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Article

Control of Neopestalotiopsis zimbabwana Using Origanum vulgare L. Essential Oil: Combined In Vitro, In Vivo and In Silico Approaches

by
Héctor Gómez-Yáñez
1,
Ramón Marcos Soto-Hernández
1,*,
Lucero del Mar Ruiz-Posadas
1,*,
Guadalupe Valdovinos-Ponce
2,
Irving Israel Ruiz-López
3,
Cecilia Beatriz Peña-Valdivia
1 and
Guadalupe Mora-Báez
4
1
Botánica, Colegio de Postgraduados, Campus Montecillo, Carretera México-Texcoco Km. 36.5, Texcoco 56264, Mexico
2
Fitosanidad–Fitopatología, Colegio de Postgraduados, Campus Montecillo, Carretera México-Texcoco Km. 36.5, Texcoco 56264, Mexico
3
Facultad de Ingeniería Química, Benemérita Universidad Autónoma de Puebla, Av. San Claudio y 18 Sur, Col. Jardines de San Manuel, Puebla 72570, Mexico
4
Instituto Tecnológico Superior de Tlatlauquitepec, Carretera Amozoc-Nautla Km. 122.6, Almoloni, Tlatlauquitepec, Puebla 73907, Mexico
*
Authors to whom correspondence should be addressed.
Horticulturae 2025, 11(10), 1232; https://doi.org/10.3390/horticulturae11101232
Submission received: 11 August 2025 / Revised: 17 September 2025 / Accepted: 19 September 2025 / Published: 13 October 2025
(This article belongs to the Special Issue Fungal Diseases and Pathogens Affecting Horticultural Crops: New Challenges)
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Abstract

Neopestalotiopsis zimbabwana is an emerging phytopathogen with multiple hosts. Considering the environmental, toxicological, and resistance issues linked to synthetic fungicides, Origanum vulgare L. essential oil (OEO) was evaluated through in vitro, in vivo, and in silico approaches. The pathogen, isolated from Watsonia borbonica L., was molecularly identified. Gas chromatography–mass spectrometry (GC–MS) analysis showed hexadecanoic acid (15.98%), dodecanoic acid (15.74%), terpinen-4-ol (11.61%), and thymol (7.65%) as the main components. In vitro assays determined a minimum inhibitory concentration (MIC) of 30% OEO and a minimal fungicidal concentration (MFC) of 60% OEO. Growth chamber trials demonstrated that preventive sprays maintained 0% foliar damage—similar to Captan®—while controls reached ≈98%; suspending applications after week 4 resulted in ≈45% damage by week 8. These results confirm that OEO lacks systemic residual activity, acting only as a protectant within preventive integrated pest management (IPM) schemes. Docking to cytochrome b (protein data bank, PDB: 5TL8) indicated strong binding of α-farnesene (−7.638 kcal·mol−1), isoterpinolene (−6.944), and α-terpineol (−6.918), suggesting disruption of mitochondrial respiration via Complex III. OEO represents a promising eco-friendly alternative for managing N. zimbabwana under controlled conditions and reducing reliance on synthetic fungicides.

