Chemical composition and in vitro/in vivo antisaprolegniosis efficacy of Satureja Bachtiarica and Achillea Talagonica essential oils on rainbow trout eggs

Abstract

The rising prevalence of fungal infections in aquaculture, combined with the high cost, adverse side effects, and increasing resistance associated with synthetic antifungal agents, has stimulated interest in natural alternatives. This study investigated the chemical composition and antifungal activity of essential oils derived from Satureja bachtiarica Bunge and Achillea talagonica Boiss against Saprolegnia parasitica, the causative agent of saprolegniosis. Gas chromatography- mass spectrometry analysis revealed that the predominant constituents of the S. bachtiarica essential oil were carvacrol (71.61%), γ-terpinene (12.71%), p-cymene (7.01%), α-terpinene (1.74%), and β-bisabolene (1.39%). In contrast, the main components of A. talagonica oil included camphor (25.26%), chrysanthenone (23.68%), α-pinene (11.85%), 1,8-cineole (6.25%), caryophyllene oxide (5.20%), and camphene (3.27%). Both essential oils demonstrated significant antifungal activity. The minimum inhibitory concentrations were 1.56 µL/mL and 3.12 µL/mL, while the minimum fungicidal concentrations were 3.12 µL/mL and 6.25 µL/mL for S. bachtiarica and A. talagonica, respectively. In vivo assays demonstrated that both essential oils significantly inhibited the transmission of S. parasitica from contaminated to healthy eggs, yielding survival rates of 88.84% for S. bachtiarica and 80.40% for A. talagonica, compared with 75.83% in the untreated control group. During incubation, the essential oils also prevented contamination, resulting in survival rates of 89.40% and 87.43%, respectively, relative to 85.33% in the control group. These findings indicate that these essential oils are promising natural alternatives to conventional antifungal agents such as malachite green and formalin. However, further studies are necessary to fully evaluate their environmental safety and potential impact on non-target organisms.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-33870-2.

Introduction

Aquaculture, a rapidly growing global food production sector, faces ongoing challenges from infectious diseases, especially those caused by waterborne fungal pathogens. Among these, Saprolegnia parasitica, a ubiquitous oomycete, is a primary pathogen responsible for saprolegniosis in freshwater fish and their eggs^1,2. This disease leads to significant economic losses due to high mortality rates and reduced fish quality^2,3. Conventional antifungal agents, such as malachite green, formalin, hydrogen peroxide, and sodium chloride, have been employed for decades to control saprolegniosis. However, their long-term efficacy is compromised by significant environmental concerns⁴. Moreover, the use of these agents raises issues related to toxicity, potential carcinogenicity, and residues in edible fish products, which can increase susceptibility to suprainfection^5–8, Compounding these issues is the growing problem of antifungal resistance, which increasingly renders conventional treatments less effective and emphasizes the urgent need for alternative antimicrobial strategies with novel mechanisms of action⁹.

In recent years, there has been growing interest in exploring natural alternatives in aquaculture to ensure fish welfare, minimize environmental impact, and promote economic benefits¹⁰. Numerous studies have demonstrated the effectiveness of herbal medicines in controlling pathogenic fungi responsible for saprolegniasis^11–15. Plants from the Lamiaceae and Asteraceae families, known for their antimicrobial properties, are frequently utilized to combat various pathogens^16,17. Satureja bachtiarica Bunge, a member of the Lamiaceae family, is particularly notable for its antimicrobial and antifungal capabilities, mainly attributed to compounds such as p-cymene, terpinene, menthone, thymol, carvacrol^18,19. Similarly, Achillea talagonica Boiss, a prominent representive of the Asteraceae family, has attracted attention for its antioxidant, analgesic, cytotoxic, antibacterial, and antifungal properties. These beneficial effects are largely attributed to its diverse array of secondary metabolites, including acetylenes, flavonoids, phenolic acids, and mono- and sesquiterpenes²⁰. Despite the well-documented antimicrobial properties of numerous Lamiaceae and Asteraceae species, critical knowledge gaps persist, hindering the development of plant-derived antifungals for saprolegniosis control in aquaculture. First, although several Satureja and Achillea species have shown promising in vitro activity, no previous study has performed a direct comparative evaluation of a carvacrol-dominated Satureja oil with a camphor/chrysanthenone-rich Achillea oil against the same S. parasitica isolates under identical experimental conditions. Second, most prior investigations have been restricted to in vitro assays or limited-scale in vivo studies that do not simultaneously evaluate both curative (inhibition of fungal transmission from infected to healthy eggs) and preventive (protection of uncontaminated eggs during incubation) efficacy in rainbow trout eggs (Oncorhynchus mykiss), the most vulnerable and economically critical life stage. Third, no study has yet evaluated the essential oils of the regionally abundant Iranian endemic species S. bachtiarica Bunge and A. talagonica Boiss in an aquaculture setting, despite their exceptionally high contents of carvacrol and oxygenated monoterpenes, respectively.

