The study, published in the journal Acta Parasitologica, was conducted by Akshay Majare, Nandkishore Lavhe, V. K. Biradar and Tini Pillai of the College of Agriculture in Nagpur, together with colleagues at the ICAR–Central Institute for Cotton Research and the ICAR–Central Potato Research Institute. The researchers set out to answer two questions that matter enormously for anyone designing integrated pest management programmes. First, how lethal are two species of entomopathogenic nematodes, Steinernema siamkayai and Heterorhabditis indica, against third-instar fall armyworm larvae? Second, are these nematodes compatible with two commercially important entomopathogenic fungi, Metarhizium anisopliae and Beauveria bassiana, and does the answer depend on whether the organisms are applied together or one after the other?

Entomopathogenic nematodes are microscopic roundworms that hunt insect larvae in the soil. Their infective juveniles, the free-living stage used in biocontrol, carry symbiotic bacteria in their guts. When a nematode enters an insect through natural openings such as the mouth, spiracles or anus, it releases these bacteria into the haemocoel, the insect’s open circulatory cavity. The bacteria multiply rapidly, killing the host within days through septicaemia and providing the nematodes with a nutrient-rich environment in which to reproduce. Entomopathogenic fungi attack by a different route: spores germinate on the insect’s cuticle, penetrate the body wall, and proliferate internally, eventually killing the host and sporulating on its corpse. Because the two agents exploit different infection pathways and different ecological niches, scientists have long suspected that combining them could deliver a one-two punch that neither achieves alone.

To test this, the team first measured the inherent virulence of each nematode species under controlled laboratory conditions. Third-instar fall armyworm larvae were exposed to a range of infective juvenile concentrations, from five to one hundred nematodes per larva, and mortality was recorded over ninety-six hours. The results were strikingly asymmetric. Steinernema siamkayai proved consistently and substantially more virulent than Heterorhabditis indica. At doses of thirty to forty infective juveniles per larva, S. siamkayai achieved complete mortality of the test larvae within ninety-six hours, whereas H. indica required the full one hundred juveniles per larva to reach the same endpoint. Probit analysis, a standard statistical technique for quantifying dose-response relationships in toxicology, confirmed the gap: the lethal concentration needed to kill half the larvae, the LC50, was just 5.47 infective juveniles per larva for S. siamkayai, compared with 15.14 for H. indica. The LC90 values told an even starker story, at 14.23 versus 83.52 infective juveniles per larva respectively. In practical terms, the Steinernema species needed roughly one-sixth the dose of its Heterorhabditis counterpart to achieve near-total kill.

With virulence baselines established, the researchers turned to the compatibility question. They paired each nematode with each fungus in three application regimes: simultaneous inoculation, nematode applied first followed by fungus, and fungus applied first followed by nematode. Larval mortality was recorded, and the interactions were classified using co-toxicity factor analysis, a method borrowed from pesticide combination studies that quantifies whether two agents act additively, synergistically or antagonistically when combined. The outcome hinged almost entirely on the nematode species involved. Simultaneous application of any nematode-fungus pair produced additive interactions across the board, meaning the combined mortality matched what would be expected from the sum of the individual effects, with no interference between the agents. The standout combination was S. siamkayai together with Metarhizium anisopliae, which killed 98.55 percent of larvae at ninety-six hours, the highest figure recorded in the laboratory phase of the study.

The picture changed, however, when the agents were applied sequentially. When Heterorhabditis indica was introduced after the fungi had already been applied, the interaction turned antagonistic: the combined mortality fell short of expectations, suggesting that the fungus, by establishing itself in or on the host first, somehow compromised the nematode’s ability to infect or complete its development. The authors did not identify the precise mechanism in this study, but the phenomenon is consistent with competition for the host resource. Both agents ultimately depend on the same larval cadaver for reproduction, and a fungus that has colonised a host first may leave insufficient resources, or an unsuitable internal environment, for the nematode’s symbiotic bacteria to flourish. Notably, Steinernema siamkayai was immune to this sequencing effect, maintaining additive compatibility with both fungi regardless of whether it was applied before or after them. This robustness marks it out as the more dependable partner in a combined biocontrol programme.

