

Antimicrobial Lobophorins from Endophytic Strain Streptomyces sp. R6 Obtained from Azadirachta indica

by
Xinyuan Chen
1,
Ying Du
2,
Yunlong Ma
3,
Peibin Liu
4 and
Yan Chen
4,*
1
School of Pharmacy, Liaoning University of Traditional Chinese Medicine, Dalian 116600, China
2
Solid Waste and Chemical Management Center, Ministry of Ecology and Environment, Beijing 100029, China
3
Liaoning Lvyuan Nongfeng Agricultural Technology Service Co., Ltd., Shenyang 110136, China
4
Institute of Plant Protection, Liaoning Academy of Agricultural Sciences, Shenyang 110161, China
*
Author to whom correspondence should be addressed.
Molecules 2025, 30(3), 586; https://doi.org/10.3390/molecules30030586
Submission received: 5 December 2024 / Revised: 19 January 2025 / Accepted: 22 January 2025 / Published: 27 January 2025
(This article belongs to the Section Natural Products Chemistry)
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Abstract

:
Endophytic bacteria are an important source for developing antimicrobial substances. With the aim to find eco-friendly antimicrobial agents from natural sources, Streptomyces sp. R6 was isolated from Azadirachta indica. After that, a new spirotetronate natural product, lobophorin S (compound 2), together with lobophorin H8 (compound 1) and a known macrolide compound divergolide C (compound 3) were isolated from the cultural solution of strain R6. These compounds mark the first isolation of marine-derived microbial natural products known as lobophorins (LOBs) from endophytic bacteria. The structures of these three compounds were identified by extensive NMR and HRMS analyses. The antimicrobial activities of these three compounds against eight fungal and four bacterial phytopathogens were separately evaluated. Compound 1 demonstrated better antibacterial activity against Erwinia carotovora, Pseudomonas syringae pv. tomato, and P. syringae pv. lachrymans with MIC values of 3.91, 7.81, and 15.63 μg/mL, respectively. Additionally, compounds 1–3 all showed antifungal activity against Botrytis cinerea, with the MIC values of 1.95, 7.81, and 15.63 μg/mL, respectively. Notably, the in vivo antifungal effect of 1 against B. cinerea was up to 78.51 ± 3.80% at 1.95 µg/mL, significantly surpassing polyoxin B (70.70 ± 3.81%). These results highlight the potential of lobophorins as promising lead compounds for the development of new, sustainable agents to control plant diseases.

Graphical Abstract

1. Introduction

Plant diseases significantly threaten global agricultural production, causing billions of dollars in losses annually [1,2,3]. Despite the widespread use of chemical antimicrobial agents to control these diseases, their overuse has led to several critical issues, such as antimicrobial agent residues, environmental contamination, and the emergence of resistant strains, all of which endanger both human health and ecosystems [1,4,5,6,7]. The increasing resistance of pathogens and the adverse effects of traditional antimicrobial agents underscore the need for sustainable and ecologically friendly alternatives. In this context, natural products and biological antimicrobial agents are gaining recognition as viable substitutes for conventional chemical agents [1,8,9,10].
Plant endophytic actinomycetes are one of the most important groups among endophytes, from which many compounds have been reported [11,12,13] and utilized in agriculture and the pharmaceutical industry [14,15,16]. They are considered one of the best microbial resources for producing bioactive substances, with approximately 80% of secondary metabolites being cultured and isolated from them, among which the genus Streptomyces accounts for about 50% [17]. Within this group, it is particularly worth noting that the genus Streptomyces is a major source of antimicrobial natural compounds with a variety of chemical structures, such as polyketides (PKs), terpenoids, non-ribosomal peptides (NRPs), and alkaloids, most of which are polyketide compounds [14,16]. A good example is the spirotetronate antibiotic lobophorin (LOBs), which exhibits many types of activities like antimicrobial and anti-tumor activities [18]. LOBs were initially discovered from actinomycetes associated with algae and subsequently re-isolated from the deep-sea-derived Streptomyces SCSIO 01,127 [19,20]. LOBs have historically been regarded as marine-derived microbial natural products, and their potential in agricultural production remains unexplored.
Through cultivation and isolation, we obtained an endophytic actinomycete, Streptomyces sp. R6, from the leaves of Azadirachta indica collected in Thailand [21]. In our search for natural products, the fermentation products of this endophytic strain were extracted, separated, and purified using various phytochemical methods, leading to the isolation of three compounds. The structures of these compounds were accurately elucidated using high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR) techniques. We employed a modified broth microdilution method to assess the in vitro antibacterial effects on four plant-pathogenic bacterial strains—Pseudomonas syringae pv. lachrymans, P. syringae pv. tomato, Clavibater michiganensis, and Erwinia carotovora—and eight plant-pathogenic fungal strains, including Stemphylium solani, Fulvia fulva, Alternaria solani, Botrytis cinerea, Pythium aphanidermatum, Pyricularia grisea, Fusarium oxysporum (Schl.) f. sp. cucumerinum Owen., and Corynespora cassiicola. Additionally, we conducted in vivo pot experiments to evaluate the effects against the plant-pathogenic fungus B. cinerea, in order to provide new clues for the development of antimicrobial molecules.

