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. 2025 Oct 2;25:594. doi: 10.1186/s12866-025-04194-y

Antifungal effects of metabolites from Arthrinium sp. 2–65 and identification of main active ingredients

Huan Qu 1,2,✉, Ershuai Yang 1, Mei Zuo 3, Zongze Li 4, Ludan Dai 1, Youping Su 1, Xiu Zhang 2, Yongbin Cheng 1, Yihan Chen 1, Yang Chen 3
PMCID: PMC12492679  PMID: 41039238

Abstract

Background

Using microbes and their metabolites as material to develop new biological fungicides is still vital for pesticide development. Our preliminary study found that the endophytic fungi Arthrinium sp. 2–65 of Thymus mongolicus (Ronniger) Ronniger showed a certain inhibitory effect on pathogenic fungi.

Results

In this study, the antifungal activity of Arthrinium sp. 2–65 was evaluated. The ethyl acetate extract of Arthrinium sp. 2–65 exhibited significant inhibitory activity against pathogenic fungi, especially Botrytis cinerea. The main compounds of Arthrinium sp. 2–65 metabolites were isolated and purified, and the two compounds were identified by infrared spectroscopy (IR), high-performance liquid chromatography (HPLC), 1H nuclear magnetic resonance (NMR), 13C NMR, and high-resolution mass spectrometry (HRMS) as 2-hexyl-3-methylmaleic anhydride (A) and 2-carboxymethyl-3-n-hexylmaleic acid anhydride (B).

Conclusions

The main compounds (A and B) isolated and characterised from the fermentation broth of Arthrinium sp. 2–65 showed satisfactory inhibitory effects against pathogenic fungi, especially B. cinerea. These compounds could be used as potential molecules for the development of novel pesticides to control grey mould.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12866-025-04194-y.

Keywords: Arthrinium sp., Antifungal activity, Botrytis cinerea, Maleic anhydride derivatives

Background

According to the Food and Agriculture Organization of the United Nations (FAO), plant diseases cost the world economy more than $220 billion annually, 30% of which are caused by plant pathogenic fungi; these diseases are among the main causes of huge economic losses in agriculture [1]. Grey mould caused by B. cinerea is a common disease in agricultural production; the pathogen is characterised by easy spore production, low-temperature resistance (0 °C), rapid reproduction, ease of mutation, and strong adaptability [2], relying on mycelium and conidia to cause repeated crop infestations, which can cause a variety of fruit and vegetable diseases both pre- and post-harvest [3, 4]. Moreover, B. cinerea has a very wide host range; it has been reported that B. cinerea can cause disease in more than 1400 kinds of plants, such as tomatoes, grapes, strawberries, chili peppers, jujube, and moonshine [5, 6]. B. cinerea can infect all aboveground plant tissues, such as stems, leaves, flowers, fruits, and seeds of plants, causing white or grey mouldy spots to appear on the surface of infested fruits and vegetables, along with soft rot and browning of the infected areas [7]. It has been reported that the loss of fruits and vegetables caused by grey mould is as high as 25–55% and that the global annual economic losses caused by grey mould are as high as $100 billion; therefore, effective prevention and control strategies for grey mould are key to preserving agricultural production and income [8–10].

At present, the main means of controlling grey mould involve chemical fungicides, mainly including benzimidazoles (carbendazim, thiophanate-methyl, benomyl), dicarboximides (carbendazim, vinblastine, isobutyl urea), N-phenyl carbamates (carbendazim, etofenprox), phenyl pyrimidines (pyrimethanil, pyrimethanil, pyrimethanil cyclosporine), and protective fungicides (chlorothalonil, manganese–zinc diclofenac), which feature advantages such as high efficiency, speed, and practicality [11]. However, these chemical fungicides are gradually becoming restricted due to problems associated with food safety, environmental pollution, and easy induction of resistance to pathogens [12, 13]. Compared with the traditional chemical antifungal agents mentioned above, the use of microorganisms or their metabolites to develop microbial antifungal agents, which are in line with the world’s advocacy of strategies that are low in toxicity but highly effective in disease control, has become a current hotspot in the green control of agricultural diseases [14]. Among the 82 types of new pesticides registered in 2018–2023, 21 types were microbial pesticides [15]. Control of agricultural diseases through microorganisms and their secondary metabolites has gradually become a novel measure for disease control in modern agriculture.

