Green synthesis of silver nanoparticles and its antibacterial activity using fungus Talaromyces purpureogenus isolated from Taxus baccata Linn.

The present study is focused on the synthesis of silver nanoparticles (AgNPs) utilizing endophytic fungus Talaromyces purpureogenus , isolated from Taxus baccata Linn. Extracellular extract of Talaromyces purpureogenus has shown occurrence of secondary metabolites viz. terpenoids and phenols. Gas chromatography-mass spectroscopy analysis showed the presence of 16 compounds. Techniques like Ultraviolet–visible spectroscopy, Fourier transform infrared spectroscopy, dynamic light scattering, field emission gun scanning electron microscopy, high-resolution transmission electron microscopy, energy-dispersive X-ray spectroscopy and X-ray diffraction were employed to characterize the synthesized AgNPs. UV–Vis spectroscopy showed sharp peaks at 380–470 nm which indicates the presence of metallic silver. FTIR analysis showed the presence of various functional groups like phenols, hydroxyl groups, and primary amines. In DLS, Z-average size and PdI of synthesized AgNPs were 240.2 r.nm and 0.720 respectively, with zeta potential − 19.6 mV. In FEG-SEM and HRTEM the spherical AgNPs showed diameter in the range of 30–60 nm. In EDS analysis the weight percent of Ag is 67.26% and atomic percent is 43.13%. From XRD analysis the size of AgNPs was found to be 49.3 nm with face-centered cubic crystalline nature of fungal synthesized AgNPs. These nanoparticles have shown significant antibacterial activity against tested strains viz. Listeria monocytogenes (13 ± 0.29 mm), Escherichia coli (17 ± 0.14 mm), Shigella dysenteriae (18 ± 0.21 mm) and Salmonella typhi (14 ± 0.13 mm). These synthesized AgNPs have shown effective free radical scavenging activity against 2,2 ′ -diphenyl-1-picrylhydrazyl. The present study showed that the endophytic fungus Talaromyces purpureogenus can be used as a prominent source to synthesize AgNPs by using biological, ecofriendly, and in a non-toxic way accompanied by antibacterial and antioxidant properties which further can reduce the harvesting pressure faced by Taxus baccata.


Introduction
Gymnosperms are majorly exploited for their wood quality, resins, gums, and medicinal values [1]. Taxus baccata Linn. a gymnosperm found at a height of 2300-2800 m in the western and eastern Himalayas, has innumerable medicinal utilities such as those used in kidney disease, Alzheimer [2], bronchitis, asthma, and as an aphrodisiac [3]. Apart from these uses bark is used as salted tea by the local community [4] paste of the bark is used to cure headaches and as a plaster to repair the fractured bones [5]. The plant is supposed to be a good source of anticancerous drugs Taxol or Paclitaxel [6,7]. However, the utilitarian values of this plant are causing its degradation in several ways. Lack of good harvesting techniques and statutory procedures, the leaves and bark are harvested callously leading to the injudicious exploitation of the plants. Also, the excessive stress on this plant for its resources for commercial uses and habitat disturbances are resulting in its disappearance at a rapid rate [8][9][10]. Taxus baccata is a splendid host of many types of microorganisms such as endophytic bacteria (Burkholderia and Enterobacter) [11], endophytic fungi (Aspergillus, Fusarium, Penicillium, Trichoderma, etc. [12,13] and mycorrhizae [12,14]. Endophytic fungi reside in the plant's internal tissues without causing any visible harm to them. These fungi have been reported to synthesize a variety of chemical compounds called secondary metabolites, reported to possess antibacterial, antifungal, anti-cancerous, antioxidant, and various other important activities [15][16][17].
The tissue extracts of Taxus baccata are the source of many medicines. A further advanced step has been employed where nano-sized particles coated with tissue extracts of Taxus baccata work more precisely than the ordinary processes [18,19]. However, this dependency on the plant can be minimized by the use of fungi for the production of anti-cancerous materials encapsulated on nanoparticles [20][21][22]. Nanobiotechnology has emerged as a promising technology to develop new therapeutically active nanomaterials; nanoparticles are one of them [23]. Nanoparticles exhibit special optical, electronic, and magnetic properties [24]. Nanoparticles having exceptionally tiny size result in strong adherence to the target sites and are not easily washed away in the system, thus increasing their residence time on the targets such as cancer cells. This increase in retention time results in better drug delivery and cure to the problem [25].
Exposure of secondary metabolites reduces the metallic ions into nanosized metallic particles [26]. Among other metallic nanoparticles silver nanoparticles has been immensely worked upon due to their biocompatibility [27]. Silver nanoparticles possess variety of beneficial properties including optical, good electrical conductivity and biological properties. Applications of silver nanoparticles is increasing as antibacterial agents, sensors, food industry and as anticancerous agents. Biologically synthesized silver nanoparticles possess high solubility, stability, high yield and antimicrobial properties. The process of AgNPs synthesis is also non-toxic and cost effective [28][29][30][31]. Synthesis of many fungus-coated metallic nanoparticles has been reported [32][33][34][35][36]. A very important fungus Penicillium sp. isolated from a useful plant named Glycosmis mauritiana, has been reported to effectively produce AgNPs which are showing good quality antioxidant, antimicrobial, anti-inflammatory, and tyrosinase inhibitory activity [37]. Fungus Talaromyces pupureogenus isolated from Pinus sp. resulted in the formation of AgNPs with an average size of 25 nm and has shown strong antibacterial and antioxidant properties [22]. Taxus baccata is also considered to be a host of many fungal species [12,38]. AgNPs of size 5-70 nm with spherical shape were obtained from fungus Nemania sp. isolated from Taxus baccata. These AgNPs have shown prominent antibacterial activity against certain harmful bacteria [38]. So, in the present study fungus, Talaromyces purpureogenus isolated from a gymnosperm Taxus baccata has been utilized for the synthesis of AgNPs and the characterization of these nanoparticles has been done using various spectrophotometric techniques. Further, the activity of these AgNPs has been tested as antibacterial and antioxidant agents.

