Introduction

The water mold, Saprolegnia parasitica, is one of the most important species in the genus Saprolegnia that caused saprolegniosis in fish (Paul et al. 2015). It can infect different fish developmental stages (Eissa et al. 2013; Sformo et al. 2017); however, it has been shown that there is a strong association of S. parasitica with the adult stage of the salmonid samples (Sandoval-Sierra et al. 2014). It can be considered that at least 10% of the annual economic loss in salmonid aquaculture is caused by saprolegniosis (Sandoval-Sierra et al. 2014). In order to control the disease, there is an essential need to develop strategies for the effective management of saprolegniosis (Magray et al. 2019).

For a long time, malachite green was used for treatment and prevention of saprolegniosis (Minor et al. 2014). However, the potential teratogenic, carcinogenic, and mutagenic properties of malachite green had ended in banning its use in 2002 (Minor et al. 2014). The demand for alternative treatments has pushed many researchers towards investigating more safe and eco-friendly compounds (Tedesco et al. 2019) such as medicinal herbs (Caruana et al. 2012; Khemis et al. 2016; Nardoni et al. 2019; Emara et al. 2020).

Medicinal plants can be a suitable alternative to chemotherapy in aquaculture since they have been reported to possess a complex chemical composition that displays numerous bioactivities including antimicrobial (bacterial, fungus, virus, and ectoparasites) effects (Reverter et al. 2014). Moreover, plants have some other characteristics such as biodegradability, availability, ease of cultivation, and not residing in animal tissues making them popular among aquaculturists (Dawood et al. 2021).

The genus Sambucus L., belongs to the family Adoxaceae (Rezaei-Moshaei et al. 2021), and comprises eighteen species around the world (Ebadi and Hisoriev 2011). Sambucus ebulus L. also known as dwarf elder is the only species distributed in the northern part of Iran (Ebadi and Hisoriev 2011). Various extensive studies have been done about its antioxidant, anti-inflammatory, analgesic, antimicrobial, anticancer, wound healing, antidepressant, and antiparasitic activities (Jabbari et al. 2017).

Actinidia deliciosa Liang Ferguson (Green kiwifruit) belongs to the family Actinidiaceae and grows naturally in various regions (Satpal et al. 2021). Iran is the fourth largest kiwifruit producer in the world and the fruit is mainly cultivated in the northern part of the country (Maleki et al. 2017). Due to the presence of bioactive compounds in kiwifruit, it exhibits strong antimicrobial, antiviral activity, and immunomodulatory effects (Satpal et al. 2021). It is thought that kiwifruit peel contains higher amounts of phenols and flavonoids than flesh (Alim et al. 2019).

The aim of the present study was to determine the chemical composition of Sambucus ebulus leaves and Actinidia deliciosa peels ethanolic extracts and their antifungal effects against S. parasitica.

Materials and methods

Strain tested

The in vitro tests were carried out on a S. parasitica strain (Isolate KMG3, accession number: MW819780) isolated from infected rainbow trout eggs (unpublished data). Purified cultures were kept on Sabouraud dextrose agar (SDA) and re-inoculated every month. The inocula were obtained by cutting the advancing edge of the young colonies (3-day incubation at 18 °C) using a sterile scalpel.

Ethics statement

In the present study, S. parasitica recovered from infected fish eggs was used and ethical approval was not required for the study. According to US National Institutes of Health, Policy on Humane Care and Use of Laboratory Animals (PHS) applies to offspring of egg-laying vertebrates only after hatching (Bartlett and Silk 2016).

Plant material preparation

A. deliciosa peel and S. ebulus leaves were obtained from the north of Iran (Mazandaran and Golestan provinces, respectively), air-dried in a dark place, ground by a kitchen blender, and stored at 4 °C.

Preparation of plant extracts

The pulverized plant samples were added to distilled water (1:1 masses per volume) and subjected to boiling for 10 min. After filtration, the filtrate was mixed with 70% ethanol and remained for 1 week in a dark place with intermittent shaking. The ratio of sample to solvent was 1: 9 (volume per volume). After that, the supernatant was filtered through a filter paper Whatman No. 1 (Whatman Ltd., England). The extracts were concentrated at 55 °C and then were maintained in a closed and dark container (Salama et al. 2018).