Plant endophytic fungi are an important group of microorganisms and an important resource for the search for novel and active lead compounds to develop novel antimicrobial agents [16, 17]. In the early stage of our study, the wild plant Thymus mongolicus (Ronniger) Ronniger in the Liupan Mountain area of Ningxia was used as the material, from which more than 70 strains of endophytes were systematically isolated. Some strains were found to have good inhibitory activity against pathogenic fungi. Among them, the fermentation broth of Arthrinium sp. 2–65 showed significant inhibitory activity against B. cinerea. According to reports, Arthrinium sp. can produce abundant active compounds, and natural products with maleic anhydride (MAH) structures are among the important secondary metabolites identified. These products represent valuable antifungal agents, enzyme inhibitors, and herbicides [18, 28, 33]. Herbicides such as showdomycin, aqabamycins, and arcyriaflavins have been shown to possess good antifungal and antibacterial activity and low zootoxicity in mice [33]. Moreover, MAH compounds have been employed as important drug synthesis molecules [19, 29]. Therefore, as natural products, MAH compounds have strong potential application value in the antimicrobial field. In this article, we systematically investigated the antifungal activity of Arthrinium sp. 2–65 in vitro and in vivo and then clarified its main fungistatic substances through chemical separation (preparative thin-layer chromatography (PTLC) combined with medium-pressure liquid chromatography) and spectrum analysis (infrared spectroscopy (IR), nuclear magnetic resonance (NMR), and high-resolution mass spectrometry (HRMS)) approaches. These studies lay the foundation for the application of antifungal substances of Arthrinium sp. 2–65 in agricultural disease control.

Methods

Plant material

The Thymus mongolicus (Ronniger) Ronniger specimens used in this experiment were collected from Jingyuan County of Ningxia Province, which is not in the conservation area [20]. Moreover, the plant is not endangered, and its collection does not involve ethical issues. Voucher specimens were collected and identified by Dr. Lei Zhang and were deposited in the Herbarium of North Minzu University (NMU; Yinchuan, China) with an accession number of zlnmu2022084.

Chemicals and strain

Arthrinium sp. 2–65 was isolated from Thymus mongolicus (Ronniger) Ronniger, identified by morphology combined with molecular biological methods (internal transcribed spacer, ITS, sequencing), and registered in the National Center for Biotechnology Information (NCBI) GenBank (GenBank: MN944538.1). The test pathogenic fungi were Botrytis cinerea (FH), Fusarium graminearum (XC), Valsa mali (PF), Fusarium sulphureum (MG), Fusarium oxysporum (XK), Fusarium solani (GG), and Alternaria solani (MZ), which were preserved in the Laboratory of Natural Products Research of North Minzu University. Putrescine wettable powder (50%) was purchased from Sumitomo Chemical Corporation (Tokyo, Japan). All other reagents were of analytical grade.

Morphological observations and growth curves of Arthrinium sp. 2–65

Arthrinium sp. 2–65 was cultured on potato glucose agar (PDA) medium at 28 °C for 3–4 d, and its mycelial and spore growth characteristics were observed. The growth curve of Arthrinium sp. 2–65 was constructed using the weighing method [21]. Arthrinium sp. 2–65 was inoculated in potato glucose (PDB) medium on a rotary shaker (200 r/min, 28 °C) for 12, 24, 36, 84, 108, 132, 156, 180, and 204 h. Then, the cultures were centrifuged at 12,000 ×g for 3 min and dried for weighing. The growth curve of Arthrinium sp. 2–65 was graphed based on the change in the weight of fungal hyphae with culture time.

Screening of Arthrinium sp. 2–65 against seven pathogenic fungi in vitro

In this study, Arthrinium sp. 2–65 was screened for its antagonistic activity by the plate confrontation method [22]. The cultures were incubated at 28 °C for 5 days. The mycelial growth of the pathogens was measured on the fifth day. This experiment was repeated three times. The inhibitory effects of Arthrinium sp. 2–65 in vitro were calculated by the following (Eq. 1):

graphic file with name d33e564.gif 1

where C represents the radial growth diameter (mm) of the pathogen on untreated PDA medium, T represents the radial growth diameter (mm) of the pathogen on treated PDA medium, and 4 represents the diameter (mm) of the inoculated pathogen cake.

