ABSTRACT
In this study, light exposure and cyclic adenosine monophosphate (cAMP) supplementation were applied as cultivation‐based elicitation strategies to two algicolous Irpex species associated with the marine green alga Ulva pertusa, with the aim of facilitating the isolation of new secondary metabolites with antifungal activity. The confrontation assay demonstrated that both I. lacteus YICB‐0055 and I. laceratus YICB‐0007 exhibit inhibitory effects against Fusarium oxysporum YICB‐0025 under light exposure with cAMP supplementation. HPLC analysis revealed a rich metabolite fingerprint under continuous light exposure in the presence of 5 mM cAMP. Chemical investigation of the optimized fermentation afforded two new tremulane‐type sesquiterpenes, ceriponols N–O (1 and 2), together with eight known compounds (3–10), all of which were structurally characterized by extensive spectroscopic analyses. These compounds exhibited antifungal activity against plant‐pathogenic fungi such as F. oxysporum. The results suggest that cAMP supplementation combined with light exposure may enhance secondary metabolite production in Irpex fungi, indicating their potential as sources of antimicrobial compounds.
Keywords: algicolous fungus, biological activity, natural products, terpenoid, Ulva pertusa
Light exposure combined with 5 mM cAMP enhances secondary metabolism in Ulva pertusa‐associated Irpex fungi. Optimized cultivation of I. lacteus affords two new tremulane‐type sesquiterpenes, ceriponols N and O, with antifungal activity against plant‐pathogenic fungi. These findings highlight cultivation‐based elicitation as an effective approach to uncover bioactive metabolites from algicolous fungi.
1. Introduction
Algicolous fungi are an important source of bioactive compounds, many of which possess significant pharmaceutical and agricultural value [1]. Sesquiterpenes are an important class of fungal secondary metabolites that have attracted considerable attention owing to their diverse biological activities, including antifungal, antibacterial, and anticancer effects [2, 3]. These bioactive compounds are produced under specific environmental conditions, including light exposure, which can significantly affect fungal metabolism [4]. In recent years, signaling molecules such as cyclic adenosine monophosphate (cAMP) have been investigated as tools for regulating fungal metabolite biosynthesis [5, 6, 7]. As a second messenger, cAMP participates in diverse cellular processes, including growth, differentiation, and secondary metabolism [7]. In fungi, cAMP signaling activates protein kinase A (PKA), which modulates downstream transcriptional programs and has been associated with the production of diverse secondary metabolites, including bioactive compounds with antifungal activity [8]. Moreover, light is a key environmental signal that regulates fungal secondary metabolism through photoreceptors and light‐responsive regulatory networks. In Aspergillus nidulans, the VelB–VeA–LaeA velvet complex connects light signaling with secondary metabolism [4], while light‐dependent gene activation is mediated by photoreceptors such as phytochromes and White Collar proteins [9, 10]. In parallel, algicolous fungi are recognized as prolific sources of structurally diverse bioactive secondary metabolites [11]. Cultivation‐based elicitation strategies, such as one strain many compounds (OSMAC), co‐culture, and chemical induction, have been widely used to activate cryptic biosynthetic pathways [12]. Although direct evidence for a synergistic interaction between cAMP signaling and light exposure remains limited, their combined manipulation represents a plausible strategy to explore and potentially enhance secondary metabolite production in algicolous fungi.
In this study, we aimed to explore the potential of combining cAMP signaling and light exposure to enhance the production of secondary metabolites in fungi associated with Ulva pertusa, a marine green alga. The fungi associated with U. pertusa have been reported to produce a range of bioactive compounds, including novel sesquiterpenes [13, 14]. Given the growing interest in the use of light and cAMP signaling to manipulate fungal metabolism, we hypothesized that optimizing these factors could promote the production of bioactive compounds. To test this hypothesis, we selected two algicolous strains of Irpex, a fungal genus known to produce diverse secondary metabolites, including compounds with antifungal activity [15]. These strains were fermented under optimized culture conditions involving cAMP supplementation and light exposure, and the resulting metabolites were analyzed for their chemical structures and antifungal activities.
2. Results and Discussion
2.1. Confrontation Assay
In this study, a confrontation assay was conducted to evaluate the inhibitory effects of I. lacteus YICB‐0055 (strain A) and I. laceratus YICB‐0007 (strain B) against F. oxysporum YICB‐0025 (strain C) under different conditions. The assay was performed on PDA plates with 5 mM cAMP supplementation under continuous white light at 2800–3000 lux. The control group was incubated in the dark without cAMP. Over a 7‐day period, the growth and morphology of each strain were monitored daily (Figure 1), showing clear differences in colony expansion under different conditions. In addition, colony radius measurements from day 1 to day 7 showed that strain C grew continuously in monoculture under dark conditions without cAMP, reaching approximately 4.0 cm by day 7, whereas its radial growth was markedly restricted in coculture under both tested conditions (Figure 2).
