Botrytis cinerea, the gray mold pathogen, is among the most formidable adversaries in agriculture. It infects more than 200 plant species, kills host cells by secreting toxic compounds, and breaches plant defenses using cell wall-degrading enzymes, resulting in substantial yield losses across crops worldwide. For mulberry, the cornerstone of the silk industry and a plant of significant medicinal value due to its rich secondary metabolites, current control strategies rely heavily on chemical fungicides such as thiophanate-methyl and on resistant cultivars, approaches that are costly, environmentally damaging, and increasingly limited in effectiveness. The search for sustainable alternatives has driven attention toward endophytes, the bacteria, fungi, and actinomycetes that colonize internal plant tissues and are sometimes described as a plant’s second genome.

The research team, led by Lixue Wang, Yixuan Zhang, Wanting Li, and colleagues at Zhejiang University and Southwest University, subjected ZJU_268 to a battery of technical characterizations. Electron microscopy revealed rod-shaped cells with wrinkled outer surfaces, a feature associated with surface adhesion and stress tolerance in Bacillus species. Because 16S rRNA gene sequences are too conserved to reliably separate closely related Bacillus species, the team turned to the gyrB gene, which showed 99.7 percent similarity to B. velezensis, and confirmed the identification with average nucleotide identity analysis. Whole-genome sequencing on PacBio and Illumina platforms showed a compact circular chromosome of 3.93 megabases with no plasmids, containing 3,748 protein-coding genes. Functional annotation revealed extensive machinery for carbohydrate utilization, amino acid metabolism, cofactor and vitamin biosynthesis, biofilm formation, and stress response, along with several strain-specific biosynthetic gene clusters predicted to encode secondary metabolites, including antimicrobial lipopeptides.

Perhaps the most striking technical demonstration involved tagging ZJU_268 with a green fluorescent protein and tracking its journey through mulberry seedlings. Confocal laser scanning microscopy showed that the bacterium established stable populations on root surfaces, penetrated internal tissues, spread through the cortex and vascular system, and eventually migrated upward into stems and leaves. Quantitative recovery of GFP-labeled colonies from homogenized tissues over 35 days confirmed that roots served as the primary reservoir while aerial tissues were colonized later and at lower densities, establishing ZJU_268 as a genuine systemic endophyte rather than a transient surface dweller.

In laboratory antagonism assays, ZJU_268 proved devastatingly effective against fungi. Inhibition rates against most Ascomycetous pathogens exceeded 85 percent, and mycelial growth of Botrytis cinerea was nearly eliminated, with an inhibition rate of 98.09 percent. Scanning electron micrographs showed treated hyphae distorted, folded, twisted, and locally swollen, in sharp contrast to the smooth, intact filaments of untreated controls. The antifungal activity emerged about nine hours after incubation, peaked at 24 hours, and remained chemically robust: the cell-free culture supernatant retained its potency after heating at temperatures up to 121°C and across a pH range from 2 to 10, indicating that the active compounds are unusually stable molecules.

The greenhouse results translated this potency into disease control. In sterilized soil, mulberry seedlings treated with ZJU_268 cell suspensions showed a disease incidence of 41.67 percent, and those treated with the cell-free supernatant 66.67 percent, compared with severe disease in untreated pathogen-challenged plants and 25.00 percent in the fungicide-treated positive control. Quantitative PCR confirmed that pathogen abundance in roots was significantly lower in both bacterial treatments. Critically, efficacy persisted in non-sterilized natural soil containing intact indigenous microbial communities, with control efficacies of 47.62 percent for the live bacterium and 28.57 percent for the supernatant. Growth promotion was equally dramatic: at an optimal dose of 10⁶ CFU/mL, treated seedlings showed increases of roughly 74 percent in root length, 81 percent in shoot length, 173 percent in total fresh weight, and 136 percent in total dry weight, while seed germination rates rose by up to 24 percent.