To check that the laboratory findings survived contact with more realistic conditions, the team validated the most promising combinations in pot experiments, with mortality and co-toxicity factors assessed at one hundred and twenty hours after treatment. Alone, S. siamkayai caused the highest larval mortality at 80.3 percent, followed by H. indica at 70.5 percent. Among the combined treatments, S. siamkayai plus M. anisopliae again led the field, achieving 71.5 percent mortality, and every nematode-fungus combination tested in pots registered additive interactions by co-toxicity analysis. The somewhat lower figures under pot conditions compared with the laboratory are unsurprising, since soil structure, moisture and other environmental variables inevitably dilute infection efficiency, but the qualitative conclusion held: simultaneous application preserves compatibility, and S. siamkayai is the more forgiving and more lethal of the two nematodes.

The significance of these results extends beyond a single pest. Fall armyworm management currently leans heavily on synthetic insecticides and Bt-transgenic crops, both of which face mounting resistance problems. Field populations of the pest have already shown multiple and cross-resistance to Bt toxins and organophosphates in some regions, and the caterpillar’s polyphagous habits, documented across dozens of host plant species in the Americas, make crop rotation alone an inadequate defence. Microbial biocontrol agents offer a complementary tool that is difficult for pests to circumvent, because the selection pressures they impose differ fundamentally from those of chemical toxins. Moreover, entomopathogenic nematodes and fungi are self-replicating, leave no toxic residues, and are compatible with many other components of integrated pest management, including certain insecticides, as earlier studies on their chemical compatibility have shown.

What this study adds is a practical rule of thumb for deploying such agents together. Compatibility between biocontrol agents is often assumed rather than tested, and the assumption can be costly. Prior research has documented both synergy and antagonism in nematode-fungus combinations against other pests, including black vine weevil, wireworms and scarab grubs, and the mechanisms underlying these interactions, from volatile organic compounds emitted by fungi that influence nematode foraging to direct competition for the host cadaver, remain an active area of investigation. By demonstrating that application sequence is a decisive variable for H. indica but not for S. siamkayai, the Indian team has given practitioners a concrete, testable guideline: if in doubt, apply simultaneously, and if a sequential schedule is unavoidable, choose the nematode species that tolerates it.

The authors identify S. siamkayai, particularly in combination with Metarhizium anisopliae, as a promising microbial strategy for the integrated management of fall armyworm. Field-scale trials will be needed to confirm that the additive interactions observed in the laboratory and in pots translate into meaningful yield protection in farmers’ fields, where UV radiation, desiccation and soil heterogeneity all challenge the survival of both agents. But the core message is already actionable. In the escalating contest between growers and one of the world’s most adaptable crop pests, the details of biological control matter, and something as mundane as the order of two spray applications may be the difference between a partnership that works and one that quietly undermines itself.

Subject of Research: Compatibility and virulence of entomopathogenic nematodes and fungi against the fall armyworm Spodoptera frugiperda

Article Title: Application Sequence Determines the Compatibility and Virulence of Entomopathogenic Nematodes and Entomopathogenic Fungi Against Spodoptera frugiperda

Article References: Majare, A., Lavhe, N., Biradar, V. K., Pillai, T., Deshmukh, V., Banu, G., Fand, B. B., Shah, V., Mhatre, P. H., & Thube, S. (2026). Application Sequence Determines the Compatibility and Virulence of Entomopathogenic Nematodes and Entomopathogenic Fungi Against Spodoptera frugiperda. Acta Parasitologica, 71(5), Article 220. https://doi.org/10.1007/s11686-026-01406-x

Image Credits: AI Generated

DOI: 10.1007/s11686-026-01406-x

Keywords: fall armyworm, Spodoptera frugiperda, entomopathogenic nematodes, entomopathogenic fungi, Steinernema siamkayai, Heterorhabditis indica, Metarhizium anisopliae, Beauveria bassiana, biological control, application sequence, co-toxicity factor, integrated pest management

The review, led by Ya-Qiang Zheng of Guizhou University of Traditional Chinese Medicine together with Komivi Senyo Akutse, Song Mei, Artemio Mendoza-Mendoza and Bin Chen, is the first systematic synthesis to unite the drought, salt and heavy metal literature on endophytic entomopathogenic fungi within a single mechanistic framework. The timing could hardly be more pressing. Abiotic stresses are estimated to cause global yield losses exceeding fifty percent, and climate change is intensifying drought frequency while more than 800 million hectares of soil worldwide are already affected by salinity. Heavy metal pollution from mining, sewage irrigation and industrial emissions adds a third, largely irreversible threat to arable land.

