ANTI-ASPERGILLUS NIGER ACTION OF BIOSYNTHESIZED SILICON DIOXIDE NANOPARTICLES ALONE OR COMBINED WITH MATRICARIA CHAMOMILLA L. EXTRACT

, which cause mould infections in fruits and vegetables as well as harmful health effects. Different species, such as Aspergillus , Penicillium , Alternaria , and Fusarium were isolated from imported yellow corn samples; however, Aspergillus spp. was the most prevalent fungus. The current work attempts to synthesize novel, effective nanomaterials that are stable and antifungal by employing efficient approaches. An extract of Matricaria chamomilla L. was used in the biosynthesis of silicon dioxide nanoparticles (SiO 2 NPs) at room temperature. Ultraviolet-visible spectroscopy (UV-Vis), Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), transmission electron microscopy (TEM), and Zeta analyses were used to characterize the biosynthesized NPs. The average size of SiO 2 NPs was found to be 17-28 nm. TEM images were used to confirm the biogenesis of spherical-shaped, well-dispersed SiO 2 NPs. The zeta potential graph shows that SiO 2 NPs have a negative potential value (-31.0 mV). The antifungal activity of M. chamomilla L. extract, SiO 2 NPs, and SiO 2 combined with the extract was investigated against A. niger isolate compared to miconazole. SiO 2 NPs combined with M. chamomilla L. extract revealed higher antifungal activity than SiO 2 NPs, M. chamomilla L. extract, and miconazole with inhibition zones of 25±0.54, 17±0.37, 20±0.61 and 13±0.23 mm, respectively. This work provides a good alternate technique that is used as an antifungal agent, M. chamomilla L. extract supplemented with SiO 2 NPs, against A. niger , the pathogen for humans and crop plants.

with diameters ranging from 1 to 100 nm change dramatically, which makes them more effective than their bulk materials (El-Fallal et al., 2023;El-Zahed et al., 2022b).Silicon dioxide nanoparticles (SiO2 NPs) were reported to be more effective fungicides against A. flavus, Fusarium oxysporum, and A. niger than bulk SiO2 (Tran et al., 2023;Goswami & Mathur, 2022).Unfortunately, Etefagh et al. (2013) reported the quickly developed resistance of A. niger fungi to both NPs and nanolayers.Thus, this study aimed to isolate A. niger from Z. mays L. and treat it using M. chamomilla L. extract alone or combined with SiO2 NPs as new alternative antifungal agents.

MATERIALS AND METHODS
Isolation, purification, and identification of fungi from imported Zea mays L. seeds Unpackaged cereal grains of yellow corn were purchased from various stores in Damietta governorate, Egypt.Cereal grain samples were transported to the laboratory and kept in polythene bags under refrigeration (4°C).About 10 seeds of Zea mays L. were sterilized by putting them into 0.001% mercury chloride solution (HgCl2) for 3 minutes and washed with sterilized distilled water (3 times).Then sterilized grains were blotted dry between sterile Whatman No. 1 filter papers, inoculated into potato dextrose agar (PDA) amended with chloramphenicol (0.1 g/l) plates, and incubated at 30ºC for 5-7 days.After the incubation period, Aspergillus spp.growth with black fungal spore appearance was taken and transferred into new PDA agar plates and incubated at 30°C for 3-5 days.Fungal colonies were sub-cultivated on PDA slants stored at 4°C for further use (Choi et al., 1999).

Identification of Aspergillus spp. isolates
The fungal colonies were examined and recognized using the morphological, macroscopic, and microscopic criteria based on the keys of Pitt & Hocking (2009), and Silva et al. ( 2011).The colour of mycelium, reverse colour, size, shape, colour, and surface of conidia, conidiophores, vesicles, hyphae, and mycelia were determined as identification characteristics.

Preparation of M. chamomilla L. extract
The M. chamomilla L. extract was prepared according to the method described by Srivastava & Gupta (2009) with modification.In brief, M. chamomilla L. flowers were collected, dried in a shadow area at 25°C, and ground using a grinder machine.Aliquot 5 g from the dried powder was soaked into 50 ml of 70% ethanol (PioChem, Egypt) and put in a shaker (120 rpm, at room temperature; 25°C) for 24 hours.The plant extract was obtained by filtering the suspension using sterile Whatman No. 1 filter papers and then the pure extract was stored at 4°C for further use.