GC-MS analysis

Constituents of S. ebulus ethanolic extract were analyzed by GC-MS as previously reported (Khosravi et al. 2018). A 6890 N Agilent gas chromatograph coupled to a 5975 C Agilent mass-selective detector (Agilent Technologies, Avondale, PA, USA) with a 7683 Agilent auto sampler was employed and 1.0 µL of the sample was injected in the split less mode at 250 °C into a 30 m × 0.25 mm × 0.5 μm DB-5 MS capillary column and operated by MSD Chemstation Software (Agilent Technologies). The temperature program used for the chromatographic separation was as follows: 50 °C for 2 min, the temperature increase at 25 °C.min −1 to 100 °C and hold for 2 min, then temperature increase at 5 °C.min −1 to 290 °C where it was finally held for 5 min. The carrier gas was helium (99.999%) and was kept at a constant flux of 1.0 mL.min −1. The mass spectrometer was run in the electron impact ionization mode and the energy of the electrons was retained at 70 eV. After injection of sample to GC/MS, some unknown peaks were detected. The impact mass spectra of these obtained peaks were searched for in our computer library (Khosravi et al. 2018).

Determination of total phenolic and flavonoid contents

The phenolic contents of A. deliciosa were assessed using the Folin-Ciocalteu method described previously with minor modification (Ainsworth and Gillespie 2007). In brief, 25 µl of the extract was added to 95 µl of 2n Folin-Ciocalteu reagent and then 300 µl sodium carbonate solution was added. The final volume reached 1800 µl using distilled water and then incubated at 37 °C for 45 min. The absorbance of the solution was calculated at 758 nm by spectrophotometry and expressed as mg gallic acid equivalents (GAE)/g dry weight sample.

Flavonoid contents of A. deliciosa were determined using aluminum chloride in a colorimetric method; the result was derived from the calibration curve of quercetin (QE) and expressed as mg QE /g dry weight sample (Aryal et al. 2019).

In vitro assays

Tests were performed following the HeMP (Stueland et al. 2005) and agar dilution (Ali et al. 2019) methods with some modifications. For the HeMP method, agar plugs (5 mm) with actively growing mycelia were inoculated in the center of yeast extract glucose chloramphenicol (YGC) agar plate (8 cm) and incubated at 18 °C for 1 day. The sterilized hemp seeds were then placed in a circle around the Saprolegnia colony and the agar plates were incubated at 18 °C for another 2 days. Then, the Saprolegnia-colonized hemp seeds were transferred with sterile forceps into wells of a 48-well flat-bottom tissue culture plate (NEST, China) and 1 mL of each test extract was added. Stock solutions at 30% of herbal extracts were diluted into sterile distilled water (SDW) to obtain concentrations ranging from 0.1 to 10%, to find a MIC value, and from 0.5 to 30% to find a MLC value expressed as the lowest concentration, at which fungal growth was not noticeable in two replicates. All concentrations were tested twice. SDW without extract and malachite green at 10 ppm were set as controls. In order to ensure good growth of Saprolegnia, 2% GY broth (D (+)-glucose monohydrate 10 g, yeast 2 g, potassium dihydrogen orthophosphate (KH2PO4) 2.04 g, and (disodium hydrogen phosphate anhydrous) Na2HPO4 0.596 g (in 1 L of distilled water and autoclaving) (Willoughby and Pickering 1977) were included in each test concentration. To find MIC, Saprolegnia-colonized hemp seeds were exposed to different herbal extract concentrations for 48 h. To find the MLC, they were kept in contact with the solutions for 60 min, followed by rinsing of the exposed hempseeds in water and the transfer of the seed to pure water. The wells on the tissue culture plates were examined by the use of a stereomicroscope (Olympus, Japan) 48 h after the start of the exposure. The mycelial growth of Saprolegnia was graded as 0 for complete inhibition of the growth and 1 for a partial or full growth of the mycelium on the hemp seeds (Stueland et al. 2005) (Fig. 1).

Fig. 1
figure 1

The mycelial growth of Saprolegnia was graded as 0 for complete inhibition of the growth (a) and 1 for a partial (b) or full growth of the mycelium on the hemp seeds (c)

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For the agar dilution method (Ali et al. 2019), the extracts were added at 1, 2.5, 5, and 10% concentration to sterilized molten SDA held at 45 °C. SDA without herbal extracts was used as a negative control treatment. Then, the plates were inoculated with Saprolegnia-colonized agar plugs (5 mm) and/or hemp seeds and the mean radial growth of Saprolegnia hyphae was calculated after 3 days of incubation at 18 °C. All the concentrations were examined in triplicates. All chemicals used, unless otherwise stated, were purchased from Merck Company (Darmstadt, Germany).

Statistical analysis

One-way ANOVA followed by Duncan’s comparison test was performed using SPSS version 15 for differences in radial growth of Saprolegnia at different herbal extract concentrations. Differences between treated and controls were evaluated at a p value < 0.05.