The authors begin with an evolutionary observation that reframes how these fungi should be seen. Rather than being mere insect parasites that occasionally wander into plants, entomopathogenic fungi appear deeply entwined with the plant root mycobiome. Genomic analyses show that Metarhizium and Beauveria species are close relatives of established endophytes and plant-associated fungi such as Fusarium. The Metarhizium lineage may even have travelled the opposite evolutionary route: starting as saprophytes attracted to root exudates, becoming plant endophytes, and only later acquiring the machinery to kill insects. Many Metarhizium species retain the flexibility to switch between all three lifestyles, which is precisely what makes them so attractive as dual-purpose agricultural agents.

Under drought, the mechanisms these fungi deploy are remarkably layered. In red oak seedlings, B. bassiana colonisation maintained higher leaf relative water content and stomatal conductance while promoting root growth, giving trees better access to scarce water. In tomato, the same fungus pushed plants toward what researchers call a water spender strategy, growing larger and drawing more water through improved root function and stomatal regulation. In onion, colonisation enhanced uptake of phosphorus, calcium, magnesium and iron, nutrients central to energy transfer, membrane stability and chlorophyll synthesis. Wheat seeds primed with M. anisopliae MetA1 showed improved photosynthesis, growth and yield under drought, while maize plants colonised by M. robertsii grew taller under water stress, although the authors caution that results in maize have been inconsistent, likely because inoculation method strongly determines whether the fungus establishes at all.

Beneath these visible improvements lies a concerted molecular campaign against oxidative damage. Drought triggers the accumulation of reactive oxygen species, which shred membranes and proteins. The review documents how endophytic entomopathogenic fungi consistently boost the plant’s enzymatic arsenal, elevating superoxide dismutase, catalase, peroxidase and ascorbate peroxidase activity across species as diverse as tomato, onion, wheat and mallow. These enzymatic gains are mirrored by reductions in malondialdehyde, a chemical fingerprint of lipid peroxidation, indicating genuinely preserved membrane integrity. In parallel, the fungi stimulate non-enzymatic defences: polyphenols, flavonoids and ascorbic acid accumulate, most dramatically under severe drought, when fungal support matters most.

Osmotic adjustment, the accumulation of compatible solutes such as proline, soluble sugars and free amino acids, forms a third pillar of drought resilience, though the review is careful to note that this response is species-specific. Proline rose in colonised tomato, onion and wheat, helping cells retain turgor pressure, yet in red oak the fungus conferred drought tolerance without raising proline at all, suggesting alternative strategies such as enhanced root growth can substitute. Hormonal rewiring completes the picture: B. bassiana upregulated genes governing stomatal behaviour and abscisic acid signalling in tomato, elevated ABA in colonised maize, and in mallow increased gibberellins while suppressing ethylene, the senescence hormone that accelerates tissue damage under stress. The precise molecular dialogue between fungus and host under drought, the authors stress, remains largely unmapped and represents a major frontier.

Under salinity, the fungal playbook shifts toward ion management. The central challenge for salt-stressed plants is maintaining a favourable potassium-to-sodium ratio, and here the evidence is striking. Rice seeds primed with M. anisopliae MetA1 accumulated less sodium and more potassium in both roots and shoots, while B. bassiana strain BeauA1 improved the same ratio under both saline and non-saline conditions. The fungi also promoted proline and carbohydrate accumulation to maintain cell turgor, activated antioxidant enzymes including glutathione S-transferase, and in soybean reprogrammed hormone balance by lowering abscisic acid while raising jasmonic acid. Growth gains followed: improved shoot length, chlorophyll content, leaf area and even stolon production in potato, all under salt concentrations that would normally suppress them. One Metarhizium pinghaense strain even sustained indole-3-acetic acid production at up to 200 millimolar sodium chloride in vitro, sustaining root growth when it mattered most.