FIGURE 1.
Colony morphology of I. lacteus (strain A) and I. laceratus (strain B) during confrontation culture with F. oxysporum (strain C) on PDA plates under different conditions over a 7‐day period.
FIGURE 2.
Changes in the colony radius of F. oxysporum (strain C) under monoculture and co‐culture conditions over 7 days. (a) Co‐culture with I. lacteus (strain A). (b) Co‐culture with I. laceratus (strain B).
The inhibitory effects were further evaluated based on the inhibition rates determined on day 7 (Table S1). Under light conditions with cAMP supplementation, I. lacteus showed a day‐7 inhibition rate of 71.11% against F. oxysporum, while I. laceratus exhibited a day‐7 inhibition rate of 66.67%. Under dark conditions without cAMP, the day‐7 inhibition rates were 62.50% for I. lacteus and 42.50% for I. laceratus. These results indicate that both I. lacteus and I. laceratus inhibited the growth of F. oxysporum, with stronger inhibitory effects observed under continuous light with cAMP supplementation.
2.2. Structural Elucidation of Metabolites
A total of 10 compounds (1–10, Figure 3), including two new compounds (1 and 2), were isolated from the fermentation extracts of I. lacteus and I. laceratus. The remaining eight compounds were identified as known compounds, and they are as follows: 11,12‐dihydroxy‐1‐tremulen‐5‐one (3) [16], ceriponol C (4) [17], 15α‐hydroxytrametenolic acid (5) [18], 3β‐hydroxylanosta‐8,24‐dien‐21‐oic acid (6) [19], 3β‐acetoxylanosta‐7,9(11),24‐trien‐21‐oic acid (7) [20], tyrosamic E (8) [21], 6‐amino‐3‐(4‐hydroxy‐benzyl)‐1,4‐diazonane‐2,5‐dione (9) [22], and cyclo(Pro‐Tyr) (10) [23]. The structures of the isolated compounds were elucidated using a combination of modern spectroscopic techniques, including infrared spectroscopy (IR), ultraviolet spectroscopy (UV), nuclear magnetic resonance (NMR), and high‐resolution electrospray ionization mass spectrometry (HRESIMS). In particular, the structures of the two new compounds (1 and 2) were elucidated by extensive 1D and 2D NMR spectroscopic analyses, with the corresponding spectra provided in the Supporting Information (Figures S2–S15), whereas the known compounds were identified by comparison of their NMR data with those reported in the literature, confirming their structural assignments.
FIGURE 3.
Structures of compounds 1–10.
Compound 1 was obtained as a white amorphous powder, and its molecular formula was determined as C16H26O4 by HRESIMS analysis (m/z 283.1917, [M + H]+). The UV spectrum exhibited absorption at λ max at 200 nm. The IR spectrum of compound 1 showed absorption bands at 3425 and 1638 cm−1, arising from hydroxy and carbon–carbon double bond stretching vibrations, respectively. The 1H NMR spectrum (Table 1) showed three methyl groups [δ H 0.90 (3H, d, J = 8.0 Hz, H‐13), 1.01 (3H, s, H‐14), 1.08 (3H, s, H‐15)], one oxygenated methyl group at δ H 3.34 (3H, s, H‐16), three methylenes [δ H 1.43 (2H, m, H‐8), 1.90 (1H, m, H‐10b), 2.02 (1H, m, H‐10a), 4.23 (1H, m, H‐11b), 4.31 (1H, ddt, J = 12.5, 2.5, 2.5, H‐11a)], and six methine protons [δ H 1.92 (1H, m, H‐6), 2.65 (1H, m, H‐3), 3.17 (1H, m, H‐7), 3.44 (1H, br d, J = 7.9 Hz, H‐5), 3.77 (1H, dd, J = 7.9, 11.5 Hz, H‐4), 4.97 (1H, d, J = 4.5 Hz, H‐12)]. The 13C NMR data (Table 1) displayed a total of 16 carbons, including two olefinic carbon signals [δ C 135.3 (C‐1) and 128.2 (C‐2)], a sp 3‐type quaternary carbon at δ C 38.2 (C‐9), two methylenes [δ C 47.6 (C‐10) and 47.0 (C‐8)], an oxygenated methylene at δ C 69.9 (C‐11), three methines [δ C 51.7 (C‐3), 41.6 (C‐6), 41.1 (C‐7)], three oxygenated methines [δ C 106.7 (C‐12), 83.1 (C‐5), 75.3 (C‐4)], a methoxy group at δ C 54.8 (C‐16), and three methyl groups [δ C 29.6 (C‐15), 28.1 (C‐14), 14.5 (C‐13)]. The above functionalities account for one of the four degrees of unsaturation, suggesting a tricyclic structure in compound 1.
