Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 77(1): 65-82, 2024 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-2424 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Luțu, O.A., Soare, L.C., Fierăscu, I., Fierăscu, R.-C., Dobres- cu, C.M., Păunescu, A., Ponepal, C.M., Topală, C.M., Vîjan, L.E., Deliu, I., Negrea, D., Vîlcoci, D.Ș., Cîrstea, G., Aldea, F., Honțaru, S.O., & Șuțan, N.A. (2024). Phytotoxicity, cytogenotoxicity and antimicrobial potential of extracts with gold-silver bimetallic nanopar- ticles obtained from pteridophyte spores. Caryologia 77(1): 65-82. doi: 10.36253/caryologia-2424 Received: December 13, 2023 Accepted: May 21, 2024 Published: July 8, 2024 Copyright: © 2024 Authors. This is an open access, peer-reviewed article published by Firenze University Press (ht tps://www.fupress.com/caryolo - gia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. ORCID OAL: 0000-0001-8351-0919 LCS: 0000-0002-2874-3135 IF: 0000-0002-1834-9812 RCF: 0000-0003-4224-9157 CMD: 0000-0001-7972-3612 AP: 0000-0002-0228-4524 CMP: 0000-0002-0391-7629 CMT: 0000-0002-9117-4983 LEV: 0000-0003-1607-4947 DN: 0000-0001-7525-1056 DSV: 0000-0002-9777-6782 GC: 0000-0002-7442-7109 NAS: 0000-0001-7459-628X Phytotoxicity, cytogenotoxicity and antimicrobial potential of extracts with gold- silver bimetallic nanoparticles obtained from pteridophyte spores Oana Alexandra Luțu1, Liliana Cristina Soare1,*, Irina Fierăscu2,3, Radu-Claudiu Fierăscu2,4, Codruța Mihaela Dobrescu1, Alina Păunescu1, Cristina Maria Ponepal1, Carmen Mihaela Topală1, Lore- dana Elena Vîjan1, Ionica Deliu1, Denis Negrea5, Denisa Ștefania Vîlcoci5, Georgiana Cîrstea5, Florentina Aldea6, Sorina Octavia Honțaru7, Nicoleta Anca Șuțan1 1 Department of Natural Sciences, Faculty of Science, Physical Education and Informatics, National University of Science and Technology Politehnica Bucharest, Pitesti University Center, Romania, 1 Targu din Vale Str., 110040 Pitesti, Romania 2 The National Institute for Research & Development in Chemistry and Petrochemistry, ICECHIM, 202 Spl. Independentei, 060021 Bucharest, Romania 3 Faculty of Horticulture, University of Agronomic Sciences and Veterinary Medicine of Bucharest, 59 Marasti Bvd., 011464 Bucharest, Romania 4 Faculty of Chemical Engineering and Biotechnology, National University of Science and Technology Politehnica Bucharest, 1-7 Gheorghe Polizu St., 011061 Bucharest, Romania 5 Regional Research and Development Center for Innovative Materials, Processes and Products for the Automotive Industry (CRC&D-Auto), National University of Science and Technology Politehnica Bucharest, Pitesti University Center, Romania, 1 Targu din Vale Str., 110040 Pitesti, Romania 6 Department of Developmental Biology, Institute of Biology-Bucharest, Romanian Acad- emy, 296 Splaiul Independentei Street, 060031 Bucharest, Romania 7 Department of Health Care and Physiotherapy, Faculty of Sciences, Physical Education and Informatics, National University of Science and Technology Politehnica Bucharest, Pitesti University Center, Romania, 1 Targu din Vale Str., 110040 Pitesti, Romania *Corresponding author. Email: liliana.soare@upb.ro Abstract. Investigating the toxicity of naturally occurring or synthesized nanoparticles for various applications is absolutely necessary for environmental protection and safety use. The aim of these research was to investigated the phytotoxicity, cytogenotoxic- ity and antibacterial potential of the extracts with gold-silver bimetallic nanoparticles (Au-Ag NPs) obtained from green synthesis in Asplenium scolopendrium L. and Dry- opteris filix-mas (L.) Schott spores extracts. To our knowledge, this is the first report of the Au-Ag NPs phytosynthesis based on extracts obtained from fern spores. UV-Vis spectroscopy analysis of the samples revealed the maximum absorbance, characteristic of samples with bimetallic nanoparticles, which varied depending on the Au:Ag ratio. Energy-dispersive X-ray spectroscopy confirmed the presence and distribution of Au, Ag and other chemical elements. The presence of specific secondary metabolites in the extracts that helped in NPs biosynthesis stimulated growth processes. Good results http://www.fupress.com/caryologia https://doi.org/10.36253/caryologia-2424 https://doi.org/10.36253/caryologia-2424 https://www.fupress.com/caryologia https://www.fupress.com/caryologia https://orcid.org/0000-0001-8351-0919 https://orcid.org/0000-0002-2874-3135 https://orcid.org/0000-0002-1834-9812 https://orcid.org/0000-0003-4224-9157 https://orcid.org/0000-0001-7972-3612 https://orcid.org/0000-0002-0228-4524 https://orcid.org/0000-0002-0391-7629 https://orcid.org/0000-0002-9117-4983 https://orcid.org/0000-0003-1607-4947 https://orcid.org/0000-0001-7525-1056 https://orcid.org/0000-0002-9777-6782 https://orcid.org/0000-0002-7442-7109 https://orcid.org/0000-0001-7459-628X mailto:liliana.soare@upb.ro 66 Oana Alexandra Luțu et al. were recorded for some Dryopteris filix-mas samples, correlated with a significantly increased mitotic index. Cell viability decreased significantly in three of the nanoformulations. Only extracts with Au-Ag NPs showed antimicrobial effect against Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633 and Escherichia coli ATCC 8739. The testing of the antibacterial potential of these extracts must be extended to other bacterial strains and other microorganisms, the search of new antimicrobial resources being an urgent necessity nowadays. Keywords: spore extracts, Asplenium scolopendrium L., Dryopteris filix-mas (L.) Schott, phytosynthesis, gold-silver nanoparticles, phytotoxicity, cell viability, cytogenotoxicity, antibacterial potential. INTRODUCTION Green synthesis is a promising substitute for tradi- tional synthesis methods. Among different green syn- thesis methods, the use of unicellular