2. Results

2.1. Elucidation of Chemical Structures

The extract obtained from the fermentation of strain R6 was fractionated by silica gel column chromatography, Sephadex LH-20 gel chromatography and high-performance liquid chromatography (HPLC), resulting in the isolation of compound 1 (lobophorin H8), compound 2 (lobophorin S, which is characterized as a novel spirotetronate natural product), and a previously identified macrolide compound, compound 3 (divergolide C) (Figure 1) [22].
Compound 1 was obtained as a yellowish-colored solid. Its HRESIMS analysis, in association with 13C NMR, and HSQC spectra suggested a molecular formula C33H44O6 with a degree of unsaturation of 12. The 1H NMR of compound 1 (Table 1) revealed the presence of five olefinic protons (δH 5.14, br s, H-21; δH 5.26, d, J = 10.5 Hz, H-19; δH 5.22, d, J = 10.0 Hz, H-15; δH 5.43, m, H-12; δH 6.03, br d, J = 10.0 Hz, H-11) and four midfield protons (δH 3.66, dd, J = 10.5, 5.3 Hz, H-9; δH 3.53, m, H-13; δH 4.19, br s, H-17; δH 3.58, br d, J = 10.5 Hz, H-20). Four singlets and three doublets of methyl (δH 1.28, d, J = 6.8, H-33; δH 0.65, d, J = 7.0 Hz, H-28; δH 1.04, d, J = 6.8, H-29) were also observed in the 1H NMR. The 13C NMR of compound 1 (Table 1) indicated the presence of 33 carbon atoms. 13C NMR and various HSQC spectra suggested that these 33 carbon atoms were distributed as: seven methyl, three methylene, fourteen methane (five sp2 and nine sp3) and nine quaternary carbon atoms (two carbonyl, one ketone, four olefinic, and two oxygenated carbons). These 33 typically skeletal carbons suggested that compound 1 existed as apentacyclic aglycon featuring atetronate moiety spiro-linked with a cyclohexene ring and belonged to a large class of spirotetronate antibiotics [23]. Moreover, the NMR and mass spectra of compound 1 were consistent with literature reports, and its structure was identified to be lobophorin H8 [24].
The NOESY spectrum of compound 1 provided the following information regarding its relative configuration and interactions: H-5 (δ 2.01) to H-9 (δ 3.66) and H-5 to H-28 (δ 0.65), as shown in Figure 2. This implies that the methyl group at site 28 (H-28) is in close proximity to H-9 and H-5, exhibiting identical orientation. Furthermore, the NOESY correlation was observed between H-10 (δ 2.07) and H-29 (δ 1.04), demonstrating that H-10 and the 29-CH3 group are on the same side of the ring. There are two double bonds in the macrocyclic structure. The NOESY interaction between H-17/H-19 and H-13/H-15 indicate that both pairs are transoriented. Additionally, ∆11(12) was identified as Z configuration due to the 3J(H-C(11), H-C(12) = 10.0 Hz. Thus, the relative structure of compound 1 was finally established and named as lobophorin H8. To the best of our knowledge, this study represents the first report of the isolation of lobophorin H8 from a natural source [20]. Its structure is illustrated in Figure 2.
Compound 2 was obtained as a yellowish solid. Its HRESIMS, along with 13C NMR, and HSQC spectra revealed a molecular formula C39H54O9 with a degree of unsaturation of 13. The 1H NMR spectrum of compound 2 contained five olefinic protons (δH 5.14, br s, H-21; δH 5.27, d, J = 10.5 Hz, H-19; δH 5.23, d, J = 10.0 Hz, H-15; δH 5.45, m, H-12; δH 5.74, br d, J = 10.0 Hz, H-11) (Table 1). The 13C NMR and HSQC spectroscopic data (Table 1) of compound 2 indicated 39 carbon resonances, including eight methyl, four methylene, eighteen methane (five sp2 and thirteen sp3) and nine quaternary carbon atoms (two carbonyl, one ketone, four olefinic and two oxygenated). From these characteristic signals, it can be deduced that compound 2 belonged to a large class of spirotetronate antibiotics and was highly similar in structure to compound 1 according to the NMR data (Table 1). The only difference between compounds 2 and 1 is a substitution of glycosidic moiety in 2, which was found according to six carbon signals (including one methyl at δC 17.7, one methylene at δC 34.9, and four oxygenated methine at δC 99.6, 72.6, 67.1, and 65.3) in the 13C NMR spectra of 2. The key HMBC correlations (Figure 3) of H-1′ with C-9 (δC 85.6) and C-5′ (δC 65.3) confirmed that a digitoxose unit was established located at C-9 in 2, which was further verified by the correlations of H-11 (δH 5.74) with H-9 (δH 3.57) from 1H-1H COSY spectrum (Figure 3).
The relative configuration of compound 2 was determined through NOESY experiments. In the NOESY spectrum (Figure 2), the correlations of H-17 with H-23 suggested that 17-OH in 2 might be α-oriented. An α-orientation for 9-O-L-digitoxose was deduced from the NOESY correlations of H-1′ with H-3′/H-4′. Therefore, the structure of compound 2 was established and designated as lobophorin S.

