The study set out to answer a deceptively simple question. Both vermicompost and Piriformospora indica, a root-colonizing endophytic fungus sometimes classified in the genus Serendipita, are known individually to promote plant growth and reshape soil microbial populations. What remained largely unknown was what happens when the two are applied together, and whether their combined effect on apple seedlings is simply additive or something more synergistic. To find out, the researchers grew apple seedlings under four regimes: an untreated control, vermicompost alone, fungal inoculation alone, and the two combined. They then measured everything from seedling biomass and root architecture to photosynthetic capacity, soil chemistry, enzyme activity, and the composition of the rhizosphere microbiome—the teeming microbial ecosystem hugging the root surface.

The results were striking in their consistency. Every treatment that included either vermicompost or the fungus promoted seedling growth, encouraged root development, and enhanced photosynthetic capacity compared with untreated controls. But the combination outperformed both single treatments, delivering the strongest gains across nearly every metric the team tracked. Seedlings receiving both the fungus and the worm castings grew larger, built more extensive root systems, and photosynthesized more efficiently than their counterparts in any other group. For orchardists and nursery operators, the implication is that these two inputs, often marketed and applied separately, may work best as a paired prescription.

Nutrient uptake told a more nuanced story. The combined treatment significantly increased both the uptake fluxes—the rate at which phosphorus and potassium streamed into the roots—and the total contents of those two essential macronutrients in plant tissue. Vermicompost alone, by contrast, was the standout for nitrogen uptake, improving the seedlings’ acquisition of that nutrient without the fungus needing to be present. This divergence suggests that the two amendments operate through partially distinct physiological channels: the fungus appears to amplify phosphorus and potassium mobilization, while the organic matter in vermicompost feeds the nitrogen cycle directly. Growers tailoring fertilization strategies to specific nutrient deficiencies could, in principle, select one or both inputs accordingly.

Soil health metrics followed a similar pattern. Both vermicompost and the combined treatment improved soil fertility and boosted the activity of soil enzymes, the biological catalysts that drive decomposition, nutrient mineralization, and organic matter turnover. Notably, the fungus on its own showed no significant benefit to these soil properties. That asymmetry is scientifically telling: it implies that Piriformospora indica does not so much improve the soil directly as it reprograms the plant and its microbial entourage to exploit the soil more effectively. The vermicompost, meanwhile, supplies the raw biochemical substrate—the carbon, nitrogen, and micronutrients—that enzymes and microbes need to do their work. Together, the two create a feedback loop in which better soil feeds better roots, and better roots cultivate better soil.

The heart of the study, however, lies in its microbiome analysis. Using high-throughput sequencing of rhizosphere soil, the researchers found that all three treatments enriched taxa classified as plant-growth-promoting, or PGP, microbes—organisms known to fix nitrogen, solubilize minerals, produce plant hormones, or suppress pathogens. But which of these enriched taxa actually mattered for the growth response? To move beyond correlation, the team deployed random-forest analysis, a machine-learning method that ranks microbial features by their predictive power for a given outcome. The algorithm’s verdict was unambiguous: the bacterial phylum Actinobacteriota, and within it especially the genus Streptomyces, emerged as the key contributor distinguishing high-performing seedlings from the rest.

Streptomyces are the workhorses of the actinomycete world, famous both for their earthy smell in freshly turned soil and for their prodigious output of antibiotics, including the compounds behind many clinical drugs. In the rhizosphere, they are prized for their ability to solubilize phosphorus, produce indole-3-acetic acid and other auxin-type hormones that stimulate root growth, and outcompete pathogens. The Shanxi team did not stop at statistical association. In a crucial experimental step, they isolated four Streptomyces strains directly from fresh vermicompost and inoculated apple seedlings with them. The purified bacteria reproduced the growth-promoting effects observed with the full vermicompost treatment, enhancing seedling vigor and soil fertility on their own. That causal demonstration elevates the finding from an intriguing correlation to a functional mechanism: actinomycetes recruited or enriched by vermicompost and the fungus are not passive bystanders but active agents of plant growth.

The study’s design reflects a broader shift in plant science toward viewing crops as holobionts—plants plus their associated microbiomes—as integrated biological units. Earlier work cited by the authors has shown that vermicompost can suppress apple replant disease, that Piriformospora indica reshapes rhizosphere communities in willows and other species, and that specific bacterial strains such as Pseudomonas sp. CM11 can induce lateral root formation in apple rootstocks. What this new research adds is a mechanistic bridge: a specific bacterial taxon, identifiable by machine-learning analysis and verifiable through strain isolation, that links a widely used organic amendment and a model endophytic fungus to concrete physiological outcomes in a major fruit crop. The four Streptomyces strains have been deposited in GenBank, and the full microbiome dataset is available in the NCBI Sequence Read Archive, giving other researchers the raw material to build on the work.

