Essential oils of Lavandula angustifolia and Thymus vulgaris as biocontrol agents: managing Dactylopius opuntiae infestation and associated bacterial infections

Abstract

Dactylopius opuntiae, the invasive cactus cochineal, is a major pest threatening Opuntia ficus-indica cultivation, especially in arid regions. This study assesses the insecticidal and antibacterial properties of essential oils (EOs) from Lavandula angustifolia (lavender) and Thymus vulgaris (thyme) as eco-friendly biocontrol agents. Laboratory (in vitro) and greenhouse (in vivo) tests were performed using emulsified EOs at 1%, 5%, and 10% concentrations. In vitro tests showed that lavender EO caused 95.0 ± 3.0% mortality in nymphs and adult females of D. opuntiae at 10% after 8 days, with an LT₅₀ of 3.0 days. Thyme EO was slightly less effective (90.0 ± 3.5%, LT₅₀ = 3.5 days). Greenhouse experiments indicated reduced but significant efficacy (85.5 ± 4.0% and 80.0 ± 4.5% mortality for lavender and thyme EOs at 10%, respectively). Disk diffusion and broth microdilution tests against Proteus penneri showed that thyme EO had stronger antibacterial activity (MIC = 0.625%, MBC = 1.25%) than lavender EO (MIC = 1.25%, MBC = 2.5%), both with bactericidal effects. These findings demonstrate the dual insecticidal and antibacterial potential of lavender and thyme EOs, supporting their inclusion in Integrated Pest Management (IPM) programs. Further research is needed to evaluate their field persistence, effects on non-target organisms, and formulation stability for sustainable cactus pest control.

Introduction

Cactophagous insect pests, particularly Dactylopius opuntiae (Hemiptera: Dactylopiidae), have emerged as a major biotic threat to Opuntia ficus-indica, a cactus species of significant agroecological importance in arid and semi-arid regions. Originally native to the Americas and introduced elsewhere for biological control of invasive cacti, D. opuntiae has become a highly invasive and destructive pest in North Africa, especially in Morocco. Since its first detection in 2014, the pest has rapidly infested major cactus-growing zones, decimating more than 50,000 hectares of plantations and causing extensive ecological and socioeconomic damage^1–3.

This alarming spread is attributed to its high reproductive capacity, absence of effective natural enemies, and ease of dispersal via wind or contaminated plant materials. The cochineal feeds on cactus cladodes by extracting sap and injecting phytotoxic substances that induce tissue necrosis, reduced fruiting, and ultimately plant death⁴. Moreover, cochineal-induced injuries create openings for opportunistic phytopathogens, including Proteus penneri, a bacterium associated with decaying plant tissues that further accelerates plant decline through secondary infections^5,6.

As a resilient and multifunctional crop, O. ficus-indica plays a strategic role in dryland agriculture, serving purposes ranging from human nutrition and animal feed to pharmaceutical, cosmetic, and land restoration applications⁷. The collapse of cactus-based systems due to D. opuntiae not only results in biodiversity loss and yield reduction but also undermines the livelihoods of rural communities dependent on cactus farming. Therefore, developing effective and sustainable pest control solutions is an urgent priority.

Although chemical insecticides remain the dominant control strategy, their widespread and repeated use has led to pesticide resistance, off-target toxicity, environmental pollution, and concerns about food safety due to chemical residues^8,9. Moreover, access to these products is limited for smallholder farmers due to high costs and regulatory constraints. These limitations underscore the need for eco-friendly, accessible, and sustainable alternatives.

In this context, essential oils (EOs) derived from aromatic plants have garnered attention as promising botanical biopesticides. Their insecticidal, antibacterial, antifungal, and antioxidant properties stem from complex mixtures of terpenoids, phenolics, and alcohols^10,11. Among these, Thymus vulgaris (thyme) and Lavandula angustifolia (lavender) are particularly valued for their rich content of thymol, carvacrol, linalool, and linalyl acetate—compounds known to disrupt microbial membranes, interfere with neurotransmission, and impair insect physiology^12–14.

