Endophytic fungi inhabit living internal plant tissues for at least part of their life cycle without causing apparent disease symptoms. Within host tissues, they coexist with complex microbial communities and engage in continuous ecological interactions, including competition for space, nutrients, and other limiting resources. These interactions are accompanied by substantial biosynthetic capacity and an extensive repertoire of secondary metabolites, making endophytic fungi a rich reservoir of structurally diverse bioactive compounds, including antimicrobial natural products. Among endophytic fungi, species of Diaporthe have attracted considerable attention because of their broad host range and exceptional biosynthetic potential. Members of the genus produce a wide array of secondary metabolites with antifungal and other biological activities, highlighting Diaporthe as an important source of bioactive natural products.

Fungal pathogens pose an increasing threat to human health, agricultural productivity, and global food security, while the rapid emergence of antifungal resistance continues to compromise the efficacy of existing control strategies. Moreover, the limited chemical diversity and restricted range of cellular targets represented by current antifungal drugs highlight the need for new molecules with novel structural scaffolds. Against this backdrop, plant-associated endophytic fungi have emerged as a promising source of structurally diverse antifungal natural products. Despite extensive investigations into the chemistry and bioactivity of Diaporthe, it remains unclear whether competitive interactions are reflected in cell-free extracellular antifungal activity and the composition of extracellular metabolites. Growth inhibition observed during dual-culture confrontation may arise from multiple, potentially overlapping processes, whereas metabolomic annotation alone cannot establish causal relationships between individual metabolites and the observed biological activity.

In the new study, the research team first characterized the growth of Diaporthe sp. PBS30-7 under different temperatures, nutrients, and pH conditions using a two-stage experimental design. Colony growth was assessed at 23, 27, and 33 °C on potato dextrose agar, malt extract agar, and oatmeal agar. Growth kinetics were quantified by estimating the maximum specific growth rate and lag phase duration using a modified Gompertz model fitted to colony diameter data by non-linear regression. The results showed that culture medium explained the largest proportion of the variation in colony growth, followed by temperature and the temperature × medium interaction. Colony expansion was generally greater at 23 and 27 °C than at 33 °C. Compared with temperature and culture medium, environmental pH had a smaller influence on colony expansion, with a modest but significant reduction observed at pH 8.

Time-resolved dual-culture assays then demonstrated that Diaporthe sp. PBS30-7 consistently maintained colony expansion while restricting the growth of both Penicillium sumatrense and P. citrinum, forming a Type F interaction pattern with each target fungus. Against P. sumatrense, colony diameter was reduced from 3.07 cm in monoculture to 1.33 cm in dual culture by Day 8, corresponding to a mean mycelial growth inhibition of 65.14 ± 2.29%. During the first 3 days of incubation, both fungi exhibited progressive radial expansion. Thereafter, expansion of P. sumatrense became minimal, whereas Diaporthe sp. PBS30-7 continued to expand from 1.32 cm on Day 3 to 5.30 cm by Day 8. A comparable competitive pattern was observed against P. citrinum, with colony diameter decreasing from 3.63 cm in monoculture to 1.20 cm in dual culture by Day 8, corresponding to a mean mycelial growth inhibition of 56.70 ± 1.42%.

Microscopic examination further distinguished the interaction zones from the corresponding control cultures. In the absence of Diaporthe sp. PBS30-7, both P. sumatrense and P. citrinum produced intact hyphae and normally developed conidiophores. In contrast, dual-culture interaction with Diaporthe sp. PBS30-7 was associated with distinct morphological alterations in both pathogens. In P. sumatrense, conidiophores were deformed, localized pigment accumulation was evident, and Diaporthe sp. PBS30-7 hyphae overgrew and formed extensive hyphal interweaving within the interaction zone. In P. citrinum, hyphae were deformed and accompanied by reduced conidiation together with localized hyphal lysis. These observations suggest that several ecological processes may operate concurrently during fungal confrontation, although the study was careful to note that overgrowth cannot be interpreted as definitive mycoparasitism without comparable evidence of penetration or intracellular colonization.

Cell-free culture filtrates prepared from 7-day liquid cultures of Diaporthe sp. PBS30-7 retained measurable antifungal activity against both Penicillium species in the absence of living mycelia and direct fungal contact. The culture filtrate inhibited mycelial growth of both pathogens, with mean mycelial growth inhibition values of 41.32% for P. sumatrense and 45.12% for P. citrinum. Under the same experimental conditions, itraconazole produced higher inhibition, with values of 54.91% and 77.41%, respectively. Morphological responses differed among treatments. Compared with the untreated controls, P. sumatrense exposed to the culture filtrate exhibited altered conidiophore morphology together with reduced conidiation, whereas P. citrinum primarily showed reduced conidiation. These findings support a contribution of extracellular chemical factors to the overall antagonistic phenotype.