Biosynthesis of SiO2 NPs
Sodium silicate (Na2SiO3) solution was prepared by dissolving 0.0366 g Na2SiO3 (purity >98.8%,Sigma-Aldrich) in 100 ml distilled water.10 ml from the previous solution was mixed with 2 ml NaOH solution at pH 9 (5M, PioChem, Egypt), and then added to 10 ml from M. chamomile L. extract.The final mixture was shaken at 120 rpm and 25°C (room temperature) for 3 days.As a result, the mixture was filtered, and the resulting precipitate was thoroughly washed three times with distilled water and methanol.Centrifugation at 8000 rpm for 20 minutes was used to remove the residues from the mixture.The precipitate was then heated in a furnace at 550°C for 45 minutes.The residue was then dried in an oven and stored for future characterization and application operations (Rahimzadeh et al., 2022).

Characterization of the biosynthesized SiO2NPs
Double beam spectrum UV-Vis spectrophotometer V-760 (JASCO, UK) was used to monitor the production of SiO2 NPs.The FT/IR-4100typeA was used to analyze the Fourier transform infrared spectroscopy (FT-IR) of SiO2 NPs.An X-ray diffractometer (model LabX XRD-6000, Shimadzu, Japan) was also used to record the SiO2 NPs X-ray diffraction patterns (XRD).Transmission electron microscopic (TEM) investigation was performed using TEM JEOL JEM-2100, Japan (200 kV).The surface charge and stability of SiO2 NPs were measured using a Zeta potential analyzer (Malvern Zetasizer, Nano-ZS90, UK).

Antifungal activity agar well diffusion method
The fungicidal action of SiO2 NPs alone or combined with the M. chamomilla L. extract against A. niger was demonstrated in vitro using an agar well diffusion test according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI, 2006).100 μl from M. chamomilla L. extract, SiO2 NPs, SiO2 combined with the extract, Na2SiO3, and miconazole were prepared and added separately under aseptic conditions into 5mm wells in PDA plates before incubation at 30°C for 5 days.The inhibition zones were calculated in millimetres (mm).The increase in fold area for SiO2 NPs combined with chamomile extract was calculated according to the equation: (B 2 -A 2 )/A 2 ; A is the inhibition zone of antifungal agents (SiO2 NPs or chamomile extract) alone and B is SiO2 NPs combined with chamomile extract, respectively (El-Dein et al., 2021).

Radial mycelial growth inhibition
The inhibition of fungal radial growth was investigated according to Quiroga et al. ( 2004).Briefly, a 5 mm disc of 7-day culture was cut and placed upside down in the centre of PDA plates amended with 200 μl of the tested antifungal agents (100 μg/ml) dissolved in 70% of ethanol; M. chamomilla L. extract, SiO2 NPs, and SiO2 NPs combined with the extract.Inoculated PDA plates without antifungal agents were used as a control.The plates were incubated at 30°C for 7 days.The average diameter of the growth was measured in mm and percent inhibition rates were calculated through the following formula: Inhibition rate (%) = (Rr) / R Where R represents the fungal radial growth on the control.In contrast, r represents the fungal radial growth on the treated plate.

Statistical analysis
The ANOVA test was performed to analyse the findings with SPSS software version 18.The significance level was set at 0.05.The trials were repeated three times.All results were provided as the mean and standard deviation (SD) (O'Connor, 2000).

