The study addressed a fundamental constraint of biological control in soil: the spatial mismatch between stationary fungal formulations and mobile wireworm larvae. Even when a highly virulent fungal strain is identified, its field performance depends on whether larvae actually encounter the inoculum. Wireworms navigate a hierarchical sequence of chemical cues, orienting toward carbon dioxide gradients produced by respiring roots at long range, then discriminating among potential hosts using root-emitted volatile organic compounds such as hexanal at intermediate distances. Aggregation signals like 2-pentylfuran guide larvae to suitable feeding sites, while close-range feeding stimulants including sugars, lipids, and proteins prolong residence time near a resource. The Belgian team hypothesized that fungal strains capable of emitting attractive volatiles could function as active lethal sinks, recruiting foraging wireworms to an edible particle loaded with infective conidia rather than relying on accidental contact alone.

To identify suitable candidates, the researchers assembled a diverse panel of entomopathogenic fungi from three sources. Reference strains were acquired from the BCCM/MUCL collection in Belgium following a comprehensive literature review, with priority given to strains recovered from insect cadavers. Two novel strains were isolated from naturally infected wireworm larvae collected in agricultural fields in Gedinne, Belgium, and Sauvagnon, France, and molecular identification based on sequencing of the internal transcribed spacer region, beta-tubulin, and elongation factor genes confirmed them as Metarhizium anisopliae MUCL 58549 and Ophiocordyceps sp. MUCL 58550. Two commercially available Metarhizium strains were included solely as methodological benchmarks to calibrate larval susceptibility. The non-pathogenic biological control fungus Trichoderma harzianum MUCL 29707 served as a reference treatment to control for non-pathogenic effects associated with bead presence or fungal growth within the formulation.

The experimental framework proceeded in four steps. First, in vitro virulence was assessed by direct fungal contact: starved late-instar larvae of Agriotes lineatus and A. obscurus were exposed for five hours to standardized conidial colonies of approximately 10 million conidia per well, then monitored weekly for twelve weeks. A commercial lambda-cyhalothrin solution served as the insecticide control and induced rapid, complete mortality within the first week, confirming the sensitivity of the bioassay. Second, the most virulent candidates were incorporated into calcium-alginate beads enriched with 30 percent starch, a phagostimulant chosen because carbohydrate-rich substrates elicit strong biting responses in wireworms. Beads were produced by dispensing 100-microliter droplets of a matrix containing 2 percent sodium alginate and 3 × 10⁸ conidia per milliliter into sterile 0.1 M calcium chloride solution under continuous agitation. Forty beads were introduced into each sterilized soil microcosm together with a single larva and a potato food cube, and mortality was recorded every ten days over twelve weeks.

The initial screening revealed a striking hierarchy of pathogenicity. Most reference strains, including Beauveria bassiana MUCL 39817, Beauveria brongniartii MUCL 131 and MUCL 34853, Lecanicillium lecanii MUCL 8115, Paecilomyces fumosoroseus MUCL 15122, Purpureocillium lilacinum MUCL 38701, and Aspergillus flavus MUCL 55276, did not significantly affect larval survival relative to the untreated control, which retained approximately 92 percent survival. Ophiocordyceps sp. MUCL 58550 emerged as the most virulent strain, significantly reducing larval survival to approximately 43 percent with a hazard ratio of 11.7, meaning exposure increased the mortality risk nearly twelvefold. The commercial benchmark Metarhizium brunneum strain B also significantly increased mortality risk with a hazard ratio of 9.2. Metarhizium anisopliae MUCL 58549, although not statistically significant in this direct-contact assay, exhibited substantial biological activity with a hazard ratio of 4.1, and was selected for subsequent soil trials based on the magnitude of this observed effect.