Study of the antifungal activity of the fermentation broth and the crude extract of Arthrinium sp. 2–65

The mycelial growth rate method was used to evaluate the antifungal activity of Arthrinium sp. 2–65, and fermentation broth samples from three fermentation periods (96, 108, and 180 h) were selected for this experiment based on the prior growth curve established for Arthrinium sp. 2–65. One portion of the fermentation broth was filtered through a 0.22-µm filter membrane and sufficiently mixed with PDA medium (1:9 v/v) to obtain toxic culture medium. The other portion of the fermentation broth was concentrated to 1/4 of its original volume by freeze-drying. Then, the fermentation extract was concentrated with ethyl acetate three times. The extracts were combined, dried with anhydrous sodium sulfate, and then rotary evaporated to obtain the crude extract. The crude extract (42 mg) was dissolved in acetone (5 mL), filtered through a 0.22-µm filter membrane, then mixed with 420 mL of PDA medium to obtain toxic culture medium with a concentration of 0.1 mg/mL. The two different types of toxic culture medium were centrally inoculated with pathogenic fungal cake. In the fermentation broth inhibition experiment, medium without fermentation broth was used as a blank control. In the crude extract inhibition experiment, medium with acetone (5 mL) was used as a blank control. Each treatment was repeated three times. The above-mentioned plates were incubated at 28 °C, and the growth of the colonies was observed and measured. The inhibition rate was calculated using Eq. 1.

Inhibitory effects of the fermentation broth and the crude extract on tomato grey mould

Healthy and green tomatoes with fruit stalks were chosen, and their surfaces were disinfected by wiping with 75% ethanol and were air-dried before use. After fermentation, mycelia from Arthrinium sp. 2–65 were removed by filtration to obtain a fermentation broth. A portion of the fermentation broth was filtered through a 0.22-µm filter membrane, and another portion was treated according to the previous method to obtain the crude extract. The crude extract was fully dissolved in acetone and Tween 80 (1:4 v/v). After that, the mixture was diluted with sterile water to a concentration of 0.15 mg/mL and filtered through a 0.22-µm filter membrane for the following experiments. The tomato surface was punctured with a vaccination needle at each fruit equator (4 mm ×1 mm), followed by spraying with the fermentation broth and the 0.15 mg/mL crude extract. After 24 h, 2 µL of B. cinerea spore suspension (1 × 106/mL–1) was inoculated into the tomato wound and then cultured in a 28 °C incubator. Treatment with sterile water was used as the blank control, and treatment with 1000-fold-diluted 50% procymidone wettable powder was used as the drug control. Each treatment was repeated eight times. The control efficacy was investigated after 5 days, and the inhibition rate was calculated using Eq. 2:

graphic file with name d33e613.gif 2

where C represents the disease spot diameter (mm) on the tomato samples in the blank control, and T represents the disease spot diameter (mm) on the tomato samples treated with fermentation broth, crude extract, or 50% procymidone wettable powder, and 4 represents the diameter (mm) of the inoculated pathogen cake.

Isolation and structural identification of compounds from the crude extract of Arthrinium sp. 2–65

Arthrinium sp. 2–65 was inoculated in PDB medium and then shaken for 180 h. The fermentation broth was filtered to separate the mycelia to obtain the fungal liquid. The fungal liquid was then freeze-dried for 24 h to concentrate it to one-third of its original volume. Then, the mycelia and concentrated fungal liquid were extracted with ethyl acetate, and the extracts were combined, dried with anhydrous sodium sulfate, and evaporated to remove the solvent to obtain the crude product. The composition of Arthrinium sp. 2–65 was analysed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The chromatographic conditions of HPLC were as follows: ZORBAX SB-C18 chromatographic column (250 mm x 4.6 mm, 5 μm), mobile phase (CH3CN: H2O = 4:1), detection wavelength (280 nm), column temperature (35 °C), flow rate (1 mL/min), and injection volume (10 µL). Two compounds (A and B) were obtained through separation of the crude product with dichloromethane and dichloromethane–methanol (24:1, v/v) as the eluting solvent by PTLC combined with medium-pressure liquid chromatography, and structural analyses of A and B were performed using IR, 1H NMR, 13C NMR, and HRMS.