Thanks to their broad-spectrum activity, low toxicity to mammals, and environmental biodegradability, essential oils are considered ideal candidates for integration into Integrated Pest Management (IPM) programs—especially in sensitive agroecosystems, such as cactus plantations^15,16. However, despite this potential, few studies have evaluated their dual-action effectiveness—namely, their simultaneous insecticidal and antibacterial activity—against cactus-specific pest–pathogen complexes such as D. opuntiae and P. penneri, especially under greenhouse or semi-field conditions.

Recent studies have also revealed that endophytic and rhizospheric bacteria associated with O. ficus-indica can suppress cochineal populations via antagonistic mechanisms¹⁷. This study demonstrated that certain endorhizosphere-associated strains significantly reduce the survival of D. opuntiae, indicating the potential for synergistic formulations that combine microbial and botanical agents.

Given the growing pressure of D. opuntiae infestations and the secondary bacterial infections caused by P. penneri, there is a pressing need for biorational solutions that can target both insect and pathogen simultaneously. Essential oils from L. angustifolia and T. vulgaris may offer this dual functionality, but their combined effects under controlled and semi-natural conditions remain poorly understood. We hypothesize that the essential oils of L. angustifolia and T. vulgaris, owing to their rich bioactive compositions, exert both insecticidal and bactericidal effects, thereby representing a viable dual-action strategy for controlling cactus cochineal infestations and their associated bacterial infections.

This study was designed to:

• Evaluate the insecticidal efficacy of lavender and thyme EOs against D. opuntiae (L1 nymphs and adult females) at three concentrations (1%, 5%, and 10%) under laboratory and greenhouse conditions;

• Determine the median lethal time (LT₅₀) for each treatment;

• Assess the antibacterial activity of both oils against P. penneri using disk diffusion, MIC, and MBC methods;

• Compare the biocidal effectiveness of these EOs with standard chemical controls, in order to inform their potential inclusion in eco-friendly pest and disease management frameworks tailored to cactus cultivation.

Materials and methods

Plant material and essential oil extraction

Fresh aerial parts (leaves, stems, and inflorescences) of Thymus vulgaris and Lavandula angustifolia were harvested at the full flowering stage from organically managed cultivation plots in the Beni Mellal region (32° 20′ N, 6° 30′ W, 465 m altitude), Morocco, in April 2023. This period corresponds to the peak biosynthesis of monoterpenoids and sesquiterpenes, as reported in previous phytochemical studies^18,19.

Plant material was collected manually in the early morning (08:00–10:00), placed in sterile paper bags, and transported to the laboratory within 2 h to minimize losses of volatile components. Only undamaged, healthy tissues were selected, and foreign material or decaying parts were discarded.

Essential oils were extracted by hydro distillation using a 2 L Clevenger-type apparatus (Labglass, Germany), as recommended by the European Pharmacopoeia (2020). For each batch, 500 g of fresh material was immersed in 5 L of distilled water, resulting in a plant-to-water ratio of 1:10 (w/v). The distillation process was carried out for 3 h from the point of boiling. The distillate was collected, and the oil phase was separated. It was then dried over anhydrous sodium sulfate (Sigma-Aldrich, purity ≥ 99%), filtered through a 0.45 µm membrane, and stored in sterile 10 mL amber glass vials (Wheaton, USA). Samples were kept at 20 ± 2 °C in the dark, without refrigeration, to avoid crystallization or precipitation of heavier fractions. All oils were used within 30 days post-extraction.

The yield (v/w %) of essential oil was calculated by dividing the volume of oil obtained by the fresh weight of plant material and expressed in mL per 100 g of biomass.

Emulsion preparation for bioassays

For biological tests, the essential oils were diluted in sterile distilled water containing 0.1% Tween 80 (polyoxyethylene sorbitan monooleate; Sigma-Aldrich), a non-ionic surfactant selected for its low toxicity and capacity to form stable emulsions without altering EO bioactivity^20,21. Emulsions were freshly prepared before each bioassay.

