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

The research, conducted by a team at Hunan University of Humanities, Science and Technology in Loudi, China, took the crucial step of moving beyond greenhouse pots and into real, metal-contaminated paddy fields. Field trials of microbial inoculants are notoriously difficult because introduced microbes must compete with established soil communities, survive fluctuating moisture and temperature, and maintain their functional effects amid enormous ecological complexity. Many promising laboratory strains fail this test entirely. In this study, the researchers inoculated rice plants with a functional Penicillium sp. strain and compared the results against control plants that received only a sterile growth medium, allowing them to isolate the effect of the fungus itself rather than the medium it was cultured in.

The headline result was striking in its breadth. Concentrations of all four tested heavy metals, chromium, cadmium, lead, and antimony, were significantly reduced in the roots, shoots, and grains of the inoculated rice plants relative to controls. That matters because most bioremediation studies report effects on a single metal, typically cadmium, and often only in one plant compartment. A single fungal treatment that suppresses uptake of four chemically distinct metals, spanning transition metals and metalloids with different soil chemistries and uptake pathways, suggests the fungus is not merely blocking one transport protein but is instead altering the broader conditions that govern metal mobility and plant access. The authors also examined grain weight and soil metal availability as part of their measurements, building a picture of how the treatment influenced both plant performance and the pool of metals that plants can actually access.

To understand how a soil fungus could produce such sweeping changes, the team turned to amplicon sequencing of the rhizosphere, the narrow zone of soil immediately surrounding the roots where plant and microbial activity intersect most intensely. The rhizosphere is one of the most densely populated microbial habitats on Earth, and its composition strongly influences which nutrients and contaminants reach the root surface. Sequencing revealed that Penicillium inoculation markedly decreased the relative abundance of Proteobacteria, a bacterial phylum that includes many metal-tolerant species, while substantially enriching Acidobacteriota, Chloroflexi, and Methylomirabilota. These shifts are not cosmetic. Changes in the dominant bacterial lineages of the rhizosphere can alter pH, redox conditions, organic acid fluxes, and the formation of iron plaques on root surfaces, all of which are known levers on metal solubility and uptake.

The enrichment of Acidobacteriota and Chloroflexi is particularly intriguing from a soil chemistry standpoint. Both phyla are associated with distinct metabolic lifestyles compared with the fast-growing copiotrophic bacteria that often dominate disturbed soils, and their expansion under fungal inoculation hints at a reorganization of the rhizosphere carbon economy. Methylomirabilota, meanwhile, are linked to methane and one-carbon compound cycling, processes tightly coupled to the flooded, oxygen-limited conditions of paddy soils. While the study is explicitly framed as exploratory and correlative rather than a demonstration of causation, the consistent directional shifts across these lineages provide a plausible microbial mechanism through which the fungus could reduce the bioavailability of metals to the plant.

The second half of the mechanistic story comes from the plant itself. Using untargeted liquid chromatography–mass spectrometry metabolomics, the researchers profiled the metabolic fingerprints of rice roots with and without the fungal partner. The treatment drove prominent accumulation of sucrose, guanosine, and inosine, while malic acid and L-aspartic acid were significantly depleted. Each of these changes is chemically meaningful. Sucrose accumulation points to altered carbon allocation and sugar signaling, processes that plants use both to fuel stress responses and to communicate with root-associated microbes. Guanosine and inosine are purine nucleosides, and their buildup suggests heightened nucleotide turnover, a hallmark of active signaling and stress adaptation. Malic acid, conversely, is a well-documented root exudate that can chelate metals and recruit specific rhizobacteria, so its depletion could directly reduce the chemical routes by which metals are solubilized and delivered to the root.