Data analysis

Analysis of variance (ANOVA) and Duncan’s multiple comparison tests were performed with IBM SPSS (version 26.0), and the standard deviation of each mean was calculated (p < 0.05). All data in the graphs are presented as the means ± standard deviations (SDs) of at least three duplicate samples (n = 3). The charts were generated with WPS Office (Beijing Kingsoft Office Software, Inc., China).

Results

Colony characterisation and morphological observation of mycelia and spores of Arthrinium sp. 2–65

As shown in Fig. 1a, in a colony assay on PDA medium, Arthrinium sp. 2–65 grew quickly and produced rich, fluffy hyphae, with white hyphae that grew outward radially. As shown in Fig. 1b, the conidia were yellowish-brown, ovoid, or subglobose [23]. The growth curve of Arthrinium sp. 2–65 is shown in Fig. 1c. The biomass of the mycelia increased gradually in the first 108 h and then decreased gradually over time, stabilising after 180 h. Therefore, fermentation broth and fermentation crude extract samples from the logarithmic period (96 h), stabilisation period (108 h), and recession period (180 h) of mycelium growth were chosen for rescreening to determine their inhibitory activities against fungal pathogens.

Fig. 1.

Fig. 1

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Growth traits of Arthrinium sp. 2-65. Data are expressed as the means ± SDs (n = 3). (a) Colony characteristics of Arthrinium sp. 2-65. (b) Mycelial and spore morphologies of Arthrinium sp. 2-65. (c) Growth curve of Arthrinium sp. 2-65

Studies on the antifungal activity of Arthrinium sp. 2–65

Antagonistic activity

The antagonistic activity levels of Arthrinium sp. 2–65 against seven plant pathogenic fungi were determined by the plate confrontation method [22], and the results of the antagonistic activity assays at 96 h are shown in Fig. 2a. Arthrinium sp. 2–65 showed strong and broad-spectrum antagonistic activity against all plant pathogenic fungi tested except A. solani and F. oxysporum, with inhibition rates that were generally greater than 60%. As shown in Fig. 2b, the mycelial growth of the plant pathogenic fungi was significantly inhibited by Arthrinium sp. 2–65 compared with the control group.

Fig. 2.

Fig. 2

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Antagonistic effect of Arthrinium sp. 2-65 against test pathogens. Data are expressed as the means ± SDs (n = 3). Lowercase letters (a, b, c) denote significant differences between groups (Tukey’s HSD test, p < 0.05). Groups sharing the same letter are not statistically different. (a) Inhibition rates of Arthrinium sp. 2-65 against the seven pathogenic fungi. (b) Antagonistic effects of Arthrinium sp. 2-65 against F. graminearum (XC), B. cinerea (FH), V. mali (PF), and F. sulphureum (MG)

Antifungal activity levels of the fermentation broth and crude extract

The results of the antifungal activity assay of the fermentation broth of Arthrinium sp. 2–65 at different stages are shown in Fig. 3a. The fermentation broth of Arthrinium sp. 2–65 showed little inhibitory effect on six test pathogens but presented relatively good inhibitory activity against B. cinerea, with inhibition rates of 34.62%, 34.62%, and 44.87% at 96, 108, and 180 h, respectively.

Fig. 3.

Fig. 3

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Inhibitory effects of Arthrinium sp. 2-65 on pathogenic fungi. Data are expressed as the means ± SDs (n= 3). Lowercase letters (a–m) denote significant differences between groups (Tukey’s HSD test, p < 0.05). Groups sharing the same letter are not statistically different. (a) Inhibitory effects of the fermentation broth (FJ) ofArthrinium sp. 2-65 on pathogenic fungi. (b) Inhibitory effects of the crude extract (CT) of Arthrinium sp. 2-65 on pathogenic fungi. (c) Inhibitory effects of the crude extract of Arthrinium sp. 2-65 on A. solani (MZ), B. cinerea (FH), and F. oxysporum (XK) on PDA culture medium. (d) Inhibitory effects of the fermentation broth (FJ) and crude extract (CT) of Arthrinium sp. 2-65 on B. cinereain vivo. (From left to right: blank control group (CK), drug control group (50% putrescine wettable powder, FML), 0.15 mg/mL crude extract of Arthrinium sp. 2-65 (CT), and fermentation broth of Arthrinium sp. 2-65 (FJ).)

