Fresh seaweed was gathered by hand from the Mehdia coast (latitude: 9,440 N and longitude: 79,000 E), cleaned thoroughly in seawater to remove external matter such as sand, epiphytes, and other algae contaminants; and then placed in polyethylene plastic bags and transported to the laboratory promptly to minimize deterioration (Muthukumar et al., 2015). In the laboratory, the samples were rewashed three times with tap water and one more time with distilled water. The samples were dried at 40 ± 2°C for 78 h, and then kept in dark bottles at 4°C, after being mechanically blended into a fine powder for future analysis and applications.

A measure of 100 mL of distilled water and 5 g of seaweed were mixed, stirred for 1 h at 6,000 rpm, and permitted to sit at ambient temperature for 24 h. Following filtration, the extract was kept in the refrigerator in dark bottles for later use (Amin, 2019).

A measure of 10 mL of U. lactuca’s aqueous extract and 1 mM of silver nitrate (AgNO₃) were combined, stirred, and heated to 100°C for 24 h. When the reaction’s color shifts from translucent to brown, the reaction is deemed successful (Bhimba and Kumari, 2014).

The effectiveness of the bioreduction of AgNO₃ by U. lactuca’s aqueous extract into AgNPs was demonstrated using UV–Vis (Schimatzu UV-1800). At intervals of 100 nm, the absorbance spectrum was measured over a wavelength range of 200–1000 nm.

The Bruker FT-IR instrument was utilized to analyze the sample-synthesized NPs to ascertain the functional groups of the AgNPs. The RockSolid permanently aligned interferometer and the DigiTect integrated detection system, with a 24-bit analog-to-digital converter (ADC), was utilized at the University Center of Analysis, Expertise, Technology Transfer and Incubator within the Faculty of Science at Ibn Tofail University, Kenitra, Morocco. The spectral analysis was focused on the range of 400–4,000 cm⁻¹.

The crystallographic data of the dried silver nitrate solution were obtained after bioreduction. The X’Pert³ materials research diffractometer was used to record the XRD patterns. The samples were scanned using a 2θ range of 10°–70°.

The SEM analysis was carried out on an SEM JEOL. The Center for Innovation and technological by technology: Center of Innovation and Technology Transfer of Moulay Ismail University, CITT -UMI in Meknes City, Morocco established the size and shape of the AgNPs.

The reducing power of the extracts was ascertained using the Oyaizu method (Oyaizu, 1986). Potassium ferricyanide (1%, w/v) was incorporated into 2.5 mL of phosphate buffer (0.2 M, pH 6.6) after the extracted sample (1 mL) had been combined with it. For 20 min, the combined solution was incubated at 50°C. Then, 2.5 mL of 10% w/v trichloroacetic acid was incorporated, and the mixture was centrifuged at 3000 rpm for 10 min. A measure of 2.5 mL of the supernatant was combined with 0.5 mL of ferric chloride (0.1%, w/v) and 2.5 mL of distilled water. After 10 min of incubation, the absorbance was determined at 700 nm. The reaction mixture’s increased absorbance demonstrated a higher reducing power.

The MIC is the lowest inhibitory concentration (MIC) at which no visible growth was seen. This experiment was performed in triplicate, employing the macrodilution method described by Tarfaoui et al. (2022) to determine the MICs. The extracts were dissolved in DMSO, a solvent with no known antibacterial properties (Rahmi and Putri, 2020). Serial dilutions of the extracts were prepared in agar solutions (2–100 μL/mL).