The present investigation directly addresses these critical gaps by (i) comprehensively characterizing the chemical composition of essential oils derived from two understudied Iranian plants, (ii) evaluating their in vitro and in vivo antisaprolegniosis activity using standardized and replicated challenge models in rainbow trout (O. mykiss) eggs, and (iii) performing the first systematic comparative assessment of a highly carvacrol-rich versus a camphor/chrysanthenone-rich essential oil profile in both transmission-inhibition and prophylactic contexts. Collectively, this work provides novel and directly comparable evidence on the potential of these native plant resources as safe, effective, and sustainable alternatives to conventional antifungal agents such as malachite green and formalin in commercial trout hatcheries.

Materials and methods

Plant collection and preparation

The aerial and flowering branches of S. bachtiarica and A. talagonica were collected during their flowering period (June and August 2023) from Chaharmahal and Bakhtiari Province, Iran. The specimens were formally identified by Dr. Hamze Ali Shirmardi, a botanist at the Research Center for Agricultural and Natural Resources of Chaharmahal and Bakhtiari province in Shahrekord, Iran, Identification was confirmed by morphologically examining the specimens reproductive and vegetative characteristics (e.g., inflorescence, floral structures, leaf morphology, and indumentum) and comparison with authentic botanical samples and monographs, including the Flora of Iran²¹. Voucher specimens were deposited at the herbarium of the same center with the following deposition numbers: No. 7586 for S. bachtiarica and No. 7587 for A. talagonica. As these are native, naturally growing, and non-endangered species in the region, no specific permits were required for their collection. Following authentication, the plant materials were dried at room temperature in the shade to prevent direct sunlight exposure and preserve their quality. Subsequently, the dried materials were ground into a fine powder using an Endecott food processor. To prevent thermal degradation, grinding was performed in short, low-speed intervals. The resulting powder was then sieved through a 16-mesh screen to achieve uniform particle size and eliminate debris²². Finally, the powder was stored in airtight, dark containers at 4 °C for no longer than two weeks prior to hydrodistillation.

Extraction procedures

Essential oils were extracted from the powdered plant materials by steam distillation using a Clevenger apparatus (British Pharmacopoeia) for 3 h¹⁹. The distillation rate was maintained at 2.5–3.0 mL distillate/min. The extracted oils were dehydrated over anhydrous sodium sulfate and stored at 4 °C in the dark for a maximum of one month prior to GC–MS analysis and antifungal assays. All chemical composition analyses and biological assays were completed within this one-month period, ensuring no degradation of volatile constituents or loss of antifungal activity. The essential oil yield was calculated based on the dry weight of the plant material and is expressed as mL/100 g. The formula used for this calculation was:

Essential oil yield (mL/100 g) = (Volume of essential oil obtained (mL) / Weight of dry plant material (g)) × 100.

Identification of essential oils components

The chemical composition of each essential oil was determined using both gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). Since only a single measurement was performed for each oil, replicate values and standard deviations were not applicable. Gas chromatography (GC) analysis was performed using an Agilent 7890 A system with a flame ionization detector (FID) and an HP-5 capillary column (30 m × 0.25 mm id × 0.25 μm film thickness). Helium was used as the carrier gas at a constant flow rate of 0.8 mL/min. The oven temperature was programmed as follows: an initial temperature of 60 °C was held for 4 min, then increased at a rate of 4 °C/min to 280 °C, and finally held at 280 °C for 10 min. The injection temperature was 290 °C, with 1 µL sample injected at a 1:10 split ratio. Gas chromatography-mass spectrometry (GC–MS) analysis was conducted using an Agilent 5975 system, equipped with an HP-5MS column (30 m × 0.25 mm id, 0.25 μm film thickness). The same oven temperature program and carrier gas flow rate as in the GC-FID analysis were used. Injector and interface temperatures were 290 °C and 300 °C, respectively. Mass spectra were obtained using electron impact (EI) ionization at 70 eV, with a mass range of 50–550 m/z. The ion source and detector temperatures were maintained at 250 °C and 150 °C, respectively.

The identification of chromatographic peaks was achieved by comparing their calculated Kovats retention indices with those of genuine standards. Kovats retention indices were calculated by linear interpolation based on the retention times of a homologous series of n-alkanes (C₅ to C₂₄). The accuracy of the results was further verified by comparing the mass spectra of each component with those in the widely recognized Wiley 7.0 and Adams mass spectral-Kovats index libraries²³. Compound identification was confirmed only when the mass spectral match similarity to library standards exceeded 90%. Peak area percentages were calculated from the chromatograms obtained with the HP-5 column, without applying FID response factors. Relative standard deviations for peak areas were maintained below 5%.

Isolation and Identification of S. parasitica

To isolate and identify S. parasitica, the shells of infected fish eggs were cultured on Yeast Glucose Chloramphenicol (YGC) agar (20 g glucose, 5 g yeast extract, 0.1 g chloramphenicol, 14.9 g agar per liter). The plates were incubated at 18 °C for 8 days. Pure cultures were achieved by transferring a small portion of the mycelium growth margin to a fresh culture medium, a process that was repeated three times²⁴. Isolates were characterized based on macroscopic features (colony, morphology, pigmentation, growth pattern) and microscopic features (absence of septa, sexual and asexual structures)²⁵. To induce zoospore formation, a 0.5 cm disk from the edge of a 3-day-old mycelial culture was transferred to 30 mL Potato Dextrose Broth (PDB) and incubated at 20 °C. After 24 h, the mycelium was rinsed twice with sterile water and incubated for an additional 24 h. Mature sporangia and zoospores were then observed under a light microscope. Secondary zoospores were identified using lactophenol-stained wet mounts and standard identification keys^24,25.