and multicellular biological entities for obtaining nanoparticles (NPs) represents one of the most promising routes (Ettadili et al. 2022). Plants produce alkaloids, flavonoids, carbohy- drates, polymers, proteins, and numerous antioxidants that are efficiently used in NPs synthesis (Patel et al. 2021), because they are involved in the bioreduction of metal salts (Nasrollahzadeh et al. 2019). According to Chatterjee et al. (2019), among Crypto- games, algae and bryophytes are the most used to obtain nanoparticles, while ferns are little investigated. Various organs of ferns can be used to obtain the extracts need- ed for phytosynthesis, the spores being considered in recent years (Soare and Șuțan 2018). The antioxidant potential and medicinal value of fern species determinate their selection for the synthesis of AgNPs and AuNPs (Makarov et al. 2013). By reducing AgNO3 and HAuCl4 in Adiantum philippense extracts, AuNPs and AgNPs were obtained (Sant et al. 2013) and Kunjiappan et al. (2015) reported the synthesis of AuNPs in Azolla microphylla extract. The investigation of the phytotoxicity of NPs contrib- utes to the establishment of their toxicity profile (USEPA 2005). Seed germination, root and stem growth, and seedling biomass are the morphophysiological param- eters frequently used in such studies (Drăghiceanu et al. 2019; Pathipati et al. 2018). The biochemical param- eters and those related to the cytogenotoxicity of NPs (Drăghiceanu et al. 2019) are often added to the morpho- physiological ones, because if the NPs or the aggregates of NPs are small, they can penetrate the cell and interact with different cellular components and induce metabolic or genetic changes (Pathipati et al. 2018). Silver has been considered an antimicrobial agent since ancient times. The antimicrobial activity of AgNP is influenced by two important factors: the high stability of dispersion and the release of Ag ions (Harada et al. 2018). AgNPs can effectively eliminate pathogenic bacteria, and by forming alloys with other noble metals (e.g Au) the stability of these materials can be significantly improved while maintaining antibacterial activity (Qin et al. 2021). Au-Ag NPs showed increased antimicrobial, anti- oxidant and anticancer activities (Godipurge et al. 2016) compared to monometallic NPs due to the interactions between the two metals that occur in fine structures determining surfaces with new characteristics (Latif-ur- Rahman et al. 2015). The first aim of this study was to determine the abil- ity of aqueous extracts obtained from fern spores to pro- duce bimetallic nanoparticles (Au-Ag NPs). Secondly, we sought to characterize the extracts with NPs by UV-Vis spectroscopy (UV-Vis), scanning transmission electron microscopy coupled with energy dispersive spectros- copy (STEM-EDX), X-Ray diffraction (XRD), and test to establish their phytotoxicity, cytogenotoxicity, and anti- bacterial potential. MATERIALS AND METHODS Obtaining extracts from fern spores The spores used for the extracts were obtained from different mature individuals plants of Asplenium scolo- pendrium L. (A) and Dryopteris filix-mas (L.) Schott (D) from Vâlsan Valley (Argeș, Romania). The voucher specimens were recorded in the herbarium collection of the Argeș County Museum (Asplenium scolopendri- um – 11.331, Dryopteris filix-mas – 11.330) (Soare et al. 2021). The ratio between plant material (spores) and sol- vent (distilled water) was 1:100 (g/mL). The micromet- ric dimensions of the spores which form a fine powder did not require grinding of the biological material. The spores and solvent were maintained in contact at room temperature (15 oC) for 5 days then filtered. For AuNPs and AgNPs phytosynthesis, we used plant extracts (DAM, AAM), 0.1 mM HAuCl4 and 1 mM AgNO3, the two reagents being added in 1:1 and 1:10 proportions (AA 1:1, AA 1:10, DA 1:1, DA 1:10) (Fierăscu et al. 2017b). The experimental variants are presented in Table 1. 67Phytotoxicity, cytogenotoxicity, and antimicrobial potential of gold-silver nanoparticle extracts from pteridophyte spores Physicochemical characterization of extracts Physicochemical characterization of extracts is performed by UV-Vis, STEM-EDX (HITACHI SU8230 microscope) and XRD (Rigaku SmartLab). The formation of the Au-Ag NPs was examined using the PerkinElmer Lambda25 UV-Vis Spectro- photometer, in the range 370-600 nm for Au-Ag NPs (Fierăscu et al. 2017b) using a 10 mm quartz cuvette with optical path. STEM-EDX was used to confirm the presence of Au and Ag, and to investigate particles shape and size dis- tribution. For each extract a drop was poured on STEM sample holder (Ni grid with carbon support film) and dried for 24 hours in a desiccator. For each sample EDX area scans were performed in order to obtain chemical elemental information and confirm the Au and Ag pres- ence. Also, EDX mapping have been obtained in order to investigate Au and Ag presence and distribution. STEM images provided information about particle’s shape and size distribution (Soare and Șuțan 2018). X-Ray diffraction. The solutions containing nano- particles dispersions were prepared for analysis by dep- osition on the surface of the sample holder and evapo- rated at room temperature for 10-15 minutes before being subjected to analysis. The XRD analysis were per- formed using a 9 kW Rigaku SmartLab diffractometer (Rigaku Corp., Tokyo, Japan, 45 kV and 200 mA, CuKα radiation-1.54059 Å), in scanning mode 2θ/θ, between 7° and 90° (2θ). The analyzes were performed using the Rigaku Data Analysis Software PDXL 2, database pro- vided by ICDD. Crystallite size was determined using the Debye- Scherrer equation: 𝐷𝐷! = 𝐾𝐾 × 𝜆𝜆 𝛽𝛽 × 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 (1) where Dp represents the average size of the crystallites, K - Scherrer constant (for cubic structures, K = 0.94), β - the width at half-height of the diffraction maximum, θ - Bragg angle, λ - wavelength (1.54059 Å in our case). Assessment of the phytotoxic effect by Triticum test The seeds of Triticum aestivum L., Miranda variety, were provided by Agricultural Research and Develop- ment Station Pitești, Albota, Romania. The seeds were hydrated in distilled water and immersed in the test solu- tion for 1 hour. Then, the seeds were placed in Petri dish- es on filter paper and periodically watered with distilled water. The Petri dishes were kept in the dark at 20±2 °C temperature and 20±3% humidity, until the measure- ments were made. Ten seeds were used for each variant. After 4 days from the beginning of the experiment the root and stem length were measured and fresh and dry weight were determinate. The dry weight was established after keeping the plant material in the oven, at 80 °C, until a constant weight was obtained (Azooz et al. 2012). The inhibition rate of the length of root and stem seedlings was calculated using formulas (2) provided by Ma et al. (2019): The inhibition rate = [(Gm-Gx)/Gm]×100 (2) where Gm - values reached for the Control determined parameters, Gx - values reached for the determined parameters for the variants with extracts. Evaluation of cytogenotoxic effects by Allium test After removing the cataphylls and old roots, onion bulbs (Allium cepa L.) of about 3.5 cm diameter were placed with the discoidal stem in distilled water and kept in the dark for 48 h at room temperature. The bulbs with new roots were transferred to the test solution for 48 hours (Table 1). The roots were stored in 70% etha- nol at 2-4 °C, after they were fixed 24 hours in Farm- er’s solution. The squash technique was used to display in a single layer the root tips cells hydrolysed with 1N HCl and stained with 2% aceto-orcein. To evaluate the cytogenetic activity of the extracts, approximately 3000 cells/experimental variant were analysed and the mitotic Table 1. Experimental variants. Variants Content Dilution Control Distilled water - DAM D10 Aqueous extract of D spores 10 DAM D100 100 DA1:1 D10 Aqueous extract of D spores with bimetallic nanoparticles (Au-Ag 1:1) 10 DA1:1 D100 100 DA1:10 D10 Aqueous extract of D spores with bimetallic nanoparticles (Au-Ag 1:10) 10 DA1:10 D100 100 AAM D10 Aqueous extract of A spores 10 AAM D100 100 AA1:1 D10 Aqueous extract of A spores with bimetallic nanoparticles (Au-Ag 1:1) 10 AA1:1 D100 100 AA1:10 D10 Aqueous extract of A spores with bimetallic nanoparticles (Au-Ag 1:10) 10 AA1:10 D100 100 Note: A - Asplenium scolopendrium L., D - Dryopteris filix-mas (L.) Schott. 68 Oana Alexandra Luțu et al. index (MI), mitosis phase indices and the frequency of chromosomal aberrations were determined (Soare and Șuțan 2018). Evaluation of cell viability by Evans Blue test To establish the cell viability with Evans Blue stain- ing, we used the protocols proposed by Chen et al. (2008), Vijayaraghavareddy et al. (2017) and Adamakis et al. (2019) with minor modifications. After the experi- mental treatment, 10 roots from each onion bulb were randomly selected. These were immersed for 15 min- utes in 2 ml of 0.25% aqueous Evans Blue solution and then rinsed with distilled water to remove excess dye. The roots were kept in distilled water overnight at room temperature. To extract the dye, the apical parts of the roots (5 mm) were excised on the next day and placed in 2 ml of 1% aqueous solution of sodium dodecyl sul- fate and kept in a water bath at 50 °C for one hour. For the quantitative determination of the absorbed dye, the absorbance at 600 nm was measured using T70+ UV-Vis Spectrophotometer. Evaluation of antimicrobial activity The antibacterial activity of the undiluted extracts with and without Au-Ag NPs was tested against stand- ard bacterial strains (Escherichia coli ATCC 8739, Staph- ylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633) (LTA, Italy) by Kirby-Bauer protocol on Mueller Hinton agar, according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (Radji et al. 2013). For the sterilized discs (6 mm diameter) 7 µl of extract was added and after the incubation period (24 h, 37 °C), the diameter of inhibition zones around the discs was determined. The measurements were performed consid- ering the negative (distilled water, AD) and the positive (ampicillin, ATB) control. To determine minimal inhibi- tory concentration (MIC), binary serial dilutions of the tested extracts were performed (according to the CLSI standard, adapted broth dilution method), in which equal amounts of microbial inoculum were inoculated (Radji et al. 2013). Equal amounts of microbial inocu- lum (0,5 McFarland) and broth with decreasing amounts of extracts were incubated at 37 °C for 24 h. The results were appreciated by the unaided eye, considering the control (broth tube without extract, inoculated). The interpretation of the results was made the next day considering the following aspects: clear culture medium – without bacterial development, hazy medium – bacterial development. The MIC value was determined by calculating the arithmetic mean of the last dilution with clear culture medium and the first dilution with hazy culture medium. Statistical analysis Data obtained after three repetitions of Triticum and Allium tests, Evans Blue and Kirby-Bauer protocols, were statistical analysed using IBM SPSS Statistics 23. The mean and standard error (SE) were calculated, and the averages were compared with Duncan’s multiple com- parison test. RESULTS AND DISCUSSION UV-Vis Spectroscopy UV-Vis spectroscopy is a technique used to char- acterize nanoparticles of noble metals and is easy to apply (Hu and Xianyu 2021). UV-Vis analysis of the samples revealed that in the case of the extracts with bimetallic nanoparticles (Figure 1), the following peaks were obtained: 533 nm (AA1:1), 521 nm (DA1:1), and 441 nm (AA1:10, DA1:10). The peak value recorded for AA1:10 and DA1:10 is closer to monometallic Ag due to the higher proportion of Ag compared to Au. Our results are confirmed by other research. The forma- tion of bimetallic NPs is highlighted by the appearance of a single band whose peak is located between that of the AuNPs and AgNPs (Tamuly et al. 2013; Garcia et al. 