Antibacterial, antibiofilm, and metabolomic profiling of the novel freshwater fungi Longipedicellata megafusiformis and Wicklowia fusiformispora

Abstract

Freshwater lignicolous fungi represent an underexplored reservoir of bioactive secondary metabolites, yet their chemical diversity and antimicrobial potential remain insufficiently characterized. In this study, we investigated the antibacterial and antibiofilm properties of two recently described species, Longipedicellata megafusiformis and Wicklowia fusiformispora, by integrating in vitro biological assays with high-resolution LC–QQQ–MS–based metabolomic profiling. Crude mycelial ethyl acetate (EtOAc) extracts of both fungi exhibited inhibitory activity against pathogenic bacteria, while L. megafusiformis additionally displayed measurable biofilm-preventive effects. Untargeted LC–MS annotation revealed 27 metabolites in L. megafusiformis and 33 in W. fusiformispora, spanning alkaloids, peptides, polyketides, terpenoid derivatives, saponins, and carotenoid-related pigments. Several annotated metabolites in L. megafusiformis have well-documented antibacterial activities, including glucosinalbin, distomadine B, agelasidine A, sugikurojin B, 6,8-diprenylorobol, septacidin, ambiguine isonitrile, bachitrocin C, epipachysamine D, spirilloxanthin, nodulisporic acid E, and anhydrorhodovibrin, providing a coherent chemical rationale for the observed antibacterial phenotype. Notably, compounds with validated antibiofilm activity were restricted to spiroleucidine-type scaffolds and carotenoid derivatives such as spirilloxanthin. Importantly, the present study does not assign antibiofilm activity to individual compounds, and the observed effects should be interpreted at the crude-extract level. The metabolome of W. fusiformispora was dominated by structurally distinct antibacterial metabolites, including betaine, bengamide E, aspidocarpine, cyanocycline B, jantithrem G, chrysosporide, ajudazol A, plakinamine F, and several saponin-type compounds. However, no metabolites with experimentally confirmed antibiofilm function were detected in this species. The combined datasets reveal two complementary antimicrobial chemotypes: one enriched in multi-scaffold antibacterial and antibiofilm constituents (L. megafusiformis), and another characterized by a broad yet exclusively antibacterial metabolome (W. fusiformispora). These findings highlight freshwater fungi as promising sources of chemically diverse antimicrobial agents and demonstrate the value of integrating untargeted metabolomics with classical bioassays as a foundation for future isolation-guided and mechanistic studies.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-36637-5.

Introduction

Freshwater fungi are ecologically important decomposers that contribute to organic matter turnover and nutrient cycling in aquatic ecosystems¹. Among these, saprobic lignicolous fungi colonizing submerged decaying wood play a central role in degrading lignocellulosic substrates through diverse hydrolytic and oxidative enzymes^2–4. In addition to enzymatic degradation, these fungi synthesize a broad spectrum of secondary metabolites that mediate chemical defense, competition, and ecological interactions⁵. Despite their ecological significance, the metabolite diversity of freshwater fungi remains poorly characterized, particularly in tropical regions where warm, humid environments support high fungal diversity and specialization^6,7.

The global rise of multidrug-resistant (MDR) pathogens has intensified efforts to identify new antimicrobial agents from natural sources⁸. Fungi have historically yielded transformative drugs including penicillins, cephalosporins, and echinocandins demonstrating their pharmaceutical potential^9–11. However, chemical investigations have focused primarily on terrestrial and marine fungi, leaving freshwater saprobes comparatively understudied^12,13. Recent studies indicate that fungi inhabiting submerged wood can produce structurally diverse polyketides, terpenoids, peptides, and alkaloids with antimicrobial activities^14–16, suggesting that freshwater habitats constitute underexplored reservoirs of bioactive metabolites. Beyond inhibiting planktonic bacterial cells, natural products capable of interfering with biofilm formation are urgently needed due to the clinical challenges posed by biofilm-associated infections¹⁷. Biofilms, consisting of microbial cells embedded within an extracellular polymeric matrix, confer increased resistance to antibiotics and host immune responses, contributing to persistent infections and medical device failures¹⁸. Fungal natural products especially those from unstudied taxa, represent promising antibiofilm agents, yet only a small proportion of freshwater fungi have been assessed for such activity^19,20.

During a survey conducted by the Center of Excellence in Fungal Research (CEFR), Mae Fah Luang University, two previously undescribed lignicolous fungal species, L. megafusiformis and W. fusiformispora, were isolated from submerged decaying wood in northern Thailand. Multigene phylogenetic analyses (ITS, LSU, SSU, TEF1-α) confirmed both as new taxa within Pleosporales²¹. No prior studies have examined their chemical constituents or biological activities, leaving their biosynthetic potential completely unexplored. To investigate their capacity for bioactive metabolite production, mycelial and broth crude EtOAc extracts of both ex-type cultures were evaluated for antibacterial and antibiofilm activity against clinically relevant gram-positive and gram-negative pathogens. These assays targeted both planktonic bacterial growth and biofilm formation to provide an integrative antimicrobial assessment. Because pathogens such as Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli are major contributors to biofilm-related infections, identifying fungal metabolites capable of preventing biofilm development holds significant biomedical relevance. Complementing the biological assays, untargeted LC–QQQ–MS metabolomic profiling was performed to characterize the semi-polar metabolite composition of the mycelial extracts. The resulting profiles revealed a chemically rich assemblage including alkaloids, peptides, terpenoid-related compounds, fatty acid derivatives, and nitrogen- or sulfur-containing metabolites structural classes frequently associated with antimicrobial or quorum-sensing-modulating activity in fungi. This study provides the first integrated assessment of the antibacterial, antibiofilm, and metabolomic profiles of L. megafusiformis and W. fusiformispora, expanding current knowledge of freshwater fungal chemistry and highlighting the considerable biosynthetic potential of these newly described taxa. These findings underscore freshwater habitats as promising yet underinvestigated sources of bioactive natural products.