Isolation and identification of endophytic fungi
Healthy tissues (leaf, stem, and roots) of Taxus baccata were collected from the Choordhar region of Shimla District of Himachal Pradesh, India. The tissues were sterilized in the lab by using the method of Schulz et al. [39]. The plant tissues were properly washed under running tap water. The surface sterilization was done by first immersing in 70% ethanol for 60 s, followed by sodium hypochlorite (4% of available chlorine) for 3 min and then dipped in 75% of ethanol for 30 s. For the sterilization of leaves, the method of Suryanarayan and Thennarasan [40] was followed. The leaf samples were cut into pieces of different sizes. Finally, sterilized samples were rinsed with autoclaved distilled water three times and were allowed to dry in laminar airflow. The plant samples such as root, bark, stem, and leaves were cut into small pieces and placed on the Petri plates containing potato dextrose agar (PDA, Himedia Pvt. Ltd. India). The PDA was also supplied with streptomycin (100 mg/L, Himedia Pvt. Ltd. India) to prevent endophytic bacterial growth. Petri plates were incubated at 28 ± 2 °C. These Petri plates were observed on regular basis for endophytic fungal growth. A pure culture of the endophytic fungi was obtained after continuous transfer of hyphal tips on fresh PDA plates. Morphological identification of the isolated endophytic fungi was performed at ARI Pune, India (Agharkar Research Institute). Out of 08 endophytic fungi 07 (Aspergillus flavus, A. nidulans, A. niger, Penicillium viridicatum etc.) belonged to family Aspergillaceae and one (Talaromyces purpureogenus) to Trichomaceae. Molecular-level identification of isolated fungi was carried by PCR using ITS4 and ITS5 primer pair. The identification was being carried out at ARI Pune, India (Agharkar Research Institute). T. purpureogenus formed soft and cottony ascocarps with interwoven hyphae. The fruiting body of this particular fungus were yellowish in colour. Structures like conidia, metulae and phialides were observed carefully. The phialides were supported by the metulae. Further, identification of the fungus was done when the genomic DNA was isolated in pure form from the culture. The ITS-rDNA partial gene was successfully amplified using ITS4 and ITS5. The sequencing PCR was set up with ABI-BigDye ® Terminatorv 3.1 Cycle Sequencing Kit. The raw sequence obtained from ABI 3100 automated DNA sequencer was manually edited for inconsistency. The sequence data was aligned with publicly available sequences and analyzed to reach identity. All isolated endophytic fungi were screened for antibacterial activity and T. purpureogenus showed significant activity against tested bacterial strains. Therefore, it was further selected for the synthesis of AgNPs.