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 350 400 450 500 550 600 A bs or ba nc e (a .u .) nm AA1:1 AA1:10 DA1:10 DA1:1 Figure 1. UV-Vis spectra of Asplenium scolopendrium L. and Dry- opteris filix-mas (L.) Schott spores extracts with Au-Ag nanoparti- cles (AA1:1, AA1:10, DA1:1, DA1:10 sample). The arrow indicates the maximum absorbance obtained for the investigated samples. 69Phytotoxicity, cytogenotoxicity, and antimicrobial potential of gold-silver nanoparticle extracts from pteridophyte spores 2014; Malathi et al. 2014). An example is for monome- tallic nanoparticles (AgNPs, AuNPs), as well as bimetal- lic ones (Ag-AuNPs) biosynthesized in extract obtained from the root of the medicinal plant Plumbago zeylanica, Salunke et al. (2014) obtained the maximum absorb- ance at 440 nm for Ag monometallics, at 570 nm for Au monometallics and at 540 nm for bimetallic ones. Çıplak et al. (2018) obtained a maximum absorbance at 410 nm for AgNPs and at 534 nm for AuNPs. In the case of bimetallic nanoparticles Ag67Au33, Ag50Au50 and Ag33Au67, the authors obtained different values of absorbance, depending on the Au:Ag ratio, respectively 412 nm, 519 nm and 523 nm. In extracts with nanoparticles, the carbonyl group at 1635 cm-1 shows an increased intensity as a result of the capture/reduction of the metals (Drăghiceanu et al. 2021). It was also confirmed that the carbonyl group had stronger ability to bind with metal nanoparticles or act as stabilizing agents (Huleihel et al. 2002). STEM-EDX Analysis The investigation of the samples with EDX (Energy- dispersive X-ray spectroscopy) revealed the elemental com- position (Su 2017), besides the elements added for obtain- ing the nanoparticles being highlighted and others (Cu, Ni, O, Al, Cl, Ca, Sn, K, and Mg), characteristic of the extracts, as it is observed from the Figures 2 and 3. The EDS mapping exposed the 2D presence and distribution of chemical elements within the investigated areas for all the samples, as can be seen for Au and Ag in A. scolopendrium (AA1:1) and D. filix-mas extracts (DA1:1) (Figure 4). STEM analysis was used to investigate nanoparticles shape and size (Su 2017). The Au-Ag NPs obtained in the A. scolopendrium and D. filix-mas extracts had sizes between 5-39 nm for AA1:1 sample, 8-39 nm for AA1:10, 13-35 nm for DA1:1 and 15-35 nm for DA1:10 (Figure 5). Particle size between 4-94 nm and 2-78 nm, respectively, have been reported for AgNPs and AuNPs obtained by phytosynthesis assisted by various pteridophytes (Rao et al. 2021). XRD Analysis The crystallographic characteristics of the materials were evaluated from the diffraction pattern of the sam- ples. Figure 6 presents the normalized spectra obtained for the four samples. Figure 2. EDX spectra obtained for the Asplenium scolopendrium L. spores extract with Au-Ag nanoparticles (AA1:1 sample). 70 Oana Alexandra Luțu et al. The recorded spectra were interpreted using the ded- icated software and the present phases were identified by comparison with corresponding ICDD entries. The results obtained are presented in Table 2. The identified phases were Au (ICDD card no. 00-004-0784), Ag (ICDD card no. 01-071-4613), Ag2O (ICDD card no. 00-012- 0793, marked with * on figure 6), and A3O4 (ICDD card no. 03-065-9750, marked with # on figure 6). From XRD data it can be observed that all samples have a similar composition (although much well defined for sample DAA 1:10, while sample DAA 1:1 presents a much poorer defined spectra). Regarding the phases identified in the NPs solutions, it must be stated that the discrimination between Ag and Au is difficult, as the two metals exhibit similar diffraction patterns (the dif- fraction peaks overlapping). Also, the presence of differ- ent types of silver oxides was previously suspected to be due to oxidation of the NPs (Fierăscu et al. 2020; Șuțan et al. 2021). Also, the identification must also consider the results obtained by other methods (especially UV-Vis and STEM-EDX). As such, samples with a lower Au:Ag ratio (AA 1:10 and DA 1:10) exhibit in the UV-Vis spec- tra a peak around 441 nm, which could be assigned to the presence of Ag2O (Abouhaswa et al. 2022; Shume et al. 2020), although in our case the spectrum appears to have a small hypsocromic shift, which could be assigned to the presence of AgNPs (Bhui et al. 2009); other authors assign for the presence of Ag2O a peak around 430 nm (Shume et al. 2020), which would imply a batho- cromic shift of our spectra, which could be explained by a very small contribution of the AuNPs (suggested also by the shape of the UV-Vis spectra, slightly deformed towards higher wavelengths. The double silver oxide (Ag3O4) is most probably a secondary phase, formed either during reaction or during sample preparation for analysis, as other authors also noticed (Rajalakshmi et al. 2023) Samples AA and DA 1:1, presents specific UV-Vis peaks for AuNPs (above 520 nm). However, in the XRD spectra, these samples exhibit similar diffrac- tion peaks as the other two samples. The presence of Au in the samples is also confirmed by the EDX map- ping performed on the samples, as such, the most prob- ably explanation for these two samples is that the silver / silver oxide phases are either masked by the AuNPs (in the form of core-shell structure, with the shell formed by AuNPs), which would allow the proheminent presence of AuNPs in the UV-Vis spectra, or by the oxidation of the AgNPs core during sample preparation. Our opin- ion, based on the analytic results, is that, for the samples Figure 3. EDX spectra obtained for the Dryopteris filix-mas (L.) Schott spores extract with Au-Ag nanoparticles (DA1:1 sample). 