Antifungal Activity of Garlic (Allium sativum L.) Essential Oil and Hydrosol Against Zymoseptoria tritici, the Causal Agent of Septoria Tritici Blotch in Bread Wheat

Antifungal Activity of Garlic (Allium sativum L.) Essential Oil and Hydrosol Against Zymoseptoria tritici, the Causal Agent of Septoria Tritici Blotch in Bread Wheat | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Antifungal Activity of Garlic (Allium sativum L.) Essential Oil and Hydrosol Against Zymoseptoria tritici, the Causal Agent of Septoria Tritici Blotch in Bread Wheat Roumeissa Djerboua, Laid Benderradji, Ibtissem Sanah, Fairouz Djeghim, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8612946/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Septoria tritici blotch (STB), caused by Zymoseptoria tritici , is one of the most damaging foliar diseases of bread wheat ( Triticum aestivum L.), leading to severe yield losses and increasing reliance on synthetic fungicides. The emergence of fungicide-resistant populations and growing environmental concerns highlight the need for sustainable alternative control strategies. In this study, the antifungal activity of essential oil (EO) and hydrosol obtained from a local Algerian red garlic ( Allium sativum L.) variety was evaluated in vitro against Z. tritici. Garlic extracts were characterized through morphological, physicochemical, and chemical analyses, with EO volatile composition determined by GC–MS. Twenty-one compounds accounting for 99.97% of the total EO composition were identified, dominated by organosulfur compounds, particularly diallyl trisulfide (36.99%) and diallyl disulfide (35.78%). Antifungal assays revealed a strong dose-dependent inhibition of mycelial growth. Garlic EO completely inhibited fungal growth at 0.30 µL mL⁻¹, although a reduction in efficacy over time suggested limited stability. Notably, garlic hydrosol achieved complete and stable inhibition at 150 µL mL⁻¹, despite its lower sulfur content, representing a novel and underexplored approach for STB control. These findings demonstrate the potential of garlic-derived EO and hydrosol as eco-friendly antifungal agents and support their possible integration into sustainable disease management strategies for wheat, contributing to the reduction of chemical fungicide inputs. Zymoseptoria tritici Septoria tritici blotch antifungal activity garlic essential oil garlic hydrosol sustainable plant protection Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Septoria tritici blotch (STB), caused by the heterothallic ascomycete Zymoseptoria tritici (syn. Septoria tritici or Mycosphaerella graminicola ), is one of the most destructive foliar diseases affecting bread wheat ( Triticum aestivum L.) worldwide. Severe epidemics can result in yield losses of 35–50%, with high incidence levels reported in Algeria, where STB was detected in 80% of surveyed wheat fields across 11 localities between 2010 and 2012 (Allioui et al. 2021 ). Owing to its global distribution and economic impact, Z. tritici is ranked among the ten most devastating plant pathogenic fungi (Damiens et al. 2021 ). The pathogen follows a hemibiotrophic infection strategy, initiating infection with an asymptomatic biotrophic phase followed by a necrotrophic phase characterized by extensive leaf necrosis (Platel et al. 2022 ). Bread wheat ( Triticum aestivum L.) is one of the most important staple crops worldwide and a fundamental component of global food security, particularly in developing regions. Global demand for wheat is projected to increase by nearly 60% by 2050 to meet the nutritional needs of a growing population (Atwa et al. 2025 ). In Algeria, wheat plays a strategic role in national food security as both a dietary cornerstone and a central focus of agricultural policy (Ouzani et al. 2025 ). However, wheat production is increasingly threatened by biotic stresses, particularly fungal diseases, which significantly reduce grain yield and quality (Suarez-Fernandez and De Francesco 2024 ). Current management of STB relies heavily on repeated applications of synthetic fungicides. Nevertheless, their intensive use has raised serious concerns related to environmental contamination, phytotoxic effects, risks to human health, and the rapid emergence of fungicide-resistant Z. tritici populations (Sahli et al. 2018 ; Allioui et al. 2021 ). These limitations highlight the urgent need for sustainable, safe, and locally sourced alternatives for effective wheat disease control. In this context, increasing attention has been directed toward bio-based disease management strategies. Plant-derived products, including essential oils (EOs) and hydrosols, have emerged as promising alternatives to conventional fungicides due to their biodegradability, low toxicity, and broad-spectrum antimicrobial activity (Okorska et al. 2023 ). Among these, garlic ( Allium sativum L.) extracts are particularly notable for their strong antifungal properties, largely attributed to sulfur-containing compounds such as allicin, S-allyl cysteine, thiosulfates, and other organosulfur derivatives (Lawson et al. 1991 ; Verma et al. 2023 ). In Algeria, garlic cultivation has steadily increased, providing an abundant and locally available resource for the development of plant-based antifungal agents. Beyond their biological activity, the characterization of garlic germplasm is essential for identifying varieties best adapted to specific agro-ecological conditions and for maximizing the yield of bioactive metabolites (Salahuddin et al. 2019 ). Morphological characterization supports breeding programs, diversity assessments, and genotype selection, which are crucial since antifungal efficacy can be influenced by varietal origin (Panthee et al. 2006 ; Ragas et al. 2019 ). Despite numerous studies reporting the antimicrobial potential of garlic EO, comprehensive physicochemical characterization—particularly of garlic hydrosol, a by-product of hydrodistillation—remains limited. To date, no study has reported the combined antifungal activity of both garlic EO and hydrosol against Z. tritici , nor has the hydrosol obtained from a locally cultivated Algerian garlic variety been characterized. The present study aimed to address this knowledge gap by investigating the antifungal potential of essential oil and hydrosol derived from a local Algerian red garlic variety against Z. tritici , the causal agent of STB. Specifically, we (i) conducted morphological characterization of the garlic genotype, (ii) performed physicochemical analyses of EO and hydrosol, (iii) identified the volatile constituents of EO using gas chromatography–mass spectrometry (GC–MS), and (iv) assessed the temporal stability of EO antifungal activity. This multidisciplinary approach seeks to contribute to the development of bio-based, eco-friendly, and locally available solutions for sustainable wheat disease management. 2. Materials and Methods 2.1 Plant Material Garlic ( Allium sativum L.) bulbs of a local red variety (RL) were used in this study. The plants were cultivated under open-field conditions and harvested in July 2021 from a non-irrigated experimental plot at the Debbah Pilot Farm, located in Didouche Mourad in northern Constantine, Algeria. The site is situated at an elevation of 500 m above sea level (36°29′26″ N, 6°37′14″ E) and is characterized by a semi-arid Mediterranean climate. During the 2020–2021 growing season, the total annual rainfall was 313.97 mm, with mean air temperatures ranging from 8.2°C to 28°C and relative humidity between 36.2% and 75%. Bulbs were harvested at full maturity, air-dried under ambient conditions, and stored in a dark, ventilated environment at room temperature until further use. 2.2 Fungal Strain A pathogenic strain of Zymoseptoria tritici (teleomorph: Mycosphaerella graminicola ), the causal agent of Septoria tritici blotch (STB) in bread wheat, was employed for antifungal bioassays. The isolate was originally obtained from infected wheat ( Triticum aestivum L.) leaves collected from experimental fields and was kindly provided by the National Institute of Agronomic Research of Algeria (INRAA). The strain had been previously identified based on its morphological and cultural characteristics and maintained as part of the INRAA fungal collection. Colonies were cultured on potato dextrose agar (PDA) and incubated at 20°C in the dark to preserve their pathogenicity prior to experimental use. 2.3. Morphological Characterization of Plant Material Morphological traits of the garlic ( Allium sativum L.) bulbs were assessed following the official descriptor for Allium sativum published by the International Union for the Protection of New Varieties of Plants (UPOV 2001 ). A comprehensive set of qualitative traits was evaluated, including: bulb size; bulb shape in longitudinal and cross sections; position of cloves at the tip of the bulb; clove distribution; external clove morphology; compactness of cloves; position of the root disc; shape of the bulb base; ground color; presence and intensity of anthocyanin stripes; thickness and adherence of the outer dry scales; clove size; clove color and color intensity; anthocyanin stripes on clove scales; flesh color; and time of harvest maturity. Quantitative traits measured included the number of bulbs per kilogram, average bulb weight, bulb diameter (cm), number of cloves per bulb, and average clove weight. All morphological evaluations were conducted on a representative sample of 30 garlic bulbs randomly selected from the harvested batch to ensure statistical reliability. 2.4. Essential Oil and Hydrosol Extraction Essential oil (EO) and hydrosol were extracted from bulbs of the local red garlic ( Allium sativum L.) variety under industrial-scale conditions using the hydrodistillation with a Clevenger- type apparatus for 2 h 30 min under controlled conditions. The extraction was performed at the private distillation facility “Arom’Est,” specialized in essential and vegetable oil production, located in Annaba, Algeria. Fresh garlic cloves were peeled, chopped into small pieces, and placed in a stainless-steel industrial distillation unit with distilled water. The hydrodistillation process was carried out. Upon completion, the EO and hydrosol fractions were separated based on their density differences using a separating funnel. Both extracts were stored in amber glass bottles at 4°C in the dark until further analyses to prevent degradation. The EO yield (%) was calculated using the following Eq. 1 : $$\:\mathbf{E}\mathbf{o}\:\mathbf{Y}\mathbf{i}\mathbf{e}\mathbf{l}\mathbf{d}\:\left(\mathbf{\%}\right)=\left(\frac{\mathbf{w}\mathbf{e}\mathbf{i}\mathbf{g}\mathbf{h}\mathbf{t}\:\mathbf{o}\mathbf{f}\:\mathbf{e}\mathbf{x}\mathbf{t}\mathbf{r}\mathbf{a}\mathbf{c}\mathbf{t}\mathbf{e}\mathbf{d}\:\mathbf{o}\mathbf{i}\mathbf{l}}{\mathbf{w}\mathbf{e}\mathbf{i}\mathbf{g}\mathbf{h}\mathbf{t}\:\mathbf{o}\mathbf{f}\:\mathbf{g}\mathbf{a}\mathbf{r}\mathbf{l}\mathbf{i}\mathbf{c}}\right)\times\:100$$ 1 2.5. Physicochemical and Organoleptic Characterization of Garlic Extracts The physicochemical properties of garlic essential oil (EO) and hydrosol were analyzed in accordance with the standards of the European Pharmacopoeia, the International Organization for Standardization ISO 3061:2008. Organoleptic Analysis : the organoleptic characteristics of EO and hydrosol were evaluated assessing appearance, color, and odor. Physicochemical Analysis of Essential Oil : t he refractive index was measured at 20°C using a REICHERT AR6 automatic refractometer previously calibrated with distilled water. Relative density was determined with a METTLER TOLEDO Densito 30 PX electronic densimeter. The acid value was quantified by titration with 0.05 N alcoholic KOH according to and calculated using standard equations. Optical rotation was determined following ISO 592:1998 using a 10 mL polarimeter tube filled with a dilute EO–ethanol solution. The pH of EO samples was measured at room temperature using pH indicator strips. Physicochemical Analysis of Hydrosol : several parameters were measured to assess the composition and potential bioactivity of the garlic hydrosol. Turbidity was determined at 25 ± 2°C using a Lovibond® infrared turbidimeter. Electrical conductivity was measured with a Jenway 4510 conductivity meter after electrode stabilization. Viscosity was determined using a VISCO TM-895 rotational viscometer Density was measured with a standard hydrometer, reading specific gravity directly from a graduated cylinder. The pH was determined using a calibrated digital pH by immersing the electrode directly in the sample until the reading stabilized. 2.6. Gas Chromatography–Mass Spectrometry (GC–MS) Analysis The chemical composition of the garlic essential oil (EO) was determined using a gas chromatograph (Agilent 6890 Plus, Hewlett Packard, USA) coupled to a mass spectrometer (Agilent 5973, Hewlett Packard, USA). Separation was achieved on a HP-5MS capillary column (30 m × 0.25 mm i.d., 0.25 µm film thickness) coated with 5% phenyl-95% dimethylpolysiloxane as the stationary phase. The injector temperature was set at 250°C, and samples (0.2 µL) were injected in split mode (1:20). High-purity helium (N6.0) was used as the carrier gas at a constant flow rate of 0.5 mL min⁻¹. The oven temperature program was as follows: initial temperature 60°C (held for 8 min), increased at 2°C min⁻¹ to 250°C, and then held isothermally for 10 min, for a total run time of 113 min. The mass spectrometer operated in total ion current (TIC) scan mode over an m/z range of 30–550. Ionization was performed by electron impact (EI) at 70 eV. The ion source and interface temperatures were maintained at 230°C and 280°C, respectively. A solvent delay of 3.5 min was applied. The quadrupole mass analyzer was used for detection. Identification of EO constituents was achieved by comparing their mass spectra and retention times with those available in the NIST and Wiley mass spectral libraries. Additionally, Kovats retention indices (RI) were calculated relative to a homologous series of n -alkanes (C 8 –C 29 ) analyzed under the same chromatographic conditions, and the results were compared with published literature data to confirm compound identity. 2.7. Antifungal Activity Assay The antifungal activity of garlic essential oil (EO) and hydrosol against Zymoseptoria tritici was evaluated at the Biotechnology Research Center (CRBT), Constantine, Algeria, following the protocols described by Hammer et al ( 1999 ), with minor modifications. Appropriate volumes of EO and hydrosol were first dissolved in a maximum of 1 mL of dimethyl sulfoxide (DMSO) and then added to 100 mL of sterile potato dextrose agar (PDA) medium (after autoclaving and cooling to ~ 45°C) to obtain the following final concentrations: 0.10, 0.15, 0.22, and 0.30 µL/mL for EO, and 50, 100, 150, and 200 µL/mL for hydrosol. Each treatment was poured into four replicate Petri dishes. Two control treatments were included: (i) positive control – PDA medium supplemented with 1 mL of DMSO (final DMSO concentration ≤ 1%); and (ii) negative control – PDA medium without any additives. After medium solidification, each plate was inoculated with 5 µL of a 1 × 10⁶ spores/mL suspension of Z. tritici and incubated in the dark at 20°C for 10 days. Antifungal efficacy was expressed as the percentage of growth inhibition (I%) relative to the control, calculated according to Eq. 2 $$\:\mathbf{I}=\left(\frac{\mathbf{C}-\mathbf{T}}{\mathbf{C}}\right)\times\:100$$ 2 Where: C (mm) = radial growth of the pathogen on control plates; T (mm) = radial growth on treatment plates. 2.8. Stability Assay of Garlic Essential Oil To assess the short-term stability of garlic essential oil (EO) and its persistence in inhibiting Zymoseptoria tritici growth, an additional experiment was performed using the same methodology described for the antifungal activity assay (Section 2.7 ). The experiment was designed to evaluate the effect of different time intervals between EO incorporation into the medium and fungal inoculation. Three experimental groups were established: Group 1 (Day 0): Inoculation was performed immediately after the solidification of the EO-supplemented PDA medium. Group 2 (Day 3): Inoculation was delayed by three days. During this period, the EO-containing plates were stored under sterile conditions at room temperature. Group 3 (Day 6): Inoculation was delayed by six days, with plates maintained under the same conditions. Each plate was inoculated with 5 µL of a Z. tritici spore suspension (1 × 10⁶ spores/mL) and incubated in the dark at 20°C for 10 days. All treatments were performed in triplicate. Fungal growth was evaluated by measuring radial colony expansion, and the percentage of growth inhibition was calculated according to the formula described in Section 2.7 . 2.9. Statistical Analysis All data obtained in this study were subjected to statistical analysis at a significance level of p < 0.05. Descriptive statistics (mean, minimum, maximum, and coefficient of variation, CV) were first calculated for morphological and physicochemical traits to evaluate variability within the garlic samples. The antifungal activity of the essential oil (EO) was analyzed using a two-way analysis of variance (ANOVA) to assess the effects of concentration and treatment duration, while the antifungal activity of the hydrosol was evaluated using a one-way ANOVA. When significant differences were detected, Fisher’s least significant difference (LSD) post-hoc test was applied to compare mean values. All statistical analyses were performed using JMP Trial 17 software (SAS Institute Inc., Cary, NC, USA). 3. Results 3.1. Morphological Characterization Bulb and clove traits are the principal economic organs of garlic ( Allium sativum L.), widely used for culinary, medicinal, and propagation purposes (Ragas et al. 2019 ). They also serve as fundamental selection criteria in breeding programs aimed at improving yield, quality, and adaptability (Panthee et al. 2006 ). In this study, morphological characterization was conducted to define the specific phenotypic features of the local Algerian red garlic variety (RL) (Fig. 1 ), for which limited information is currently available in the literature. 3.1.1 Quantitative Traits The quantitative morphological characteristics of the RL variety are summarized in Table 1 . The number of bulbs per kilogram varied from 40 to 55, with an average of 47.35 ± 3.81, while the mean bulb dry weight was 25.52 ± 6.35 g. Bulb diameter averaged 3.7 ± 0.2 cm, and the number of cloves per bulb ranged from 9 to 17. The average clove dry weight was 2.17 ± 0.67 g. The coefficients of variation (CV) for the five traits ranged from 0.05% to 24.91%. The highest variability was observed for bulb dry weight (24.91%), indicating considerable heterogeneity for this trait (Shrestha et al. 2022 ). In contrast, the number of cloves per bulb (14.28%), bulbs per kilogram (8.05%), clove dry weight (0.31%), and bulb diameter (0.05%) exhibited lower variability (CV < 20%), indicating relative uniformity within the population. These results are in line with those reported (Boukeria 2016 ) for the same variety and are within the range described by Pasupula et al ( 2024 ), and Popa and Cosmulescu ( 2024 ) across diverse garlic germplasm. Such differences in morphological traits are often attributed to genetic variation as well as environmental influences including soil type, climatic conditions, and agronomic practices (Panthee et al. 2006 ; Akan 2022 ; Baswarsiati et al. 2024 ). Bulb weight, in particular, is a key agronomic trait that significantly influences garlic yield, often correlating with clove size and planting material quality (Popa and Cosmulescu 2024 ). Table 1 Quantitative morphological traits of the local Algerian red garlic (RL) variety Quantitative parameters RL variety (mean ± SD) Min Max CV (%) Number of bulbs per kg 47.35 ± 3.81 40 55 8.05 Bulb dry weight (g) 25.52 ± 6.35 10.8 37.89 24.91 Bulb diameter (cm) 3.70 ± 0.20 3.5 3.9 0.05 Number of cloves per bulb 13.10 ± 1.87 9 17 14.28 Clove dry weight (g) 2.17 ± 0.67 1.29 3.26 0.31 Values are expressed as mean ± standard deviation (SD) along with minimum (Min), maximum (Max), and coefficient of variation (CV%) values. 