Results

Antibacterial activity

Crude EtOAc extracts from the mycelial and broth cultures of L. megafusiformis and W. fusiformispora exhibited measurable antibacterial activity against all four reference pathogens, as determined by both disc diffusion and broth microdilution assays (Fig. S1 and Fig. 1). Clear zones of growth inhibition were observed around all test discs, indicating that secondary metabolites present in both fungal species effectively suppressed bacterial proliferation. In the disc diffusion assay, extracts of L. megafusiformis produced inhibition zones ranging from 12.4 ± 0.3 to 18.6 ± 0.7 mm. The mycelial extract generated inhibition zones of 15.2 ± 0.3 mm against S. aureus, 14.2 ± 0.5 mm against B. subtilis, 18.6 ± 0.7 mm against E. coli, and *15.8 ± 0.4 mm against P. aeruginosa. The broth extract showed slightly smaller inhibition zones across all strains, with values of 13.5 ± 0.4 mm (S. aureus), 12.4 ± 0.3 mm (B. subtilis), 16.1 ± 0.5 mm (E. coli), and 13.8 ± 0.6 mm (P. aeruginosa).

Fig. 1.

Inhibition zones (mm) of crude EtOAc extracts from mycelial and broth cultures of L. megafusiformis and W. fusiformispora, and ampicillin against selected pathogens All extracts were tested at 50 µg/mL, whereas ampicillin was tested at 10 µg/mL. Values are expressed as mean ± SD. Letters indicate significant differences (p < 0.05) among means according to one-way ANOVA followed by Tukey’s HSD test. Statistical differences represent species-dependent responses to the crude EtOAc extracts rather than selective or mechanistic effects.

Extracts obtained from W. fusiformispora displayed inhibition zones in a similar range, spanning 10.8 ± 0.3 to 16.3 ± 0.3 mm. The mycelial extract produced clear zones of 16.3 ± 0.3 mm (S. aureus), 13.4 ± 0.3 mm (B. subtilis), 14.2 ± 0.6 mm(E. coli), and 11.7 ± 0.4 mm (P. aeruginosa). Broth extracts yielded smaller but consistent zones of 15.6 ± 0.5 mm, 12.6 ± 0.5 mm, 12.8 ± 0.4 mm, and 10.8 ± 0.3 mm against the respective pathogens. The positive control, ampicillin, produced larger inhibition zones ranging from 20.2 ± 0.4 to 22.3 ± 0.4 mm, confirming the susceptibility of the test strains. MIC and MBC values provided further quantification of antibacterial efficacy (Fig. 2). For L. megafusiformis, MIC values varied between 6.25 and 50.00 µg/mL, depending on the pathogen and extract type. The mycelial extract exhibited MICs of 25.00 µg/mL against S. aureus, 12.50 µg/mL against B. subtilis, 6.25 µg/mL against E. coli, and 12.50 µg/mL against P. aeruginosa. The corresponding MBC values ranged from 12.50 to 50.00 µg/mL. Broth extracts showed MIC values of 6.25–50.00 µg/mL and MBC values extending up to 100.00 µg/mL, with S. aureus and P. aeruginosa requiring higher concentrations for bactericidal action.

Fig. 2.

Minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations (µg/mL) of crude EtOAc extracts from mycelial, broth cultures of L. megafusiformis and W. fusiformispora, and ampicillin against selected pathogens. Letters indicate significant differences (p < 0.05) among means according to one-way ANOVA followed by Tukey’s HSD test. Statistical differences represent species-dependent responses to the crude EtOAc extracts rather than selective or mechanistic effects.

For W. fusiformispora, both extracts demonstrated MIC values in the range of 3.12–25.00 µg/mL, indicating activity against all test pathogens. The mycelial extract showed MIC values of 6.25 µg/mL (S. aureus), 12.50 µg/mL (B. subtilis), 3.12 µg/mL (E. coli), and 12.50 µg/mL (P. aeruginosa). MBC values for this extract ranged from 12.50 to 100.00 µg/mL. The broth extract showed MIC values of 6.25–25.00 µg/mL, with corresponding MBC values extending to 100.00 µg/mL for some pathogens.