The heavy metal findings are arguably the most surprising, revealing the fungi as both detoxification engines and ecological regulators. On the fungal side, B. bassiana immobilises cadmium, lead, zinc and copper on its cell wall through carboxyl, phosphate, hydroxyl and amino groups, removing up to 84.5 percent of total metals from multi-metal wastewater. Inside fungal cells, cadmium entering through calcium channels is bound by glutathione and processed by upregulated cytochrome P450 enzymes, whose inhibition cuts cadmium removal by 45 percent. Some Metarhizium species acquired a bacterial gene, methylmercury demethylase, through horizontal gene transfer, allowing M. robertsii to convert highly toxic methylmercury into volatile elemental mercury that escapes the rhizosphere. Others, such as Beauveria caledonica, excrete oxalic acid that precipitates metals as insoluble oxalate crystals, locking them away from plant roots.

On the plant side, colonised hosts activate their own cadmium efflux pumps and metal-binding proteins while suppressing uptake transporters: in rice, M. robertsii silenced the cadmium importer OsNramp5, cutting cadmium in roots by up to 44.3 percent and in grains by 24.7 percent. In an especially timely result, M. anisopliae seed treatment disrupted what researchers describe as a Trojan Horse effect, in which nanoplastics act as carriers that smuggle lead into rice roots, reducing the soil-to-root transfer of lead by roughly a third while restoring antioxidant balance and rebuilding a beneficial rhizosphere microbiome rich in Sphingomonas and Burkholderia. The fungi even stabilise metals in soil itself, with B. bassiana FE14 cutting bioavailable cadmium from 26.23 to 5.41 milligrams per kilogram through organic acid secretion.

The review closes with a sober assessment of what stands between laboratory promise and field reality. Effectiveness depends on specific plant-fungus combinations and environmental contexts, so systematic strain screening across crops, climates and soils is paramount. Stable endophytic colonisation under fluctuating temperature, humidity, ultraviolet radiation and microbial competition remains difficult to achieve, demanding better formulations such as microencapsulation and optimised seed coating methods. The authors also urge rigorous ecological risk assessment: these fungi are potent insect pathogens, and their effects on pollinators and other beneficial insects must be evaluated before deployment, while growing concern frames all microbial inoculants as potential neomicrobiota that could disrupt native ecosystems. Yet if molecular tools such as CRISPR gene editing, multi-omics profiling and improved regulatory frameworks can unlock these fungi’s full potential, the authors argue, agriculture could gain a single microbial ally that fights pests, buffers climate extremes and cleans contaminated soils simultaneously, a combination no chemical input has ever offered.

Subject of Research: Mechanisms by which endophytic entomopathogenic fungi enhance plant tolerance to abiotic stresses including drought, salinity and heavy metal toxicity

Article Title: Entomopathogenic fungi: beyond biocontrol-unravelling mechanisms of enhanced plant abiotic stress tolerance

Article References: Zheng, Y.-Q., Akutse, K. S., Mei, S., Mendoza-Mendoza, A., & Chen, B. (2026). Entomopathogenic fungi: beyond biocontrol-unravelling mechanisms of enhanced plant abiotic stress tolerance. Crop Health, 4(1), Article 14. https://doi.org/10.1007/s44297-026-00077-4

Image Credits: AI Generated

DOI: 10.1007/s44297-026-00077-4

Keywords: entomopathogenic fungi, fungal endophytes, abiotic stress, drought tolerance, salinity, heavy metals, Beauveria bassiana, Metarhizium, plant-microbe interaction, bioremediation, sustainable agriculture, phytohormones

The urgency behind the work is hard to overstate. Mosquito-borne pathogens impose a global burden measured in hundreds of millions of infections every year, and Aedes aegypti has expanded its footprint dramatically over recent decades, carried along by urbanization, international trade and a warming climate. The conventional response—synthetic chemical insecticides—is showing its age. Resistance to the organophosphate larvicide temephos has been documented across Southeast Asia and beyond, with resistant larvae overproducing detoxification enzymes such as cytochrome P450 monooxygenases and esterases. Residual insecticides can persist in soils and sediments, poison non-target aquatic organisms and accumulate along food chains, prompting health agencies to call for larvicides that are biodegradable, targeted and affordable. Researchers have responded by mining the living world for alternatives: plant extracts, bacterial metabolites, actinobacterial filtrates and, increasingly, the secretions of fungi that spend their lives attacking insects. The new study argues that these entomopathogenic fungi, long valued as living biocontrol agents, are also superb chemical factories for green nanotechnology.