The inhibition rates of the crude extract of Arthrinium sp. 2–65 on plant pathogens are shown in Fig. 3b. The antifungal activity of the crude extract of Arthrinium sp. 2–65 showed a gradually increasing trend at 96, 108, and 180 h and showed good inhibitory activity levels against B. cinerea, V. mali, and (A) solani, with inhibitory rates of 87.65%, 40.54%, and 52.05%, respectively, at 180 h. Among all the pathogens tested, the inhibitory effect on (B) cinerea was marked and showed an increasing trend with time. For example, the inhibition rate was only 28.4% at 96 h, but the rates were 76.13% and 87.65% at 108 and 180 h, respectively. The mycelial inhibition of the test plant pathogens by the crude extract of Arthrinium sp. 2–65 at 180 h is shown in Fig. 3c. The most prominent inhibitory effect on hyphal growth was demonstrated against B. cinerea.

Based on the above-mentioned results, both the fermentation broth and crude extract showed good inhibitory activities on B. cinerea, which prompted us to further explore the inhibitory activity of Arthrinium sp. 2–65 against B. cinerea in vivo.

Inhibitory activity of arthrinium sp. 2–65 against B. cinerea in vivo

Based on the good inhibitory activity in vitro, we investigated the inhibitory effect of the fermentation broth (FJ) and the 0.15 mg/mL crude extract (CT) of Arthrinium sp. 2–65 on tomato fruit tissue in vivo, using 50% putrescine wettable powder (FML, 1000×) as the positive control and distilled water as the blank control (CK) (Fig. 3d). The results showed that B. cinerea mycelia grew vigorously outward from the inoculation site in the blank control, and a large number of grey–black spores were produced on the surface of the mycelium. Compared with the blank control, both the fermentation broth and crude extract demonstrated good inhibitory effects on B. cinerea. The inhibitory effect of the crude extract was most marked, with only a very small amount of mycelium around the inoculation site, and the inhibitory effect was comparable to that of the commercial 50% putrescine wettable powder. Furthermore, compared with the 50% putrescine wettable powder, the crude extract-treated tomatoes maintained their original colour (the colour of tomatoes treated with 50% putrescine wettable powder changed from green to red, whereas the crude extract-treated tomatoes maintained a green colour), indicating a certain fresh-keeping effect.

Analysis of the antifungal active compounds of Arthrinium sp. 2–65

Arthrinium sp. 2–65 was inoculated in PDB medium and incubated in a shaker at 28 °C and 150 r/min for 180 h. The mixture was filtered to obtain the fungal liquid. Then, the fungal liquid was extracted with ethyl acetate and evaporated to obtain the crude extract. The main components of the crude extract were analysed by TLC and HPLC, separated by PTLC, and identified by IR, HRMS, and NMR. The results for the fermentation broth of Arthrinium sp. 2–65 after incubation for 180 h are shown in Fig. 4a. A large number of mycelial microspheres were formed in the PDB medium with the increase in incubation time; the mycelial microspheres were milky white, and the fermentation broth was light yellow with a light wine flavour. The TLC analysis revealed three main spots at 254 nm (Fig. 4b).

Fig. 4.

Fig. 4

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The fermentation broth of Arthrinium sp. 2-65 in PDB medium and analysis of its components. (a) Mycelial microspheres of Arthrinium sp. 2-65 in PDB medium at 180 h. (b) TLC spectrogram of theArthrinium sp. 2-65 crude extract and the structural formulae of compounds A and B

The HPLC spectrum of Arthrinium sp. 2–65 is shown in Fig. 5. Two main peaks (1.853 and 4.990 min) indicated that there were at least two main compounds in the crude extract of Arthrinium sp. 2–65 (Fig. 5a). Combined with the TLC results (Fig. 4b), compounds A and B were obtained using PTLC and medium-pressure liquid chromatography, affording yields of 11.00% and 21.18%, respectively. Compounds A and B were then analysed by HPLC. The retention times of compounds A and B were 4.993 min (Fig. 5b) and 1.850 min (Fig. 5c), respectively.

Fig. 5.