The transition from petri dish to soil microcosm produced what the authors describe as a virulence reversal. Encapsulated Metarhizium anisopliae MUCL 58549 significantly reduced larval survival to approximately 38 percent in soil, tripling the mortality risk with a hazard ratio of 3.08. In contrast, Ophiocordyceps sp. MUCL 58550, the most lethal strain in vitro, showed only a moderate and statistically insignificant effect in soil, with survival remaining around 46 percent. Koch’s postulates were fulfilled for both pathogens: all surface-sterilized cadavers from the pathogen treatments yielded outgrowth of the respective inoculated fungi, confirmed microscopically by conidial morphology, demonstrating that mortality resulted from successful internal infection rather than surface contamination. The authors attribute the discrepancy to behavioral defenses. Wireworms are not passive targets; they can actively reduce infection risk through grooming and increased molting frequency to physically shed conidia before germ tubes can breach the cuticle. In a three-dimensional soil matrix, only transient or accidental contact occurs unless an attractive cue bridges the spatial gap, creating a race against time between pathogen penetration and host evasion.

Behavioral assays using a glass Y-tube olfactometer partially filled with moistened vermiculite revealed that the two surviving candidates differed sharply in their capacity to recruit larvae. Overall responsiveness averaged 63.1 percent across treatments. Larvae were significantly attracted to beads containing Trichoderma harzianum MUCL 29707, with 70 percent of responding individuals selecting the fungal arm over the blank-bead control, yielding an odds ratio of 2.33. Metarhizium anisopliae MUCL 58549 beads elicited a similar positive chemotaxis, with 65 percent choice and an odds ratio of 1.85. Ophiocordyceps sp. MUCL 58550 produced no directional response, with larvae distributed equally between arms. The strong attraction to the non-pathogenic Trichoderma highlights a critical constraint of attract-and-kill systems: behavioral recruitment without lethality risks creating pest safe havens where wireworms congregate on a resource that cannot infect them.

To understand the chemical basis of these behavioral differences, the researchers performed volatolomic profiling by thermal desorption gas chromatography coupled with mass spectrometry, sampling headspace from airtight chambers containing formulated beads of the same age and production batches used in the behavioral assays. Dynamic headspace collection over four hours on Tenax TA/Carbograph sorbent tubes, with butylbenzene as internal standard, detected 80 volatile organic compounds spanning six chemical classes: alcohols, ketones, aldehydes, terpenes, alkanes, and aromatic compounds. Permutational multivariate analysis of variance confirmed a significant effect of fungal strain on overall volatile composition, explaining 76.8 percent of the variance, and a supervised partial least squares discriminant analysis achieved 100 percent cross-validated accuracy with a 1,000-iteration permutation test confirming significance at p < 0.001. Twenty compounds with variable importance in projection scores exceeding 1.0 served as principal discriminant biomarkers.

The volatile signatures aligned closely with behavioral outcomes. Metarhizium anisopliae MUCL 58549 emitted a profile dominated by sesquiterpene hydrocarbons, led by putatively identified aristolochene at 20.1 percent and eremophilene at 19.0 percent of total emissions, compounds structurally similar to root-derived terpenes that function as host-location cues for subterranean herbivores. By emitting these compounds, the fungus may mimic an active root system, increasing its attractiveness to foraging wireworms through an active farnesyl pyrophosphate biosynthetic pathway. Trichoderma harzianum MUCL 29707 was uniquely characterized by elevated C8 oxylipins, particularly 1-octen-3-ol at 18.6 percent and 3-octanone at 17.6 percent, compounds widely recognized as biologically active fungal volatiles with dose-dependent effects on wireworms. Ophiocordyceps sp. MUCL 58550, by contrast, was dominated by ubiquitous short-chain ketones and alkanes such as 2-pentanone, heptane, and butanone, widespread products of general microbial metabolism that lack specialized attractant properties, likely explaining the strain’s chemical neutrality.