2.2. In Vitro Antimicrobial Activity Assay

The 96-well plate method was employed to evaluate the in vitro inhibitory activities of the three compounds against four bacterial and eight fungal plant pathogens [25,26,27]. The test results of the inhibitory activities were presented in terms of their Minimum Inhibitory Concentration (MIC). As shown in Table 2, compounds 1–3 exhibited moderate to good antimicrobial activity and exhibited a relatively broad antimicrobial spectrum against various pathogenic bacteria. Compound 1 displayed significant antibacterial effect against all tested pathogenic bacteria, particularly against Erwinia carotovora with an MIC value of 3.91 μg/mL, which was much lower than that of streptomycin (31.25 μg/mL). Compound 3 was also effective against E. carotovora with an MIC value of 31.25 μg/mL. Additionally, compound 2 demonstrated significant growth inhibition for Pseudomonas syringae pv. tomato, with an MIC value of 15.63 μg/mL. All three compounds exhibited antifungal activity against Botrytis cinerea with MIC values of 1.95, 7.81, and 15.63 μg/mL, respectively, which were similar to the positive control polyoxin B (3.91 μg/mL) but higher than that of carbendazim (31.25 μg/mL) and amphotericin B (125 μg/mL). Additionally, compound 1 and 3 showed good inhibitory activity against Fulvia fulva, with an MIC value of 31.25 μg/mL (Table 3).
In the in vitro antimicrobial activity assay, the three compounds exhibited varying degrees of activity against a range of plant pathogens. This variability can be attributed to two primary factors: firstly, the distinct species of each strain, which possess varying levels of drug susceptibility and resistance; secondly, the differing mechanisms of action and targets of the compounds themselves. These factors, among others, contribute to the significant differences in activity observed across different strains. Furthermore, even the positive controls demonstrated considerable variation in their inhibitory effects.

2.3. Antifungal Activity Against Botrytis Cinerea In Vivo

We further investigated the inhibitory activity of compound 1 against gray mold in tomatoes using pot experiments [28,29]. The spray method was selected to inoculate uniformly growing tomato seedlings, and then, compound 1 was applied to the inoculated tomato plants at five gradient concentrations. The experimental results show that as the concentration of compound 1 increased, a dose-dependent control effect on the gray mold pathogen was observed (Table 4). Notably, at a concentration of 7.81 µg/mL, the in vivo control effect on B. cinerea reached as high as 85.44 ± 3.29%. When the concentration was reduced to 3.91 µg/mL and 1.95 µg/mL, the control effects were 80.73 ± 2.42% and 78.51 ± 3.80%, respectively. At the 0.05 level, there was no statistically significant difference between the two treatments. At a concentration of 1.95 µg/mL, the in vivo control effect of polyoxin B on B. cinerea was 70.70 ± 3.81%, which was significantly lower than that of compound 1.