Silver’s antimicrobial pedigree reaches back to antiquity, but its nanoscale incarnation behaves very differently from the metal in jewelry and tableware. Shrink silver to a few dozen nanometers and the proportion of atoms sitting on the particle surface skyrockets; those surface atoms interact directly with biological membranes and steadily release silver ions, the species responsible for much of the toxicity. Chemists can force this transformation with reagents such as sodium borohydride or citrate, but those routes typically demand hazardous chemicals, elevated temperatures and organic solvents. Biosynthesis sidesteps the problem. Filamentous fungi secrete a rich cocktail of extracellular enzymes, proteins and polysaccharides; when their cell-free culture filtrate meets an aqueous silver nitrate solution, those biomolecules reduce silver ions (Ag+) to neutral metallic atoms (Ag0), which nucleate into nanoparticles. Other proteins then adsorb onto the nascent particle surfaces and cap them, arresting further growth and stabilizing the colloid against aggregation. The fungus, in effect, performs the reduction, the shaping and the stabilization in a single, room-temperature step, using nothing more exotic than its own metabolism.

The two species were not chosen at random. Trichoderma asperellum is a soil-dwelling workhorse of agricultural biocontrol, famed for parasitizing plant pathogens and secreting an arsenal of secondary metabolites. Metarhizium anisopliae, the agent of the so-called green muscardine disease in beetles, actively infects insects and has served for more than a century as a mycoinsecticide. Where most previous mycosynthesis studies tested a single organism, the team also combined the two cultures, harvesting a mixed filtrate containing the proteins and metabolites of both before the reduction step. This combined system, designated Ta Ma AgNPs, yielded particles with properties distinct from either single-species batch—and, as the bioassays later confirmed, distinct biological behavior. The rationale, the authors suggest, is that pooling two different fungal secretomes can produce a more diverse biomolecular coating on the nanoparticle surfaces, potentially enhancing colloidal stability and biological activity in ways that neither organism achieves alone.

Characterization followed a demanding, multi-instrument protocol. Ultraviolet–visible spectroscopy detected the characteristic surface plasmon resonance of silver nanoparticles—the collective oscillation of conduction-band electrons that absorbs light in the visible spectrum—confirming that reduction had succeeded in all three systems. Attenuated total reflectance Fourier-transform infrared spectroscopy, or ATR-FTIR, mapped the functional groups of the proteins, carbohydrates and other biomolecules adsorbed onto the particle surfaces, direct evidence that fungal metabolites were acting as capping agents. Cryogenic field-emission scanning electron microscopy and cryogenic transmission electron microscopy—techniques that image flash-frozen specimens close to their native, hydrated state—revealed uniformly spherical particles in every preparation. The mean diameters measured by TEM were 9.17 nanometers for the Trichoderma-derived particles (Ta AgNPs), 11.25 nanometers for the Metarhizium-derived particles (Ma AgNPs) and 12.54 nanometers for the combined-fungal particles (Ta Ma AgNPs), all squarely within the size range associated with potent biological activity. Dynamic light scattering characterized the hydrodynamic dimensions of the particles in suspension, while zeta potential measurements, which quantify the effective surface charge of a colloid, ranged from −23 to −26.9 millivolts. Values of that magnitude imply strong electrostatic repulsion between particles, keeping them dispersed in water rather than clumping and settling—an indispensable property for any nanoparticle intended to be applied to mosquito breeding habitats.