The practical implications extend well beyond the apple orchard. Vermicompost is already a cornerstone of organic and low-input agriculture worldwide, valued for improving soil structure, water retention, and nutrient availability. Piriformospora indica, meanwhile, is increasingly marketed as a biostimulant for horticultural crops, prized for its broad host range and its capacity to confer tolerance to drought, salinity, and heavy metals. If the growth benefits of both depend substantially on recruiting actinomycetes, then management practices that favor these bacteria—such as maintaining organic matter inputs, minimizing disruptive tillage, and avoiding broad-spectrum biocides—could amplify the returns on either investment. Conversely, the finding that the fungus alone did not improve soil fertility cautions against expecting microbial inoculants to substitute for organic amendments; the two appear to be complements, not substitutes.

There are, of course, caveats. The experiments were conducted on apple seedlings under controlled conditions, and translating seedling responses to mature orchard trees across seasons and variable field soils remains a challenge that the authors themselves acknowledge in framing their conclusions. The random-forest approach identifies predictive taxa but cannot fully disentangle whether Streptomyces abundance is a cause or a consequence of vigorous growth, though the strain-inoculation experiment goes a long way toward resolving that ambiguity. Still, the convergence of evidence—community profiling, machine-learning attribution, and functional validation with isolated strains—makes a compelling case that actinomycetes are central players in vermicompost- and fungus-mediated plant growth. For a discipline often accused of cataloging microbes without explaining them, the study offers a template: find the keystone taxon, isolate it, and prove it works. In the rhizosphere of a young apple tree, at least, the earthworm’s gift and the fungus’s partnership appear to converge on the same microscopic allies, quietly manufacturing the growth that growers see above ground.

Subject of Research: The role of rhizosphere Actinomycetes in vermicompost- and Piriformospora indica-mediated apple seedling growth and nutrient uptake

Article Title: Rhizosphere microbiome analysis revealed the role of Actinomycetes in vermicompost- and Piriformospora indica-mediated apple growth

Article References: Zhao, Q., Li, Q., Ma, X., Li, H., Qin, M., Zhang, Y., Chen, M., Cheng, C., & Li, L. (2026). Rhizosphere microbiome analysis revealed the role of Actinomycetes in vermicompost- and Piriformospora indica-mediated apple growth. Plant and Soil. https://doi.org/10.1007/s11104-026-09099-x

Image Credits: AI Generated

DOI: 10.1007/s11104-026-09099-x

Keywords: apple, vermicompost, Piriformospora indica, rhizosphere microbiome, Actinomycetes, Streptomyces, plant growth promotion, soil fertility, nutrient uptake, soil enzymes, endophytic fungus, random forest analysis

Nitrogen is the nutrient that most often limits crop productivity, and modern agriculture supplies it overwhelmingly through synthetic fertilizers produced by the energy-intensive Haber-Bosch process. Legumes such as soybeans and peas solve part of the problem naturally, hosting rhizobia bacteria in root nodules that convert atmospheric dinitrogen gas into biologically usable ammonia. Cereals, however, including rice, wheat and maize, lack this symbiotic machinery, so they depend almost entirely on applied nitrogen. Global nitrogen budgets for cereal systems show that a large share of applied fertilizer is never captured by the crop, escaping into waterways or the atmosphere as potent pollutants. For decades, scientists have therefore pursued associative nitrogen fixation, the idea that free-living soil bacteria could be encouraged to colonize the roots of cereals and share some of the nitrogen they fix. The new study adds a striking piece to that puzzle by showing that a fungal endophyte can reshape the soil environment to favor exactly those bacteria.

The research team, led by Jun-Feng Ouyang and Chuan-Chao Dai, worked with two genetically distinct rice subspecies: a japonica variety called WYJ23 and an indica variety called HLY996. Each variety was grown under low-nitrogen conditions both with and without inoculation by the endophytic fungus B3, a strain of Phomopsis liquidambaris that the group had previously shown to improve nitrogen cycling in the rice rhizosphere. The central question was deceptively simple: if the fungus changes which nitrogen-transforming microbes gather around rice roots, does it do so by changing the chemistry of the rhizosphere, the narrow zone of soil directly influenced by root secretions? To answer it, the researchers collected the metabolites exuded into the rhizosphere soil and subjected them to untargeted metabolome analysis, a technique that profiles the full spectrum of small molecules present in a sample without preselecting which compounds to look for.

The metabolomic comparison revealed that infection with B3 substantially reshaped the rhizosphere metabolite profiles of both rice subspecies. Among the many compounds whose abundance shifted, one stood out for its consistency: p-hydroxyphenylacetic acid, a phenolic acid, was upregulated in the rhizosphere of both japonica WYJ23 and indica HLY996 plants carrying the endophyte. That cross-subspecies consistency matters, because it suggests the fungus is not merely nudging the idiosyncratic chemistry of one rice genotype but is instead steering a shared metabolic pathway that operates across rice diversity. Phenolic acids are well known players in root-soil signaling, and previous studies have implicated related compounds, such as benzoic acid in maize and flavones in rice and poplar, in the recruitment of diazotrophic, or nitrogen-fixing, bacteria. The new findings place p-hydroxyphenylacetic acid squarely within this growing catalog of root-derived molecules that function as microbial attractants.