3. Discussion

Lobophorins (LOBs) are a subset of spirotetronate antibiotics with over 60 naturally occurring variants [30] that share a similar skeletal structure with kijanimicin [31]. LOBs have achieved certain therapeutic value in clinical aspects [31,32]. Historically reported LOBs have been isolated from marine microorganism algae and streptomycetes [19,20,33,34,35,36,37,38]. Interestingly, three compounds have been isolated and identified from the plant endophytic actinomycete strain R6, including one new and one known LOB, marking a departure from the traditional marine sources. Therefore, our research adds to a chemical diversity in LOBs.
Crop diseases caused by phytopathogenic fungi and bacteria represent a significant threat to agricultural production, leading to substantial losses in crop yield and quality annually [1,2,3,39,40]. Among these, B. cinerea, a serious plant pathogen [41], has been classified as a high-risk of resistance to many known fungicides [42,43]. The excessive use of high-concentration fungicides can result in the formation of toxic residues, increased drug resistance [44], and also poses environmental hazards [45,46]. The growing resistance of plant pathogens to conventional treatments and the environmental concerns associated with chemical fungicides have driven people to seek novel and effective solutions. LOBs exhibit a wide range of biological activities, and their development and utilization have significant medical value [47]. As of now, LOBs that have been isolated exhibit antibacterial activity against a variety of bacteria. LOBs F, H, and K demonstrate antimicrobial effects against Staphylococcus aureus [20,35,37]. LOB F also shows activity against Enterococcus faecalis [20] and Bacillus subtilis [35]. LOBs G, A, and B are effective against B. Calmette-Guerin (BCG), Mycobacterium tuberculosis H37Rv, and B. subtilis [41], and are also effective against Mycobacterium smegmatis MC2 155 [48]. LOB L shows significant activity against Micrococcus luteus and B. thuringiensis [38]. LOBs H, I [35] and H8 display activity against B. subtilis, with LOB H8 also showing activity against M. smegmatis MC2 155 [48]. To date, the isolated LOBs have demonstrated antibacterial activity against a multitude of bacterial strains. Regarding antifungal activity, specific reports on LOBs are notably absent. In this study, both LOBs showed a broad spectrum of inhibitory effects against plant pathogenic bacteria and fungi. In vitro tests demonstrated that LOB H8 effectively inhibited three bacterial growths. Furthermore, in vivo tests confirmed the bioactivity of the compound against B. cinerea. The isolation of LOBs from endophytic plant actinomycetes not only diversifies the structural landscape of polyketide compounds but also highlights the potential of natural products as a source for developing new and efficient active ingredients to combat plant diseases [15,49]. We will continue to explore the mechanisms of the antimicrobial activity of these compounds, providing a foundation for their application in agricultural production.

4. Materials and Methods

4.1. Isolation of Streptomyces sp. R6

Streptomyces sp. R6 was isolated from the neem plant by the standard technique and tissue homogenate technique [50,51], and it was identified by phylogenetic analysis and comparison of its 16S rDNA sequence with the sequences available in the EzTaxon database. It was found that the strain R6 exhibits the highest similarity with Streptomyces pactum NBRC 13433T and Streptomyces olivaceus NRRL B-3009T, with homologies of 99.09%. Consequently, the strain was named Streptomyces sp. R6 (Genbank accession no. MF375233). Strain R6 was preserved in the Laboratory of Pesticide Science, College of Plant Protection at Shenyang Agricultural University, China.