To understand how these effects arose, the researchers sequenced the root microbiome of treated plants using 16S rRNA and ITS amplicon profiling. Bacterial communities clustered distinctly between treated and control plants on Bray-Curtis ordination, whereas fungal communities shifted less in composition but changed markedly in their ecological structure: fungal co-occurrence networks lost connectivity, with fewer nodes linked and fewer positive associations, while bacterial network topology remained largely intact. Taxonomically, the treatments depleted several potentially harmful taxa, including the bacterial genera Pantoea, Afipia, and Rhizorhapis and the fungal genera Verruconis and Exobasidium, while enriching beneficial bacteria such as Streptomyces, Flavobacterium, Rhizobium, Pseudomonas, and Novosphingobium, and fungi including Cladosporium, Arthrobotrys, Meyerozyma, and Xenoacremonium. The team then cultured 223 bacterial and 42 fungal isolates from treated roots and validated their functions directly. Selected Pseudomonadaceae strains inhibited B. cinerea by 85 to 96 percent in dual cultures, reduced lesion sizes on detached mulberry leaves, and boosted seedling fresh weight and shoot height, while fungal isolates from the Cladosporiaceae and Nectriaceae families suppressed the pathogen by roughly 50 percent and promoted plant growth. Compatibility assays showed ZJU_268 could work synergistically with these enriched partners, suggesting a path toward engineered microbial consortia.

Metabolomics added the final layer of the mechanism. Untargeted liquid chromatography–mass spectrometry detected 3,431 metabolites in mulberry roots and identified 625 that changed significantly across treatments. Both the live bacterium and its supernatant elevated a suite of compounds, including the cytokinin trans-zeatin, thioinosine monophosphate, L-galactose, uridine, L-serine, and the fungicide carbendazim. When these purified metabolites were fed to representative enriched isolates in vitro, bacterial growth measured by optical density and fungal colony expansion both increased significantly. Tellingly, none of the same metabolites stimulated Botrytis cinerea; L-serine and uridine actually inhibited it. This selectivity suggests the root itself becomes a chemical gatekeeper, feeding friendly microbes while starving or repelling the pathogen. Quantitative PCR of plant genes supported this interpretation, with upregulation of CYP735A, consistent with zeatin accumulation, and of PSP and PAL, reflecting shifts in amino acid and phenylpropanoid metabolism. Live cells and supernatant also diverged in their metabolic signatures: the supernatant drove a large accumulation of ascorbate, an antioxidant buffer, whereas live colonization promoted dynamic turnover of ascorbate metabolism, hinting at complementary modes of protection, rapid chemical defense from secreted metabolites and sustained physiological reprogramming from an established endophyte.

The authors frame their findings at the level of the holobiont, the plant plus its microbial constellation, arguing that ZJU_268 functions less as a lone assassin of fungi and more as a microbiome architect, reshaping who lives in the root, rewiring interaction networks, and editing the chemical environment so that the resulting community suppresses disease and fuels growth. With its efficacy demonstrated in natural soil, its stability under heat and pH extremes, and its dual biocontrol and growth-promoting repertoire encoded on a single plasmid-free chromosome, ZJU_268 represents a promising candidate for biological products aimed at sustainable sericulture. For an industry seeking to cut pesticide use without sacrificing yield, a bacterium plucked from the roots of a tree that has stood for a thousand years may prove an unexpectedly modern ally.

Botrytis cinerea is a generalist plant pathogen capable of infecting numerous fruits, vegetables and ornamental crops. Its gray mold disease commonly develops when tissues are wounded, aging or exposed to humid conditions, and infections can spread rapidly during transport and storage. The fungus produces abundant spores, allowing it to move between plants and commodities, while its flexible lifestyle helps it colonize living and dead plant material. Repeated use of chemical fungicides has also contributed to resistance in some Botrytis populations, increasing interest in complementary approaches. Essential oils are complex mixtures of volatile compounds produced by plants, often including terpenes and oxygen-containing derivatives. These molecules can affect microbial membranes, disrupt cellular functions or interfere with fungal development, although their activity depends on the particular compounds, their concentrations and how they interact. Because essential oils vary with species, geography, plant tissue and environmental conditions, chemical characterization is essential before biological results can be interpreted or reproduced.