TABLE 1.
1H (500 MHz) and 13C NMR (125 MHz) data for 1 and 2 (in CD3OD, δ in ppm).
| Pos. | 1 | 2 | ||
|---|---|---|---|---|
| δ C, type | δ H (J in Hz) | δ C, type | δ H (J in Hz) | |
| 1 | 135.3, C | 148.7, C | ||
| 2 | 128.2, C | 129.4, C | ||
| 3 | 51.7, CH | 2.65, m | 41.0, CH | 2.64, m |
| 4a | 75.3, CH | 3.77, dd (7.9, 11,5) | 25.1, CH2 | 1.70, m |
| 4b | 1.83, m | |||
| 5a | 83.1, CH | 3.44, br d (7.9) | 42.0, CH2 | 1.72, overlapping |
| 5b | 1.97, dd (14.2, 3.0) | |||
| 6 | 41.6, CH | 1.92, m | 72.9, C | |
| 7 | 41.1, CH | 3.17, m | 53.4, CH | 3.12, t (9.2) |
| 8a | 47.0, CH2 | 1.43, m | 43.8, CH2 | 1.59, overlapping |
| 8b | 1.65, overlapping | |||
| 9 | 38.2, C | 37.0, C | ||
| 10a | 47.6, CH2 | 2.02, d (16.6) | 48.5, CH2 | 2.00, overlapping |
| 10b | 1.90, m | 2.35, dd (15.2, 2.3) | ||
| 11a | 69.9, CH2 | 4.31, ddt (12.5, 2.5, 2.5) | 69.1, CH2 | 4.54, s |
| 11b | 4.23, m | |||
| 12 | 106.7, CH | 4.97, d (4.5) | 63.5, CH2 | 4.26, d (8.8) |
| 13 | 14.5, CH3 | 0.90, d (8.0) | 19.9, CH3 | 1.05, s |
| 14 | 28.1, CH3 | 1.01, s | 27.0, CH3 | 0.86, s |
| 15 | 29.6, CH3 | 1.08, s | 28.8, CH3 | 1.11, s |
| 16 | 54.8, CH3 | 3.34, s | 173.0, C | |
| 17 | 20.8, CH3 | 2.04, s | ||
| 18 | 172.8, C | |||
| 19 | 20.8, CH3 | 2.04, s | ||
The planar framework of compound 1 was elucidated by an extensive analysis of COSY correlations and HMBC spectra (Figure 4). The COSY correlations of H2‐8/H1‐7/H1‐6/H3‐13/H1‐5/H1‐4/H1‐3/H1‐12 established a spin system, together with the HMBC correlations from H1‐10a [δ H 2.02 (1H, m)] to C‐7 (δ C 41.1), and from H2‐8 [δ H 1.43 (2H, m)] to C‐1 (δ C 135.3), revealing the presence of five‐membered ring A. Based on the two olefinic carbon signals [δ C 135.3 (C‐1) and 128.2 (C‐2)], it could be deduced that ring A was connected to the other ring by a carbon–carbon double bond (C‐1/C‐2). Based on the chemical shift of C‐11 (δ C 69.9)/C‐12 (δ C 106.7), it could be deduced that C‐11/C‐12 was connected to one/two oxygen atoms. The HMBC correlations from H3‐16 [δ H 3.34 (3H, s)] to C‐12 (δ C 106.7) established that a methoxy group was attached to the C‐12 position. Then, based on the molecular formula, degree of unsaturation, COSY correlations, and HMBC correlations from H1‐12 [δ H 4.97 (1H, d, J = 7.9 Hz)] to C‐2 (δ C 128.2)/C‐11 (δ C 69.9), it could be deduced that C‐11, C‐2, C‐3, C‐12, and O constituted a five‐membered ring C. The HMBC correlations from H3‐13 [δ H 0.90 (3H, d, J = 8.0 Hz)] to C‐7 (δ C 41.1)/C‐6 (δ C 41.6)/C‐5 (δ C 83.1) indicated that a methyl group (C‐13) was attached to the C‐6 position. Based on the COSY correlations, HMBC correlations from H1‐7 [δ H 3.17 (1H, m)] to C‐2 (δ C 128.2), and from H1‐3 [δ H 2.65 (1H, m)] to C‐1 (δ C 135.3), it could be deduced that the C‐1, C‐2, C‐3, C‐4, C‐5, C‐6, and C‐7 constituted a seven‐membered ring B. The HMBC correlations from H3‐14 [δ H 1.01 (3H, s)] to C‐8 (δ C 47.0)/C‐9 (δ C 38.2)/C‐10 (δ C 47.6), and from H3‐15 [δ H 1.08 (3H, s)] to C‐8 (δ C 47.0)/C‐9 (δ C 38.2)/C‐10 (δ C 47.6), suggested that both C‐14 and C‐15 were attached to C‐9. Thus, a sesquiterpene with a 5/7/5 tricyclic skeleton was identified.