Preparation of zoospore suspension

A zoospore suspension was prepared as described by Tandel et al²⁶. Actively growing mycelial disks (7 mm in diameter) of S. parasitica were immersed in 50 mL sterile aquarium water supplemented with chloramphenicol (0.5 mg/L) and Tween 20 (0.1%), and incubated at 20 ± 2 °C for 18 h. Zoospores were then collected, and their concentration was determined using a hemocytometer to achieve a final concentration of 1 × 10⁶ zoospores/mL. To verify accuracy, serial dilutions were prepared and inoculated onto three YGC agar plates, which were subsequently incubated at 20 °C for one week^25,27.

In vitro antifungal assays

The Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC) of the essential oils were determined using serial macro- and microdilution Assays.

Macrodilution assay

The essential oils were initially diluted in a solution of 1% dimethyl sulfoxide (DMSO) and 0.1% Tween 80. To assess the antifungal effect of the essential oils, a two-fold serial dilution of each oil was prepared. A 1 mL aliquot of each dilution was then added to seven separate tubes, each containing 1 mL 2X YGC broth with a final concentration of 1 × 10⁶ zoospores/mL. This resulted in final essential oil concentrations of 50, 25, 12.5, 6.25, 3.12, 1.56, and 0.78 µL/mL^28,29. As positive control, malachite green was similarly diluted to achieve final concentrations ranging from 50 µg/mL to 0.78 µg/mL. A placebo control was prepared by adding 1 mL of the 1% DMSO and 0.1% Tween 80 mixture to a series of three tubes without essential oils. To ensure the sterility of the process, a negative control was included, consisting of three tubes containing sterile water and sterile culture medium.

Following incubation, the MIC was defined as the lowest oil concentration that prevented visible fungal growth. To determine the MFC, samples from the MIC and all higher concentrations were plated on YGC agar. After 6 days of incubation at 18 °C, the MFC was recorded as the lowest concentration that resulted in complete absence of fungal growth.

Microdilution assay

The microdilution method was used to evaluate the antifungal activity of the essential oils²⁶. This assay was performed in triplicate on separate 96-well plates for each essential oil and each reference compound to eliminate any possibility of cross-contamination or volatile interactions between oils. A stock solution of each essential oil (1% in YGC broth with 1% DMSO and 0.1% Tween 80) was prepared. Each plate contained two-fold serial dilutions of a single test substance (S. bachtiarica oil, A. talagonica oil, malachite green, or formalin) ranging from 100 to 1.56 µL/mL (or µg/mL for reference compounds). In each plate, columns 9–10 contained solvent control (1% DMSO and 0.1% Tween 80), and columns 11–12 contained fungal growth control (fungal culture only). Each well was inoculated with 100 µL of zoospore suspension (2 × 10⁶ zoospores/mL). Plates were incubated at 18 °C for 6 days. The MIC was determined as the lowest concentration with no visible fungal growth. For MFC determination, samples from the MIC and higher concentrations were plated on YGC agar and incubated at 18 °C for 6 days, and the MFC was defined as the lowest concentration that showed no fungal growth.

In vivo antifungal assays

Inhibition of infection transmission

The efficacy of essential oils in inhibiting the transmission of infection was assessed as described by Sharifpour et al³⁰. Five hundred uncontaminated fertilized trout eggs were placed in each of six baskets (4 × 12 × 12 cm), each containing 10 infected eggs wrapped in cotton netting. The baskets were then transferred to six single-row California troughs. Infection was monitored daily. To ensure uniform contamination, eggs were exchanged between trays once the infection reached approximately 10% (after around 3 days). To accelerate the infection process, water flow was stopped for 2 h, twice daily. After 72 h, each trough was treated daily for 1 h with either essential oils or controls. Essential oil treatments were applied at twice the MFC determined by macrodilution (6.25 µL/L for S. bachtiarica and 12.5 µL/L for A. talagonica), a standard practice when moving from in vitro assay to short term bath applications of plant essential oils in salmonid egg incubation^13–15,31. Malachite green (12.5 mg/L) and formalin (50 mg/L) were used at established hatchery doses correspond to 2 × their reported MFCs in flow through systems³.

The treatments were as follows: the first trough (control) received no antifungal or disinfectant; the second trough (placebo) contained 10 mg/L of DMSO and 1 mg/L of Tween 80; the third trough contained 50 mg/L of formalin; the fourth trough received 12.5 mg/L of malachite green; the fifth trough contained 12.5 mL/L of A. talagonica essential oil with 1% DMSO and 0.1% Tween 80; and the sixth trough contained 6.25 mL/L of S. bachtiarica essential oil with 1% DMSO and 0.1% Tween 80. All treatments were conducted in triplicate and continued for 18 to 20 days until the eggs reached the eyed stage. Throughout the experiment, constant conditions were maintained: incoming water flow at 12 L/min, a temperature of 7–11 °C, dissolved oxygen around 8 ppm, and a pH of approximately 7.7. At the study’s conclusion, the hatching percentage and survival rate of larvae weighing up to 1 g were calculated²⁷. The following formulas were used:

Fungal infected eggs = (Number of infected eggs/Total number of eggs) × 100.

