Antifungal, Insecticidal, and Repellent Activities of Rosmarinus officinalis Essential Oil and Molecular Docking of Its Constituents against Acetylcholinesterase and β-Tubulin

The aim of this study was to determine the phytochemical composition and evaluate the antifungal and insecticidal properties of Rosmarinus officinalis essential oil (EO). GC-MS was employed to analyze the phytochemical profile of the EO. The antifungal activity of the EO was assessed by calculating growth inhibition rates for Alternaria alternata, Fusarium oxysporum, and Botrytis cinerea. Repellent capacity and toxicity were evaluated through inhalation and contact tests on Callosobruchus maculatus. Molecular docking techniques were utilized to test the insecticidal and antifungal activities of rosemary EO. The analysis revealed a total of sixteen components in R. officinalis EO, with 1,8-cineole (40.80%) being the major constituent, followed by α-pinene (26.18%) and camphor (19.53%). Antifungal evaluation demonstrated a significant inhibitory impact on the mycelial growth of the tested fungi, with complete inhibition observed against B. cinerea. In terms of insecticidal capacity, the EO induced complete mortality of C. maculatus adults at a concentration of 1 μL/L air, with an inhalation test LC50 value of 0.62 μL/L air. Concentration-dependent reductions were observed in the number of both laid eggs and emerged insects, reaching a 99.36% reduction. The EO also exhibited a moderate effectiveness in repelling insects, with an average repellency rate of 50.83%. In silico analysis identified borneol as the most active molecule against insect acetylcholinesterase (PDB: 6ARY) with a Glide score of −7.254 kcal/mol. α-Caryophyllene showed the highest activity against B. cinerea β-tubulin (PDB: 3N2G) with a Glide score of −7.025 kcal/mol. These findings suggest that the EO derived from Moroccan Rosmarinus officinalis has potential as an effective natural agent against pathogenic fungi and could serve as a sustainable and environmentally friendly alternative as a bioinsecticide.


Introduction
Chickpea (Cicer arietinum), globally renowned for its nutritional richness in dietary protein, minerals, and vitamins, faces considerable challenges in storage due to yield losses inficted by various pests, notably bruchids.Among these, Callosobruchus maculatus, commonly known as the chickpea weevil, emerges as a particularly destructive insect.Tis species has the ability to lay eggs in cultivated felds and storage warehouses.Te larvae, feeding internally on the seeds, present formidable challenges for efective management using chemical insecticides [1].Te loss of yield in leguminous crops during storage, primarily caused by various insects, particularly bruchids, poses a signifcant challenge for traders and farmers [2].C. maculatus, known as the chickpea weevil, stands out as one of the most destructive pests afecting chickpeas.Tis insect exhibits the ability to deposit eggs in both cultivated felds and storage sites, and its larvae, which internally consume the seeds, prove challenging to manage using chemical insecticides [1].Synthetic insecticides are frequently used to control pests in agricultural crops.Consequently, their excessive and widespread use has resulted in adverse impacts on human health, environmental contamination, pesticide residues on fruits, vegetables, and seeds, as well as the development of pest resistance and weed resistance.At present, biopesticides derived from essential oils (EOs) are gaining prominence as a promising alternative to alleviate the negative impacts of synthetic pesticides.Tese biopesticides are known for their specifcity in targeting agricultural pests and their biodegradable nature [3].
Filamentous fungi represent the most widespread group of pathogenic microorganisms afecting crops, leading to a decline in agricultural productivity, deterioration in quality, and substantial economic losses [4].Among these, mycotoxigenic fungi are renowned for their capability to infest various cereals, vegetables, spices, and fruits.Tese molds generate various mycotoxins known for their genotoxic, carcinogenic, and immunosuppressive efects on human health [5].Currently, the main methods for preventing fungal infections involve the adoption of resistant cultivars and phytosanitary treatments [6].However, to limit the accumulation of residues in the soil and combat the constant evolution of new fungal strains resistant to chemical treatments, it is imperative to adopt new, environmentally friendly approaches to crop protection [7].In this situation, EOs are emerging as potential substitutes for synthetic fungicides, thereby enhancing the means of protection against these plant pathogens [8].
Recently, EOs derived from aromatic and medicinal plants have garnered signifcant interest due to their diverse biological efects [9].Rosmarinus ofcinalis (Lamiaceae), native to the Mediterranean basin, possesses an essential oil that contributes to its remarkable properties.Tis oil is acknowledged for its antioxidant, antidiabetic, antiinfammatory, anticarcinogenic, antimicrobial, hepatoprotective, antinociceptive, antiulcerogenic, antithrombotic, and diuretic efects [10].Furthermore, rosemary essential oil comprises several monoterpenes like limonene and 1,8-cineole, which have exhibited antifungal properties and have demonstrated success in the control of insects [8,11,12].Despite this, there remains a paucity of data and research regarding the bioactivities of Moroccan chemotypes of R. ofcinalis EO, particularly concerning phytopathogenic fungi and agricultural pests.Terefore, the purpose of the present study was to characterize and assess the antifungal activity of the essential oil extracted from the aerial parts of R. ofcinalis against specifc phytopathogenic fungi responsible for leguminous contamination.Moreover, this study explores the insecticidal and repellent capacities of the EO derived from this emblematic plant of the Moroccan pharmacopoeia against Callosobruchus maculatus, a notorious pest afecting chickpea seeds.Additionally, to understand the mechanism of action of Rosmarinus ofcinalis EO during insecticidal and antifungal activities, molecular docking studies, in silico, against insect acetylcholinesterase and Botrytis cinerea β-tubulin were conducted.

