Bioactive eremophilane meroterpenoids produced by the soil fungus Stchigelomyces brunnescens gen. et comb. nov.

ABSTRACT

The emergence of antimicrobial resistance has created an urgent demand for novel antibiotics. Fungi in the order Sordariales represent a rich source of chemically diverse and bioactive secondary metabolites. However, many taxa within this order remain insufficiently explored. During our ongoing search for novel taxa and bioactive compounds, we isolated a soil-borne fungus initially identified as Cladorrhinum brunnescens. The multigene phylogenetic inference using four DNA loci (ITS, LSU, rpb2, and tub2) revealed that this species represents a distinct lineage within the family Podosporaceae, herein transferred to the new genus Stchigelomyces. Investigation of its secondary metabolites led to the discovery of four novel eremophilane meroterpenoids (1–4) and two previously undescribed salicyloid derivatives (6–7), together with a known triene fatty acid (5). The structures of all compounds were elucidated by comprehensive spectroscopic analyses, and their antimicrobial properties were assessed. Among the tested compounds, stchigelomycin C (3) exhibited strong inhibitory effects against some Gram-positive bacteria, yeast, and filamentous fungi, particularly Bacillus subtilis and Mucor hiemalis, at a concentration of 8.3 μg/mL. These findings further demonstrate the potential of taxa belonging to the Sordariales as promising sources for the discovery of new bioactive compounds.

GRAPHICAL ABSTRACT

1. Introduction

Fungi are widely recognized as a rich reservoir of bioactive natural compounds with structural diversity and novelty (Atanasov et al. 2021; Mapook et al. 2022). They are sophisticated poducers of secondary metabolites, which are characterized by diverse, complex, and structurally unique chemical scaffolds with a vast array of biological activities. Numerous fungal metabolites together with their semisynthetic derivatives, such as penicillins, cephalosporins, and pleuromutilins, have been successfully developed into antibiotics to treat infectious diseases (Schrey et al. 2025). However, the effectiveness of many existing therapeutics has been diminished due to their inappropriate use (Mittal et al. 2020). Over the past decades, the fast emergence and global spread of multidrug-resistant pathogens have posed a serious threat to public health and modern medicine (Gadar and McCarthy 2023; Marino et al. 2025). Consequently, research and development of novel antimicrobial drugs are urgently required to expand the range of therapeutic options.

Among the Sordariomycetes, the order Sordariales stands out for its remarkable morphological diversity and taxonomic complexity. Whole-genome sequences from 99 Sordariales species indicated that this order comprises nine distinct families, including Chaetomiaceae, Podosporaceae, Sordariaceae, Lasiosphaeriaceae, Lasiosphaeridaceae, Schizotheciaceae, Naviculisporiaceae, Bombardiaceae, and Diplogelasinosporaceae (Hensen et al. 2023). The Sordariales comprises over 1500 taxa exhibiting various ecological niches, including lignicolous, terricolous, coprophilous, and herbicolous taxa (Lundqvist 1972; Thiyagaraja et al. 2025). Sordarialean fungi are distinguished by their capacity to synthesize diverse secondary metabolites that exhibit promising pharmacological properties (Charria-Girón et al. 2022). For example, sordarins isolated from Sordaria araneosa exhibited great antifungal activity by interacting with the elongation factor 2 in eukaryotes (Domínguez and Martín 1998). Up to 2022, a total of 174 compounds had been reported from taxa of the Sordariales, excluding those from the family Chaetomiaceae (Charria-Girón et al. 2022). In contrast, Chaetomiaceae, the most extensively investigated group within the Sordariales, yielded 191 new compounds with diverse bioactivities between 2016 and 2021 (Ibrahim et al. 2021). The number of compounds isolated from the Sordariales has continued to grow in recent years. Integrative approaches combining taxonomic and chemical screening afforded novel natural products with diverse bioactivities. For example, Charria-Girón et al. (2023) introduced a previously undescribed species within the genus Amesia, Amesia hispanica (Chaetomiaceae), based on a multilocus phylogenetic analysis and morphological data. This species produced novel dactylfungin derivatives featuring potent antifungal properties. Similarly, a group of previously undescribed azaphilones was reported from a root-associated endophytic fungus of Aster tataricus. Integrated morphological observations and molecular data confirmed this fungus as a previously undescribed species of Tengochaeta, Tengochaeta bulbillosa (Chaetomiaceae) (Barrera-Adame et al. 2025). Furthermore, exploration of less-studied taxa continues to reveal their chemical diversity. For instance, four thiodiketopiperazine derivatives were firstly reported from Morinagamyces vermicularis (Schizotheciaceae) using Feature-based Molecular Networking (Harms et al. 2024). Additionally, six previously undescribed tetramic acids, arcopilins A–F, were isolated from Arcopilus navicularis (Chaetomiaceae) and exhibited promising disruptive activity against preformed Staphylococcus aureus biofilms (Charria-Girón et al. 2024).

In our continuing exploration of novel and bioactive compounds from sordarialean fungi, we isolated a soil fungus previously named Cladorrhinum brunnescens, which is herein transferred to the new genus Stchigelomyces (Podosporaceae) based on the phylogenetic analyses applying four DNA loci namely: ITS (internal transcribed spacer), LSU [large subunit of the nuclear ribosomal RNA (rRNA) gene], rpb2 (the second largest subunit of DNA directed RNA polymerase II), and tub2 (beta-tubulin) gene sequences. In addition, four novel eremophilane meroterpenoids (1–4), and two previously undescribed salicyloid derivatives (6–7) along with one known triene fatty acid (5) were isolated from the newly introduced taxon. Herein, the structure elucidation and bioactivity assessment of all purified compounds, including their antimicrobial activity and cytotoxicity, are described.

2. Materials and methods

2.1. Fungal isolation

Soil samples were collected during a Westerdijk citizen science campaign in 2019 in the Netherlands. Axenic cultures were prepared following the procedures described by Giraldo et al. (2019) and incubated at approximately 21 °C. All isolates were deposited at the CBS Culture Collection of the Westerdijk Fungal Biodiversity Institute in Utrecht, The Netherlands.

2.2. Morphological characterization

Phenotypic characteristics are assessed as described by Marin-Felix et al. (2020), using the Royal Horticultural Society (1996) color chart to evaluate the colony colors. Methods used for micromorphological descriptions are provided in the Supplemental material.

2.3. DNA isolation, amplification, and phylogenetic study

DNA isolation, PCR amplification, and sequencing were applied to the experimental workflow previously described by Charria-Girón et al. (2023). The internal transcribed spacer (ITS) regions (primers used ITS4 and ITS5), the large subunit (LSU) of the nuclear ribosomal RNA (rRNA) gene complex (primers used LROR and LR7), the partial fragments of the second largest subunit of DNA directed RNA polymerase II (rpb2) (primers used RPB2-5F and RPB2-7cR), and beta-tubulin (tub2) (primers used BT1819R and BT2916) genes were amplified and sequenced for phylogenetic analysis. Phylogenetic analyses were conducted using a combined four-loci dataset from our isolates together with corresponding sequences from type and reference strains of Podosporaceae, and Sordaria fimicola SMH 4106 and Pseudoneurospora canariensis FMR 12156 as outgroups (Table S1). Each locus was first aligned independently using MAFFT v. 7 (Katoh and Standley 2013) and then manually optimized using MEGA v. 10.2.4 (Kumar et al. 2018). The four loci were subsequently concatenated after verifying that no phylogenetic conflicts existed. The Maximum Likelihood (ML) analysis was conducted on the CIPRES portal (www.phylo.org) using RAxML-HPC BlackBox v. 8.2.12 under default parameters (Stamatakis 2014). Bayesian Inference (BI) was performed using MrBayes v. 3.2.1 (Ronquist et al. 2012), employing the Markov chain Monte Carlo sampling (MCMC) analysis of four parallel runs of 10 million generations, starting from a randomly generated tree topology and ending when the average standard deviation of split frequencies reaches < 0.01. Trees were sampled every 1000 generations with 25% of the initial trees removed as ‘burn-in’ after likelihood values reached stationary, and posterior probabilities (pp) were calculated from the remaining trees. Bootstrap support (bs) ≥ 70% and pp ≥ 0.95 were regarded as statistically significant. The sequences generated in this study were deposited in GenBank (CBS 150394 – LSU: PX115911, ITS: PX115910, rpb2: PX117582, tub2: PX117583; CBS 643.75A – rpb2: PX204737), and the alignment is available in Table S1.

A second phylogenetic study was done using the alignments available in Marin-Felix and Miller (2022) and adding the ITS and LSU sequences of Podospora bizantiorum and P. dennisiae (Tan and Shivas 2022) to correctly classify both taxa within the Sordariales, since these were not located in the clade of the family Podosporaceae in our phylogenetic study. The phylogenetic tree is available in Figure S57.