The authors emphasize that their study demonstrates strong correlations between volatile profiles and larval attraction, but the causal role of individual compounds such as aristolochene or specific blends remains to be validated through dose-response bioassays using synthetic pure compounds. They also note that soil experiments were conducted under sterilized microcosm conditions that do not capture the influence of native microbial communities, fluctuating abiotic conditions, and heterogeneous soil structures characterizing natural agroecosystems. Whether the starch-enriched matrix elicits biting responses of sufficient frequency to contribute to observed mortality remains to be confirmed in vivo, and the effectiveness of the formulation against younger instars, eggs, or adults is an open question. From an agronomic perspective, the volatolomic insights support a stimulate-and-deter push-pull strategy in which high-concentration oxylipin formulations could repel larvae from susceptible crops while attractive starch-enriched beads pull and eliminate the pest in the inter-row, creating a lethal sink in the soil matrix. The dual-infection capability of entomopathogenic fungi, through both oral ingestion and cuticular penetration, could increase the likelihood that even brief encounters with the formulation result in infection. Ongoing studies are evaluating the precise impact of the formulation on soil microbial communities and non-target arthropods, an essential step toward assessing ecological safety and field applicability of this behaviorally targeted wireworm management framework.

Subject of Research: Integrating fungal virulence, formulation, and volatile-mediated attraction for an effective attract-and-kill strategy against Agriotes spp.

Article Title: Integrating fungal virulence, formulation, and volatile-mediated attraction for an effective attract-and-kill strategy against Agriotes spp.

Article References: Lalaymia, I., Ruhland, F., Regnier, N., Delahaut, E., Boullis, A., Martin, C., Verheggen, F. J., & Declerck, S. (2026). Integrating fungal virulence, formulation, and volatile-mediated attraction for an effective attract-and-kill strategy against Agriotes spp.. Frontiers in Agronomy, 8, Article 1941150. https://doi.org/10.3389/fagro.2026.1941150

Image Credits: AI Generated

DOI: 10.3389/fagro.2026.1941150

Keywords: Integrating, fungal, virulence, formulation, volatile-mediated, attraction, effective, attract-and-kill, strategy, against, Agriotes, scientific research

The study, published in the Journal of Agriculture and Food Research, focused on Fusarium oxysporum f. sp. fabae, a soil-borne fungus that causes devastating wilt in faba bean. Farmers in Yunnan Province have long observed that growing wheat alongside faba bean reduces wilt incidence, but the underlying chemistry remained murky. The new work, led by Bijie Hu and colleagues, connects the dots across three levels: the signals released by sick faba bean roots, the defensive compounds those signals induce in wheat, and the lethal effects of those compounds on the pathogen itself.

The team began with hydroponic experiments in which faba bean plants were grown alone or alongside wheat, and then subjected to three kinds of stress: the fungal pathogen itself, cinnamic acid as a stand-in for abiotic stress, and both together. Using untargeted metabolomics of root exudates, they found dramatic shifts in the chemical composition of what stressed faba bean roots released into their surroundings. Under combined stress, more than 770 differential metabolites were detected compared with unstressed monoculture plants, with the vast majority upregulated.

Pathway analysis pointed consistently toward secondary metabolism, including flavonoid biosynthesis and plant hormone signal transduction. When the researchers screened for metabolites that were upregulated across every stress treatment, four candidates stood out: jasmonic acid, salicylic acid, allantoin, and luteolin. Quantitative measurements confirmed that faba bean boosted secretion of these compounds under biotic and abiotic stress alike, and that intercropping pushed the levels even higher. In field plots, intercropping raised jasmonic acid in the root zone by nearly 64 percent, salicylic acid by about 46 percent, and allantoin by roughly 15 percent compared with faba bean grown alone.

These molecules are well-known players in plant stress biology. Jasmonic acid and salicylic acid are classic defense hormones, while allantoin is a nitrogen-rich metabolite linked to oxidative stress responses. The authors interpret their coordinated release as a broadcast distress call, a chemical SOS that neighboring plants can detect and respond to. Analogous systems have been documented elsewhere: chili plants under attack induce benzoxazinoid production in adjacent corn, and barnyard grass releases jasmonic acid that prompts rice to pump out allelopathic compounds.

To test whether wheat actually listens, the team applied jasmonic acid, salicylic acid, and allantoin exogenously to wheat at concentrations matching those found in faba bean exudates, then measured benzoxazinoid levels in wheat leaves and rhizosphere soil. The response was clear. Salicylic acid treatment raised DIMBOA content in wheat leaf tissue by up to 96 percent and MBOA by nearly 60 percent. Jasmonic acid and allantoin produced similarly robust, dose-dependent increases. Crucially, the benzoxazinoids also accumulated in the rhizosphere soil, confirming that wheat not only synthesized the compounds but secreted them into the soil where the pathogen lives.