Antibiofilm activity

Crude EtOAc extracts obtained from both mycelial and broth cultures of L. megafusiformis and W. fusiformispora exhibited measurable antibiofilm activity against all four bacterial pathogens tested, as summarized in Fig. 3. All extracts produced notable reductions in biofilm biomass relative to the untreated controls, demonstrating that biofilm formation was consistently inhibited across both gram-positive and gram-negative strains. For L. megafusiformis, the mycelial extract showed inhibition values ranging from 58.45 ± 3.51% to 68.34 ± 4.37%, depending on the pathogen. Biofilm inhibition reached 65.33 ± 5.33% in S. aureus, 58.45 ± 3.51% in B. subtilis, 68.34 ± 4.37% in E. coli, and 63.24 ± 4.33% in P. aeruginosa. The broth extract demonstrated lower but still quantifiable antibiofilm activity, with inhibition values of 44.65 ± 2.24% (S. aureus), 42.23 ± 4.34% (B. subtilis), 43.45 ± 5.38% (E. coli), and 40.03 ± 1.54% (P. aeruginosa). Across all strains, the mycelial extract consistently exhibited higher inhibition percentages than the broth extract. Extracts of W. fusiformispora produced higher overall biofilm inhibition compared with L. megafusiformis. The mycelial extract displayed inhibition values of 72.34 ± 3.56% against S. aureus, 67.06 ± 3.48% against B. subtilis, 77.34 ± 1.09%against E. coli, and 51.23 ± 2.87% against P. aeruginosa. The broth extracts also inhibited biofilm formation, though to a lesser extent, with inhibition values of 56.11 ± 2.35% (S. aureus), 53.21 ± 4.22% (B. subtilis), 61.17 ± 1.15% (E. coli), and 42.22 ± 3.83% (P. aeruginosa). The numerical pattern observed across pathogens indicated that the mycelial extract consistently yielded higher inhibition than the broth extract in this species as well. The reference antibiotic ampicillin exhibited the highest levels of biofilm inhibition across all pathogens, with values ranging from 71.29 ± 1.14% to 89.39 ± 0.98%, confirming the suitability of the assay conditions and the responsiveness of each bacterial strain to antibiofilm assessment.

Fig. 3.

Biofilm inhibition (%) of crude EtOAc extracts from mycelial and broth cultures of L. megafusiformis and W. fusiformispora, and ampicillin against selected pathogens. All extracts were tested at 50 µg/mL, whereas ampicillin was tested at 10 µg/mL. Values are expressed as mean ± SD. Letters indicate significant differences (p < 0.05) among means according to one-way ANOVA followed by Tukey’s HSD test. Statistical differences represent species-dependent responses to the crude EtOAc extracts rather than selective or mechanistic effects.

Metabolomic profiling

Untargeted LC–QQQ–MS analysis in positive electrospray ionization mode revealed 27 metabolites in the mycelial crude EtOAc extract of L. megafusiformis (Table 1). The detected compounds exhibited retention times ranging from 9.351 to 24.772 min, with molecular masses between 355.23 and 962.48 Da and mass errors (DB values) within − 3.56 to + 4.23 ppm. The annotated metabolites encompassed structurally diverse groups, including alkaloids, polyketide-derived compounds, peptide-like metabolites, terpenoid-related structures, saponin-like molecules, and highly oxygenated natural products. Representative alkaloid-type metabolites included glucosinlabin, distomadine B, agelasidine A, cycloshermilamine D, naamidine I, ambiguine isonitrile, and himeradine A. Several polyketide-related or aromatic compounds were also detected, such as 3-strictosidine, antillatoxin A, sugikurojin B, 6,8-diprenylorobol, spirilloxanthin, and nodulisporic acid E. Peptide-like or cyclic peptide metabolites detected included phakellistatin, acidissiminin, septacidin, hovenine A, and bactithrocin C. Additional metabolites included diacethylchitin, terrelactone A, trunkamide, taxumain A, taiwachelin, okeneone, epipachsamine D, and anhydrorhodovibrin. All features showed high-confidence annotation based on accurate mass and retention time characteristics.

Table 1.

Putatively annotated metabolites detected in the mycelial crude EtOAc extract of L. megafusiformis by LC-QQQ-MS in positive ion mode.

Retention time(min)	Name	Chemical class	Formula	Mass	Score	DB (ppm)
9.351	Glucosinalbin	sulfur-containing compound	C₁₄H₁₉NO₁₀S₂	425.0450	92.38	-0.04
17.447	Distomadine B	Alkaloid	C₁₇H₁₄N₄O₆	370.0919	83.58	1.64
17.539	3-Strictosidine	Indole alkaloid	C₂₇H₃₄N₂O₉	530.2269	81.79	0.85
19.246	Antillatoxin A	Lipopeptide	C₂₈H₄₅N₃O₅	503.3369	94.36	1.97
19.852	Agelasidine A	Alkaloid	C₁₈H₃₃N₃O₂S	355.2289	91.53	-1.16
19.897	Diacetylchitin	Carbohydrate derivative	C₃₂H₅₃N₃O₁₈	767.3308	92.58	-2.17
20.113	Terrelactone A	Polyketide	C₂₆H₂₈O₈	468.1781	90.94	-0.78
20.136	Cycloshermilamine D	Alkaloid	C₂₁H₁₆N₄OS	372.1036	84.72	-2.41
20.148	Trunkamide	Cyclic peptide	C₄₃H₆₃N₇O₈S	837.4442	92.54	-1.97
20.365	Taxumain A	Alkaloid	C₃₀H₄₄O₁₃	612.2783	97.59	0.18
20.847	Naamidine I	Alkaloid	C₂₆H₃₀N₆O₅	506.2280	96.81	0.53
21.158	Taiwachelin	Siderophore	C₄₁H₇₀N₈O₁₈	962.4812	98.61	0.46
21.352	Phakellistatin	Cyclic peptide	C₃₅H₅₂N₈O₈S	744.3661	83.67	4.23
21.566	Acidissiminin	Peptide	C₄₃H₆₅NO₄	659.4902	83.58	-1.81
21.617	Sugikurojin B	Alkaloid	C₄₁H₅₈O₃	598.4373	89.44	-2.13
21.711	Spiroleucidine	Alkaloid	C₃₀H₄₉N₃O₂	483.3839	88.95	2.91
21.926	6,8-Diprenylorobol	Prenylated flavonoid	C₂₅H₂₆O₆	422.1718	87.93	-2.72
22.349	Septacidin	Nucleoside antibiotic	C₃₀H₅₁N₇O₇	621.3828	84.12	-3.56
22.364	Ambiguine isonitrile	Indole alkaloid	C₂₆H₃₀N₂O₂	402.2319	89.84	2.85
22.844	Hovenine A	Alkaloid	C₂₇H₄₂N₄O₄	486.3221	80.86	3.11
22.851	Himeradine A	Alkaloid	C₂₈H₄₃N₃O	437.3407	80.22	0.29
23.005	Okenone	Carotenoid	C₄₁H₅₈O₂	582.4424	86.22	-2.15
23.024	Bacithrocin C	Peptide	C₁₈H₂₇N₅O₃	361.2105	81.86	-2.51
23.109	Epipachysamine D	Steroidal alkaloid	C₃₀H₄₆N₂O	450.3629	85.14	4.12
23.224	Spirilloxanthin	Carotenoid	C₄₂H₆₀O₂	596.4577	81.31	-2.82
23.751	Nodulisporic acid E	Indole diterpene	C₃₇H₅₁NO₃	557.3870	91.22	0.17
24.772	Anhydrorhodovibrin	Carotenoid	C₄₁H₅₈O	566.4469	87.32	-3.21