Eyed egg percent = (Number of eyed eggs/ (Total number of eggs-Total number of primary mortalities)) × 100.

Hatchability percent = (Number of hatched eggs/Total number of eggs) × 100.

Prevention of contamination

To examine the efficacy of S. bachtiarica and A. talagonica essential oils in preventing contamination of freshly fertilized trout eggs by S. parasitica during incubation, newly fertilized eggs were placed in fiberglass troughs (six troughs, each containing three trays of 7,000 eggs). The experimental groups included: (1) negative control (no treatment), (2) placebo (10 mg/L DMSO + 1 mg/L Tween 80), (3–4) essential oils at twice the MIC, (5–6) positive controls (malachite green and formalin). Each treatment was replicated three times and applied daily for 30 min. Dead eggs were carefully siphoned and counted post-hatching. At the conclusion of the study, live larvae weighing up to 1 g were counted²⁷.

Statistical analyses

Data are reported as the mean ± standard deviation (SD). All statistical analyses were performed using GraphPad Prism 10.0 (GraphPad Software, San Diego, CA, USA). A one-way analysis of variance (ANOVA) followed by Tukey’s HSD post hoc test was used to identify significant differences at p ≤ 0.05. Prior to analysis, the assumptions of normality (using the Shapiro-Wilk test) and homogeneity of variance (using Levene’s test) were validated.

Results

Essential oils extraction yield

The extraction yields of the essential oils from the aerial parts of S. bachtiarica and A. talagonica were 2.0 mL/100 g and 0.18 mL/100 g of dry plant material, respectively.

Essential oil s’ chemical composition

The phytochemical constituents identified in the essential oils of S. bachtiarica and A. talagonica by GC–MS analysis are presented in Tables 1 and 2. Eighteen compounds accounting for 99.82% were identified in S. bachtiarica essential oil samples, and the major constituents were Carvacrol (71.61%), γ-Terpinene (12.71%), Cymene (7.01%), α-Terpinene (1.74%), and β-bisabolene (1.39%). In the A. talagonica essential oil, thirty-two compounds were identified, representing 99.13% of the total composition. The principal constituents included Camphor (25.26%), Chrysanthenone (23.68%), α-Pinene (11.85%), 1,8-Cineole (6.25%), Caryophyllene oxide (5.2%), and Camphene (3.27%).

Table 1.

Chemical composition of S. bachtiarica essential oil.

Constituents	Kovats index (KI)	Percentage
α-Thujene	926	0.32
α-Pinene	934	0.63
Camphene	949	0.15
β-Pinene	978	0.25
β-Myrcene	989	0.43
α-Phellandrene	1006	0.12
α-Terpinene	1017	1.74
Cymene	1025	7.01
Limonene	1029	0.23
1,8-Cineole	1031	0.09
γ-Terpinene	1058	12.71
Borneol	1169	0.14
Dihydrocarvone	1201	0.12
Carvacrol	1307	71.61
Carvacryl acetate	1374	0.97
β-caryophellene	1424	0.95
β-bisabolene	1509	1.39
Caryophyllene oxide	1588	0.96
Total		99.82

Kovats index (KI) calculated against n-alkanes.

Table 2.

Chemical composition of A. talagonica essential oil.

Constituents	Kovats index (KI)	Percentage
α-Pinene	934	11.85
Camphene	949	3.27
β-Sabinene	974	0.58
β-Pinene	978	0.67
Myrcene	991	0.48
α-Phellandrene	1013	0.49
α-Terpinolene	1017	0.44
p-Cymene	1024	2.32
Limonene	1028	0.36
1,8-Cineole	1031	6.25
γ-Terpinene	1058	0.64
Terpinolene	1089	0.48
trans-Sabinene hydrate	1099	0.5
α-Thujone	1102	1.34
β-Thujone	1105	1.42
cis-p-Menth-2-en-1-ol	1106	0.67
Chrysanthenone	1127	23.68
Camphor	1148	25.26
Isoborneol	1158	1.37
Borneol	1165	1.1
Myrtenal	1213	0.65
Chavicol	1263	2.48
α-Copaene	1285	0.4
cis-Carveol	1339	0.35
Carvone	1346	0.91
α-Ylangene	1363	0.42
β-Caryophyllene	1424	0.73
β-Selinene	1483	0.35
Germacrene D	1515	2.43
Bicyclogermacrene	1580	0.53
Caryophyllene oxide	1588	5.2
α-Cadinol	1656	1.51
Total		99.13

Kovats index (KI) calculated against n-alkanes

Identification of S. parasitica

Microscopic examination confirmed the identity of the isolated fungus as S. parasitica. Observations revealed long, branched, aseptate hyphae, along with cylindrical and elongated zoosporangia on fertile hyphae. Furthermore, spherical zoospores within double walls were clearly visible (Fig. 1).