FIGURE 4.
COSY and HMBC correlations of 1–2.
The relative configuration of 1 was determined by NOESY spectroscopy. The NOESY (Figure 5) cross peaks from H‐7 to H‐3/H‐4/H‐5, H‐3 to H‐12, H‐7 to H‐14/CH3‐13, suggested the β‐orientation of H‐7, H‐3, H‐4, H‐5, H‐14, H‐12, and CH3‐13. The presence of NOE correlation between H‐6 and H‐15 suggested the α‐orientation of H‐6 and H‐15. The 1H and 13C NMR spectral data of 1 were very similar to those of 3α,3β,10β‐trimethyl‐decahydroazuleno[6,7]furan‐8,9,14‐triol [24]. The only difference was that the hydroxy group at C‐12 in 3α,3β,10β‐trimethyl‐decahydroazuleno[6,7]furan‐8,9,14‐triol is replaced by a methoxy group at C‐12 in compound 1. This was revealed from the HMBC correlations from CH3‐16 to C‐12 and H‐12 to C‐2 (Figure 4). Based on the above analysis, the structure of 1 was established as in Figure 3. The absolute configuration of compound 1 was assigned as 3S, 4S, 5R, 6S, 7S, 12S by comparison of its experimental and calculated ECD spectra (Figure 6).
FIGURE 5.
NOESY correlations of 1 and 2.
FIGURE 6.
Experimental and calculated ECD spectra of 1 in MeOH.
Compound 2 was obtained as a colorless oil. Its molecular formula C19H30O5 was established by the HRESIMS analysis (m/z 339.2184, [M + H]+). The IR spectrum of compound 2 showed absorption bands at 3446, 1736, and 1630 cm−1, arising from hydroxy, carbonyl, and carbon–carbon double bond stretching vibrations, respectively. The 1H NMR spectrum (Table 1) showed five methyl groups [δ H 0.86 (3H, s, H‐14), 1.05 (3H, s, H‐13), 1.11 (3H, s, H‐15), 2.04 (3H, s, H‐17), 2.04 (3H, s, H‐19)], four methylenes [δ H 1.59 (1H, overlapping, H‐8a), 1.65 (1H, overlapping, H‐8b), 1.70 (1H, m, H‐4a), 1.72 (1H, overlapping, H‐5a), 1.83 (1H, m, H‐4b), 1.97 (1H, dd, J = 14.2, 3.0 Hz, H‐5b), 2.00 (1H, overlapping, H‐10a), 2.35 (1H, dd, J = 15.2, 2.3 Hz, H‐10b)], two oxygenated methylenes [δ H 4.26 (2H, d, J = 8.8 Hz, H‐12), 4.54 (2H, s, H‐11)], two methine protons [δ H 2.64 (1H, m, H‐3), 3.12 (1H, t, J = 9.2 Hz, H‐7)]. The 13C NMR data (Table 1) displayed a total of 19 carbons, including two olefinic carbon signals [δ C 148.7 (C‐1) and 129.4 (C‐2)], two sp 3‐type quaternary carbon [δ C 37.0 (C‐9) and 72.9 (C‐6)], two carboxyl/ester carbons [δ C 173.0 (C‐16) and 172.8 (C‐18)], four methylenes [δ C 48.5 (C‐10), 43.8 (C‐8), 42.0 (C‐5), and 25.1 (C‐4)], two oxygenated methylenes [δ C 69.1 (C‐11) and 63.5 (C‐12)], two methines [δ C 53.4 (C‐7) and 41.0 (C‐3)], and five methyl groups [δ C 28.8 (C‐15), 27.0 (C‐14), 20.8 (C‐17), 20.8 (C‐19), 19.9 (C‐13)]. The above functionalities account for three of the five degrees of unsaturation, suggesting a bicyclic structure in compound 2.