3.1.2 Qualitative Traits Phenotypic observations of qualitative morphological traits are shown in Fig. 1 and summarized in Table 2 . The RL variety produced small- to medium-sized bulbs with white to yellowish outer scales lacking anthocyanin stripes, while the inner scales exhibited pink to red pigmentation, supporting its classification as a “red” variety. The bulb base was flat, and the shape in longitudinal section was transverse broad elliptic, while the cross-section was elliptic. Cloves were radially arranged, with no external cloves, and exhibited a compact configuration. The root disc was flat, and clove scales were pink to purple with strong pigmentation and anthocyanin stripes, whereas the clove flesh was white to yellowish. The variety showed late dormancy and late harvest maturity. These findings are consistent with (Boukeria 2016 ) and highlight distinct phenotypic features, particularly clove arrangement and pigmentation, which can aid in germplasm identification and breeding (Panthee et al. 2006 ; Akan 2022 ). Such qualitative traits are critical for marketability and consumer preference (Baswarsiati et al. 2024 ) and play a vital role in genotype differentiation. The absence of external cloves, often associated with reduced commercial quality (Kıraç et al. 2022 ), further enhances the market potential of the RL variety. Table 2 Qualitative morphological characteristics of bulb and cloves of the local red (RL) garlic variety Parameters RL Variety Bulb size Small – Medium Bulb shape in longitudinal section Transverse broad elliptic Bulb shape in cross section Elliptic Position of cloves at tip of bulb At same level Distribution of cloves Radial External cloves Absent Compactness of cloves Compact Position of root disc Flat Shape of bulb base Flat Ground color of dry external scales White–yellowish Anthocyanin stripes on bulb dry external scales Absent Thickness of dry external scales Medium Skin adherence of dry bulb external scales Medium Clove size Medium Color of clove scale Pink–purple Intensity of color of clove scale Strong Anthocyanin stripes on clove scale Present Color of flesh White–yellowish End of dormancy of clove in bulb Late Time of harvest maturity Late Phenotypic traits were assessed following the UPOV ( 2001 ) and IPGRI (2001) descriptors for Allium sativum . 3.2. Essential Oil Yield The essential oil (EO) yield obtained from the local Algerian red garlic ( Allium sativum L.) variety in this study was 0.075% (w/w). This value is comparable to that reported by (Shalaby et al. 2011 ) for an Egyptian garlic cultivar (0.073%) extracted using the same hydrodistillation method. Similarly, Nazzaro et al ( 2022 ) documented yields ranging from 0.03% to 0.08% across two different cultivars, with our results being closer to the upper end of this range. However, the yield reported here is lower than those observed in several other studies. For instance, Sarangi et al ( 2024 ) reported a yield of 0.17%, while Boukeria ( 2016 ) documented yields of 0.46%, 0.61%, and 0.51% for the Messidrom, Germidour, and Mocpta Bulguar varieties, respectively, and even reported a maximum yield of 0.72% for the same Local Red variety analyzed in this work. Variability in EO yield among garlic varieties is influenced by a combination of genetic, physiological, methodological, and environmental factors. According to some authors (Mugao 2004 ; Ezeorba et al. 2022 ), several parameters, including the plant organ used for extraction, harvest period, post-harvest handling, drying method, plant maturity stage, and extraction conditions, can significantly impact EO yield. Moreover, genotypic variation, plant density, and cultivation practices (e.g., irrigation and fertilization) further modulate EO biosynthesis and accumulation. Environmental conditions during the 2020–2021 growing season likely contributed to the relatively low yield reported in this study. The experimental site experienced a total annual rainfall of 313.97 mm and relative humidity as low as 36.2%, conditions that may have imposed moderate abiotic stress and limited the biosynthetic potential of secondary metabolites. These findings underscore the importance of optimizing both agronomic practices and environmental conditions to enhance EO yield in future cultivation and extraction strategies. 3.3. Physicochemical and Organoleptic Characterization of Garlic Extracts The physicochemical characterization of essential oils (EOs) and hydrosols is a critical step for assessing their functional properties, standardizing quality control, and determining their suitability for various applications in food, cosmetics, pharmaceuticals, and agricultural formulations (Barkatullah et al. 2012 ; Belkhodja et al. 2021 ). The key organoleptic and physicochemical properties of garlic EO and hydrosol are summarized in Tables 3 and 4 , respectively. 3.3.1 Organoleptic Properties The garlic EO obtained from the local red variety exhibited a liquid consistency and a yellow coloration, typical of extracts rich in volatile organosulfur compounds (Oliveira et al. 2024 ).Its pungent, intense, and characteristic odor is primarily attributed to diallyl sulfides and related sulfurous constituents (Borlinghaus et al. 2021 ). In contrast, the hydrosol presented as a colorless to very pale yellow aromatic water with a less intense odor, reflecting its lower volatile content (generally < 0.1% w/v) (Aćimović et al. 2020 ). This compositional difference aligns with expectations, as hydrosols primarily consist of water-soluble compounds and trace amounts of EO constituents. 3.3.2 Physicochemical Properties of Essential Oil The refractive index (RI) of the garlic EO was 1.51 ± 0.00, which falls within the range reported in previous studies (1.43–1.52) (Rafe and Nadjafi 2014 ; Dehariya et al. 2021 ). RI is strongly influenced by oil composition, particularly the degree of unsaturation, the cis/trans isomer ratio, and the abundance of oxygenated monoterpenes. A relatively high RI value suggests the presence of heavier or more polar compounds, which may enhance the bioactivity and stability of the oil (Boukeria, 2016 ). The acid value was 6.62 ± 0.10 mg/g, significantly higher than those reported by Lemma et al ( 2022 ) (1.54 ± 0.30 mg/g) and El Faqer et al ( 2024 ) (1.86 ± 0.10 mg/g). Acid value is an established indicator of oil quality and hydrolytic stability; values below 2 mg/g generally indicate good preservation and low levels of free fatty acids (Lemma et al. 2022 ). The elevated acid value in our sample suggests partial degradation, likely due to hydrolysis of ester bonds during storage or sample handling. The optical rotation was 0° ± 0.00, consistent with a racemic or balanced mixture of chiral molecules. This result is consistent with commercial garlic oils (typically − 2° to + 5°) and reflects the optical purity and stereochemical composition of the extract (Do et al. 2015 ) Optical rotation is a useful analytical parameter for detecting adulteration and confirming geographical or varietal origin. The density of the garlic EO was 1.10 ± 0.02 g/cm³, higher than values reported by Dehariya et al ( 2021 ) (0.873 ± 0.003 g/cm³) and El Faqer et al ( 2024 ) (0.978 ± 0.03 g/cm³), but comparable to the range (1.025–1.029 g/cm³) observed by Boukeria et al ( 2016 ). Density correlates with the molecular weight distribution and polarity of oil constituents; values above 1.0 g/cm³ suggest the presence of relatively heavy or complex sulfur-containing molecules. This property also explains the EO’s tendency to settle below the aqueous phase during separation. The pH of the EO was 5.00 ± 0.00, indicating a slightly acidic nature consistent with previous reports (Boukeria et al. 2016 ). This property is relevant for determining the oil’s stability and compatibility with certain formulations. 3.3.3 Physicochemical Properties of Hydrosol The physicochemical analysis of garlic hydrosol provides original data not previously reported in the literature and contributes to a deeper understanding of its compositional and functional profile. The turbidity was 20.22 ± 0.34 NTU, indicating a moderate level of suspended particulates, likely composed of microdroplets of aromatic compounds or colloidal organic matter. Although slight turbidity is typical of plant hydrosols, its magnitude may serve as a useful indicator of volatile content and process efficiency. The viscosity averaged 3.37 ± 0.06 mPa·s, a value slightly higher than that of pure water, suggesting the presence of dissolved phytochemicals or colloidal components. This parameter is relevant for understanding hydrosol structure and its behavior in formulations (Barkatullah et al. 2012 ). The electrical conductivity (EC) was 198.25 ± 0.5 µS, reflecting a moderate concentration of ionizable solutes, such as sulfur-containing compounds and trace minerals, released during hydrodistillation (ISO 7888:1985). These findings are consistent with previously reported conductivity ranges for botanical hydrosols (25.2–730.0 µS) (Acheampong et al. 2015 ). Finally, the pH of the hydrosol was 7.51 ± 0.03, indicating a slightly alkaline nature, and its density was 1.00 ± 0.00 g/mL, closely matching that of water. As noted by Tavares et al ( 2022 ) hydrosols generally exhibit densities near 1.0 g/mL due to their low solute concentration. Table 3 Organoleptic characteristics of garlic essential oil and garlic hydrosol Extract Garlic essential oil Garlic hydrosol Appearance Liquid Liquid Color Yellow Colorless to very pale yellow Odor Strong, pungent, characteristic Less intense, characteristic Table 4 Physicochemical properties of garlic ( Allium sativum L.) essential oil and hydrosol Garlic essential oil Mean ± SD CV (%) Garlic hydrosol Mean ± SD CV (%) Refractive index at 20°C 1.51 ± 0.00 0.11 Turbidity (NTU) 20.22 ± 0.34 1.68 Acid value (mg/g) 6.62 ± 0.10 1.44 Viscosity (mPa·s) 3.37 ± 0.06 1.78 Optical rotation (°) 0.00 ± 0.00 0.00 Conductivity (µS) 198.25 ± 0.50 0.25 Density (g/cm³) 1.10 ± 0.02 1.58 Density (g/mL) 1.00 ± 0.00 0.00 pH 5.00 ± 0.00 0.00 pH 7.51 ± 0.03 0.41 Data are presented as mean ± standard deviation (SD, n = 3). CV (%) = coefficient of variation calculated as (SD/mean) × 100. 3.4. Gas Chromatography–Mass Spectrometry (GC–MS) Analysis The chemical composition of the essential oil (EO) obtained from the local Algerian red garlic ( Allium sativum L.) variety, along with the retention times (Rt) and relative abundances of the identified compounds, is presented in Table 5 . The corresponding chromatographic profile is shown in Fig. 2 GC–MS analysis allowed the identification of 21 volatile compounds, representing 99.97% of the total EO composition. The EO profile was dominated by organosulfur compounds, which accounted for 83.32% of the total composition. The principal constituents were diallyl trisulfide (36.99%) and diallyl disulfide (35.78%), followed by diallyl sulfide (5.49%) and methyl allyl disulfide (2.65%). Other sulfur-based volatiles present in lower concentrations included dimethyl trisulfide (0.55%), 2-vinyl-4H-1,3-dithiin (1.23%), 2-methyl-1,3-dithiolane (0.46%), and 1,3-dithiane (0.17%). These results are consistent with previous reports on garlic EO composition (Jan et al. 2012 ; Molina-Calle et al. 2016 ). Similarly, Sarangi et al ( 2022 ) reported diallyl trisulfide (37%) and diallyl disulfide (25.9%) as the main constituents, while Herrera-Calderon et al ( 2021 ) found diallyl trisulfide (44.21%) and diallyl disulfide (22.08%) as major components. The relative abundance of these compounds tends to vary with factors such as genotype, growth stage, post-harvest handling, drying conditions, and extraction parameters, all of which can influence the volatile profile (Modafferi et al. 2025 ). The predominance of sulfur-containing compounds observed in the EO of Algerian red garlic aligns with its well-documented biological functionality. These volatile organosulfur molecules are widely recognized for their antimicrobial, antifungal, antioxidant, cardioprotective, anti-atherosclerotic, immunomodulatory, and antihypertensive activities (Gong et al. 2021 ; Ozoude and Chukwu 2025 ). Notably, diallyl disulfide and diallyl trisulfide have been extensively reported as key bioactive compounds contributing to garlic’s fungistatic and fungicidal activity, as well as its capacity to modulate oxidative stress and inflammatory responses. Their prevalence in the EO composition thus underlines the potential of this extract as a natural biocontrol agent for plant pathogens such as Zymoseptoria tritici . Table 5 Volatile composition of essential oil from the local Algerian red garlic ( Allium sativum L.) determined by GC–MS analysis Peak Compound Rt (min) RI Relative abundance (%) 1 2-Phenyl-4,6-di(4-acetylaminophenyl)pyrimidine 4.705 925 0.122 2 Acetonitrile, 2-(5-chloro-3-oxo-3H-1,2-dithiol-4-yl)propylamino 5.065 940 0.252 3 Diallyl sulfide (1-Propene, 3,3′-thiobis) 5.390 960 5.485 4 Methyl allyl disulfide 7.779 1008 2.654 5 1,3-Dithiane 8.894 1025 0.168 6 2-Hydroxy-3-methoxy-succinic acid, dimethyl ester 10.220 1045 0.125 7 Dimethyl trisulfide 10.740 1063 0.547 8 Diallyl disulfide 18.907 1172 35.778 9 Benzene, 1,3-dichloro 19.382 1186 1.085 10 Hexopyranosid-3-ulose, methyl 2,4,6-tris-O-(trimethylsilyl)-, dimethyl acetal 19.942 1200 3.225 11 Heptane, 4-methoxy-3-(methoxymethyl) 22.794 1245 8.252 12 1H-Imidazole, 1-phenyl 26.149 1288 0.634 13 Benzenamine, 3,5-dinitro 27.018 1300 0.323 14 2-Vinyl-4H-1,3-dithiin 28.144 1318 1.230 15 Diallyl trisulfide (Trisulfide, di-2-propenyl) 34.602 1395 36.990 16 Tetramethyl 1,1′-(1,8-naphthylene) bis(1,2,3-triazole-4,5-dicarboxylate) 34.854 1402 0.171 17 7-Amino-7H-S-triazolo[5,1-c]-S-triazole-3-thiol 38.597 1438 0.410 18 2-Methyl-1,3-dithiolane 39.106 1445 0.460 19 tert-Butyl isopropyl disulfide, perfluoro 49.119 1540 1.729 20 2-Docosenoic acid, 2,4,21,21-tetramethyl-, methyl ester, (E) 62.231 1670 0.021 21 3-Iodo-4-methoxy-phenyl)-morpholin-4-yl-methanethione 75.039 1782 0.249 Retention time (Rt), retention index (RI), and relative abundance (%) of individual compounds were determined by GC–MS. Compound identification was based on comparison of mass spectra with NIST and Wiley libraries and confirmed by Kovats retention indices calculated relative to a homologous series of n-alkanes (C 8 –C 29 ). Compounds are listed in order of elution. The chromatogram illustrates the volatile composition of the essential oil, highlighting the predominance of sulfur-containing compounds such as diallyl trisulfide (peak 1) and diallyl disulfide (peak 2), which together accounted for over 70% of the total composition. Peak identification was performed by mass spectral matching with reference libraries and retention index comparison. 3.5 Antifungal Activity Septoria tritici blotch (STB), caused by the heterothallic ascomycete Zymoseptoria tritici , is one of the most economically significant foliar diseases of wheat worldwide, leading to severe yield reductions and quality losses (Allioui et al. 2021 ) .Current management strategies largely rely on repeated fungicide applications, yet the rapid emergence of fungicide-resistant strains, combined with environmental and health concerns, highlights the urgent need for sustainable alternatives. In this context, plant-derived natural products, such as essential oils (EOs) and hydrosols, represent promising biofungicidal agents with broad-spectrum activity and low ecological impact (Galisteo et al. 2022 ). In the present study, the antifungal activity of garlic ( Allium sativum L.) EO and hydrosol was evaluated in vitro against the radial mycelial growth of Z. tritici . The results, summarized in Table 6 and illustrated in Fig. 3 – 5 , clearly demonstrate significant antifungal potential for both extracts, with pronounced concentration-dependent effects. 3.5.1 Essential Oil Activity and Minimum Inhibitory Concentration (MIC) Garlic EO exhibited a strong antifungal effect at all tested concentrations (0.10–0.30 µL/mL) compared to the untreated and DMSO-treated controls (p < 0.001). At lower concentrations (0.10 and 0.15 µL/mL), inhibition rates were modest (26.67 ± 9.88% and 30.00 ± 0.00%, respectively), whereas 0.22 µL/mL produced a significant increase in antifungal efficacy (42.86 ± 0.00%). Complete suppression of fungal growth (100 ± 0.00%) was achieved at 0.30 µL/mL, indicating this concentration as the minimum inhibitory concentration (MIC) under the experimental conditions. The dose-dependent response suggests that bioactive components within the EO interact synergistically to disrupt fungal physiology as their concentration increases. These findings are consistent with previous reports demonstrating potent antifungal activity of garlic EO against a range of phytopathogens. Perello et al ( 2013 ) reported complete inhibition of Z. tritici conidial germination by allicin at 120 µg/mL, while Taheri et al ( 2023 ) observed comparable growth suppression in other fungal species. The superior efficacy of EO at lower concentrations compared with hydrosol is likely attributable to its higher content of volatile organosulfur compounds, as confirmed by our GC–MS analysis (see Table 5 ), where diallyl trisulfide (36.99%) and diallyl disulfide (35.78%) predominated. These compounds are well-documented for their membrane-disrupting and enzyme-inhibiting activities (Krid et al. 2025 ). 3.5.2 Hydrosol Bioactivity and Mode of Action. Garlic hydrosol also displayed antifungal activity, albeit at higher concentrations (50–200 µL/mL). A moderate inhibition of 17.12 ± 5.20% was recorded at 50 µL/mL, increasing to 45.65 ± 0.00% at 100 µL/mL. Complete inhibition occurred at 150 µL/mL and persisted at 200 µL/mL, indicating a MIC ≤ 150 µL/mL. The lower efficacy relative to EO reflects the reduced concentration of hydrophobic sulfur compounds in the aqueous distillate. Nevertheless, the results demonstrate that hydrosol retains sufficient bioactivity to exert significant antifungal effects, likely due to the presence of water-soluble sulfur volatiles and other polar metabolites. Hydrosols, though less studied, are gaining attention for their potential as sustainable, low-toxicity plant protection agents (Roanisca et al. 2024 ). Their ability to inhibit Z. tritici growth suggests they could serve as complementary biofungicidal tools, especially in integrated pest management (IPM) strategies where safety and environmental compatibility are prioritized. 3.5.3 Mechanistic Insights and Structure–Activity Considerations The antifungal efficacy of garlic extracts is primarily attributed to their rich profile of organosulfur compounds, including diallyl sulfide, diallyl disulfide, diallyl trisulfide, 2-vinyl-4H-1,3-dithiin, and dimethyl trisulfide. These bioactive molecules act through multiple, complementary mechanisms that collectively impair fungal development and survival. Owing to their lipophilic nature, organosulfur compounds readily integrate into fungal cell membranes, increasing permeability and compromising membrane integrity, which leads to leakage of intracellular contents and disruption of essential ion gradients (Taheri et al. 2023 ). They also interfere with critical metabolic enzymes and signaling pathways involved in respiration, cell wall biosynthesis, and fungal growth, thereby perturbing key physiological processes (Krid et al. 2025 ). Furthermore, garlic volatiles are capable of modulating redox homeostasis by inducing oxidative stress, which triggers apoptosis-like cell death. At the molecular level, transcriptomic evidence suggests that essential oil components can alter the expression of genes associated with stress response, membrane transport, and nutrient uptake, thereby further inhibiting fungal survival and adaptability (Taheri et al. 2023 ). In addition to their biochemical mechanisms, the physicochemical properties of garlic EO likely enhance its bioactivity. The relatively acidic pH and high refractive index of the oil facilitate its diffusion into lipid membranes, while the ionic content and viscosity of the hydrosol may improve its stability and diffusion dynamics at higher concentrations. Together, these structural and physicochemical attributes underpin the potent antifungal action of garlic-derived extracts, providing a strong rationale for their development as natural biofungicidal agents in sustainable crop protection strategies. 