Fig. 1.

S. parasitica (100X). (A) arrow indicates branched aseptate hyphae; (B) Arrow indicates an elongated zoosporangium.

In vitro antifungal activity (MIC and MFC values of essential oils against S. parasitica)

The results of the in vitro macro- and microdilution assays against S. parasitica are summarized in Table 3. The essential oil from S. bachtiarica demonstrated superior antifungal efficacy compared to both malachite green and formalin, with Its MIC and MFC values were 1.56 and 3.12 µL/mL, respectively. A. talagonica essential oil also exhibited strong activity, with MIC and MFC values of 3.12 and 6.25 µL/mL, respectively. These values were comparable to those of malachite green (MIC = 3.12 µg/mL, and MFC = 6.25 µg/mL) and superior to formalin (MIC = 12.5 and MFC = 25 µg/mL). The solvent control (1% DMSO + 0.1% Tween 80) showed no antifungal activity, confirming that the observed effects were attributable solely to the essential oils.

Table 3.

Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values of different experimental groups against S. parasitica (Mean ± SD, n = 3).

Groups	MIC	MFC
Control	–	–
Placebo	> 100	> 100
Formalin	12.5 ± 0.00 (µg/mL)	25.00 ± 0.00 (µg/mL)
Malachite green	3.12 ± 0.00 (µg/mL)	6.25 ± 0.00 (µg/mL)
A. talagonica	3.12 ± 0.00 (µL/mL)	6.25 ± 0.00 (µL/mL)
S. bachtiarica	1.56 ± 0.00 (µL/mL)	3.12 ± 0.00 (µL/mL)

MIC , Minimum inhibitory concentration; MFC , Minimum fungicidal concentration

Values are from three fully independent biological replicates performed on separate days with freshly prepared zoospore suspensions and essential-oil dilutions. In all three repetitions the results were exactly identical, therefore SD = 0.00 µL/mL or µg/mL

Effects of essential oils on inhibiting infection transmission

Both S. bachtiarica and A. talagonica essential oils significantly inhibited the transmission of S. parasitica infections from contaminated to healthy eggs. As detailed in Table 4, the survival rates from the eyed stage to hatching were highest in the S. bachtiarica group, comparable to the malachite green group, and significantly higher than the control (p ≤ 0.05). Although A. talagonica was slightly less effective, it still provided a marked improvement over the control, with a survival rate of 87.43 ± 0.09%. Post-hatching, the survival rates of larvae weighing up to 1 g followed a similar pattern. The S. bachtiarica group yielded the highest survival, closely followed by malachite green. The A. talagonica essential oil resulted in a larval survival rate of 97.83 ± 0.04%, which, while lower than S. bachtiarica, remained significantly higher compared to the control group (p ≤ 0.05). These results indicate that both essential oils are effective in controlling infection transmission during critical development stages, with S. bachtiarica demonstrating performance comparable to malachite green.

Table 4.

The efficiency (Mean ± SD) of S. bachtiarica and A. talagonica essential oils against S. parasitica on the survival rate (%) of rainbow trout eggs and larvae.

Survival rate (%)
Groups	Inhibiting saprolegniasis transmission			Protective effects against saprolegniasis
	Up to eyed stage	Eyed stage-hatching stage	Up to 1 g	Up to eyed stage	Eyed stage-hatching stage	Up to 1 g
Control	43.6 ± 1.9a	75.83 ± 0.63a	92.12 ± 0.36a	75.01 ± 0.34a	85.33 ± 0.25a	97.73 ± 0.04a
Placebo	43.53 ± 1.33a	75.95 ± 0.04a	92.13 ± 0.24a	75.03 ± 0.31a	85.27 ± 0.17a	97.78 ± 0.03ab
Formalin	56.93 ± 0.9b	81.97 ± 0.23b	94 ± 0.35b	81.84 ± 0.28b	88.21 ± 0.17b	98.04 ± 0.11ab
Malachite green	66.47 ± 1.41c	88.66 ± 0.27c	96.04 ± 0.13c	84.77 ± 0.26c	89.45 ± 0.25c	98.07 ± 0.09ab
A. talagonica	53.8 ± 1.44b	80.4 ± 1.28 d	93.06 ± 0.17d	79.9 ± 0.28d	87.43 ± 0.09d	97.83 ± 0.31ab
S. bachtiarica	64.53 ± 1.33c	88.84 ± 0.31c	96.16 ± 0.34c	84.74 ± 0.27c	89.04 ± 0.22c	98.14 ± 0.18b

Different superscript letters (^{a, b, c, d}) within a column indicate significant differences among the groups (P ≤ 0.05)

Effects of essential oil on preventing contamination

The preventive effects of S. bachtiarica and A. talagonica essential oils against S. parasitica contamination during the incubation period were notable. As shown in Table 4, the hatching rates in the S. bachtiarica group were the highest, comparable to those observed with malachite green, and significantly higher than the untreated control (p ≤ 0.05). A. talagonica was slightly less effective but still demonstrated considerable efficacy, showing a significant improvement over the control group. Both essential oils effectively reduced contamination and enhanced egg survival during incubation. At the post-hatching stage, survival rates of larvae up to 1 g body weight were also significantly improved in the S. bachtiarica group compared to the control, and were closely followed by malachite green (p ≤ 0.05). A. talagonica essential oil also maintained a strong survival rate, which was higher than the control group. These results further emphasize the potential of these essential oils, particularly S. bachtiarica, as safe, natural alternatives to synthetic antifungal treatments in preventing contamination during egg incubation.