2.4. Cultivation and metabolite extraction

Fermentation and extraction procedures for strain CBS 150394 are described in the Supplemental material. The mycelial extract (130 mg) was separated using a PLC 2250 preparative HPLC system (Gilson, Middleton, WI, USA) equipped with a Luna C₁₈ column (250 × 50 mm, 10 µm, Phenomenex®, Torrance, CA, USA) applying the following conditions: solvent A: deionized water (H₂O) + 0.1% formic acid; solvent B: acetonitrile (MeCN) + 0.1% formic acid; flow: 50 mL/min; collected fraction volume: 20 mL; gradient: from 5% to 30% B in 20 min, an increase from 30% to 60% B in 60 min, then solvent B increased to 100% in 25 min and decreased to 5% in 5 min, to end with an isocratic elution of 5% B for 10 min. This gradient afforded five pure compounds: stchigelomycin A (1) (2.62 mg, t_R = 89.44 min), stchigelomycin B (2) (1.01 mg, t_R = 96.33 min), stchigelomycin C (3) (6.95 mg, t_R = 85.05 min), stchigelomycin D (4) (0.57 mg, t_R = 102.01 min), and compound (5) (1.44 mg, t_R = 74.19 min).

The supernatant extract (205 mg × 2) was separated using a PLC 2250 preparative HPLC system (Gilson, Middleton, WI, USA) equipped with a Gemini C₁₈ column (250 × 50 mm, 10 µm, Phenomenex®, Torrance, CA, USA) applying the following conditions: solvent A: deionized water (H₂O) + 0.1% formic acid; solvent B: acetonitrile (MeCN) + 0.1% formic acid; flow: 50 mL/min; collected fraction volume: 20 mL; gradient: from 10% to 25% B in 15 min, then an increase from 25% to 70% B in 45 min, then solvent B increased to 100% in 10 min, to end with an isocratic elution of 100% B for 10 min. These separation conditions resulted in the separation of two pure compounds: vaccinol T (6) (6.46 mg, t_R = 50.51 min), and vaccinols U₁/U₂ (7a/7b, 2:3) (16.23 mg, t_R = 43.27 min).

Stchigelomycin A (1): Dark-brown amorphous solid; ${\left[\alpha \right]}_{\mathrm{D}}^{20}$ −71.0 (c 0.1, MeOH); UV/Vis (MeOH): λ_max (log ε) = 313, 223 nm; NMR data (¹H NMR: 500 MHz, ¹³C NMR: 125 MHz, methanol-d₄) see Table 1; HR-(+)ESI-MS: m/z 455.2421 [M – H₂O + H]⁺ (calcd. 455.2428 for C₂₇H₃₅O₆⁺), 473.2526 [M + H]⁺ (calcd. 473.2534 for C₂₇H₃₇O₇⁺), 495.2343 [M + Na]⁺ (calcd. 495.2353 for C₂₇H₃₆NaO₇⁺); t_R = 12.50 min (LC-ESI-MS).

Table 1.

¹³C and ¹H NMR data of 1–4.

Position	1		2		3		4
	δC,a type	δHa multi (J [Hz])	δC,b type	δHb multi (J [Hz])	δC,a type	δHa multi (J [Hz])	δC,c type	δHc multi (J [Hz])
1	76.3, CH	4.68 t (2.9)	76.4, CH	4.65 t (3.0)	77.2, CH	4.66 t (3.0)	77.0, CH	4.65 t (2.8)
2	30.5, CH2	α 1.78 tt (13.5, 3.0) β 2.06 dq (13.5, 2.9)	30.8, CH2	α 1.80 tt (13.9, 3.4) β 1.97 dq (14.4, 3.4, 2.9)	30.6, CH2	α 1.77 tt (14.3, 3.5) β 2.05 dq (14.3, 3.0)	30.6, CH2	α 1.79 tt (13.8, 3.1) β 1.98 dq (14.0, 2.6)
3	21.6, CH2	α 1.69 td (13.3, 3.2) β 1.93 d (13.3)	26.7, CH2	α 1.51 dq (13.8, 3.7) β 1.71 m	21.4, CH2	α 1.69 td (13.0, 3.3) β 1.85 dd (13.0, 3.2)	26.2, CH2	α 1.51 m β 1.75 m
4	45.7, CH	1.56 ddd (11.8, 8.3, 3.5)	37.9, CH	1.58 dqd (13.1, 6.7, 3.7)	44.3, CH	1.91 ddt (12.9, 9.0, 4.8)	36.1, CH	1.89 dqd (13.1, 7.0, 3.3)
5	39.1, C		39.9, C		38.3, C		38.7, C
6	35.7, CH2	α 2.46 dd (16.0, 2.8) β 2.66 d (16.0)	35.9, CH2	α 2.40 dd (15.9, 2.8) β 2.56 d (15.9)	36.2, CH2	α 1.26 dd (14.4, 14.2) β 1.62 dd (14.4, 5.3)	35.9, CH2	α 1.25 dd (14.2, 14.0) β 1.43 (overlapped)
7	147.9, C		148.2, C		45.6, CH	2.67 dd (14.2, 5.2)	45.3, CH	2.52 dd (13.9, 5.2)
8	197.1, CO		197.3, CO		102.7, C		102.5, C
9	63.1, CH	3.25 s	63.1, CH	3.22 s	63.6, CH	3.10 s	63.2, CH	3.09 s
10	68.9, C		69.0, C		65.3, C		65.3, C
11	126.7, C		126.4, C		70.0, CH2	α 4.41 d (13.0) β 4.50 d (13.0)	69.7, CH2	α 4.41 dq (13.1, 2.1) β 4.50 m
12	174.6, CO		174.8, CO		152.2, C		152.0, C
13	18.4, CH3	2.28 d (2.6)	18.4, CH3	2.27 d (2.6)	105.5, CH2	α 4.99 br s β 5.00 br s	105.2, CH2	4.99 m
14	62.9, CH2	α 3.31 overlapped β 3.83 dd (11.0, 3.6)	15.6, CH3	0.93 d (6.7)	63.3, CH2	α 3.41 dd (11.1, 7.8) β 3.83 dd (11.1, 4.6)	14.9, CH3	0.99 d (6.3)
15	20.5, CH3	1.28 s	19.5, CH3	1.25 s	20.4, CH3	1.18 s	19.1, CH3	1.15 s
1’	168.0, CO		167.9, CO		168.0, CO		167.7, CO
2’	120.1, CH	5.94 d (15.2)	120.1, CH	5.94 d (15.2)	120.3, CH	5.92 d (15.2)	120.0, CH	5.92 d (15.3)
3’	147.8, CH	7.38 dd (15.2, 11.1)	147.8, CH	7.37 dd (15.2, 11.0)	147.6, CH	7.36 dd (15.2, 11.0)	147.3, CH	7.36 dd (15.3, 11.1)
4’	125.0, CH	6.38 dd (15.2, 11.1)	125.0, CH	6.38 dd (15.2, 11.0)	125.0, CH	6.36 dd (15.3, 11.0)	124.7, CH	6.37 dd (15.2, 11.1)
5’	148.2, CH	6.67 d (15.2)	148.1, CH	6.67 d (15.2)	148.0, CH	6.65 d (15.3)	147.7, CH	6.65 d (15.2)
6’	134.1, C		134.1, C		134.1, C		133.8, C
7’	146.3, CH	5.55 d (9.7)	146.3, CH	5.55 d (9.7)	146.2, CH	5.54 d (9.7)	145.9, CH	5.54 d (9.7)
8’	36.0, CH	2.48 overlapped	36.0, CH	2.48 dddd (9.9, 8.4, 6.7, 5.5)	36.0, CH	2.48 dp (14.4, 6.6)	35.7, CH	2.48 m
9’	31.3, CH2	α 1.30 m β 1.43 dtd (14.9, 7.5, 5.6)	31.3, CH2	α 1.30 m β 1.43 dqd (13.1, 7.4, 5.5)	31.3, CH2	α 1.31 m β 1.42 m	31.0, CH2	α 1.31 m β 1.43 m
10’	12.3, CH3	0.87 t (7.4)	12.3, CH3	0.87 t (7.4)	12.3, CH3	0.86 t (7.4)	12.1, CH3	0.87 t (7.4)
11’	12.7, CH3	1.82 d (1.2)	12.7, CH3	1.82 d (1.3)	12.7, CH3	1.82 d (1.5)	12.4, CH3	1.82 d (1.3)
12’	20.7, CH3	0.99 d (6.6)	20.7, CH3	0.99 d (6.7)	20.7, CH3	0.99 d (6.6)	20.4, CH3	0.99 d (6.3)

^aMeasured in methanol-d₄ at 125 MHz for ¹³C and 500 MHz for ¹H. ^bMeasured in methanol-d₄ at 175 MHz for ¹³C and 700 MHz for ¹H. ^cMeasured in methanol-d₄ at 150 MHz for ¹³C and 600 MHz for ¹H.

Stchigelomycin B (2): Pale-brown amorphous solid; ${\left[\alpha \right]}_{\mathrm{D}}^{20}$ −67.0 (c 0.1, MeOH); UV/Vis (MeOH): λ_max (log ε) = 312, 224 nm; NMR data (¹H NMR: 700 MHz, ¹³C NMR: 175 MHz, methanol-d₄) see Table 1; HR-(+)ESI-MS: m/z 457.2576 [M + H]⁺ (calcd. 457.2585 for C₂₇H₃₇O₆⁺), 479.2392 [M + Na]⁺ (calcd. 479.2404 for C₂₇H₃₆NaO₆⁺); t_R = 14.49 min (LC-ESI-MS).