Preparation of endophytic fungal extracts
T. purpureogenus was inoculated in potato dextrose broth (PDB) and was incubated at 28 ± 2 °C on an incubator shaker with 150 rpm for 6 days. The growth of the fungus was observed regularly. This fungal biomass was filtered with the help of Whatman filter paper no. 1. This filtered mass was washed 3 times with distilled water to remove the media. For the preparation of endophytic fungal extracts, two standard procedures [41,42] were used. The harvested fungal mass was weighed and 20 g of it was transferred to a 500 mL Erlenmeyer flask containing 100 mL of distilled water. These flasks were incubated at 28 ± 2 °C for 24 h with the 180 rpm shaking for the extraction. At the end of this period, the cell-free filtrate was filtered using Whatman No. 1 filter paper and used in further experimental processes.

Screening for secondary metabolites
For the screening of secondary metabolites, the method of Radji et al. [43] was followed with slight modifications. Small rectangular blocks from the agar plates containing fungal mycelium were cut and inoculated into a 250 mL Erlenmeyer flask containing 100 mL of (PDB). After pouring the flasks were incubated at 25-27 °C for 21 days with occasional shaking. After the defined period, the broth culture was filtered to separate mycelium and filtrate. The extraction was performed with majorly 05 organic solvents and after standardization ethyl acetate was preferred as extraction solvent because of better results as compared to other organic solvents. Since, ethyl acetate is less polar than alcoholic extract, it dissolves the hydrocarbon rich secondary metabolites much better than alcohols. Insights into previous works also substantiated the selection of the solvent [44,45]. An equal volume of ethyl acetate was added to the filtrate. The solution was mixed well for 10-15 min and then kept for 5-10 min. The formation of two clear immiscible layers was observed. The upper layer of ethyl acetate was separated using a separating funnel as it contains the extracted compounds. Rotary evaporation supplemented with reduced pressure at 35-40 °C was done to remove the solvents and resulting in a concentrated extract. Further, the extract was dissolved in DMSO and stored in the refrigerator. The qualitative screening of phytochemicals present in the fungal extract was done by following the standard procedure of Devi et al. [46].

GCMS analysis of fungal extract
Mass spectrum of compounds was done by gas chromatography-mass spectroscopy (GCMS analyser-Thermo Scientific TSQ 8000 Gas Chromatograph-Mass Spectrometer). It was paired with GC TRACE-1300 GC. The ion source was EI source programmable to 350 °C with mass range 2.1100 a.m.u and column temperature 400 °C. The column used was Rtx-5 of 30 m in length, 0.25 mm column inside diameter with 0.25 µm film coating. The sample was filtered through Whatman Filter Device (0.2 µm). Helium (99.99%) gas was used as the carrier with a flow rate of 1 mL/min in split mode. A volume of 1 µL of the fungal extract was injected into the column with 280 °C inlet temperature. The temperature of the oven was initially set at 70 °C for 2 min and then it was elevated at a rate of 7 °C/min up to 320 °C. The temperature of ion sources was maintained at 250 °C. The mass spectrum of compounds present in the fungal extract was obtained by electron ionization at 70 eV and the detector operates in scan mode 30-500 Da atomic units. The total running time was 22.77 min with 6694 scans. The obtained spectrum of the extract was compared with the database of the National Institute of Science and Technology (NIST) library [47].