3.5.4 Implications for Sustainable Disease Management The demonstrated antifungal activity of both garlic EO and hydrosol against Z. tritici represents a significant step toward developing natural, eco-friendly biofungicides for sustainable wheat production. The strong inhibitory effects, particularly the complete growth suppression at low EO concentrations, highlight their potential utility as either stand-alone treatments or as components of integrated disease management programs aimed at reducing synthetic fungicide dependency. Furthermore, the use of locally produced garlic-based extracts aligns with circular bioeconomy principles, leveraging agricultural biodiversity to enhance crop resilience while minimizing environmental impact. Future studies should focus on field trials, formulation optimization, and synergistic combinations with other biological control agents to fully exploit their potential in commercial agriculture. Table 6 In vitro antifungal activity of garlic ( Allium sativum L.) essential oil and hydrosol against Zymoseptoria tritici mycelial growth Essential oil (µL/mL) Inhibition rate (I%) Hydrosol (µL/mL) Inhibition rate (I%) 0.10 26.67 ± 9.88 CD 50 17.12 ± 5.20 C 0.15 30.00 ± 0.00 C 100 45.65 ± 0.00 B 0.22 42.86 ± 0.00 B 150 100.00 ± 0.00 A 0.30 100.00 ± 0.00 A 200 100.00 ± 0.00 A Control + 0.00 ± 0.00 H Control + 0.00 ± 0.00 D Control − 0.00 ± 0.00 H Control − 0.00 ± 0.00 D Results are expressed as mean ± standard deviation (n = 3). Different letters (A–D, H) indicate statistically significant differences among treatments (p < 0.001, Fisher’s post-hoc test). Control (+): medium containing 1% DMSO; Control (−): untreated medium. Representative PDA plates showing growth inhibition at different treatment levels: C+: positive control (1% DMSO); C−: negative control (untreated); C1–C4: essential oil concentrations of 0.10, 0.15, 0.22, and 0.30 µL mL⁻¹, respectively; C5–C8: hydrosol concentrations of 50, 100, 150, and 200 µL mL⁻¹, respectively. Progressive growth suppression is observed with increasing concentrations, culminating in complete inhibition at 0.30 µL mL⁻¹ EO and ≥ 150 µL mL⁻¹ hydrosol. Inhibition rates (I%) were quantified at four essential oil concentrations (0.10, 0.15, 0.22, and 0.30 µL mL⁻¹). Data are presented as mean ± standard deviation (n = 3). Different letters (A–D, H) indicate significant differences among treatments (p < 0.001). The concentration of 0.30 µL mL⁻¹ resulted in complete growth suppression, representing the minimum inhibitory concentration (MIC). Inhibition rates (I%) were determined at four hydrosol concentrations (50, 100, 150, and 200 µL mL⁻¹). Results are expressed as mean ± standard deviation (n = 3). Different letters (A–D) indicate statistically significant differences (p < 0.001). Complete inhibition was achieved at concentrations ≥ 150 µL mL⁻¹, suggesting a potent antifungal effect even in the aqueous distillation byproduct. 3.6 Stability Assay of Garlic Essential Oil The temporal stability of the antifungal activity of garlic ( Allium sativum L.) essential oil (EO) against Zymoseptoria tritici was evaluated by assessing the inhibition rate (I%) at three defined inoculation times following EO incorporation into PDA medium: immediately after preparation (Day 0), after 3 days (Day 3), and after 6 days (Day 6). The results are summarized in Table 7 and illustrated in Fig. 6 3.6.1 Temporal Dynamics of Antifungal Efficacy At Day 0, freshly incorporated EO displayed a robust, concentration-dependent antifungal activity. Complete inhibition (100.00 ± 0.00%) was observed at 0.30 µL/mL, while significant growth suppression was also recorded at 0.22 µL/mL (42.86 ± 0.00%) and 0.15 µL/mL (30.00 ± 0.00%). Even the lowest concentration (0.10 µL/mL) significantly reduced mycelial development by 26.67 ± 9.88% compared to the controls (p < 0.001). This strong initial activity highlights the potency of the bioactive volatile compounds in freshly prepared EO formulations. However, a substantial decline in antifungal efficacy was evident by Day 3 across all concentrations. The inhibition rates dropped markedly relative to Day 0, and the differences between 0.15, 0.22, and 0.30 µL/mL were no longer statistically significant, indicating a loss of the previously observed dose-dependent effect. The lowest concentration (0.10 µL/mL) exhibited minimal antifungal activity (5.00 ± 0.00%), underscoring the pronounced loss of bioactivity upon storage in the medium. By Day 6, the antifungal potential of garlic EO was severely diminished. Although 0.30 µL/mL remained the most effective concentration, its inhibition capacity decreased dramatically to 23.96 ± 8.46%, reflecting a 76% reduction relative to Day 0. The other concentrations displayed uniformly low and statistically indistinguishable inhibition levels, indicating near-complete functional degradation of antifungal activity after six days. 3.6.2 Mechanistic Basis of EO Instability The progressive decline in antifungal efficacy over time is likely linked to the physicochemical instability of organosulfur constituents, the principal bioactive agents in garlic EO. Compounds such as diallyl disulfide, diallyl trisulfide, and 2-vinyl-4H-1,3-dithiin are highly volatile, thermolabile, and prone to oxidative degradation under ambient conditions (Zhao et al. 2021 ). When incorporated into a semi-solid medium like PDA and stored at room temperature, these volatiles may evaporate, oxidize, or undergo structural transformations, resulting in reduced antifungal potency and altered biochemical activity profiles. This temporal instability has critical implications for the practical application of garlic EO in plant disease management. The results clearly demonstrate that maximum antifungal efficacy is achieved only when EO is freshly prepared and applied immediately. Even short-term delays in application (e.g., 3 days) significantly compromise bioactivity, emphasizing the necessity of formulation strategies that stabilize active components and prevent premature degradation. 3.6.3 Strategies for Enhancing Stability Improving the shelf-life and bioefficacy of garlic EO-based formulations requires targeted stabilization approaches. Zhao et al ( 2021 ) reported that co-formulating garlic EO with lipid carriers such as vegetable oils (corn, soybean, or olive oil) significantly enhances its stability by reducing volatilization and oxidation rates. Such formulations preserved higher concentrations of diallyl disulfide and diallyl trisulfide over time, maintaining antifungal activity for extended periods. Other promising approaches include microencapsulation, emulsification, or incorporation into biodegradable polymer matrices, all of which could provide controlled release and protection against environmental degradation. These findings underscore the importance of integrating formulation science with natural product research to optimize the practical application of EO-based biopesticides. The rapid decline in antifungal activity observed here is not merely a limitation but an opportunity to innovate delivery systems that retain the inherent potency of plant-derived bioactives for sustainable crop protection. Table 7 Stability of the antifungal activity of garlic ( Allium sativum L.) essential oil in PDA medium against Zymoseptoria tritici after short-term storage and inoculation at three defined intervals (Day 0, Day 3, and Day 6) EO concentration (µL/mL) I% Day 0 I% Day 3 I% Day 6 0.10 26.67 ± 9.88 CD 5.00 ± 0.00 G 9.72 ± 6.36 FG 0.15 30.00 ± 0.00 C 17.50 ± 3.54 E 11.11 ± 4.81 F 0.22 42.86 ± 0.00 B 20.00 ± 0.00 E 8.33 ± 1.41 FG 0.30 100.00 ± 0.00 A 20.00 ± 0.00 E 23.96 ± 8.46 D Control + 0.00 ± 0.00 H 0.00 ± 0.00 H 0.00 ± 0.00 H Control − 0.00 ± 0.00 H 0.00 ± 0.00 H 0.00 ± 0.00 H Values are expressed as inhibition rate (I%) ± standard deviation (SD). EO: essential oil. Different letters (A–H) indicate statistically significant differences among treatments at p < 0.001 (Fisher’s LSD). Control (+): treated with 1% DMSO. Control (–): untreated. 4. Discussion The present study demonstrates that bio-based extracts obtained from a locally cultivated Algerian red garlic ( Allium sativum L.) variety exhibit strong antifungal activity against Zymoseptoria tritici , the causal agent of Septoria tritici blotch (STB), one of the most economically damaging diseases of bread wheat. Both garlic essential oil (EO) and hydrosol were able to significantly inhibit mycelial growth in vitro , with complete growth suppression achieved at relatively low concentrations. These findings confirm the relevance of garlic-derived products as promising alternatives to synthetic fungicides within sustainable wheat disease management strategies. Previous studies have widely documented the antifungal activity of garlic-derived essential oils against a range of plant pathogenic fungi, including agents of foliar and soil-borne diseases. In most cases, growth inhibition has been reported at relatively high concentrations, with efficacy strongly influenced by garlic genotype, extraction method, and fungal species tested. Muy-Rangel et al ( 2018 ) supported the antifungal potential of garlic essential oil, reporting a strong inhibitory effect against Alternaria tenuissima , with a marked reduction in mycelial growth from 250 ppm and complete inhibition at 1,000 ppm (CI₅₀ = 229 ppm; MIC = 1,023 ppm). Similarly, Hassan et al ( 2024 ) reported complete inhibition of colony growth of Fusarium proliferatum and Macrophomina phaseolina at high concentrations of garlic essential oil. These findings are in agreement with the antifungal activity observed in the present study, although differences in effective concentrations may be attributed to variations in fungal species and the chemical composition of the essential oil used. In addition, several studies have confirmed the antifungal activity of garlic essential oil against various phytopathogenic fungi, although at different concentrations and using diverse application methods (Wang et al. 2019 ; Dabodhia et al. 2022 ; Deshmukh et al. 2026 ). In comparison, the complete inhibition of Zymoseptoria tritici observed in the present study at low EO concentrations suggests a particularly strong antifungal potential of the local Algerian red garlic variety used. This enhanced activity may be related to the high relative abundance of diallyl trisulfide and diallyl disulfide identified in the essential oil, compounds that have been repeatedly associated with strong fungistatic and fungicidal effects. Similar sulfur-containing compounds were reported by Chen et al ( 2024 ) to be the primary contributors to antifungal activity in garlic-derived products, with diallyl trisulfide exhibiting the highest potency. Although their study focused on isolated compounds, these findings support the hypothesis that diallyl sulfides play a central role in the antifungal effects observed in the present work. In contrast to previous investigations that primarily focused on garlic essential oil, very limited attention has been given to garlic hydrosol as an antifungal agent. Where hydrosols have been evaluated, their biological activity has often been reported as weaker or inconsistent when compared to essential oils. Notably, the present findings demonstrate that garlic hydrosol can achieve complete and stable inhibition of Z. tritici at higher concentrations, highlighting a clear divergence from earlier observations, and indicating that hydrosol may represent an underexploited resource for disease control. This difference may be attributed to the presence of water-soluble sulfur compounds and other polar metabolites that are not retained in the oil fraction but are preserved in the aqueous distillate. Similarly, comparable studies on the antifungal activity of garlic hydrosol have confirmed its effectiveness against various fungal pathogens, supporting its potential as a bio-based antifungal agent. Tagoe et al ( 2009 ) reported that aqueous extracts of garlic exhibited marked growth inhibition against several phytopathogenic fungi, including Aspergillus flavus , Aspergillus niger , and Cladosporium herbarum , suggesting that water-soluble garlic constituents can contribute significantly to antifungal activity. Further insights into the chemical basis of this activity were provided by Galisteo et al ( 2022 ), who identified a total of eight compounds in the organic fraction of garlic hydrolate. The most abundant compounds were diallyl disulfide, diallyl trisulfide, p -methylpyridine, and methyl allyl trisulfide. Although these compounds are also present in garlic essential oil at higher relative proportions, their occurrence in the hydrolate supports the existence of antifungal-active constituents in the aqueous distillate, which may contribute to the distinct biological effects observed for hydrosols. More recently, Galisteo et al ( 2025 ) highlighted the antifungal relevance of garlic hydrolate beyond its minor volatile composition. In their study, four novel oxygenated organosulfur compounds, termed garlicinals A–D, were isolated from garlic hydrolate obtained from industrial agrowaste. These compounds, characterized by an α,β-unsaturated aldehyde structural motif, exhibited pronounced fungicidal activity, thereby reinforcing the potential of garlic hydrolate as an effective and underexploited agent for crop protection. The antifungal efficacy of garlic EO can be largely attributed to its high content of organosulfur compounds, as revealed by GC–MS analysis. Diallyl trisulfide and diallyl disulfide were the dominant constituents, together accounting for more than 70% of the total volatile profile. These compounds are widely recognized for their strong antimicrobial properties as consistently demonstrated in previous studies by Perello et al ( 2013 ), Sarfraz et al (2022), and Barbu et al ( 2023 ), and their ability to disrupt fungal cell membranes, inhibit key metabolic enzymes, and interfere with cellular redox balance (Wang et al. 2019 ; Krid et al. 2025 ). The dose-dependent inhibition observed in this study supports the hypothesis that synergistic interactions among sulfur-containing volatiles play a central role in suppressing Z. tritici growth. Although the hydrosol contained a markedly lower proportion of volatile sulfur compounds, it nevertheless achieved complete and stable inhibition of fungal growth at higher concentrations. This result is particularly noteworthy, as garlic hydrosol has received far less attention than essential oil in the context of plant disease control. The antifungal activity of the hydrosol may be associated with the presence of water-soluble sulfur compounds and other polar metabolites released during hydrodistillation Galisteo et al ( 2025 ). Unlike EO, whose activity declined rapidly over time, the hydrosol displayed sustained inhibitory effects, suggesting a greater stability under the experimental conditions. This characteristic could represent a practical advantage for agricultural applications, especially where ease of handling, safety, and formulation stability are critical considerations. The stability of garlic-derived bioactive compounds is strongly influenced by the environment in which they are present. Studies on allicin from Allium sativum have demonstrated a markedly longer persistence in aqueous solutions than in oil-based ones, where rapid degradation occurs, with allicin remaining active for several days in water but only a few hours in vegetable oil (Fujisawa et al. 2008 ). This observation aligns with broader studies on hydrosols (aromatic waters), which report that their chemical composition—particularly oxygenated compounds—can remain relatively stable under typical storage conditions and in acidic aqueous environments (Garneau et al. 2014 ). Moreover, hydrosols have been shown to exhibit good physicochemical stability under thermal stress and pH variation, further supporting their potential for sustained biological activity (Almeida et al. 2024 ). Together, these findings suggest that aqueous garlic extracts and hydrosols may provide a more stable environment for bioactive sulfur compounds, which may contribute to the persistent antifungal effects observed in the present study. The temporal stability assay highlighted an important limitation of garlic EO, namely the rapid loss of antifungal efficacy when incorporated into the growth medium and stored prior to inoculation. This decline is most likely due to the high volatility and oxidative sensitivity of organosulfur compounds, which may evaporate or degrade during storage (Zhao et al. 2021 ). These findings emphasize that, while EO is highly potent when freshly applied, its practical use in crop protection would require appropriate formulation strategies to enhance stability and prolong bioactivity. Approaches such as encapsulation, emulsification, or incorporation into carrier systems may help overcome these limitations and improve field applicability (Mossa et al. 2018 ; Gong et al. 2021 ), Bouqellah et al ( 2025 ) reported that encapsulation of Allium sativum essential oil in silver nanoparticles (AgNPs) enhances its stability and antifungal activity. The AgNPs act as carrier systems, improving the persistence and controlled release of volatile compounds, which results in increased inhibition of fungal growth. From a broader perspective, the use of garlic-derived extracts aligns well with current efforts to reduce reliance on chemical fungicides and mitigate the development of fungicide-resistant Z. tritici populations. The strong antifungal activity observed, combined with the local availability of garlic and the potential valorization of hydrosol as a low-cost by-product, supports the integration of these natural products into sustainable and circular bioeconomy-based plant protection systems. However, further research is required to validate these findings under field conditions, to optimize formulations, and to assess possible synergistic effects with other biological or reduced-risk control strategies. 