Pathway enrichment analysis tied these metabolite shifts into a coherent map. Six core pathways were substantially reprogrammed by the fungal inoculation: glyoxylate and dicarboxylate metabolism, Calvin cycle-mediated carbon fixation, purine metabolism, glycine, serine and threonine metabolism, aminoacyl-tRNA biosynthesis, and starch and sucrose metabolism. The involvement of glyoxylate and dicarboxylate metabolism is especially notable because this pathway intersects directly with the tricarboxylic acid cycle and governs the flux of organic acids, the same compounds that mediate metal chelation in the rhizosphere. Reprogramming of Calvin cycle-related carbon fixation and starch and sucrose metabolism indicates that the fungus influences how photosynthetically fixed carbon is partitioned below ground, potentially redirecting sugars away from exudation patterns that favor metal mobilization. Meanwhile, changes in aminoacyl-tRNA biosynthesis and amino acid metabolism suggest altered protein synthesis demands consistent with a plant recalibrating its stress physiology.

The authors are careful about what their data can and cannot show. The study is presented as an exploratory correlative framework, linking inoculation to reduced metal accumulation, reshaped bacterial communities, and reprogrammed root metabolism, rather than proving direct causal chains between each element. That caution is scientifically appropriate. Distinguishing whether the fungal partner directly immobilizes metals, whether the bacterial community shifts do the heavy lifting, or whether the plant’s own metabolic rewiring is the dominant factor will require follow-up experiments, potentially including sterile and gnotobiotic systems, isotope tracing, and targeted perturbation of individual community members. Still, the convergence of three independent lines of evidence, metal concentrations, community composition, and metabolomics, all pointing in the same direction, is exactly the kind of multi-omics consistency that makes a correlative framework credible and worth pursuing.

The practical implications extend well beyond rice paddies in Hunan Province. Heavy metal contamination of agricultural soil is a global problem, affecting major grain-producing regions from South and Southeast Asia to parts of Europe and the Americas, and food safety standards for cadmium and lead in rice are tightening in many markets. Microbial inoculants based on Penicillium and related fungi fit neatly into the growing movement toward microbiota-mediated bioremediation, which the authors describe as a sustainable and eco-friendly strategy. Unlike chemical amendments, a living fungal inoculant can potentially establish, persist, and adapt, and unlike soil replacement it requires no excavation. The challenges that remain are the classic ones for inoculant science: consistency across soil types, climates, and agronomic practices; regulatory pathways for deliberate microbial release; and scaling production of viable inoculum. But this study demonstrates that the field-scale hurdle, often the point where such approaches fail, can be cleared.

What makes the work resonant beyond its immediate application is the picture it paints of the rhizosphere as an integrated system that can be steered. A single fungal introduction did not act in isolation; it cascaded through the bacterial community, through the pool of bioavailable metals, and through the plant’s own central metabolism, ultimately changing how much toxin reached the grain. That systems-level view, grounded in amplicon sequencing and untargeted metabolomics, reflects a broader shift in soil science away from single-factor fixes and toward ecological engineering of the plant microbiome. If subsequent work can confirm the causal mechanisms and refine the inoculant for reliable deployment, the humble Penicillium strain studied here could become part of a practical toolkit for growing safer food on contaminated land, turning one of agriculture’s most stubborn contamination problems into a manageable biological conversation between roots, fungi, and soil.

Subject of Research: Fungal inoculation to reduce heavy metal accumulation in rice through rhizosphere microbiome and root metabolism changes

Article Title: Penicillium sp. inoculation alters rhizosphere microorganisms and root metabolism to reduce heavy metal accumulation in field rice

Article References: Dai, Q., Yang, H., Duan, R., Fu, J., Zhou, W., & Yang, L. (2026). Penicillium sp. inoculation alters rhizosphere microorganisms and root metabolism to reduce heavy metal accumulation in field rice. BMC Plant Biology. https://doi.org/10.1186/s12870-026-10009-7

Image Credits: AI Generated

DOI: 10.1186/s12870-026-10009-7

Keywords: bioremediation, heavy metals, rice, Penicillium, rhizosphere microbiome, metabolomics, cadmium, soil microbiology, plant metabolism, fungal inoculant, food safety, paddy soil