With the materials verified, the team turned to the mosquito. Following World Health Organization guidelines for laboratory larvicidal testing, they exposed Aedes aegypti larvae to each formulation at four concentrations—25, 50, 100 and 150 micrograms per milliliter—and scored mortality at 24 and 48 hours, correcting for control mortality with Abbott’s formula and computing lethal concentrations by probit analysis. All three preparations proved potently larvicidal, but the combined-fungal particles led at every time point. Their 24-hour median lethal concentration (LC50) was 52.32 micrograms per milliliter, against 58.61 for Ta AgNPs and 63.39 for Ma AgNPs; by 48 hours the values had fallen to 39.50, 43.60 and 51.46 micrograms per milliliter, respectively. Equally telling, the nanoparticles outperformed the raw fungal filtrates from which they were made: mortality reached 82.50 percent for Ta AgNPs, 80.44 percent for Ma AgNPs and 87.69 percent for Ta Ma AgNPs, all higher than the killing achieved by unmodified fungal extracts. The silver was no passive carrier of fungal toxins; the particles themselves were the weapons.

How exactly do these particles dispatch a larva? The leading mechanisms assemble into a coherent picture. At nine to thirteen nanometers, the spheres can adhere to and penetrate the larval cuticle, the chitinous armor through which the insect breathes and senses its world. Once inside, silver nanoparticles and the ions they liberate attack a battery of cellular targets simultaneously: they bind sulfur- and phosphorus-rich biomolecules such as proteins and DNA, inhibit respiratory and antioxidant enzymes, and catalyze the generation of reactive oxygen species that oxidize membrane lipids and structural proteins. Studies of biosynthesized silver particles in mosquito larvae have documented disruption of the midgut epithelium, deranged ion regulation and characteristic aberrant swimming before death, typically within hours to days of exposure. The negative zeta potentials measured here suggest each particle carries a corona of anionic fungal proteins, which may promote adhesion to positively charged membrane surfaces—and may help explain why the dual-species corona of the combined batch proved most lethal of all. Mortality climbed steadily with both dose and exposure time, a pattern consistent with progressive, cumulative toxicity rather than any single-point kill mechanism.

The same particles were then pitted against four bacterial pathogens representing both major cell-envelope architectures: the Gram-negatives Escherichia coli and Pseudomonas aeruginosa and the Gram-positives Bacillus megaterium and Staphylococcus aureus. Using the zone-of-inhibition well diffusion method and benchmarking against the antibiotic chloramphenicol, the researchers found that all three formulations inhibited P. aeruginosa and B. megaterium, with the clearest effect at the highest dose of 150 micrograms per milliliter. Activity against Pseudomonas is particularly noteworthy. P. aeruginosa is an opportunistic pathogen shielded by a restrictive outer membrane and a formidable talent for biofilm formation, and it ranks among the multidrug-resistant bacteria for which new agents are most urgently sought. Silver nanoparticles are believed to act through multiple, simultaneous mechanisms—membrane disruption, ion release, protein denaturation, oxidative stress and interference with DNA replication—which together make comprehensive bacterial resistance far harder to evolve than it is against single-target antibiotics. The differences observed among the four species likely reflect their contrasting cell-wall structures and the distinct biomolecular coatings of each nanoparticle batch, and the authors suggest that this dual larvicidal and antibacterial profile positions the formulations as a two-in-one tool for mosquito-borne disease control and antimicrobial applications alike.

None of this means silver-spiked water will be sprayed across tropical neighborhoods tomorrow. The work remains laboratory-scale, and the road from beaker to breeding ground is long. Silver nanoparticles can themselves harm non-target aquatic organisms, and questions of environmental persistence, accumulation and dose escalation in real water bodies must be resolved before any field deployment; the formulations would also need to survive sunlight, dilution and microbial degradation in open habitats, and to be manufactured at scale at tolerable cost. The authors do not claim otherwise. What the study does establish is that fungal biosynthesis can reliably deliver nanoparticles of tightly controlled size, negative surface charge and robust colloidal stability—and that combining two entomopathogenic species produces a particle that outperforms those made by either fungus alone, both as a larvicide and as an antibacterial. As dengue incidence climbs across Asia, Africa and Latin America and insecticide resistance erodes the frontline tools of vector control, the image of two humble molds, grown side by side in a culture flask, quietly manufacturing a dual-purpose weapon against mosquitoes and bacteria alike is precisely the kind of biological ingenuity the field has been hunting for. Corresponding author Titik Kartika and colleagues conclude that the combined Ta Ma nanoparticles, above all, stand out as a candidate alternative strategy for mosquito-borne disease control—and, from the same flask, a possible ally in the broader fight against antimicrobial resistance.