5. Conclusions This study demonstrates the strong antifungal potential of bio-based extracts derived from a locally cultivated Algerian red garlic ( Allium sativum L.) variety against Zymoseptoria tritic i, the causal agent of Septoria tritici blotch (STB) in wheat. Comprehensive morphological, physicochemical, and chemical analyses revealed a volatile profile dominated by organosulfur compounds, particularly diallyl disulfide and diallyl trisulfide, that are responsible for the extracts’ biological activity. In vitro assays showed that the essential oil (EO) achieved complete growth inhibition at remarkably low concentrations (0.30 µL/mL), whereas the hydrosol, despite its lower sulfur content, displayed full antifungal efficacy at 150 µL/mL, marking the first report of such activity for this aqueous distillation byproduct. Mechanistic insights suggest that these bioactive molecules act synergistically to disrupt fungal membranes, inhibit key metabolic enzymes, perturb redox homeostasis, and modulate gene expression, thereby compromising fungal survival. However, the EO’s bioactivity declined significantly over time, underscoring the need for formulation strategies, such as encapsulation or carrier-based delivery systems, to improve stability and prolong field efficacy. Overall, the findings highlight the potential of garlic-derived EO and hydrosol as natural, eco-friendly biofungicides that could reduce reliance on synthetic chemicals in wheat disease management. Their local availability, low toxicity, and multifaceted modes of action make them promising candidates for integration into sustainable crop protection strategies and circular bioeconomy models. Future research should prioritize formulation optimization, field validation, and the exploration of synergistic combinations with other biocontrol agents to translate these laboratory findings into effective agricultural solutions. Declarations Conflicts of Interest: The authors declare no conflicts of interest Funding: This study was supported within the scope of the Project also funded under the National Recovery and Resilience Plan (NRRP), Mission 4 Component 2 Investment 1.4 - Call for tender No. 3138 of December 16, 2021, rectified by Decree n.3175 of December 18, 2021 of Italian Ministry of University and Research funded by the European Union – NextGenerationEU; Award Number: Project code CN_00000033, Concession Decree No. 1034 of June 17, 2022 adopted by the Italian Ministry of University and Research, CUP: D43C22001260001, Project title “National Biodiversity Future Center - NBFC”. DGRSDT (mesrs): General Directorate of Scientific Research and Technological Development - Ministry of Higher Education and Scientific Research, Algeria. Author Contributions: Conceptualization, R.D., I.S. and A.D.; methodology, R.D., I.S., A.D. and F.D.; software, F.D., C.B. and MDE; formal analysis, R.D., F.D. and C.B.; investigation, R.D.; resources, R.D. A.B. and I.S.; data curation, F.D., M.D.E., L.R; and F.B. writing—original draft preparation, R.D. M.D.E and L.R.; writing—review and editing, I.S. M.D.E, and L.R.; visualization, R.D.; supervision, L.B.; project administration, L.B., L.R. and F.B. ; funding acquisition, R.D. and L.R. All authors have read and agreed to the published version of the manuscript. Acknowledgments: The authors would like to thank the Technical Institute for Vegetable and Industrial Crops (ITCMI), the National Institute of Agronomic Research of Algeria (INRAA) especially Mrs. Meamiche Hayat, and the Biotechnology Research Center (CRBt), Algeria, for their valuable contribution to this work. 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Foods 10:1637. https://doi.org/10.3390/foods10071637 Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revisions 12 Mar, 2026 Reviewers agreed at journal 03 Feb, 2026 Reviewers invited by journal 03 Feb, 2026 Editor invited by journal 21 Jan, 2026 Editor assigned by journal 19 Jan, 2026 First submitted to journal 15 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8612946","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":585184564,"identity":"cd8a44e9-f57b-4fff-9779-176b7e5ad5cf","order_by":0,"name":"Roumeissa Djerboua","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Roumeissa","middleName":"","lastName":"Djerboua","suffix":""},{"id":585184565,"identity":"4320f223-9365-452d-a44c-250e45576e4e","order_by":1,"name":"Laid Benderradji","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Laid","middleName":"","lastName":"Benderradji","suffix":""},{"id":585184566,"identity":"73dea1ea-61b2-478f-8e47-18b4fee17a3c","order_by":2,"name":"Ibtissem Sanah","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Ibtissem","middleName":"","lastName":"Sanah","suffix":""},{"id":585184567,"identity":"419a347d-8eed-4b70-8832-fccf11092550","order_by":3,"name":"Fairouz Djeghim","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Fairouz","middleName":"","lastName":"Djeghim","suffix":""},{"id":585184568,"identity":"5b383617-2ade-4839-b5ba-4e1bf22f6632","order_by":4,"name":"Ali Debbi","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Ali","middleName":"","lastName":"Debbi","suffix":""},{"id":585184569,"identity":"673f92af-e7e5-4630-bd82-0a8f62ae543a","order_by":5,"name":"Chawki Bensouici","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Chawki","middleName":"","lastName":"Bensouici","suffix":""},{"id":585184570,"identity":"1220b9ac-93a3-44a1-909e-288a84845169","order_by":6,"name":"Abdelkader Benbelkacem","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Abdelkader","middleName":"","lastName":"Benbelkacem","suffix":""},{"id":585184571,"identity":"d45db651-203e-4264-a0eb-8587a72a5b0e","order_by":7,"name":"Maria D’Elia","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"","lastName":"D’Elia","suffix":""},{"id":585184572,"identity":"8c55622f-2be8-49bd-b9c3-c84c11696973","order_by":8,"name":"Luca Rastrelli","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyUlEQVRIiWNgGAWjYPACCxk29gYGhgQgk40I5YxAtRI8bDwHoFqI0APRwiCRAOUT0mLefvz544oKCR4+yTdmDx78sWPgk2/Ar0XmTI5h45kzQIdJ55gbJLYlE3aYBEMOY2NjG1iLmURiAzMRWvifP2xs/AfUInnGTCLhTz0RWiQSDBsbG4BaJHiAWtgOE6PljeHMhmOgQE4rk0hsO87DxpZAyGHpDz421NjIybcf3ib540+1nHzzAQLWoAMeEtWPglEwCkbBKMAGAKOsM1T+TRaJAAAAAElFTkSuQmCC","orcid":"","institution":"University of Salerno: Universita degli Studi di Salerno","correspondingAuthor":true,"prefix":"","firstName":"Luca","middleName":"","lastName":"Rastrelli","suffix":""},{"id":585184573,"identity":"ceb9168c-cec4-4757-8de2-3104f07c41ef","order_by":9,"name":"Faical Brini","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Faical","middleName":"","lastName":"Brini","suffix":""}],"badges":[],"createdAt":"2026-01-15 18:13:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8612946/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8612946/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101956295,"identity":"ec86ea1c-1d57-4aae-93e2-5db0ad20182d","added_by":"auto","created_at":"2026-02-05 11:44:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":167190,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological features of the local Algerian red garlic (RL) variety. (a) Whole bulb showing external morphology; (b) individual cloves highlighting size and pigmentation; (c) bulk harvest collected from the Constantine region\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8612946/v1/2c6a83025b02f71f3565c7e9.png"},{"id":102295082,"identity":"dd791351-7858-4ea3-b017-5c3201ae5b64","added_by":"auto","created_at":"2026-02-10 10:08:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":119538,"visible":true,"origin":"","legend":"\u003cp\u003eGC–MS chromatographic profile of essential oil from the local Algerian red garlic (\u003cem\u003eAllium sativum\u003c/em\u003e L.)\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8612946/v1/8322d8aad6efadb877391a8f.png"},{"id":101956300,"identity":"237c0693-80da-4dbb-b25f-4646ef5c760e","added_by":"auto","created_at":"2026-02-05 11:44:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":176659,"visible":true,"origin":"","legend":"\u003cp\u003eMacroscopic effects of garlic (Allium sativum L.) essential oil and hydrosol on the radial mycelial growth of Zymoseptoria tritici\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8612946/v1/298738e24229f2464fbdbdee.png"},{"id":102295030,"identity":"f43839dc-efbd-4e39-8d89-ee5bd6252b4e","added_by":"auto","created_at":"2026-02-10 10:07:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":158459,"visible":true,"origin":"","legend":"\u003cp\u003eInhibitory effect of garlic (\u003cem\u003eAllium sativum\u003c/em\u003e L.) essential oil on radial growth of \u003cem\u003eZymoseptoria tritici\u003c/em\u003e \u003cem\u003ein vitro\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8612946/v1/bb813b145488b58032e311fe.png"},{"id":101956297,"identity":"4de39be8-1b0f-40cf-964b-9b430ada1e51","added_by":"auto","created_at":"2026-02-05 11:44:35","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":230244,"visible":true,"origin":"","legend":"\u003cp\u003eInhibitory effect of garlic hydrosol on radial growth of \u003cem\u003eZymoseptoria tritici\u003c/em\u003e \u003cem\u003ein vitro\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8612946/v1/ec10940b4559de8d6aac1b44.png"},{"id":102295025,"identity":"1d207cc9-27d8-4fcb-814c-1551e1434239","added_by":"auto","created_at":"2026-02-10 10:07:37","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":130841,"visible":true,"origin":"","legend":"\u003cp\u003eAntifungal activity of garlic (Allium sativum L.) essential oil against Zymoseptoria tritici in PDA medium following inoculation at three different time intervals (Day 0, Day 3, and Day 6)\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8612946/v1/3fb441efa67465b1b6fbe1b1.png"},{"id":102298774,"identity":"53344977-de32-4a91-817b-b9edaa1a96ee","added_by":"auto","created_at":"2026-02-10 10:59:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2521241,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8612946/v1/d358a2dd-aa34-426f-b1f9-5d5047b7ca1c.pdf"}],"financialInterests":"","formattedTitle":"Antifungal Activity of Garlic (Allium sativum L.) Essential Oil and Hydrosol Against Zymoseptoria tritici, the Causal Agent of Septoria Tritici Blotch in Bread Wheat","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eSeptoria tritici blotch (STB), caused by the heterothallic ascomycete \u003cem\u003eZymoseptoria tritici\u003c/em\u003e (syn. \u003cem\u003eSeptoria tritici\u003c/em\u003e or \u003cem\u003eMycosphaerella graminicola\u003c/em\u003e), is one of the most destructive foliar diseases affecting bread wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e L.) worldwide. Severe epidemics can result in yield losses of 35\u0026ndash;50%, with high incidence levels reported in Algeria, where STB was detected in 80% of surveyed wheat fields across 11 localities between 2010 and 2012 (Allioui et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Owing to its global distribution and economic impact, \u003cem\u003eZ. tritici\u003c/em\u003e is ranked among the ten most devastating plant pathogenic fungi (Damiens et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The pathogen follows a hemibiotrophic infection strategy, initiating infection with an asymptomatic biotrophic phase followed by a necrotrophic phase characterized by extensive leaf necrosis (Platel et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBread wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e L.) is one of the most important staple crops worldwide and a fundamental component of global food security, particularly in developing regions. Global demand for wheat is projected to increase by nearly 60% by 2050 to meet the nutritional needs of a growing population (Atwa et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In Algeria, wheat plays a strategic role in national food security as both a dietary cornerstone and a central focus of agricultural policy (Ouzani et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). However, wheat production is increasingly threatened by biotic stresses, particularly fungal diseases, which significantly reduce grain yield and quality (Suarez-Fernandez and De Francesco \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCurrent management of STB relies heavily on repeated applications of synthetic fungicides. Nevertheless, their intensive use has raised serious concerns related to environmental contamination, phytotoxic effects, risks to human health, and the rapid emergence of fungicide-resistant \u003cem\u003eZ. tritici\u003c/em\u003e populations (Sahli et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Allioui et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These limitations highlight the urgent need for sustainable, safe, and locally sourced alternatives for effective wheat disease control.\u003c/p\u003e \u003cp\u003eIn this context, increasing attention has been directed toward bio-based disease management strategies. Plant-derived products, including essential oils (EOs) and hydrosols, have emerged as promising alternatives to conventional fungicides due to their biodegradability, low toxicity, and broad-spectrum antimicrobial activity (Okorska et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Among these, garlic (\u003cem\u003eAllium sativum\u003c/em\u003e L.) extracts are particularly notable for their strong antifungal properties, largely attributed to sulfur-containing compounds such as allicin, S-allyl cysteine, thiosulfates, and other organosulfur derivatives (Lawson et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Verma et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In Algeria, garlic cultivation has steadily increased, providing an abundant and locally available resource for the development of plant-based antifungal agents.\u003c/p\u003e \u003cp\u003eBeyond their biological activity, the characterization of garlic germplasm is essential for identifying varieties best adapted to specific agro-ecological conditions and for maximizing the yield of bioactive metabolites (Salahuddin et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Morphological characterization supports breeding programs, diversity assessments, and genotype selection, which are crucial since antifungal efficacy can be influenced by varietal origin (Panthee et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Ragas et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Despite numerous studies reporting the antimicrobial potential of garlic EO, comprehensive physicochemical characterization\u0026mdash;particularly of garlic hydrosol, a by-product of hydrodistillation\u0026mdash;remains limited. To date, no study has reported the combined antifungal activity of both garlic EO and hydrosol against \u003cem\u003eZ. tritici\u003c/em\u003e, nor has the hydrosol obtained from a locally cultivated Algerian garlic variety been characterized.\u003c/p\u003e \u003cp\u003eThe present study aimed to address this knowledge gap by investigating the antifungal potential of essential oil and hydrosol derived from a local Algerian red garlic variety against \u003cem\u003eZ. tritici\u003c/em\u003e, the causal agent of STB. Specifically, we (i) conducted morphological characterization of the garlic genotype, (ii) performed physicochemical analyses of EO and hydrosol, (iii) identified the volatile constituents of EO using gas chromatography\u0026ndash;mass spectrometry (GC\u0026ndash;MS), and (iv) assessed the temporal stability of EO antifungal activity. This multidisciplinary approach seeks to contribute to the development of bio-based, eco-friendly, and locally available solutions for sustainable wheat disease management.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Plant Material\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eGarlic (\u003cem\u003eAllium sativum\u003c/em\u003e L.) bulbs of a local red variety (RL) were used in this study. The plants were cultivated under open-field conditions and harvested in July 2021 from a non-irrigated experimental plot at the Debbah Pilot Farm, located in Didouche Mourad in northern Constantine, Algeria. The site is situated at an elevation of 500 m above sea level (36\u0026deg;29\u0026prime;26\u0026Prime; N, 6\u0026deg;37\u0026prime;14\u0026Prime; E) and is characterized by a semi-arid Mediterranean climate. During the 2020\u0026ndash;2021 growing season, the total annual rainfall was 313.97 mm, with mean air temperatures ranging from 8.2\u0026deg;C to 28\u0026deg;C and relative humidity between 36.2% and 75%. Bulbs were harvested at full maturity, air-dried under ambient conditions, and stored in a dark, ventilated environment at room temperature until further use.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Fungal Strain\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eA pathogenic strain of \u003cem\u003eZymoseptoria tritici\u003c/em\u003e (teleomorph: \u003cem\u003eMycosphaerella graminicola\u003c/em\u003e), the causal agent of Septoria tritici blotch (STB) in bread wheat, was employed for antifungal bioassays. The isolate was originally obtained from infected wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e L.) leaves collected from experimental fields and was kindly provided by the National Institute of Agronomic Research of Algeria (INRAA). The strain had been previously identified based on its morphological and cultural characteristics and maintained as part of the INRAA fungal collection. Colonies were cultured on potato dextrose agar (PDA) and incubated at 20\u0026deg;C in the dark to preserve their pathogenicity prior to experimental use.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Morphological Characterization of Plant Material\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eMorphological traits of the garlic (\u003cem\u003eAllium sativum\u003c/em\u003e L.) bulbs were assessed following the official descriptor for \u003cem\u003eAllium sativum\u003c/em\u003e published by the International Union for the Protection of New Varieties of Plants (UPOV \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). A comprehensive set of qualitative traits was evaluated, including: bulb size; bulb shape in longitudinal and cross sections; position of cloves at the tip of the bulb; clove distribution; external clove morphology; compactness of cloves; position of the root disc; shape of the bulb base; ground color; presence and intensity of anthocyanin stripes; thickness and adherence of the outer dry scales; clove size; clove color and color intensity; anthocyanin stripes on clove scales; flesh color; and time of harvest maturity. Quantitative traits measured included the number of bulbs per kilogram, average bulb weight, bulb diameter (cm), number of cloves per bulb, and average clove weight. All morphological evaluations were conducted on a representative sample of 30 garlic bulbs randomly selected from the harvested batch to ensure statistical reliability.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Essential Oil and Hydrosol Extraction\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eEssential oil (EO) and hydrosol were extracted from bulbs of the local red garlic (\u003cem\u003eAllium sativum\u003c/em\u003e L.) variety under industrial-scale conditions using the hydrodistillation with a Clevenger- type apparatus for 2 h 30 min under controlled conditions. The extraction was performed at the private distillation facility \u0026ldquo;Arom\u0026rsquo;Est,\u0026rdquo; specialized in essential and vegetable oil production, located in Annaba, Algeria. Fresh garlic cloves were peeled, chopped into small pieces, and placed in a stainless-steel industrial distillation unit with distilled water. The hydrodistillation process was carried out. Upon completion, the EO and hydrosol fractions were separated based on their density differences using a separating funnel. Both extracts were stored in amber glass bottles at 4\u0026deg;C in the dark until further analyses to prevent degradation. The EO yield (%) was calculated using the following Eq.