Three concentrations were tested: 1%, 5%, and 10% (v/v). These levels were chosen based on prior in-house dose-range screening for sublethal and lethal thresholds and were consistent with concentrations reported in literature for insecticidal and antimicrobial effectiveness^22,23. Emulsions were vortexed at 2,000 rpm for 2 min (VWR^® Vortex Mixer) and applied immediately. No preservatives or co-solvents were used to avoid interference with biological activity. The homogeneity of emulsions was visually inspected prior to use.

Target organisms

Dactylopius opuntiae

Field populations of D. opuntiae were collected from infested Opuntia ficus-indica plants in the Oulad Ayad locality (32° 23′ N; 6° 48′ W), during the active reproductive season²⁴. Cladodes harboring mature colonies were clipped and enclosed in aerated polyethylene boxes for transport.

Insects were reared under controlled laboratory conditions (26 ± 2 °C, 60 ± 10% RH, photoperiod 14:10 h light: dark) using pesticide-free O. ficus-indica cladodes placed on sterile, moistened sand in 50 × 40 × 30 cm transparent boxes. The colony was maintained for at least two generations before use in experiments to ensure physiological uniformity.

Morphological identification was confirmed using a stereomicroscope (Zeiss Stemi 305) following Cruz-Rodríguez et al.¹. Voucher specimens were preserved in 70% ethanol and deposited at the Polyvalent Laboratory for Research and Development (LPRD), Faculty of Polydisciplinary Studies, Sultan Moulay Slimane University, Morocco.

Proteus penneri

Necrotic tissues were excised from symptomatic O. ficus-indica cladodes and surface-sterilized with 70% ethanol for 1 min, rinsed in sterile water, and homogenized. Aliquots were streaked onto nutrient agar and incubated at 37 °C for 24 h. Single colonies exhibiting swarming motility and characteristic odor were isolated, purified, and maintained on slants at 4 °C.

Identification was carried out using API 20E strips (bioMérieux) and confirmed by 16S rRNA gene sequencing²⁵. The isolate was stored in LB-glycerol (25%) stocks at − 80 °C.

Insecticidal bioassays

Laboratory assays (in vitro)

To evaluate how well the essential oils could kill D. opuntiae, we set up controlled laboratory bioassays. We placed sections of Opuntia ficus-indica cladodes (60 cm² each) inside Petri dishes (14.5 cm in diameter) to mimic the insects’ natural environment. Ten first-instar nymphs or adult females were carefully introduced into each dish.

We applied lavender and thyme essential oils at concentrations of 1%, 5%, and 10% using a fine mist sprayer to ensure even coverage. Tap water was used as a control to check for natural mortality. We then monitored the insects at 1, 3, and 8 days after treatment, considering them dead if they did not respond to gentle prodding²⁶. Each treatment was repeated five times, and the entire experiment was conducted twice to confirm reliability²¹.

Mortality was calculated according to Abbott’s formula:

Greenhouse trials (in vivo)

To simulate field conditions, greenhouse trials were conducted on 6-month-old potted O. ficus-indica plants (grown in 10 L pots with a sterilized mixture of soil, sand, and compost, in a 2:1:1 ratio). Each plant was infested with 20 L1 nymphs using a fine brush. After 24 h of acclimation, EO emulsions were applied using a calibrated 2-L backpack sprayer (nozzle diameter, 1.0 mm; pressure, 1.5 bar) until the runoff point was reached.

Plants were arranged in a randomized complete block design (RCBD), with four replicates per treatment, and maintained at 26 ± 2 °C with natural daylight and 60% RH. Mortality was assessed after 10 days by direct count and confirmation of immobility. Plants were not caged to allow natural airflow and volatilization dynamics.

Antibacterial assays

Disk diffusion test

The antibacterial activity of the essential oil emulsions was initially evaluated using the agar disk diffusion method, as outlined in the Clinical and Laboratory Standards Institute²⁷ guidelines. The bacterial strain Proteus penneri, isolated from infected Opuntia ficus-indica cladodes and confirmed through 16S rRNA gene sequencing, was grown in nutrient broth to mid-log phase. The suspension was adjusted to a 0.5 McFarland standard (1.5 × 10⁸ CFU/mL) and uniformly spread on Mueller–Hinton agar plates using sterile cotton swabs.