Morphological identification of fungal isolates
Aspergillus spp. was found to be the most prevalent fungus in yellow corn samples (70.2%), followed by Penicillium (14.19%),Alternaria (10.3%), and Fusarium spp.(5.31%) (Figure 1).Similarly, Abe et al. ( 2015) reported that the majority of the isolated and detected fungal genera in maize were Aspergillus, Fusarium, Penicillium, Alternaria, Cladosporium, Curvularia, Mucor, and Trichoderma.Identification of fungal isolates based on the morphological characteristics of different fungal genera.Aspergillus section Nigri was likewise the most common among other Aspergillus species, with only one species identified, A. niger.Whereas Penicillium, Alternaria, and Fusarium, were recognized by genus rather than species.A. niger exhibited a black colony that propagated radially from the spot of inoculation, according to macroscopic examination.The colony gradually grew to fill the whole Petri dish, with underlying, white mycelium topped by a layer of densely packed.Black conidial heads and reverse pallid were also observed.A. niger colonies indicated that it is originally white, but soon becomes black with conidial production.Microscopic morphology revealed conidiophores borne from surface hyphae, with hyaline, heavy, smooth walls; vesicles spherical, containing densely packed metulae and phialides across the whole surface.The conidia were brown, spherical, with visibly roughened walls, radiating heads and borne in big.SiO2 NPs FT-IR spectroscopic investigation revealed different peaks: 3402 cm -1 and 3306 cm -1 correspond to the -OH bond, 2925.48 cm -1 , which corresponds to the -C-CH3-bond, 2857.02cm -1 corresponds to the -C-CH2-bond, 1635 cm −1 substituted cis-tri and vinyl absorption in addition to 1418 cm -1 and 1234.1 cm -1 corresponds to amines stretching vibrations.The C-O bond stretching appeared at 1034.2 cm −1 .While 1012.36 cm -1 and 530.13 cm -1 correspond to Si-O bending vibration, in which the oxygen atom moves perpendicular to the Si-O-Si plane (very strong), and 705.21 cm -1 corresponds to -C-CH2-bond (Figure 3).These phytochemicals stabilize the NPs and prevent them from clumping together.
Goswami & Mathur (2022) confirmed the existence of chemical composition and functional groups of the bio-generated SiO2 NPs using the FT-IR spectrum.Aside from that, a band at 1645 cm −1 was found in the plant extract spectrum, which corresponded to silanol OH group adsorption.Furthermore, the FT-IR data revealed the presence of several proteins that act as capping and stabilizing agents (Elazab et al., 2023).
Figure 3 FT-IR spectra of plant extract and SiO2 NPs.
Zeta potential examination of the produced SiO2 NPs indicated a zeta potential value of -31.0 mV (Figure 4).The previous findings showed the good stability of biosynthesized SiO2 NPs due to substantial repulsion forces between the particles caused by their high surface negative charge values, which inhibit aggregation and agglomeration.2022) used Rhus coriaria L. extract in the green synthesis of SiO2 NPs and compared them with chemically synthesized SiO2 NPs.In both cases, the results revealed that the SiO2 NPs had a spherical form.The chemically synthesized SiO2 NPs had an average size of approximately 30 nm, whereas the green synthesized SiO2 NPs had an average size of around 55 nm, and the NPs were amorphous.Figure 6 depicts the XRD pattern of SiO2 NPs; significant diffraction peaks of SiO2 NPs were found at 2θ = 32°, 46°, 57°, 66°, and 76°.The recorded diffraction peaks were similar to Goswami & Mathur (2022) results that proved the crystallographic structure of SiO2 NPs.Debye-Scherrer equation was used to determine the size of the SiO2 NPs (D = kλ/βcosθ; D; average crystalline particle size, k; Scherer's constant (0.9), λ; wavelength of x-ray (1.5406 Å), β; XRD peak full width at half maximum, and θ; diffraction angle).The mean size was found to be 22.7 nm, which resembled the TEM data.

Antifungal efficiency of M. chamomilla L. extract alone or combined with SiO2 NPs
The biosynthesized M. chamomilla L. extract, SiO2 NPs and SiO2 NPs combined with M. chamomilla L. extract showed higher antifungal activity against A. niger than Miconazole as shown in Figures 7 &8.The biosynthesized SiO2 NPs had substantial synergistic effects when combined with chamomile extract, in addition to its antifungal properties, which increased its fold area by 1.16.These findings confirmed the potential for employing biosynthesized SiO2 NPs as promising alone or mixed antifungal agents in various medicinal and industrial applications.According to EL-Hefny et al. ( 2019), essential and recovery oils derived from M. chamomilla fresh flowers have been found to have antifungal activity against fungi related to biodeterioration of cultural assets (A. niger, A. terreus, A. flavus, and F. culmorum).The study also documented that the antifungal action of essential oils of M. chamomilla was dose-dependent, with the best results against A. niger.