Plant Material.
In this study, the plant material used consists of Rosmarinus ofcinalis leaves, harvested in the Boulemane (Skoura) region of Morocco in May 2021.Laboratory botanists identifed the plant samples by referring to various botanical works and plant catalogs.Subsequently, the samples were subjected to a cleaning process and left to dry in the shade and open air for 15 days before the extraction process began.

Essential Oil Extraction.
A quantity of 200 grams of dried R. ofcinalis leaves was subjected to hydrodistillation for 3 hours using a Clevenger-type apparatus with 1000 mL of distilled water, following the standard procedure outlined in the European Pharmacopoeia [13].Te resulting oils were subsequently dried using anhydrous sodium sulfate and then stored in dark conditions at 4-5 °C until testing and analysis.Te EO yield, calculated relative to the weight of the dried plant material, was expressed as a percentage (v/w) [14].Tese specifc fungal strains have garnered recognition for their dual attributes of mycotoxigenicity and phytopathogenicity, thus assuming pivotal roles in the attenuation of agricultural yields and the compromise of overall production quality [8].It is imperative to note that the inclusion of these fungal strains in the study was facilitated through collaboration with the esteemed Hassan II Institute of Agronomy and Veterinary Sciences situated in Rabat (Morocco).Te meticulous preparation of spore suspensions involved the cultivation of 7-day-old cultures of the fungi on potato dextrose agar (PDA) medium within tubes infused with a 0.9% NaCl solution.Following this cultivation period, the quantifcation of spore counts was methodically executed using a Malassez cell, and the resultant suspensions underwent judicious dilution to attain an inoculum concentration of approximately 10 6 spores/mL [17].Tis rigorous methodology not only ensured the precision of spore suspension preparation but also laid the foundation for subsequent phases of the study, thereby enhancing the scientifc rigor and reliability of the experimental procedures.

Disk Difusion Method.
Te examination of the antifungal properties intrinsic to Rosmarinus ofcinalis EO was systematically conducted employing the disk difusion assay, a technique well-established in prior scientifc endeavors [3].In the initial phase, Petri plates with a standardized diameter of 90 mm were meticulously prepared, housing a uniform distribution of the Czapek-Dox agar medium.Te agar medium underwent central inoculation with a precisely measured 100 μL spot containing an inoculum of established concentration at 10 6 spores/mL.After the inoculation, sterile Whatman flter paper disks, measuring 5 mm in diameter, were thoroughly impregnated with R. ofcinalis EO at 5, 10, and 40 μg/mL concentrations, utilizing dimethyl sulfoxide (DMSO) as the solvent.Tese EO-laden disks were judiciously positioned on the surface of the agar medium.Additionally, control Petri plates, inoculated with 10 μL per disk of DMSO, were included to function as negative controls for fungal growth.To uphold the experimental integrity, Petri plates were hermetically sealed with paraflm and subjected to a carefully regulated incubation period of 6 days at 25 °C [8].Noteworthy is the implementation of three replicates for each concentration of the EO, a critical practice to ensure the statistical robustness and reliability of the empirical fndings.Troughout the incubation period, meticulous daily observations were recorded, capturing precise measurements of mycelial growth.Te quantifcation of the inhibitory efect on fungal growth, relative to the control, was accomplished through the application of the following formula: In this formulation, dc and dt represent the average diameter (in millimeters) of fungal mycelial growth in the control and treated fungal strains, respectively.Tis comprehensive and systematic approach not only facilitated a nuanced exploration of the antifungal efcacy of Rosmarinus ofcinalis EO but also ensured the methodological rigor and replicability of the experimental fndings within an academic context.

Insect Collection and Breeding Conditions.
Te investigation into insecticidal activity intricately focused on the examination of Callosobruchus maculatus, a signifcant pest exerting its impact on chickpeas.Te cultivation of bruchids, the larvae of this insect, was purposefully undertaken within glass jars, utilizing chickpea (Cicer arietinum) seeds as the substrate.A paramount consideration in this study was the meticulous maintenance of controlled environmental conditions to ensure the reliability and consistency of the experimental outcomes.Tese glass jars served as microcosms for the cultivation of the insect population, sustaining a carefully regulated temperature of 25 °C.Te relative humidity levels were maintained at 65 ± 5%, and a standardized photoperiod of 14 hours of illumination followed by 10 hours of darkness was rigorously imposed.Tis level of environmental control persisted across multiple generations of the insect population, Scientifca imparting a level of stability crucial for the systematic study of their behavior and susceptibility to insecticidal agents [15].Such precision in environmental control not only mirrors the natural habitat of the insect but also establishes a robust foundation for the comprehensive assessment of insecticidal efcacy against Callosobruchus maculatus.