4.2. Production and Extraction of Antimicrobial Compounds

The production and extraction of Streptomyces sp. R6 were conducted in accordance with the methods described below [52]. Fermentation of strain R6 was carried out using an optimized formula of F medium, i.e., glucose, 10.0 g; soluble starch, 20.0 g; peptone, 5.0 g; NaCl 4.0 g; yeast extract, 5.0 g; MgSO4·7H2O, 0.5 g; K2HPO4, 0.5 g and CaCO3, 2.0 g per liter of medium with pH of 8.0. The starter (OD600 = 0.9) of Streptomyces sp. R6 was placed in a 250 mL flask, which was then incubated at 28 °C for 48 h in a rotary shaker (Zhichu, Shanghai, China) at 180 rpm. For mass fermentation, a total of 90 L of the medium was used, and 400 mL of medium was inoculated into each 2 L Erlenmeyer flask with 20 mL of starter culture. All the flasks were incubated under identical conditions for 7 days. Mycelia were removed by centrifugation at 6000 rpm and 4 °C for 30 min. The aliquot broth was shaken with 4% Amberlite XAD-16 (ROHM and HAAS, Philadelphia, PA, USA) resin for 4 h at 25 °C. The resins were harvested by filtration through a double layer of muslin cloth and dried at 28 °C. The fully dried resins were transferred to a separation funnel and extracted with methanol (500 mL). The methanol extract was concentrated using a rotary evaporator (Buchi, Switzerland) under reduced pressure. This MeOH extract was concentrated using a rotary evaporator at 28 °C to yield 11.58 g of crude solid residue.

4.3. Purification

The purification of the fermentation products was conducted following the methods outlined below [25]. The MeOH extract was divided into six fractions (fractions C1–C6) using various ratios of CH2Cl2−MeOH (100:0, 100:2, 100:4, 100:8, 100:16, and 0:100, 2 L each) as the mobile phase with a silica gel column (100–200 mesh, 350 mm × 25 mm i.d.). The compound 1 (48.2 mg, about 98% purity) and a mixture were obtained from fraction C1, which was collected using CH2Cl2-MeOH (100:1.6) as the mobile phase in another silica gel column (200–300 mesh, 350 mm × 15 mm i.d.). The compound 2 (tR = 35.6 min, 24.4 mg, about 96% purity) was isolated from the mixture by reverse-phase semipreparative HPLC (Agilent, Santa Clara, CA, USA), equipped with a 250 mm × 9.4 mm, 5 μm, ZORBAX Eclipse XDB C18 column (Agilent, Santa Clara, CA, USA) eluted with 85% CH3OH + 0.1% HCOOH as mobile phase at a flow rate of 3 mL/min for 40 min and UV detector at 210 nm. Fraction C2 which was collected using CH2Cl2-MeOH (100:2) as the mobile phase was subsequently chromatographed on a Sephadex LH-20 (GE Healthcare, Uppsala, Sweden) column (500 mm × 20 mm i.d.) using CH2Cl2-CH3OH (1:1) as mobile phase to obtain fractions C2a and C2b. Further purification C2b was performed by reverse-phase semipreparative HPLC (flow rate: 3 mL/min for 38 min, UV detection: 210 nm) (Agilent, Santa Clara, CA, USA), eluted with 45% CH3CN + 0.1% HCOOH to obtain 81.6 mg, about 97% purity, of compound 3 (tR = 31.6 min).
For structural elucidation, NMR spectra were recorded using an NMR spectrometer (Avance-600) (Bruker, Karlsruhe, Germany) operated at room temperature. The instrument was calibrated with carbon signals of CDCl3 (δC 77.0 and δH 7.26) and residual proton signals. AP-300 polarimeter (Atago, Tokyo, Japan) was utilized to measure optical rotations. A 6500 series quadrupole-time-of-flight (Q-TOF) mass spectrometer (Agilent, Santa Clara, CA, USA) was used to record high-resolution electrospray ionization mass spectrometry (HRESIMS) data.