Establishing that the fungus changes root chemistry was only the first step. The team then asked whether those changed chemicals were actually responsible for recruiting the nitrogen-fixing community. In a soil conditioning experiment, they treated soil with the rhizosphere metabolites harvested from B3-inoculated rice and from uninoculated controls, then measured the resulting microbial communities. Soils receiving metabolites from endophyte-colonized plants exhibited markedly higher abundances of nitrogen-fixing bacteria. High-throughput sequencing of the conditioned soils sharpened the picture further: metabolites from B3-inoculated plants of both rice subspecies significantly enriched the bacterial taxon Enterobacter, a genus that includes well-documented plant-associated diazotrophs. In other words, the chemical signature left by the fungus-colonized roots was sufficient, on its own, to summon the very bacteria capable of fixing atmospheric nitrogen.

To test the mechanism at the level of individual molecules and microbes, the researchers turned to co-culture assays, growing nitrogen-fixing bacterial strains in the presence of p-hydroxyphenylacetic acid. The compound proved to be a highly bioavailable substrate for the diazotrophs that B3 inoculation had enriched, meaning the bacteria could readily take it up and use it to fuel their growth. Among the tested taxa, strains of Enterobacter showed the most pronounced responses, with measurable increases in both growth and biofilm formation. Biofilm formation is particularly significant in this context: dense bacterial films on root surfaces are thought to create the oxygen-limited microenvironments that nitrogenase, the oxygen-sensitive enzyme responsible for nitrogen fixation, requires to function. A molecule that simultaneously feeds diazotrophs and encourages them to coat the root in a biofilm is therefore doing double duty as both a nutrient and an architectural signal.

The final and arguably most consequential phase of the study asked whether this fungal-chemical-bacterial cascade actually benefits the plant. In greenhouse assays, the team applied Enterobacter strains and a microbiome modified by p-hydroxyphenylacetic acid to rice plants and measured growth and nitrogen accumulation. Both treatments significantly promoted rice biomass and nitrogen content, confirming that the recruited bacteria deliver more than mere company. The standout performer was Enterobacter strain T-9, which produced the largest gains of any isolate tested: increases of 30.05 percent in dry biomass and 24.6 percent in nitrogen accumulation relative to controls. Those are substantial effect sizes for a single bacterial inoculant, and they demonstrate that the enrichment strategy does not merely shift community composition but translates into measurable plant performance under nitrogen-limited conditions.

The study’s design deserves attention because it carefully disentangles correlation from causation at each step. Metabolomics identified candidate compounds; soil conditioning showed that those compounds reshape communities; sequencing pinpointed the enriched taxa; co-culture verified substrate preference and growth responses; and plant assays confirmed functional benefits. This chain of evidence addresses a persistent weakness in rhizosphere research, where studies often stop at showing that a treatment and a community shift co-occur. By isolating p-hydroxyphenylacetic acid and demonstrating its direct effects on Enterobacter physiology, the researchers provide a mechanistic link rather than a statistical association. The work also builds on a broader literature showing that root exudates act as signals and nutrients in plant-microbe interactions, and that specific exudate compounds can be manipulated, whether by endophytes, by breeding, or by genetic engineering of flavone biosynthesis, to enhance associative nitrogen fixation in cereals.

The agricultural implications are considerable. If a single, chemically defined compound can be applied to soil to recruit nitrogen-fixing bacteria around cereal roots, growers could potentially reduce fertilizer inputs without sacrificing yield, cutting both costs and the environmental externalities of nitrogen pollution. The compound itself, p-hydroxyphenylacetic acid, is a small organic acid that could in principle be manufactured and applied as a soil amendment, or its production could be encouraged by inoculating crops with endophytes such as B3. The finding that the mechanism worked across both japonica and indica subspecies suggests a degree of generality that would be essential for any practical deployment across the enormous diversity of cultivated rice. At the same time, the authors emphasize the tight association between rhizosphere metabolites and nitrogen-fixing bacteria as a general principle, hinting that similar metabolite-mediated recruitment may operate in other crops and with other beneficial taxa, a possibility already supported by studies of peach, soybean, maize and poplar systems.

There remain, of course, the usual distances between greenhouse results and open-field reality. Soil conditioning experiments and co-culture assays are controlled settings, and the behavior of p-hydroxyphenylacetic acid in complex paddy soils, with their distinctive flooded chemistry and competing microbial populations, will need direct testing. The stability, dosage and timing of any metabolite-based amendment would also require optimization, and the ecological consequences of systematically enriching Enterobacter populations in paddies would need assessment. Still, the study represents a compelling proof of concept: an invisible fungal partner inside the root can reprogram the chemical conversation between plant and soil, and that reprogrammed conversation recruits bacteria that pull nitrogen from the atmosphere and hand it to the crop. As the search for nitrogen-efficient cereals accelerates, the most transformative tools may prove to be not genes or fertilizers, but the small molecules that mediate who gets invited to the root’s dinner table.

Subject of Research: How an endophytic fungus alters rice rhizosphere metabolites to recruit nitrogen-fixing bacteria

Article Title: Endophytic Phomopsis liquidambaris recruits nitrogen-fixing taxa by altering the rhizosphere metabolites of Oryza sativa L.