Essential Oil's Toxicity against C. maculatus
(1) Toxicity of the Essential Oil by Contact Test.Te quantifcation of essential oil concentrations, expressed in microliters per liter (μL/L) relative to the enclosed air volume in the experimental jars, has been rigorously established.Each Petri plate, serving as a controlled experimental unit, was meticulously equipped with 100 grams of Cicer arietinum seeds and featured a flter paper disk infused with distinct concentrations (1, 5, 10, and 20 μL/L) of R. ofcinalis EO.To ensure a representative insect sample, ten adult C. maculatus insects comprising fve males and fve females were judiciously selected from their rearing environment.Tese insects, aged up to 24 hours postseed emergence, were deposited into each plate, and the plates were promptly resealed postintroduction.Tis carefully orchestrated experimental design was systematically replicated three times for each concentration, adhering to stringent research practices [18].
Over a span of four consecutive days, at 24-hour intervals, a meticulous count of deceased insects was systematically recorded to discern and evaluate the mortality rate within the respective concentration levels.Additionally, a thorough examination of eggs laid on the seeds and plate walls was conducted, employing a binocular magnifying glass for enhanced precision.Furthermore, a consistent count of insects emerged was conducted commencing from the 28th day following their initial confnement.Tese detailed observations and counts were systematically juxtaposed with those from the control groups, forming the basis for determining reduction rates in both eggs laid and emergence [18].
Te mortality rate, corrected by Abbott's formula, was computed through the expression: where Po and Pc represent the observed mortality in the test and control, respectively.Te egg-laying reduction rate was calculated using the following formula: where Nt and Nc represent the number of eggs in the test and control jars, respectively.Te reduction rate of emerged insects was determined using the formula as follows: where Nc and Nt signify the number of insects hatched in the control and test jars, respectively.Tis meticulous experimental framework, coupled with a rigorous analytical approach, aims to contribute valuable insights into the feld of insecticidal research, specifcally in understanding the impact of Rosmarinus ofcinalis EO on Callosobruchus maculatus.
(2) Toxicity of the Essential Oil by Inhalation.In summary, the experimental setup involved suspending miniature cotton loops within glass jars, to which varying concentrations of Rosmarinus ofcinalis essential oil (1 μL, 5 μL, 10 μL, and 20 μL) were meticulously applied using a micropipette.Subsequently, a total of 10 Callosobruchus maculatus bruchids (comprising both males and females) aged no more than 48 hours were introduced into each jar.Te jars were then hermetically sealed to create a controlled environment.Tree replicates were meticulously conducted for each experimental condition.To provide a baseline for comparison, a control group was established, consisting of cotton loops without the essential oil solution.Te subsequent computation of the mortality rate was performed using previously described and validated methodologies.Tis experimental design aimed to systematically evaluate the impact of varying concentrations of Rosmarinus ofcinalis EO on the mortality of Callosobruchus maculatus, contributing to a nuanced understanding of its insecticidal efcacy [18].

Essential Oil's Repellent Activity against C. maculatus.
In this investigative study, the evaluation of the repellent properties demonstrated by the essential oil derived from R. ofcinalis against C. maculatus insects was conducted utilizing the preferential area method, as elucidated in previous scholarly research [8].Te experimental protocol involved the strategic placement of flter paper disks within Petri plates.Each disk (9 cm of diameter) was meticulously divided into equal halves, resulting in sections measuring 31.80 cm 2 each.Te halves were subjected to diferent treatments: one received a uniform application of 0.5 mL of varying EO concentrations (5, 10, and 20 μL/mL) prepared in acetone, corresponding to doses of 0.079, 0.157, and 0.315 μL/cm 2 per disk, respectively, while the other half was exclusively impregnated with 0.5 mL of acetone.Tereafter, 10 pairs of C. maculatus adult bruchids, each less than a day old, were carefully positioned in the central region of each Petri plate.To maintain a controlled environment, the plates were securely sealed using paraflm.To ensure the robustness and reliability of the results, each experimental run was meticulously repeated three times, ensuring consistency with the conditions prevailing in the insect-rearing environment.
Following a precisely controlled 30-minute exposure period, the enumeration of bruchids present on the EOtreated section of the disk was conducted and meticulously compared with the control treated solely with acetone.Te repulsion percentage, a key metric in assessing the efcacy of the EO as a repellent, was calculated using the formula [15]: where NC and NT represent the numbers of C. maculatus insects in the control zone and treated zone, respectively.Tis quantitative approach facilitated a comprehensive evaluation of the repellent efectiveness of R. ofcinalis EO against Callosobruchus maculatus.Furthermore, the obtained average repellency percentage was subjected to categorization into specifc repellency classes ranging from 0 to 100% [19].Tis classifcation provided a nuanced understanding of the repellent potential of R. ofcinalis EO, contributing to the broader knowledge base concerning its applications in insect management.Te systematic and thorough nature of this experimental design enhances the reliability and applicability of the fndings, thereby advancing our comprehension of the insectrepelling attributes of R. ofcinalis EO.