Sterile 6 mm filter paper disks (Whatman No. 1) were impregnated with 10 µL of each essential oil emulsion at 1%, 5%, and 10% (v/v), respectively. Disks containing 0.1% Tween 80 and ampicillin (10 µg) served as negative and positive controls. The plates were incubated at 37 °C for 24 h. Zones of inhibition were measured in millimeters using a digital Vernier caliper. Each treatment was tested in triplicate.

Minimum inhibitory concentration (MIC)

MIC values were determined using the broth microdilution method in 96-well microplates, as described in the CLSI M07-A9 (2020) guidelines. Essential oils were diluted two-fold in Mueller–Hinton broth containing 0.1% Tween 80, generating concentrations ranging from 0.078% to 10%. Each well received 100 µL of bacterial inoculum (approximately 5 × 10⁵ CFU/mL).

Control wells contained broth with Tween 80 (negative control) and broth with ampicillin (10 µg/mL, positive control). After 24 h of incubation at 37 °C, bacterial growth was visually examined and confirmed by measuring the optical density at 600 nm with a microplate reader (BioTek ELx800). The MIC was determined as the lowest concentration at which no visible growth was observed. All assays were performed in triplicate.

Minimum bactericidal concentration (MBC)

The MBC was determined according to the Clinical and Laboratory Standards Institute (CLSI) guidelines²⁸. After MIC determination, 10 µL aliquots from wells showing no visible growth were subcultured onto nutrient agar plates and incubated at 37 °C for 24 h.

The MBC was defined as the lowest concentration of the essential oil at which no visible bacterial colony was observed, indicating 99.9% killing of the initial inoculum. Each treatment was performed in triplicate. The MBC/MIC ratio was calculated to differentiate between bacteriostatic (MBC/MIC > 4) and bactericidal (MBC/MIC ≤ 4) effects²⁹.

Statistical analysis

Insect mortality was corrected using Abbott’s formula³⁰. Normality and homoscedasticity were verified using Shapiro–Wilk and Levene’s tests, respectively.

One-way ANOVA followed by Tukey’s HSD post hoc test (α = 0.05) was used to compare means among treatments. LT₅₀ values were calculated via probit regression³¹. For antibacterial data, inhibition zones and MIC values were analyzed using ANOVA. All statistical analyses were performed in R³², and results were expressed as mean ± standard deviation (SD).

Results

Insecticidal activity of essential oils under laboratory conditions

The application of Lavandula angustifolia and Thymus vulgaris essential oils (EOs) induced significant mortality in Dactylopius opuntiae under controlled laboratory conditions. A precise dose- and time-dependent effect was observed for both L1 nymphs and adult females (Tables 1 and 2; Fig. 1). Lavender oil consistently exhibited higher efficacy than thyme oil across all concentrations and time points²³.

Table 1.

Mortality of Dactylopius opuntiae first-instar nymphs (laboratory trials).

Essential oil	Concentration (%)	1-Day mortality (%) ± SE	3-Day mortality (%) ± SE	8-Day mortality (%) ± SE	P-value	LT₅₀ (days ± SE)	95% CI
Lavender EO	1	20.0 ± 2.5d	35.0 ± 3.0de	50.0 ± 4.0d	< 0.0001	8.0 ± 0.2	7.8–8.2
	5	40.0 ± 3.5c	65.0 ± 4.5c	85.0 ± 5.0b	< 0.0001	5.0 ± 0.3	4.8–5.2
	10	70.0 ± 5.0a	90.0 ± 5.5a	95.0 ± 3.0a	< 0.0001	3.0 ± 0.2	2.8–3.2
Thyme EO	1	15.0 ± 2.0d	30.0 ± 3.5e	45.0 ± 4.5d	< 0.0001	8.5 ± 0.3	8.3–8.7
	5	35.0 ± 3.0c	60.0 ± 5.0c	80.0 ± 6.0b,c	< 0.0001	5.5 ± 0.3	5.3–5.7
	10	65.0 ± 4.0ab	85.0 ± 5.0a,b	90.0 ± 3.0a,b	< 0.0001	3.5 ± 0.2	3.3–3.7
Control (tap water)	0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	–	–	–

Values are means ± standard error (SE) of five replicates. Different letters in the same column indicate significant differences (Tukey’s HSD test, α = 0.05).