The Solanum lycopersicum variety was chosen for the experiment. In a greenhouse, tomato seedlings were planted in 15 by 15 cm plastic pots filled with 1:1 black dirt and peat soil for the vegetable mix, under 16 h of photoperiod. When necessary, plants were watered (Yousef et al., 2021). In the Moulay Bouselham region, we purchased strawberry plants from a nearby nursery. Two months after planting, tomato plants were moved (among the strawberry plants) into a controlled environment with a temperature of 21°C and 70% humidity for acclimatization. To provide ideal circumstances for the inoculation of gray mold, the humidity was raised to 100%, and the plants were covered with agrofilm 24 h prior to the inoculation. The plants were treated by spraying the leaves with 1 mL of aqueous and U-AgNP extracts after 24 h. Each tomato and strawberry trifoliate had three wounds made on its center leaves. On each wound, 2 mm of B. cinerea mycelium was applied. The plants were once more wrapped with agrofilm and stored for 48 h at 21°C and 100% humidity. After 48 h, the agrofilm was removed. The plants were frequently watered, and the severity and emergence of infection signs were observed. Teldor 50% sc was used as a standard antifungal. Utilizing DMSO (% 5) served as the negative control. Each treatment has three duplicates or three plants. All evaluations were conducted 3, 6, and 8 days after inoculation.

The uniform and healthy fruits of tomatoes (harvested from the developing plants) and strawberries (picked from a local store), were sterilized with ethanol at 70° and dried using a sterile filter paper after being rinsed three times with sterile distilled water. At three distinct locations throughout the equatorial zone, 2-mm diameter needles were used to create 3-mm deep wounds. A 20-μL drop of each extract was then applied to the wounds (Hassan et al., 2021). A 2-mm diameter mycelial explant was also inserted into the incisions after 2 h. The same protocol was applied to tomato and strawberry leaves. After that, the fruits and leaves were coated with plastic covers and retained in a dark and humid environment.

The scale of infection was assessed 8 days after treatment of the plants intentionally infected with B. cinerea mycelium. The percentage of affected leaf area on randomly chosen plants was used to determine the severity of the disease. It was estimated using a disease rating scale of 1–9, recommended by Notteghem et al. (1980). Table 1 presents the results.

Equation 3 was used to convert the severity scale into a Percentage Severity Index (SI) for analysis:

where Nt is the total number of plants noticed, Xi is the disease severity scale, ni is the number of damaged plants (leaves or fruits) with a rating of i, and 9 is the maximum disease severity scale.

The data were analyzed using a one-way analysis of variance (ANOVA) followed by Tukey’s test (α = 0.05) to determine significance levels. The results of three repetitions are presented by mean ± SD. A p < 0.05 was considered statistically significant.

The reaction mixture’s color shifted from light green to dark brown, which was the first sign that AgNPs were being formed (Hashemabadi et al., 2021).

After being refined, AgNPs were tested using UV spectrophotometry. Figure 1 shows a distinct and unique characteristic peak at 440 nm, which corresponds to the plasmon absorbance of the AgNP. In contrast, the characteristic peak was registered at 401 nm for the aqueous extract.

The chemical composition of the AgNPs’ surface is examined using FT-IR spectroscopy, which also permits the identification of potential biomolecules that may be in charge of the Ag²⁺ ion reduction and capping of the bioreduced AgNPs produced by U. lactuca extract. The absorption bands were visible in the FT-IR spectrum at 465.35, 1082.33, 1408.34, 1536.40¹, 1631.31, 2348.97, 2916.90, and 3271.40 cm⁻¹ (Figure 2).

The peak at 465.35 cm⁻¹ is attributed to the coupling of O–Si–O bending and K–O stretching vibrations (Theodosoglou et al., 2010). The peak at 1082 cm⁻¹ is assigned to the C–N stretch of aliphatic amines, while the peak at 1408 cm⁻¹ is associated with C–N stretching vibrations of aromatic and aliphatic amines. The peak at 1536 cm⁻¹ is linked to the N–O asymmetric stretching of nitro compounds, and the peak at 1631 cm⁻¹ is attributed to the protein amide I bond, resulting from a carbonyl stretch in proteins. This peak was relatively close to the native protein reported by Macdonald and Smith (1996), which implies that although proteins and AgNPs are conversing, their secondary structure is unaffected by the binding process or the subsequent Ag nanoparticle-protein interaction (Fayaz et al., 2010).