The planar framework of compound 2 was elucidated by an extensive analysis of COSY correlations and HMBC spectra (Figure 4). The HMBC correlations from H3‐14 [δ H 0.86 (3H, s)] to C‐8 (δ C 43.8)/C‐9 (δ C 37.0)/C‐10 (δ C 48.5) and H3‐15 [δ H 1.11 (3H, s)] to C‐8 (δ C 43.8)/C‐9 (δ C 37.0)/C‐10 (δ C 48.5), indicated that two methyl groups (C‐14/C‐15) was attached to the C‐9 position. The COSY correlations of H1‐7/H2‐8 and H2‐5/H2‐4/H1‐3/H2‐12, established two spin systems, together with the HMBC correlations from H1‐10b [δ H 2.35 (1H, dd, J = 15.2, 2.3 Hz)] to C‐7 (δ C 53.4)/C‐8 (δ C 43.8)/C‐14 (δ C 27.0), H1‐8a [δ H 1.59 (1H, overlapping)] to C‐15 (δ C 28.8), H1‐8b [δ H 1.65 (1H, overlapping)] to C‐1 (δ C 148.7)/C‐14 (δ C 27.0), revealing the presence of five‐membered ring A. Based on the two olefinic carbon signals [δ C 148.7 (C‐1) and 129.4 (C‐2)], it could be deduced that ring A was connected to the other ring by a carbon–carbon double bond (C‐1/C‐2). Based on the COSY correlations and the HMBC correlations from H1‐7 [δ H 3.12 (1H, t, J = 9.2 Hz)] to C‐2 (δ C 129.4), H1‐5a [δ H 1.72 (1H, overlapping)] to C‐7 (δ C 53.4), H1‐10a [δ H 2.00 (1H, overlapping)] to C‐2 (δ C 129.4), H1‐8a [δ H 1.59 (1H, overlapping)] to C‐6 (δ C 72.9), H2‐12 [δ H 4.26 (2H, d, J = 8.8 Hz)] to C‐2 (δ C 129.4)/C‐3 (δ C 41.0)/C‐4 (δ C 25.1), it could be inferred that C‐1, C‐2, C‐3, C‐4, C‐5, C‐6, and C‐7 together form a seven‐membered ring, designated as B. The HMBC correlations from H3‐17 [δ H 2.04 (3H, s)] to C‐16 (δ C 173.0), H3‐19 [δ H 2.04 (3H, s)] to C‐18 (δ C 172.8), H2‐11 [δ H 4.54 (2H, s)] to C‐1 (δ C 148.7)/C‐2 (δ C 129.4)/C‐3 (δ C 41.0)/C‐16 (δ C 173.0), and H2‐12 to C‐2 (δ C 129.4)/C‐3 (δ C 41.0)/C‐4 (δ C 25.1)/C‐18 (δ C 172.8), suggest the attachment of two acetoxy groups to C‐11 and C‐12, respectively. Based on the HMBC correlations from H3‐13 [δ H 1.05 (3H, s)] to C‐5 (δ C 42.0)/C‐6 (δ C 72.9)/C‐7 (δ C 53.4), it could be inferred that the methyl group (CH3‐13) was attached to C‐6. Thus, a sesquiterpene with a 5/7 bicyclic skeleton was identified.
The relative configuration of 2 was determined by NOESY spectroscopy. The NOESY (Figure 5) cross‐peaks from H‐7 to H2‐12/CH3‐14 suggested the β‐orientation of H‐7, H2‐12, and CH3‐14. The presence of NOE correlation between CH3‐13 and CH3‐15 suggested the α‐orientation of CH3‐13 and CH3‐15. Compound 2 exhibited spectroscopic data closely resembling those of 11‐O‐acetyl‐5β,11,12‐trihydroxy‐1‐tremulene, except for an additional acetoxy group [δ H 2.04 (3H, s, H‐19), δ C 20.8 (C‐19), and 172.8 (C‐18)] and a shift of the hydroxyl group from C‐5 in 11‐O‐acetyl‐5β,11,12‐trihydroxy‐1‐tremulene to C‐6 in compound 2 [25]. Compound 2 was isolated from the same fungal strain as compound 1, indicating that they share a common biosynthetic origin. Since the absolute configuration of compound 1 has been established by ECD calculations, the absolute configuration of compound 2 was deduced as 3S, 6R, 7R by analogy with the biosynthetic pathway and the stereochemistry of compound 1. Based on the above analysis, the structure of 2 was established as in Figure 3.
2.3. HPLC Analysis of Metabolic Profiles Under Different Cultivation Conditions
A small‐scale fermentation experiment was performed to analyze the secondary metabolites produced by I. lacteus and I. laceratus under different cultivation conditions. The metabolic fingerprints were analyzed using high‐performance liquid chromatography (HPLC). The combination of continuous light exposure and 5 mM cAMP treatment resulted in an abundant and diverse metabolite profile, as evidenced by the rich HPLC fingerprint. The identified compounds were also assigned within the fingerprint chromatogram (Figure 7). Based on preliminary HPLC profiling of small‐scale cultures under different cultivation conditions, continuous light exposure in the presence of 5 mM cAMP produced a rich and diverse chromatographic fingerprint. Therefore, this condition was selected for subsequent large‐scale fermentation.