\u0026nbsp;\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e:\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Equ1\" class=\"Equation\"\u003e \u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:\\mathbf{E}\\mathbf{o}\\:\\mathbf{Y}\\mathbf{i}\\mathbf{e}\\mathbf{l}\\mathbf{d}\\:\\left(\\mathbf{\\%}\\right)=\\left(\\frac{\\mathbf{w}\\mathbf{e}\\mathbf{i}\\mathbf{g}\\mathbf{h}\\mathbf{t}\\:\\mathbf{o}\\mathbf{f}\\:\\mathbf{e}\\mathbf{x}\\mathbf{t}\\mathbf{r}\\mathbf{a}\\mathbf{c}\\mathbf{t}\\mathbf{e}\\mathbf{d}\\:\\mathbf{o}\\mathbf{i}\\mathbf{l}}{\\mathbf{w}\\mathbf{e}\\mathbf{i}\\mathbf{g}\\mathbf{h}\\mathbf{t}\\:\\mathbf{o}\\mathbf{f}\\:\\mathbf{g}\\mathbf{a}\\mathbf{r}\\mathbf{l}\\mathbf{i}\\mathbf{c}}\\right)\\times\\:100$$\u003c/div\u003e \u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Physicochemical and Organoleptic Characterization of Garlic Extracts\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe physicochemical properties of garlic essential oil (EO) and hydrosol were analyzed in accordance with the standards of the European Pharmacopoeia, the International Organization for Standardization ISO 3061:2008. \u003cem\u003eOrganoleptic Analysis\u003c/em\u003e: the organoleptic characteristics of EO and hydrosol were evaluated assessing appearance, color, and odor. \u003cem\u003ePhysicochemical Analysis of Essential Oil\u003c/em\u003e: \u003cb\u003et\u003c/b\u003ehe refractive index was measured at 20\u0026deg;C using a REICHERT AR6 automatic refractometer previously calibrated with distilled water. Relative density was determined with a METTLER TOLEDO Densito 30 PX electronic densimeter. The acid value was quantified by titration with 0.05 N alcoholic KOH according to and calculated using standard equations. Optical rotation was determined following ISO 592:1998 using a 10 mL polarimeter tube filled with a dilute EO\u0026ndash;ethanol solution. The pH of EO samples was measured at room temperature using pH indicator strips. \u003cem\u003ePhysicochemical Analysis of Hydrosol\u003c/em\u003e: several parameters were measured to assess the composition and potential bioactivity of the garlic hydrosol. Turbidity was determined at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C using a Lovibond\u0026reg; infrared turbidimeter. Electrical conductivity was measured with a Jenway 4510 conductivity meter after electrode stabilization. Viscosity was determined using a VISCO TM-895 rotational viscometer Density was measured with a standard hydrometer, reading specific gravity directly from a graduated cylinder. The pH was determined using a calibrated digital pH by immersing the electrode directly in the sample until the reading stabilized.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Gas Chromatography\u0026ndash;Mass Spectrometry (GC\u0026ndash;MS) Analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe chemical composition of the garlic essential oil (EO) was determined using a gas chromatograph (Agilent 6890 Plus, Hewlett Packard, USA) coupled to a mass spectrometer (Agilent 5973, Hewlett Packard, USA). Separation was achieved on a HP-5MS capillary column (30 m \u0026times; 0.25 mm i.d., 0.25 \u0026micro;m film thickness) coated with 5% phenyl-95% dimethylpolysiloxane as the stationary phase. The injector temperature was set at 250\u0026deg;C, and samples (0.2 \u0026micro;L) were injected in split mode (1:20). High-purity helium (N6.0) was used as the carrier gas at a constant flow rate of 0.5 mL min⁻\u0026sup1;. The oven temperature program was as follows: initial temperature 60\u0026deg;C (held for 8 min), increased at 2\u0026deg;C min⁻\u0026sup1; to 250\u0026deg;C, and then held isothermally for 10 min, for a total run time of 113 min. The mass spectrometer operated in total ion current (TIC) scan mode over an m/z range of 30\u0026ndash;550. Ionization was performed by electron impact (EI) at 70 eV. The ion source and interface temperatures were maintained at 230\u0026deg;C and 280\u0026deg;C, respectively. A solvent delay of 3.5 min was applied. The quadrupole mass analyzer was used for detection. Identification of EO constituents was achieved by comparing their mass spectra and retention times with those available in the NIST and Wiley mass spectral libraries. Additionally, Kovats retention indices (RI) were calculated relative to a homologous series of \u003cem\u003en\u003c/em\u003e-alkanes (C\u003csub\u003e8\u003c/sub\u003e\u0026ndash;C\u003csub\u003e29\u003c/sub\u003e) analyzed under the same chromatographic conditions, and the results were compared with published literature data to confirm compound identity.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Antifungal Activity Assay\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe antifungal activity of garlic essential oil (EO) and hydrosol against \u003cem\u003eZymoseptoria tritici\u003c/em\u003e was evaluated at the Biotechnology Research Center (CRBT), Constantine, Algeria, following the protocols described by Hammer et al (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1999\u003c/span\u003e), with minor modifications. Appropriate volumes of EO and hydrosol were first dissolved in a maximum of 1 mL of dimethyl sulfoxide (DMSO) and then added to 100 mL of sterile potato dextrose agar (PDA) medium (after autoclaving and cooling to ~\u0026thinsp;45\u0026deg;C) to obtain the following final concentrations: 0.10, 0.15, 0.22, and 0.30 \u0026micro;L/mL for EO, and 50, 100, 150, and 200 \u0026micro;L/mL for hydrosol. Each treatment was poured into four replicate Petri dishes. Two control treatments were included: (i) positive control \u0026ndash; PDA medium supplemented with 1 mL of DMSO (final DMSO concentration\u0026thinsp;\u0026le;\u0026thinsp;1%); and (ii) negative control \u0026ndash; PDA medium without any additives. After medium solidification, each plate was inoculated with 5 \u0026micro;L of a 1 \u0026times; 10⁶ spores/mL suspension of \u003cem\u003eZ. tritici\u003c/em\u003e and incubated in the dark at 20\u0026deg;C for 10 days.\u003c/p\u003e \u003cp\u003eAntifungal efficacy was expressed as the percentage of growth inhibition (I%) relative to the control, calculated according to Eq.\u0026nbsp;\u003cspan refid=\"Equ2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Equ2\" class=\"Equation\"\u003e \u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$\\:\\mathbf{I}=\\left(\\frac{\\mathbf{C}-\\mathbf{T}}{\\mathbf{C}}\\right)\\times\\:100$$\u003c/div\u003e \u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eWhere: C (mm)\u0026thinsp;=\u0026thinsp;radial growth of the pathogen on control plates; T (mm)\u0026thinsp;=\u0026thinsp;radial growth on treatment plates.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Stability Assay of Garlic Essential Oil\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo assess the short-term stability of garlic essential oil (EO) and its persistence in inhibiting \u003cem\u003eZymoseptoria tritici\u003c/em\u003e growth, an additional experiment was performed using the same methodology described for the antifungal activity assay (Section \u003cspan refid=\"Sec9\" class=\"InternalRef\"\u003e2.7\u003c/span\u003e). The experiment was designed to evaluate the effect of different time intervals between EO incorporation into the medium and fungal inoculation.\u003c/p\u003e \u003cp\u003eThree experimental groups were established:\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eGroup 1 (Day 0): Inoculation was performed immediately after the solidification of the EO-supplemented PDA medium.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eGroup 2 (Day 3): Inoculation was delayed by three days. During this period, the EO-containing plates were stored under sterile conditions at room temperature.\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eGroup 3 (Day 6): Inoculation was delayed by six days, with plates maintained under the same conditions.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eEach plate was inoculated with 5 \u0026micro;L of a \u003cem\u003eZ. tritici\u003c/em\u003e spore suspension (1 \u0026times; 10⁶ spores/mL) and incubated in the dark at 20\u0026deg;C for 10 days. All treatments were performed in triplicate. Fungal growth was evaluated by measuring radial colony expansion, and the percentage of growth inhibition was calculated according to the formula described in Section \u003cspan refid=\"Sec9\" class=\"InternalRef\"\u003e2.7\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Statistical Analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAll data obtained in this study were subjected to statistical analysis at a significance level of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Descriptive statistics (mean, minimum, maximum, and coefficient of variation, CV) were first calculated for morphological and physicochemical traits to evaluate variability within the garlic samples. The antifungal activity of the essential oil (EO) was analyzed using a two-way analysis of variance (ANOVA) to assess the effects of concentration and treatment duration, while the antifungal activity of the hydrosol was evaluated using a one-way ANOVA. When significant differences were detected, Fisher\u0026rsquo;s least significant difference (LSD) post-hoc test was applied to compare mean values. All statistical analyses were performed using JMP Trial 17 software (SAS Institute Inc., Cary, NC, USA).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Morphological Characterization\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eBulb and clove traits are the principal economic organs of garlic (\u003cem\u003eAllium sativum\u003c/em\u003e L.), widely used for culinary, medicinal, and propagation purposes (Ragas et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). They also serve as fundamental selection criteria in breeding programs aimed at improving yield, quality, and adaptability (Panthee et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). In this study, morphological characterization was conducted to define the specific phenotypic features of the local Algerian red garlic variety (RL) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), for which limited information is currently available in the literature.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e3.1.1 Quantitative Traits\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe quantitative morphological characteristics of the RL variety are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The number of bulbs per kilogram varied from 40 to 55, with an average of 47.35\u0026thinsp;\u0026plusmn;\u0026thinsp;3.81, while the mean bulb dry weight was 25.52\u0026thinsp;\u0026plusmn;\u0026thinsp;6.35 g. Bulb diameter averaged 3.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 cm, and the number of cloves per bulb ranged from 9 to 17. The average clove dry weight was 2.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67 g.\u003c/p\u003e \u003cp\u003eThe coefficients of variation (CV) for the five traits ranged from 0.05% to 24.91%. The highest variability was observed for bulb dry weight (24.91%), indicating considerable heterogeneity for this trait (Shrestha et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In contrast, the number of cloves per bulb (14.28%), bulbs per kilogram (8.05%), clove dry weight (0.31%), and bulb diameter (0.05%) exhibited lower variability (CV\u0026thinsp;\u0026lt;\u0026thinsp;20%), indicating relative uniformity within the population.\u003c/p\u003e \u003cp\u003eThese results are in line with those reported (Boukeria \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) for the same variety and are within the range described by Pasupula et al (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), and Popa and Cosmulescu (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) across diverse garlic germplasm. Such differences in morphological traits are often attributed to genetic variation as well as environmental influences including soil type, climatic conditions, and agronomic practices (Panthee et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Akan \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Baswarsiati et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Bulb weight, in particular, is a key agronomic trait that significantly influences garlic yield, often correlating with clove size and planting material quality (Popa and Cosmulescu \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eQuantitative morphological traits of the local Algerian red garlic (RL) variety\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQuantitative parameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRL variety (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMin\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMax\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCV (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber of bulbs per kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e47.35\u0026thinsp;\u0026plusmn;\u0026thinsp;3.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBulb dry weight (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e25.52\u0026thinsp;\u0026plusmn;\u0026thinsp;6.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e37.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e24.91\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBulb diameter (cm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber of cloves per bulb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e13.10\u0026thinsp;\u0026plusmn;\u0026thinsp;1.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e14.28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClove dry weight (g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.31\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eValues are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) along with minimum (Min), maximum (Max), and coefficient of variation (CV%) values.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e3.1.2 Qualitative Traits\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003ePhenotypic observations of qualitative morphological traits are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The RL variety produced small- to medium-sized bulbs with white to yellowish outer scales lacking anthocyanin stripes, while the inner scales exhibited pink to red pigmentation, supporting its classification as a \u0026ldquo;red\u0026rdquo; variety. The bulb base was flat, and the shape in longitudinal section was transverse broad elliptic, while the cross-section was elliptic.\u003c/p\u003e \u003cp\u003eCloves were radially arranged, with no external cloves, and exhibited a compact configuration. The root disc was flat, and clove scales were pink to purple with strong pigmentation and anthocyanin stripes, whereas the clove flesh was white to yellowish. The variety showed late dormancy and late harvest maturity. These findings are consistent with (Boukeria \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and highlight distinct phenotypic features, particularly clove arrangement and pigmentation, which can aid in germplasm identification and breeding (Panthee et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Akan \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSuch qualitative traits are critical for marketability and consumer preference (Baswarsiati et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and play a vital role in genotype differentiation. The absence of external cloves, often associated with reduced commercial quality (Kıra\u0026ccedil; et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), further enhances the market potential of the RL variety.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eQualitative morphological characteristics of bulb and cloves of the local red (RL) garlic variety\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRL Variety\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBulb size\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSmall \u0026ndash; Medium\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBulb shape in longitudinal section\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTransverse broad elliptic\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBulb shape in cross section\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eElliptic\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePosition of cloves at tip of bulb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAt same level\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDistribution of cloves\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRadial\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExternal cloves\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompactness of cloves\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCompact\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePosition of root disc\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFlat\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eShape of bulb base\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFlat\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGround color of dry external scales\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWhite\u0026ndash;yellowish\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnthocyanin stripes on bulb dry external scales\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbsent\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThickness of dry external scales\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedium\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSkin adherence of dry bulb external scales\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedium\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eClove size\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMedium\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eColor of clove scale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePink\u0026ndash;purple\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIntensity of color of clove scale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStrong\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnthocyanin stripes on clove scale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePresent\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eColor of flesh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWhite\u0026ndash;yellowish\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEnd of dormancy of clove in bulb\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLate\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTime of harvest maturity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLate\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003ePhenotypic traits were assessed following the UPOV (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) and IPGRI (2001) descriptors for \u003cem\u003eAllium sativum\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Essential Oil Yield\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe essential oil (EO) yield obtained from the local Algerian red garlic (\u003cem\u003eAllium sativum\u003c/em\u003e L.) variety in this study was 0.075% (w/w). This value is comparable to that reported by (Shalaby et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) for an Egyptian garlic cultivar (0.073%) extracted using the same hydrodistillation method. Similarly, Nazzaro et al (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) documented yields ranging from 0.03% to 0.08% across two different cultivars, with our results being closer to the upper end of this range.\u003c/p\u003e \u003cp\u003eHowever, the yield reported here is lower than those observed in several other studies. For instance, Sarangi et al (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) reported a yield of 0.17%, while Boukeria (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) documented yields of 0.46%, 0.61%, and 0.51% for the Messidrom, Germidour, and Mocpta Bulguar varieties, respectively, and even reported a maximum yield of 0.72% for the same Local Red variety analyzed in this work. Variability in EO yield among garlic varieties is influenced by a combination of genetic, physiological, methodological, and environmental factors. According to some authors (Mugao \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Ezeorba et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), several parameters, including the plant organ used for extraction, harvest period, post-harvest handling, drying method, plant maturity stage, and extraction conditions, can significantly impact EO yield. Moreover, genotypic variation, plant density, and cultivation practices (e.g., irrigation and fertilization) further modulate EO biosynthesis and accumulation. Environmental conditions during the 2020\u0026ndash;2021 growing season likely contributed to the relatively low yield reported in this study. The experimental site experienced a total annual rainfall of 313.97 mm and relative humidity as low as 36.2%, conditions that may have imposed moderate abiotic stress and limited the biosynthetic potential of secondary metabolites. These findings underscore the importance of optimizing both agronomic practices and environmental conditions to enhance EO yield in future cultivation and extraction strategies.