Article References: Ouyang, J.-F., Zhang, Y., Fei, Y.-J., Jiang, L., & Dai, C.-C. (2026). Endophytic Phomopsis liquidambaris recruits nitrogen-fixing taxa by altering the rhizosphere metabolites of Oryza sativa L.. Plant and Soil. https://doi.org/10.1007/s11104-026-09087-1

Image Credits: AI Generated

DOI: 10.1007/s11104-026-09087-1

Keywords: rice, endophytic fungus, Phomopsis liquidambaris, nitrogen fixation, rhizosphere metabolites, p-hydroxyphenylacetic acid, Enterobacter, soil microbiome, root exudates, metabolomics, sustainable agriculture, Plant and Soil

The scientific rationale for looking inside plants rather than at them rests on a decades-old insight: endophytes, the fungi and bacteria that colonize plant tissues without causing visible disease, are often the true chemical factories behind a plant’s medicinal reputation. These microorganisms can synthesize bioactive secondary metabolites that help the host defend itself against pathogens, and many of those molecules have proven promising starting points for drug and agrochemical discovery. Medicinal plants, in particular, tend to harbor one or more endophytic fungi capable of producing such compounds. Ginger has a rich ethnopharmacological pedigree, used traditionally across Asia and Africa against ailments ranging from catarrh and rheumatism to asthma and diabetes, and its well-known pungent principles, including gingerols, shogaols, paradols and zingerone, have been extensively studied. Yet the microbes living inside the rhizome remain far less explored, and earlier work has already yielded intriguing metabolites such as epidithiodiketopiperazine, epicorazine A and pretrichodermamide A from fungi associated with ginger.

The urgency of the search is difficult to overstate. According to the World Health Organization, an estimated 263 million malaria cases and 597,000 deaths were recorded globally in 2023, with the African region shouldering roughly 94 percent of cases and 95 percent of deaths. Children under five account for about 76 percent of malaria deaths in that region. More than 400 species of Anopheles are known to affect humans, but only around 30 play a major role in transmitting the Plasmodium parasite, and Anopheles gambiae sits at the top of that short list in Nigeria. Widespread resistance to synthetic insecticides has made vector control increasingly difficult, which is precisely why natural products with larvicidal activity are attracting renewed attention from researchers and public health planners alike.

The journey from farm to molecule began in Kafanchan, in Nigeria’s Kaduna State, where fresh, healthy ginger rhizomes were collected and authenticated at the herbarium of Bayero University Kano, with a voucher specimen deposited for reference. Back in the laboratory, the plant material was washed, surface sterilized through a sequential bath of 70 percent ethanol, 0.5 percent sodium hypochlorite and 96 percent ethanol, then rinsed with sterile distilled water. Small segments of tissue were plated onto potato dextrose agar supplemented with chloramphenicol to suppress bacterial growth and incubated for a week at 27 degrees Celsius. Fungal hyphae were purified by hyphal tipping, and the isolate was identified through colony morphology and microscopic examination of its reproductive structures. The culture showed white to cream colonies with a yellowish-brown reverse, septate antler hyphae, intercalary chlamydospores, and clavate to cylindrical macroconidia with rounded ends, features consistent with an Arthroderma species.

With the fungus in hand, the team turned to fermentation chemistry. Mycelial plugs were inoculated into potato dextrose broth and incubated at room temperature for three weeks, after which the culture was filtered and extracted with ethyl acetate, while the mycelial mat was separately macerated with methanol. The combined organic extract, weighing 15 grams, was purified by column chromatography on silica gel using an escalating polarity gradient from n-hexane through chloroform to ethyl acetate. From the chloroform-ethyl acetate fraction at a 9:1 ratio emerged a brown amorphous solid, coded TC23, which would turn out to be the study’s headline compound. The entire pipeline, from a few grams of rhizome to a purified natural product, illustrates why endophytes are increasingly viewed as a sustainable alternative to harvesting large quantities of plant biomass, a particular advantage for slow-growing medicinal species where land and harvest time impose real constraints.

Characterizing TC23 required a full battery of spectroscopic techniques. Electrospray ionization mass spectrometry in negative mode produced a pseudo-molecular ion at m/z 269.2578, matching the calculated value of 269.2587 for the formula C17H34O2. Carbon-13 NMR revealed seventeen carbon signals, dominated by a carbonyl carbon at 179.15 parts per million downfield, while the DEPT 135 experiment resolved one methyl and fifteen methylene carbons. Proton NMR showed the classic signature of a long saturated fatty acid: a triplet at 0.88 parts per million for the terminal methyl group, a dense multiplet at 1.28 for the methylene bulk, a beta-position multiplet at 1.63, and a triplet at 2.34 for the methylene adjacent to the carbonyl. Two-dimensional experiments, including COSY, HSQC and HMBC, mapped the proton-proton and long-range proton-carbon connectivities along the chain. Infrared spectroscopy sealed the identification, with a broad carboxylic acid O-H stretch spanning 2500 to 3500 inverse centimeters, a strong carbonyl peak at 1707, and C-H stretches at 2920 and 2851. The data matched heptadecanoic acid, or margaric acid, reported in the literature.