The study demonstrated that amino acid residues’ carbonyl groups have a great affinity for metal, which may lead to the creation of a layer that covers metal NPs and serves as a capping agent to prevent agglomeration and stabilize the medium (Al-Fa’ouri et al., 2021). These findings support the hypothesis that certain proteins function as stabilizing and reducing agents for AgNPs. The peak at 2348 cm⁻¹ refers to the asymmetric vibration of C–H, while the peak at 2916 cm⁻¹ is caused by the symmetric and asymmetric stretching of alkenes along the C–H bond. The peak at 3271 cm⁻¹ is associated with the O–H stretching of water molecules, alcohols, or phenols, as well as the presence of N–H amides stretching vibration. Thus, it can be assumed that the plant extract’s saponins, tannins, glycosides, flavonoids, and polyphenols give the NPs their additional stability and are compatible with the findings of Khalifa et al. (2016). The existence of organic molecules such as alkenes, alkanes, primary amines, ester, and nitro groups is confirmed by FT-IR spectral analysis, which also demonstrates the significance of U. lactuca extract in the environmentally friendly synthesis of AgNPs.

The XRD examination indicates the formation of crystalline silver particles, wherein nanocrystals are created, as indicated by the peaks at 2θ values of 27.93°, 32.41°, 38.40°, 46.29°, 57.51°, and 78.6° corresponding to (110), (111), (122), (200), (220), and (311) crystal planes of the crystalline structure (Figure 3).

Green-produced AgNPs were examined for surface morphology using a scanning electron microscope. The SEM of AgNPs revealed a spherical shape with a size range of 45–61 nm, involving agglomeration caused by U. lactuca’s organics acting as stabilizing agents (Figure 4; Harinee et al., 2019).

Energy dispersive X-ray spectroscopy (EDAX) analysis was performed to verify the existence of AgNPs and offer the chemical purity of the produced NPs (Figure 5), revealing peaks between 0 and 4 kV. The optical absorption peak indicates that nanocrystalline elemental silver is occurring, which was noticed at approximately 3 keV, which is characteristic of the metallic silver nanocrystals’ absorption owing to surface plasma resonance. Strong silver signals and weak carbon and oxygen peaks are visible in the spectrum; these could result from biomolecules attached to the surface of the AgNPs. The presence of salt or protein residue in the marine environment may lead to the discovery of additional substances such as Si and Ca (Amin, 2019).

The MIC value, which is the lowest concentration of an antimicrobial agent (MIC) that stops a microorganism from developing, can be used to assess the effectiveness of NPs (Parvekar et al., 2020). The findings in Table 3 show that up to 2 μL/mL of U-AgNP extract impeded the development of S. aureus and E. coli. In comparison, this extract at a concentration of 4 μL/mL was able to block the development of S. epidermidis. AgNPs were examined for antibacterial activity, and according to Kamble et al. (2023), K. pneumoniae revealed a MIC value of 25 μL/mL, and E. coli demonstrated a MIC value of 50 μL/mL.

The results of the study indicate that U-AgNPs exhibited a greater significant antibacterial ability compared to the aqueous extract, indicating that Ag ions are better agents for antimicrobial activity (Tamayo et al., 2014; Rather et al., 2019). U-AgNPs were also found to have greater bacterial potency, as well as various S. aureus and E. coli strains. This may be due to their enhanced encounters with the proteins and polysaccharides on cell walls (Prasher et al., 2018).

It is interesting to note that the functions of bacteria can be altered by the concentration of silver nitrate (Pandian et al., 2010). At a concentration of 1 mM silver nitrate, AgNPs were biosynthesized, according to Kalimuthu et al. (2008). Furthermore, the bacteria continued to survive while the AgNPs were being synthesized and started growing again after the silver was taken out of their surroundings (Kalimuthu et al., 2008).

A. alternata, A. versicolor, B. cinerea, F. equiseti, and Penicillium spp. were the antifungal agents against which aqueous and U-AgNPs were tested in vitro (Table 4). The antifungal activity of different concentrations, including 2.5, 1.25, and 0.62 mg/mL, was examined. The growth capacity of several fungi was dose-dependently reduced by aqueous or U-AgNP extracts. Additionally, AgNPs have shown efficacy and provide the most promising antifungal action against B. cinerea, F. equiseti, A. alternata, Penicillium spp., and A. versicolor, with inhibition rates of 100, 88, 68.45, 58.33, and 50%, respectively, at the dosage of 2.5 mg/mL (Table 4). When compared to the standard reference antifungal drug, Teldor 50% sc, we found good antifungal activity, when compared to the negative control, all the tested extract dosages demonstrated an antifungal effect on the tested pathogens. Aqueous extract showed the lowest activity against A. versicolor and Penicillium spp.; A. alternata was moderately sensitive to aqueous extract.