Discussion

Saprolegniosis, a major fungal infection, poses significant challenges during rainbow trout egg incubation. In this study, S. parasitica was identified based on morphological criteria, including the presence of long, branched, aseptate hyphae, cylindrical and elongated zoosporangia, and spherical zoospores within double walls, features characteristic of this pathogen^24,25. For decades, hatchery management has relied on laborious manual removal of dead eggs or the application of chemical agents to control fungal outbreaks. Common antifungal chemicals, such as malachite green, formalin, hydrogen peroxide, sodium chloride, copper sulfate, potassium permanganate, and methylene blue, have been widely used. However, many of these substances are either insufficiently effective or pose significant risks, including the accumulation of chemical residues in fish tissues, potential carcinogenicity, teratogenic effects, the development of resistant fungal strains, and disruption of aquatic ecosystems^4–8. These significant concerns have prompted the search for safe and environmentally friendly alternatives that minimize reliance on hazardous chemicals, thereby ensuring both consumer and environmental safety. In this context, plant-derived products, particularly essential oils, have attracted increasing attention as potential antifungal agents in aquaculture.

Chemical composition and yield variability

In the current study, the essential oils from S. bachtiarica and A. talagonica were obtained by hydro-distillation, yielding 2 mL/100 g and 0.18 mL/100 g dry matter, respectively. Previous studies have reported varying yields for S. bachtiarica essential oil, including 1.4–2.1 mL/100 g¹⁹, 1.65–2.15%³², 1.8 mL/100 g²², and 2.7%³³. Similarly, the yield of A. talagonica essential oil has been documented by various researchers, including Saeidnia et al.³⁴ at 0.2 mL/100 g, and Niazpoor et al.³⁵ at 0.236 mL/100 g. These results are consistent with the findings of the present study. However, variations in essential oil yields can arise from several factors, such as the plant’s geographical origin, age at extraction, environmental and seasonal conditions, cultivation practices, and harvesting time^36,37. Importantly, the extraction technique itself can significantly influence both the quality and yield of active compounds. While traditional methods like hydro-distillation, steam distillation, cold pressing, solvent extraction, and simultaneous distillation-extraction are widely used^38,39, they present challenges such as the loss of volatile components, reduced extraction efficiency, degradation of unsaturated compounds, and potential toxic residues^39,40. To address these issues, advanced methods such as supercritical fluid extraction, subcritical liquid extraction, solvent-free microwave extraction, and ultrasound extraction have been developed, offering improved efficiency and the potential to capture a broader spectrum of bioactive compounds³⁷.

In this study, GC-MS analysis of S. bachtiarica essential oil identified 18 compounds, with the phenolic monoterpene carvacrol as the major component, constituting 71.61% of the total composition. Carvacrol is well-known for its broad-spectrum antimicrobial and antifungal properties, making it a key driver of the oil’s efficacy. In addition, other monoterpenes such as γ-terpinene (12.71%) and p-cymene (7.01%) were identified. Although present in smaller amounts, these compounds are also recognized for their antifungal activity. Together, these three compounds constitute over 90% of the essential oil’s composition, providing a strong indication that these are the primary contributors to the oil’s potency against S. parasitica. In line with our results, many researchers have reported that the main compounds of different Satureja species are various monoterpenes like carvacrol and thymol, along with other substances such as γ-terpinene, p-cymene, and α-terpinene, albeit in different percentages^18,32,33. Similarly, the essential oil of A. talagonica was rich in camphor (25.26%) and chrysanthenone (23.68%), both of which are known for their antifungal properties. These two compounds together constitute nearly 50% of the total oil composition and are likely responsible for the majority of the oil’s activity. Consistently, Saeidnia et al.³⁴ also recognized the same compounds as major components in the essential oil of A. talagonica.

Antifungal mechanisms and comparisons

The potent antifungal activity of S. bachtiarica and A. talagonica essential oils against S. parasitica is primarily attributed to their major constituents, carvacrol (71.61%, a phenolic monoterpene) and camphor (25.26%, bicyclic monoterpene ketone), respectively. These compounds disrupt fungal cell membranes integrity and interfere with metabolic processes^10,34. However, minor constituents, though present in lower concentrations, likely contribute to the overall efficacy through synergistic interactions⁴¹.