Table 2.

Mortality of Dactylopius opuntiae adult females (laboratory trials).

Essential Oil	Concentration (%)	1-Day mortality (%) ± SE	3-Day mortality (%) ± SE	8-Day mortality (%) ± SE	P-value	LT₅₀ (days ± SE)	95% CI
Lavender EO	1	10.0 ± 1.5d	25.0 ± 3.0d	40.0 ± 4.0d	< 0.0001	8.5 ± 0.3	8.3–8.7
	5	35.0 ± 4.0c	60.0 ± 5.5b,c	85.0 ± 3.0a,b	< 0.0001	5.5 ± 0.3	5.3–5.7
	10	60.0 ± 5.0a,b	85.0 ± 4.5a	95.0 ± 3.0a	< 0.0001	3.5 ± 0.2	3.3–3.7
Thyme EO	1	8.0 ± 1.2d	20.0 ± 2.5d	35.0 ± 3.5d	< 0.0001	9.0 ± 0.3	8.8–9.2
	5	30.0 ± 4.0c	55.0 ± 5.0c	80.0 ± 4.5b	< 0.0001	6.0 ± 0.3	5.8–6.2
	10	55.0 ± 4.5b	80.0 ± 5.0a,b	90.0 ± 3.5a,b	< 0.0001	4.0 ± 0.2	3.8–4.2
Control (tap water)	0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	–	–	–

Fig. 1.

Adult females of Dactylopius opuntiae were treated with essential oils of Thymus vulgaris and Lavandula angustifolia at different concentrations. (A) Untreated control; (B) Thyme EO at 1%; (C) Thyme EO at 5%; (D) Thyme EO at 10%; (E) Lavender EO at 10%

At 10% concentration, lavender EO induced 95.0 ± 3.0% mortality in nymphs and 95.0 ± 3.0% in adults at 8 days post-treatment, compared to 90.0 ± 3.0% and 90.0 ± 3.5% with thyme EO, respectively. Even at the lowest concentration (1%), lavender oil showed higher efficacy (50.0 ± 4.0% in nymphs; 40.0 ± 4.0% in adults) than thyme EO (45.0 ± 4.5% in nymphs; 35.0 ± 3.5% in adults). Control groups treated with tap water exhibited negligible mortality (< 5%).

Signs of desiccation, melanization, and disintegration of the waxy covering are visible, particularly at higher doses, confirming the vigorous insecticidal activity of the essential oils (Fig. 1).

Insecticidal efficacy under greenhouse trials

Greenhouse experiments confirmed the insecticidal efficacy of both oils, although the mortality rates were slightly reduced due to environmental variables (Table 3). At 10% concentration, lavender oil caused 85.5 ± 4.0% mortality in L1 nymphs, while thyme oil achieved 80.0 ± 4.5%. At 5% and 1%, lavender EO maintained higher efficacy (50.5 ± 3.0% and 20.5 ± 2.0%) compared to thyme (45.0 ± 3.5% and 18.0 ± 1.5%). Differences among treatments were statistically significant²³, confirming a consistent trend observed under laboratory conditions.

Table 3 .

Mortality of first-instar nymphs in greenhouse trials (10 days).

Essential oil	Concentration (%)	Mortality (%) ± SE	P-value
Lavender EO	1	20.5 ± 2.0 c	< 0.0001
	5	50.5 ± 3.0 b
	10	85.5 ± 4.0 a
Thyme EO	1	18.0 ± 1.5 c
	5	45.0 ± 3.5 b
	10	80.0 ± 4.5 a
Control (tap water)	0	0.0 ± 0.0	–

Values are means ± SE of four replicates. Different letters indicate significant differences (Tukey’s HSD test, α = 0.05).

These findings demonstrate that lavender EO remains effective in vivo, although it shows some decline, possibly due to environmental factors: volatilization, plant surface interactions, and climate variability.