Stchigelomycin C (3): Dark-brown amorphous solid; ${\left[\alpha \right]}_{\mathrm{D}}^{20}$ −90.0 (c 0.1, MeOH); UV/Vis (MeOH): λ_max (log ε) = 314, 221, 200 nm; NMR data (¹H NMR: 500 MHz, ¹³C NMR: 125 MHz, methanol-d₄) see Table 1; HR-(+)ESI-MS: m/z 441.2628 [M – H₂O + H]⁺ (calcd. 441.2636 for C₂₇H₃₇O₅⁺), 459.2732 [M + H]⁺ (calcd. 459.2741 for C₂₇H₃₉O₆⁺), 481.2549 [M + Na]⁺ (calcd. 481.2561 for C₂₇H₃₈NaO₆⁺); t_R = 12.52 min (LC-ESI-MS).

Stchigelomycin D (4): Pale-brown amorphous solid; ${\left[\alpha \right]}_{\mathrm{D}}^{20}$ −22.9 (c 0.1, MeOH); UV/Vis (MeOH): λ_max (log ε) = 311, 224 nm; NMR data (¹H NMR: 600 MHz, ¹³C NMR: 150 MHz, methanol-d₄) see Table 1; HR-(+)ESI-MS: m/z 425.2680 [M – H₂O + H]⁺ (calcd. 425.2686 for C₂₇H₃₇O₄⁺), 443.2785 [M + H]⁺ (calcd. 443.2792 for C₂₇H₃₉O₅⁺), 465.2602 [M + Na]⁺ (calcd. 465.2611 for C₂₇H₃₈NaO₅⁺); t_R = 15.79 min (LC-ESI-MS).

Compound (5): White amorphous solid; UV/Vis (MeOH): λ_max (log ε) = 305, 220 nm; NMR data (¹H NMR: 700 MHz, ¹³C NMR: 175 MHz, methanol-d₄) comparable to those in the reported literature (Dörfelt et al. 2000); t_R = 10.94 min (LC-ESI-MS).

Vaccinol T (6): Dark-brown amorphous solid; ${\left[\alpha \right]}_{\mathrm{D}}^{20}$ +16.0 (c 0.1, MeOH); UV/Vis (MeOH): λ_max (log ε) = 303, 228, 217 nm; NMR data (¹H NMR: 500 MHz, ¹³C NMR: 125 MHz, DMSO-d₆) see Table 1; HR-(+)ESI-MS: m/z 291.1568 [M – H₂O + H]⁺ (calcd. 291.1591 for C₁₇H₂₃O₄⁺), 309.1689 [M + H]⁺ (calcd. 309.1697 for C₁₇H₂₅O₅⁺), 331.1506 [M + Na]⁺ (calcd. 331.1516 for C₁₇H₂₄NaO₅⁺); t_R = 8.26 min (LC-ESI-MS).

Vaccinols U₁/U₂ (7a/7b, 2:3): Pale-brown amorphous solid; UV/Vis (MeOH): λ_max (log ε) = 276, 221, 202 nm; NMR data (¹H NMR: 500 MHz, ¹³C NMR: 125 MHz, DMSO-d₆) see Table 1; HR-(+)ESI-MS: m/z 275.1633 [M – H₂O + H]⁺ (calcd. 275.1642 for C₁₇H₂₃O₃⁺), 293.1740 [M + H]⁺ (calcd. 293.1747 for C₁₇H₂₅O₄⁺), 315.1558 [M + Na]⁺ (calcd. 315.1567 for C₁₇H₂₄NaO₄⁺); t_R = 7.16 min (LC-ESI-MS).

2.5. Chromatography and spectral methods

ESI-MS (electrospray ionization mass) spectra of crude extracts and pure compounds were recorded at 4.5 and 1.0 mg/mL concentrations, respectively, using an UltiMate 3000 Series uHPLC (Thermo Scientific Inc., Waltham, MA, USA) connected to an amaZon speed ESI-iontrap-MS spectrometer (Bruker Daltonics, Bremen, Germany) (Charria-Girón et al. 2023). The high-resolution electrospray ionization mass (HR-ESI-MS) spectra were acquired using a maXis ESI-Time-of-flight (TOF) mass spectrometer (Bruker Daltonics, Bremen, Germany) (Wennrich et al. 2025). The isolated compounds were dissolved in methanol (Uvasol, Merck, Darmstadt, Germany) for the measurement of UV-Vis spectra, optical rotations, and electronic circular dichroism (ECD) spectra. Compounds were prepared in deuterated methanol-d₄ or DMSO-d₆ for 1D/2D NMR spectral analyses. All measurements were performed using the same instruments and conditions as described by Charria-Girón et al. (2023, 2024).

2.6. Computational section

The possible conformations of compounds 1–6 were analyzed using the Omega2 software (OMEGA 2021) with an energy window of 10 kcal/mol based on the ECD data. Geometry optimization and TDDFT (time-dependent density functional theory)-ECD calculations were conducted indicating methanol as a solvent using the integral equation formalism variant (IEFPCM) model implemented in Gaussian09 software (Frisch et al. 2009). Detailed computational procedures are provided in the Supplemental material.

2.7. Biological assays

The cytotoxic and antimicrobial assays of isolated compounds were conducted implementing the previously described methods by Charria-Girón et al. (2023). All assessed compounds in the bioassays were of > 95% purity, as confirmed by NMR analysis (see Supplemental material).

3. Results

3.1. Phylogenetic study

The combined dataset comprises individual alignments of 605 bp for ITS, 885 bp for LSU, 948 bp for rpb2, and 618 bp for tub2. Figure 1 shows the phylogenetic tree obtained from the RAxML analysis of the combined dataset, whose topology agreed with the one generated by the Bayesian analysis, including bs ≥ 70 and pp ≥ 0.95 at the nodes. Our strain CBS 150394 was assigned to the family Podosporaceae, clustering with CBS 643.75A, the type strain of C. brunnescens, in a fully supported monophyletic lineage distant from the generic type of Cladorrhinum and as a sister clade of Podospora, suggesting that it represents a different genus in the Podosporaceae, introduced herein as Stchigelomyces. Moreover, after including all Cladorrhinum species in our phylogenetic analysis, a recently described species C. carnegieae turned to belong to Podospora, and we propose herein the new combination. On the other hand, “C. yunnanense” could not be confirmed as a member of Cladorrhinum and is therefore excluded.

Figure 1.

Randomized accelerated maximum likelihood (RAxML) phylogram retrieved from the combined sequences of the ITS, LSU, rpb2, and tub2 genes of selected strains belonging to the Podosporaceae, using Sordaria fimicola SMH 4106 and Pseudoneurospora canariensis FMR 12156^T as outgroups. Bootstrap support (bs) values ≥ 70 and Bayesian posterior probability (pp) scores ≥ 0.95 are indicated along the branches. Branch lengths are proportional to distance. Novel taxa proposed in the present study are in bold. Ex-epitype, ex-isotype, and ex-type strains of the different species are indicated with ^{ET, IsoT,} and ^T, respectively.

Furthermore, P. bizantiorum and P. dennisiae, the most recent species described in the genus (Tan and Shivas 2022) apart from P. sichuanensis (Pan et al. 2025), are grounded outside Podosporaceae, and both are here excluded from Podospora (Figure S57). Podospora dennisiae is clustering with Pseudorhypophila (Naviculisporaceae), suggesting that this species could belong to the latter genus, while P. bizantiorum is located in Schizotheciaceae possibly representing a new genus. However, further studies including morphological characteristics and the sequencing of the correct regions of rpb2 and tub2 are necessary to verify those hypotheses.

3.2. Taxonomy

Podospora carnegieae (Crous & Jurjević) Y. Marín, comb. nov.

MycoBank: MB860418.

Basionym: Cladorrhinum carnegieae Crous & Jurjević, Fungal Systematics and Evolution 13: 394. 2024.

Holotype: USA, Arizona, Tortilla Flat (Apache trail), on Carnegiea gigantea (Cactaceae), Mar. 2023, Z. Jurjević, 5817, CBS H-25332; culture ex-type CPC 45918 = CBS 150814.

Notes: Cladorrhinum carnegieae was the last species introduced in the genus to accommodate a species producing cladorrhinum-like anamorph and sterile teleomorph consisting of protoperithecia (Crous et al. 2024). This species is here transferred to Podospora based on its phylogenetic affinity.

Stchigelomyces Y. Marín & Hern.-Restr., gen. nov.

MycoBank: MB860416.

Type species: Stchigelomyces brunnescens (W. Gams) Y. Marín & Hern.-Restr., comb. nov.

Etymology: Named in honor of the mycologist Alberto M. Stchigel Glikman, expert in sordarialean taxa.

Vegetative hyphae septate, branched, hyaline to subhyaline, smooth- and thin-walled. Anamorph. Conidiophores aggregated in tufts, septate, profusely branched, branches usually flexuous and densely entangled, often ending in sterile cells with obtuse apices, subhyaline to pale brown, smooth, cell walls generally thicker than those of the vegetative hyphae. Conidiogenous cells phialidic, intercalary, terminal or lateral, cylindrical, subcylindrical, to lageniform, subhyaline to pale brown, with one or rarely two conidiogenous openings, bearing conspicuous collarettes. Conidia one-celled, hyaline to subhyaline, smooth, globose to obovoid, guttulate, often truncate at the base or bearing a subcylindrical hilum, aggregated in slimy masses. Teleomorph not observed.