The LC–QQQ–MS profiling of the mycelial extract of W. fusiformispora in positive ion mode detected 33 putatively annotated metabolites (Table 2). Retention times ranged from 1.711 to 27.497 min, with molecular masses spanning 117.08 to 744.36 Da and DB values between − 1.51 and + 5.11 ppm.

Table 2.

Putatively annotated metabolites detected in the mycelial crude EtOAc extract of W. fusiformispora by LC-QQQ-MS in positive ion mode.

Retention time (min)	Name	Chemical class	Formula	Mass	Score	DB (ppm)
1.711	Betaine	Quaternary ammonium compound	C₅H₁₁NO₂	117.0818	97.63	2.79
10.627	Aspidocarpine	Indole alkaloid	C₂₂H₃₀N₂O₃	370.2251	87.54	-0.58
17.551	Dimoracin	Prenylated flavonoid	C₃₈H₃₂O₈	616.2127	84.82	2.98
17.698	Bengamide E	Polyketide–amide	C₁₇H₃₀N₂O₆	358.2101	83.26	-0.27
18.081	Cyanocycline B	Cyclic peptide	C₂₁H₂₄N₄O₅	412.1764	89.59	1.72
18.658	Glycitein	Isoflavone	C₁₆H₁₂O₅	284.0729	93.69	4.46
18.853	Biorobin	Flavonoid glycoside	C₂₇H₃₀O₁₅	594.1588	80.41	0.31
19.426	Janthitrem G	Indole diterpene	C₃₉H₅₁NO₆	629.3711	92.64	-0.67
19.428	Disinomenine	Alkaloid	C₃₈H₄₄N₂O₈	656.3083	91.08	-1.51
19.541	Chrysosporide	Glycoside	C₂₆H₄₇N₅O₅	509.3565	89.37	-1.27
20.042	Ajudazol A	Polyketide	C₃₄H₄₂N₂O₇	590.2995	94.02	0.32
20.064	Asterosterol	Sterol	C₂₆H₄₂O	370.3245	89.47	0.96
20.147	Glaucasterol	Sterol	C₂₇H₄₂O	382.3247	83.75	1.17
20.169	Plakinamine F	Steroidal alkaloid	C₃₁H₄₈N₂O	464.3778	90.73	1.17
20.176	Calycanthidine	Alkaloid	C₂₃H₂₈N₄	360.2319	83.64	0.52
20.437	Triedimycin A	Macrolide polyketide	C₃₈H₅₅N₃O₁₀	713.3921	84.55	3.21
20.453	Basellasaponin A	Triterpenoid saponin	C₄₇H₇₀O₂₁	970.4449	86.94	3.93
20.595	Lokysterolamine A	Steroidal alkaloid	C₃₁H₅₀N₂O	466.3929	92.22	0.62
20.844	Naamidine I	Imidazole alkaloid	C₂₆H₃₀N₆O₅	506.2265	90.43	-1.25
21.119	Granulatamide B	Peptide	C₂₄H₃₄N₂O	366.2669	84.06	-0.18
21.333	Phakellistatin 5	Cyclic peptide	C₃₅H₅₂N₈O₈S	744.3641	94.46	1.21
22.372	Ambiguine isonitrile	Indole alkaloid	C₂₆H₃₀N₂O₂	402.2306	88.32	-0.12
22.929	13Z-Docosenamide	Fatty acid amide	C₂₂H₄₃NO	337.3396	85.57	5.11
22.967	4-Acetoxyplakinamine B	Steroidal alkaloid	C₃₃H₅₂N₂O₂	508.4032	95.49	0.33
23.108	Epipachysamine D	Steroidal alkaloid	C₃₀H₄₆N₂O	450.3616	93.21	0.62
24.317	Cyclopassifloside X	Triterpenoid glycoside	C₃₇H₆₂O₁₂	698.4236	94.83	-0.55
25.236	Glucoconvallasaponin A	Steroidal saponin	C₃₈H₆₂O₁₄	742.4128	88.54	-1.15
26.811	Fusicoccin A	Diterpene glycoside	C₃₆H₅₈O₁₂	682.3924	90.98	-0.43
27.267	Acinetoferrin	Siderophore	C₂₈H₄₈N₄O₉	584.3407	94.23	-1.43
27.497	3-Methylthiopropanamine	Sulfur-containing amine	C₄H₁₁NS	105.0608	92.21	-0.37