Toxicity of essential oil to nymphs and adult females: (LT₅₀ values)

Probit analysis of LT₅₀ values revealed that lavender EO killed D. opuntiae significantly faster than thyme EO (Table 4). At 10% concentration, lavender EO had an LT₅₀ of 3.0 ± 0.2 days for nymphs and 3.5 ± 0.2 days for adults, while thyme EO required 3.5 ± 0.2 and 4.0 ± 0.2 days, respectively.

Table 4.

LT₅₀ values (days) for nymphs and adult females of D. opuntiae (laboratory trials).

Essential oil	Cochineal stage	Concentration (%)	LT₅₀ (days ± SE)	95% confidence interval
Lavender EO	Nymphs	10	3.0 ± 0.2	2.8–3.2
	Adult females	10	3.5 ± 0.2	3.3–3.7
Thyme EO	Nymphs	10	3.5 ± 0.2	3.3–3.7
	Adult females	10	4.0 ± 0.2	3.8–4.2

LT₅₀ values calculated using probit regression analysis. Values represent means ± SE from five replicates.

At 5%, LT₅₀ values were moderately longer (lavender: 5.0–5.5 days; thyme: 5.5–6.0 days), and at 1%, both oils showed delayed effects, with LT₅₀ values exceeding 8 days. These results further support the superior potency and rapid mode of action of lavender oil at higher doses.

A consistent dose–response relationship was evident in all bioassays (Fig. 1), with increasing EO concentrations correlating with higher mortality rates. This trend was statistically supported by one-way ANOVA followed by post hoc analysis²³. The 10% treatments were significantly more effective than the 5% and 1% in both test systems and life stages. The relationship plateaued beyond the 10% dose, indicating a saturation effect for both EOs at this concentration.

Antibacterial activity against Proteus penneri

Both lavender and thyme essential oils exhibited significant inhibitory activity against Proteus penneri, a phytopathogenic bacterium co-isolated from necrotic cladodes (Fig. 2; Table 5). Disk diffusion assays showed clear concentration-dependent inhibition zones. Thyme EO at 10% induced the largest mean zone (22.5 ± 1.3 mm), followed by lavender EO (18.7 ± 1.1 mm). At 5%, thyme and lavender oils showed inhibition zones of 15.8 ± 1.0 mm and 12.4 ± 0.9 mm, respectively. Limited activity was recorded at 1%.

Table 5 .

Antibacterial activity of essential oils against Proteus penneri.

Essential oil	Concentration (%)	Inhibition zone (mm ± SE)	P-value
Thyme EO	1	7.2 ± 0.5c	< 0.0001
	5	15.8 ± 1.0b	< 0.0001
	10	22.5 ± 1.3a	< 0.0001
Lavender EO	1	5.5 ± 0.4d	< 0.0001
	5	12.4 ± 0.9b,c	< 0.0001
	10	18.7 ± 1.1a,b	< 0.0001
Ampicillin (10 µg)	–	27.5 ± 1.2a	–
Tween 80 (0.1%)	–	0.0 ± 0.0e	–

Values are mean inhibition diameters (mm ± SE). Different letters indicate significant differences (Tukey’s HSD test, α = 0.05, n = 3).

Statistical analysis confirmed the significant effect of oil type and concentration on inhibition zones²³. The positive control (ampicillin 10 µg) produced a 27.5 ± 1.2 mm zone, while the negative control (Tween 80, 0.1%) showed no inhibition.

Minimum inhibitory and bactericidal concentrations (MIC and MBC)

The results of the MIC and MBC assays further confirmed the potent antibacterial activity of Thymus vulgaris and Lavandula angustifolia essential oils against Proteus penneri. As shown in Table 6, thyme essential oil exhibited the lowest MIC value (0.625 ± 0.05% v/v), indicating a strong bacteriostatic effect, while lavender oil had a slightly higher MIC of 1.25 ± 0.08%, suggesting moderate antibacterial activity. The reference antibiotic, ampicillin (10 µg/mL), demonstrated the highest efficacy, with a MIC of less than 0.015% (w/v), whereas Tween 80 (0.1%) used as a negative control had no inhibitory effect.

