Bio-mediated SeNPs synthesis and its characterization

Searching for effective and safe alternatives to chemical fungicides is needed globally as chemicals have been associated with negative health impacts. In this respect, bio-mediated SeNPs using Fenugreek seeds aqueous extract was used for the first time as potential antifungal bioagent against two Fusarium spp. The current study describes the biosynthesis of SeNPs using Fenugreek seed aqueous extract as an ecofriendly method. Fenugreek seed extract had several naturally occurring bioactive compounds, such as alkaloids, flavonoids, phenols, amino acids, glycosides, and polysaccharides with reducing bioactivity (Ramamurthy et al. 2013). These biomolecules successfully bio-mediated the synthesis of SeNPs by reducing selenite salt to SeNPs. The low cytotoxic effect of biosynthesized SeNPs is probably attributed to the various functional biomolecules found in the bio-extract used for synthesis. The treatment of tomato fruits with SeNPs completely protected it from any infection signs (100% reduction) and preserved the fresh-like appearance and color when stored at 5 °C or 25 °C, indicating possible application of SeNPs at its MIC during storage, and transportation. SeNPs characterization was primarily done using UV–VIS, which detected the biosynthesized SeNPs at 360 nm with brick red color. Similarly, Al-Qaraleh et al. (2022) reported the color change of reaction mixture from colorless to brick red with maximum absorption between 260 and 350 nm. This was caused by Surface Plasmon Resonance of the formed NPs, confirming the bio-reduction of Na2SeO3 solution to Se0 element by Moringa peregrine aqueous extract. The XRD patterns verified seven intense peaks that correspond to the crystallographic planes of Se crystals, thus confirming the nano-crystalline nature of biosynthesized SeNPs as compared with standard file no. 06-0362 (Ingole et al. 2010). Similarly, SeNPs crystal structure and phase composition were identified by Srivastava and Mukhopadhyay (2015), where the reflections of pure Se crystal at 23.6°, 29.9°, 41.4°, 43.8°, 51.8°, 55.9°, 61.8°, 65.3°, and 68.3° were attributed to the Bragg reflection peaks at (100), (101), (110), (102), (111), (201), (003), (202), (210), and (211). The FTIR analysis demonstrated various absorption peaks corresponding to different biomolecules present in the biological extract used for biosynthesis. The bands at 2918.58 cm−1 are typical bands for polysaccharides as determined by C-H symmetric/asymmetric stretching (Salem et al. 2022a). The C=C alkene group was detected at 1634 cm−1 (Anu et al. 2017). The C–N stretching of amines was detected at 1040.93 cm−1 and the C–N–C bending bands at 517.62 cm−1 and 463.22 cm−1 (Alagesan and Venugopal 2019; Al-Qaraleh et al. 2022). Typical absorption peaks for OH stretching and C–H vibration were detected at 3274.28 cm−1 and 1445.93 cm−1, respectively (Mellinas et al. 2019). The N–O stretching group is responsible for the peaks at 1423.47 and 1382.33 cm−1. The carboxyl group (C=O) stretching vibration peaks was detected at 1229.25 cm−1 (Satgurunathan et al. 2017). Effective stabilizing and/or reducing agents in the bioextract are due to the existence of important functional groups, such as O–H, N–H, C–N, C–H, N–O, C–N–C, and C=O as previously proposed by Elnady et al. (2022a, b) that is probably responsible for the bio-reduction and stability of SeNPs.

Antifungal activity of biosynthesized SeNPs and its mode of action

The antifungal potentialities of diverse nanometals such as, Se, Ag, Cu, and TiO2 have been confirmed against some phytopathogenic fungi such as, P. digitatum, A. alternata, and Aspergillus spp. (Ouda 2014; Sánchez-López et al. 2020).

In the current study, biosynthesized SeNPs using Fenugreek seeds extract showed high antifungal effect against F. oxysporum and F. moniliforme as detected by MIC and MFC. The MICs of SeNPs were 0.25 and 1.7 mg/mL, and the MFCs were 0.27 and 2.9 mg/mL againt F. oxysporum and F. moniliforme, respectively. These values were much lower than those determined by Salem et al. (2022a) who reported that the composite SeNPs/pomegranate peel extract revealed MFC that ranged 22.5–25 mg/mL against Penicillium digitatum.