Notes: The genus Cladorrhinum was introduced to accommodate C. foecundissinum, a species isolated from dung collected in the Ardennes region in Belgium (Saccardo and Marchal 1885). This genus is characterized by the production of intercalary phialides with flaring collarettes and one-celled conidia in slimy masses (Mouchacca and Gams 1993). Cladorrhinum was then connected to the teleomorphic genera Apiosordaria and Cercophora in the Lasiosphaeriaceae since some species produced both morphs simultaneously (Mouchacca and Gams 1993). In the last years, the family Lasiosphaeriaceae as well as the genera belonging to it have gone through multiple taxonomic rearrangements based on polyphasic studies combining morphological and sequence data (Wang et al. 2019; Marin-Felix et al. 2020; Marin-Felix and Miller 2022). Wang et al. (2019) redefined the genus Cladorrhinum including three species, i.e., the type species and two teleomorphic species previously identified as Thielavia hyalocarpa and T. intermedia (Chaetomiaceae). Therefore, the description of the genus was emended to incorporate the production of non-ostiolate ascomata and 1-celled ascospores. Moreover, this genus was located in the newly introduced family Podosporaceae, which encompassed three different genera, i.e., Cladorrhinum, Podospora, and Triangularia, all characterized by the production of cladorrhinum-like anamorph. Subsequently, Marin-Felix et al. (2020) emended once again the description of Cladorrhinum since Cercophora coprophila, producing ostiolate ascomata and two-celled ascospores, and Arnium olerum and A. tomentosum, characterized by ostiolate ascomata and 1-celled ascospores, were transferred to this genus. While the three genera of the Podosporaceae show very similar anamorphs, they differ in the sexual morph. The biggest issue is that species with ostiolate and non-ostiolate ascomata, as well as 1- and 2-celled ascospores, can be found in Cladorrhinum, Podospora, and Triangularia, with many overlapping features among them (Cai et al. 2006; Marin-Felix et al. 2020). This requires us to use sequence data to be able to delimitate these genera. In our phylogenetic study, taxa belonging to the Podosporaceae were divided into four well-supported lineages representing four different genera. Therefore, Stchigelomyces is herein introduced to accommodate C. brunnescens, which was located in a lineage far from the other three already known genera.

Stchigelomyces brunnescens (W. Gams) Y. Marín & Hern.-Restr., comb. nov. Figure 2

Figure 2.

Stchigelomyces brunnescens (CBS 150394). (a) Conidiophores, phialides, adelophialides (arrows), and conidia. (b) Lateral conidiogenous cells and adelophialides (arrows). (c) Terminal conidiogenous cells and adelophialides (arrows). (d) Lateral conidiogenous cell with slimy mass of conidia. (e, f) Intercalary lageniform conidiogenous cells. (g) Conidia. Scale bars: a–g = 10 µm.

MycoBank: MB860417.

Basionym: Cladorrhinum brunnescens W. Gams, Mycotaxon 48: 435. 1993.

Synonym: Podospora brunnescens (W. Gams) S.K. Huang & K.D. Hyde, Fungal Diversity 111: 514. 2021.

Holotype: The Netherlands, North Holland Province, isolated from cocos fibers buried in soil, isol. J. Antheunisse, No. 34; ex-type strain CBS 643.75A.

Vegetative hyphae septate, branched, hyaline to subhyaline, smooth- and thin-walled, 2–3.5 µm wide. Anamorph. Conidiophores aggregated in tufts, semi-macronematous, septate, profusely branched, branches usually flexuous and densely entangled, often ending in sterile cells with obtuse apices, subhyaline to pale brown, smooth, cell walls generally thicker than those of the vegetative hyphae, 2.7–4 µm wide, length indeterminate. Conidiogenous cells phialidic, intercalary, terminal or lateral, cylindrical, subcylindrical to lageniform, commonly flexuous, subhyaline to pale brown, with one or rarely two conidiogenous openings, bearing conspicuous collarettes 1.3–3.5 µm wide, 0.5–2.5 µm deep; terminal and lateral phialides subcylindrical to lageniform, 6–20 × 2.5–3 µm, apex 1.7–2 µm; adelophialides cylindrical, subcylindrical or lageniform, 2.5–8 × 1–2.5 µm. Conidia one-celled, hyaline to subhyaline, smooth- and thin-walled, subglobose to obovoid, 2.5–4 × 2–3 µm, guttulate, often truncate at the base or bearing a subcylindrical hilum 0.5–1 µm, aggregated in slimy masses. Teleomorph not observed.

Cultural characterization: Colonies on OA covering all plate (90 mm) at 25 °C in 14 d, cottony, raised, margins fringed to fimbriate, greyed-orange (166A–D) with grey (201A–B) to greyed-white (156A–C) mycelium; reverse greyed-orange (165A–D). Colonies on PCA reaching 48–69 mm at 25 °C in 14 d, cottony, raised, slightly lobulated, margins fringed to fimbriate, orange white (159A–D) with greyed-orange (168D) circular ring around center; reverse greyed-orange (164A–C) with orange white (159A–D) margins. Colonies on PDA attaining 9–21 mm at 25 °C in 14 d, velvety to slightly cottony, raised, lobulated, margins regular, greyed green (197A–D) to white with greyed orange (163A–D and 166A–C) margins; reverse greyed-orange (166A–C) with paler greyed-orange (163A–D) margins. Colonies on MEA attaining 11–17 mm at 25 °C in 14 d, cottony, raised, slightly lobulated, margins regular to fringed, greyed-green (197A–D) with white mycelium and yellow white (158A–B) margins; reverse greyed-orange (165A–D) with yellow white (158A–B) margins.

Additional specimen examined: The Netherlands, Groningen Province, Warffum, isolated from soil, 06 Jun. 2019, coll. C. Dijkstra & L. Kruit, isol. M. Hernández-Restrepo, NL19-25013 = CBS 150394.

Distribution: Germany and The Netherlands. According to globalfungi database (https://globalfungi.com/, accessed on 26 August 2025), similar ITS sequences are reported from Estonia (soil).

Notes: This species was introduced as C. brunnescens to accommodate a fungus isolated from cocos fibres buried in soil in the Netherlands and from wheat-field soil in Germany (Mouchacca and Gams 1993). Afterwards, it was transferred to Podospora based only on ITS and LSU sequence data (Huang et al. 2021). However, Marin-Felix and Miller (2022) already doubted the validity of the new combinations based on the lack of phylogenetic support of this species within the Podospora clade and the long phylogenetic distance in their tree. In the present study, our phylogenetic analysis based on ITS, LSU, rpb2, and tub2 and morphological data corroborate that this species represents a new genus named Stchigelomyces in the Podosporaceae.

Excluded species

Cladorrhinum yunnanense Y. Pan, J.W. Xia, X.G. Zhang & Z. Li, Mycotaxon 136(3): 602. 2021.

Holotype: China, Yunnan, on dead branches of Angiosperma, coll. Y. Pan, 16 Apr. 2019, HSAUP 0525.

Notes: Cladorrhinum yunnanense is here excluded from the genus since its introduction presents several incongruences. First of all, in the description and figure legend, the presence of sporodochia is mentioned, which has never been reported for this genus. Furthermore, the illustrations (Figure 2 in Pan et al. 2021) is clearly showing the exudate droplets typically formed by stachybotrys-like fungi in culture. On the other hand, the sequences linked to the strain are related to Podosporaceae, and according to our phylogenetic study, it belongs to Podospora instead of Cladorrhinum (Figure 1). It should be clarified by verifying the morphology of the holotype specimen, but this was not possible during this study.

Podospora bizantiorum Y.P. Tan, Index of Australian Fungi 3: 14. 2022.

Holotype: Australia, Queensland, Barwidgi, from soil, 20 April 2021, Y.P. Tan, BRIP 72548b.

Notes: This species is not located in the Podosporaceae. According to our phylogenetic study (Figure S57), this species belongs to the Schizotheciaceae, probably representing a new genus. Regrettably, the fungus was not examined morphologically, probably because of a lack of sporulation.

Podospora dennisiae Y.P. Tan, Bishop-Hurley, Marney & R.G. Shivas, Index of Australian Fungi 3: 14. 2022.

Holotype: Australia, Queensland, Rossville, from soil, 13 May 2022, coll. M.D.E. Shivas, R.G. Shivas & T.S. Marney, BRIP 74887a.

Notes: This species was recently introduced based only on ITS and LSU sequence data and wrongly assigned to Podospora (Tan and Shivas 2022). According to our analysis, P. dennisiae is located in the family Naviculisporaceae, forming a well-supported clade (83 bs) with other species of Pseudorhypophila (Figure S57). Further studies should be done including morphological data and sequences of rpb2 and tub2, to verify if it should be transferred to this later genus.

3.3. Isolation and identification of compounds 1–7

Compound 1 (Figure 3) was purified as a dark-brown amorphous solid. The HR-ESI-MS of 1 (Figure S2) determined its molecular formula as C₂₇H₃₆O₇ based on the revealed protonated molecule and sodium adduct at m/z 473.2526 [M + H]⁺ (calculated 473.2534) and 495.2343 [M + H]⁺ (calculated 495.2353), respectively, thus indicating ten degrees of unsaturation.