The detected metabolites included a wide range of structural classes, such as alkaloids, terpenoids, polyketides, lactone-containing metabolites, sterols, fatty amides, saponins, and peptide-like compounds. Alkaloid-type compounds included betaine, aspidocarpine, cyanocycline B, jantithrem G, calycinathine, ambiguine isonitrile, and 3-methylthiopropanamine. Terpenoid- or sterol-related metabolites deetected were biorobin, asterosterol, glaucasterol, and lokysterolamine A. Several polyketide-derived or lactone-containing compounds were detected, including dimoracin, disinommecine, triedimycin A, granulatamide B, and fusicoocin A. Peptide-like structures included bengamide E, plakinamine F, and phakellistatin 5, while saponin-type metabolites comprised basellasaponin A, cyclopassifloside X, and glucoconvallasaponin A. Additional detected compounds included naamidine I, acinetoferrin, 13Z-docosenamide, and 4-acetoxyplakinamine B. All features were supported by accurate mass measurement and retention characteristics.

Discussion

The present study provides the first comprehensive characterization of the antibacterial, biofilm-preventive, and metabolomic profiles of the newly described freshwater fungi L. megafusiformis and W. fusiformispora. Both species demonstrated clear inhibitory activity against clinically significant gram-positive and gram-negative pathogens, while inhibition of biofilm formation during early developmental stages was observed for selected extracts, thereby positioning these fungi as promising reservoirs of bioactive secondary metabolites. Despite the rapidly increasing recognition that freshwater fungi constitute an important source of structurally unique natural products, they remain disproportionately underrepresented in natural-product drug discovery^22–24. The findings reported here therefore contribute meaningful new insight into the chemical and biological potential of lignicolous fungi inhabiting submerged wood, an ecological niche known to foster high biosynthetic diversity due to persistent competition for space and resources^25,26.

Both fungal species inhibited all four test bacteria, though W. fusiformispora consistently produced stronger activity, particularly against E. coli and S. aureus. This pattern aligns with earlier reports demonstrating that freshwater-derived fungi frequently biosynthesize potent anti-infective molecules, including polyketides, alkaloids, terpenoids, peptides, and amide-based metabolites^27–30. Moreover, the observation that mycelial EtOAc extracts exhibited markedly stronger activity than broth extracts supports the widely documented notion that filamentous fungi typically accumulate their most potent secondary metabolites intracellularly. This results from the spatial organization of key biosynthetic enzymes such as polyketide synthases and nonribosomal peptide synthetases, which are primarily cell-associated and not efficiently secreted into the culture medium^31–33.

The biofilm-preventive activities, observed frequently exceeding 70% inhibition, carry particular biomedical relevance. Biofilm-associated infections remain exceptionally difficult to manage because biofilm-embedded cells display enhanced tolerance to conventional antibiotics³⁴. Consequently, natural products capable of interfering with early biofilm formation, including initial adhesion or matrix development, are increasingly explored as anti-virulence or infection-prevention strategies^35,36. In this study, strong inhibition of biofilm formation was observed even when antibacterial activity was moderate, consistent with previous observations that biofilm-prevention effects can occur independently of bactericidal activity³⁷. It should be emphasized that the antibiofilm assay employed here assessed prevention of biofilm development rather than eradication of mature biofilms, and the observed effects should therefore be interpreted within this context^38–40. Untargeted LC–QQQ–MS profiling revealed that L. megafusiformis and W. fusiformisporapossess chemically rich and structurally diverse metabolomes consistent with their ecological roles as freshwater lignicolous fungi. The extracts contained a broad suite of alkaloids, peptides, terpenoids, carotenoid derivatives, and polyketide-associated scaffolds, reflecting the metabolic versatility required to compete on submerged wood. Integrating LC–MS annotations with the literature compiled in both tables provides strong chemical support for the antibacterial activity of both fungi and clarifies the more limited distribution of compounds with documented antibiofilm effects.