Fig. 5

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HPLC analysis of the main components of Arthrinium sp. 2-65. (a) HPLC analysis of the main components of the crude extract. (b) HPLC spectrum of compound A. (c) HPLC spectrum of compound B

Data for compound A: light yellow liquid; IR (cm–1): 2955, 2927, 2856, 1760, 1672, 1457, 1385, 1274, 1117, 917, 896, 733. 1H NMR (500 MHz, CDCl3) δ: 0.80 (t, 3 H, J = 6.5 Hz, –CH3), 1.21–1.29 (m, 6 H, –(CH2)3–), 1.47–1.52 (m, 2 H), 2.00 (s, 3 H, =C–CH3), 2.36 (t, J = 7.5 Hz, 2 H,=CCH2–); 13C NMR (125 MHz, CDCl3) δ: 9.46, 13.95, 22.43, 24.43, 27.53, 29.07, 31.33, 140.41, 144.75, 165.85, 166.24. HRMS (ESI) m/z for C11H16O3 [M + H]+calcd 197.1172, found 197.1189. According to the above-mentioned data and literature [24], compound A was identified as 2-hexyl-3-methylmaleic anhydride, and its molecular formula is shown in Fig. 4. The IR, MS, 1H-NMR, and 13C-NMR spectra are shown in Supplementary Figures S1–S7.

Data for compound B: yellow liquid; 1H NMR (500 MHz, CDCl3) δ: 0.87(t, J = 6.5 Hz, 3 H, –CH3), 1.25–1.37 (m, 6 H, –(CH2)3–), 1.56–1.62 (m, 2 H), 2.48 (t, J = 7.5 Hz, 2 H, =CCH2–), 3.57 (s, 2 H, –CH2–COOH); 13C NMR (125 MHz, CDCl3) δ: 13.99, 22.43, 24.93, 27.53, 29.14, 29.16, 31.28, 135.59, 148.09, 165.17, 172.75. HRMS (ESI) m/z for C12H16O5 [M-CO2H]+ calcd 195.1016, found 195.1023. According to the above-mentioned data and literature [25], compound B was identified as 2-carboxymethyl-3-n-hexylmaleic acid anhydride, and its molecular formula is shown in Fig. 4. The IR, MS, 1H-NMR, and 13C-NMR spectra are shown in Supplementary Figures S8–S14.

Discussion

Arthrinium sp. is a class of fungi that are widely distributed in natural environments [26]. In this study, using Thymus mongolicus (Ronniger) Ronniger as the starting material, multiple strains of endophytic fungi were isolated in our early research stage. Among them, the endophytic fungus numbered 2–65 exhibited broad-spectrum antagonistic activity against the seven tested pathogens, with inhibition rates of over 60% against six of the tested pathogens. Therefore, 2–65 was identified by ITS analysis combined with morphological assessment, and it was determined that it belongs to a member of Arthrinium sp. and was named Arthrinium sp. 2–65.

Arthrinium species are known to produce various classes of secondary metabolites, including alkaloids, terpenes, coumarins, xanthones, and pyrones. These compounds exhibit a wide range of promising biological activities, such as antimicrobial, anti-inflammatory, and anticancer properties [27]. In this experiment, Arthrinium sp. 2–65 demonstrated broad-spectrum antagonistic activity against seven pathogenic fungi, and the inhibition rate against most pathogenic fungi reached more than 60%. Therefore, whether the good antagonistic effect of Arthrinium sp. 2–65 was related to its special metabolites, as reported in the literature, was determined. The antifungal activity experiment revealed that compared with the fermentation broth, the crude extract of Arthrinium sp. 2–65 showed better inhibitory activity against B. cinerea, V. mali, and (A) solani at 180 h, with inhibition rates of 87.65%, 40.54%, and 52.05%, respectively. Using tomato as the test material, compared with the fermentation broth, the crude product of Arthrinium sp. 2–65 showed a more significant inhibitory effect on (B) cinerea in vivo, with an inhibitory effect that was comparable to that of the positive control agent. These results suggested that some antifungal substances were present in the fermentation broth of Arthrinium sp. 2–65, and these antifungal substances might exist in the crude extract of Arthrinium sp. 2–65. Therefore, the crude extract was analysed by TLC, and three different spots were observed at 254 nm. Determining the precise structures of compounds is an important prerequisite for studying the functions of natural products; therefore, in this experiment, compounds A and B were separated by PTLC combined with medium-pressure liquid chromatography, and their structures were characterised by IR, 1H NMR, 13C NMR, and HRMS .