Table 6.

MIC and MBC values of essential oils and controls against Proteus penneri.

Treatment	MIC (% v/v) ± SE	MBC (% v/v)	MBC/MIC ratio	Interpretation
Thyme essential oil	0.625 ± 0.05	1.25	2.0	Strong bactericidal effect
Lavender essential oil	1.25 ± 0.08	2.5	2.0	Moderate bactericidal effect
Ampicillin (10 µg/mL)	< 0.015	< 0.03	2.0	Reference antibiotic control
Tween 80 (0.1%)	0.0	0.0	–	Negative control

The corresponding MBC values were 1.25% for thyme oil and 2.5% for lavender oil, yielding MBC/MIC ratios of 2 in both cases. According to the established interpretive criteria²⁹, an MBC/MIC ratio of ≤ 4 indicates that the agent exerts bactericidal activity rather than merely inhibiting growth. Therefore, both essential oils demonstrated bactericidal potential, with thyme oil being more potent.

These findings align with earlier disk diffusion data (“Minimum inhibitory and bactericidal concentrations (MIC and MBC)”). They are consistent with the presence of major bioactive components such as thymol and carvacrol in thyme oil and linalool in lavender oil, all of which are known to disrupt bacterial membranes and induce cellular leakage^12,16,27.

Discussion

This study provides compelling evidence for the dual bioactivity of Lavandula angustifolia and Thymus vulgaris essential oils, validating their potential as environmentally sound agents for managing Dactylopius opuntiae infestations and associated bacterial infections caused by Proteus penneri. These findings contribute to the increasing body of literature advocating for the use of plant-derived biopesticides in Integrated Pest Management (IPM) strategies, particularly within arid and semi-arid agroecosystems where conventional chemical control faces ecological, economic, and regulatory limitations^10,14.

The insecticidal assays demonstrated a clear concentration- and time-dependent effect on both L1 nymphs and adult females of D. opuntiae, with lavender essential oil exhibiting superior efficacy at all tested concentrations. At 10%, lavender EO induced 95% mortality in both developmental stages within eight days, with LT₅₀ values of 3.0 and 3.5 days, respectively. These results are consistent with previous reports highlighting the rapid neurotoxic and cytotoxic effects of essential oil constituents¹³.

The observed efficacy may be attributed to the bioactive compounds within the essential oils. Linalool and linalyl acetate, major constituents of L. angustifolia, are known to affect insect physiology through interference with the octopaminergic system, inhibition of acetylcholinesterase, and disruption of cellular respiration^14,27. Thymol and carvacrol, phenolic compounds present in high concentrations in T. vulgaris, act by compromising membrane integrity, leading to ion leakage, oxidative stress, and mitochondrial dysfunction^12,33. Together, these Compounds contribute to rapid insect mortality through multimodal physiological disruption. Meanwhile, both essential oils showed significant antibacterial activity against P. penneri, an opportunistic phytopathogen often associated with necrotic tissues of O. ficus-indica. Thyme EO demonstrated the most potent antibacterial effect, with an inhibition zone of 22.5 mm at 10% concentration, and MIC and MBC values of 0.625% and 1.25%, respectively. Lavender EO was moderately less effective, yet retained bactericidal capacity (MIC = 1.25%, MBC = 2.5%), as indicated by MBC/MIC ratios ≤ 2.

These bactericidal effects can also be linked to the bioactive constituents of the oils. Thymol and carvacrol have been shown to cause membrane destabilization, protein denaturation, and inhibition of ATP synthesis in Gram-negative bacteria such as Proteus spp.^16,33. Linalool, while less potent, induces membrane depolarization and intracellular leakage, thereby contributing to antimicrobial action¹². The results of this study thus reinforce the relevance of essential oils as dual-function agents that target both insect pests and bacterial pathogens.