1. Introduction

Microbial pathogens continue to harm global agricultural systems, resulting in reduced crop production and threatening global food security. Phytopathogenic fungi are of significant importance because they successfully infect plant tissues, thereby destroying productive agricultural crops [1,2].
Neopestalotiopsis spp. have established themselves as significant phytopathogens affecting high-value crops such as strawberry (Fragaria × ananassa). Among them, Neopestalotiopsis rosae has been primarily associated with this crop, causing foliar lesions and yield reduction [3,4,5]. In contrast, Neopestalotiopsis zimbabwana has gained prominence due to its ability to infect multiple hosts, including avocado (Persea americana L.), guava (Psidium guajava L.), jackfruit (Artocarpus heterophyllus L.), and ornamental species such as Watsonia borbonica L. [4,6,7].
The phytopathological threat posed by Neopestalotiopsis spp.—now compounded by the emergence of the multi-host species Neopestalotiopsis zimbabwana—presents substantial management challenges. Members of the genus are responsible for crown and root rot, fruit necrosis, and leaf-spot diseases that markedly reduce yield and market quality in strawberry and other high-value crops [8]. The strawberry industry in California faces annual losses of $1.99 billion because of the damage caused by reappearing or new fungal diseases [8].
Current control programs emphasize preventive use of Captan® (contact, protectant) and the relatively greater curative action of Prochloraz®, recognizing that curative treatments are typically less reliable and more difficult to manage. These chemical treatments offer short-term efficacy against phytopathogenic fungi [1,9,10,11]. Fungicides harm pollinators and natural enemies [12,13] and select resistance via target site mutations. Agricultural workers face high exposure during application, risking toxicity [14]. These risks require strategies sustaining efficacy while protecting ecosystems, food safety, and public health.
In this context, integrating pre-treatment fungicide applications within a broader integrated pest management (IPM) strategy represents a more sustainable long-term approach. Framing fungicide use as targeted, preventive applications within IPM reduces reliance on curative interventions and aligns with the goal of minimizing residues, non-target effects, and the selection of resistant strains. Within such an IPM program, lower-risk preventive tools (including botanical extracts) are positioned as complementary components, provided that efficacy and phytotoxicity are jointly optimized [15,16,17].
A promising avenue is the use of botanical extracts obtained from medicinal and aromatic plants. These biodegradable plant-derived preparations owe their antifungal activity to a diverse array of secondary metabolites—phenols, fatty acids, flavonoids, alkaloids, glycosides, tannins, and terpenes—and are therefore classified broadly as botanical pesticides, if the use is specific to fungi botanical fungicides (BP) [18,19]. Within this group, essential oils (EOs) stand out for their high terpene and fatty acid content, which confers broad spectrum antimicrobial, insecticidal, and antioxidant properties [20,21,22].
In strawberries, low-dose EO regimens can be both effective and well tolerated. For example, Rasiukevičiūtė et al. [23] applied Thymus vulgaris L. essential oil (TEO) at 0.06% volume/volume (v/v) every 7–10 d (days; four sprays, starting at 10% flowering). This regimen produced healthier plants compared with the untreated control, with no yield penalty; the treatment was aimed at gray mold (Botrytis cinerea), another major strawberry disease.
In particular, Origanum vulgare essential oil (OEO) stands out for its high content of bioactive compounds, especially the phenolic monoterpenes thymol and carvacrol. Studies by Abd et al. [20] and Jan et al. [21] have confirmed the antimicrobial efficacy of OEO against fungal pathogens, supporting its use as a natural preservative in the food and pharmaceutical industries. From an agricultural perspective, OEO has demonstrated the capacity to suppress phytopathogens effectively while posing reduced risks to non-target organisms and showing minimal environmental persistence compared with synthetic chemicals.
However, EOs can injure host foliage, a phenomenon known as phytotoxicity, defined as a negative impact on plant tissues associated with cellular dysfunction [24] when used at high concentrations or excessive frequency: in field trials a 5% v/v TEO spray combined with 2.5% v/v and adjuvant 92.5% v/v water, applied twice weekly, caused severe phytotoxic burns in arugula (Eruca sativa) within one day of the first application [25]. Therefore, phytotoxicity should be evaluated alongside efficacy when optimizing EO-based treatments [24,26].
Beyond EOs, hydrosols are obtained during steam distillation as the aqueous co-product; they contain much lower concentrations of phenolic compounds than the essential oil yet retain trace volatiles (<1 g·L−1), typically enriched in oxygenated terpenes, that confer measurable bioactivity. The HOV exhibits inhibitory activity against pathogenic fungi, as reported for Origanum vulgare hydrosol (HOV) [27].
The evaluation of natural antifungal agents has become more effective through integrated testing methods that combine in vitro, in vivo, and in silico approaches. The basic antifungal activity needs to be determined by in vitro screenings for minimum inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) but in vivo assays test field or greenhouse performance by studying plant physiology and microclimate effects and disease progression in real hosts [28].
In silico methods through molecular docking help enhance empirical data by showing potential binding interactions at a molecular level to understand mechanisms of action and discover essential structural components for compound optimization, i.e., the study of Yao et al. [29] implemented a combination of in vitro and in vivo tests, along with in silico methods, to demonstrate that isolated natural compounds exhibited significant antifungal activity. Their research highlights how the integration of multiple experimental approaches can provide strong laboratory evidence for promising natural antifungal agents.
Building on this perspective, the present study evaluated the hypothesis that OEO contains bioactive components capable of inhibiting N. zimbabwana under in vitro and growth-chamber conditions. We further hypothesized that key OEO constituents would exhibit high-affinity binding to fungal cytochrome b, as assessed by molecular docking analyses.
We pursued five objectives: (i) isolate and identify the causal phytopathogen; (ii) extract OEO and characterize its composition by gas chromatography–mass spectrometry (GC–MS); (iii) evaluate the in vitro antifungal activity against the causal agent (subsequently identified as N. zimbabwana); (iv) assess a preventive OEO-based formulation on strawberry plants under growth chamber conditions; and (v) perform molecular docking to identify OEO constituents with high binding affinity to cytochrome b.

2. Materials and Methods

2.1. Morphological and Molecular Identification

Symptomatic leaves of W. borbonica L. showing necrotic lesions with dark acervuli were collected in Tehuitzinapa, Mixteca region, Puebla, México (18°19′55″ N, 98°16′32″ W). Samples were placed in paper bags and transported to the Bioassay Laboratory for Medicinal Plants, Colegio de Postgraduados, Texcoco, State of México. In the laboratory, leaf pieces (5 × 5 mm) were surface-sterilized with 1% NaOCl for 3 min [8,30], rinsed with sterile distilled water, and plated on potato dextrose agar (PDA; BD BIOXON®, Becton Dickinson, Mexico City, México) for fungal isolation [31]. At the time of sampling, the causal agent was unknown.
All isolation procedures were conducted inside a Labconco® Purifier Cell Logic+ Class II biological safety cabinet equipped with a high-efficiency particulate air filter (HEPA; Labconco, Kansas City, MO, USA) to minimize cross-contamination. Plates were incubated in a Thermo Fisher Scientific® incubator (Waltham, MA, USA) at 25 °C for 7 d under a 12 h light/12 h dark photoperiod [32], illuminated at ≈1500 lx (lux; measured at plate level with a Steren LUX-900 digital lux meter); light emitting diode (LED) strips were controlled by a timer.
Causal pathogen identification was initially performed via visual observation of conidia and acervuli under a stereomicroscope (Carl Zeiss Microscopy GmbH, Jena, Germany) at 40× magnification. Colonies exhibiting morphological characteristics consistent with Neopestalotiopsis spp. were selected, performing multiple subcultures until purity was confirmed through uniform colony morphology and absence of visible contaminants [32]. Once purity was established, fungal cultures were maintained for subsequent experimental analyses, including genetic characterization. Fresh PDA plates were inoculated with the purified isolates under identical incubation conditions to ensure consistent fungal development throughout the study.
Genomic DNA was extracted from 80 mg of mycelium from 7–10-day-old cultures using the DNeasy Plant Mini Kit (QIAGEN, Hilden, Germany; cat. no. 69104) and eluted in 50 µL AE buffer. DNA purity (absorbance ratio A260/A280 ≈ 1.8–2.0) was verified spectrophotometrically. Polymerase chain reaction (PCR) was conducted in 25 µL reactions containing 1× PCR buffer (1.5–2.0 mM MgCl2), 200 µM deoxynucleoside triphosphates (dNTPs), 0.4 µM each primer, 1 U Taq polymerase and 10–50 ng template DNA. Thermocycling: 95 °C for 3 min; 35 cycles of 95 °C for 30 s, annealing [33,34].
PCR amplicons were purified with the QIAquick® PCR Purification Kit (QIAGEN, cat. nos. 28104/28106) and eluted in 30 µL nuclease-free water prior to Sanger sequencing (QIAGEN; cat. nos. 28104/28106) [35]. Chromatograms were quality-checked, trimmed, and assembled. Sequences were aligned in MEGA 11 [36] and queried against GenBank using Basic Local Alignment Search Tool—nucleotide (BLASTn) to confirm species identity [37].