FIGURE 7.
HPLC analysis of extracts under different culture conditions: (a) ethyl acetate extracts of fermentation broth of I. lacteus; (b) ethanol extracts of mycelia of I. lacteus; (c) ethyl acetate extracts of fermentation broth of I. laceratus; (d) ethanol extracts of mycelia of I. laceratus.
2.4. Assessment of Antifungal Activity
All isolated compounds were tested for antifungal activity. The results showed that compounds 1, 2, 4, 6, and 7 exhibited inhibitory effects under the experimental conditions, while the other compounds did not show inhibition at a concentration of 100 µg·mL−1. The detailed antifungal activity data are summarized in Table 2. Compound 1 exhibited MIC values of 100 µg·mL−1 against Fusarium oxysporum, Fusarium graminearum, and Gaeumannomyces graminis, whereas compound 2 showed MIC values of 100 µg·mL−1 against F. graminearum and 25 µg·mL−1 against G. graminis.
TABLE 2.
The antifungal activities of 1, 2, 4, 6, and 7.
| MIC (µg·mL− 1) | ||||
|---|---|---|---|---|
| Compounds | F. oxysporum YICB‐0025 | F. graminearum ACCC 38864 | G. graminis ACCC 38864 | G. cingulata bio‐24525 |
| 1 | 100 | 100 | 100 | >100 |
| 2 | >100 | 100 | 25 | >100 |
| 4 | >100 | >100 | >100 | 100 |
| 6 | >100 | >100 | 100 | >100 |
| 7 | >100 | >100 | 100 | >100 |
| Carbendazim | <0.8 | <0.8 | <0.8 | <0.8 |
3. Conclusion
This study suggests that cAMP signaling and light exposure may be involved in the regulation of fungal metabolism and may influence the metabolite profiles of algicolous fungi, providing a preliminary basis for the cultivation‐based modulation of fungal secondary metabolism. HPLC analysis revealed a rich and diverse metabolite profile under the selected fermentation conditions, from which two new tremulane‐type sesquiterpenes, ceriponols N–O (1 and 2), together with eight known compounds (3–10), were isolated. These metabolites exhibited antifungal activity, supporting the potential of combining light exposure and cAMP supplementation to promote the synthesis of bioactive compounds. This approach highlights the potential of algicolous fungi as a valuable source of antifungal agents.
4. Experimental Section
4.1. General Experimental Procedures
All solvents used were of analytical grade, except for HPLC‐grade acetonitrile (ACN) and methanol (MeOH) in HPLC analysis and separation. Optical rotation was measured using an Autopol VI polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA). UV and ECD spectra were recorded on a Chirascan CD spectrometer (Applied Photophysics Ltd., Surrey, UK). IR spectra were obtained using a Nicolet iS50 FT‐IR spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). NMR spectra were recorded on a Bruker Avance Neo 500 MHz NMR spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany), operating at 500 MHz for 1H and 125 MHz for 13C. ESI mass spectra were acquired using an Xevo G2‐XS QTof mass spectrometer (Waters Corp., Milford, MA, USA). Compound separation was performed using an Agilent 1260 HPLC system (Agilent Technologies Inc., Santa Clara, CA, USA) with an Eclipse SB‐C18 column (250 mm × 9.4 mm, 5 µm). Column chromatography was carried out with silica gel (200–300 mesh, Qingdao Haiyang Chemical Co., Qingdao, China), RP‐18 (YMC Co., Ltd., Kyoto, Japan), and Sephadex LH‐20 (GE Healthcare, Uppsala, Sweden). Potato Dextrose Agar (PDA; Qingdao Hi‐Tech Industrial Park Hope Bio‐Technology Co., Ltd., Qingdao, China) was used for both the confrontation assay and large‐scale fermentation. The PDA medium was prepared by dissolving 40.01 g of the commercial PDA powder in 1 L of distilled water, followed by autoclaving at 121°C for 15 min. Cyclic adenosine monophosphate (cAMP; CAS No. 60‐92‐4; purity 98%; Shanghai Yuanye Bio‐Technology Co., Ltd., Shanghai, China) was stored at −20°C until use. After autoclaving, cAMP was aseptically added to the PDA medium to a final concentration of 5 mM, and the medium was used for both confrontation assays and large‐scale static fermentation.
4.2. Fungal Materials
I. Lacteus YICB‐0055 and I. laceratus YICB‐0007 were isolated in July 2024 from the intertidal zone along the coastal region of Yantai, Shandong Province, China, where they were associated with the marine alga U. pertusa. Both strains are algicolous fungi and are taxonomically classified within the phylum Basidiomycota, class Agaricomycetes, order Polyporales, family Meruliaceae, and genus Irpex. The identification of these two strains was conducted based on morphological characteristics and internal transcribed spacer (ITS) sequence analysis. The corresponding ITS sequence data have been deposited in the GenBank database under the accession numbers PQ385870 and PQ386401 [26].