Biosynthesis and characterization of AgNPs
For the synthesis of AgNPs from Talaromyces purpureogenus, method of Sandhu et al. [48] was opted with little modifications. An aqueous solution of silver nitrate (80 mL; 2 mM) was mixed with 20 mL of fungal extract and incubated at room temperature. Flasks were kept covered with aluminium foil to reduce the chances of photo-oxidation. Prior standardization of reaction mixture was done before proceeding towards the final process of nanoparticle synthesis. According to the standardization the time limits were finalized which were corresponding to the color change and AgNPs synthesis. The aliquots of samples were withdrawn subsequently after 12 h, 24 h, 36 h, 48 h, 72 h, and 96 h for observing the color change from transparent to dark brown.
For the determination of successful synthesis of AgNPs UV-Vis spectrophotometry (Shimadzu UV-Vis Spectrophotometer) was done by measuring the absorbance of the solution at different time intervals (1,12,24,48,72, and 96 h) at 300-700 nm. After 48-72 h of reaction, the mixture was centrifuged at 12,000 rpm for 15 min. The unbounded capping material was removed by repeated washing (4 times) with double distilled water. Thereafter, the pellet was lyophilized to get dry powder of the material. There are different types of functional groups involved in the stabilization and capping of synthesized nanoparticles which were detected by using FTIR. Characteristic peaks were recorded in 400-4000 cm −1 at resolution 4 cm −1 . The sample was analyzed twice. The size was analyzed with the help of Dynamic Light Scattering analysis (DLS-Malvern).Surface morphological studies were done using FEGSEM technique. However, for the elemental analysis, FEGSEM-EDS was used. For the analysis of the shape and size of synthesized nanoparticles the AgNPs powder was suspended in ethanol for High-Resolution Transmission Electron Microscopy (HRTEM) and the presence of specific metallic groups was found with the help of HRTEM-EDX (energy-dispersive X-ray spectroscopy. Finally, XRD was used for the analysis of morphology and crystalline nature of AgNPs in a wide range of Bragg angles 2θ at scanning rate 30-80 at 0.041/min with a time constant of 2 s.

Antibacterial activity
To determine the antibacterial activity of AgNPs disc diffusion method was used as described by Brumfitt et al. [49]. This method was preferred over other methods based on literature, its promising and precise results [22]. All the bacterial strains (Listeria monocytogenes, Shigella dysenteriae, E. coli, and Salmonella typhi) were cultured in10 mL of MHB in 50 mL Erlenmeyer flask for 24 h at 37 °C. Fifty µL of the bacterial cell suspension was spread over the MHA plates. Further, discs of 6 mm diameter were prepared from Whatman filter paper-3 which was further autoclaved and sterilized. These discs were dipped in different concentrations of solution of AgNPs. For positive control discs were supplied with antibiotics (Vanomycin and Ampicillin, 0.1 mg/mL). The plates with antibiotics were used as positive control and those with DMSO were used as a negative control. These paper discs were placed on each plate which was further incubated at 37 °C for 24 h. The antibacterial activity of AgNPs was measured as a clear zone of inhibition (mm) using a vernier caliper.
The lowest concentration of an antibacterial agent to inhibit/prevent the growth of bacterial growth is called minimum inhibitory concentration (MIC). For the determination of MIC values of synthesized AgNPs microbroth dilution method of Sarkar et al. [50] was followed. Fifty µL of each sterile nutrient broth and normal saline was added to each well of the micro-titre plate. Now 50 µL of nanoparticle solution was dissolved in DMSO (25 mg/mL). This 50 µL was added to the first row of the micro-titre plate followed by serial dilution across the plate. Ten µl of each resazurin dye (indicator) and bacterial inoculums were added to each well. Plates were wrapped properly in a cling film to prevent the process of dehydration of bacteria and incubated at 37 °C for 24 h. These experiments were performed in duplicates to avoid any sort of error. A change in the color of the solution in the well was observed. If the color changes from purple to pink or colorless it indicated that bacteria have grown in it. The lowest concentration at which no color change was observed was considered as MIC value of AgNPs.

Antioxidant assays
For the determination of antioxidant potential of T. purpureogenus and synthesized AgNP's two assays viz. DPPH assay and reducing power assay were performed.

DPPH (1, 1-diphenyl-2-picrylhydrazyl) free radical scavenging activity
The antioxidant activity of the synthesized silver nanoparticles was determined by DPPH (1,1-diphenyl-2-picryl-hydrazyl) free radical scavenging assay as given by Blois [51]. One mL of extract prepared in 10% DMSO of different concentrations (30 µg/mL, 60 µg/mL, 90 µg/mL, 120 µg/mL and 150 µg/mL) along with standard made up of DPPH + DMSO. One mL of 0.3 mM DPPH was added to it under dark conditions and was incubated for 30 min in dark. The absorbance was taken at 517 nm by UV-Vis spectrophotometer (UV-1800 Shimadzu). Ascorbic acid was used as reference or standard with same concentrations (30 µg/mL, 60 µg/mL, 90 µg/mL, 120 µg/mL and 150 µg/mL). The percent inhibition was calculated by the formula given below: The amount of extract that quenches 50% free radicals of DPPH is called its IC 50 value. IC 50 value (µg/mL) was determined by plotting a graph of % inhibition against different concentrations of extracts.