The biological results were the most eye-catching part of the study. In a brine shrimp lethality test against larvae of Artemia salina, margaric acid achieved a median lethal concentration of just 3.010 micrograms per milliliter, with mortality rising from 70 percent at 10 micrograms per milliliter to 100 percent at 1000, and no deaths in the solvent control. Under the widely used Meyer’s criteria, extracts with LC50 values below 1000 micrograms per milliliter are considered toxic, placing margaric acid firmly in the highly toxic category. The authors contrasted this with earlier work on endophytic fungi from red ginger, in which isolates resembling Mucor and Trichoderma species were essentially nontoxic to brine shrimp, with LC50 values of 2300 and 1747 micrograms per milliliter respectively, a discrepancy they attribute to differences in fungal species and environmental conditions.

Against the malaria vector itself, the compound performed respectably. Following the World Health Organization’s standard larvicidal testing protocol, third instar larvae of Anopheles gambiae were exposed to concentrations of 1000, 500, 250 and 125 micrograms per milliliter in triplicate. Margaric acid achieved 70 percent mortality at the highest dose after 24 hours and an LC50 of 79.63 micrograms per milliliter. The crude fungal extract actually outperformed the pure compound, with an LC50 of 51.13 micrograms per milliliter and complete mortality at 1000 micrograms per milliliter, hinting at synergistic effects among the extract’s constituents. For context, the synthetic larvicide pylarvex used as a positive control killed 100 percent of larvae at 125 micrograms per milliliter, where margaric acid managed 45 percent. The compound also compared favorably with other natural products: it was more potent than the anthraquinone derivative 13-hydroxyversicolorin B from an endophytic Podospora species, which had an LC50 of 294.5 micrograms per milliliter, and outperformed an Aspergillus extract with an LC50 of 143.88, though it fell short of sterigmatocystin, that study’s most potent metabolite at 13.3 micrograms per milliliter.

The authors are candid about the study’s limitations. Identification of the fungus relied on macroscopic and microscopic morphology, which constrains resolution to roughly the genus level; molecular barcoding would be needed for a definitive species assignment. The mechanism by which margaric acid kills brine shrimp larvae also remains unexplored, leaving open questions about whether the fatty acid disrupts membranes, interferes with molting, or acts through some other pathway. Whether the compound’s high cytotoxicity in the brine shrimp assay would translate to hazards for non-target aquatic organisms, or conversely to useful pharmaceutical activity, will require dedicated follow-up. These caveats are typical of early natural product chemistry, where a single paper establishes identity and preliminary bioactivity while leaving the deeper biology to future work.

Even so, the findings carry a certain viral appeal: a saturated fatty acid, chemically unremarkable at first glance, produced by a fungus hiding inside the world’s most familiar spice, capable of killing the larvae of the mosquito that transmits Africa’s deadliest disease. The authors suggest that margaric acid could serve as a potential biodegradable natural larvicide in mosquito control programs, and that its pronounced cytotoxicity warrants investigation for other bioactivities. As resistance to synthetic insecticides continues to erode conventional vector control, molecules sourced from the endophytic microbiome of medicinal plants offer a renewable and environmentally gentler pipeline. Ginger’s hidden fungus may be only the first of many such discoveries waiting inside the tissues of the world’s medicinal flora.

Subject of Research: Larvicidal and cytotoxic margaric acid isolated from an endophytic fungus of ginger rhizome

Article Title: Isolation and characterization of margaric acid from an endophytic fungus of Zingiber officinale rhizome, its cytotoxicity and larvicidal activity against Anopheles gambiae

Article References: Muhammad, N. A., Muhammad, A. J., Adoum, O. A., Abubakar, S., & Salihu, A. S. (2026). Isolation and characterization of margaric acid from an endophytic fungus of Zingiber officinale rhizome, its cytotoxicity and larvicidal activity against Anopheles gambiae. Discover Chemistry, 3(1), Article 561. https://doi.org/10.1007/s44371-026-01009-5

Image Credits: AI Generated

DOI: 10.1007/s44371-026-01009-5

Keywords: margaric acid, endophytic fungus, Zingiber officinale, Anopheles gambiae, malaria, larvicide, natural products, Arthroderma, brine shrimp lethality, heptadecanoic acid, vector control, NMR characterization

The urgency behind the search is difficult to overstate. According to recent estimates from the World Health Organization, bacterial antimicrobial resistance was associated with approximately 1.14 million deaths in 2021 alone. Between 1990 and 2021, deaths linked to antimicrobial resistance among adults aged 70 and older rose by more than 80 percent, and projections suggest that by 2050 resistance could be directly responsible for roughly 1.91 million deaths annually, with a further 8.22 million deaths associated with resistant infections. Gram-negative bacteria such as E. coli, Acinetobacter baumannii, Pseudomonas aeruginosa and Shigella are among the worst offenders, wielding structurally dynamic cell envelopes, antibiotic-inactivating enzymes, remodeled porins, efflux pumps and altered drug targets that render many existing drugs ineffective.