In Silico Molecular Docking of Essential Oil's Antifungal and Insecticidal Activities.
In this research, the utilization of computational methods aimed to evaluate the biological activities of R. ofcinalis EO involved a comprehensive investigation into the inhibition of acetylcholinesterase for assessing insecticidal potential and the inhibition of Botrytis cinerea β-tubulin to evaluate antifungal activity.For the preparation of ligands, we meticulously compiled all the compounds identifed in R. ofcinalis EO through GC/MS from PUBCHEM in Structure Data File (SDF) format.Subsequently, these diverse ligands were subjected to a complete pretreatment phase for the docking calculations using the LigPrep tool within the Schrödinger Software program (v. 11.5).Tis process involved applying the OPLS3 force feld, the generation of up to 32 stereoisomers for each ligand, and specifcation of ionization states at pH 7.0 ± 2.0 [9].
Te preparation of proteins involved obtaining threedimensional crystal structures of acetylcholinesterase (PDB: 6ARY) [20] and Botrytis cinerea β-tubulin (PDB: 3N2G) [21] from the Protein Data Bank (PDB) in PDB format.Tese structures underwent meticulous construction and refnement by the use of the Protein Preparation Wizard in Schrödinger-Maestro (v.11.5).Te process involved introducing hydrogens (H) to heavy atoms, converting selenomethionines to methionines, and eliminating all water molecules.Following these steps, the proteins underwent minimization utilizing the OPLS3 force feld, with the maximum.Te creation of the receptor grid commenced with the initiation of the creation module, wherein a ligand atom was chosen, resulting to the establishment of a default grid box.Subsequently, the ligand was connected to the grid box generated from the protein by the use of the Standard Precision.
Executing the Standard Precision, fexible ligand docking procedure was conducted through the Glide module within Schrödinger-Maestro (v.11.5).Tis involved incorporating penalties for non-cis/trans amide bonds.Specifc parameters for ligand atoms, such as the Van der Waals scaling factor and the partial charge cutof, were carefully programmed to 0.80 and 0.15, respectively.Te fnal score, determined based on energy-minimized poses, was presented as a Glide score.Te optimal docked pose for each ligand was identifed as the one with the lowest Glide score value [22].Tis rigorous computational approach aimed to unveil the potential molecular interactions of Rosmarinus ofcinalis EO compounds with acetylcholinesterase and Botrytis cinerea β-tubulin, providing valuable insights into their insecticidal and antifungal mechanisms of action.

Statistical Analysis.
Te computation of mean values and standard deviations (SD) was carried out using GraphPad Prism 8, a software developed by Microsoft.Te antifungal and insecticidal test results were statistically analyzed using the same software platform.A one-way ANOVA and a Tukey test were then performed.A statistically signifcant diference between groups was shown by a result below the predetermined signifcance level of p < 0.05.Moreover, lethal concentrations (LC 50 and LC 95 ), along with their corresponding confdence intervals, were calculated through the application of the probit method [23], after ftting a probit model to the data obtained.Quantile functions corresponding to probabilities of 0.50 and 0.95, respectively, were used to calculate LC 50 and LC 95 doses, in order to assess the degree of efcacy of the rosemary EO studied.Tese statistical analyses were instrumental in providing a robust and comprehensive evaluation of the experimental data, facilitating meaningful comparisons and inferences drawn from the study.

Results and Discussion
3.1.Essential Oil Yield.Te leaves of R. ofcinalis collected from Boulemane (Morocco), produced a clear yellow EO possessing a distinctive aroma, and rendered a yield of 2.44% (v/w).It is widely recognized that the yield of EO is infuenced by several factors, including the plant species, the geographical distribution, and the organ used.Te collection period and the extraction method were also involved [24].For instance, the EOs extracted from R. ofcinalis leaves in studies conducted by El-Demerdash et al.Al Zuhairi et al. and Rezouki et al. yielded 3.2%, 0.98%, and 1.25%, respectively [25][26][27].Tis discrepancy in yield is often due to the geographic origin of the plant collected [9].

Essential Oil's Phytochemical Composition.
Te EO extracted from R. ofcinalis underwent thorough examination and identifcation of its chemical composition through the utilization of GC-MS technique.Te identifed constituents are comprehensively detailed in Table 1.According to the data obtained, a total of sixteen compounds were discerned in the Rosmarinus ofcinalis EO, collectively representing 98.82% of the total essence.Predominant among these compounds were 1,8-cineole (40.80%), α-pinene (26.18%), and camphor (19.53%), all belonging to the class of monoterpenes.Notably, these monoterpenes constituted the major components of the investigated EO.Other compounds were present in yields of less than 3%.