Reducing power assay
For the estimation of reductive potential of the fungal extract and AgNPs the method of Oyaizu [52] was followed. Ascorbic acid was taken as standard. One mL of distilled water was mixed with fungal extracts/AgNPs and standard (1 mg/mL). This solution was further mixed with phosphate buffer (2.5 mL, 0.2 mol/L, pH 6.6) and potassium ferricyanide (2.5 mL, 1% w/v) was added to the mixture. The solution was mixed properly and centrifuged for 10 min at 3000 rpm. Then, 2.5 mL of supernatant was mixed with 2.5 mL of distilled water and 0.5 mL of FeCl 3 (0.1% w/v). The absorbance was measured at 700 nm in UV-Vis spectrophotometer (UV-1800 Shimadzu). The experiment was performed in triplicates. The high value of the absorbance in reaction mixture indicated a greater reducing power of the fungal extract and AgNPs.

Results and discussion
Molecular identification of the fungal strain was done using ITS4 and ITS5 sequencing. The tested fungal strain showed 100% sequence similarity with Talaromyces purpureogenus. Sequence analysis with NCBI accession number MK108916.1, Talaromyces purpureogenus strain KNUPD2 resulted in the following alignment statistics. The colour of the reaction mixture i.e., mycelial extract and AgNO 3 was observed at regular intervals which changed from white to orangish brown after 48 h (Fig. 1b,  c). This color change can be correlated with the process of excitation of surface plasmon vibration within the biologically produced AgNPs [53]. In UV-vis spectroscopy a peak was obtained at 390-460 nm by UV-visible spectroscope (Fig. 2).
During the spectral analysis, spectroscopy was done at regular intervals predefined during standardization. Maximum absorbance was observed at 450 nm which is a characteristic surface plasmon resonance peak of mycosynthesized AgNPs [54], the results are similar to previous findings [55,56]. The peaks obtained at 438 nm wavelength probably give nanoparticles with size a size range of 60-80 nm [57]. These results are in accordance with the previous results [19,[58][59][60][61]. FTIR (Fig. 3) showed the bands at 3428.4 cm −1 confirming the presence of hydroxyl group, H-bonded -OH stretch [62]. A peak at 1621.9 cm −1 showed the presence of amides or primary amine with N-H bend. A peak at 1383.9 cm −1 confirmed the presence of phenols or tertiary alcohol -OH bend. A peak at 1076.1 cm −1 corresponds to -C-N primary amine stretch or cyclic ether with large rings, C-O stretch. Peaks at 1022.0 cm −1 correspond to aromatic C-H in-plane bend. The peak at 516.0 cm −1 may corresponds to the presence of aryl disulfide (S-S) or Polysulfides (S-S stretch) or C-Br stretch [63]. This concludes that these functional groups are somehow involved in the stabilization of AgNPs and act as a capping agent [64]. These results were in accordance with the previous results [38,65].
DLS (dynamic light scattering) measurement with zeta potential was used to find out the average size of AgNPs (Fig. 4). The Z-average was 240.2 r.nm with 0.720, PdI value (Fig. 4a). Zeta potential having either high negative value or high positive value, show a tendency to repel each other thus declining the agglomeration [65,66]. The polydispersed nature of the nanoparticles is a result of highly negative zeta potential avoiding the formation of agglomerates leading to a concise stability. The obtained nanoparticles have zeta potential equal to − 19.5 mV (Fig. 4b) attesting the repulsive nature of the silver nanoparticles leading to the stability [67,68]. Present X-ray diffraction analysis demonstrated that the nature of synthesized AgNPs is crystalline (Fig. 5). The XRD pattern of synthesized AgNPs is shown in Fig. 5. The result of XRD analysis indicated the 2θ value of 38°, 46°, 67° and 78° correspond to 111, 200, 220, and 311 respectively to the planes of silver. The XRD results confirmed the occurrence of AgNPs. These results also confirmed the nature of AgNPs as crystalline face-centered cubic which is in agreement with the database of JCPDS (now