Untargeted UHPLC–Orbitrap HRMS/MS coupled with feature-based molecular networking resolved the metabolite profile of Diaporthe sp. PBS30-7 into multiple structurally related molecular families. Spectral similarity organized molecular features into discrete clusters, among which beauvericin-type cyclodepsipeptides and diketopiperazine-related metabolites represented two of the most prominent molecular families. The beauvericin molecular family comprised a series of closely connected features sharing conserved product ions at m/z 134.0963 and 91.0546. The cluster contained beauvericin together with several structurally related analogs, including beauvericin A, B, C, F, and a beauvericin K-type feature. A second prominent molecular family comprised diketopiperazine-related metabolites, with tentative annotations including cyclo(D-Val-L-Pro), cyclo(Leu-Pro), cyclo(Pro-Thr), cyclo(L-Pro-4-hydroxy-L-Leu), and cyclo(Tyr-Pro). Because authentic standards were not analyzed, library matches were treated as putative annotations.

Molecular docking revealed distinct target-dependent binding profiles across the annotated metabolites, with the complete docking results provided through a supplementary data link in the Data Availability. Overall, beauvericin derivatives exhibited the most prominent docking profiles, with multiple analogs showing favorable docking scores against CYP51, Fks1, and the ABC transporter, highlighting these proteins as candidate targets warranting further investigation. At the individual-compound level, Beauvericin K showed the most favorable docking score against CYP51 at −11.1 kcal/mol, Beauvericin H2 showed a favorable score against Fks1 at −10.5 kcal/mol, and Beauvericin C showed a favorable score against the ABC transporter at −9.1 kcal/mol. In comparison, the diketopiperazines generally exhibited more moderate docking profiles. Among them, cyclo(L-Tyr-L-Tyr) was the most notable candidate, showing relatively favorable scores across multiple targets, with a score of −8.6 kcal/mol against Hsp90. Erg1 generally yielded only weak-to-moderate docking scores and was therefore not prioritized for subsequent structural analysis.

Representative docking poses further illustrated the predicted binding environments of the four prioritized ligand–target pairs. Beauvericin K occupied the predicted CYP51 binding pocket, where its docking pose involved a hydrogen bond with Y127 and additional contacts with I141 and I366. Beauvericin H2 was accommodated within the predicted Fks1 binding region and interacted with multiple surrounding residues, including K1124, N1146, K1285, D1288, N1296, F1297, and K1300. Within the ABC transporter, Beauvericin C was positioned in proximity to R605, F1084, and Q1236, with its predicted pose involving hydrogen-bonding and aromatic interactions. Cyclo(L-Tyr-L-Tyr) occupied a localized binding region of Hsp90 surrounded by residues including E32, N36, I116, G117, G122, and F123, with the predicted pose displaying multiple polar and aromatic interactions. The research team emphasized that docking scores and static poses do not demonstrate target engagement, binding stability, or functional inhibition under biological conditions, particularly where modeled receptor structures were used. The prioritized metabolite–target pairs should therefore be regarded as testable mechanistic hypotheses rather than validated antifungal mechanisms.

Several limitations define the scope of the present conclusions. Chemical profiling was conducted on a pooled agar–mycelium extract from solid culture, whereas antifungal activity was evaluated using cell-free filtrates from liquid culture. These distinct cultivation systems preclude a direct association between the annotated features and filtrate activity. Moreover, profiling was limited to the ethyl acetate fraction and may have excluded highly polar, volatile, or poorly recovered bioactive constituents. Future studies should therefore apply bioassay-guided fractionation directly to active filtrates, using complementary extraction strategies across a broader polarity range. Isolation, structural elucidation, quantitative analysis, and standardized antifungal testing of individual compounds and defined combinations will be necessary to resolve their respective contributions and potential interactions. The chemical analysis was exploratory, and annotations based on accurate mass, MS/MS fragmentation, and molecular-network relationships remain putative and require confirmation with authentic standards or isolated compounds characterized by orthogonal spectroscopic methods.

Diaporthe sp. PBS30-7 exhibited a dynamic, target-dependent competitive phenotype against two Penicillium species, while its cell-free culture filtrate retained antifungal activity in the absence of direct fungal contact. Feature-based molecular networking analysis of the culture-associated extract prioritized several putative metabolite families, and molecular docking generated testable metabolite–target hypotheses; however, neither the active constituents nor their mechanisms were established. The study extends current knowledge of Diaporthe by documenting strain-specific environmental responses, resolving temporal and target-dependent differences in fungal confrontation, and establishing an integrated framework for prioritizing candidate metabolites without conflating chemical association with mechanistic causation. These findings provide a foundation for bioassay-guided isolation from active culture filtrates, followed by structural confirmation and functional validation. The research team noted that integrating these approaches with whole-genome sequencing, biosynthetic-gene-cluster analysis, and targeted metabolomics could subsequently connect biosynthetic potential with confirmed metabolite production and biological function.

Subject of Research: Competitive phenotype and metabolite repertoire of the endophytic Diaporthe sp. PBS30-7

Article Title: Competitive phenotype and metabolite repertoire of the endophytic Diaporthe sp. PBS30-7

Article References: Competitive phenotype and metabolite repertoire of the endophytic Diaporthe sp. PBS30-7. (n.d.). https://doi.org/10.3389/fmicb.2026.1943254

Image Credits: AI Generated

DOI: 10.3389/fmicb.2026.1943254

Keywords: endophytic fungi, Diaporthe, Pereskia bleo, Penicillium, beauvericin, diketopiperazines, molecular docking, feature-based molecular networking, antifungal metabolites, fungal competition, CYP51, Fks1