2.2. Plant Material and Hydrodistillation: OEO/HOV Co-Collection, Separation

Origanum vulgare was cultivated at the Experimental Field of the Colegio de Postgraduados (Montecillos campus). Taxonomic identity was confirmed at the CHAPA Herbarium, via comparison with certified records and standard taxonomic keys.
The plant was harvested in the early morning [38], a time chosen to ensure maximum preservation of volatile compounds, which tend to degrade under strong sunlight and high temperatures [39,40]. Immediately after cutting, the stems were manually separated from the leaves to focus the extraction on the leaf tissue, known to contain the highest concentration of EO.
The leaves were then allowed to air-dry under shaded conditions for approximately 24 h, a process that gently reduced moisture content while minimizing the loss of thermolabile aromatic compounds [41,42]. Leaves of Origanum vulgare were hydrodistilled in water (≈1:12 w/v; 500 g in 6 L). The first 5 min of distillate was discarded. The OEO was separated by decantation, collected with a separatory funnel, and—together with the HOV—stored in amber glass vials at 4 °C [43].

2.3. GC–MS Analysis of OEO

GC–MS analysis was performed exclusively on the OEO to characterize its volatile composition by GC–MS system (HP 6890 GC coupled to an HP 5973 mass selective detector, MSD; Agilent) [44]. Separation used an HP-5MS fused-silica capillary column, 30 m × 0.25 mm i.d., 0.25 µm film [45]. The OEO sample (1 µL) was injected in split mode (10:1) with the inlet at 60 °C. Oven: 60 °C; ramp 5 °C · min−1 to 200 °C; hold 1 min. Helium (99.999%) served as the carrier at 1.0 mL · min−1 (constant flow). The mass spectrometer (MS) operated in electron ionization (EI) mode at 70 eV, with a scan range from mass-to-charge ratio (m/z) 50–550. Compounds were identified by comparing their mass spectra with the National Institute of Standards and Technology (NIST) using the 2011 library, as commonly applied in EO studies [22].
The HOV was not subjected to GC–MS or other chromatographic analyses. Instead, it was characterized by pH, electrical conductivity (EC), and total dissolved solids (TDS). pH was measured with a Hanna Instruments HI98129.

2.4. In Vitro Assay (PDA), MIC and MFC Definitions

A conidial dilution was prepared from pure N. zimbabwana cultures by washing the surface of a PDA plate with sterile distilled water and scraping off with a sterilized glass rod with rounded ends. The dilution was filtered through two layers of sterile cheesecloth to remove hyphal fragments [46]. Conidial concentration was quantified using a Neubauer chamber and adjusted to 1 × 105 conidia · mL−1 [46].
For inoculation, 10 µL of the adjusted suspension was applied in duplicate onto PDA plates, with two equidistant droplets placed at opposite ends of each dish to allow independent colony growth and prevent overlap. OEO was dissolved in dichloromethane (DCM, Baker®, ≥99.9% purity). Solutions were prepared with increasing concentrations of OEO in DCM, corresponding to the following proportions: 0:100 (0.0 µL OEO); 10:90 (1.0 µL OEO); 20:80 (2.0 µL OEO); 30:70 (3.0 µL OEO), up to 100:0 (10 µL OEO).
For uniform application, 4 mm diameter Whatman® No. 3, sterile filter paper discs (Cytiva, Little Chalfont, UK) were impregnated with 10 µL of each solution and placed at the center of PDA plates previously inoculated with N. zimbabwana [47].
Appropriate controls were included to validate the assay. Negative control treatments consisted of (a) filter paper discs moistened with sterile distilled water and (b) discs impregnated only with pure DCM, corresponding to the previously specified 0:100 EO to DCM ratio (modified from Macías et al. [32]. For the positive control, Captan (Captan 50 WDG, ADAMA) was prepared as a 2500 mg·L−1 stock suspension (equivalent to 2500 µg·mL−1) and pipetted onto sterile filter-paper discs using the same disc-diffusion protocol as the OEO dichloromethane (DCM) treatments; this concentration falls within the in vitro efficacy range reported for Captan against Neopestalotiopsis spp. [48,49]. Both controls and the Captan positive control were applied at 10 µL per disc using the same disc diffusion protocol as the OEO–DCM treatments, adapted from disc-diffusion EO assays in filamentous fungi [47] with a center–edges arrangement similar to Beasley et al. [50].
After application of the respective treatments, the Petri dishes were incubated under the same conditions used during phytopathogen purification for a 14-day period. Each dish was sealed with Parafilm® (Bemis Company, Inc., Neenah, WI, USA). Fungal mycelium growth was monitored over 14 d, documenting growth patterns via digital imaging. High-resolution scans of Petri dishes were obtained every 3 d using an HP® 2025 scanner, and inhibition zones were quantified using ImageJ® software (version 1.54g) [51]. After the initial incubation period, mycelial plugs from treatments showing inhibitory effects were subcultured onto fresh PDA plates and incubated for an additional 14 d (totaling 28 d), and during this second phase, growth recovery was assessed visually without digital imaging.
The experiment employed a completely randomized design (CRD) with 17 treatments replicated five times (85 total experimental units). Each unit consisted of a PDA plate inoculated with N. zimbabwana, a filter paper disc, and the assigned treatment. Data were analyzed using Analysis of Variance (ANOVA), and treatment means were compared by Tukey’s honest significant difference (HSD) test (p < 0.05) in R (v.4.1). Plates were randomly distributed in the incubator to minimize positional bias. The MIC was defined as the lowest OEO concentration showing statistically significant inhibition after 14 d; the MFC corresponding to the lowest OEO concentration preventing mycelial regrowth after 28 d.