4.3. Confrontation
The confrontation assay was performed to assess the inhibitory effects of I. lacteus and I. laceratus against F. oxysporum. The selected strains were cultured on media supplemented with 5 mM cAMP. Continuous white light was supplied throughout the cultivation period by 14‐W LED lamps (2800–3000 lux). The control group was incubated in the dark without the addition of cAMP. All strains were cultivated for 7 days, and their growth was monitored daily. The inhibition effect was assessed by observing the growth suppression of F. oxysporum in the presence of the test strains.
4.4. Fermentation, Extraction, and Isolation
Large‐scale static fermentation was performed on I. lacteus and I. laceratus, with continuous white light exposure and the addition of 5 mM cAMP in the fungal medium. Continuous white light was provided by 14‐W LED lamps at an intensity of approximately 2800–3000 lux. I. lacteus and I. laceratus were fermented separately using 100 flasks for each strain (1 L flasks, 300 mL working volume per flask), corresponding to a total culture volume of 30 L per strain. The culture was maintained at room temperature for 21 days. After 21 days of cultivation, 300 mL of ethyl acetate was added to each culture flask and allowed to stand to ensure the inactivation of mycelia and spores. Mycelium and fermentation broth were separated by vacuum filtration. The fermentation broth was subjected to three rounds of ethyl acetate liquid–liquid extraction, with the ethyl acetate fractions combined and concentrated by rotary evaporation under reduced pressure, yielding the ethyl acetate extract. The mycelium was naturally air‐dried, then ground and subjected to three rounds of ethanol soaking extraction, with 24 h intervals between each extraction. After vacuum filtration, the extract solutions were combined and concentrated under reduced pressure, yielding the ethanol crude extract. The final yields of ethyl acetate extracts from I. lacteus and I. laceratus were 18.9 and 15.5 g, respectively, compared with 3.3 and 10.6 g for the corresponding ethanol extracts.
The ethyl acetate extract from I. lacteus was initially fractionated by silica gel column chromatography (CC), eluted with a gradient CH2Cl2/MeOH (5:1 to 0:1) to yield fractions A and B. Fraction A was further separated by C18 gel CC and eluted with a MeOH‐H2O mixture (4:1) to obtain subfraction A1. Subfraction A1 was then purified through silica gel CC (CH2Cl2/MeOH, 50:1 to 1:1) and Sephadex LH‐20 (MeOH), yielding compound 9 (6.0 mg). Fraction B was further separated on C18 gel CC and eluted with a MeOH‐H2O mixture (1:1 to 1:0) to obtain subfractions B1–B3. Subfraction B1 was subsequently purified by silica gel CC (CH2Cl2/MeOH, 50:1) and Sephadex LH‐20 (MeOH), affording compound 3 (3.1 mg). Subfraction B2 was subsequently purified by silica gel CC (CH2Cl2/MeOH, 4:1) and Sephadex LH‐20 (MeOH), affording compound 2 (2.2 mg). Subfraction B3 was subsequently purified by silica gel CC (CH2Cl2/MeOH, 1:1) and Sephadex LH‐20 (MeOH), affording compounds 1 (2.2 mg) and 5 (3.1 mg). The ethanol extract from I. lacteus was separated by C18 gel CC and eluted with a MeOH‐H2O mixture (4:1) to obtain fraction C. Fraction C was then purified through Sephadex LH‐20 (MeOH), yielding compound 5 (4.3 mg).
The ethyl acetate extract from I. laceratus was initially fractionated by silica gel CC, eluted with a gradient of petroleum ether (PE)/EtOAc (5:1, 3:1, and 1:1), followed by elution with CH2Cl2/MeOH (5:1), resulting in the collection of four fractions (Frs. 1–4). Fr. 1 was subsequently purified by silica gel CC (CH2Cl2/MeOH, 50:1 to 1:1), Sephadex LH‐20 (MeOH), and semipreparative HPLC (ACN/H2O, 3:7, 2.0 mL/min), affording compound 7 (18.0 mg). Fr. 2 was further separated by C18 gel CC and eluted with a MeOH‐H2O mixture (4:1 to 1:0), then purified through Sephadex LH‐20 (MeOH), affording compound 4 (3.0 mg). Fr. 4 was purified through C18 gel CC (MeOH‐H2O, 3:2 to 1:0) and Sephadex LH‐20 (MeOH), affording compounds 6 (19.6 mg) and 10 (2.3 mg). The ethanol extract from I. laceratus was separated by C18 gel CC and eluted with a MeOH‐H2O mixture (4:1), then purified through Sephadex LH‐20 (MeOH), affording compound 8 (5.0 mg).