4.4. In Vitro Antimicrobial Assay

A modified broth microdilution method [25,26,27] was employed to evaluate the in vitro antimicrobial activities of purified compounds against four bacterial strains (Pseudomonas syringae pv. lachrymans, P. syringae pv. tomato, Clavibater michiganensis, Erwinia carotovora) and eight fungal strains (Stemphylium solani, Fulvia fulva, Alternaria solani, Botrytis cinerea, Pythium aphanidermatum, Pyricularia grisea, Fusarium oxysporum (Schl.) f. sp. cucumerinum Owen., Corynespora cassiicola) in 96-wells microtiter plates. Bacterial and fungal strains were obtained from the Laboratory of Plant Science, College of Plant Protection, Shenyang Agricultural University, China.
Bacteria and fungi were cultured in a nutrient broth (NB) and potato dextrose broth (PDB), respectively, and subsequently prepared as mixed suspensions [25,26,27]. The medium volume per well was 100 μL. These fungi and bacteria were inoculated in Roswell Park Memorial Institute 1640 medium and Mueller–Hinton broth medium. Solutions of three purified compounds and positive controls (amphotericin B, streptomycin, carbendazim, and polyoxin B) were prepared in Dimethyl sulfoxide (DMSO) with a two-fold step dilution ranging from 0.98 to 500 µg/mL. A 1% DMSO was used as the negative control, representing the highest concentration used in the assay. A total of 10 µL (106 cfu/mL) of bacterial and fungal pathogen suspensions were added to each well. After mixing, the bacterial cultures were incubated at 24 °C for 24 h and the fungi at 30 °C for 48 h. The experiment was repeated three times. A microplate reader (Molecular Devices, San Jose, CA, USA) was used to record the optical density (OD) of each well at 600 nm. The lowest concentration to show 100% inhibition of the growth of the pathogen was defined as the minimum inhibitory concentration (MIC). The percentage inhibition of each dilution was calculated using the following formula:
Inhibition (%) = 100 × [(1 − Treatment OD)/negative control OD]

4.5. In Vivo Antifungal Activity Against Botrytis Cinerea by Greenhouse Pot Experiments

The in vivo activity of compound 1 against B. cinerea was tested under greenhouse conditions, with references to and improvements upon the tomato pot experiment [28,29]. Disinfected tomato seeds were treated with warm water to promote germination, and seeds with good germination were grown in plastic pots containing a 1:1 (v/v) mixture of vermiculite and peat. When the tomato plants reached the 6-leaf stage, they were inoculated with a mycelial suspension of B. cinerea (5 × 108 cfu/mL), applying 3 mL per plant. After 24 h, the plants were treated with Compound 1 at various concentrations (0.49, 0.98, 1.95, 3.91, and 7.81 µg/mL), applying 1 mL per plant. Polyoxin B was used as the positive control and distilled water as the negative control. After treatment, the tomato plants were placed in a sealed space at 20 °C and 85% relative humidity (RH), and a light/dark cycle of 16/8 h for 7 d. All treatments were replicated three times. Polyoxin B was established as the positive control, while distilled water served as the negative control. Antifungal activity data were recorded when the negative control (CK) showed significant symptoms of disease. The disease index (DI) and control effect (I) were calculated as follows:
DI = [∑ (number of diseased leaves at each grade × level of disease)/ (total number of leaves × 9)] × 100
I (%) = [(CK − T)/CK] × 100
where CK refers to the DI value of negative control and T refers to the DI value of compound 1 and Polyoxin B.

4.6. Data Analysis

Analysis of variance (ANOVA) was conducted using SPSS Statistics 22.0 (IBM/SPSS, Chicago, IL, USA). Treatment means were separated by Tukey’s Honestly Significant Difference test (p = 0.05).

5. Conclusions

Three compounds were isolated and identified from the plant endophytic actinomycete strain R6, including one new and one known LOB. Unlike previous related studies [19,20,33,34,35,36,37,38], the LOBs isolated in this study originate from plant endophytic actinomycetes and exhibit certain inhibitory activities against both plant pathogenic bacteria and fungi. In vitro tests demonstrated that LOB H8 effectively inhibited three bacterial strains. Furthermore, in vivo tests confirmed the bioactivity of LOB H8 against B. cinerea. These results highlight the potential of lobophorins as promising lead compounds for the development of new, sustainable agents to control plant diseases. On this basis, further studies will be conducted to elucidate its mechanism of action, providing a theoretical foundation and basis for the development of new antifungal agents to prevent and treat gray mold disease and to enrich the variety of antifungal agents available.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules30030586/s1, 16S rDNA sequence of Streptomyces sp. R6 isolated from Azadirachta indica; Figure S1: Phylogenetic tree of actinomycete R6; Figures S2–S9: HPLC, HRESIMS, 1H NMR, 13C NMR, HSQC, HMBC, 1H-1H COSY, and NOESY spectrum of lobophorin H8 (1); Figures S2 and S10–S16: HPLC, HRESIMS, 1H NMR, 13C NMR, HSQC, HMBC, 1H-1H COSY, and NOESY spectrum of lobophorin S (2); Figures S17–S20: HPLC, HRESIMS, 1H NMR, and 13C NMR spectrum of divergolide C (3); Figure S21: Inhibitory effects of lobophorin H8 (1) on Botrytis cinerea.