Recent research has highlighted the role of rhizospheric and endophytic bacteria in suppressing D. opuntiae, providing complementary or synergistic mechanisms to botanical biopesticides. Qessaoui et al.³⁴ reported that Bacillus and Pseudomonas strains isolated from the rhizosphere of O. ficus-indica induced significant mortality in cochineal nymphs under laboratory conditions. El-Aalaoui et al.³⁵ further demonstrated that Pseudomonas koreensis, in combination with natural adjuvants, achieved over 90% mortality in greenhouse trials. Such microbes exert their effects through multiple pathways, including the production of insecticidal metabolites, competition for ecological niches, and the induction of systemic resistance in plants.

The integration of essential oils with these beneficial microbes represents a promising strategy to enhance pest suppression and broaden the spectrum of action. Essential oils exhibit rapid contact toxicity and antimicrobial effects, whereas microbial agents possess persistence and the potential for colonization. However, compatibility between the two agents must be evaluated to avoid antagonistic interactions, particularly given the known antimicrobial properties of EOs¹⁵. Carefully designed formulations and dose optimization may help circumvent such limitations.

Despite their promising bioactivity, the potential phytotoxic and environmental impacts of essential oils must be carefully considered before their large-scale application. Although no visible phytotoxic effects were observed in the current study, previous investigations have reported chlorosis, necrosis, and growth inhibition in various plant species exposed to high EO concentrations^5,21. Moreover, the volatile nature of essential oils raises concerns regarding their effects on non-target organisms, including beneficial insects, soil microbiota, and aquatic systems.

In this context, the development of advanced formulations, such as nanoemulsions, encapsulated carriers, or slow-release systems, may enhance the stability, adherence, and specificity of essential oils while mitigating off-target toxicity^10,36. Furthermore, regulatory and safety assessments—including ecotoxicological and residue studies—are essential to ensure the responsible implementation of EO-based products in IPM frameworks.

To advance the practical implementation of essential oils as biopesticides, future research should focus on:

• Large-scale field validation under diverse agroecological zones

• Phytotoxicity and ecotoxicology assessments on non-target flora and fauna

• Formulation engineering, such as biodegradable carriers or nano-emulsions to improve adherence and residual activity

• Economic feasibility studies, comparing EO production and application costs to conventional pesticides

• Combination strategies, integrating essential oils with microbial biocontrol agents (e.g., Bacillus subtilis, Beauveria bassiana) for synergistic effects and enhanced resistance management²³

In summary, the current study validates the dual biocidal efficacy of L. angustifolia and T. vulgaris essential oils, confirming their potential integration into sustainable frameworks for pest and disease control. While their performance under laboratory and semi-controlled conditions is encouraging, practical deployment requires refinement of the formulation, field-level trials, and comprehensive safety assessments to meet the standards of modern agroecological practice.

Conclusion

This study confirms the promising biocidal efficacy of Lavandula angustifolia and Thymus vulgaris essential oils as natural alternatives to synthetic pesticides for managing Dactylopius opuntiae and its associated phytopathogen Proteus penneri. Both oils exhibited strong, concentration-dependent effects, with lavender oil demonstrating rapid insecticidal action and thyme oil showing superior antimicrobial properties. These dual activities position essential oils as multifunctional tools within integrated pest and disease management strategies.

In conclusion, thyme and lavender essential oils offer a promising and sustainable approach to controlling pests and diseases. However, their practical use relies on translational research that connects laboratory results with real-world farming conditions. Therefore, they deserve further study within multidisciplinary approaches that combine phytochemistry, entomology, plant pathology, and agroecology to develop comprehensive, eco-friendly pest management solutions.

Author contributions

Conceptualization: B.E.; Data curation: S.A. and S.S.A.; Formal analysis: B.E.; Investigation: all authors; Methodology: B.E.; Supervision S.S.A.; Visualization: all authors; writing—original draft: B.E. and S.S.A.; Writing—review and editing: B.E. All authors read and approved the final manuscript. The authors declare that there are no conflicts of interest related to this article. Supervision: S.S.A.; Visualization: all authors; Writing—original draft: B.E. and S.S.A.; Writing—review and editing: B.E. All authors read and approved the final manuscript.

Funding

No external funding was received for this research project."

Data availability

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.