2.5. Leaf Phytotoxicity Screen and Formulations

For the in vivo assays in strawberry cv. Festival host plants were acquired as plug plants from a certified nursery in Ciudad Guzmán, Jalisco, México [52] and acclimatized in a greenhouse at Colegio de Postgraduados. Plants were maintained in 1 L pots filled with substrate and irrigated with Steiner’s hydroponic nutrient solution (prepared in the laboratory) until root systems matured [53,54].
After acclimation, plants were transferred to a growth chamber at 25 ± 3 °C, 70% Relative Humidity (RH), under a 12 h photoperiod.
Following confirmation of the antifungal activity of OEO in vitro, a leaf phytotoxicity (plant safety) assay was performed to determine the highest concentration that produced no visible injury, thereby establishing the no-observed-adverse-effect level (NOAEL) and the lowest-observed-adverse-effect level (LOAEL). Candidate formulations were prepared by dissolving OEO in a fixed vehicle composed of 67.7% v/v HOV, 0.6% v/v Tween 20 (stabilizer/adjuvant), 21.1% v/v ethanol (96% purity; solubilizing agent), and 10% v/v distilled water; OEO concentrations were adjusted so that the total volume equaled 100% v/v.
A 10 µL droplet of each formulation was applied to the adaxial (upper) surface of a single leaflet, with one leaflet treated per strawberry leaf. Each concentration, together with its respective vehicle, was applied to a separate strawberry plant to ensure independent assessment of phytotoxic effects. Each treatment was evaluated across four replicates per concentration over a 7-day observation period. The inclusion of HOV was justified by previous findings showing antifungal activity only at concentrations ≥ 60% v/v [43]. These thresholds guided the selection of the working dilution for subsequent in planta assays.

2.6. In Vivo Assay in Strawberry Plants, Treatments, Inoculation and Disease Assessment

Once the botanical fungicide formulation was established based on the leaf phytotoxicity evaluation, the in vivo preventive assays were conducted on strawberry plants. The inoculum was prepared as in the in vitro assays (Section 2.4), maintaining the same concentration (1 × 105 conidia · mL−1). Two leaves per plant were selected for inoculation, and both adaxial and abaxial surfaces of each leaflet were evenly sprayed with 1 mL of the conidial suspension.
Prior to inoculation, four foliar treatments were applied preventively (Figure 1), and treatments were distributed across three experimental groups. Group 1 received weekly applications of the Botanical Fungicide (BF, 1A) or Captan (1B) for eight consecutive weeks. Group 2 received the same treatments during the first four weeks only (2A and 2B), after which applications were discontinued to assess residual protection.
Group 3 was inoculated at the start of the experiment but received neither the botanical formulation nor Captan, serving as the positive control. This group also included non-inoculated plants that received no treatment or application, serving as untreated controls without intervention.
The BF (OEO-based) was applied as a preventive foliar spray prepared according to the leaflet methodology. The commercial fungicide Captan was diluted in distilled water to match the concentration used in the in vitro assays. Both treatments were evenly sprayed onto the adaxial and abaxial surfaces of each leaflet to ensure uniform coverage.
The study followed a CRD. For each condition, five plants were randomly assigned, and pots were randomly arranged within the growth area. On each plant, two trifoliate leaves were selected and all three leaflets per leaf were inoculated (6 leaflets/plant), yielding 30 leaflets per condition (5 plants × 2 leaves × 3 leaflets). Across the four treatments (1A, 1B, 2A, 2B), this accounted for 120 inoculated leaflets; the positive control (inoculated, untreated) contributed 30 leaflets. The negative control (non-inoculated) comprised 30 leaflets. In total, 180 leaflets were assessed (60 leaves from 30 plants).
To quantify strawberry leaf damage, a virtual grid method adapted from Bhattarai et al. [46] was employed to calculate the proportion of necrotic area relative to total leaflets area. First, photographic evidence of each infected leaf was obtained. A calibrated 5 mm diameter white circle was overlaid on each image to standardize scale, followed by superimposition of a virtual grid. This grid enabled the identification of necrotic tissue, spots, or lesions caused by N. zimbabwana. Equation (1) shows the damage percentage calculation:
Damage = (Number of damaged cells/Total number of cells) × 100
Preventive sprays were applied weekly under two schedules: weeks 1–8 (continuous) or weeks 1–4 only (then discontinued). Disease severity was assessed weekly for up to 8 weeks post inoculation and expressed as the percentage of leaf area damaged.