Essential Oil's Antifungal
Activity.Filamentous fungi, in particular species of the genera Fusarium, Alternaria, and Botrytis, are commonly associated with the contamination of legume and fruit crops during harvesting and storage, thus constituting a signifcant threat [40].Tese molds, known for their capacity to produce mycotoxins, are dangerous and pathogenic for humans [5].In the context of this investigation, two distinct concentrations of R. ofcinalis EO underwent testing to assess their, in vitro, antifungal efects against B. cinerea, F. oxysporum, and A. alternata growths (Figures 1, 2, and 3).
Te results revealed pronounced antifungal efects of rosemary EO against the investigated fungi, displaying a dose-dependent inhibition of their mycelial growth.Troughout the entire incubation period, R. ofcinalis oil completely inhibited B. cinerea mycelial growth across all tested concentrations.Furthermore, the concentration of 10 μg/mL of oil was inefective against A. alternata mycelial growth, while the 40 μg/mL concentration demonstrated partial antifungal activity.In the case of F. oxysporum, both concentrations, with particular emphasis on the 40 μg/mL concentration, demonstrated inhibitory efects compared to the control.Tese efects were noted in terms of delayed kinetics of fungal mycelial growth, which commenced on the frst day of observation.
Tese fndings underscore the potential of R. ofcinalis EO as an efective bio-antifungal agent against signifcant plant-pathogenic fungi, suggesting its utility in mitigating the risks associated with fungal contamination in crops, particularly those caused by B. cinerea, F. oxysporum, and A. alternata.
Te growth inhibition rate (%) of the three fungal strains tested was evaluated during the 6 th day of incubation for both concentrations of R. ofcinalis EO.Te outcomes depicted in Figure 4 indicate that B. cinerea displayed the highest sensitivity to rosemary EO, with its growth completely inhibited (100%) by both concentrations of the oil.In contrast, F. oxysporum and A. alternata exhibited lower Our results align with a recent investigation, which demonstrated that R. ofcinalis EO, containing primarily 1,8-cineole (52.20%), camphor (15.20%), and α-pinene (12.40%), displayed a reduction of 15.3% in Aspergillus favus mycelial growth when used at 250 μg/mL.Te study also revealed minimum inhibitory and fungicidal concentrations of 500 μg/mL for the same fungal strain.Additionally, the R. ofcinalis EO exhibited an anti-afatoxigenic efect by inhibiting the synthesis of afatoxins B 1 and B 2 after treatment with 250 μg/mL concentration [41].Additionally, the study reported alterations in the morphology of A. favus, such as reduced conidiophore size and hyphal thickness when treated with R. ofcinalis EO (250 μg/mL) as observed through scanning electron microscopy [41].Moreover, our fndings surpassed those reported by Šernaitė et al.where application of R. ofcinalis EO at a concentration of 2000 μl/ mL reduced B. cinerea mycelial growth by 31.91%[42].Other study reported that the minimum inhibitory concentration (MIC) observed when rosemary EO was applied against Fusarium oxysporum f.sp.Albedinis was 0.2 g/L [43].
Te antifungal efcacy of EOs is often linked to their chemical composition, potentially due to the individual or synergistic efects of major and minor components.Compounds like linalool, borneol, 1,8-cineole, and camphor detected in the studied R. ofcinalis EO could contribute to its observed fungicidal activity.Studies have highlighted the potency of linalool, followed by 1,8-cineole, against A. pullulans, D. hansenii, and the genus Penicillium, Scientifca exhibiting strong inhibitory efects on their mycelial growth [34].Moreover, in vitro assessments have indicated a signifcant reduction in A. alternata mycelial growth upon exposure to chemical compounds found in rosemary oil, such as camphor, α-pinene, and borneol [35].Additionally, various monoterpenes present in rosemary EO, including limonene and 1,8-cineole, have demonstrated antifungal properties [12].Terefore, the wide range of compounds present in the investigated EO, coupled with their diverse antifungal capacities, poses challenges in identifying the specifc active components responsible for the antifungal action.
Various mechanisms have been proposed to elucidate the toxic efect of EOs against fungi.For instance, the antifungal efects of rosemary may be associated with the terpenes and phenolic compounds in the EO.Tese components are recognized for their ability to disrupt cell membranes, leading to the leakage of cellular contents, hindering electron transport, ATPase activity within mitochondria, and ultimately resulting in microbial death [44].In related studies, certain EOs were noted to signifcantly diminish the synthesis of phospholipase enzymes in C. albicans strains, thereby substantially reducing their virulence [45].

Essential Oil's Toxicity against C. maculatus.
Te toxicity of R. ofcinalis EO against C. maculatus insect was assessed via two various tests, focusing on inhalation and contact, to evaluate its insecticidal capacity against this famous chickpea pest.Results, detailed in Figures 5 and 6, revealed a signifcant insecticidal efect of the studied rosemary EO in both contact and inhalation tests.Te mortality of C. maculatus adults increased with higher EO doses and prolonged exposure durations.At the lowest concentration (1 μL/L), R. ofcinalis EO induced mortality rates of 56.66 ± 5.77% and 63.33 ± 5.77% in C. maculatus adults after 24 hours in the contact and inhalation tests, respectively.Over time, these rates signifcantly escalated (p < 0.05) with exposure duration, resulting in total insect mortality after 96 hours.Notably, at higher concentrations (10 μL/L and 20 μL/L), complete mortality (100%) was noted within the initial 24 hours in chickpea bruchid adults exposed to the EO in both tests.Te control jar showed a mortality rate of 0%.
Te lethal concentrations (LC 50 and LC 95 ) of rosemary EO causing 50% and 95% mortality, respectively, in C. maculatus adults within 24 hours were determined and are listed in Table 2. Te fndings indicate a more pronounced biocidal efect in the inhalation test, evidenced by a lower LC 50 value (0.62 μL/L air) compared to the contact test (LC 50 � 1.15 μL/L air).Tis observation was also confrmed by LC 95 data (Table 2).