Figure 3.

Chemical structures of 1–7.

The DEPTQ and HSQC spectral data of 1 (Table 1, Figures S4, S7) unravelled twenty-seven carbon signals, categorized into eight unprotonated including three carbonyls (δ_C 197.1, 174.6, 168.0), three olefinics (δ_C 147.9, 134.1, 126.7), and two aliphatics (δ_C 68.9, 39.1); nine methines divided into five olefninc (δ_C 148.2, 147.8, 146.3, 125.0, 120.1) and four aliphatic (δC 76.3, 63.1, 45.7, 36.0) carbon atoms. In addition, the DEPTQ and HSQC spectral data of 1 revealed five methylenes (δ_C 62.9, 35.7, 30.5, 31.3, 21.6) and five methyls (δ_C 20.7, 20.5, 18.4, 12.7, 12.3). The obtained results clarified for seven degrees of unsaturation and hence concluding that 1 features a tricyclic structure.

The ¹H NMR and ¹H–¹H COSY spectra of 1 (Table 1, Figures 4, S3, S5) revealed three spin systems: 1) between four trans olefinic proton signals at δ_H 5.94 (d, J = 15.2 Hz, H-2’), 7.38 (dd, J = 15.2, 11.1 Hz, H-3’), 6.38 (dd, J = 15.2, 11.1 Hz, H-4’), and 6.67 (d, J = 15.2 Hz, H-5’); 2) a spin system extending along one olefinic proton at δ_H 5.55 (d, J = 9.7 Hz, H-7’), an aliphatic methine at δ_H 2.48 (overlapped, H-8’) coupled to a doublet methyl group at δ_H 0.99 (d, J = 6.6 Hz, H₃-12’), a diastereotopic methylene group at δ_H 1.30/1.43 (H₂-9’) ending at a triplet terminal methyl group at δ_H 0.87 (t, J = 7.4 Hz, H₃-10’); 3) from an oxygenated aliphatic methine proton at δ_H 4.68 (t, J = 2.0 Hz, H-1) to two diastereotopic methylene groups at δ_H 1.78/2.06 (H₂-2) and 1.69/1.93 (H₂-3) to another methine proton at δ_H 1.56 (ddd, J = 11.8, 8.3, 3.5 Hz, H-4) ending at an oxygenated diastereotopic methylene group at δ_H 3.31/3.83 (H₂-14). Based on the obtained results, a literature search of 1 revealed its structural similarity to dictyopanines A–C, antibacterial metabolites previously reported from the tropical fungus Dictyopanus sp. HKI 0181 (Dörfelt et al. 2000). A detailed comparison of ¹³C/¹H NMR spectral data of 1 (Table 1) and those reported for dictyopanine C revealed that 1 similarly featured an ester formed of a triene fatty acid moiety and an eremophilane sesquiterpene alcohol.

Figure 4.

Key ¹H–¹H COSY and HMBC correlations of 1–4.

To further confirm the depicted structure of 1, its HMBC spectrum was acquired and the results (Figures 4 and S6) revealed key correlations from H-1 and H-3’ to C-1’ (δ_C 168.0) confirming that the ester linkage is present at C-1 of the eremophilane moiety and from H-1 to two other carbon atoms, one methine at δ_C 63.1 (C-9) and one unprotonated at δ_C 68.9 (C-10) suggesting the presence of an epoxide ring between C-9 and C-10. In addition, the HMBC spectrum of 1 revealed key correlations from an olefinic methyl group at δ_H 2.28 (d, J = 2.6 Hz, H₃-13) to C-7 (δ_C 147.9), C-11 (δ_C 126.7), and C-12 (δ_C 174.6) suggesting its binding at C-11 and to be neighbouring a terminal carboxylic acid moiety; from a diastereotopic methylene H₂-6 at δ_H 2.46 (dd, J = 16.0, 2.8 Hz)/2.66 (d, J = 16.0 Hz) to a ketocarbonyl carbon at δ_C 197.1 (C-8); from a hydroxymethylene moiety (H₂-14) to C-3 (δ_C 21.6)/C-4 (δ_C 45.7)/C-5 (δ_C 39.1) suggesting its binding at C-4; and from a singlet methyl group at δ_H 1.28 (s, H₃-15) to C-4, C-5, and C-6 (δ_C 35.7) suggesting its binding at C-5. These HMBC correlations confirmed the depicted structure of 1 (Figure 4) as a tricylic eremophilane sesquiterpene alcohol esterified to a triene fatty acid. The relative configuration of 1 was determined via its ROESY spectrum (Figures 5, S8) that revealed key ROE correlations between H-1/H-9 indicating their presence toward the same face of the molecule and from H₃-15/Hα-6/Hα-14 indicating their projection toward the opposite face of the molecule.

Figure 5.

Key ROESY correlations of 1–4.

The absolute configuration of 1 was established from the similarity between its measured and calculated TDDFT-ECD spectra (Figure 6). As shown in Figure 6, a close coherence was found between the experimental ECD spectrum and that predicted for the (1R,4S,5R,9R,10S,8’R) configuration throughout the entire range. From these findings, compound 1 was identified as a previously undescribed eremophilane sesquiterpene ester named stchigelomycin A.

Figure 6.

Measured and calculated ECD spectra of 1 in MeOH.

Compound 2 was isolated as a pale-brown amorphous solid. The HR-ESI-MS spectrum of 2 (Figure S10) displayed a protonated molecular ion peak and a sodium adduct at m/z 457.2576 [M + H]⁺ (calculated 457.2585) and 479.2392 [M + Na]⁺ (calculated 479.2404), respectively, establishing its molecular formula as C₂₇H₃₆O₆ indicating ten degrees of unsaturation as in 1. Comparison of the molecular formulas of 1 and 2 indicated that compound 2 is a deoxygenated derivative of 1. A careful investigation of ¹³C and ¹H NMR spectral data of 2 compared to 1 (Table 1, Figures S11, S12) revealed the presence of a doublet methyl group at δ_H 0.93 (d, J = 6.7 Hz, H₃-14; δ_C 15.6) in 2 replacing the diastereotopic hydroxymethylene moiety at δ_H 3.31/3.83 (H₂-14) in 1. Apart from this sole difference between 1 and 2, their ¹³C and ¹H NMR spectral data revealed a close similarity suggesting a related chemical structure of 2 (Figure 3) as an eremophilane sesquiterpene alcohol esterified to a triene fatty acid. The ¹H–¹H COSY spectrum of 2 (Figures 4, S13) revealed three spin systems comparable to those recognized in 1.

Further confirmation to the depicted structure of 2 (Figure 3) was obtained through its HMBC spectrum that revealed comparable key correlations to 1 in addition to key correlations from H₃-14 to C-3 (δ_C 26.7), C4 (δ_C 37.9), and C-5 (δ_C 39.9), confirming its binding at C-4. The HMBC spectrum of 2, similar to 1, revealed key correlations from H-1 at δ_H 4.65 (t, J = 3.0 Hz) and H-3’ at δ_H 7.37 (dd, J = 15.2, 11.0 Hz) to C-1’ (δ_C 167.9) confirming that the ester linkage is present at C-1 of the eremophilane moiety and from H-1 to another two carbon atoms, one methine at δ_C 63.1 (C-9) and one unprotonated at δ_C 69.0 (C-10) suggesting the presence of an epoxide ring between C-9 and C-10. The relative configuration of 2 was determined via its ROESY spectrum (Figures 5, S16) that correlated H-1 and H-9 (δ_H 3.22, s), thus indicating their projection toward the same face of the molecule, whereas H₃-14 and H₃-15 were differently correlated indicating their presence toward the opposite face of the molecule. The absolute configuration of 2 was established based on the comparison of its experimental ECD and the calculated TDDFT-ECD (Figure 7) that revealed a good fitting with (1R,4S,5R,9R,10S,8’R) configuration. Accordingly, compound 2 was characterized as a previously undescribed eremophilane sesquiterpene ester named stchigelomycin B.

Figure 7.

Measured and calculated ECD spectra of 2 in MeOH.