2.7. In Silico Docking Against Cytochrome b (PDB: 5TL8)

To assess the hypothesis that OEO constituents target mitochondrial respiration, we performed docking against cytochrome b (protein data bank, PDB 5TL8) [55]—the catalytic subunit of Complex III in the electron-transport chain—as the receptor. Protein sequences and structural data were retrieved from the PDB; the 5TL8 structure was curated (chain selection, removal of crystallographic waters and non-essential heteroatoms, rebuilding of missing side chains, addition of hydrogens and Gasteiger charges) and converted to AutoDock PDB format with partial charges and atom types (PDBQT). All GC–MS-identified OEO compounds were docked as flexible ligands to a rigid receptor with AutoDock Vina (exhaustiveness = 64; four replicates per ligand). Interactions were characterized with Protein–Ligand Interaction Profiler (PLIP) and visualized in PyMOL. Simulations were interpreted explicitly within a respiration-centered framework; no enzymes of the ergosterol biosynthesis pathway were modeled, and therefore no direct effects on that pathway are inferred.
Molecular docking—using a rigid receptor and flexible ligands—was performed with AutoDock Vina v1.2.7. Binding affinities are reported as Vina scores (binding free energy, ΔG, kcal·mol−1); lower values indicate stronger predicted binding. The workflow followed established Vina/PLIP pipelines for essential-oil constituents [56].
For receptor preparation, the crystallographic structure (PDB ID 5TL8) was curated as follows: (i) retained the biologically relevant chain(s); (ii) removed crystallographic waters, buffers/cryoprotectants, and other non-essential heteroatoms, except structural ions or cofactors when present; (iii) built missing side chains and resolved alternate locations; (iv) added all hydrogens and Gasteiger charges; and (v) converted to PDBQT using AutoDockTools 1.5.1. The receptor was held rigid during docking.
Once the compounds were identified by GC–MS, the corresponding ligands were retrieved from PubChem [57] in 3D Structure-Data File (SDF) format; geometries were minimized and exported to PDBQT using Open Babel. Blind docking was performed for each ligand we report the best-scoring mode (lowest ΔG). Protein–ligand contacts for the three highest-affinity ligands were characterized with PLIP v2.4.0 (hydrogen bonds and hydrophobic contacts), and PyMOL was used to generate 3D pocket views with residue labels and a distinct ligand coloration.

3. Results

3.1. Morphological and Molecular Identification

On PDA, colonies from our samples were white and cottony with regular margins; over time, numerous black conidiomata developed across the surface. Conidia were ellipsoid, 4 septate (five-celled), with hyaline terminal cells bearing 2–3 apical appendages. Mean conidial size was 28 × 7.1 µm.
The TEF1-α consensus from the isolate (≈293 bp) matched N. zimbabwana strain CBS 111495 [58] with 100.0% identity across 275 no mismatches (nt). Alignment to N. rosae strain CBS 101057 [59] showed 96.67% identity across 270 nt.

3.2. Chemical Composition of O. vulgare Essential Oil (GC–MS)

The GC–MS analysis of the OEO revealed a total of 18 constituents (Table 1). The most abundant components were hexadecanoic acid (#18, 15.98%), dodecanoic acid (#16, 15.74%), and (−)-terpinen-4-ol (#7, 11.61%), followed by nonanoic acid (#11, 9.36%), (Z)-sabinene hydrate (#3, 8.17%), tetradecanoic acid (#17, 7.87%), and thymol (#12, 7.65%). Minor constituents such as p-cymene (#1, 4.20%), isoterpinolene (#4, 2.56%), and α-farnesene (#14, 2.11%) were also present in lower proportions.

3.3. In Vitro Antifungal Activity of Origanum vulgare Essential Oil

In the in vitro assay corresponding to MIC (Figure 2), water, ethanol, Tween 20, and DCM (0% OEO) produced small inhibition. Captan showed similarly low activity, whereas the O. vulgare hydrosol exhibited a moderate effect. The BF reached ≈50% inhibition. Pure OEO treatments displayed a dose-dependent inhibition. At day 14, the MIC 30% v/v OEO (3 µL OEO + 7 µL DCM per 10 µL disc).
After the initial 14-day incubation, mycelial plugs from inhibitory treatments were subcultured onto fresh PDA and incubated for an additional 14 d (total = 28 d) to assess regrowth. The MFC was 60% v/v OEO (6 µL OEO + 4 µL DCM per 10 µL disc); at this concentration and at all higher concentrations, no regrowth of Neopestalotiopsis zimbabwana was observed through day 28. At concentrations < 60% v/v, colonies developed on PDA at growth rates comparable to the control.

3.4. Leaf Phytotoxicity Screen and Formulations

Four OEO concentrations (0.3, 0.6, 1.2, and 2.4% v/v), together with a vehicle control (0% OEO), were evaluated. After 7 d, no foliar injury was observed with vehicle, 0.3%, or 0.6%, whereas 1.2% and 2.4% produced visible discoloration. The NOAEL was 0.6% v/v OEO, and the LOAEL was 1.2% v/v OEO.

3.5. In Vivo Efficacy of the OEO-Based Formulation in Strawberry Plants

Foliar damage was assessed at week 4 and at the endpoint (week 8) in the strawberry preventive assay (Figure 3). At week 4, the non-inoculated plant control and all sprayed treatments (1A, 1B, 2A and 2B) showed 0% foliar damage, whereas the inoculated, untreated plant control had already reached ≈50% foliar damage. By week 8, continuous weekly applications (1A and 1B) maintained 0% damage, indistinguishable from the non-inoculated plant control, while discontinuation after week 4 (2A and 2B) allowed damage to progress to ≈45%; the inoculated plant, untreated control (Group 3) reached ≈98%.

3.6. In Silico Docking Analysis Against Cytochrome b

Docking analysis against cytochrome b (PDB: 5TL8) revealed marked differences in binding affinities across the evaluated compounds (Table 2). Among the twenty ligands tested, α-farnesene exhibited the most favorable binding free energy (ΔG = −7.638 kcal·mol−1), outperforming the synthetic reference fungicide Captan (#19, −7.254 kcal·mol−1), isoterpinolene (#18, −6.944 kcal·mol−1) and α-terpineol (#17, −6.918 kcal·mol−1).
For the representative target, Protein–Ligand Interaction Profiler (PLIP v2.4.0) indicated that the three top-scoring terpenes are stabilized predominantly by hydrophobic contacts, with no hydrogen bonds detected under default cutoffs. In Figure 4, the highest-affinity OEO compound, α-farnesene (orange), occupies the binding pocket and engages hydrophobic contacts with residues phenylalanine (PHE50, PHE53, PHE109, PHE217), proline (PRO213), isoleucine (ILE359), methionine (MET360), and leucine (LEU467) at distances between hydrophobic ligand and residue atoms ranging from 3.64 to 3.92 Ångström (Å).