71Phytotoxicity, cytogenotoxicity, and antimicrobial potential of gold-silver nanoparticle extracts from pteridophyte spores a b Figure 4. EDX-mapping - presence and distribution of chemical elements within the investigated areas (left): Au (right top) and Ag (right bottom) mapping in Asplenium scolopendrium L. spores extract with Au-Ag nanoparticles (AA1:1 sample) (a) and in Dryopteris filix-mas (L.) Schott (DA1:1 sample) (b). 72 Oana Alexandra Luțu et al. with lower Au content (AA and DA 1:10) the silver/silver oxide nanoparticles with lower AuNPs content reprezent a majority phase, in the samples with a higher Au con- tent (samples AA and DA 1:1), clusters of nanoparticles are formed, in which the AuNPs are found on the outer layer (thus contributing to the UV-Vis spectra), while the mixture of silver/silver oxide NPs found in the inner lay- er are re-arranged during sample preparation for XRD, which allows them to exhibit a much intense specific XRD peaks. Assessment of the phytotoxic effects The evaluation of phytotoxicity can be made by following some morphological, genetic, biochemical, physiological parameters, etc. Triticum test is frequently used for phytotoxicity studies in higher plants due to its advantages: quick results, simplified operative procedure, good reproducibility and repeatability and reduced costs (Drăghiceanu et al. 2019). The extracts obtained, with and without NPs, stimulated the growth of the root and stem. Statis- tically significant differences were noticed for the root incubated in the DA1:10 D10, DA1:10 D100 sam- ples and for the stem defined by DA1:10 D100 sample (Table 3). The insignificant growth inhibition observed in the DAM D100 sample, may be due to the decreased amount of bioactive substances, following the dilu- tion of the sample. Fern spores contain many sub- stances that also play a reserve role and in combating stress, such as lipids, proteins, and amino acids, such as proline, arginine, and some LEA-type proteins (late embryogenesis abundant), that promote embryo growth (López-Pozo et al. 2018). Except for the two abovementioned variants, the presence of NPs in extracts did not induce significant changes in root and stem growth. The presence of secondary metabolites in extracts cancels out the effect of nanoparticles (Zhang et al. 2021). Wet and dry weight were not significantly influ- enced by the tested extracts. According to Jahn et al. (2010) the understanding of the extent of genetic vari- ation for biomass traits in plants is limited. In a gen- Figure 5. Asplenium scolopendrium L. extract with Au-Ag nanoparticles, AA1:1 sample. Au-AgNPs analysis in BFSTEM (x150k magnifica- tion) (left). Dryopteris filix-mas (L.) Schott extract with Au-Ag nanoparticles, DA1:1 sample. Au-AgNPs analysis in BFSTEM (x200k magni- fication) (right). 20 30 40 50 60 70 80 90 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 *( 31 1) #( 13 2) *( 22 0) #( 14 0)#( 11 1) *( 11 0) (2 22 ) (3 11 ) (2 00 ) N or m al iz ed in te ns ity 2q (degrees) AA 1:10 AA 1:1 DA 1:10 DA 1:1 (1 11 ) Figure 6. XRD spectra of the obtaining nanoparticles in Asplenium scolopendrium L. and Dryopteris filix-mas (L.) Schott spores extracts (blue AA 1:1, green AA 1:10, black DA1:1, red DA1:10). 73Phytotoxicity, cytogenotoxicity, and antimicrobial potential of gold-silver nanoparticle extracts from pteridophyte spores otype, wheat seed size and protein content are cor- related with vigorous seedlings and higher yields bio- mass (Ries and Everson 1973). The seed we used in the experiment were from one wheat variety (Trivale), and the differences that appeared in dry biomass due to seed size and protein content are minimum. Hilty et al. (2021) consider that at the organ level and on short time scales, in our case wheat seeds and 4 days of growth, we can speak about growth in terms of one-dimensional elongation (roots, stems, leaves – for monocots) while growth as biomass accumulation should be used when we talk about plants and longer time scales. The biomass is the result of the photosyn- thesis minus photorespiration. Therefore, the biomass production can be enhanced by reducing photores- piratory losses (Peterhansel and Maurino 2010) or by increasing the photosynthesis rate and thus leaf area (Usuda 2004). Also, the leaf traits (thickness, size, shape, number etc.) are key factors in biomass produc- tion (Yang and Hwa 2008). In this study, in the short period of the experiment, the leaves didǹ t appeared and the quantity of biomass produced by the stem was small. The small differences in biomass that were regis- tered can be attributed to the depletion of the storage compounds Hilty et al. (2021), which was necessary for the root and stem growth. Table 2. XRD results obtained for the analyzed samples and the corresponding Miller indices. Peak position (approx., 2q) Sample/attribution Crystallite size (determined using eq. (1)), nm AA 1:1 AA 1:10 DA1:1 DA1:10 AA 1:1 AA 1:10 DA1:1 DA1:10 27.5 Ag2O (110) Ag2O (110) Ag2O (110) Ag2O (110) 5.03 4.76 6.38 4.96 32.2 A3O4 (111) A3O4 (111) A3O4 (111) A3O4 (111) 6.42 4.88 7.05 6.81 38 Au/Ag (111) Au/Ag (111) Au/Ag (111) Au/Ag (111) 7.91 5.06 8.04 9.05 44.1 Au/Ag (200) Au/Ag (111) Au/Ag (111) - - - - - 46.2 A3O4 (140) A3O4 (140) A3O4 (140) A3O4 (140) - - - - 55 Ag2O (220) Ag2O (220) Ag2O (220) - - - - - 57.5 A3O4 (132) A3O4 (132) A3O4 (132) A3O4 (132) - - - - 65.2 Ag2O (311) Ag2O (311) Ag2O (311) Ag2O (311) - - - - 76.8 Au/Ag (311) Au/Ag (311) Au/Ag (311) Au/Ag (311) - - - - 81.4 Au/Ag (222) - - Au/Ag (222) - - - - Table 3. The