Author Contributions

Conceptualization, X.C. and Y.C.; methodology, X.C. and Y.C.; software, X.C., Y.D. and P.L.; validation, X.C., Y.D. and P.L.; formal analysis, X.C., Y.D., Y.M. and P.L.; investigation, X.C., Y.D. and P.L.; resources, Y.C.; data curation, X.C. and Y.M.; writing—original draft preparation, X.C. and Y.M.; writing—review and editing, X.C., Y.M. and Y.C.; visualization, X.C. and Y.M.; supervision, Y.C.; project administration, Y.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data are contained within the article and Supplementary Materials.

Acknowledgments

The authors are grateful to Zhiguo Yu and Mingshan Ji (College of Plant Protection, Shenyang Agricultural University, China) for their technical support during preparation of this manuscript.

Conflicts of Interest

Author Yunlong Ma was employed by the company Liaoning Lvyuan Nongfeng Agricultural Technology Service Co., Ltd. The remaining 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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Molecules 30 00586 g001
Figure 1. The chemical structures of compounds 1–3.
Figure 1. The chemical structures of compounds 1–3.
Molecules 30 00586 g001
Molecules 30 00586 g002
Figure 2. The 1H-1H COSY, HMBC and NOESY correlations of compound 1.
Figure 2. The 1H-1H COSY, HMBC and NOESY correlations of compound 1.
Molecules 30 00586 g002
Molecules 30 00586 g003
Figure 3. The 1H-1H COSY, HMBC, and NOESY correlations of compound 2.
Figure 3. The 1H-1H COSY, HMBC, and NOESY correlations of compound 2.
Molecules 30 00586 g003
Table 1. The 1H (600 MHz) and 13C (150 MHz) NMR data of compounds 1 and 2 in CDCl3 (δ, ppm).
Table 1. The 1H (600 MHz) and 13C (150 MHz) NMR data of compounds 1 and 2 in CDCl3 (δ, ppm).
No.1No.2
δH, (mult, J in Hz)δC, multδH, (mult, J in Hz)δC, mult
1-167.2 C1-167.1 C
2-102.2 C2-102.1 C
3-206.4 C3-206.1 C
4-50.9 C4-50.8 C
52.01 (t-like, 9.1)42.7 CH52.01 (t-like, 9.5)43.2 CH
61.60 (m)31.1 CH61.62 (m)31.2 CH
71.60 (overlap), 1.54 (m)41.6 CH271.64 (m), 1.54 (m)41.7 CH2
82.26 (m)34.6 CH82.27 (m)34.0 CH
93.66 (dd, 10.5, 5.3)76.1 CH93.57 (m)85.6 CH
102.07 (td, 10.0, 2.4)39.1 C102.18 (m)38.3 CH
116.03 (br d, 10.0)125.3 CH115.74 (br d, 10.0)124.7 CH
125.43 (m)126.6 CH125.45 (m)127.3 CH
133.53 (m)53.1 CH133.56 (m)52.9 CH
14-135.7 C14-135.2 C
155.22 (d, 10.0)122.9 CH155.23 (d, 10.0)123.2 CH
162.37 (overlap), 2.22 (m)31.8 CH2162.37 (overlap), 2.20 (m)31.8 CH2
174.19 (br s)72.9 CH174.19 (br s)72.9 CH
18-140.7 C18-140.7 C
195.26 (d, 10.5)118.2 CH195.27 (d, 10.5)118.2 CH
203.58 (br d, 10.5)40.2 CH203.58 (br d, 10.5)40.2 CH
215.14 (br s)120.6 CH215.14 (br s)120.5 CH
22-137.6 C22-137.7 C
232.37 (overlap)31.8 CH232.37 (overlap)31.8 CH
242.37 (m), 1.80 (m)35.2 CH2242.38 (m), 1.80 (m)35.2 CH2
25-83.0 C25-83.0 C
26-201.1 C26-201.0 C
271.61 (s)15.1 CH3271.60 (s)15.0 CH3
280.65 (d, 7.0)22.2 CH3280.65 (d, 7.0)22.1 CH3
291.04 (d, 6.8)12.9 CH3291.08 (d, 6.8)14.4 CH3
301.38 (s)13.7 CH3301.36 (s)13.7 CH3