The researchers used gas chromatography–mass spectrometry, commonly abbreviated GC–MS, to examine the oils’ chemical profiles. In this technique, gas chromatography separates volatile molecules according to properties such as volatility and their interactions with the column, while mass spectrometry records characteristic fragmentation patterns that help identify them. The approach allowed the team to compare the dominant constituents of oils obtained from the nine selected native species. The plants produced markedly different quantities of oil. The yield ranged from 0.25 percent for B. genistelloides to 1.28 percent for P. inaequalifolia, indicating that extraction efficiency differed substantially among species. The chemical profiles also fell into distinct broad patterns: some were dominated by monoterpene hydrocarbons, others by oxygenated monoterpenes, and others by sesquiterpene hydrocarbons. Such differences are important because two oils can share the same general botanical origin yet behave very differently against a pathogen when their constituent mixtures differ.

The most striking result came from P. inaequalifolia, whose oil was rich in oxygenated monoterpenes and contained eucalyptol as its principal identified component, at 35.22 percent. In the laboratory, the oil completely inhibited fungal mycelial growth at a concentration of 500 microliters per liter. Its median effective dose, or ED₅₀, was 200.63 microliters per liter. ED₅₀ represents the concentration estimated to reduce the measured biological response by half, so lower values generally indicate greater potency under the tested conditions. The oil also had an ED₉₀/ED₅₀ ratio of 1.31. This relatively small ratio indicates a steep concentration–response relationship: a modest increase above the concentration associated with half-maximal inhibition produced a much stronger effect. The result makes P. inaequalifolia an especially interesting candidate for follow-up studies, while also emphasizing that laboratory potency alone does not establish safety, stability or practical effectiveness on harvested produce.

Baccharis genistelloides produced the lowest ED₅₀ among the oils tested, at 71.50 microliters per liter, making it the strongest performer by that measure. Its chemical profile differed sharply from that of P. inaequalifolia. Rather than being dominated by oxygenated monoterpenes, the B. genistelloides oil was characterized by sesquiterpenes, particularly gamma-muurolene and delta-cadinene. The contrast suggests that strong antifungal activity may arise through more than one chemical route. A single abundant constituent may contribute substantially, but activity can also reflect additive or synergistic effects among several compounds present at lower concentrations. The study’s results do not identify which individual molecule, or combination of molecules, is responsible for inhibiting B. cinerea. Determining that mechanism will require experiments with purified compounds, reconstructed mixtures and tests designed to distinguish effects on fungal membranes, respiration, spore germination and mycelial growth.

Several other oils showed intermediate activity. These came from Gynoxys malcabalensis, Piper acutifolium, Piper lanceifolium and Siparuna muricata. The study provides the first chemical characterization and antifungal evaluation reported for the essential oil of G. malcabalensis, adding a new entry to the phytochemical record of an understudied Andean plant. By contrast, oils from Baccharis latifolia and the Purple and Yellow cultivars of Arracacia xanthorrhiza were comparatively weak in the assay, with ED₅₀ values above 900 microliters per liter. That range of responses is scientifically useful. It shows that “essential oil” is not a single type of treatment and that closely related or locally available plants cannot be assumed to have equivalent antifungal properties. Chemical composition must be measured alongside biological activity, and the performance of each oil must be evaluated under the conditions relevant to its intended use.

The research is relevant to postharvest disease management because essential oils can potentially be applied to crop surfaces, packaging materials or storage environments. Their volatility may allow active compounds to contact fungal growth without requiring the same application strategy as a conventional liquid fungicide. However, translating an in vitro result into a usable treatment involves multiple hurdles. An oil must remain effective on a real commodity, where waxes, moisture, temperature and surface texture can alter its distribution. It must not damage the fruit or vegetable, change its flavor or aroma undesirably, or create unacceptable residues. Formulation is another challenge: volatile compounds can evaporate, oxidize or separate from water-based preparations. Encapsulation and controlled-release systems may improve stability, but these approaches require independent testing. Dose, exposure time and application method must also be optimized, and any treatment would need evaluation for effects on beneficial microorganisms and other organisms in the production system.