4. Discussion

GC–MS of our OEO revealed an atypical profile dominated by saturated fatty acids (57.3% of the total), hexadecanoic (15.98%) and dodecanoic (15.74%) acids. Among oxygenated monoterpenes, terpinen-4-ol (11.61%) and thymol (7.65%) were the most abundant constituents, whereas carvacrol was not detected in our chromatograms. Monoterpene hydrocarbons (e.g., p-cymene 4.2%, isoterpinolene 2.56%, (E)-β-ocimene 0.4%) and sesquiterpenes (e.g., α-farnesene 2.11%, cis-muurola-4(14), 5-diene 0.84%) comprised a minor fraction.
This composition contrasts sharply with the carvacrol chemotype typically reported for Origanum vulgare subsp. hirtum, where carvacrol consistently dominates the oil, across growth phases, as documented by Baranauskienė et al. [39]. It also differs from the recent GC–MS analysis of O. vulgare essential oil by Hou et al. [60], who found carvacrol 89.98%, with β-caryophyllene 3.34% and thymol 2.39%. Several factors could explain this divergence: (i) botanical/chemotype differences (our material may not be subsp. hirtum); (ii) phenological stage (pre-flowering sampling can shift the monoterpenoid profile); (iii) analytical conditions, as our GC oven maximum of 200 °C is unusually low for EO analysis relative to the 50 → 280 °C (5 °C · min−1) programs commonly used in comparative work, which can under-elute higher-boiling phenolic monoterpenes and sesquiterpenes.
In the case of HOV, our assays with a surface application of 10 µL/plate yielded ≈ 20% inhibition. By contrast, Darapanit et al. [61] reported that hydrosol using the agar-dilution method (the compound dispersed throughout PDA) achieved 100% inhibition at 10,000 mg·L−1 (1% w/v) against Neopestalotiopsis/Pseudopestalotiopsis. Although the materials and experimental conditions differ, both studies target the same phytopathogen, supporting the consideration of plant-derived products as potential alternatives for its control.
Our in vitro data showed a comparably low efficacy of the hydrosol against N. zimbabwana (slightly below 20%). Consistent with this trend, the hydrosol from Mexican oregano (Poliomintha longiflora) showed only mild antimicrobial activity against bacteria (MIC ≈ 3000 mg·L−1) in Cid-Pérez et al. [62]. Given species/chemotype differences, we cite this as a cross-species indicator rather than a direct equivalence to O. vulgare. Still, it supports the working hypothesis that, when combined with OEO, the hydrosol may act additively or even synergistically to provide preventive protection.
In our in vitro assays, we determined that MIC was 30% v/v (equivalent to 300 µL·mL−1). In a related study, Ye et al. [63] evaluated TEO against Neopestalotiopsis spp. and reported a MIC of 100 mg·L−1. Expressed as % v/v (assuming a density of TEO, ρ = 0.80 g·mL−1), this corresponds to 125 µL·L−1 (0.0125% v/v) meaning that the effective concentration was ≈24-fold lower than ours. Their assay employed the way agar-dilution method, with the mycelial plug placed at the center of the plate. Under these conditions, the pathogen is exposed to a low but uniform concentration throughout the medium, which explains why the treatment was more effective than a punctual application on the Petri dish surface; under such conditions, the pathogen is continuously exposed to a low but uniform concentration, thereby slowing its growth.
In the scenario of in planta pre-treatment assay, we observed that, when strawberry leaves were pretreated before inoculation, N. zimbabwana did not develop, revealing a preventive and growth-delaying effect. However, when weekly applications were suspended (2A and 2B), the plants lost protection; since oregano oils at the applied concentration did not display fungicidal activity, rapid recurrence occurred, with the pathogen resuming growth and spreading over the injured tissue. For comparison, Captan showed the same trend, consistent with the findings of Mackenzie et al. [9], where preventive pre-treatment in vivo suppressed disease development, but interruption of the schedule led to re-emergence, with foliar damage reaching ≈24%.
This rapid recurrence confirms that OEO functions solely as a preventive protectant, which agrees with our in vitro observation of BP, and underscores the need for continuous applications, aligning with the preventive and monitoring-based framework of IPM. Importantly, below the MFC, OEO acts only in a fungistatic manner: it suppresses pathogen growth under continuous exposure but does not eradicate it, which explains the rapid recrudescence once treatments are suspended. This interpretation is consistent with Remolif et al. [64], who also reported that EO vapors control postharvest rots in nectarines only during continuous exposure, confirming their fungistatic rather than fungicidal mode of action.
Docking indicated that the top-ranked terpenes (α-farnesene, isoterpinolene, α-terpineol) preferentially occupy a hydrophobic pocket on a membrane-embedded target. For thymol, predicted affinities were comparable—ΔG ≈ −6.8 kcal·mol−1 as reported by Daniel et al. [65] versus −6.509 kcal·mol−1 in our model—with small shifts attributable to routine differences in grid definition (blind versus directed docking) and Vina search settings. Such a hydrophobic binding mode is mechanistically compatible with a mitochondria-centered effect: inhibition within the respiratory chain would be expected to decrease ATP and elevate ROS. Independent phenotypic data in Saccharomyces cerevisiae show that terpenoids can trigger mitochondrial dysfunction, ROS accumulation, and—depending on the compound—ATP depletion [66]. While cytochrome b (Complex III) remains a plausible site of action, we regard this as a working hypothesis consistent with the mitochondrial readouts rather than a proven molecular target.