In similar studies, the fungi used in our study have been tested by different algae species extracts for identifying antifungal activities by several researchers, such as Fusarium sp. (Shanmugam, 2014), A. alternata (Vehapi et al., 2020), Penicillium spp. (Lagrouh et al., 2017) and B. cinerea (El-Bilawy et al., 2022). According to published research, this study is the first to look into the antifungal properties of the aqueous and AgNP extracts of U. lactuca against A. versicolor and F. equiseti. This feature makes the study more original.

The findings point to the existence of bioactive substances in both kinds of extracts that can prevent the growth of certain fungi. Since it contains phytochemical components, which include protein, lipids, pigments, polyphenols, polysaccharides, alkaloids, terpenes, chlorophylls, and carotenoids that may be utilized as antioxidants and antimicrobials, U. lactuca has wide range of biological activity (Chojnacka et al., 2012; Sakthieaswari and Srisudha, 2016; Manchu et al., 2014). However, the effectiveness of AgNPs against fungal infections is only partially supported by research Vivek et al. (2011) investigated the red alga Gelidiella acerosa extract’s role in the creation, characterization, and use of biologically produced nanomaterials. They found that AgNPs had a significant antifungal effect when compared to conventional antifungal medications. While extensive research has been conducted on AgNPs’ effects on bacteria, their function in antifungal effects has not garnered as much attention (Bruna et al., 2021; Khaldoun et al., 2024). On the contrary, AgNPs’ microbial agent mechanism is still up for debate and is not entirely understood (Rafińska et al., 2019); It is commonly known that when fungal cells are exposed to AgNP solution, pits form on the membrane’s surface, destroying the integrity of the cells (Wen et al., 2022). Moreover, the Ag NPs’ antifungal activity is probably closely related to their smaller size and shape because of their increased surface area, which enhances their antimicrobial activity (Osonga et al., 2020). Additionally, based on experimental evidence, it has been demonstrated that exposure to silver ions causes DNA to lose its ability to replicate, leading to a reduction in cell viability and, ultimately, death (Takáč et al., 2023).

To investigate whether the extracts include any characteristics that could shield tomato and strawberry leaves, fruits, and petioles against B. cinerea, the treatments with aqueous and U-AgNP extracts were applied before pathogen challenge at various doses (0.62, 1.25, and 2.5 mg/mL).

At concentrations of 1.25, and 2.5 mg/mL, U-AgNPs considerably inhibited the growth of B. cinerea’s mycelia, but at 0.62 mg/mL, the effect of the aqueous extract was significantly diminished. Based on the suppressed effects on mycelia growth and the antifungal effects of U-AgNPs on PDA, we selected a concentration of 1.25 and 2.5 mg/mL for our next fungal infection trials on detached leaves, fruits, and plants.

The aqueous extract did not produce any decrease in the degree of damage in leaves following infection by pathogens. On tomato leaves, the aqueous extract showed no considerable inhibition; however, at concentrations of 2.5 and 1.25 mg/mL, strawberry leaves showed moderate inhibition (77 and 79%) compared to the negative control (100%). However, U-AgNP extract appears to decrease the injury in tomato and strawberry leaves inoculated with B. cinerea (Figure 7). U-AgNP extract appears to be more effective at producing the protective effect, leading to a decrease in the extent of damages created after the pathogen infection compared to the negative control.