CONCLUSION
A. niger was isolated, identified and selected for studying the antifungal activity.SiO2 NPs were biosynthesized using the extract of M. chamomilla L. as a bioreducing agent.The provided biosynthesis approach was rapid, simple, cheap, and eco-friendly with high stability.SiO2 NPs production was confirmed and characterized using UV-Vis, FT-IR and XRD spectral analyses and TEM and Zeta potential analysis.To the best of our knowledge, this is the first report on the antimicrobial study of a combination of M. chamomilla L. extract and SiO2 NPs.The biosynthesized NPs combined with chamomile extract had higher antifungal activity against A. niger than individual SiO2 NPs or chamomile extract.

Figure 1
Figure 1 Colonies of A. niger, A. flavus, Alternaria spp., Penicillium spp., and Fusarium spp.isolated from yellow corn on PDA incubated at 30°C for two; (A) and five days; (B &C).Purification of A. niger at 30°C for seven days; (D).
Biosynthesis and characterization of SiO2NPsEthnomedicinal extract of M. chamomilla L. was preferred to aqueous extraction and used in phytochemistry and pharmacological applications due to the successful extraction of the existence of major bioactive compounds (Alhazmi et al., 2022).Moreover, M. chamomilla L. extract is rich in flavonoids, polyphenols, terpenoids, and sugars that act as bio-reducing agents during the biosynthesis of the NPs process (Alshehri & Malik, 2020; Dogru et al., 2017).The formation of a white precipitate during the biosynthesis process was the first indication of the biosynthesis of SiO2 NPs.UV-Vis spectrophotometer was used to analyse these solutions, which revealed strong three peaks at 266, 313, and 358 nm due to the surface plasmon resonance characteristic of the SiO2 NPs in the reaction medium (Figure 2).Park et al. (2010) recorded two strong peaks of SiO2 NPs at 215 and 280 nm while Babu et al. (2018) reported that SiO2 NPs showed a broad adsorption peak at 450 nm.

Figure 2
Figure 2 UV-visible spectroscopy of SiO2 NPs compared to plant extract and Na2SiO3.

Figure 4
Figure 4 Zeta potential analysis of SiO2 NPs.The TEM micrograph revealed a good dispersion of the biosynthesized sphericalshaped SiO2 NPs with an average size of 17-28 nm (Figure 5).Al-Azawi et al. (2019) attained spherical-shaped SiO2 NPs with a 33.94 nm range utilising an aqueous extract of Thuja orientalis leaf.Rahimzadeh et al. (2022) used Rhus coriaria L. extract in the green synthesis of SiO2 NPs and compared them with chemically synthesized SiO2 NPs.In both cases, the results revealed that the SiO2 NPs had a spherical form.The chemically synthesized SiO2 NPs had an average size of approximately 30 nm, whereas the green synthesized SiO2 NPs had an average size of around 55 nm, and the NPs were amorphous.

Figure 6
Figure 6 XRD spectrum of the biosynthesized SiO2 NPs using M. chamomilla L. extract.

Figure 9
Figure 9 displayed the A. niger mycelium radial growth and inhibition rate percent during treatment using tested antifungal agents, including chamomile extract alone and chamomile extract combined with SiO2 NPs.SiO2 NPs combined with chamomile extract showed higher antifungal action against A. niger with an inhibition rate of 34% than chamomile extract alone which inhibited the fungal growth by 16%.Goswami & Mathur (2022) reported that A. niger and F. oxysporum were suppressed by 1000 µg/l of SiO2 NPs derived from agricultural waste (Sugarcane bagasse and corn cob) with mycelia growth inhibition 58.92% and 73.42%, respectively.In comparison to conventional emulsions and free oils, SiO2 NPs act as a stabilizer, preventing the simple escape of essential oils from the emulsion system (Mariyate & Bera 2021).Masoud et al. (2022) said that the combination of bio-silica NPs and Cassia nodosa extract increased the inhibition action of some fungal strains like F. oxysporum and R. solani.

Figure 9
Figure 9 Radial mycelial growth of A. niger in the presence or absence of chamomile extract alone or chamomile extract + SiO2 NPs at a concentration of 100μg/ml.