5. Conclusions

Future research will be directed toward validating the efficacy of the proposed botanical formulation under greenhouse and field conditions, specifically to evaluate its persistence and residual activity under production-relevant scenarios. Additionally, efforts will focus on exploring the potential synergy between OEO and its HOV, applying combined in vitro and in silico approaches—including molecular docking and, potentially, molecular dynamics—to predict and assess interactive effects between their respective bioactive components.
The mechanism of action suggested by docking simulations—namely, interference with mitochondrial respiration via cytochrome b inhibition—remains hypothetical and demands biochemical verification through specific assays, such as cytochrome b enzyme inhibition and mitochondrial respiration measurements.
We acknowledge that the absence of compositional data for the HOV constitutes a current limitation. Future studies should conduct targeted chemical analyses of the HOV to confirm its phytochemical profile and clarify its role within the formulation. All these investigations will strengthen the positioning of OEO as a promising candidate for integrated disease management programs, offering a sustainable alternative to single-site synthetic fungicides.

Author Contributions

Conceptualization, L.d.M.R.-P. and I.I.R.-L.; Methodology, H.G.-Y., R.M.S.-H., L.d.M.R.-P., G.V.-P., I.I.R.-L. and C.B.P.-V.; Software, H.G.-Y.; Validation, H.G.-Y.; Formal analysis, H.G.-Y.; Investigation, H.G.-Y.; Resources, R.M.S.-H., L.d.M.R.-P. and G.M.-B.; Data curation, H.G.-Y.; Writing—original draft, H.G.-Y.; Writing—review and editing, L.d.M.R.-P.; Supervision, R.M.S.-H., L.d.M.R.-P., G.V.-P., I.I.R.-L. and C.B.P.-V. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by a doctoral scholarship awarded to the first author by the Secretaría de Ciencia, Humanidades, Tecnología e Innovation (SECIHTI) of México, application number 2022-000002-01NACF-05976.

Data Availability Statement

Dataset available on request from the authors. The raw data supporting the conclusions of this article will be made available by the authors on request.

Conflicts of Interest

The first author declares no conflict of interest. The funder had no role in the design of the study; in the collection, analysis, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Horticulturae 11 01232 g001
Figure 1. Treatment allocation. A = botanical pesticide; B = Captan. Group 1 (1A, 1B): treatment applied weekly and continuously for the entire 8-week experimental period (solid border). Group 2 (2A, 2B): treatment applied during weeks 1–4 and then discontinued (dashed border). Positive control: inoculated plants with no treatment. Negative control: non-inoculated plants with no treatment (no border coding).
Figure 1. Treatment allocation. A = botanical pesticide; B = Captan. Group 1 (1A, 1B): treatment applied weekly and continuously for the entire 8-week experimental period (solid border). Group 2 (2A, 2B): treatment applied during weeks 1–4 and then discontinued (dashed border). Positive control: inoculated plants with no treatment. Negative control: non-inoculated plants with no treatment (no border coding).
Horticulturae 11 01232 g001
Horticulturae 11 01232 g002
Figure 2. Inhibition percentages of Neopestalotiopsis zimbabwana in vitro under Origanum vulgare essential oil (OEO) treatments after 14 d. Abbreviations (with bar colors, in the order they appear): distilled water (H2O, very light blue), ethanol (EtOH, light gray), Tween 20 (Tw 20, pale lavender), Captan (bright yellow), HOV (Origanum vulgare hydrosol, dark olive green), botanical fungicide (BP, medium olive green). 10–100% OEO—shown as a green gradient: very pale green (10%), light green (20–40%), medium green (50–60%), vivid green (70–80%), very dark/forest green (90–100%)—correspond to the v/v proportion of OEO in dichloromethane. Bars show mean ± SE, Standard Error. Letters above the bars indicate significant differences among treatments according to Tukey’s HSD, Honestly Significant Difference (p < 0.05, n = 5).
Figure 2. Inhibition percentages of Neopestalotiopsis zimbabwana in vitro under Origanum vulgare essential oil (OEO) treatments after 14 d. Abbreviations (with bar colors, in the order they appear): distilled water (H2O, very light blue), ethanol (EtOH, light gray), Tween 20 (Tw 20, pale lavender), Captan (bright yellow), HOV (Origanum vulgare hydrosol, dark olive green), botanical fungicide (BP, medium olive green). 10–100% OEO—shown as a green gradient: very pale green (10%), light green (20–40%), medium green (50–60%), vivid green (70–80%), very dark/forest green (90–100%)—correspond to the v/v proportion of OEO in dichloromethane. Bars show mean ± SE, Standard Error. Letters above the bars indicate significant differences among treatments according to Tukey’s HSD, Honestly Significant Difference (p < 0.05, n = 5).
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Figure 3. In vivo disease severity on strawberry leaves. Percent foliar damage caused by Neopestalotiopsis zimbabwana at W-4 (week 4 evaluation) and W-8 (week 8, endpoint evaluation). A = Botanical fungicide, OEO-based, 0.6% v/v; B = Captan. 1A/1B: weekly applications during weeks 1–8 (continuous). 2A and 2B: weekly applications during weeks 1–4 only (discontinued thereafter). Color coding: A = Olive Green; B = Bright Yellow; inoculated, untreated (positive control) = Rust Red; non-inoculated (negative control) = Kelly Green. Bars show mean ± SE. Different letters within a given time point indicate significant differences among treatments by Tukey’s HSD (p < 0.05; n = 5). Lowercase letters (a, b and c) correspond to the W-4 analysis; uppercase letters (A, B and C) correspond to the W-8 analysis. Tukey’s tests at W-4 and W-8 were run independently, so letters are not compared across time points.
Figure 3. In vivo disease severity on strawberry leaves. Percent foliar damage caused by Neopestalotiopsis zimbabwana at W-4 (week 4 evaluation) and W-8 (week 8, endpoint evaluation). A = Botanical fungicide, OEO-based, 0.6% v/v; B = Captan. 1A/1B: weekly applications during weeks 1–8 (continuous). 2A and 2B: weekly applications during weeks 1–4 only (discontinued thereafter). Color coding: A = Olive Green; B = Bright Yellow; inoculated, untreated (positive control) = Rust Red; non-inoculated (negative control) = Kelly Green. Bars show mean ± SE. Different letters within a given time point indicate significant differences among treatments by Tukey’s HSD (p < 0.05; n = 5). Lowercase letters (a, b and c) correspond to the W-4 analysis; uppercase letters (A, B and C) correspond to the W-8 analysis. Tukey’s tests at W-4 and W-8 were run independently, so letters are not compared across time points.
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Figure 4. Protein Data Bank (PDB) entry 5TL8 binding pocket with α-farnesene (orange). Protein backbone, grey cartoon. Hydrophobic contacts are shown as dashed lines toward the ligand centroid. Visualization generated in PyMOL.
Figure 4. Protein Data Bank (PDB) entry 5TL8 binding pocket with α-farnesene (orange). Protein backbone, grey cartoon. Hydrophobic contacts are shown as dashed lines toward the ligand centroid. Visualization generated in PyMOL.
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Table 1. Compounds and their percentages detected in Origanum vulgare essential oil by GC–MS analysis.
Table 1. Compounds and their percentages detected in Origanum vulgare essential oil by GC–MS analysis.
#CompoundFormulaRetention Time
(min)		Score Match (%)Percentages (%)
1p-cymeneC10H146.35874.2
2trans-β-ocimeneC10H166.62770.4
3(Z)-sabinene hydrateC10H18O7.35868.17
4isoterpinoleneC10H167.87862.56
5(E)-trans-2-menthenolC10H18O8.71750.46
6octanoic acidC8H16O29.66803.16
7(−)-terpinen-4-olC10H18O10.188411.61
8α-terpineol C10H18O10.5831.05
9thymol methyl etherC11H16O11.89863.14
10linalyl propionateC13H22O212.18750.5
11nonanoic acidC9H18O212.79839.36
12thymolC10H14O13.39827.65
13decanoic acidC10H20O215.21805.21
14α-farneseneC15H2416.47802.11
15cis-muurola-4(14),5-dieneC15H2417.99780.84
16dodecanoic acidC12H24O220.138315.74
17tetradecanoic acidC14H28O224.4797.87
18hexadecanoic acidC16H32O228.478215.98
Table 2. Docking binding energies (ΔG, kcal·mol−1) against cytochrome b (PDB: 5TL8).
Table 2. Docking binding energies (ΔG, kcal·mol−1) against cytochrome b (PDB: 5TL8).
#CompoundAffinity
(kcal·mol−1)		Docking Energy Comments
1dichloromethane−2.723Moderate-low potential
2octanoic acid−4.946Moderate-low potential
3decanoic acid−5.267Moderate-low potential
4nonanoic acid−5.277Moderate-low potential
5thymol methyl ether−5.500Medium-low potential
6tetradecanoic acid−5.767Medium-low potential
7dodecanoic acid−5.789Modest binding affinity, medium-low potential
8linalyl propionate−5.834Medium-low potential
9trans-β-ocimene−5.958Medium-low potential
10(−)-terpinen-4-ol−6.053Moderate potential
11hexadecanoic acid−6.082Moderate potential
12(Z)-sabinene hydrate−6.116Moderate potential
13(E)-trans-2-menthenol−6.158Moderate potential
14thymol−6.509Moderate potential
15p-cymene−6.788Moderate potential
16cis-muurola-4(14),5-diene−6.900High-moderate potential, noteworthy
17α-terpineol−6.918High-moderate potential, noteworthy
18isoterpinolene−6.944High-moderate potential, noteworthy
19Captan−7.254Synthetic reference, robust standard
20α-farnesene−7.638Promising metabolite, high binding affinity
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MDPI and ACS Style