Compound 3 was purified as a dark-brown amorphous solid with a molecular formula determined as C₂₇H₃₈O₆ based on its HR-ESI-MS results that revealed a protonated molecular ion peak at m/z 459.2732 [M + H]⁺ (calculated 459.2741) and a sodium adduct at m/z 481.2549 [M + Na]⁺ (calculated 481.2561) indicating nine degrees of unsaturation. The DEPTQ and HSQC spectral data of 3 (Table 1, Figures S20 and S23) revealed twenty-seven carbon signals, categorized into six unprotonated, including one carbonyl (δ_C 168.0), two olefinics (δ_C 152.2, 134.1), and three aliphatics (δ_C 102.7, 65.3, 38.3), eleven methines divided into six olefinic including one exocyclic methylene (δ_C 148.0, 147.6, 146.1, 125.0, 120.3, 105.5) and five aliphatic (77.2, 63.6, 45.6, 44.3, 36.0) carbon atoms. In addition, the DEPTQ and HSQC spectral data of 3 also revealed six methylenes (δ_C 70.0, 63.3, 36.2, 31.3, 30.6, 21.4) and four methyls (δ_C 20.7, 20.4, 12.7, 12.3). The obtained results accounted for five degrees of unsaturation and thus indicated that compound 3 comprises a tetracyclic structure. The ¹H NMR and ¹H–¹H COSY spectra of 3 (Table 1, Figures 4, S19, S21) revealed four spin systems: 1) between four trans olefinic proton signals at δ_H 5.92 (d, J = 15.2 Hz, H-2’), 7.36 (dd, J = 15.2, 11.0 Hz, H-3’), 6.36 (dd, J = 15.3, 11.0 Hz, H-4’), and 6.65 (d, J = 15.3 Hz, H-5’); 2) a spin system extending along one olefinic proton at δ_H 5.54 (d, J = 9.7 Hz, H-7’), an aliphatic methine at δ_H 2.48 (dp, J = 14.4, 6.6 Hz, H-8’) coupled to a doublet methyl group at δ_H 0.99 (d, J = 6.6 Hz, H₃-12’), a diastereotopic methylene group at δ_H 1.31/1.42 (H₂-9’) ending at a triplet terminal methyl group at δ_H 0.86 (t, J = 7.4 Hz, H₃-10’); 3) from an oxygenated aliphatic methine proton at δ_H 4.66 (t, J = 3.0 Hz, H-1) to two diastereotopic methylene groups at δ_H 1.77/2.05 (H₂-2) and 1.69/1.85 (H₂-3) to another methine proton at δ_H 1.91 (ddd, J = 12.9, 9.0, 4.8 Hz, H-4) ending at an oxygenated diastereotopic methylene group at δ_H 3.41/3.83 (H₂-14); 4) between a diasteretopic methylene group at δ_H 1.26/1.62 (H₂-6) and a methine proton at δ_H 2.67 (dd, J = 14.2, 5.2 Hz, H-7). According to the obtained results, compound 3 was suggested to be an ester derivative of an eremophilane sesquiterpene alcohol and a triene fatty acid similar to 1 and 2. To confirm the depicted structure of 3 (Figure 3), its HMBC spectrum (Figures 4 and S22) was acquired and it revealed key correlations from H-1 and H-3’ to C-1’ (δ_C 168.0) confirming that the ester linkage is present at C-1 of the eremophilane moiety and from H-1 to two other carbon atoms, one methine at δ_C 63.6 (C-9) and one unprotonated at δ_C 65.3 (C-10) suggesting the presence of an epoxide ring between C-9 and C-10. The HMBC spectrum of 3 also revealed key correlations from a terminal olefinic methylene group at δ_H 4.99/5.00 to C-7 (δ_C 45.6), C-12 (δ_C 152.2), and C-11 (δ_C 70.0) suggesting its binding at C-12; from a diastereotopic methylene H₂-6 at δ_H 1.26 (dd, J = 14.4, 14.2 Hz)/1.62 (dd, J = 14.4, 5.3 Hz) to C-5 (δ_C 38.3), C-7, C-8 (δ_C 102.7), and C-10; from a hydroxymethylene moiety (H₂-14) to C-3 (δ_C 21.4)/C-4 (δ_C 44.3)/C-5 (δ_C 38.3) suggesting its binding at C-4; and from a singlet methyl group at δ_H 1.18 (s, H₃-15) to C-4, C-5, and C-6 (δ_C 36.2) suggesting its binding at C-5. These HMBC correlations confirmed the depicted structure of 3 (Figures 3 and 4) as a tetracylic eremophilane sesquiterpene alcohol esterified to a triene fatty acid. The relative configuration of 3 was determined by acquiring its ROESY spectrum (Figures 5, S24). The obtained results revealed key ROE correlations from H-1/H-9 and H-7/H-4 indicating their presence toward the same plane of the molecule, whereas a key ROE correlation was identified between H₃-14/H₃-15, indicating their presence toward the opposite face of the molecule. The absolute configuration of 3 was established by comparing its experimental results with the calculated ECD spectra (Figure 8), showing a pattern consistent with the conformer featuring (1R, 4S,5R,7R,8R,9R,10S,8’R) configuration. Accordingly, compound 3 was identified as a previously undescribed eremophilane sesquiterpene ester that was trivially named stchigelomycin C.

Figure 8.

Measured and calculated ECD spectra of 3 in MeOH.

Compound 4 was isolated as a pale-brown amorphous solid. Its molecular formula was established as C₂₇H₃₈O₅ supported by the HR-ESI-MS (Figure S26) that displayed a protonated molecular ion peak at m/z 443.2785 [M + H]⁺ (calculated 443.2792) and a sodium adduct at m/z 465.2602 [M + Na]⁺ (calculated 465.2611), hence suggesting nine degrees of unsaturation as in 3. Comparison of the molecular formulas of 3 and 4 indicated that the latter is a deoxygenated derivative of 3 interpreting its lessened molecular weight by 16 Da. A thorough investigation of ¹³C and ¹H NMR spectral data of 4 compared to 3 (Table 1) suggested the presence of a doublet methyl group at δ_H 0.99 (d, J = 6.3 Hz, H₃-14; δ_C 14.9) in 4 replacing the diastereotopic hydroxymethylene moiety at δ_H 3.41/3.83 (H₂-14) in 3. Apart from this only difference between 3 and 4, their ¹³C and ¹H NMR spectral data (Table 1) showed a close coherence, suggesting a similar chemical scaffold of 4 (Figure 3) as an eremophilane sesquiterpene alcohol esterified to a triene fatty acid. The ¹H–¹H COSY spectrum of 4 (Figures 4, S29) revealed three spin systems comparable to those recognized in 3. Further confirmation for the proposed structure of 4 (Figure 3) was obtained through its HMBC spectrum (Figures 4, S30) that suggested comparable key correlations to 3 in addition to key correlations from H₃-14 to C-3 (δ_C 26.2), C4 (δ_C 36.1), and C-5 (δ_C 38.7), confirming its binding at C-4. The HMBC spectrum of 4, similar to 3, revealed key correlations from H-1 at δ_H 4.65 (t, J = 2.8 Hz) and H-3’ at δ_H 7.36 (dd, J = 15.3, 11.1 Hz) to C-1’ (δ_C 167.7) confirming that the ester linkage is present at C-1 of the eremophilane moiety and from H-1 to another two carbon atoms, one methine at δ_C 63.2 (C-9), and one unprotonated at δ_C 65.3 (C-10) suggesting the presence of an epoxide ring between C-9 and C-10. The relative configuration of 4 was determined via its ROESY spectrum (Figures 5, S32) that correlated H-1 and H-9 (δ_H 3.09, s), thus indicating their projection toward the same face of the molecule, whereas H₃-14 and H₃-15 were differently correlated indicating their presence toward the opposite face of the molecule. The absolute configuration of 4 was determined by comparing its experimental results with the calculated ECD spectra (Figure 9), showing a pattern consistent with the conformer featuring (1R,4S,5R,7R,8R,9R,10S,8’R) configuration. Accordingly, compound 4 was identified as a previously undescribed eremophilane sesquiterpene ester named stchigelomycin D.

Figure 9.

Measured and calculated ECD spectra of 4 in MeOH.

Compound 6 was yielded as a dark-brown amorphous solid. The HR-ESI-MS of 6 (Figure S42) established its molecular formula as C₁₇H₂₄O₅ suggesting six degrees of unsaturation by revealing a protonated molecular ion peak and a sodium adduct at m/z 309.1689 [M + H]⁺ (calculated 309.1697) and 331.1506 [M + Na]⁺ (calculated 331.1516), respectively. The DEPTQ and HSQC spectra of 6 (Table 2, Figures S44, S47) revealed seventeen carbon resonances that were differentiated into six unprotonated including one carbonyl (δ_C 169.1, 155.2, 149.6, 128.5, 111.4, 71.6), four methines (δ_C 137.8, 112.0, 79.8, 78.3), four methylenes (δ_C 33.8, 32.0, 26.35, 21.9), and three methyls (δ_C 26.42, 24.4, 13.8). These findings accounted for four degrees of unsaturation revealing that compound 6 features a bicyclic structure. The ¹H NMR and ¹H–¹H COSY spectral data of 6 (Table 2, Figures 10, S43, S45) revealed three distinguished spin systems: 1) between two doublet ortho-coupled aromatic proton signals at δ_H 6.91 and 7.46 with an equal coupling constant (J value) of 7.5 Hz ascribed to H-4 and H-5, respectively; 2) from an oxygenated methine at δ_H 3.39 (dd, J = 10.1, 1.8 Hz, H-2’) to a diastereotopic methylene group at δ_H 2.52 (dd, J = 14.3, 10.1 Hz, Hα-1’) and 2.94 (dd, J = 14.3, 1.8 Hz, Hβ-1’); 3) from an oxygenated methine at δ_H 5.44 (dd, J = 7.6, 3.9 Hz, H-8) to three consecutive methylene groups at δ_H 1.65/1.98 (H₂-9), 1.34 (H₂-10), and 1.32 (H₂-11) ending at a terminal triplet methyl group at δ_H 0.87 (t, J = 7.0 Hz, H₃-12). Supported by the obtained results, a literature search of 6 revealed its structural relation to vaccinols and emerfurans, prenylated phthalide derivatives, previously reported from Pestalotiopsis vaccinii (Wang et al. 2014, 2015, 2017) and Aspergillus (syn: Emericella) sp. IFM57991 (Saito et al. 2016), respectively. A detailed investigation of ¹H and ¹³C NMR spectral data of 6 revealed its close similarity to vaccinol Q (Wang et al. 2017) apart from being deoxygenated at C-10/C11 and deprived of the oxygenated methyl group at C-2’. Further confirmation for the depicted structure of 6 (Figure 3) was attained via its HMBC spectrum (Figures 10, S46) that revealed key correlations from H-8 to C-1 (δ_C 169.1), C-2 (δ_C 111.4), C-7 (δ_C 149.6), C-9 (δ_C 33.8), and C-10 (δ_C 26.35), confirming the binding of the unbranched aliphatic side chain at C-8. The HMBC spectrum of 6 also revealed key correlations from a diastereotopic methylene group (H₂-1’) to C-5 (δ_C 137.8), C-6 (δ_C 128.5), C-7 (δ_C 149.6), C-2’ (δ_C 78.3), and C-3’ (δ_C 71.6). In addition, two singlet methyl groups H₃-4’/H₃-5’ revealed key correlations to C-2’/C-3’ indicating the presence of 2,5-dihydroxy-3-isopentyl moiety binding at C-6. The relative configuration of 6 was elucidated through its ROESY spectrum (Figures 10, S48) that revealed key ROE correlations from H-8/Hα-10 and from Hα-1’/H-2’ suggesting the projection of each pair toward the same face of the molecule.