Compound A was obtained as a yellow liquid. The molecular formula of compound A was assigned as C11H16O3 based on its HRMS spectroscopic data [M + H]+, m/z 197.1189, (Calcd. for [C11H17O3]+, 197.1172). Its IR (KBr) spectrum exhibited absorptions at 2957/2856 cm–1 (saturated alkyl) and 1760 cm–1(carbonyl). According to 1D NMR and HMQC data, compound A displayed two methyl groups (δH/δC 0.80/13.95, 2.00/9.46) and five methylene groups (δH/δC 1.24/22.43, 29.07 and 31.33; 1.56/27.53; 2.40/24.43) (Table 1 and S3, S4, and S6). In the 13C spectrum (Table 1), two ketone carbonyl groups [δC 165.85(C5), 166.24(C2)], two olefinic groups [δC 144.75(C4), 140.41(C3)], and seven aliphatic carbons [δC 31.33, 29.07, 27.53, 24.43, 22.43, 13.95, 9.46] were observed. The carbons [δC 13.95, 9.46] indicated the presence of two methyl groups. Combined with DEPT135 data (S5), these results indicated the presence of five methylene groups. The key HMBC correlations (Fig. 6, S7) from H-12 (δH 2.00) to C-3 (δC 140.41)/C-4(δC 144.75)/C-5(δC 165.85)/C-2(δC 166.24) confirmed the position of the enomethyl in compound A; H-6 (δH 2.40) to C-3 (δC 140.41)/C-4(δC 144.75)/C-2(δC 166.24) confirmed the position of the methylene in compound A. Therefore, compound A was identified as 2-hexyl-3-methylmaleic anhydride.

Table 1.

1H (500 MHz) and 13C (125 MHz) NMR data for compounds A and B

Atomic Position Compound A Compound B
δH (J in Hz) δC δH (J in Hz) δC
2 - 165.85 - 165.17
3 - 144.75 - 148.09
4 - 140.41 - 135.59
5 - 166.24 - 165.17
6 2.36, t (7.5) 24.43 2.48, t (7.5) 24.93
7 1.47–1.52, m 27.53 1.56–1.62, m 27.53
8 1.21–1.29, m 31.33 1.25–1.37, m 29.14
9 1.21–1.29, m 29.07 1.25–1.37, m 31.28
10 1.21–1.29, m 22.43 1.25–1.37, m 22.43
11 0.80, t (6.5) 13.95 0.87, t (6.5) 13.99
12 2.00, s 9.46 3.57, s 29.16
13 - - - 172.75
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“-” indicates there is no H or C atom at this atomic position

Fig. 6.

Fig. 6

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Selected 2D NMR correlations (HMBC) for A and B

Compound B was obtained as a dark yellow liquid. The molecular formula of compound B was assigned as C12H16O5 based on its HRMS spectroscopic data ([M-CO2H]+, m/z 195.1023, Calcd. for [C12H16O5]–, 195.1016). Its IR (KBr) spectrum exhibited absorptions at 2930/2855 cm–1 (saturated alkyl) and 1765/1715cm–1 (carbonyl). According to 1D NMR and HMQC data, compound B displayed only one methyl group (δH/δC 0.87/13.99) and six methylene groups (δH/δC 1.25/22.43, 29.14 and 31.28; 1.56/27.53; 2.48/24.93; 3.57/29.16), which were extremely similar to those of compound A (Table 1 and S10, S11, and S13). The methyl group at the C12 position became an acetoxy compared with that in compound A. In the 13 C spectrum (Table 1), one carboxy group (δC 172.75 (C-13)), two ketone carbonyl groups [δC 165.17 (C-2, C-5)], two olefinic groups (δC 135.59, 148.10), and seven aliphatic carbons (δC 31.28, 29.16, 29.14, 27.53, 24.93, 22.43, 13.99) were observed. The carbon [δC 13.99(C11)] indicated the presence of one methyl group. Combined with DEPT135 data (S12), these results indicated the presence of six methylene groups. The key HMBC correlations (Fig. 6, S14) from H-11(δH 0.87) to C-10 (δC 22.43)/C-9(δC 31.28) confirmed the position of the methyl in compound B; H-6 (δH 2.48) to C-7 (δC 27.53)/C-4(δC 135.59)/C-3(δC 148.09)/C-2, C-5(δC 165.17) confirmed the position of one methylene in compound B; H-12 (δH 3.57) to C-4(δC 135.59)/C-3(δC 148.09)/C-2, C-5(δC 165.17) and C-13(δC 172.75) confirmed that a carboxymethyl group attached to the C-4 position of compound B. Therefore, compound B was identified as 2-carboxymethyl-3-n-hexylmaleic acid anhydride [28].