Results

GC-MS analysis

A total of seven components were identified from GC-MS of S. ebulus leaf extract that together constitute 99% of the compound. The main constituents in the extract were monophthalate (66.17%), palmitic acid (13.83%), isovaleric acid (6.28%), α-linoleic acid (5.24%), phytol (4.25%), acetic acid (2.11%), and pentanoic acid (1.12%) (Table 1).

Table 1 Chemical composition of ethanolic extract of S. ebulus measured by GC-MS analysis
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Total phenolic and flavonoid contents

A. deliciosa ethanolic extract contained total phenolic (TP) contents at 162 mg (GAE)/g DW and total flavonoid (TF) content at 2.31 mg (QE)/g DW.

In vitro assay

Based on the HeMP method, the ethanolic extract of S. ebulus showed an antimycotic activity with MIC and MLC values of 2.5% and 20%, respectively. Regarding A. deliciosa, none of the tested doses had a fungicidal effect; nevertheless, it appeared clearly to have a fungistatic effect with an observed MIC corresponding to a dose of 5%.

We also tested the effect on mycelial growth quantitatively by using the agar dilution method. Compared to the control group, the growth of S. parasitica was inhibited by S. ebulus in a dose-dependent manner (Fig. 2). In this way, treatment with 1% concentration resulted in an incomplete but statistically significant (p <0.05) inhibition of mycelial growth. A concentration of 2.5% gave a reduction of close to 57% (p < 0.05) and at 5% concentration of S. ebulus, no hyphal growth was observed (Figs. 2 and 3). On the contrary, the radial growth of S. parasitica did not show statistically significant differences comparable with the control group up to 10% concentration of A. deliciosa (p >0.05) (Fig. 2). Ten percent concentration yielded an inhibition of 68% (p < 0.05) (Figs. 2 and 4).

Fig. 2
figure 2

Inhibition of S. parasitica growth on Sabouraud dextrose agar (SDA) following treatment with different Sambucus ebulus (S. ebulus) and Actinidia deliciosa (A. deliciosa) ethanolic extract concentrations. Different superscript letters indicate the existence of statistical difference among groups (p < 0.05)

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Fig. 3
figure 3

 S. parasitica grown on SDA plates with different concentrations of Sambucus ebulus ethanolic extracts. The concentration from left to right was 0, 1, 2.5, 5, and 10%. The pictures were taken after 3 days of incubation at 18 °C

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Fig. 4
figure 4

 S. parasitica grown on SDA plates with different concentrations of Actinidia deliciosa ethanolic extracts. The concentration from left to right was 0, 1, 2.5, 5, and 10%. The pictures were taken after 3 days of incubation at 18 °C

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Discussion

In the present study, the hyphal growth of S. parasitica was inhibited completely by S. ebulus at 5% concentration, and the growth inhibition was dose-dependent (p < 0.05). This finding is in agreement with the study of Rodino et al. (2015), who observed mycelial growth inhibition to 50 and 84% in Phytophthora infestans (the oomycete), when using S. ebulus fruit hydroalcoholic extract at the concentration of 2 and 4% in the medium, respectively. Also, Shahiri Tabarestani et al. (2017) reported that different concentrations (10, 20, and 30%) of alcoholic extracts of S. ebulus leaves significantly inhibited the mycelial growth and formation of sclerotia of the Macrophomina phaseolina (the agent of soybean charcoal rot). Interestingly, the chemical composition of S. ebulus leaves extracts in our study was similar to Shahiri Tabarestani et al. (2017) results and in both works, phthalates were the most constituents (concentration >50%). Phthalates are synthetic chemical compounds used as plasticizers (Kumari and Kaur 2021). However, in recent years, several natural phthalates were extracted from microorganisms, algae, and plants (Kilani-Feki et al. 2011), and their antifungal activity was demonstrated by many authors (Harikrishnan et al. 2010; Akpuaka et al. 2012). Phthalates were found to alter the activity of various enzymes including purified yeast glucose 6-phosphate dehydrogenase (Ohyama 1977) and PINK1, a serine/threonine kinase in human neuroblastoma cells (Xu et al. 2021). These enzymes were also reported from S. parasitica, e.g., glucose 6-phosphate dehydrogenase (SPRG_0526) and protein-serine/threonine kinase (SPRG_10674). The observed inhibitory activity of S. ebulus in the present study may be attributable to alteration in the activity of such enzymes, but it needs to be investigated more detail in the future.