Table 2.

¹³C and ¹H NMR data of 6 and 7.

Position	6		7a		7b
	δC,a type	δHb multi (J [Hz])	δC,a type	δHb multi (J [Hz])	δC,a type	δHb multi (J [Hz])
1	169.1, CO		106.9, CH	6.09 br s	106.5, CH	6.17 d (2.0)
2	111.4, C		113.32, C		113.05, C
3	155.2, C		152.54, C		152.59, C
4	112.0, CH	6.91 d (7.5)	113.3, CH	6.674 d (7.5)	113.0, CH	6.669 d (7.5)
5	137.8, CH	7.46 d (7.5)	134.13, CH	7.15 d (7.5)	134.15, CH	7.16 d (7.5)
6	128.5, C		127.9, C		128.0, C
7	149.6, C		145.26, C		145.10, C
8	79.8, CH	5.44 dd (7.6, 3.9)	85.13, CH	5.03 dd (8.4, 4.0)	84.26, CH	5.29 dt (6.3, 2.8)
9	33.8, CH2	α 1.65 ddt (14.2, 9.9, 6.2) β 1.98 m	38.74, CH2	α 1.70 m β 1.81 m	36.44, CH2	α 1.61 m β 1.90 m
10	26.35, CH2	1.34 m	23.71, CH2	1.38 m	23.77, CH2	1.38 m
11	21.9, CH2	1.32 m	14.4, CH3	0.94 t (7.0)	14.4, CH3	0.91 t (7.0)
12	13.8, CH3	0.87 t (7.0)	54.67, CH3	3.44 s	54.22, CH3	3.38 s
1’	32.0, CH2	α 2.52 dd (14.3, 10.1) β 2.94 dd (14.3, 1.8)	34.24, CH2	α 2.68 dd (14.3, 10.0) β 2.94 dd (14.3, 1.8)	34.21, CH2	α 2.71 dd (14.3, 10.2) β 2.92 dd (14.3, 1.7)
2’	78.3, CH	3.39 dd (10.1, 1.8)	81.07, CH	3.56 dd (10.0, 1.8)	81.19, CH	3.55 dd (10.2, 1.7)
3’	71.6, C		73.73, C		73.70, C
4’	24.4, CH3	1.10 s	24.63, CH3	1.10 s	24.58, CH3	1.10 s
5’	26.42, CH3	1.12 s	25.64, CH3	1.11 s	25.68, CH3	1.11 s

Measured in DMSO-d₆ at ^a125 MHz for ¹³C and ^b500 MHz for ¹H.

Figure 10.

Key ¹H–¹H COSY, HMBC, and ROESY correlations of 6 and 7.

The absolute configuration of 6 was established by comparing its experimental and calculated TDDFT-ECD spectra (Figure 11), and these findings indicated a close coherence of the measured ECD spectrum to that calculated for the conformer having (8S,2’S) configuration. According to these results, compound 6 was characterized as a previously undescribed prenylated phthalide derivative that was trivially named vaccinol T.

Figure 11.

Measured and calculated ECD spectra of 6 in MeOH.

Compounds 7a and 7b were obtained as an inseparable mixture that appeared as a pale-brown amorphous solid. The HR-ESI-MS spectrum of 7a/7b (Figure S50) showed a protonated molecular ion peak at m/z 293.1740 [M + H]⁺ (calculated 293.1747) and a sodium adduct at m/z 315.1558 [M + Na]⁺ (calculated 315.1567) that established its molecular formula as C₁₇H₂₄O₄ indicating six degrees of unsaturation. Despite revealing a single peak in LR- and HR-ESI-MS (Figures S49, S50), the ¹H NMR and DEPTQ spectral data of 7 (Table 2, Figures S51, S52) revealed two comparable sets of proton and carbon signals that suggested being an inseparable mixture of two epimeric compounds (7a/b) in 2:3 ratio. The ¹H–¹H COSY spectrum of 7 (Figures 10, S53) revealed a pair of three comparable spin systems and each of them corresponds to one stereoisomer as follows: 1) between two aromatic proton signals H-4/H-5; 2) from a methine signal at H-8 to a diastereotopic methylene group (H₂-9) followed by a second methylene group (H₂-10) and ending by a terminal triplet methyl group (H₃-11); 3) between a diastereotopic methylene group (H₂-1’) to a methine signal (H-2’). Based on the results obtained, a literature search of 7 indicated being a pair of isomeric prenylated phthalide derivatives also structurally related to vaccinols (Wang et al. 2014, 2015, 2017) and emerfurans (Saito et al. 2016). The HMBC spectrum of 7 (Figures 10, S54) further confirmed the depicted structure of 7 through revealing key correlations from H₂-1’, H₃-4’, and H₃-5’ to C-2’ (δ_C 81.07/81.19) and C-3’ (δ_C 73.73/73.70) suggesting the presence of an epoxide ring between C-2’ and C-3’ by comparing to the reported literature (Pripdeevech et al. 2024). The HMBC spectrum of 7 (Figures 10, S54) further revealed key correlations from H₂-1’ to C-5 (δ_C 134.13/134.15), C-6 (δ_C 127.9/128.0), and C-7 (δ_C 145.26/145.10) confirming the binding of 2,5-epoxy-3-isopentyl moiety at C-6. In addition, the key HMBC correlations from H-8 and OCH₃-12 to C-1 (δ_C 106.9/106.5) confirm the presence of 1-methoxyfuran moiety fused to an aromatic ring, resulting in the depicted structure of 7 as a prenylated phthalide derivative.

The relative configuration of 7 was established based on acquiring its ROESY spectrum (Figures 10, S56) that revealed comparable key ROE correlations of both isomers such as those from H-2’ to H-5, H₂-1’, and Me-4’. These ROE correlations, together with revealing similar coupling constants (J value), support that H-2’ adopts an axial orientation in both 7a and 7b. The ROESY spectrum (Figures 10, S55) revealed key correlation between H-1 at δ_H 6.17 (d, J = 2.0 Hz) and H-8 at δ_H 5.29 (dt, J = 6.3, 2.8 Hz) in 7b suggesting their orientation toward the same face of the molecule while their counterparts in 7a didn’t reveal any correlation suggesting their opposite orientation. The higher coupling constants (J value) of H-8 in 7a compared to 7b suggesting its axial and equatorial orientations, respectively. In conclusion, the ROESY spectrum together with the interpretation of coupling constants (J value) of H-1, H-8, and H-2’ assigned the absolute configurations of 7a and 7b as (1R,8S,2’S) and (1R,8R,2’S), respectively. According to the data presented above, compound 7 was identified as an inseparable mixture of two isomeric phtalide derivatives (7a and 7b) that were named vaccinols U₁ and U₂, respectively.

3.4. Antimicrobial and cytotoxic activities

Compounds 1–7 were assessed for their antimicrobial activities against a panel of fungal and bacterial pathogens, including Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis, Mycolicibacterium smegmatis), Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumanni, Chromobacterium violaceum), yeast (Wickerhamomyces anomalus, Candida albicans, Schizosaccharomyces pombe, Rhodotorula glutinis), and filamentous fungi (Mucor hiemalis) (Table 3). Among the isolated compounds, stchigelomycin C (3) exhibited strong inhibitory effects against tested Gram-positive bacteria, yeast, and filamentous fungi, particularly B. subtilis and M. hiemails, at a concentration of 8.3 μg/mL. Notably, its inhibitory effect on B. subtilis was more pronounced than that observed for the positive control, oxytetracycline (MIC = 16.6 μg/mL). In contrast, stchigelomycin B (2) showed lower activity than stchigelomycin C (3), with MIC values of 16.6 μg/mL against B. subtilis and 33.3 μg/mL against M. hiemails. Stchigelomycin A (1), stchigelomycin D (4), and compound 5 exhibited rather weak or negligible antimicrobial activities (MIC ≥ 66.6 μg/mL). Neither vaccinol T (6) nor vaccinols U1/U2 (7a/7b, 2:3) exhibited significant antimicrobial activity.