Structural identification revealed that both compounds A and B were MAH derivatives. Natural products with MAH structures are valuable antifungal agents, enzyme inhibitors, and herbicides, and some MAH derivatives have been reported to have little or no animal toxicity [29]. Due to the excellent biological activity of MAH and its derivatives as well as the modifiability of their structures, they have become commonly used important organic raw materials with great biological potential [30]. The first synthesis of MAH derivatives can be traced back to two centuries ago [29]. At present, many naturally occurring MAH derivatives have been identified from plants and microorganisms [29, 31, 32]. More interestingly, many MAH derivatives, such as zopellin, 2,3-dimethyl maleic anhydride, and n−3,5-dichloroaniline-3,4-dimethylmaleimide, have exhibited excellent inhibitory effects against B. cinerea, with minimum inhibitory concentrations (MICs) of 0.78, 3, and 0.1 µg/mL, respectively [33]. These results were consistent with the findings in this study that the crude extract of Arthrinium sp. 2–65 showed specific and significant inhibitory activity against B. cinerea, and the main compounds of the crude extract were the MAH derivatives A and B, providing a potential new mode for the control of B. cinerea in practice

Conclusions

In summary, based on the good inhibitory rate of the crude extract of Arthrinium sp. 2–65 against B. cinerea in vitro and in vivo, two compounds were isolated and identified by TLC, HPLC, PTLC, NMR, and HRMS, and were determined to be 2-hexyl-3-methylmaleic anhydride (A) and 2-carboxymethyl-3-n-hexylmaleic acid anhydride (B). However, the low production yields of compounds A and B restricted systematic investigations of their antifungal activities in this article. Chemical synthesis is a potential method to expand material sources effectively and produce large amounts of such compounds. Moreover, among all test pathogens, the specific and significant inhibitory effect of Arthrinium sp. 2–65 secondary metabolites against B. cinerea suggested that there might be specific targets in B. cinerea for the compounds containing MAH groups. This question will be explored in future studies. The results of this study provide good guidance for further studies on the inhibitory effects of compounds A and B against B. cinerea for the discovery of other potential antifungal agents.

Supplementary Information

Supplementary Material 1. (658.1KB, docx)

Acknowledgements

This work was supported by the Natural Science Foundation of Ningxia Province (2023AAC03295), Science and Technology Leading Talent Project of Ningxia (2022GKLRLX06), National Natural Science Foundation of China (31960551), Open Subject of North Minzu University Ningxia Key Laboratory for the Development and Application of Microbial Resources in Extreme Environments (NXTSL2505). We would like to acknowledge Dr. Tian Liwen (School of Pharmaceutical Sciences, Southern Medical University) for structural identification and Dr. Zhang Lei (School of Biological Science and Engineering, North Minzu University) for Thymus mongolicus (Ronniger) Ronniger identification.

Authors’ contributions

H.Q. investigation, supervision, project administration, funding acquisition, writing – original draft. E.Y. data curation, writing & editing. M.Z. validation. Z.Z. L. conceptualization, resources. L.D.D. data curation. Y.P.S. graphics drawing. Y.B. C. validation. Y.H.C. validation. X.Z. conceptualization, resources. Y.C. review & editing.

Funding

This work was supported by the Natural Science Foundation of Ningxia Province (2023AAC03295), Science and Technology Leading Talent Project of Ningxia (2022GKLRLX06), National Natural Science Foundation of China (31960551), Open Subject of North Minzu University Ningxia Key Laboratory for the Development and Application of Microbial Resources in Extreme Environments (NXTSL2505).

Data availability

The datasets used and analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1. (658.1KB, docx)

Data Availability Statement

The datasets used and analysed during the current study are available from the corresponding author on reasonable request.

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