What makes the new study particularly compelling is its focus on biofilms, the dominant form of microbial growth in nature. Biofilms are structured communities of bacteria encased in a self-produced extracellular matrix of polysaccharides, proteins, nucleic acids and lipids. This matrix shields the inhabitants from antibiotics and immune attack, restricts drug penetration, and supports cell-to-cell communication, making biofilm-associated infections notoriously difficult to eradicate. Both E. coli and Shigella form robust biofilms that dramatically amplify their resistance. Most previous screens of natural products have tested only free-floating, planktonic bacteria, leaving antibiofilm activity largely unexplored. The Indian research team set out to close that gap by examining not just whether their fungal extract killed bacteria, but whether it could prevent biofilms from forming and dismantle those already established.

The journey began with leaves collected from healthy A. officinalis trees. After thorough washing and surface sterilization with ethanol and sodium hypochlorite, leaf segments were placed on potato dextrose agar supplemented with antibiotics to suppress bacterial contaminants. Fungal hyphae emerged only from the cut edges of the sterilized segments, confirming that the fungus truly lived inside the plant tissue rather than on its surface. Morphological examination under light and field-emission scanning electron microscopes revealed dense, septate, branched hyphae and a gray to grayish-green, velvety colony. Molecular identification followed: the researchers amplified the internal transcribed spacer region of the fungal genome with the universal primers ITS1 and ITS4, sequenced the product, and compared it against the Unite 9.0 database. The result was a 100 percent match with Daldinia sp., confirmed by a maximum-likelihood phylogenetic analysis built from the closest neighbors in the NCBI database.

To coax the fungus into releasing its chemical defenses, the team grew it in potato dextrose broth for 21 days in the dark, then extracted the culture filtrate with ethyl acetate and methanol. The ethyl acetate fraction, designated AO-EA, proved the more potent of the two and became the focus of all subsequent experiments. In agar well diffusion assays, the crude extract produced clear zones of inhibition ranging from 13.67 to 17.33 millimeters against E. coli and Shigella. Those zones are smaller than the 30 to 33 millimeters produced by the fluoroquinolone antibiotic ciprofloxacin, but the comparison is not straightforward. Ciprofloxacin is a purified single compound with a well-characterized target, whereas AO-EA is a crude mixture in which any individual active molecule is present at relatively low concentration, diluted among inactive constituents. Purification and enrichment of the active principles could substantially improve potency.

Quantitative testing reinforced the promise. Broth microdilution following Clinical and Laboratory Standards Institute guidelines yielded minimum inhibitory concentrations of 625 micrograms per milliliter for E. coli and 312.5 micrograms per milliliter for Shigella, with minimum bactericidal concentrations of 625 micrograms per milliliter for both organisms, indicating that the extract does not merely stall growth but kills the cells outright. Time-kill kinetics over 24 hours showed a significant, progressive decline in viable counts of both pathogens at the MIC, while untreated controls grew normally. Field-emission scanning electron microscopy then revealed what the killing looked like at the cellular level: after three hours of exposure, treated cells appeared deformed, wrinkled and broken, with altered shape and size, in stark contrast to the smooth, intact rods of the untreated controls.

The mechanism of action came into sharper focus through two complementary techniques. Flow cytometry with propidium iodide, a red fluorescent dye that enters only cells with compromised membranes, showed that after three hours at twice the MIC, 84.04 percent of E. coli cells and 62.15 percent of Shigella cells had lost membrane integrity, a rightward shift in fluorescence that signals catastrophic permeability failure. Confocal laser scanning microscopy confirmed the same pattern visually, with treated cells glowing red in the propidium iodide channel. The confocal experiments also used the dye DCFDA to probe for reactive oxygen species, and the results were striking: treated cells displayed a surge in fluorescence comparable to that produced by hydrogen peroxide, indicating that the extract floods the bacteria with oxidative stress. Membrane disruption and ROS-mediated damage together appear to form a two-pronged killing strategy.

The antibiofilm results may be the study’s most impressive contribution. In crystal violet staining assays, AO-EA at its MIC prevented new biofilm formation by 93.03 percent for E. coli and 89.38 percent for Shigella. More remarkably, when the researchers allowed mature 24-hour biofilms to establish first and then added the extract, biomass fell by 86.20 percent for E. coli and 83.02 percent for Shigella. Published criteria consider inhibition above 50 percent to be good antibiofilm activity, and the extract not only cleared that bar decisively but approached the performance of ciprofloxacin, which achieved just over 90 percent inhibition. An MTT assay, which measures the metabolic activity of living cells through a colorimetric reaction, showed that only around 10 to 15 percent of biofilm cells remained viable and metabolically active after treatment. Scanning electron micrographs of treated biofilms revealed scattered, loosely associated cells instead of the dense, layered architecture of untreated communities, pointing to impaired adhesion and destabilized cell-to-surface interactions.