In S. bachtiarica, minor compounds such as γ-terpinene (12.71%, monoterpene hydrocarbon), p-cymene (7.01%, monoterpene hydrocarbon), and β-bisabolene (1.39%, sesquiterpene) may enhance carvacrol’s activity by increasing membrane permeability or inhibiting fungal enzymes, as suggested by studies on similar Lamiaceae oils²⁹. For A. talagonica, minor components like α-pinene (11.85%, monoterpene hydrocarbon), 1,8-cineole (6.25%, monoterpene oxide), and caryophyllene oxide (5.20%, sesquiterpene oxide) likely complement chrysanthenone (23.68%, monoterpene ketone) by targeting multiple fungal pathways, such as cell wall synthesis and oxidative stress induction²⁰. The chemical families of these compounds, predominantly monoterpenes, sesquiterpenes, and their oxygenated derivatives, exhibit diverse mechanisms of action, potentially amplifying antifungal effects through synergistic interactions. For instance, Angane et al.³⁶ noted that monoterpene hydrocarbons such as γ-terpinene can destabilize fungal membranes, facilitating the penetration of oxygenated compounds like carvacrol. Conversely, antagonistic interactions, including competition for binding sites, cannot be excluded, particularly in A. talagonica, where the moderate efficacy (MIC 3.12 µL/mL vs. 1.56 µL/mL for S. bachtiarica) may reflect lower synergy among its diverse constituents. Future studies using fractional inhibitory concentration indices or isobolographic analyses are needed to quantify these interactions and to confirm the roles of minor compounds in enhancing or modulating antisaprolegniosis activity.

The MIC and MFC values for the essential oils of S. bachtiarica and A. talagonica were 1.56 µL/mL and 3.12 µL/mL, and 3.12 µL/mL and 6.25 µL/mL, respectively. These values were comparable to those of malachite green (MIC = 3.12 µL/mL; MFC = 6.25 µL/mL) and superior to formalin (MIC = 12.5 µL/mL; MFC = 25 µL/mL), indicating that the antifungal effects of these essential oils are comparable to those of standard fungicides under in vitro conditions. It is noteworthy that no significant differences in fungal proliferation were observed in the placebo group containing solubilizers (1% DMSO and 0.1% Tween 80) compared to the intervention groups. This confirms that the antifungal properties observed can be confidently attributed solely to the essential oils themselves. In agreement with our findings, Kumar et al.⁴² indicated that 1% DMSO exhibited negligible inhibitory effects on the fish pathogen S. parasitica. Similarly, Tedesco et al.⁴³ and Takao et al.⁴⁴ documented that DMSO and tween 80, respectively, at the concentrations used, lacked discernible antifungal properties.

In vivo efficacy and implications

The ability of the S. bachtiarica essential oil to inhibit the transmission of S. parasitica from contaminated to healthy fish eggs and its protective effect were similar to those observed with malachite green (p > 0.05). The S. bachtiarica essential oil exhibited the highest inhibitory and protective abilities compared to all other groups (p ≤ 0.05). Regarding the performance of the A. talagonica essential oil, our results showed that its antifungal effects in inhibiting transmission and protecting fish eggs were generally similar to those of the formalin group (p > 0.05). However, the differences were statistically significant compared with the control and placebo groups (p ≤ 0.05). These findings revealed that the efficacy of this essential oil was not comparable with that of S. bachtiarica and malachite green under in vivo conditions (p ≤ 0.05). Many medicinal plants and their constituents have been studied for their antimicrobial properties against various microorganisms. Plant-derived essential oils can inhibit mycelial growth or even induce fungal cell death, including S. parasitica, at relatively low concentrations^19,29. Tampieri et al.⁴⁵ examined the antifungal effects of essential oils on S. parasitica, demonstrating that Origanum vulgare and Thymus vulgaris exhibited fungistatic activity at 100 µL/mL and fungicidal activity at 200 µL/mL. Gormez & Diler, (2014)²⁹ also reported that the MIC value of Origanum onites essential oil for S. parasitica was 10 µL/mL. Another study by Miljanović et al.⁴⁶ investigated the impact of sage, bay laurel, and rosemary essential oils on S. parasitica and Aphanomyces astaci. Among these, only sage essential oil significantly inhibited the mycelial growth of S. parasitica. The use of essential oils to treat S. parasitica-infected rainbow trout eggs has shown promising outcomes. In particular, repeated treatments of rainbow trout eggs with certain essential oils, such as those from Zataria multiflora and Satureja cuneifolia at concentrations of 5 ppm or more, have been shown to enhance hatching rates^14,15. A particularly relevant comparison can be made with the study of Metin et al.¹⁵, who evaluated the essential oil of Satureja cuneifolia, a congeneric species also belonging to the Lamiaceae family, against S. parasitica strains isolated from rainbow trout eggs. The major constituents of S. cuneifolia oil were carvacrol (40.3%), thymol (20.5%), γ-terpinene (11.2%), and p-cymene (9.1%), yielding a phenolic monoterpene-rich profile broadly similar to S. bachtiarica. In vitro, S. cuneifolia oil exhibited MIC and MFC values of 50 µL/mL and 100 µL/mL, respectively, against S. parasitica. In comparison, the essential oil of S. bachtiarica in the present study demonstrated markedly higher potency, with an MIC of 1.56 µL/mL and an MFC of 3.12 µL/mL. In vivo, Metın et al.¹⁵ showed that repeated treatments with S. cuneifolia improved egg survival and reduced fungal transmission, findings consistent with our observations for S. bachtiarica. However, the superior performance of S. bachtiarica, comparable to malachite green in both the infection-transmission and contamination-prevention assays, suggests that species-specific phytochemical differences within the genus Satureja (higher carvacrol content of S. bachtiarica (71.6% vs. 40.3%) and the synergistic contribution of γ-terpinene and p-cymene, which together exceed 90% of the oil composition) may significantly influence antifungal efficacy. This comparative evidence underscores the potential of Lamiaceae essential oils as promising natural antifungal agents while highlighting the exceptional potency of S. bachtiarica among related taxa.