Table 3.

Cytotoxicity and antimicrobial activity of compounds 1–7.

	IC50 (µmol/L)							Positive control
Test cell line	1	2	3	4	5	6	7	Epothilone B (µg/mL)
Mouse fibroblast (L929)	23	14	6.8	–	21	–	–	0.00098
Human endocervival adenocarcinoma (KB3.1)	14	5.8	5.4	22	21	–	–	0.000028
Human prostate carcinoma (PC-3)	n.d	n.d	2.6	n.d	n.d	n.d	n.d	0.00075
Human breast adenocarcinoma (MCF-7)	n.d	n.d	7.2	n.d	n.d	n.d	n.d	0.0026
Human ovarian cancer (SKOV-3)	n.d	n.d	6.3	n.d	n.d	n.d	n.d	0.0016
Human epidermoid carcinoma (A431)	n.d	n.d	2.3	n.d	n.d	n.d	n.d	0.000041
Human lung carcinoma (A549)	n.d	n.d	5.1	n.d	n.d	n.d	n.d	0.000078
Test microorganism	MIC (µg/mL)							Positive control (µg/mL)
Acinetobacter baumanni (DSM 30008)	–	–	–	n.d	–	–	–	0.53C
Bacillus subtilis (DSM 10)	66.6	16.6	8.3	66.6	66.6	–	–	16.6O
Candida albicans (DSM 1665)	–	–	–	–	–	–	–	2.1N
Chromobacterium violaceum (DSM 30191)	–	–	–	n.d	–	–	–	0.83G
Escherichia coli (DSM 116)	–	–	–	–	–	–	–	0.42G
Mucor hiemalis (DSM 2656)	66.6	33.3	8.3	n.d	66.6	–	–	2.10N
Mycolicibacterium smegmatis (ATCC 700084)	–	–	33.3	–	–	–	–	0.10K
Pseudomonas aeruginosa (PA 14)	–	–	–	–	–	–	–	0.21G
Rhodotorula glutinis (DSM 10134)	–	–	66.6	n.d	–	–	–	1.00N
Schizosaccharomyces pombe (DSM 70572)	–	–	–	n.d	–	–	–	4.20N
Staphylococcus aureus (DSM 346)	–	33.3	16.6	66.6	66.6	–	–	0.42G
Wickerhamomyces anomalus (DSM 6766)	–	–	–	n.d	–	–	–	0.42N

IC₅₀: Half-maximal inhibitory concentration, µg/mL. MIC: Minimum inhibitory concentration in µg/mL. “-“: No activity under test conditions (MIC > 66.6 µg/mL, IC₅₀ > 37). n.d: Not determined. G: Gentamicin. O: Oxytetracycline. N: Nystatin. C: Ciprofloxacin. K: Kanamycin.

On the other hand, cytotoxic assays revealed that stchigelomycin C (3) demonstrated the highest cytotoxic activity against seven different cell lines, aligning with its antimicrobial activities. Meanwhile, stchigelomycin A (1), stchigelomycin B (2), stchigelomycin D (4), and compound 5, presented mild cytotoxicity against the mouse fibroblast cell line L929 and the human endocervival adenocarcinoma KB 3.1. In contrast, vaccinol T (6) and vaccinols U1/U2 (7a/7b, 2:3) did not show any significant cytotoxicity against these two cell lines.

4. Discussion

Even though numerous secondary metabolites have been discovered within the order Sordariales, many taxonomic groups and even entire lineages remain insufficiently investigated. Several families, such as Podosporaceae, Diplogelasinosporaceae, Lasiosphaeriaceae, and Schizotheciaceae, remain largely neglected (Charria-Girón et al. 2022). Even within well-investigated families such as Chaetomiaceae, newly recognized lineages have yielded distinct and previously unknown metabolites. For example, investigations into the less-studied genus Tengochaeta revealed unique metabolic profiles that differ from those observed in the related genus Amesia, highlighting the chemical potential hidden within underrepresented lineages (Charria-Girón et al. 2024; Barrera-Adame et al. 2025). These findings point to the importance of conducting systematic investigations across all phylogenetic branches to fully uncover the chemical diversity of this order. Moreover, increasing evidence suggests that greater phylogenetic divergence often correlates with higher biosynthetic novelty. For instance, a novel linkage-based algorithm was employed to analyze the genomes of 101 species of Dothideomycetes, revealing limited overlap in BGC repertoires across genera and showing a linear increase in repertoire diversity with repertoire size (Gluck-Thaler et al. 2020).

In this study, compounds 1–7 exhibited distinct antimicrobial and cytotoxic profiles. Stchigelomycin C (3) displayed the broadest and most promising antimicrobial spectrum. It exhibited remarkable inhibitory effects against B. subtilis and M. hiemalis (MIC = 8.3 μg/mL), surpassing the potency of the reference antibiotic oxytetracycline (MIC = 16.6 μg/mL) against B. subtilis. Its strong activity against some Gram-positive bacteria, yeasts, and filamentous fungi suggests its potential applications as a broad-spectrum antimicrobial agent. Compared to stchigelomycin B (4), the superior activity of 3 may be attributed to the hydroxymethyl (–CH₂OH) substitution in 3, as opposed to the methyl (–CH₃) group in 4. The hydroxymethyl group in 3 serves as both a hydrogen-bond donor and acceptor, enabling stronger interactions with biological targets compared to the non-polar methyl group in 4 (Santos et al. 2021). Similar trends have been observed in other groups of fungal metabolites. For example, in antifungal polyene macrolides, the hydroxyl groups contribute to improving the stability of antibiotic-ergosterol interactions in membrane, strengthening bioactivity (Qiao et al. 2023). However, mild to moderate cytotoxic effects of stchigelomycins might limit their applicability, their bioactivity could still be improved by chemical derivatization on future projects. While vaccinol T (6) and vaccinols U₁/U₂ (7a/7b) showed no significant antimicrobial or cytotoxic activities, their phenolic and lactone-rich scaffold suggests the potential for other biological functions, such as antioxidant or enzyme-modulatory activities, which have been reported for similar structural classes (Platzer et al. 2022). It was reported that vaccinol J isolated from Pestalotiopsis vaccinii possesses inhibitory effect against enterovirus 71 without cytotoxicity (Wang et al. 2017). The absence of toxicity may also make these compounds useful as non-toxic reference structures for future synthetic modification.

5. Conclusions

This study introduces Stchigelomyces as a new genus within the Podosporaceae and demonstrates its capacity to produce structurally unique secondary metabolites, including four novel eremophilane meroterpenoids with promising antimicrobial activity. The discovery of this bioactive scaffold within a neglected family highlights the need to investigate closely related taxa to determine whether the production of stchigelomycins is restricted to the newly established genus or more widespread across the Podosporaceae. By linking fungal systematics with secondary metabolite screening, this work underscores the potential of unexplored lineages to expand the accessible chemical space and to contribute to the discovery of novel bioactive natural products.

Funding Statement

This work was funded by the Deutsche Forschungsgemeinschaft (DFG), [490821847] (granted to Y.M.-F.), and the Landwirtschaftliche Rentenbank, Germany. This study benefited from the European Union’s Horizon 2020 research and innovation program (RISE) under the Marie Skłodowska-Curie Grant Agreement [101008129], Project Acronym “Mycobiomics”. The Alexander von Humboldt (AvH) Foundation funded S.S.E. through the Georg-Forster Fellowship for Experienced Researchers [Ref 3.4-1222288-EGY-GF-E]. The fungal isolate was obtained thanks to the “Fungi for the future” Citizen Science Project from the WI. L.Z. was supported by the China Scholarship Council (CSC). M.A.-R. was supported by a personal PhD stipend from the German Academic Exchange Service (DAAD) Program.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author contributions

Conceptualization: L.Z., E.C.-G., S.S.E., and Y.M.-F.; methodology: L.Z., M.H.-R., M.A.-R., E.C.-G., M.A.A.I., S.S.E., and Y.M.-F.; software: L.Z., M.H.-R., M.A.-R., E.C.-G., S.S.E., and Y.M.-F.; formal analysis: L. Z., M.A.A.I., S.S.E., and Y.M.-F.; investigation: L.Z., M.A.-R., E.C.-G., and Y.M.-F.; data curation: L.Z., M.H.-R., M.A.-R., E.C.-G., M.A.A.I., S. S.E., and Y.M.-F.; writing-original draft: L.Z., M.H.-R., S.S.E., M.A.A.I., and Y.M.-F.; writing-review and editing: L.Z., M.H.-R., E.C.-G., M.A. A.I., S.S.E., and Y.M.-F.; resources: M.H.-R. and Y.M.-F.; project administration: S.S.E. and Y.M.-F.; supervision: S.S.E. and Y.M.-F.; funding acquisition: S.S.E. and Y.M.-F. All authors have read and agreed to the published version of the manuscript.

Data availability statement

All data related to structure elucidation, bioassays, and phylogenetic analysis are available as Supplementary Material. The DNA sequences are deposited in GenBank (https://www.ncbi.nlm.nih.gov/genbank/).

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/21501203.2026.2616893