Finally, high-resolution liquid chromatography mass spectrometry on an Orbitrap platform identified 15 distinct metabolites in the extract, spanning antimicrobial peptides, carboxylic acids, alkaloids, polyketides and phenols. Among the notable constituents were citrinin, a polyketide recently reported to show antimicrobial activity from other mangrove-derived fungi; radicinin, known to inhibit the grapevine pathogen Xylella fastidiosa; homovanillic acid, azelaic acid, xanthine, 5-hydroxyindole-3-acetic acid, gluconic acid and anthranilic acid, all of which have documented antibacterial credentials in other fungal systems; and peptide compounds including glycyl-L-leucine and two cyclic dipeptides of the octahydropyrrolo[1,2-a]pyrazine-dione family. Several of these molecules had not previously been reported from the genus Daldinia, although related phenols and carboxylic acids from Daldinia eschscholtzii have shown antibacterial and antibiofilm effects elsewhere. The extreme conditions of mangrove ecosystems, with their fluctuating salinity, tides and oxygen levels, are thought to drive these fungi toward chemically novel metabolism, and the competition among microorganisms packed into plant tissues likely selects for broad-spectrum defensive chemistry.

The authors caution that much work remains before any clinical application. The active compounds must be isolated and purified, their individual contributions disentangled, their mechanisms confirmed at the molecular level, and their safety and efficacy tested far beyond the culture dish. Nevertheless, the study demonstrates that a single fungus plucked from the leaves of an ecologically important, traditionally medicinal mangrove can simultaneously kill planktonic Gram-negative pathogens, shatter their membranes, provoke lethal oxidative stress, block nearly all new biofilm formation and dismantle the majority of established biofilms. As resistance continues to erode the value of existing antibiotics, such findings suggest that the world’s mangrove forests, among the most productive and least explored ecosystems on Earth, may hold a substantial share of the next generation of anti-infective drugs, provided they are studied before they disappear.

Subject of Research: Antimicrobial and antibiofilm activity of secondary metabolites from the mangrove endophytic fungus Daldinia sp. isolated from Avicennia officinalis

Article Title: Antimicrobial and Antibiofilm Activity of an Endophytic Fungus, Daldinia sp. Isolated From Avicennia officinalis

Article References: Dutta, N., Kumari, N., Lather, A., & Kumar, K. (2026). Antimicrobial and Antibiofilm Activity of an Endophytic Fungus, Daldinia sp. Isolated From Avicennia officinalis. MicrobiologyOpen, 15(5), Article e70422. https://doi.org/10.1002/mbo3.70422

Image Credits: AI Generated

DOI: 10.1002/mbo3.70422

Keywords: endophytic fungus, Daldinia, mangrove, Avicennia officinalis, antimicrobial resistance, antibiofilm, Escherichia coli, Shigella, secondary metabolites, LC-HRMS, membrane disruption, reactive oxygen species

The substrate in question, ent-pimara-8(14),15-dien-19-oic acid, is a pimarane-type diterpene carboxylic acid, a molecule built around a rigid tricyclic core decorated with two carbon-carbon double bonds, an internal one at position 8(14) and an external vinyl group at position 15. When the researchers added this compound to growing liquid cultures of the fungus, the organism responded by transforming it into four oxidized products. Two of these, 15,16-epoxy-ent-pimar-8(14)-en-19-oic acid and 15,16-dihydroxy-ent-pimar-8(14)-en-19-oic acid, were known compounds, but the other two, 17-hydroxy-15,16-epoxy-ent-pimar-8(14)-en-19-oic acid and 15-oxo-16-hydroxy-ent-pimar-8(14)-en-19-oic acid, had never been described before. In every case, the fungus attacked the vinyl group and left the internal double bond intact.

That selectivity is what makes the result remarkable. Monosubstituted double bonds like the vinyl group are typically more reactive toward chemical oxidants, yet when chemists treated a related pimaradiene with the classic epoxidation reagent meta-chloroperbenzoic acid, they obtained only epoxides at the internal 8(14) position along with rearranged byproducts, the opposite outcome. Previous microbial studies pointed the same way: a strain of Aspergillus niger oxidized the tricyclic ring system of the same substrate at carbons 1, 6, 7, 11 and the internal double bond while ignoring the vinyl group, and the endophytic fungus Preussia minima also hydroxylated positions away from the vinyl moiety. The new work shows that A. sclerotiorum does essentially the reverse, a biotransformation pattern the authors describe as at least uncommon.

To probe whether this apparent regioselectivity was real, the team challenged the fungus with a second diterpene, abietic acid, a resin acid bearing two endocyclic double bonds at positions 7(8) and 13(14). If the fungus simply preferred external double bonds, abietic acid, which has none, should be metabolized differently. Instead, the organism oxidized a methyl group attached at carbon 13, outside the ring system, producing 16-hydroxyabieta-7(8),13(14)-dien-19-oic acid. This mirrors what it did with the pimarane substrate, where the vinyl group and the methyl group both hang off carbon 13, suggesting the enzymes recognize the spatial region around that carbon rather than a particular bond type.

The structural detective work relied on one- and two-dimensional nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry. In the epoxide product, the characteristic ABX spin system of vinylic hydrogens near 5.4 and 4.9 parts per million in the proton spectrum vanished, replaced by new signals between 2.5 and 2.8 parts per million, while carbon-13 signals for the sp2 carbons at 147.2 and 112.9 parts per million gave way to aliphatic signals at 58.9 and 42.5. For the diol product, the carbon-15 resonance appeared at 80.4 parts per million, a deshielded position that literature correlations assign to the 15S configuration. Because an epoxide hydrolase opening the epoxide by an SN2-type attack at carbon 16 would yield only one epimer, whereas an SN1 mechanism through a carbocation would yield both, the researchers could infer that the epoxide products share that same 15S configuration. The hydroxyketone product, carrying a carbonyl signal at 214.8 parts per million, appears to arise from further oxidation of the diol’s C-15 hydroxyl group.