The L. megafusiformis extract showed abundant antibacterial metabolites supported by literature. These include glucosinolate derivatives such as glucosinalbin⁴¹, quinoline alkaloids including distomadine B⁴². Nitrogenous marine-associated molecules such as cycloshermilamine D⁴³ and phakellistatin peptides⁴⁴ further reinforce the antibacterial signature. Polyketide-related compounds including sugikurojin B⁴⁵, 6,8-diprenylorobol⁴⁶, septacidin⁴⁷, and cyanobacterial alkaloids such as ambiguine isonitrile⁴⁸ have all been reported to inhibit bacterial growth. Additional antibacterial compounds, hovenine A⁴⁹, bachitrocin C⁵⁰, epipachysamine D⁵¹, spirilloxanthin⁵², nodulisporic acid E⁵³, and anhydrorhodovibrin⁵⁴, expand the multi-scaffold antimicrobial repertoire. These combined chemical signals correspond well with the antibacterial activity recorded in the biological assays. Documented antibiofilm activity was limited to a small subset of annotated compounds. These include sulfur-containing metabolites such as agelasidine A⁵⁵, phakellistatin⁵⁶, 6,8-diprenylorobol⁵⁷, anhydrorhodovibrin⁵⁸, and carotenoid-related pigments such as spirilloxanthin⁵², reported to inhibit biofilm formation in pathogenic bacteria. Most other annotated metabolites are known antibacterial compounds but lack direct antibiofilm characterization in the cited literature. Accordingly, the biofilm-preventive phenotype observed for L. megafusiformis should be interpreted at the crude-extract level, and cannot be attributed to individual metabolites based on the present data. The extract of W. fusiformispora also showed strong antibacterial chemical architecture but with distinct structural patterns. Notable antibacterial compounds include betaine⁵⁹, bengamide E⁶⁰, and aspidocarpine alkaloids⁶¹. Cyanobacterial-type antibacterial metabolites such as cyanocycline B⁶² and jantithrem G⁶³ were also present. Additional compounds with reported antibacterial activity include chrysosporide⁶⁴, ajudazol A⁶⁵, and the steroidal alkaloid plakinamine F⁶⁶. Several other metabolites including calycinathine⁶⁷, triedimycin A⁶⁸, basellasaponin A⁶⁹, lokysterolamine A⁷⁰, granulatamide B⁷¹, ambiguine isonitrile⁴⁸, epipachysamine D⁷², and cyclopassifloside X⁷³, further contribute to the broad antibacterial profile. This diversity suggests a multi-scaffold antibacterial mechanism rather than reliance on a single dominant metabolite class. In contrast to L. megafusiformis, none of the annotated compounds detected in W. fusiformispora have been previously reported to possess antibiofilm activity based on the available literature. Although several putatively annotated metabolite classes (e.g., saponins, terpenoids, and steroidal alkaloids) are structurally associated with membrane-related or signaling-modulatory effects in other systems, no direct antibiofilm evidence exists for the specific metabolites detected, and the observed effects should therefore be interpreted cautiously. Comparison of the two fungi reveals clear functional contrasts in their metabolomes. L. megafusiformis uniquely contains compounds with documented antibiofilm activity, while W. fusiformispora displays greater structural diversity among antibacterial metabolites. These patterns may reflect distinct ecological strategies within submerged-wood habitats: L. megafusiformis may suppress initial bacterial adhesion via dedicated antibiofilm molecules, whereas W. fusiformispora may rely on broad-spectrum antibacterial metabolites to inhibit competitors at later colonization stages. The prevalence of marine-derived and cyanobacterial-associated scaffolds in both fungi also reinforces the idea that freshwater ascomycetes possess versatile PKS–NRPS and terpene biosynthetic pathways, possibly shaped by convergent evolution or adaptive pressures of resource-limited aquatic substrates^1,3,7. Together, these findings confirm that both freshwater saprobic fungi are productive sources of antibacterial metabolites, with L. megafusiformis contributing the only documented antibiofilm molecules in the dataset. The results indicate that complex metabolite mixtures contribute to the observed antibacterial activity and, in the case of L. megafusiformis, to extract-level antibiofilm effects. The demonstrated chemical diversity highlights the biotechnological potential of these fungi and underscores the value of future isolation-guided and mechanistic studies to identify individual metabolites responsible for antibacterial and antibiofilm activities.

Conclusion

This study demonstrates that L. megafusiformis and W. fusiformispora are rich sources of structurally diverse secondary metabolites with clear antibacterial potential. Untargeted LC–QQQ–MS profiling, supported by literature evidence, revealed multiple alkaloids, peptides, terpenoids, saponins, and polyketide-related metabolites previously associated with antimicrobial activity. Among these, L. megafusiformis uniquely contained compounds with documented antibiofilm properties, whereas W. fusiformispora exhibited broader structural diversity among antibacterial metabolites without any known antibiofilm constituents. These chemical distinctions parallel the biological assays and suggest that the two fungi may employ complementary ecological strategies on submerged wood, L. megafusiformis inhibiting initial adhesion and W. fusiformispora exerting broad-spectrum antibacterial pressure. Although only a limited number of metabolites in L. megafusiformis have been previously reported to exhibit antibiofilm activity, the pronounced antibiofilm effects observed in the crude EtOAc extracts from both fungi indicate that extract-level activity cannot be attributed to individual compounds based on the present data. This observation highlights the importance of evaluating whole-extract behavior as an initial screening approach, while recognizing that the specific contributors to antibiofilm activity remain unresolved. Collectively, the results position both fungi as promising reservoirs of antibacterial natural products, with L. megafusiformis additionally offering potential leads for antibiofilm development. Future work should prioritize isolation-guided purification, structure elucidation, molecular docking, and mechanistic assays to identify key bioactive metabolites and to further explore the pharmaceutical potential of freshwater fungi.

Materials and methods

Sample collection and isolation

Decaying submerged wood samples were collected from the river, a tropical freshwater river system flowing through Chiang Rai and Chiang Mai Provinces, northern Thailand. Sampling targeted wood pieces that had been partially submerged or fully immersed in river water, representing typical substrates for freshwater saprobic fungi. Two sampling sites were selected along different sections of the river: Mae Yao, Chiang Rai Province (19°56′ N, 99°49′ E), collected on 4 December 2019, where L. megafusiformis (specimen MFLU 21–0062; ex-type culture MFLUCC 21–0085) was isolated; and Tha Ton, Mae Ai District, Chiang Mai Province (20°03′ N, 99°21′ E), collected on 9 December 2019, where W. fusiformispora (specimen MFLU 21–0068; ex-type culture MFLUCC 21–0091) was obtained. Field sampling was conducted by S. Boonmee and S.K.U. Chandrasiri under the freshwater fungal diversity project of the Excellent Center of Fungal Research (ECFR), Mae Fah Luang University (MFU), with collection approved by the local environmental authority²¹. Each piece of wood (approximately 10 × 3 × 2 cm) was carefully detached from the riverbed using sterile forceps, placed into sterile polyethylene bags, labeled with date and site information, and transported to the laboratory within 24 h. Samples were gently rinsed with sterile distilled water to remove adhering debris and incubated in moist chambers (sterile plastic boxes lined with damp tissue paper) at 25–28 °C for 7–10 days to promote sporulation. Sporulating structures were examined daily under a stereo microscope (Olympus SZ61). Fruiting bodies and conidiophores were aseptically transferred to 2% malt extract agar (MEA; Difco, USA) using sterile needles. After 24 h of germination, individual spores were isolated under an Olympus BX53 compound microscope fitted with a micromanipulator and transferred to fresh MEA plates to establish pure cultures. Cultures were maintained at 25 °C in darkness, with monthly sub-culturing to sustain viability. Voucher specimens were air-dried and deposited in the Mae Fah Luang University Herbarium (MFLU), while living cultures were preserved in the Mae Fah Luang University Culture Collection (MFLUCC). Long-term preservation included both mineral-oil overlay and cryopreservation in 15% glycerol at − 80 °C. Colony morphology (color, growth rate, texture, and pigmentation) was recorded after 14 days of incubation on MEA. Microscopic features including conidiophores, conidiogenous cells, and conidia were examined in lactophenol cotton blue and distilled water. Digital photomicrographs were captured using a Canon EOS 700D camera mounted on the microscope. Measurements were taken from 30 to 50 replicates per structure using Tarosoft Image Framework software, and results were expressed as mean ± standard deviation (SD).