There are numerous reports referring to antifungal properties of fatty acids (Liu et al. 2008; Pohl et al. 2011). In the current study, palmitic acid (13.83%) was found to be the most prevalent fatty acid in the ethanol extracts of S. ebulus leaves followed by isovaleric acid (6.28%) and α-linoleic acid (5.24%). These fatty acids were also present in the alcoholic extracts of S. ebulus leaves used by the study of Shahiri Tabarestani et al. 2017, but in different ratios (8.24, 4.33, and 7.78, respectively). Also, Shah et al. (2021) identified palmitic acid as the second highest compound (11.95%) in the Thymus linearis leaf extract with a potent anti-oomycetes activity against S. parasitica. The extract showed mycelial growth inhibition to 54.45 ± 0.9% at 320 ppm and complete inhibition (100%) at 5120 ppm (Shah et al. 2021). According to Pohl et al. (2011), antifungal fatty acids elicit profound effects on the cell membrane, causing an increase in membrane permeability, which will result in leakage of the cytoplasm and cell death.

Phytol, an acyclic diterpene alcohol, was another compound with a considerable amount (4.25%) in S. ebulus leaf extract. Its antimicrobial activity was proven against several bacterial and fungal strains (Inoue et al. 2005; Pejin et al. 2014). For example, Inoue et al. (2005) reported that it could disrupt the cell membrane of Staphylococcus aureus, resulting in potassium ions discharging from the cells, and causing the bacterium to shrink. Recently, based on molecular docking, phytol was identified to target a number of proteins in S. parasitica such as plasma membrane ATPase, host target protein-1 and TKL protein kinase, and V-type protein ATPase (Shah et al. 2021).

Moreover, it was found that acetic acid has an antifungal effect on S. parasitica (MIC at 250 ppm) (Tedesco et al. 2019). In the present study, the concentration of acetic acid in the ethanolic extract of S. ebulus was 2.11% (Table 1). The amount of acetic acid in our work (i.e., 2.5% concentration of S. ebulus leaf extract) was calculated and equaled to 527.5 ppm which was about twice the MIC recorded for acetic acid for S. parasitica (Tedesco et al. 2019). Therefore, it seems that in our experience, the antimycotic activity of S. ebulus against S. parasitica could be originated from phthalates, fatty acids, phytol, and acetic acid. However, more detailed studies are needed to clarify which bioactive molecules are responsible for the observed activity.

In this study, two different MICs were recorded for A. deliciosa including 5 and 10% by the HeMP and agar dilution methods, respectively. This mismatched MIC value is most likely due to different densities and growth quality of hyphae per cell culture medium in the HeMP method (Stueland et al. 2005). Moreover, here, we applied lower number of replicates in this method compared to agar dilution method (i.e., two vs. three per concentration, respectively). Such variable results were also reported by other authors (Kumar et al. 2020). However, Salama et al. (2018) found that ethanol and acetone extracts of A. deliciosa inhibited the growth of yeasts (Saccharomyces cerevisiae and Candida albicans) and the fungus Aspergillus flavus, at concentrations of 400 and 600 ppm. In the current study, compared to the study of Salama et al. (2018), rather a high MIC value was obtained. This variability may be due to specific oomycetes cell wall composition, mainly composed of cellulosic compounds along with glycans compared to other true fungi which possess chitin in their cell walls (Nardoni et al. 2019; Magray et al. 2019). Furthermore, it has been reported that the extraction techniques and solvents could influence the bioactivity of herbal extracts (Emara et al. 2020). It is not clear which bioactive fraction is responsible for the antifungal effect of A. deliciosa, but it seems that it was related to phenolic and flavonoid components. Phenolic and flavonoid compounds are natural plant secondary metabolites depicted by aromatic rings, act as reducing agents and antioxidants, and are known to have activity against microorganisms (Caruana et al. 2012; Khemis et al. 2016; Alim et al. 2019). In another study, Khemis et al. (2016) demonstrated that the ethanolic extract of Opuntia stricta (Haworth) prickly pear with high phenolic contents (147.31 mg CATE/100 g of fruit) arrested the growth of Saprolegnia sp. both in vitro and in vivo. Likewise, inhibition in the mycelial growth of P. infestans was observed when the phenolic compounds present in the extracts of the olive plant (Olea europaea L.) were added to the culture medium (Del Rı́o et al. 2003).

Based on findings of the present study, compared to A. deliciosa, S. ebulus proved to be more effective in inhibiting the growth of S. parasitica in vitro and could be of interest for controlling Saprolegnia. However, more detailed studies are needed to evaluate its possible toxic effects on different fish developmental stages and efficacy in vivo