Essential Oil's Efect on Fecundity and Emergence of C. maculatus Individuals.
Te infuence of R. ofcinalis EO on the oviposition behavior and subsequent emergence of new C. maculatus individuals were systematically investigated, and the fndings are presented in Figures 7 and  8. Te results reveal a conspicuous inverse correlation between EO concentration and the number of eggs laid.At the lowest concentration (1 μL/L), the average oviposition decreased to 20.66 ± 4.04 eggs per female, representing a remarkable fecundity reduction rate of 91.88% relative to the control.At the highest concentration (20 μL/L), the average number of eggs laid/female signifcantly decreased to 1.00 ± 1.73 eggs, indicating a profound 99.36% reduction in oviposition.In the control jar, C. maculatus females laid an average of 184.66 ± 23.43 eggs per female.

Scientifca
Conversely, the number of emergences displayed a noteworthy decline with increasing EO concentrations, as depicted in Figures 7 and 8.At the lowest EO concentration (1 μL/L), the emergent C. maculatus larvae, after embryonic development in chickpea seeds, numbered 9.00 ± 2.00 individuals, compared to 111.66 ± 6.50 individuals in the control.Tis refected a substantial emergence inhibition rate of 91.88%.Meanwhile, at the highest concentration (20 μL/L), the studied EO exerted a profound inhibitory efect on the emergence of new individuals, resulting in a remarkable reduction of 99.36%.
Tese fndings underscore the potent impact of R. ofcinalis EO on the reproductive behavior and subsequent emergence of C. maculatus, suggesting its potential application as an efective biopesticide for controlling insect infestation in stored chickpea seeds.

Repellent Activity of Essential Oil against C. maculatus.
In addition to its biocidal efcacy, the repellent potential of R. ofcinalis EO against C. maculatus insects was systematically evaluated, and the results are listed in Table 3. Overall, the various concentrations of rosemary EO tested exhibited a moderate repellent efect, as per McDonald's (1970) classifcation [46].Te observed rate of repellence displayed a dose-dependent relationship, reaching a maximum of 80.00% after 30 minutes of exposure to the highest concentration of 0.315 μL/cm 2 EO.Te average percentage of repellence for the same duration of treatment was determined to be 50.83%.
Tese fndings highlight the noteworthy repellent properties of R. ofcinalis essential oil, signifying its potential utility in deterring and mitigating C. maculatus infestation.Te dose-dependent nature of the repellent efect further suggests the feasibility of utilizing varying concentrations of rosemary EO to optimize its repellent efcacy for efective insect management.
Te insecticidal activity of R. ofcinalis EO on Callosobruchus maculatus was studied in this work, revealing signifcant efects on this chickpea pest.Te results of these studies show a remarkable ability of rosemary essential oil to induce mortality in C. maculatus adults, while also afecting their fecundity and emergence.Inhalation and contact tests demonstrated a progressive increase in mortality with higher doses of EO and prolonged exposure times.For example, at lower concentrations, partial mortality was observed after 24 hours, while prolonged exposure resulted in total insect mortality.In addition, this study revealed that rosemary EO also had a signifcant impact on insect fecundity, reducing their ability to reproduce efectively.Te same EO also had a signifcant impact on insect fecundity, reducing their ability to reproduce efectively.Moreover, higher concentrations of the studied R. ofcinalis EO led to more rapid mortality, with complete elimination of insects within the frst few hours of exposure.