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Physicochemical and Organoleptic Characterization of Garlic Extracts\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe physicochemical characterization of essential oils (EOs) and hydrosols is a critical step for assessing their functional properties, standardizing quality control, and determining their suitability for various applications in food, cosmetics, pharmaceuticals, and agricultural formulations (Barkatullah et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Belkhodja et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The key organoleptic and physicochemical properties of garlic EO and hydrosol are summarized in Tables\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, respectively.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e3.3.1 Organoleptic Properties\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe garlic EO obtained from the local red variety exhibited a liquid consistency and a yellow coloration, typical of extracts rich in volatile organosulfur compounds (Oliveira et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).Its pungent, intense, and characteristic odor is primarily attributed to diallyl sulfides and related sulfurous constituents (Borlinghaus et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In contrast, the hydrosol presented as a colorless to very pale yellow aromatic water with a less intense odor, reflecting its lower volatile content (generally\u0026thinsp;\u0026lt;\u0026thinsp;0.1% w/v) (Aćimović et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This compositional difference aligns with expectations, as hydrosols primarily consist of water-soluble compounds and trace amounts of EO constituents.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e3.3.2 Physicochemical Properties of Essential Oil\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe refractive index (RI) of the garlic EO was 1.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00, which falls within the range reported in previous studies (1.43\u0026ndash;1.52) (Rafe and Nadjafi \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Dehariya et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). RI is strongly influenced by oil composition, particularly the degree of unsaturation, the cis/trans isomer ratio, and the abundance of oxygenated monoterpenes. A relatively high RI value suggests the presence of heavier or more polar compounds, which may enhance the bioactivity and stability of the oil (Boukeria, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe acid value was 6.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 mg/g, significantly higher than those reported by Lemma et al (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) (1.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 mg/g) and El Faqer et al (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) (1.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 mg/g). Acid value is an established indicator of oil quality and hydrolytic stability; values below 2 mg/g generally indicate good preservation and low levels of free fatty acids (Lemma et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The elevated acid value in our sample suggests partial degradation, likely due to hydrolysis of ester bonds during storage or sample handling.\u003c/p\u003e \u003cp\u003eThe optical rotation was 0\u0026deg; \u0026plusmn; 0.00, consistent with a racemic or balanced mixture of chiral molecules. This result is consistent with commercial garlic oils (typically \u0026minus;\u0026thinsp;2\u0026deg; to +\u0026thinsp;5\u0026deg;) and reflects the optical purity and stereochemical composition of the extract (Do et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) Optical rotation is a useful analytical parameter for detecting adulteration and confirming geographical or varietal origin.\u003c/p\u003e \u003cp\u003eThe density of the garlic EO was 1.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 g/cm\u0026sup3;, higher than values reported by Dehariya et al (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) (0.873\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003 g/cm\u0026sup3;) and El Faqer et al (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) (0.978\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 g/cm\u0026sup3;), but comparable to the range (1.025\u0026ndash;1.029 g/cm\u0026sup3;) observed by Boukeria et al (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Density correlates with the molecular weight distribution and polarity of oil constituents; values above 1.0 g/cm\u0026sup3; suggest the presence of relatively heavy or complex sulfur-containing molecules. This property also explains the EO\u0026rsquo;s tendency to settle below the aqueous phase during separation.\u003c/p\u003e \u003cp\u003eThe pH of the EO was 5.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00, indicating a slightly acidic nature consistent with previous reports (Boukeria et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). This property is relevant for determining the oil\u0026rsquo;s stability and compatibility with certain formulations.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e3.3.3 Physicochemical Properties of Hydrosol\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe physicochemical analysis of garlic hydrosol provides original data not previously reported in the literature and contributes to a deeper understanding of its compositional and functional profile. The turbidity was 20.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34 NTU, indicating a moderate level of suspended particulates, likely composed of microdroplets of aromatic compounds or colloidal organic matter. Although slight turbidity is typical of plant hydrosols, its magnitude may serve as a useful indicator of volatile content and process efficiency.\u003c/p\u003e \u003cp\u003eThe viscosity averaged 3.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 mPa\u0026middot;s, a value slightly higher than that of pure water, suggesting the presence of dissolved phytochemicals or colloidal components. This parameter is relevant for understanding hydrosol structure and its behavior in formulations (Barkatullah et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe electrical conductivity (EC) was 198.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 \u0026micro;S, reflecting a moderate concentration of ionizable solutes, such as sulfur-containing compounds and trace minerals, released during hydrodistillation (ISO 7888:1985). These findings are consistent with previously reported conductivity ranges for botanical hydrosols (25.2\u0026ndash;730.0 \u0026micro;S) (Acheampong et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFinally, the pH of the hydrosol was 7.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03, indicating a slightly alkaline nature, and its density was 1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 g/mL, closely matching that of water. As noted by Tavares et al (\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) hydrosols generally exhibit densities near 1.0 g/mL due to their low solute concentration.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eOrganoleptic characteristics of garlic essential oil and garlic hydrosol\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExtract\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGarlic essential oil\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGarlic hydrosol\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAppearance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLiquid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLiquid\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eColor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYellow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eColorless to very pale yellow\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOdor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStrong, pungent, characteristic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLess intense, characteristic\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePhysicochemical properties of garlic (\u003cem\u003eAllium sativum\u003c/em\u003e L.) essential oil and hydrosol\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGarlic essential oil\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCV (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGarlic hydrosol\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCV (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRefractive index at 20\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTurbidity (NTU)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e20.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.68\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAcid value (mg/g)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e6.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eViscosity (mPa\u0026middot;s)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e3.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.78\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOptical rotation (\u0026deg;)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eConductivity (\u0026micro;S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e198.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDensity (g/cm\u0026sup3;)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDensity (g/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e5.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e7.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.41\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD, n\u0026thinsp;=\u0026thinsp;3). CV (%)\u0026thinsp;=\u0026thinsp;coefficient of variation calculated as (SD/mean) \u0026times; 100.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Gas Chromatography\u0026ndash;Mass Spectrometry (GC\u0026ndash;MS) Analysis\u003c/h2\u003e \u003cp\u003eThe chemical composition of the essential oil (EO) obtained from the local Algerian red garlic (\u003cem\u003eAllium sativum\u003c/em\u003e L.) variety, along with the retention times (Rt) and relative abundances of the identified compounds, is presented in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. The corresponding chromatographic profile is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e GC\u0026ndash;MS analysis allowed the identification of 21 volatile compounds, representing 99.97% of the total EO composition. The EO profile was dominated by organosulfur compounds, which accounted for 83.32% of the total composition. The principal constituents were diallyl trisulfide (36.99%) and diallyl disulfide (35.78%), followed by diallyl sulfide (5.49%) and methyl allyl disulfide (2.65%). Other sulfur-based volatiles present in lower concentrations included dimethyl trisulfide (0.55%), 2-vinyl-4H-1,3-dithiin (1.23%), 2-methyl-1,3-dithiolane (0.46%), and 1,3-dithiane (0.17%). These results are consistent with previous reports on garlic EO composition (Jan et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Molina-Calle et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Similarly, Sarangi et al (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) reported diallyl trisulfide (37%) and diallyl disulfide (25.9%) as the main constituents, while Herrera-Calderon et al (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) found diallyl trisulfide (44.21%) and diallyl disulfide (22.08%) as major components. The relative abundance of these compounds tends to vary with factors such as genotype, growth stage, post-harvest handling, drying conditions, and extraction parameters, all of which can influence the volatile profile (Modafferi et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The predominance of sulfur-containing compounds observed in the EO of Algerian red garlic aligns with its well-documented biological functionality. These volatile organosulfur molecules are widely recognized for their antimicrobial, antifungal, antioxidant, cardioprotective, anti-atherosclerotic, immunomodulatory, and antihypertensive activities (Gong et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Ozoude and Chukwu \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Notably, diallyl disulfide and diallyl trisulfide have been extensively reported as key bioactive compounds contributing to garlic\u0026rsquo;s fungistatic and fungicidal activity, as well as its capacity to modulate oxidative stress and inflammatory responses. Their prevalence in the EO composition thus underlines the potential of this extract as a natural biocontrol agent for plant pathogens such as \u003cem\u003eZymoseptoria tritici\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eVolatile composition of essential oil from the local Algerian red garlic (\u003cem\u003eAllium sativum\u003c/em\u003e L.) determined by GC\u0026ndash;MS analysis\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePeak\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRt (min)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRelative abundance (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2-Phenyl-4,6-di(4-acetylaminophenyl)pyrimidine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.705\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e925\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.122\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAcetonitrile, 2-(5-chloro-3-oxo-3H-1,2-dithiol-4-yl)propylamino\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.065\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e940\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.252\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDiallyl sulfide (1-Propene, 3,3\u0026prime;-thiobis)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.390\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e960\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.485\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMethyl allyl disulfide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7.779\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1008\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.654\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1,3-Dithiane\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8.894\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1025\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.168\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2-Hydroxy-3-methoxy-succinic acid, dimethyl ester\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10.220\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1045\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.125\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDimethyl trisulfide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10.740\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1063\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.547\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDiallyl disulfide\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e18.907\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1172\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e35.778\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBenzene, 1,3-dichloro\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e19.382\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1186\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.085\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHexopyranosid-3-ulose, methyl 2,4,6-tris-O-(trimethylsilyl)-, dimethyl acetal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e19.942\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.225\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHeptane, 4-methoxy-3-(methoxymethyl)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e22.794\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1245\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8.252\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1H-Imidazole, 1-phenyl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e26.149\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1288\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.634\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBenzenamine, 3,5-dinitro\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e27.018\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.323\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2-Vinyl-4H-1,3-dithiin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e28.144\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1318\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.230\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDiallyl trisulfide (Trisulfide, di-2-propenyl)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e34.602\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1395\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e36.990\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTetramethyl 1,1\u0026prime;-(1,8-naphthylene) bis(1,2,3-triazole-4,5-dicarboxylate)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e34.854\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1402\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.171\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7-Amino-7H-S-triazolo[5,1-c]-S-triazole-3-thiol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e38.597\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1438\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.410\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2-Methyl-1,3-dithiolane\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e39.106\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1445\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.460\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003etert-Butyl isopropyl disulfide, perfluoro\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e49.119\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1540\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.729\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2-Docosenoic acid, 2,4,21,21-tetramethyl-, methyl ester, (E)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e62.231\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1670\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.021\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3-Iodo-4-methoxy-phenyl)-morpholin-4-yl-methanethione\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e75.039\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1782\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.249\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eRetention time (Rt), retention index (RI), and relative abundance (%) of individual compounds were determined by GC\u0026ndash;MS. Compound identification was based on comparison of mass spectra with NIST and Wiley libraries and confirmed by Kovats retention indices calculated relative to a homologous series of n-alkanes (C\u003csub\u003e8\u003c/sub\u003e\u0026ndash;C\u003csub\u003e29\u003c/sub\u003e). Compounds are listed in order of elution.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe chromatogram illustrates the volatile composition of the essential oil, highlighting the predominance of sulfur-containing compounds such as diallyl trisulfide (peak 1) and diallyl disulfide (peak 2), which together accounted for over 70% of the total composition. Peak identification was performed by mass spectral matching with reference libraries and retention index comparison.