In addition, El-Saadony et al. (2021), reported that biosynthesized SeNPs using Lactobacillus acidophilus inhibited some Fusarium spp. in the range of 20–40 µg/mL. While, wheat supplemented with 100 µg/mL of SeNPs significantly reduced the incidence of crown-root rot disease in wheat by 75% and improved its growth, grain quality and quantity by 5–40%. Furthermore, Joshi et al. (2021) reported that infected tomato plants coated with biosynthesized SeNPs exhibited a significant protection (72.9%) against late blight disease caused by Phytopthora infestans.

The biosynthesized SeNPs interact with the microbial cell wall leading to disruption and alteration in its permeability, NPs enter the cell and inhibit the proteins and DNA synthesis. The antimicrobial activity of SeNPs is probably attributed to reactive oxygen species (ROS), such as hydroxyl radicals, superoxide anions, and hydrogen peroxides. ROS induce damage to the microbial cell membrane, inhibiting the DNA replication and amino acid synthesis (Filipović et al. 2021; Elnady et al. 2022a, b).

Effect of SeNPs coating on F. oxysporum-induced post-harvest disease

The treatment with SeNPs at its MIC (0.25 mg/mL) successfully inhibited F. oxysporum growth in vitro, with 100% protection of treated tomato fruits up to 25 and 35 days when stored at 25 °C and 5 °C, respectively. The treatment of red colored tomato fruits (Group A) with biomediated SeNPs revealed a significant difference between SeNPs-treated infected fruits (T3) and untreated infected fruits (T4). Likewise, Salem et al. (2022a) reported that 0.5% and 1.0% from SeNPs-composite led to 84.6% and 97.2% reduction of A. alternata growth on persimmon fruit, respectively. In addition, Salem et al. (2022b) reported that the treatment with SeNPs-composite for 10 h was effective to decompose the fungus Penicillium digitatum.

Effect of storage temperature on disease progress and shelf-life of tomato fruits

The shelf-life of fruits is determined based on their appearance and spoilage. When 50% of fruits showed symptoms of shrinkage and/or spoilage, the fruits was considered to have reached the end of its shelf-life (Tolasa et al. 2021). In general, ripened fruits are more susceptible to pathogen infection and decay faster than green ones (Rodrigues and Kakde 2019). The main bioactive compounds in ripened tomato are flavonoids, lycopene, and carotenoids as well as soluble sugars, β-carotene, vitamins, and tomatine. During maturation, flavonoids accumulate while the chlorophyll is decreased. However, in green fruits, the content of α-tomatine is higher (500 mg/kg) as compared to ripened red ones (5 mg/kg), which is known to provide protection against pathogens (Chaudhary et al. 2018). Therefore, Fusarium infects ripened red tomato fruits causing its rot while using its ascorbic acid and soluble sugars necessary for growth (Bakar et al. 2013).

In general, storage at high temperature fasten the rate of ripening, thus fastening the rate of fruit deterioration, therefore using coolers slows the rate of ripening and extend fruit’s shelf life (Abiso et al. 2015). Tolasa et al. (2021) reported that mature green tomato fruits coated with cactus mucilage can be stored for three weeks. Similarly, Abiso et al. (2015) reported that tomato fruits decay of 16.66% starts early on day 6 for those stored at room temperature and then the decay was raised to 70% on the 12th day. Also, Melkamu et al. (2009) reported that mature green tomato fruits can be stored for 16 days at room temperature. Overall, the treatment of tomato fruits with SeNPs gives an alternative approach for prolonging post-harvest shelf life and maintaining the quality of Cherry tomato up to 25 days at 25 °C.

Cytotoxicity assessment of SeNPs

The low cytotoxic effect of biosynthesized SeNPs is probably due to several functional biomolecules found in the bio-extract used for biosynthesis and stabilization as confirmed by FTIR analysis, such as O–H, N–H, C–N, C–H, N–O, C–N–C, and C=O. Many researchers suggested that the cytotoxic effect of NPs depends on various factors, such as its administration routes, size, aggregation; time exposure and/or concentration, as well as capping agent used for the stabilization of produced NPs (Tayel et al. 2017; Sorour et al. 2019; Elnady et al. 2022a, b). Similarly, Elnady et al. (2022b) reported lower cytotoxicity on normal cell lines for bio-mediated AgNPs using Ulva fasciata and Citrus japonica bio-extracts as compared with chemically-synthetized ones. Remarkably, SeNPs have reduced the cytotoxicity toward higher organisms e.g., human and animals within allowed limits, but are highly bioactive against microorganisms, providing many applications in biomedical and nutritional fields (Huerta-Madroñal et al. 2021). Therefore, green bio-mediated SeNPs were used as preservatives for crops, meat products and in anticancer formulations (Salem et al. 2022a, b).