Scientifca
Our fndings align with Ainane et al. [47] research, which demonstrated a strong toxic efect of R. ofcinalis EO against T. confusum resulting in total insect mortality at a dose of 12 × 10 −2 μl/cm 3 after one day of exposure to the treatment.Similarly, recent studies reported the insecticide activity of the rosemary EO against many stored product pests, including C. maculatus, T. castaneum, S. granaries, S. oryzae, and T. confusum.In the same approach, investigations into L. dentata and O. compactum EOs belonging to the same family of R. ofcinalis (Lamiaceae) revealed signifcant toxic action against the studied C. maculatus insect marking notable LC 50 value of 4.01 μL/L air and 5.3 μL/L air, respectively [8,15].Tese same EOs exhibited complete inhibition (100%) of oviposition and emergence of the insect at higher concentrations (20 μL/L air).Generally, the insecticidal efect of R. ofcinalis EO against the tested pest can be attributed to its main components, in particular, camphor and eucalyptol (1,8-cineole), which showed strong insecticidal activity against S. zeamais and T. castaneum insects of stored legume and cereal seeds [36,37].In addition, the 1,8-cineole and R-(+)-limonene are insecticidal, mainly in the ingestion and/or contact test, against two insects (R. dominica and T. castaneum), which lead to signifcant economic losses of stored wheat grain [11].In the same sense, other reports have noted that EOs rich in eucalyptol or camphor are highly toxic for phytophagous insects in general [38,39].
Although this article did not investigate the precise mechanism of action of rosemary EO toxicity on insects, previous studies have looked at the efects of terpenoids, such as 1,8-cineole and camphor.Tese compounds were found to have an impact on the nervous system by inhibiting the activity of the enzyme acetylcholinesterase (AChE) in insects [48][49][50][51].Moreover, a study evaluating the efect of M. arvensis EO on S. granarius adults after contact reported rapid paralysis in addition to an altered walking behavior in the pests after treatment [19,36].Tis study also noted signifcant physiological changes caused by the essences of M. arvensis in the treated insects, resulting in an upregulation of various diferentially expressed proteins (DEPs).Tese proteins play a role in the development and functioning of the nervous and muscular systems, cellular respiration, protein synthesis, and detoxifcation [52].Tese results underline the considerable infuence of EO on various biological processes and elucidate the mechanisms utilized by surviving insects to recover from damage.
A remarkable decrease in both fecundity and emergence rates was evident in the C. maculatus insect, indicating the potent ovicidal and larvicidal efects of R. ofcinalis EO studied.Tis observed ovicidal efect of the EO tested could be attributed to the hindering of embryonic development following penetration of oil vapors into the eggs via the respiratory tract of the C. maculatus insect, as suggested by several researchers [53,54].Tis phenomenon is thought to be linked to the direct toxicity of EO components, which inhibit or limit metabolic activity inside the eggs.Tis efect was observed with compounds such as piperitone, isolated from C. schoenanthus EO tested on C. maculatus eggs [55], and ß-asarone, identifed in A. calamus EO applicated on C. chinensis, S. oryzae, and S. granarius eggs [56].Furthermore, research conducted on another bruchid species revealed variations in the sensitivity and vulnerability of eggs to the vapors of three EOs, including R. ofcinalis, depending on egg age and stage of embryonic development [57].Moreover, some investigations have highlighted the sterilizing efect of EOs on eggs [56].
Our investigation revealed a substantial decrease in emergence rates, evident even at the 1 μL/L dose of rosemary EO.Tis reduction might be attributable to the larvicidal efects of EO's monoterpenic compounds.A similar fnding was supported by the work of Kheloul and colleagues, who noted that young larvae (L 1 ) of T. confusum, a cereal seed pest, exhibited heightened sensitivity to the toxic impact of both L. spica EO and linalool (compound of the studied EO).Tey reported LC 50 values of 19.535 μL/L air and 14.198 μL/L air, respectively, after 24 hours of exposure time [58].Additionally, their study revealed that linalool induced greater egg mortality compared to L. spica EO at the same concentrations, consequently reducing the emergence of surviving adults, larvae, and pupae [58].
Te repellent efect observed with R. ofcinalis EO appeared moderate across all tested concentrations.Typically, the efectiveness of repellents derived from EOs is transient, largely owing to their volatility.In comparison, synthetic repellents often demonstrate greater efcacy and persistence than natural counterparts [59].Te duration of repellence exhibited by EOs is primarily infuenced by their chemical composition.Many monoterpenes like camphor, α-pinene, and cineole, commonly found in various EOs, have been highlighted in the literature for their signifcant mosquito-repellent properties, as reported by some researchers [60,61].
Overall, the fndings of the current study underline the strong insecticidal potential of the examined EO in the control of C. maculatus insect and suggest that it could be used as an efective alternative to synthetic chemicals in the protection of chickpea crops.However, further studies are needed to deepen our understanding of the mechanisms of action involved, including their impact on insect fecundity and emergence, as well as to assess the impact of these essential oils on other nontarget organisms and on the environment as a whole.
Given the known functions of EOs in suppressing fungi, bacteria, viruses, pests, and weeds, several studies have reported their potential applications in agriculture at low doses or in combination with conventional insecticides and fungicides [62].Recently, there have been promising results using nanoformulations or bioinformatics tools [63][64][65].However, their high volatility, low stability, poor water solubility, signifcant infuence on organoleptic properties, and phytotoxic efects remain obstacles to overcome in the hope of developing biofungicides and bioinsecticides derived from EOs [62].

In Silico Molecular Docking of Essential Oil's Antifungal and Insecticidal Activities.
In contemporary research endeavors, molecular modeling has become an essential tool, using computer-aided drug design (CADD) technologies to study and predict the interactions of various compounds, whether stable or volatile, such as essential oils (HE), with molecular targets linked to various biological activities [66].In the current research, molecular docking was employed to assess the inhibitory action of compounds present in R. ofcinalis EO against β-tubulin, a key component in the vitality of Botrytis cinerea.β-tubulin, in association with α-tubulin, forms microtubules, crucial components of the cytoskeleton.Microtubules play pivotal roles in cellular processes, including maintaining cell structure, facilitating cell division, and infuencing fungal virulence [21].In silico evaluation of the antifungal activity revealed that α-caryophyllene, β-caryophyllene, and verbenone, constituents of R. ofcinalis EO, exhibited signifcant antifungal activity against Botrytis cinerea β-tubulin (PDB: 3N2G), with scores of −7.025, −6.772, and −6.213 kcal/mol, respectively (Table 4).Tese in silico data confrm the antifungal properties of caryophyllene, as reported in previous studies [67].
On another front, acetylcholinesterase is a pivotal enzyme for the proper functioning of the insect nervous system.Consequently, its inhibition is a critical factor in achieving insecticidal efects.In silico data pertaining to R. ofcinalis EO indicated that borneol, camphor, and α-caryophyllene were the molecules displaying the highest efectiveness against acetylcholinesterase (PDB: 6ARY), with Glide scores of −7.254, −7.023, and −6.276 kcal/mol, respectively.Tese fndings afrm the insecticidal impact of several monoterpenes identifed in the studied rosemary EO and their infuence on the nervous system by inhibiting the activity of the acetylcholinesterase enzyme in various insects [49][50][51].Moreover, 2D and 3D viewers of R. ofcinalis EO docked in the active site of acetylcholinesterase (PDB: 6ARY) revealed that borneol established a single hydrogen bond with residue CYS 447 (Figures 9(a Previous studies have examined the way by which EOs act on insects, revealing in particular their impact on the enzyme acetylcholinesterase. Tis enzyme plays a key role in the insect nervous system, being responsible for the degradation of acetylcholine, a neurotransmitter crucial to the transmission of nerve impulses.Te action of EOs on acetylcholinesterase can disrupt this process, leading to neurological dysfunction in insects.Tis disruption can result in paralysis or rapid death of the insects, making it an important mechanism in the insecticidal activity of essential oils.However, it should be noted that EOs can also act on other biological targets, such as ion channels or respiratory systems [68][69][70].Our results indicate that the molecule with the highest afnity for acetylcholinesterase is α-caryophyllene.Other experimental and theoretical works on other insects have also confrmed the antiacetylcholinesterase efect of α-and β-caryophyllene [71,72].Other compounds, such as 1,8-cineole [73][74][75],     α-pinene [76], and δ-and c-cadinene [77], present in EOs, have also demonstrated acetylcholinesterase-inhibiting activity.EOs composed of a mixture of bioactive molecules, mainly sesquiterpenes and monoterpenes, appear to have a more pronounced acetylcholinesterase inhibitory efect.It is possible that the synergistic combination of these molecules is responsible for their inhibitory action.Tese molecular docking results provide valuable insights into the potential mechanisms underlying the antifungal and insecticidal activities of specifc compounds in Rosmarinus ofcinalis EO, contributing to a comprehensive understanding of their bioactive properties at the molecular level.