In another study, Sakhaie et al.⁴⁷ investigated the antifungal properties of Artemisia annua essential oil against three fish pathogenic fungi: Saprolegnia spp., Fusarium solani, and Aspergillus flavus, reporting the strongest inhibitory activity against Saprolegnia, followed by F. solani and A. flavus. In an in vivo experiment by Mousavi et al.⁴⁸, the antifungal properties of essential oils from Thymus vulgaris, Salvia officinalis, Eucalyptus globulus, and Mentha piperita were tested on rainbow trout eggs, with a combination of essential oils at 10 mg/L effectively preventing fungal infections. A recent study assessed thyme and cinnamon essential oil nanoemulsions on rainbow trout eggs infected with S. parasitica strains. The results showed the highest survival rates for eggs treated with thyme nanoemulsions at 50 mg/L, comparable to the formalin positive control group³¹. Beyond their antifungal effects, essential oils offer a range of practical benefits, including appetite enhancement, improved microbial balance, growth stimulation, and optimized feed efficiency⁴⁹. For instance, they positively influence gut bacterial communities, enhancing nutrient digestion and absorption, which in turn boosts fish growth through increased amino acid availability for protein synthesis and muscle development., Essential oils can also modify gut microflora by inhibiting pathogenic bacterial groups, thereby promoting the dominance of beneficial microflora^50,51. In this context, Giannenas et al.⁵² investigated the effects of Thymus vulgaris essential oil on the intestinal microbiota of rainbow trout. Their findings demonstrated a significant modulation of the gut microbiota, characterized by a reduction in total anaerobic bacteria. Beyond their antifungal activity, essential oils possess well-documented antioxidant, anti-inflammatory, anti-stress, immunostimulant, and analgesic properties⁴⁹. In this regard, essential oils derived from basil and ginger have been shown to enhance the immune system of Nile tilapia, increasing their resistance to Streptococcus agalactiae and boosting phagocytic activity through an elevated presence of immune cells⁵³. Furthermore, dos Santos et al. (2016)⁵⁴ reported that cinnamon essential oil improved the immune response in Nile tilapia subjected to acute hypoxic stress, aiding in the maintenance of blood homeostasis post-stress.

Limitations and future directions

Despite the promising results, several limitations should be considered when interpreting this study. First, the inherent hydrophobicity and low water solubility of essential oils can limit their uniform dispersion and bioavailability in aquaculture systems. Although emulsifiers were used in this study, more efficient delivery approaches, such as nanoemulsions or encapsulation, may enhance stability and reduce the required effective dose^31,55. Second, variability in essential-oil composition caused by environmental and methodological factors may affect reproducibility and large-scale application. Advanced extraction techniques may help improve consistency and reduce production costs^55,56. Third, while the tested concentrations were well tolerated under controlled experimental conditions, essential oils can exert dose-dependent toxicity, and their effects on water quality, microbiota, and non-target organisms warrant further investigation^49,57,58.

Future studies should therefore focus on optimizing delivery systems, evaluating environmentally safe application strategies, conducting ecotoxicological assessments, and validating efficacy under commercial hatchery conditions. These efforts will be essential for translating essential oil based antifungal treatments into practical and sustainable aquaculture applications.

Conclusion

In a global context of escalating antimicrobial resistance and increasing consumer demand for sustainable practices, this study demonstrates the high efficacy and significance of essential oils from S. bachtiarica and A. talagonica as natural alternatives to synthetic antifungal agents. Our findings provide a strong scientific foundation for the valorization of these regionally abundant Iranian plants. Results from both in vitro and in vivo assays unequivocally show that these essential oils are highly effective against S. parasitica, the primary causative agent of saprolegniosis in rainbow trout eggs.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

S. Habibian Dehkordi conceived the study, conducted statistical analyses, and was the principal author of the manuscript. S. Habibian Dehkordi, S.H. Shafiei, and A. Mokhtari performed clinical aspects of the study. S. Habibian Dehkordi and S. Shahrookh developed the assay for fungus recognition and determination of MIC and MFC values. All authors contributed to refining the study, editing the manuscript, and approving the final version.

Funding

This work is based upon research funded by Iran National Science Foundation (INSF) under project No. 4014510.

Data availability

The data that support the findings of this study are available from the corresponding author (upon reasonable request).

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval

The experimental procedures were approved by the Animal Research Ethics Committee at Shahrekord University (Ethical No: IR.SKU.REC.1403.025). All methods were carried out in accordance with relevant guidelines and regulations, and the study is reported in accordance with the ARRIVE guidelines⁵⁹.