Additionally, our research looked into U-AgNPs’ possible ability to prevent gray mold on strawberries and tomatoes (Figure 8). On the outer layer of the infected tomato and strawberry fruits treated with U-AgNPs at the examined concentrations, we did not find any B. cinerea lesions during this experiment (Figure 8), at concentrations of 1.25 and 2.5 mg/mL, however, a percentage of 33% of disease severity were seen in contrast when it comes to the fruits sprayed with the commercial fungicide. This indicates that U-AgNPs, at concentrations of 1.25 and 2.5 mg/mL, greatly controlled or decreased the damage that B. cinerea does to fruits. Conventional fungicide treatment of tomato and strawberry fruits also decreased disease symptoms, although not to the same “a lesser extent” as the U-AgNP treatment, at concentrations of 1.25 and 2.5 mg/mL. However, the aqueous extract had practically an inhibitory effect on B. cinerea in tomato fruits despite significant deviations from the negative control; on strawberry fruits, however, the aqueous extract had a disease severity percentage that was slightly similar to the negative control (94%), particularly at the concentration of 1.25 mg/mL (88%) and a lower value at the concentration of 2.5 mg/mL (66%). This implies that B. cinerea induced lesions on fruits, which U-AgNPs greatly reduced.

Moreover, the tomato and strawberry plants were used to assess U-AgNPs’ ability to prevent gray mold. In strawberry plants, the U-AgNP extract was more effective than the commercial fungicide; however, in tomato plants, the U-AgNP extract outperformed the commercial fungicide. The results showed 100% efficiency (0% disease severity percentage) against B. cinerea at the concentration of 2.5 mg/mL, which was more potency than the commercial fungicide (Figure 9).

The protective capacity of the U-AgNP extract was more effective at 2.5 mg/mL, obtaining a 100% level of protection across the whole plant, including fruits and leaves. At a lower concentration of 1.25 mg/mL, the extract decreased the amount of damage to the leaves (1 and 11% for tomato and strawberry leaves, respectively) compared to 100% damage in the negative control (Figure 7). The findings indicate that U-AgNP extract contains one or more active principles that, in a dose-dependent manner, may protect leaves from the pathogenic fungus B. cinerea.

Using fungicides is the main strategy for managing plant disease. However, research is necessary to produce novel, biodegradable, effective, and safe products; nevertheless, this pathogen has developed resistance to single-site inhibitors. In this study, we conducted both in vivo and in vitro experiments to investigate the antifungal activity of U-AgNPs against B. cinerea, the primary cause of the gray mold. Nevertheless, there has not been much research conducted on AgNPs’ in vivo antifungal activity. According to Elmer et al., NPs may penetrate root cells and migrate to various areas of the treated plants leading to resistance throughout the system (Elmer and White, 2016).

Due to their unique qualities, NPs have sparked a lot of concern last 10 years. These particles can swiftly pierce the matrix because they are reactive. To create green NPs, non-toxic and ecologically safe processes are employed. For NP biosynthesis, plant extracts are utilized in the bioreduction of metal ions, which is a quick, inexpensive, bacteria-free, and easily scaled-up process (Qayyum and Khan, 2016). As a result, the findings of our in vivo antifungal assay strongly support the creation of agrochemical NP agents on a platform that is both affordable and environmentally friendly. Overall, U. lactuca extract-based “green synthesis” of AgNPs eliminates the risks associated with using toxic reducing/capping agents and is a viable, affordable, and ecologically friendly method (Haseeb et al., 2019). This would significantly help reduce the use of dangerous pesticides, which are extremely detrimental when used near food crops such as fruits and vegetables (Venkatesan et al., 2016). According to current evidence, U-AgNPs can effectively replace conventional fungicides in agricultural pest management.

Ensuring the safe application of AgNPs in agricultural contexts requires thorough risk evaluations and continuous monitoring of AgNPs’ impact on human health and the environment. Regulations and guidelines can be implemented to help minimize the hazards and maximize the advantages of these cutting-edge nanomaterials. Although biosynthesized AgNPs have benefits, the environmental and health effects of using them in agrochemicals must be carefully considered. The biosynthesis process should be optimized to create AgNPs that exhibit decreased toxicity while retaining their antimicrobial efficacy. Our future goal is to reduce negative interactions with non-target organisms and minimize potential dangers to ecosystems and animal health by concentrating on developing safer AgNP formulations that can support sustainable farming practices through thorough testing and assessment.