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Antifungal Activity\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eSeptoria tritici blotch (STB), caused by the heterothallic ascomycete \u003cem\u003eZymoseptoria tritici\u003c/em\u003e, is one of the most economically significant foliar diseases of wheat worldwide, leading to severe yield reductions and quality losses (Allioui et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) .Current management strategies largely rely on repeated fungicide applications, yet the rapid emergence of fungicide-resistant strains, combined with environmental and health concerns, highlights the urgent need for sustainable alternatives. In this context, plant-derived natural products, such as essential oils (EOs) and hydrosols, represent promising biofungicidal agents with broad-spectrum activity and low ecological impact (Galisteo et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In the present study, the antifungal activity of garlic (\u003cem\u003eAllium sativum\u003c/em\u003e L.) EO and hydrosol was evaluated \u003cem\u003ein vitro\u003c/em\u003e against the radial mycelial growth of \u003cem\u003eZ. tritici\u003c/em\u003e. The results, summarized in Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, clearly demonstrate significant antifungal potential for both extracts, with pronounced concentration-dependent effects.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003e3.5.1 Essential Oil Activity and Minimum Inhibitory Concentration (MIC)\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eGarlic EO exhibited a strong antifungal effect at all tested concentrations (0.10\u0026ndash;0.30 \u0026micro;L/mL) compared to the untreated and DMSO-treated controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). At lower concentrations (0.10 and 0.15 \u0026micro;L/mL), inhibition rates were modest (26.67\u0026thinsp;\u0026plusmn;\u0026thinsp;9.88% and 30.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00%, respectively), whereas 0.22 \u0026micro;L/mL produced a significant increase in antifungal efficacy (42.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00%). Complete suppression of fungal growth (100\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00%) was achieved at 0.30 \u0026micro;L/mL, indicating this concentration as the minimum inhibitory concentration (MIC) under the experimental conditions. The dose-dependent response suggests that bioactive components within the EO interact synergistically to disrupt fungal physiology as their concentration increases. These findings are consistent with previous reports demonstrating potent antifungal activity of garlic EO against a range of phytopathogens. Perello et al (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) reported complete inhibition of \u003cem\u003eZ. tritici\u003c/em\u003e conidial germination by allicin at 120 \u0026micro;g/mL, while Taheri et al (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) observed comparable growth suppression in other fungal species. The superior efficacy of EO at lower concentrations compared with hydrosol is likely attributable to its higher content of volatile organosulfur compounds, as confirmed by our GC\u0026ndash;MS analysis (see Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), where diallyl trisulfide (36.99%) and diallyl disulfide (35.78%) predominated. These compounds are well-documented for their membrane-disrupting and enzyme-inhibiting activities (Krid et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e \u003ch2\u003e3.5.2 Hydrosol Bioactivity and Mode of Action.\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eGarlic hydrosol also displayed antifungal activity, albeit at higher concentrations (50\u0026ndash;200 \u0026micro;L/mL). A moderate inhibition of 17.12\u0026thinsp;\u0026plusmn;\u0026thinsp;5.20% was recorded at 50 \u0026micro;L/mL, increasing to 45.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00% at 100 \u0026micro;L/mL. Complete inhibition occurred at 150 \u0026micro;L/mL and persisted at 200 \u0026micro;L/mL, indicating a MIC\u0026thinsp;\u0026le;\u0026thinsp;150 \u0026micro;L/mL. The lower efficacy relative to EO reflects the reduced concentration of hydrophobic sulfur compounds in the aqueous distillate. Nevertheless, the results demonstrate that hydrosol retains sufficient bioactivity to exert significant antifungal effects, likely due to the presence of water-soluble sulfur volatiles and other polar metabolites.\u003c/p\u003e \u003cp\u003eHydrosols, though less studied, are gaining attention for their potential as sustainable, low-toxicity plant protection agents (Roanisca et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Their ability to inhibit \u003cem\u003eZ. tritici\u003c/em\u003e growth suggests they could serve as complementary biofungicidal tools, especially in integrated pest management (IPM) strategies where safety and environmental compatibility are prioritized.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003e3.5.3 Mechanistic Insights and Structure\u0026ndash;Activity Considerations\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe antifungal efficacy of garlic extracts is primarily attributed to their rich profile of organosulfur compounds, including diallyl sulfide, diallyl disulfide, diallyl trisulfide, 2-vinyl-4H-1,3-dithiin, and dimethyl trisulfide. These bioactive molecules act through multiple, complementary mechanisms that collectively impair fungal development and survival. Owing to their lipophilic nature, organosulfur compounds readily integrate into fungal cell membranes, increasing permeability and compromising membrane integrity, which leads to leakage of intracellular contents and disruption of essential ion gradients (Taheri et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). They also interfere with critical metabolic enzymes and signaling pathways involved in respiration, cell wall biosynthesis, and fungal growth, thereby perturbing key physiological processes (Krid et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Furthermore, garlic volatiles are capable of modulating redox homeostasis by inducing oxidative stress, which triggers apoptosis-like cell death. At the molecular level, transcriptomic evidence suggests that essential oil components can alter the expression of genes associated with stress response, membrane transport, and nutrient uptake, thereby further inhibiting fungal survival and adaptability (Taheri et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In addition to their biochemical mechanisms, the physicochemical properties of garlic EO likely enhance its bioactivity. The relatively acidic pH and high refractive index of the oil facilitate its diffusion into lipid membranes, while the ionic content and viscosity of the hydrosol may improve its stability and diffusion dynamics at higher concentrations. Together, these structural and physicochemical attributes underpin the potent antifungal action of garlic-derived extracts, providing a strong rationale for their development as natural biofungicidal agents in sustainable crop protection strategies.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003e3.5.4 Implications for Sustainable Disease Management\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe demonstrated antifungal activity of both garlic EO and hydrosol against \u003cem\u003eZ. tritici\u003c/em\u003e represents a significant step toward developing natural, eco-friendly biofungicides for sustainable wheat production. The strong inhibitory effects, particularly the complete growth suppression at low EO concentrations, highlight their potential utility as either stand-alone treatments or as components of integrated disease management programs aimed at reducing synthetic fungicide dependency. Furthermore, the use of locally produced garlic-based extracts aligns with circular bioeconomy principles, leveraging agricultural biodiversity to enhance crop resilience while minimizing environmental impact. Future studies should focus on field trials, formulation optimization, and synergistic combinations with other biological control agents to fully exploit their potential in commercial agriculture.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e antifungal activity of garlic (\u003cem\u003eAllium sativum\u003c/em\u003e L.) essential oil and hydrosol against \u003cem\u003eZymoseptoria tritici\u003c/em\u003e mycelial growth\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEssential oil (\u0026micro;L/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInhibition rate (I%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHydrosol (\u0026micro;L/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInhibition rate (I%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e26.67\u0026thinsp;\u0026plusmn;\u0026thinsp;9.88 CD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17.12\u0026thinsp;\u0026plusmn;\u0026thinsp;5.20 C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e45.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 B\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e42.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 A\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 A\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl +\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eControl +\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 D\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl \u0026minus;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eControl \u0026minus;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 D\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eResults are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (n\u0026thinsp;=\u0026thinsp;3). Different letters (A\u0026ndash;D, H) indicate statistically significant differences among treatments (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fisher\u0026rsquo;s post-hoc test). Control (+): medium containing 1% DMSO; Control (\u0026minus;): untreated medium.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eRepresentative PDA plates showing growth inhibition at different treatment levels: C+: positive control (1% DMSO); C\u0026minus;: negative control (untreated); C1\u0026ndash;C4: essential oil concentrations of 0.10, 0.15, 0.22, and 0.30 \u0026micro;L mL⁻\u0026sup1;, respectively; C5\u0026ndash;C8: hydrosol concentrations of 50, 100, 150, and 200 \u0026micro;L mL⁻\u0026sup1;, respectively. Progressive growth suppression is observed with increasing concentrations, culminating in complete inhibition at 0.30 \u0026micro;L mL⁻\u0026sup1; EO and \u0026ge;\u0026thinsp;150 \u0026micro;L mL⁻\u0026sup1; hydrosol.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eInhibition rates (I%) were quantified at four essential oil concentrations (0.10, 0.15, 0.22, and 0.30 \u0026micro;L mL⁻\u0026sup1;). Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (n\u0026thinsp;=\u0026thinsp;3). Different letters (A\u0026ndash;D, H) indicate significant differences among treatments (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The concentration of 0.30 \u0026micro;L mL⁻\u0026sup1; resulted in complete growth suppression, representing the minimum inhibitory concentration (MIC).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eInhibition rates (I%) were determined at four hydrosol concentrations (50, 100, 150, and 200 \u0026micro;L mL⁻\u0026sup1;). Results are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (n\u0026thinsp;=\u0026thinsp;3). Different letters (A\u0026ndash;D) indicate statistically significant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Complete inhibition was achieved at concentrations\u0026thinsp;\u0026ge;\u0026thinsp;150 \u0026micro;L mL⁻\u0026sup1;, suggesting a potent antifungal effect even in the aqueous distillation byproduct.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Stability Assay of Garlic Essential Oil\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe temporal stability of the antifungal activity of garlic (\u003cem\u003eAllium sativum\u003c/em\u003e L.) essential oil (EO) against \u003cem\u003eZymoseptoria tritici\u003c/em\u003e was evaluated by assessing the inhibition rate (I%) at three defined inoculation times following EO incorporation into PDA medium: immediately after preparation (Day 0), after 3 days (Day 3), and after 6 days (Day 6). The results are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e and illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec28\" class=\"Section3\"\u003e \u003ch2\u003e3.6.1 Temporal Dynamics of Antifungal Efficacy\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAt Day 0, freshly incorporated EO displayed a robust, concentration-dependent antifungal activity. Complete inhibition (100.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00%) was observed at 0.30 \u0026micro;L/mL, while significant growth suppression was also recorded at 0.22 \u0026micro;L/mL (42.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00%) and 0.15 \u0026micro;L/mL (30.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00%). Even the lowest concentration (0.10 \u0026micro;L/mL) significantly reduced mycelial development by 26.67\u0026thinsp;\u0026plusmn;\u0026thinsp;9.88% compared to the controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). This strong initial activity highlights the potency of the bioactive volatile compounds in freshly prepared EO formulations. However, a substantial decline in antifungal efficacy was evident by Day 3 across all concentrations. The inhibition rates dropped markedly relative to Day 0, and the differences between 0.15, 0.22, and 0.30 \u0026micro;L/mL were no longer statistically significant, indicating a loss of the previously observed dose-dependent effect. The lowest concentration (0.10 \u0026micro;L/mL) exhibited minimal antifungal activity (5.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00%), underscoring the pronounced loss of bioactivity upon storage in the medium. By Day 6, the antifungal potential of garlic EO was severely diminished. Although 0.30 \u0026micro;L/mL remained the most effective concentration, its inhibition capacity decreased dramatically to 23.96\u0026thinsp;\u0026plusmn;\u0026thinsp;8.46%, reflecting a 76% reduction relative to Day 0. The other concentrations displayed uniformly low and statistically indistinguishable inhibition levels, indicating near-complete functional degradation of antifungal activity after six days.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section3\"\u003e \u003ch2\u003e3.6.2 Mechanistic Basis of EO Instability\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe progressive decline in antifungal efficacy over time is likely linked to the physicochemical instability of organosulfur constituents, the principal bioactive agents in garlic EO. Compounds such as diallyl disulfide, diallyl trisulfide, and 2-vinyl-4H-1,3-dithiin are highly volatile, thermolabile, and prone to oxidative degradation under ambient conditions (Zhao et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). When incorporated into a semi-solid medium like PDA and stored at room temperature, these volatiles may evaporate, oxidize, or undergo structural transformations, resulting in reduced antifungal potency and altered biochemical activity profiles. This temporal instability has critical implications for the practical application of garlic EO in plant disease management. The results clearly demonstrate that maximum antifungal efficacy is achieved only when EO is freshly prepared and applied immediately. Even short-term delays in application (e.g., 3 days) significantly compromise bioactivity, emphasizing the necessity of formulation strategies that stabilize active components and prevent premature degradation.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section3\"\u003e \u003ch2\u003e3.6.3 Strategies for Enhancing Stability\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eImproving the shelf-life and bioefficacy of garlic EO-based formulations requires targeted stabilization approaches. Zhao et al (\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) reported that co-formulating garlic EO with lipid carriers such as vegetable oils (corn, soybean, or olive oil) significantly enhances its stability by reducing volatilization and oxidation rates. Such formulations preserved higher concentrations of diallyl disulfide and diallyl trisulfide over time, maintaining antifungal activity for extended periods. Other promising approaches include microencapsulation, emulsification, or incorporation into biodegradable polymer matrices, all of which could provide controlled release and protection against environmental degradation. These findings underscore the importance of integrating formulation science with natural product research to optimize the practical application of EO-based biopesticides. The rapid decline in antifungal activity observed here is not merely a limitation but an opportunity to innovate delivery systems that retain the inherent potency of plant-derived bioactives for sustainable crop protection.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStability of the antifungal activity of garlic (\u003cem\u003eAllium sativum\u003c/em\u003e L.) essential oil in PDA medium against \u003cem\u003eZymoseptoria tritici\u003c/em\u003e after short-term storage and inoculation at three defined intervals (Day 0, Day 3, and Day 6)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEO concentration (\u0026micro;L/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eI% Day 0\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eI% Day 3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eI% Day 6\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e0.10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e26.67\u0026thinsp;\u0026plusmn;\u0026thinsp;9.88 CD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.72\u0026thinsp;\u0026plusmn;\u0026thinsp;6.36 FG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e0.15\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17.50\u0026thinsp;\u0026plusmn;\u0026thinsp;3.54 E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.11\u0026thinsp;\u0026plusmn;\u0026thinsp;4.81 F\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e0.22\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e42.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.41 FG\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e0.30\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23.96\u0026thinsp;\u0026plusmn;\u0026thinsp;8.46 D\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eControl +\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 H\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eControl \u0026minus;\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 H\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eValues are expressed as inhibition rate (I%)\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). EO: essential oil. Different letters (A\u0026ndash;H) indicate statistically significant differences among treatments at p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 (Fisher\u0026rsquo;s LSD). Control (+): treated with 1% DMSO. Control (\u0026ndash;): untreated.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe present study demonstrates that bio-based extracts obtained from a locally cultivated Algerian red garlic (\u003cem\u003eAllium sativum\u003c/em\u003e L.) variety exhibit strong antifungal activity against \u003cem\u003eZymoseptoria tritici\u003c/em\u003e, the causal agent of Septoria tritici blotch (STB), one of the most economically damaging diseases of bread wheat. Both garlic essential oil (EO) and hydrosol were able to significantly inhibit mycelial growth \u003cem\u003ein vitro\u003c/em\u003e, with complete growth suppression achieved at relatively low concentrations. These findings confirm the relevance of garlic-derived products as promising alternatives to synthetic fungicides within sustainable wheat disease management strategies.