Benzoxazinoids such as DIMBOA and MBOA are defensive secondary metabolites produced almost exclusively by grasses, the Poaceae family that includes wheat, maize, and their relatives. The next question was what these compounds do to the fungus. In a clever microscopy setup using capillary grooves on glass slides, the researchers observed that FOF spores actively accumulate in the wheat rhizosphere, with numbers rising over 20 minutes of observation. Rather than avoiding wheat, the pathogen is drawn toward it, a positive chemotactic response that previous work suggests is driven in part by the benzoxazinoids themselves.

That attraction sets up what the authors describe as an “attract-and-kill” strategy. In liquid culture, DIMBOA and MBOA reduced FOF mycelial biomass in a concentration-dependent manner, with the strongest suppression at 150 micrograms per milliliter, cutting fungal growth by roughly 38 percent. Fluorescent staining revealed that the compounds also crippled biofilm formation, a key step in fungal establishment, and induced apoptotic vesicles in FOF spores, hallmarks of programmed cell death. The mechanism, the authors suggest, involves reactive oxygen species: benzoxazinoids trigger bursts of intracellular ROS that overwhelm the fungus’s antioxidant defenses, damaging mitochondrial and cytoplasmic function until the cells die.

Perhaps the most surprising finding came from germination tests. Far from being toxic to the crop it protects, the wheat-derived benzoxazinoids actually stimulated faba bean seed germination. DIMBOA treatment increased germination potential, germination index, vigor index, and seedling length, with vigor index gains reaching nearly 900 percent under some conditions, and the effects held even when seeds were simultaneously challenged with the pathogen. The authors speculate that benzoxazinoids may act somewhat like hormones, stimulating cell division and metabolic activity in germinating seeds, though they caution that the precise mechanism remains to be established.

Taken together, the study sketches a complete chemical circuit: a stressed faba bean root releases jasmonic acid, salicylic acid, and allantoin; wheat senses these signals and ramps up benzoxazinoid biosynthesis and secretion; the pathogen, lured by chemotactic cues, gathers at the wheat rhizosphere where the accumulated DIMBOA and MBOA suppress its growth, dismantle its biofilms, and push its cells into programmed death. The authors are careful to note that the links between exudate composition, benzoxazinoid induction, and fungal viability are associations, and that further work is needed to fully establish the causal mechanisms, including quantifying spore apoptosis with flow cytometry. Even so, the findings offer a compelling blueprint for disease-suppressive agriculture, suggesting that the right crop combinations can turn a field into a coordinated chemical defense network, reducing reliance on fungicides while potentially giving seedlings a head start. In an era when crop diversity is increasingly promoted for sustainable intensification, this underground conversation between a legume and a grass shows just how sophisticated plant-plant communication can be, and how much agronomic value may be hiding in the chemistry of the rhizosphere.

Subject of Research: Chemical communication between faba bean and wheat roots that induces benzoxazinoid defenses suppressing Fusarium wilt

Article Title: Root exudate-mediated plant chemical communication induces benzoxazinoid synthesis in wheat and suppresses Fusarium oxysporum f. sp. fabae activity in faba bean

Article References: Hu, B., Li, T., Yang, S., & Dong, Y. (2026). Root exudate-mediated plant chemical communication induces benzoxazinoid synthesis in wheat and suppresses Fusarium oxysporum f. sp. fabae activity in faba bean. Journal of Agriculture and Food Research, 31, Article 103324. https://doi.org/10.1016/j.jafr.2026.103324

Image Credits: AI Generated

DOI: 10.1016/j.jafr.2026.103324

Keywords: intercropping, root exudates, benzoxazinoids, DIMBOA, MBOA, jasmonic acid, salicylic acid, Fusarium oxysporum, faba bean wilt, plant chemical communication, attract-and-kill, rhizosphere