Gómez-Yáñez, H.; Soto-Hernández, R.M.; Ruiz-Posadas, L.d.M.; Valdovinos-Ponce, G.; Ruiz-López, I.I.; Peña-Valdivia, C.B.; Mora-Báez, G. Control of Neopestalotiopsis zimbabwana Using Origanum vulgare L. Essential Oil: Combined In Vitro, In Vivo and In Silico Approaches. Horticulturae 2025, 11, 1232. https://doi.org/10.3390/horticulturae11101232

AMA Style

Gómez-Yáñez H, Soto-Hernández RM, Ruiz-Posadas LdM, Valdovinos-Ponce G, Ruiz-López II, Peña-Valdivia CB, Mora-Báez G. Control of Neopestalotiopsis zimbabwana Using Origanum vulgare L. Essential Oil: Combined In Vitro, In Vivo and In Silico Approaches. Horticulturae. 2025; 11(10):1232. https://doi.org/10.3390/horticulturae11101232

Chicago/Turabian Style

Gómez-Yáñez, Héctor, Ramón Marcos Soto-Hernández, Lucero del Mar Ruiz-Posadas, Guadalupe Valdovinos-Ponce, Irving Israel Ruiz-López, Cecilia Beatriz Peña-Valdivia, and Guadalupe Mora-Báez. 2025. "Control of Neopestalotiopsis zimbabwana Using Origanum vulgare L. Essential Oil: Combined In Vitro, In Vivo and In Silico Approaches" Horticulturae 11, no. 10: 1232. https://doi.org/10.3390/horticulturae11101232

APA Style

Gómez-Yáñez, H., Soto-Hernández, R. M., Ruiz-Posadas, L. d. M., Valdovinos-Ponce, G., Ruiz-López, I. I., Peña-Valdivia, C. B., & Mora-Báez, G. (2025). Control of Neopestalotiopsis zimbabwana Using Origanum vulgare L. Essential Oil: Combined In Vitro, In Vivo and In Silico Approaches. Horticulturae, 11(10), 1232. https://doi.org/10.3390/horticulturae11101232

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