Hunting for the enzyme behind this chemistry, the team scanned publicly available whole-genome shotgun contigs of A. sclerotiorum using tblastn, searching for cytochrome P450 sequences similar to CYP105A1 from Streptomyces griseolus, a bacterial enzyme previously shown to oxidize the C-15 isopropyl group of abietic acid and to epoxidize the vinyl group of an isopimarane diterpene. The search returned two candidate open reading frames, one of 1083 nucleotides encoding 360 amino acids and another of 1641 nucleotides encoding 546 amino acids, with sequence identities of 25 and 24 percent and query coverages of 37 and 35 percent respectively. Crucially, both proteins carry the canonical P450 signature motif FxxGxxxCxG, including the cysteine residue that binds the heme iron, confirming they belong to the cytochrome P450 superfamily.

To visualize how such an enzyme might steer the substrate, the researchers turned to molecular docking using the crystal structure of a bacterial cytochrome P450 as a proxy, since no structure exists for the Aspergillus enzyme. In the docked complex, the diterpene sits in the active site with its vinyl group and the C-13 methyl group positioned closest to the heme catalytic center, exactly where an oxygenating iron-oxo species would deliver its atom. Basic amino acids, including arginine and asparagine, appear to anchor the molecule through hydrogen bonding and electrostatic interactions with the carboxylic acid at carbon 4, holding the substrate in the orientation that favors oxygenation at the side chain rather than the ring system. The docking picture closely resembles calculations published for CYP105A1 acting on abietic acid, reinforcing the idea that a similar P450 in the fungus drives the observed regioselectivity.

The practical payoff came from antiparasitic testing. The parent diterpene and its four derivatives were evaluated against promastigote forms of Leishmania amazonensis and epimastigote forms of Trypanosoma cruzi, the parasites responsible for cutaneous leishmaniasis and Chagas disease, both neglected tropical diseases that affect millions of people in Latin America and beyond. Oxidation consistently improved activity. Against L. amazonensis, the epoxide and the hydroxyketone derivatives showed IC50 values less than half that of the parent compound. Most strikingly, the hydroxyketone inhibited the trypomastigote stage of T. cruzi with an IC50 of 21.4 micromolar, slightly better than the reference drug benznidazole, which came in at 34.5 micromolar under the same assay conditions. Equally important, cytotoxicity assays against healthy Vero cells showed very low toxicity, particularly for the hydroxyketone, yielding favorable selectivity indices.

The structure-activity picture that emerges suggests that chemical modifications at the vinyl side chain matter more for antiparasitic potency than variations on rings A and B of the ent-pimarane skeleton. Earlier studies had found that hydroxylation near ring A did not substantially enhance activity against another T. cruzi strain, while ent-pimaranes modified at ring B showed IC50 values in the 15 to 20 micromolar range. The new derivatives, functionalized precisely at the side chain, now extend that trend and hint that the side chain is a promising handle for drug design.

Beyond the immediate antiparasitic results, the study validates a broader strategy: mining endophytic fungi from diterpene-producing plants for enzymes that recognize the very molecules their hosts biosynthesize. The intimate evolutionary relationship between endophytes and plant biochemistry may predispose their enzymatic machinery to accept plant terpenes as substrates, and expanding the pool of such microorganisms helps overcome the classic unpredictability of whole-cell biotransformation. With two new diterpenes in hand, a candidate P450 enzyme identified, and a docking model that explains the selectivity, the researchers have laid out a complete chain of evidence from genome to molecule to bioactivity, offering a template for turning humble tree-dwelling fungi into factories for medicinally relevant chemistry.

Subject of Research: Regioselective epoxidation of a plant diterpene by the endophytic fungus Aspergillus sclerotiorum and the antiparasitic activity of the resulting oxidized products.

Article Title: Epoxidation of ent-pimara-8(14),15-dien-19-oic acid by whole cells of the endophytic fungus Aspergillus sclerotiorum

Article References: Din, Z. U., de Medeiros, L. S., Abreu, L. M., Lazarin-Bidóia, D., Scariot, D. B., Garcia, F. P., de Paula, J. C., Nakamura, C. V., Fill, T. P., & Rodrigues-Filho, E. (2026). Epoxidation of ent-pimara-8(14),15-dien-19-oic acid by whole cells of the endophytic fungus Aspergillus sclerotiorum. International Microbiology. https://doi.org/10.1007/s10123-026-00895-0

Image Credits: AI Generated

DOI: 10.1007/s10123-026-00895-0

Keywords: Aspergillus sclerotiorum, biotransformation, diterpene, ent-pimaradienoic acid, epoxidation, cytochrome P450, endophytic fungus, Cupressus lusitanica, Leishmania amazonensis, Trypanosoma cruzi, antiparasitic activity, regioselectivity