Extraction of secondary metabolites

Pure cultures of L. megafusiformis and W. fusiformispora were cultivated in potato dextrose broth (PDB; Difco, USA) to obtain mycelial biomass for metabolite extraction following our previous study⁷⁴. Each fungus was inoculated into 20 Erlenmeyer flasks (500 mL) containing 250 mL of PDB and incubated statically at 28 °C for 21 days under ambient light. At the end of incubation, cultures were filtered through sterile muslin cloth followed by Whatman No. 1 filter paper to separate the mycelial biomass from the culture filtrate. Both fractions were extracted three times with analytical grade EtOAc at a 1:1 (v/v) ratio. The organic layers were pooled, dried over anhydrous sodium sulfate, and concentrated under reduced pressure using a Büchi R-300 rotary evaporator maintained at 40 °C to obtain the crude EtOAc extracts. Following solvent removal, the dried EtOAc extracts yielded 3.08 g from L. megafusiformis and 2.87 g from W. fusiformispora. The extracts were stored in amber-glass vials at − 20 °C until further analysis. A medium-only control (uninoculated PDB extracted identically with EtOAc) was processed to identify background signals. All extractions were performed from three independent cultures to ensure biological reproducibility. Unless otherwise stated, all chemical profiling and biological assays in this study were conducted using the crude EtOAc extracts.

Antimicrobial activity assay

The antibacterial activities of the crude EtOAc extracts obtained from L. megafusiformis and W. fusiformispora were assessed against four clinically relevant bacterial strains: S. aureus ATCC 25,923 and B. subtilis ATCC 6633 (gram-positive), and E. coli ATCC 25,922 and P. aeruginosa ATCC 27,853 (gram-negative). Each bacterial strain was streaked onto nutrient agar and incubated at 37 °C for 18–24 h. A standardized bacterial suspension equivalent to 0.5 McFarland (≈ 1 × 10⁸ CFU/mL) was prepared in sterile saline and uniformly swabbed onto Mueller–Hinton agar plates. Sterile 6-mm filter-paper discs were impregnated with 20 µL of crude EtOAc extract (stock concentration: 50 µg/mL in DMSO) and placed onto the inoculated agar surface. Plates were incubated at 37 °C for 24 h. Following incubation, the diameter of each inhibition zone (including the disc) was measured in millimeters. Ampicillin (10 µg/disc) served as the positive control, whereas 1% DMSO served as the negative control. All measurements were performed in triplicate and reported as mean ± SD. MICs of the crude EtOAc extracts were determined using the resazurin-based 96-well broth microdilution methods^14,41. Crude EtOAc extracts were dissolved in DMSO and serially two-fold diluted in Mueller–Hinton broth to obtain final testing concentrations ranging from 0.78 to 100 µg/mL, while maintaining DMSO below 1% (v/v). Each microplate well contained 100 µL of the extract dilution and 100 µL of bacterial suspension standardized to approximately 5 × 10⁵ CFU/mL. Plates were incubated at 37 °C for 24 h under static conditions. After incubation, 30 µL of freshly prepared 0.015% (w/v) resazurin solution was added to each well and the plates were further incubated for 2 h. Wells exhibiting metabolic activity reduced resazurin from blue to pink, whereas wells without bacterial growth remained blue. The MIC was defined as the lowest extract concentration that retained the blue (non-reduced) resazurin color, indicating ≥ 90% inhibition of bacterial metabolic activity compared with the untreated control. All assays were performed in triplicate. To determine MBC values, 10 µL aliquots from wells exhibiting no visible growth in the MIC assay were aseptically plated onto nutrient agar and incubated at 37 °C for 24 h. The MBC was defined as the lowest concentration that resulted in no colony formation (≥ 99.9% killing). Ampicillin (10 µg/mL) served as a positive control and produced complete elimination of bacterial growth, confirming assay reliability. All MIC and MBC measurements were performed in three independent biological replicates.