influence of aqueous spores extracts, with or without Au-Ag nanoparticles on Triticum aestivum L. parameters Variants Length (mm) Phytotoxicity (%) Weight (g) Root Stem on root on stem Fresh Dry Control 35.93±2.05bc 13.93±1.02cd 0.00 0.00 0.82±0.02a 0.38±0.00ab AAM D10 41.27±1.76ab 15.47±0.81bcd -14.84 -11.00 0.87±0.01a 0.39±0.01ab AAM D100 35.93±1.65bc 15.57±0.69bcd 0.00 -11.72 0.82±0.02a 0.36±0.02ab DAM D10 39.40±2.16abc 16.77±0.94bc -9.65 -20.33 0.77±0.03a 0.39±0.02ab DAM D100 34.43±2.03c 13.80±0.77d 4.17 0.96 0.78±0.04a 0.35±0.01b AA1:1 D10 38.20±1.37abc 14.90±0.80bcd -6.31 -6.94 0.77±0.03a 0.39±0.00ab AA1:1 D100 38.27±1.54abc 14.40±0.62cd -6.49 -3.35 0.82±0.06a 0.38±0.02ab DA1:1 D10 39.67±1.92abc 17.33±0.59ab -10.39 -24.40 0.80±0.02a 0.40±0.00a DA1:1 D100 38.77±2.29abc 14.13±0.97cd -7.88 -1.44 0.84±0.04a 0.38±0.02ab AA1:10 D10 39.70±2.01abc 14.50±0.86bcd -10.48 -4.07 0.80±0.05a 0.37±0.02ab AA1:10 D100 36.60±1.90bc 15.33±0.92bcd -1.86 -10.05 0.78±0.02a 0.39±0.02ab DA1:10 D10 44.17±1.44a 14.73±0.76bcd -22.91 -5.74 0.75±0.02a 0.37±0.00ab DA1:10 D100 43.63±1.75a 19.50±1.43a -21.43 -39.95 0.80±0.04a 0.36±0.01ab Data are shown as mean values ± SE of three replicates; a, b, c, d – interpretation of statistical significance and significant differences through Duncan’s test, p< 0.05). 74 Oana Alexandra Luțu et al. Assessment of cytogenotoxic effects Allium test is applied to determinate the effect of the plant extracts on the genetic material (Bonciu et al. 2018; Șuțan et al. 2016; Fierăscu et al. 2017a, b). The cytogenotoxic potential of various chemical agents can be assessed either by reducing or increasing of MI. In our study, statistical analysis revealed an insignificant increase in the frequency of mitotic cells in variants defined by aqueous extracts with or without Au-Ag NPs compared to the control. A significant increase of MI was determined by the aqueous extract of D. filix-mas spores DA1:10 D100 (Figure 7a). Similar results were reported by Șuțan et al. (2016) who found that ethanol extracts from A. scolopendrium leaves stimulated cell division in the root tips of A. cepa. In the root meris- tems of A. cepa exposed to actions of nanoparticles of iron oxide and copper, the MI increased by 10% and 5%, respectively, compared to the control, while AgNPs caused a decrease of 16% (Jasrotia et al. 2020). The stim- ulation of cell division and protein synthesis may be due to the electrostatic interaction of DNA and proteins caused by the penetration of AuNPs into the nucleo- plasm (Balalakshmi et al. 2017). The increase in the MI in direct correlation with AuNP dose and without the appearance of chromosomal aberrations in onion mer- istematic root cells has also been reported by Gopinath et al. (2013). In this context, it is important to empha- size that the stimulation of cell division can have nega- tive effects through an uncontrolled proliferation of cells (Hoshina et al. 2009). After extract exposure of meristematic cells of A. cepa, prophases were observed with a higher frequency in variants with Au-Ag NPs 1:10 samples, regardless of the tested dilution. Significant differences in metaphase frequencies were observed between control and DA1:10 D100 (Figure 7b). The anaphase index does not exceed 23% in the root tip cells treated with aqueous extracts prior to or after Au-Ag NPs biosynthesis (Figure 7b) and the telophase had the lowest distribution in the observed population cells. Also, vagrants, micronucleus, binucle- ate cells and C-metaphase were identified in different samples. In the extracts with Au-Ag NPs DA1: 1 D10 and AA1: 10 D10, all five types of chromosomal aberra- tions mentioned were identified (Table 4; Figure 8). This increase in the frequency of aberrations compared to the control can be attributed to a high concentration of the phytosynthesized NPs. Chromosomal aberrations observed by Palácio et al. (2021) in onion root meristem cells after exposure to AgNPs were delayed chromosomes, anaphase bridges, chromosome fragments and micronuclei. The authors appreciated that AgNPs disturbed the formation of the mitotic spindle, so that its partial or complete inactiva- tion would cause the appearance of delayed chromo- somes and the loss of genetic information. AgNPs can influence cell division by DNA degradation and depo- lymerization, their penetration into cells is facilitated by intracellular components (Kumari et al. 2009). Rajeshwari et al. (2016) showed that AuNPs caused chromosome fragmentation, anaphase bridges, laggards, sticky chromosomes, and others abnormalities. However, it should be noted that the results found in the literature on the cytogenotoxic effect of nanoparticles depending on their concentration are contradictory. Thus, increas- ing the concentration of AgNPs induced a diminution in MI and the occurrence of various chromosomal aber- rations, such as laggards, ring chromosomes, C-mitosis, chromosome fragmentation, nuclear membrane damage, multinuclear cells and chromatin bridges (Abdelsalam et al. 2019). It has also been noticed that the MI and various nuclear abnormalities increased with the gradual reduc- tion of the AgNPs diameter from 73 to 10 nm (Scherer et al. 2019). In our study, the higher frequency of chro- mosomal and nuclear aberrations recorded in the exper- imental AA1:1 D10 it may be due to the NPs with a diameter of 5-10 nm as the STEM-EDS analysis revealed. Ahmed et al. (2018) stated that the MI modification and the induction of chromosomal aberrations could be due to the interference of the NPs with the DNA and/or the mitotic apparatus. However, we could not find simi- lar results regarding the assessment of cytogenotoxicity of bimetallic nanoparticles on Allium assay. Evaluation of cell viability by Evans blue test The presence of Au-Ag NPs in extracts significantly influenced cell viability