Conclusion
In summary, the results unequivocally demonstrate that the EO derived from Moroccan Rosmarinus ofcinalis exhibits robust antifungal and insecticidal properties.Tese experimental fndings are corroborated by in silico analyses, which confrm the inhibitory efect of rosemary EO on insect acetylcholinesterase and fungal β-tubulin.Accordingly, these conclusions underscore its potential utility as an effcacious antifungal and bioinsecticidal agent for agricultural crops and the storage of leguminous products.Te observed biological activities can be attributed, at least in part, to the presence of monoterpene compounds within the EO.Te multifaceted benefts of Rosmarinus ofcinalis EO, as elucidated through this study, provide a solid foundation for further exploration and development of its practical applications in crop protection and storage management.

Figure 1 :Figure 2 :Figure 3 :Figure 4 :
Figure 1: Mycelial growth of A. alternata with time, following treatment with two concentrations of R. ofcinalis EO.

Figure 5 :Figure 6 :
Figure 5: Mortality rates (means ± SD) of C. maculatus adults when exposed to diferent concentrations of R. ofcinalis EO in a contact toxicity test.Value bars with diferent letters are signifcantly diferent (p < 0.05).
) and 10(a)).Similarly, camphor also formed a hydrogen bond with the same residue in the same active site (Figures9(b) and 10(b)).

Figure 9 :
Figure 9: Te 2D viewer of ligand interactions with the active site.(a, b) Borneol and camphor interactions with the active site of acetylcholinesterase; (c, d) α-caryophyllene and β-caryophyllene interactions with active site of sterol Botrytis cinerea β-tubulin.

Figure 10 :
Figure 10: Te 3D viewer of ligand interactions with the active site.(a, b) Borneol and camphor interactions with the active site of acetylcholinesterase; (c, d) α-caryophyllene and β-caryophyllene interactions with the active site of Botrytis cinerea β-tubulin.
Te present investigation employed GC-MS to meticulously discern the diverse phytochemical constituents inherent in the EO derived from R. ofcinalis.Te analytical protocol entailed the utilization of a GC Agilent Technologies 6890 N Network gas-phase chromatograph, featuring an HP-5MS capillary 2 Scientifca column (30.0 m × 0.250 mm × 0.250 μm flm thickness), strategically situated in Little Falls, CA, USA.Te fame ionization detector (FID) employed in this study was judiciously set at a precise temperature of 250.0 °C.Te injection of a 1.00 μL volume was executed with meticulous care, adopting a split mode at the designated temperature of 250.0 °C.
[15,16]nally, NIST MS Library (v.2.0) was judiciously employed as the mass spectral database to enhance the precision of compound identifcation.Trough the integration of these sophisticated analytical methodologies, the investigation achieved a comprehensive and precise characterization of the phytochemical profle within R. ofcinalis EO[15,16].2.4.Antifungal Activity of Essential Oil2.4.1.Culture Conditions and Strains Used.In the context of this research endeavor, three strains of flamentous fungi, namely, Botrytis cinerea, Fusarium oxysporum, and Alternaria alternata, were purposefully selected for scrutiny.

Table 1 :
Phytochemical compounds of the EO extracted from R. ofcinalis.

Table 2 :
Lethal concentrations of LC 50 and LC 95 (μL/L of air) induced mortality in C. maculatus adults in contact and inhalation toxicity tests after 24 h of treatment with R. ofcinalis EO.

Table 3 :
[46]lts of the repellent activity of R. ofcinalis EO against C. maculatus after 30 min of treatment.Class of repellent efect according to the classifcation of McDonald et al.[46]. *