called ICDD) file no. 04-0783. These results are as per the previous XRD results of biosynthesized AgNPs [17,49,56,60,69,70]. The morphological features of mycosynthesized AgNPs were observed in FEG-SEM (Fig. 6a) which showed a variety of shapes and sizes of AgNPs with few clump formations. AgNPs have shown cauliflower-like morphology in FEG-SEM (Fig. 6a), which are in accordance with the previous results [50]. HRTEM (Fig. 6b) results revealed that the synthesized AgNP's were round to oval/round shaped [71] and having a diameter ranging from 30 to 60 nm. The EDX analysis confirmed the presence of 0.63% of Ag atoms. The HRTEM-EDS ( Fig. 7) analysis showed the weight percent of Ag 67.26% and atomic percent 43.13%. The presence of mycosynthesized AgNPs was confirmed by the specific peak at 3.0 keV in EDS.
Peaks showing presence of carbon, oxygen and copper also appeared in EDS micrograph (Fig. 7). Generally, the samples are coated on copper-grid whose remnants may have resulted in the peak shown at 8 keV. These results were following the previous results [55,58]. The stability of the synthesized silver nanoparticles was observed for 50-60 days by three parameters viz. UV-vis spectroscopy, DLS analysis and FTIR analysis. Here, UVvis spectroscopy showed absorption peaks in range of 440-480 nm. Further, the Z-average size of AgNPs was 304.7 r.nm with PdI 0.807 (Fig. 4c). The zeta potential was observed to be − 16.6 mV, still a good quality result (Fig. 4d) The specific peaks at 3317.0, 1041.0 and 1589.0 cm −1 were observed quite similar to the points discussed earlier (Fig. 3). This stability of nanoparticles play a vital role in antimicrobial potential.
In the present study these mycosynthesized AgNPs showed a prominent antibacterial activity (Fig. 8) against a list of bacterias viz. E.coli, Shigella sp., Salmonella typhi (gram-negative), and Listeria sp. (gram-positive) which was similar to positive control vanomycin and ampicillin. The highest zone of inhibition was found to be in the case of E. coli (17.00 ± 0.14 mm), followed by Shigella sp. (18.00 ± 0.21 mm) and Salmonella typhi (14.00 ± 0.13 mm). The lowest zone of inhibition was found to be in Listeria sp. (13.00 ± 0.29 mm) ( Table 1). These results were found to be in accordance with the previous reports [72][73][74]. Further, MIC analysis was opted for testing antibacterial activity against above 4 strains of bacteria. MIC of fungal extract for the different bacterial strains was 31.25 µg/mL (Listeria and Shigella), 125 µg/mL (E.coli) and 62.5 µg/mL (S. typhi). MIC of mycosynthesized silver nanoparticles for different bacterial strains was 1.5625 µg/mL (Listeria and Shigella), 0.78125 (E. coli) and 3.125 (S. typhi).
DPPH analysis (Fig. 9a) was performed for the assessment of antioxidant properties of the fungal extract and mycosynthesized AgNPs. The percent inhibition in the

Conclusion
Synthesis of metallic nanoparticles in a biological way had proven to be a method where the process is effective and less toxic. In the present study an endophytic fungus, Talaromyces purpureogenus was isolated from Taxus baccata Linn. The fungus showed its potency to reduce the AgNO 3 to AgNPs where it produced high-quality crystalline AgNPs and its characterization was done using various techniques viz. UV-vis spectroscopy, FTIR, FEG-SEM, HRTEM, and XRD. The functional groups found in FTIR analysis have a probable role as capping agents. The particles were found to have a size of about 49.3 nm with crystalline fcc nature. The AgNPs also showed their effect on both gram-positive and gram-negative bacteria. Thus, the results conclude that isolated fungus Talaromyces purpureogenus is a distinguished producer of differently shaped silver nanoparticles having effective antibacterial and antioxidant activities, which can further accomplish biomedical and industrial purposes channelizing a way to reduce the pressure Taxus baccata is facing due to unethical harvesting techniques employed because of its huge utilitarian value.