The authors describe the oils from P. inaequalifolia and B. genistelloides as promising sources of antifungal compounds for future postharvest applications, but the evidence remains an early-stage screening result. The experiments were conducted against B. cinerea under controlled laboratory conditions rather than on infected plants, commercial fruit or stored produce. The study therefore establishes comparative activity, not a ready-to-deploy biopesticide. Future work will need to confirm the findings across fungal isolates, assess the oils’ toxicity and phytotoxicity, identify active components and examine how chemical profiles change with cultivation site, harvest stage and extraction procedure. Trials on representative crops will be particularly important because an effective concentration in culture medium may not behave similarly on a fruit surface. Even with those limitations, the study highlights a practical research strategy: combine chemical profiling with direct pathogen assays to discover locally available plant resources that could support more diverse and sustainable crop-protection systems.

The study’s comparison between oil yield and antifungal potency raises an important practical distinction. Baccharis genistelloides generated the lowest reported oil yield, yet its oil had the lowest ED₅₀ in the test system. Conversely, a higher extraction yield does not automatically predict stronger biological activity. Production planning would therefore need to consider at least two separate variables: how much oil can be obtained from plant material and how much oil is required to produce a defined inhibitory effect. A species that is highly active but produces little oil could still be valuable if its active constituents can be concentrated, reproduced through cultivation or incorporated into a formulation efficiently.

The chemical groupings reported by the researchers can also help organize subsequent experiments. Oils dominated by monoterpene hydrocarbons, oxygenated monoterpenes or sesquiterpene hydrocarbons represent different starting mixtures, but these broad categories do not by themselves explain antifungal performance. The abundance of a compound is only one consideration; volatility, chemical stability, interactions among constituents and the biological accessibility of the mixture may all influence the measured response. Comparing oils with similar major chemical classes, while also examining their minor constituents, could help determine whether activity tracks a specific molecule, a chemical family or a combination of compounds.

Interpretation of the concentration–response data will benefit from distinguishing the biological endpoint being measured. The reported inhibition concerns fungal mycelial growth in vitro, an important indicator of activity but not a complete description of the pathogen’s life cycle. A candidate oil might affect germination, spore production or establishment on plant tissue differently from established mycelium. Follow-up assays could therefore test several stages of B. cinerea development and use multiple isolates. Such comparisons would indicate whether the observed effects are broadly reproducible or depend on the particular fungal population and laboratory conditions used in the initial screening.

Reproducibility will likewise depend on documenting the plant material and extraction process in detail. Essential-oil composition can vary among botanical populations, and the study’s focus on native plants from a geographically distinctive region makes chemical reference data especially valuable. The newly characterized Gynoxys malcabalensis oil provides a baseline for comparisons with related species and future collections. Access to the underlying data upon request may support those comparisons, including reassessment of constituent identifications and activity estimates. Building such a record is an essential step before promising oils can be evaluated as consistent agricultural inputs rather than as one-time extracts from a particular collection.

Subject of Research: Antifungal activity of essential oils from native northern Peruvian plants against Botrytis cinerea

Article Title: Chemical characterization and in vitro antifungal activity of essential oils from selected native plants of the Andean-Amazonian region of northern Peru against Botrytis cinerea (Sclerotiniaceae)

Article References: Mena-Chacon, L. M., Chávez-Chacón, E., Coronel-Castro, E., Santillan-Huaman, N., Rojas-Vargas, J., Huaman-Pilco, J., Mondragon-Herrera, E., Oliva, M., & Huaman-Pilco, A. F. (2026). Chemical characterization and in vitro antifungal activity of essential oils from selected native plants of the Andean-Amazonian region of northern Peru against Botrytis cinerea (Sclerotiniaceae). Plant Biosystems, 160(5), Article 260. https://doi.org/10.1007/s44473-026-00258-7

Image Credits: AI Generated

DOI: 10.1007/s44473-026-00258-7

Keywords: essential oils, Botrytis cinerea, gray mold, Peru, plant pathology, biopesticides, GC–MS, postharvest disease, natural products, Chemical, characterization, vitro