Ceriponol N (1): white amorphous powder; [α]25 D +309.1 (c 0.044, MeOH); IR (KBr) ν max 3425, 2926, 1638, 1377, 1120, 1038, 791, 585, 428 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 283.1917 [M + H]+ (calcd. for C16H27O4, 283.1909).
Ceriponol O (2): colorless oil; [α]25 D +118.2 (c 0.044, MeOH); IR (KBr) ν max 3446, 2924, 1736, 1630, 1379, 1239, 1024, 798 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 339.2184 [M + H]+ (calcd. for C19H31O5, 339.2171).
4.5. HPLC Analysis of Metabolites
The HPLC analysis was performed to compare the secondary metabolites produced by I. lacteus and I. laceratus under four distinct cultivation conditions. The experimental conditions included: (1) continuous light exposure with 5 mM cAMP, (2) continuous light exposure without cAMP, (3) no light exposure with 5 mM cAMP, and (4) no light exposure without cAMP. In each condition, fermentation was carried out in 10 flasks (1 L, 300 mL working volume). The metabolic profiles of both fungal strains were analyzed by HPLC after fermentation. The HPLC analysis conditions included a crude extract concentration of 50 mg·mL−1 and an injection volume of 50 µL. The gradient elution program was set to increase methanol concentration from 10% to 100% over 50 min. The analysis was performed using an Agilent ZORBAX SB‐C18 column (5 µm, 4.6 × 150 mm).
4.6. Infection and Bioassay
Prior to evaluating the antifungal activity of the isolates against crop‐pathogenic fungi, the pathogenicity of F. oxysporum YICB‐0025 to wheat seedlings (Triticum aestivum cv. Jimai 22) was assessed following a previously described protocol with minor modifications [27]. The inoculated seedlings showed clear signs of root and crown rot compared to the control group (Figure S1). The antifungal activity was evaluated using the method according to a previously reported procedure [28]. The assay was performed in 96‐well microplates with a final volume of 200 µL per well. Each well contained 195 µL of fungal inoculum and 5 µL of test solution. The test compounds were evaluated using twofold serial dilutions at final concentrations ranging from 100 to 0.78 µg·mL−1. Carbendazim was used as the positive control (195 µL of inoculum and 5 µL of carbendazim solution), whereas DMSO was used as the negative control (195 µL of inoculum and 5 µL of DMSO, 2.5% v/v). Inoculum preparation and MIC endpoint determination were performed as described previously [31]. After incubation for 48 h, OD500 was measured using a microplate reader for MIC determination. All assays were performed in triplicate to ensure reproducibility. The tested crop‐pathogenic fungi included F. oxysporum YICB‐0025, F. graminearum ACCC 39334, G. graminis ACCC 38864, and Glomerella cingulata bio‐24525.
4.7. Computational Details
Conformational searches for compound 1 and subsequent quantum chemical simulations were operated as previously described, neglecting methyl, hydroxy, and methoxy rotations [29]. The optimization of preliminary conformers was performed at the B3LYP/6‐31G(d) level in MeOH via Gaussian 09 software to yield two energy‐minimized conformers (Figure S16) [30]. The Cartesian coordinates of the lowest‐energy conformer (1a) are provided in Table S2. ECD spectra of the two conformers were simulated at the same level and produced by the SpecDis software with sigma/gamma = 0.25 eV and UV‐shift = 0 nm [31]. They were further weighted according to Boltzmann populations and magnified to match the experimental values. All the quantum chemical calculations used the integral equation formalism variant (IEF) of the polarizable continuum model (PCM) as implemented in Gaussian 09.
Author Contributions
Rui Guo: writing – original draft, writing – review and editing, methodology, data curation, formal analysis, investigation, visualization, validation, conceptualization. Naiyun Ji: supervision, writing – review and editing, funding acquisition, conceptualization, project administration, methodology, resources.
Funding
This work was supported by the National Natural Science Foundation of China (No. 32470425).
Conflicts of Interest
The authors declare no conflicts of interest.
Supporting information
Supporting File 1: cbdv71754‐sup‐0001‐SuppMat.docx
Acknowledgments
This work was financially supported by the National Natural Science Foundation of China (No. 32470425).
Data Availability Statement
The data supporting the findings of this study, including those for compounds 1 and 2 (Figures S2–S16, Table S2), are available in the Supporting Information.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Supporting File 1: cbdv71754‐sup‐0001‐SuppMat.docx
Data Availability Statement
The data supporting the findings of this study, including those for compounds 1 and 2 (Figures S2–S16, Table S2), are available in the Supporting Information.