311.38 (s)14.7 CH3311.39 (s)14.8 CH3
321.77 (s)21.8 CH3321.77 (s)21.8 CH3
331.28 (d, 6.8)20.1 CH3331.29 (d, 6.8)20.1 CH3
- 1′4.95 (d, 3.6)99.6 CH
- 2′2.37 (m), 1.94 (dt, 14.6, 3.5)34.9 CH2
- 3′3.99 (m)67.1 CH
- 4′3.16 (br d, 7.0)72.6 CH
- 5′3.83 (m)65.3 CH
- 6′1.34 (d, 6.8)17.7 CH3
Table 2. Antibacterial effect of the isolated compounds.
Table 2. Antibacterial effect of the isolated compounds.
CompoundsMinimum Inhibitory Concentration (MIC, μg/mL)
PsLPsTCmEc
Lobophorin H8 (1)15.637.812503.91
Lobophorin S (2)62.515.6350062.5
Divergolide C (3)25012550031.25
Streptomycin7.813.9150031.25
PsL = Pseudomonas syringae pv. lachrymans; PsT = P. syringae pv. tomato; Cm = Clavibater michiganensis; Ec = Erwinia carotovora.
Table 3. In vitro antifungal activity of the isolated compounds.
Table 3. In vitro antifungal activity of the isolated compounds.
CompoundsMinimum Inhibitory Concentration (MIC, μg/mL)
SoFfBcAsPaPgFoCc
Lobophorin H8 (1)62.531.251.9562.5>50050062.5500
Lobophorin S (2)25062.57.81250500>500250500
Divergolide C (3)25031.2515.6312562.550062.5125
Amphotericin B50012512550062.5>500125125
Carbendazim50062.531.2512515.6331.2515.63>500
Polyoxin B12531.253.9131.2525050012562.5
So = Stemphylium solani; Ff = Fulvia fulva; Bc = Botrytis cinerea; As = Alternaria solani; Pa = Pythium aphanidermatum; Pg = Pyricularia grisea; Fo = Fusarium oxysporum; Cc = Corynespora cassiicola.
Table 4. The control effect of lobophorin H8 (1) on tomato grey mold disease.
Table 4. The control effect of lobophorin H8 (1) on tomato grey mold disease.
TreatmentDilute Multiple (µg/mL)Disease IndexControl Effect (%) a
Lobophorin H8 (1)0.4938.0035.22 ± 1.90 e
0.9828.6751.16 ± 3.25 d
1.9512.6378.51 ± 3.80 b
3.9111.3380.73 ± 2.42 ab
7.818.4485.44 ± 3.29 a
Polyoxin B1.9517.2370.70 ± 3.81 c
CK-58.63-
a Note: The different letters behind the numbers mean significant differences (p = 0.05). CK: negative control.
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Chen, X.; Du, Y.; Ma, Y.; Liu, P.; Chen, Y. Antimicrobial Lobophorins from Endophytic Strain Streptomyces sp. R6 Obtained from Azadirachta indica. Molecules 2025, 30, 586. https://doi.org/10.3390/molecules30030586

AMA Style

Chen X, Du Y, Ma Y, Liu P, Chen Y. Antimicrobial Lobophorins from Endophytic Strain Streptomyces sp. R6 Obtained from Azadirachta indica. Molecules. 2025; 30(3):586. https://doi.org/10.3390/molecules30030586

Chicago/Turabian Style

Chen, Xinyuan, Ying Du, Yunlong Ma, Peibin Liu, and Yan Chen. 2025. "Antimicrobial Lobophorins from Endophytic Strain Streptomyces sp. R6 Obtained from Azadirachta indica" Molecules 30, no. 3: 586. https://doi.org/10.3390/molecules30030586

APA Style

Chen, X., Du, Y., Ma, Y., Liu, P., & Chen, Y. (2025). Antimicrobial Lobophorins from Endophytic Strain Streptomyces sp. R6 Obtained from Azadirachta indica. Molecules, 30(3), 586. https://doi.org/10.3390/molecules30030586

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