compared with experimental var- iants defined by the extracts without NPs. In the absence of NPs, the extract significant increase the cell viabil- ity compared to Control (Figure 9). At the variants DA 1:1 and AA 1:10 (diluted 10), the amount of Evans Blue absorbed by the roots of Allium was significantly higher than the amount obtained for the control. Zhang et al. (2019) considered that the absorption of a large amount of this dye is due to the damage of the cell membrane caused by the NPs. This situation is also confirmed by our results: for the variants with bimetallic nanoparti- cles dilution 100, the absorption of the Evans blue was smaller than that at dilution 10. The extracts without NPs (AAMD10, AAMD100, DAMD10, DAMD100) signifi- cantly increased the cell viability compared to Control due to the phytocompounds found in fern spores, com- 75Phytotoxicity, cytogenotoxicity, and antimicrobial potential of gold-silver nanoparticle extracts from pteridophyte spores b b ab b ab ab ab ab b ab ab b a 0 2 4 6 8 10 12 Control AA M D10 AA M D100 DA M D10 DA M D100 AA 1:1 D10 AA 1:1 D100 DA 1:1 D10 DA 1:1 D100 AA 1:10 D10 AA 1:10 D100 DA 1:10 D10 DA 1:10 D100 N um be r o f d iv id in g ce lls / t ot al n um be r o f c el ls (% ) Variantsa 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Control AA M D10 AA M D100 DA M D10 DA M D100 AA 1:1 D10 AA 1:1 D100 DA 1:1 D10 DA 1:1 D100 AA 1:10 D10 AA 1:10 D100 DA 1:10 D10 DA 1:10 D100 e bc de de ab cd e cd e bc de bc de ab c bc de a ab cd ab bc de a ab ab ab ab ab ab ab ab ab ab ab b ab c ab c a ab c ab ab c c bc ab g bc c ab c bc de cd ef ab cd ef a de f bc d ef cd ef d bc fg bc D is tri bu tio n of th e m ito tic p ha se s ( % ) Variants Prophase Metaphase Anaphase Telophaseb Figure 7. The influence of extracts on the mitotic index (a) and on the distribution on mitosis phase (b) in meristematic root cells of Allium cepa L. Data are shown as mean values ± SE of three replicates; a, b, c, d, e, f, g - interpretation of statistical significance and significant dif- ferences through Duncan’s test, p< 0.05. 76 Oana Alexandra Luțu et al. pounds that protect cells from stressors. The decrease of the amount of Evans blue absorbed by the roots after the treatment with various chemicals is explained by Baker and Mock (1994); they considered that the treatment can cause a large flow of electrolytes, but without necessarily causing cell death. Unlike us, after staining with Evans blue, Prajitha and Thoppil (2016) observed that aqueous extracts of Amaranthus spinosus L. induced cell death at the top of the Allium root, with the potential for mem- brane damage being significant. Regarding cell viabil- ity at the variants with nanoparticles diluted 100 times – we obtained similar (AA1:1, DA1:10) or greater values (DA1:1, AA1:10) compared to control. A similar situation was reported by Kannaujia et al. (2019) who studied the cell viability of the roots of two wheat varieties (HD-2967 and DBW-17) after exposure to AgNPs. After AgNPs exposure, the viability of wheat root cells assessed by Evans Blue staining was maximum in the case of wheat roots from the HD-2967 variety treated with AgNPs, while in the DBW-17 variety, the maximum viability of root cells was observed in the control and was close to that from the variant treated with AgNPs. Table 4. Frequency of the main chromosomal aberrations in the meristematic root cells of Allium cepa L. Variants Chromosomal aberrations (%) Anaphase bridges Laggards Micronucleus Binucleate cells C-metaphase Control - 1.33±1.33a - - - AAM D10 19.17±3.63ab - 0.03±0.034a - - AAM D100 11.57±6.43ab - - - - DAM D10 11.01±2.44ab - - 0.07±0.07a - DAM D100 6.71±0.83ab - - - - AA1:1 D10 22.78±13.62a 0.07±0.07a - 0.07±0.07a 2.94±2.94b AA1:1 D100 7.50±3.82 ab 0.85±0.85a - - 0.85±0.85b DA1:1 D10 4.86±2.50 ab 2.98±1.50a 0.10±0.10a 0.21±0.06a 8.05±2.31b DA1:1 D100 - 2.90±2.90a - - 4.35±2.51b AA1:10 D10 1.75±3.03b 3.70±3.70a 0.10±0.06a 0.17±0.13a 65.74±5.63a AA1:10 D100 - - - - 7.69±7.69b DA1:10 D10 1.45±2.51b - 0.07±0.07a 0.03±0.03a - DA1:10 D100 - - 0.17±0.07a - Data are shown as mean values ± SE of three replicates; a, b, c, d, e, f, g - interpretation of statistical significance and significant differences through Duncan’s test, p< 0.05. Figure 8. Chromosomal aberrations identified in the root meristem cells of A. cepa exposed to DA1:1 D10. (a) – micronucleus; (b) – binucleate cell; (c) – C-mitosis; (d) – vagrants. cd f f f f cd d b ef a de c cd 0 0,01 0,02 0,03 0,04 0,05 0,06 0,07 0,08 Con tro l AA M D 10 AA M D 10 0 DA M D 10 DA M D 10 0 AA 1: 1 D 10 AA 1: 1 D 10 0 DA 1: 1 D 10 DA 1: 1 D 10 0 AA 1: 10 D 10 AA 1: 10 D 10 0 DA 1: 10 D 10 DA 1: 10 D 10 0 A bs or ba nc e (a .u .) Variants Figure 9. The influence of aqueous spores extracts, with or without Au-Ag nanoparticles, on cells viability. Data are shown as mean val- ues ± SE of three replicates; a, b, c, d, e, f - interpretation of statistical significance and significant differences through Duncan’s test, p< 0.05. 