Biofilm prevention

In this study, antibiofilm activity refers specifically to the prevention of biofilm formation during the initial adhesion and development stages, rather than eradication of pre-established biofilms. The antibiofilm activity of crude EtOAc extracts from L. megafusiformis and W. fusiformispora was assessed using a microtiter plate crystal violet assay, following established protocols with slight modifications^11,14. Overnight cultures of S. aureus ATCC 25,923, B. subtilis ATCC 6633, E. coli ATCC 25,922, and P. aeruginosa ATCC 27,853 were grown in tryptic soy broth (TSB) and adjusted to the 0.5 McFarland standard (≈ 1 × 10⁸ CFU/mL). The bacterial suspensions were then diluted 1:100 in TSB supplemented with 1% glucose to stimulate biofilm formation. For each assay, 100 µL of the standardized bacterial inoculum was transferred into sterile 96-well flat-bottom microplates containing 100 µL of fungal extract prepared at a final concentration of 50 µg/mL, which was the concentration used throughout this study for biofilm inhibition screening. Wells containing bacteria and 1% DMSO served as the negative control, while ampicillin (final concentration 10 µg/mL) was included as the positive control. Plates were incubated statically at 37 °C for 24 h to allow biofilm development. After incubation, nonadherent cells were removed by washing the wells three times with sterile phosphate-buffered saline (PBS). The remaining biofilm was fixed with absolute methanol for 15 min, air-dried at room temperature, and stained with 0.1% (w/v) crystal violet for 15 min. Excess stain was removed by gentle rinsing with distilled water. The bound dye was solubilized using 95% ethanol, and absorbance was recorded at 595 nm with a microplate reader. Antibiofilm activity was expressed as the percentage reduction in biomass relative to the untreated control using the formula: Biofilm inhibition (%) = [(A_control-A_sample)/A_control] x 100 where A_control represents the absorbance of the untreated biofilm and A_sample represents the absorbance of the extract-treated biofilm. All experiments were performed in triplicate, and results were presented as mean ± standard deviation (SD).

LC–MS analysis

Chemical profiling of the crude EtOAc extracts obtained from L. megafusiformis and W. fusiformispora was performed using an Agilent 1290 Infinity II UHPLC system coupled to an Agilent 6545 triple quadrupole mass spectrometer (LC–QQQ–MS) equipped with an electrospray ionization (ESI) source⁴¹. Chromatographic separation was achieved on an Agilent ZORBAX Eclipse Plus C₁₈ column (2.1 × 100 mm, 1.8 μm particle size). The mobile phase consisted of (A) water containing 0.1% formic acid and (B) acetonitrile containing 0.1% formic acid. The gradient program was as follows: 5% B (0–2 min), 5–95% B (2–25 min), held at 95% B for 5 min, then re-equilibrated to 5% B for 5 min before the next run. The flow rate was 0.3 mL/min, the column temperature was maintained at 40 °C, and the injection volume was 5 µL. The LC gradient was designed for untargeted metabolomic profiling to provide broad coverage of semi-polar secondary metabolites present in EtOAc extracts rather than optimized chromatographic separation of individual compounds. Accordingly, a substantial proportion of detectable features eluted within the mid-to-late gradient window, reflecting the typical retention behavior of semi-polar fungal metabolites on a reversed-phase C₁₈ column. The total ion chromatogram (TIC) for representative LC–MS analyses is provided in the Supporting Information (Fig. S2) to illustrate overall signal distribution and data quality across the chromatographic run.

For LC–QQQ–MS profiling, aliquots (2.0 mg) of each crude EtOAc extract were reconstituted in 1 mL of HPLC-grade methanol, vortexed for 2 min, and filtered through 0.22 μm PTFE syringe filters prior to injection. Mass spectra were acquired in positive electrospray ionization mode under the following conditions: capillary voltage = 3.5 kV, drying gas temperature = 325 °C, drying gas flow = 10 L/min, and nebulizer pressure = 35 psi. The fragmentor voltage was set to 120 V and skimmer voltage to 65 V. Data were collected over an m/z range of 50–1500 at a rate of 2 spectra/s. Internal mass calibration was performed automatically using reference ions at m/z 121.0509 and 922.0098. Each sample was analyzed in triplicate, and solvent blanks as well as medium-only controls were injected between runs to minimize carryover and background interference. To reduce false-positive annotations, LC–MS data were subjected to manual curation and filtering during data processing. Features detected in solvent blanks or medium-only controls were excluded. Only peaks with a signal-to-noise ratio > 3, relative abundance > 0.1% of the base-peak intensity, mass errors ≤ 5 ppm, and isotopic pattern fit scores ≥ 90% were retained. Data processing and feature extraction were performed using Agilent MassHunter Qualitative Analysis 10.0 and Mass Profiler Professional 15.0. Metabolite annotation was based on accurate precursor ion m/z values, isotopic pattern matching, and database comparison against METLIN, MassBank, and NIST17. As authentic reference standards and compound-specific MS/MS confirmation were not available, all compound assignments are considered putative rather than definitive identifications. Detailed mass spectrometry adduct assignments and representative spectra for all putatively annotated metabolites are provided in the supplementary data.

Statistical analysis

All quantitative experiments were performed using three independent biological replicates, and results are presented as mean ± standard deviation (SD). When significant interactions were observed, post hoc comparisons were performed using Bonferroni’s correction. A p-value < 0.05 was considered statistically significant.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

P.P. and P.K.; Conceptualization, S.K. and T.S.; methodology, validation, formal analysis, P.P.; investigation, data curation, S.K. and P.P.; writing—original draft preparation, P.P. and P.K.; writing—review and editing, P.K.; visualization and supervision, P.K. and P.P.; funding acquisition.

Funding

This research was funded by the National Research Council of Thailand (NRCT) for financial support under Contract No. N42A670689, and Mae Fah Luang University.

Data availability

All fungal strain sequence data have been deposited in the GenBank database under accession numbers MZ538512, MZ538546, and MZ567090 for L. megafusiformis, and MZ538533, MZ538567, MZ538576, and MZ567108 for W. fusiformispora . All data generated or analyzed during this study are included in this published article and its supplementary information files. Additional datasets are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.