Chemical and physical analysis of tomato fruits

The overall results indicate that post-harvest storage temperature as well as SeNPs treatment affect the characteristics of fresh tomato. However, the treatment of tomato fruits with SeNPs at its MIC positively affected the chemical properties of tomato fruits including TA%, pH, total carotenoids and lycopene, as well as decreased the physiological weight loss %. This effect is probably due to the barrier effect of SeNPs acting against microbial growth and preserving freshness, thus increasing fruits’ shelf life up to 25 and 35 days when stored at 25 °C and 5 °C, respectively.

In the current study, the treatment of tomato fruits with MIC of SeNPs increased the TA% as compared to untreated ones (T1), which will probably increase the shelf life of treated tomato fruits. The TA% is generally decreased by increasing the transportation time and repeated vibration, which increases the rate of respiration and consumption of organic acids (Al‐Dairi et al. 2021c). In addition, Endalew (2020) reported that the TA% of tomato was decreased at 25 °C due to the enhancement of tomato ripening and enzymes activity, thus affecting the fruits’ acidity. Also, Al‐Dairi et al. (2021c) reported that the storage of tomato fruits at 22 °C accelerated the TA% reduction after transportation for longer distance. But when fruits were stored at 10 °C, its TA% was increased to 0.31% and 0.29% for short and long distances, respectively, after 12 days.

For total lycopene and β-carotene, it is generally increased with fruits’ ripening and during the storage period (Al‐Dairi et al. 2021a). The development of carotenoid is rapid in tomato stored at room temperature, while it was observed to be slow for those stored in coolers. Tomato fruits stored at 22 °C had 5-folds increase in their total lycopene and carotenoids after 12 days (Al‐Dairi et al. 2021a), but when stored at 10 °C it was increased by 3.5-folds only, this increase was probably due to the accumulation of lycopene and carotenoids resulting from chloroplast conversion to chromoplast (Abiso et al. 2015). In general, harvested tomato at light-red ripening stage have a shorter shelf life as compared to those harvested at earlier stage. In addition, Opara et al. (2012) reported that the lycopene content of ‘Cherry’ cultivar ranged from 6.2 to 56.1 mg/100 g FW during ripening and was increased by 40.7% during ripening. The increase in tomato TA% when stored at 25 °C is probably due to the fungal infection which increased the acidity of infected fruits as previously reported by Jiao et al. (2022) who suggested that organic acids (e.g. citric acid, gluconic acid, or oxalic acid) were secreted by post-harvest fungi, such as Penicillium spp. and Botrytis cinerea, which are important virulence factors.

For the pH of fresh fruits, generally, it depends mainly on its organic acid contents, and it increases with the increase of storage duration, ripening, and respiration (Endalew 2020). In agreement with our results, the pH value was reported to range from 3.5 to 4.2 for ‘Cherry’, ‘Monika’, and ‘Isabella’ tomato during fruits’ maturation and ripening (Opara et al. 2012). Similarly, Teka (2013) reported that full ripe tomato fruits stored at 22 °C had a higher pH (4.63) as compared to mature green ones stored at 10 °C that had pH of 4.23.

Remarkably, the weight loss % was significantly decreased by increasing the MIC used for treatment, where treated fruits with SeNPs maintained their appearance, color, and weight during their storage at 25 °C and 5 °C for 25 and 35 days, respectively. Likewise, Al-Dairi et al. (2021b) reported that the weight loss in tomato fruits was increased from 3.5 to 6.91% when fruits were stored at 10 °C and 22 °C respectively, for 12 days. It is proposed that SeNPs exhibit antimicrobial activity, thus forming barriers against fungal infection, as well as protecting tomato fruits from moisture loss, managing respiration and over-ripening of treated fruits (Huang et al. 2021; Kumar and Prasad 2021). In addition, Abiso et al. (2015) reported an increase in weight loss (18.36%) of light-red tomato fruits stored at 25 °C for 10 days, as compared to 4.08% when stored in coolers.

Overall, the treatment of tomato fruits with SeNPs at its MIC positively affected the chemical properties of tomato fruits, as well as decreased its weight loss %, confirming the positive barrier effect of SeNPs against Fusarium infection as well as preserving freshness, thus increasing the shelf life of fruits. Therefore, SeNPs treatment gives an alternative approach for prolonging shelf-life, maintaining the quality of tomato and providing protection from post-harvest fungal invasion. In addition, biomediated SeNPs is eco-friendly valuable alternative to chemical fungicides in terms of health and food safety.