77Phytotoxicity, cytogenotoxicity, and antimicrobial potential of gold-silver nanoparticle extracts from pteridophyte spores Antimicrobial activity of the extracts The differences between the dimensions of inhibi- tion zone induced by antibiotic and those induced by the aqueous extracts with bimetallic NPs are significant. The aqueous extract without NPs did not inhibit the development of bacterial strains (Figure 10b). Also, the samples DA1:10 and DA1:1 did not inhibit the growth of the S. aureus and E. coli strains. We consider that the characteristic bioactive substances of spores have a rather protective effect at the cellular level, even in the case of bacterial cells, the results being correlated with those obtained at the cell viability investigated by the Evans blue test. LEA protein, present in spores, pro- vides protection against desiccation, osmotic, and oxida- tive stresses, the results being obtained using E. coli as an in vivo model to evaluate some LEA protein function (Saucedo et al. 2017). Bimetallic nanoparticles inhibit the growth of B. subtilis ATCC 6633. The largest zone of inhibition of 7.17 mm was observed in the extracts with Au-Ag NPs 1:10, DA 1:10 sample (Figure 10a). A similar situation was observed for E. coli ATCC 8739, where the zone of inhibition was 8.83 mm in AA1:10 sample and 8 mm in DA1:10 sample (Figure 10a). Aqueous extracts of spores of A. scolopendrium with Au-AgNPs had a higher antimicrobial efficiency in S. aureus ATCC 25923 than extracts of spores of D. filix- mas (Figure 10a). The influence of extracts on S. aureus ATCC 25923, B. subtilis ATCC 6633, E. coli ATCC 8739 are observed in Figure 10b. MIC determined only for variants that had antimi- crobial effect (Table 5) was between 0.046 ml extract for DA1:10 and AA1:10/ml medium in B. subtilis and E. coli and 0.187 ml extract for DA1:1 and AA1:1/ml medium in B. subtilis and S. aureus (Figure 11). Au-Ag NPs strongly inhibited B. subtilis growth compared to control and monometallic nanoparticles; a similar situation was observed for E. coli (Reddy et al. 2012). The increase in the number of Ag ions released from bimetallic nanoparticles indicates that Au ions influence the oxidation of Ag atoms (Harada et al. 2018). Green-synthesized Ag-Au NPs exhibited promising anti- bacterial activity against E. coli, B. subtilis, Klebsiella pneumoniae, Pseudomonas aeruginosa, and S. aureus in a dose-dependent manner (Amina et al. 2020). The anti- bacterial potential of plant extracts with bimetallic Ag- Au NPs depends on particle size, shape, area and sur- face polarity, morphology, and plant specific compounds (Amina et al. 2020). Spore-specific bioactive compounds allowed the production of green bimetallic nanoparti- cles, which have superior properties to those obtained by chemical synthesis, being less phytotoxic, biocompatible, environmentally friendly, which might be due to the capping of biomolecule onto the surface of NPs (Panick- er et al. 2020). CONCLUSIONS The aqueous extracts obtained from the spores of the native ferns A. scolopendrium and D. filix-mas con- stituted optimal media for the biosynthesis of Au-Ag Figure 10. The antibacterial potential of the aqueous spores’ extracts. Influence of extracts on Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633, Escherichia coli ATCC 8739. The samples DA1:10 and DA1:1 did not inhibit the growth of the S. aureus and E. coli strain (a). Also, the aqueous extracts with- out nanoparticles had no antimicrobial effect, as seen (arrow) (b). The extract with nanoparticle may produce zone of inhibition like AA1:10 in S. aureus, DA1:10 in B. subtilis, and E. coli or may not produce inhibition zone in the tested strains, like DA1:1 in E. coli. Table 5. Minimum inhibitory concentration (ml extract/ml medi- um). Experimental variants Bacterial strain Bacillus subtilis A TCC 6633 Escherichia coli ATCC 8739 Staphylococcus aureus ATCC 25923 DA1:1 0.187 Nt 0.046 DA1:10 0.046 0.093 Nt AA1:1 0.093 0.375 0.187 AA1:10 0.093 0.046 0.093 Note: Nt-not determined. 78 Oana Alexandra Luțu et al. NPs. The growth processes evaluated in the seedlings of Triticum aestivum were, in general, stimulated by both categories of extracts, with and without NPs, significant differences being obtained for those of D. filix-mas. The effect of stimulating the growth of axial organs was also confirmed by the results obtained in the Allium test. Extracts without NPs significantly improved cell viabil- ity, assessed by the Evans blue test, alongside the vari- ant with NPs, DA1:1 D100. An antimicrobial effect was observed just for sample with bimetallic NPs, against all three bacterial strains: S. aureus ATCC 25923, B. subti- lis ATCC 6633 and E. coli ATCC 8739. The sample with aqueous extract without NPs did not inhibit the devel- opment of bacterial strains. The increase in antibiotic resistance of microorganisms requires the discovery of new products with such properties, so it is useful to con- tinue the research of less evaluated resources, such as fern spores. ACKNOWLEDGEMENT O.A.L. thanks the University of Pitesti for the financial support through the grant no. CIPCS-2020-08. N.A.Ş. thanks the Romanian Ministry of Education and Research, CNCS-UEFISCDI, for the financial support through the Project number PN-III-P4-ID- PCE-2020-0620, within PNCDI III. IF and RCF also acknowledge the support obtained through a grant of the Ministry of Research, Innovation and Digitization, CNCS/CCCDI-UEFISCDI, project number PN-III-P2- 2.1-PED-2021-0273, grant no. 644PED/2022, within PNCDI III. FUNDING This research was funded by University of Pitesti through the grant no. CIPCS-2020-08, Romanian Min- istry of Education and Research, CNCS-UEFISCDI, for the through the Project number PN-III-P4-ID- PCE-2020-0620, and CNCS/CCCDI-UEFISCDI through the project number PN-III-P2-2.1-PED-2021-0273, grant no. 644PED/2022. AUTHOR CONTRIBUTIONS Conceptualization: OAL, LCS, NAȘ, IF, RCF, DN; experimental design and laboratory work: OAL, LCS, Control 1/16 1/8 1/4 Staphylococcus aureus ATCC 25923, DA 1:1 variant Bacillus subtilis ATCC 6633, DA 1:10 variant Escherichia coli ATCC 8739, AA 1:10 variant 1/128 1/64 1/32 Figure 11. Aspects of MIC evaluation. Control (broth tube without extract), bacterial cultures obtained in various extract dilutions (1/128, 1/64, 1/32, 1/4). 79Phytotoxicity, cytogenotoxicity, and antimicrobial potential of gold-silver nanoparticle extracts from pteridophyte spores IF, RCF, CMD, AP, CMP, CMT, LEV, ID, DN, DȘV, GC, FA, SOH, NAȘ; funding acquisition: OAL, NAȘ, IF, RCF. Writing, review and editing: all authors. 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