\u003c/p\u003e \u003cp\u003ePrevious studies have widely documented the antifungal activity of garlic-derived essential oils against a range of plant pathogenic fungi, including agents of foliar and soil-borne diseases. In most cases, growth inhibition has been reported at relatively high concentrations, with efficacy strongly influenced by garlic genotype, extraction method, and fungal species tested. Muy-Rangel et al (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) supported the antifungal potential of garlic essential oil, reporting a strong inhibitory effect against \u003cem\u003eAlternaria tenuissima\u003c/em\u003e, with a marked reduction in mycelial growth from 250 ppm and complete inhibition at 1,000 ppm (CI₅₀ = 229 ppm; MIC\u0026thinsp;=\u0026thinsp;1,023 ppm). Similarly, Hassan et al (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) reported complete inhibition of colony growth of \u003cem\u003eFusarium proliferatum\u003c/em\u003e and \u003cem\u003eMacrophomina phaseolina\u003c/em\u003e at high concentrations of garlic essential oil. These findings are in agreement with the antifungal activity observed in the present study, although differences in effective concentrations may be attributed to variations in fungal species and the chemical composition of the essential oil used. In addition, several studies have confirmed the antifungal activity of garlic essential oil against various phytopathogenic fungi, although at different concentrations and using diverse application methods (Wang et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Dabodhia et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Deshmukh et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2026\u003c/span\u003e). In comparison, the complete inhibition of \u003cem\u003eZymoseptoria tritici\u003c/em\u003e observed in the present study at low EO concentrations suggests a particularly strong antifungal potential of the local Algerian red garlic variety used. This enhanced activity may be related to the high relative abundance of diallyl trisulfide and diallyl disulfide identified in the essential oil, compounds that have been repeatedly associated with strong fungistatic and fungicidal effects. Similar sulfur-containing compounds were reported by Chen et al (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) to be the primary contributors to antifungal activity in garlic-derived products, with diallyl trisulfide exhibiting the highest potency. Although their study focused on isolated compounds, these findings support the hypothesis that diallyl sulfides play a central role in the antifungal effects observed in the present work.\u003c/p\u003e \u003cp\u003eIn contrast to previous investigations that primarily focused on garlic essential oil, very limited attention has been given to garlic hydrosol as an antifungal agent. Where hydrosols have been evaluated, their biological activity has often been reported as weaker or inconsistent when compared to essential oils. Notably, the present findings demonstrate that garlic hydrosol can achieve complete and stable inhibition of \u003cem\u003eZ. tritici\u003c/em\u003e at higher concentrations, highlighting a clear divergence from earlier observations, and indicating that hydrosol may represent an underexploited resource for disease control. This difference may be attributed to the presence of water-soluble sulfur compounds and other polar metabolites that are not retained in the oil fraction but are preserved in the aqueous distillate. Similarly, comparable studies on the antifungal activity of garlic hydrosol have confirmed its effectiveness against various fungal pathogens, supporting its potential as a bio-based antifungal agent. Tagoe et al (\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) reported that aqueous extracts of garlic exhibited marked growth inhibition against several phytopathogenic fungi, including \u003cem\u003eAspergillus flavus\u003c/em\u003e, \u003cem\u003eAspergillus niger\u003c/em\u003e, and \u003cem\u003eCladosporium herbarum\u003c/em\u003e, suggesting that water-soluble garlic constituents can contribute significantly to antifungal activity. Further insights into the chemical basis of this activity were provided by Galisteo et al (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), who identified a total of eight compounds in the organic fraction of garlic hydrolate. The most abundant compounds were diallyl disulfide, diallyl trisulfide, \u003cem\u003ep\u003c/em\u003e-methylpyridine, and methyl allyl trisulfide. Although these compounds are also present in garlic essential oil at higher relative proportions, their occurrence in the hydrolate supports the existence of antifungal-active constituents in the aqueous distillate, which may contribute to the distinct biological effects observed for hydrosols. More recently, Galisteo et al (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) highlighted the antifungal relevance of garlic hydrolate beyond its minor volatile composition. In their study, four novel oxygenated organosulfur compounds, termed garlicinals A\u0026ndash;D, were isolated from garlic hydrolate obtained from industrial agrowaste. These compounds, characterized by an α,β-unsaturated aldehyde structural motif, exhibited pronounced fungicidal activity, thereby reinforcing the potential of garlic hydrolate as an effective and underexploited agent for crop protection.\u003c/p\u003e \u003cp\u003eThe antifungal efficacy of garlic EO can be largely attributed to its high content of organosulfur compounds, as revealed by GC\u0026ndash;MS analysis. Diallyl trisulfide and diallyl disulfide were the dominant constituents, together accounting for more than 70% of the total volatile profile. These compounds are widely recognized for their strong antimicrobial properties as consistently demonstrated in previous studies by Perello et al (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), Sarfraz et al (2022), and Barbu et al (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and their ability to disrupt fungal cell membranes, inhibit key metabolic enzymes, and interfere with cellular redox balance (Wang et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Krid et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2025\u003c/span\u003e ). The dose-dependent inhibition observed in this study supports the hypothesis that synergistic interactions among sulfur-containing volatiles play a central role in suppressing \u003cem\u003eZ. tritici\u003c/em\u003e growth.\u003c/p\u003e \u003cp\u003eAlthough the hydrosol contained a markedly lower proportion of volatile sulfur compounds, it nevertheless achieved complete and stable inhibition of fungal growth at higher concentrations. This result is particularly noteworthy, as garlic hydrosol has received far less attention than essential oil in the context of plant disease control. The antifungal activity of the hydrosol may be associated with the presence of water-soluble sulfur compounds and other polar metabolites released during hydrodistillation Galisteo et al (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Unlike EO, whose activity declined rapidly over time, the hydrosol displayed sustained inhibitory effects, suggesting a greater stability under the experimental conditions. This characteristic could represent a practical advantage for agricultural applications, especially where ease of handling, safety, and formulation stability are critical considerations. The stability of garlic-derived bioactive compounds is strongly influenced by the environment in which they are present. Studies on allicin from \u003cem\u003eAllium sativum\u003c/em\u003e have demonstrated a markedly longer persistence in aqueous solutions than in oil-based ones, where rapid degradation occurs, with allicin remaining active for several days in water but only a few hours in vegetable oil (Fujisawa et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). This observation aligns with broader studies on hydrosols (aromatic waters), which report that their chemical composition\u0026mdash;particularly oxygenated compounds\u0026mdash;can remain relatively stable under typical storage conditions and in acidic aqueous environments (Garneau et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Moreover, hydrosols have been shown to exhibit good physicochemical stability under thermal stress and pH variation, further supporting their potential for sustained biological activity (Almeida et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Together, these findings suggest that aqueous garlic extracts and hydrosols may provide a more stable environment for bioactive sulfur compounds, which may contribute to the persistent antifungal effects observed in the present study.\u003c/p\u003e \u003cp\u003eThe temporal stability assay highlighted an important limitation of garlic EO, namely the rapid loss of antifungal efficacy when incorporated into the growth medium and stored prior to inoculation. This decline is most likely due to the high volatility and oxidative sensitivity of organosulfur compounds, which may evaporate or degrade during storage (Zhao et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These findings emphasize that, while EO is highly potent when freshly applied, its practical use in crop protection would require appropriate formulation strategies to enhance stability and prolong bioactivity. Approaches such as encapsulation, emulsification, or incorporation into carrier systems may help overcome these limitations and improve field applicability (Mossa et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Gong et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), Bouqellah et al (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) reported that encapsulation of \u003cem\u003eAllium sativum\u003c/em\u003e essential oil in silver nanoparticles (AgNPs) enhances its stability and antifungal activity. The AgNPs act as carrier systems, improving the persistence and controlled release of volatile compounds, which results in increased inhibition of fungal growth. From a broader perspective, the use of garlic-derived extracts aligns well with current efforts to reduce reliance on chemical fungicides and mitigate the development of fungicide-resistant \u003cem\u003eZ. tritici\u003c/em\u003e populations. The strong antifungal activity observed, combined with the local availability of garlic and the potential valorization of hydrosol as a low-cost by-product, supports the integration of these natural products into sustainable and circular bioeconomy-based plant protection systems. However, further research is required to validate these findings under field conditions, to optimize formulations, and to assess possible synergistic effects with other biological or reduced-risk control strategies.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThis study demonstrates the strong antifungal potential of bio-based extracts derived from a locally cultivated Algerian red garlic (\u003cem\u003eAllium sativum\u003c/em\u003e L.) variety against \u003cem\u003eZymoseptoria tritic\u003c/em\u003ei, the causal agent of Septoria tritici blotch (STB) in wheat. Comprehensive morphological, physicochemical, and chemical analyses revealed a volatile profile dominated by organosulfur compounds, particularly diallyl disulfide and diallyl trisulfide, that are responsible for the extracts\u0026rsquo; biological activity. \u003cem\u003eIn vitro\u003c/em\u003e assays showed that the essential oil (EO) achieved complete growth inhibition at remarkably low concentrations (0.30 \u0026micro;L/mL), whereas the hydrosol, despite its lower sulfur content, displayed full antifungal efficacy at 150 \u0026micro;L/mL, marking the first report of such activity for this aqueous distillation byproduct.\u003c/p\u003e \u003cp\u003eMechanistic insights suggest that these bioactive molecules act synergistically to disrupt fungal membranes, inhibit key metabolic enzymes, perturb redox homeostasis, and modulate gene expression, thereby compromising fungal survival. However, the EO\u0026rsquo;s bioactivity declined significantly over time, underscoring the need for formulation strategies, such as encapsulation or carrier-based delivery systems, to improve stability and prolong field efficacy.\u003c/p\u003e \u003cp\u003eOverall, the findings highlight the potential of garlic-derived EO and hydrosol as natural, eco-friendly biofungicides that could reduce reliance on synthetic chemicals in wheat disease management. Their local availability, low toxicity, and multifaceted modes of action make them promising candidates for integration into sustainable crop protection strategies and circular bioeconomy models. Future research should prioritize formulation optimization, field validation, and the exploration of synergistic combinations with other biocontrol agents to translate these laboratory findings into effective agricultural solutions.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflicts of Interest:\u003c/h2\u003e \u003cp\u003eThe authors declare no conflicts of interest\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis study was supported within the scope of the Project also funded under the National Recovery and Resilience Plan (NRRP), Mission 4 Component 2 Investment 1.4 - Call for tender No. 3138 of December 16, 2021, rectified by Decree n.3175 of December 18, 2021 of Italian Ministry of University and Research funded by the European Union \u0026ndash; NextGenerationEU; Award Number: Project code CN_00000033, Concession Decree No. 1034 of June 17, 2022 adopted by the Italian Ministry of University and Research, CUP: D43C22001260001, Project title \u0026ldquo;National Biodiversity Future Center - NBFC\u0026rdquo;. DGRSDT (mesrs): General Directorate of Scientific Research and Technological Development - Ministry of Higher Education and Scientific Research, Algeria.\u003c/p\u003e\u003ch2\u003eAuthor Contributions:\u003c/h2\u003e \u003cp\u003eConceptualization, R.D., I.S. and A.D.; methodology, R.D., I.S., A.D. and F.D.; software, F.D., C.B. and MDE; formal analysis, R.D., F.D. and C.B.; investigation, R.D.; resources, R.D. A.B. and I.S.; data curation, F.D., M.D.E., L.R; and F.B. writing\u0026mdash;original draft preparation, R.D. M.D.E and L.R.; writing\u0026mdash;review and editing, I.S. M.D.E, and L.R.; visualization, R.D.; supervision, L.B.; project administration, L.B., L.R. and F.B. ; funding acquisition, R.D. and L.R. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments:\u003c/h2\u003e \u003cp\u003eThe authors would like to thank the Technical Institute for Vegetable and Industrial Crops (ITCMI), the National Institute of Agronomic Research of Algeria (INRAA) especially Mrs. Meamiche Hayat, and the Biotechnology Research Center (CRBt), Algeria, for their valuable contribution to this work.\u003c/p\u003e\u003ch2\u003eData Availability Statement:\u003c/h2\u003e \u003cp\u003eThe original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAcheampong A, Borquaye LS, Acquaah SO, Osei-Owusu J, Tuani GK (2015) Antimicrobial activities of some leaves and fruit peels hydrosols. 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Foods 10:1637. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/foods10071637\u003c/span\u003e\u003cspan address=\"10.3390/foods10071637\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-plant-diseases-and-protection","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jpdp","sideBox":"Learn more about [Journal of Plant Diseases and Protection](https://www.springer.com/journal/41348)","snPcode":"41348","submissionUrl":"https://www.editorialmanager.com/jpdp","title":"Journal of Plant Diseases and Protection","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Zymoseptoria tritici, Septoria tritici blotch, antifungal activity, garlic essential oil, garlic hydrosol, sustainable plant protection","lastPublishedDoi":"10.21203/rs.3.rs-8612946/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8612946/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSeptoria tritici blotch (STB), caused by \u003cem\u003eZymoseptoria tritici\u003c/em\u003e, is one of the most damaging foliar diseases of bread wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e L.), leading to severe yield losses and increasing reliance on synthetic fungicides. The emergence of fungicide-resistant populations and growing environmental concerns highlight the need for sustainable alternative control strategies. In this study, the antifungal activity of essential oil (EO) and hydrosol obtained from a local Algerian red garlic (\u003cem\u003eAllium sativum\u003c/em\u003e L.) variety was evaluated \u003cem\u003ein vitro\u003c/em\u003e against \u003cem\u003eZ. tritici.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eGarlic extracts were characterized through morphological, physicochemical, and chemical analyses, with EO volatile composition determined by GC\u0026ndash;MS. Twenty-one compounds accounting for 99.97% of the total EO composition were identified, dominated by organosulfur compounds, particularly diallyl trisulfide (36.99%) and diallyl disulfide (35.78%). Antifungal assays revealed a strong dose-dependent inhibition of mycelial growth. Garlic EO completely inhibited fungal growth at 0.30 \u0026micro;L mL⁻\u0026sup1;, although a reduction in efficacy over time suggested limited stability. Notably, garlic hydrosol achieved complete and stable inhibition at 150 \u0026micro;L mL⁻\u0026sup1;, despite its lower sulfur content, representing a novel and underexplored approach for STB control.\u003c/p\u003e \u003cp\u003eThese findings demonstrate the potential of garlic-derived EO and hydrosol as eco-friendly antifungal agents and support their possible integration into sustainable disease management strategies for wheat, contributing to the reduction of chemical fungicide inputs.\u003c/p\u003e","manuscriptTitle":"Antifungal Activity of Garlic (Allium sativum L.) Essential Oil and Hydrosol Against Zymoseptoria tritici, the Causal Agent of Septoria Tritici Blotch in Bread Wheat","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-05 11:44:30","doi":"10.21203/rs.3.rs-8612946/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2026-03-12T11:15:30+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2026-02-03T17:32:33+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-03T16:32:55+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Journal of Plant Diseases and Protection","date":"2026-01-21T09:06:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-19T22:33:16+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Plant Diseases and Protection","date":"2026-01-15T13:12:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-plant-diseases-and-protection","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jpdp","sideBox":"Learn more about [Journal of Plant Diseases and Protection](https://www.springer.com/journal/41348)","snPcode":"41348","submissionUrl":"https://www.editorialmanager.com/jpdp","title":"Journal of Plant Diseases and Protection","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ec0cfb50-19c1-4da5-a8c4-c580a170c807","owner":[],"postedDate":"February 5th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-14T10:32:20+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-05 11:44:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8612946","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8612946","identity":"rs-8612946","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}