Ganoderma applanatum-mediated green synthesis of silver nanoparticles: Structural characterization, and in vitro and in vivo biomedical and agrochemical properties

2.1 Chemicals

AgNO₃ was purchased from Sigma-Aldrich (St Louis, MO, USA). L-Ascorbic acid, 1,1-diphenyl-2-picrylhydrazyl (DPPH), n-hexane, chloroform and methanol were procured from Merck (Darmstadt, Germany). Phenolic standards, including, protocatechuic acid, catechin, chlorogenic, epicatechin, ferulic acid and apigenin were obtained from Sigma-Aldrich. Millipore distilled water was used in all experiments.

2.2 Collection of basidiomycete species

A survey was conducted in the forest region of Sanakeri, Sirsi, Uttar Karnataka, India (14.6185°N 74.8354°E) from November to December 2015. The basidiomycetes free from diseases were collected and stored at 4 °C till further use. The basidiomycete species were identified by their double walled basidiospores, ornamented shape and other characteristics according to the procedure described in the literature. The identification of seven basidiomycete species, named from BT.MBKUD-1 to BT.MBKUD-7, was further confirmed by comparison to the specimen’s collection at Department of Biotechnology and Microbiology, Karnatak University (Table S1) (Jogaiah et al., 2016).

2.3 Preparation of basidiomycete extracts

Basidiomycete samples, including Polyphorus sp. (BT.MBKUD-1), Tyromyces duracinus (BT.MBKUD-2), Pleurotus florida (BT.MBKUD-3), G. lucidum (BT.MBKUD-4), G. applanatum (BT.MBKUD-7), and two unidentified basidiomycetes (BT.MBKUD-5) and (BT.MBKUD-6), were washed thoroughly 2–3 times with running tap water followed by sterilized distilled water (SDW). The samples were then cut immediately into small pieces and air-dried on sterile blotting paper under shade. Dried samples were crushed using a sterile electric blender to obtain a fine-powdered form, and stored in sterile polyethylene bags before use. The dry powder from each species was extracted separately with the following gradient solvent system: n-hexane, chloroform, methanol and water by subjecting to the Soxhlet apparatus (Supelco-64826, Sigma-Aldrich). Each solvent extraction step was repeated thrice and filtered. The filtered extracts were concentrated separately using rotary flash evaporator (Buchii R-100 rotary evaporators, vacuum pump V-100). The crude extract with a final concentration of 500 µg mL⁻¹ (w/v) was further assessed for its antimicrobial activities against clinical pathogens and phytopathogens.

2.4 Antibacterial assay of basidiomycete extracts using nutrient agar plates

The antibacterial activities of the n-hexane, chloroform, methanol and water crude extracts of the seven basidiomycete species were assessed against five clinical bacterial pathogens (Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Bacillus subtilis and Pseudomonas aeruginosa) using agar disc diffusion method. The bacterial inoculum preparation was done by a direct colony suspension method recommended by National Committee for Clinical and Laboratory Standards (NCCLS). The bacterial inoculum suspension previously adjusted to 10⁶ CFU mL⁻¹ was uniformly spread on sterile nutrient agar (NA) plates using sterile glass swab. Sterile 6-mm diameter wells were prepared equidistantly around the margin of the inoculated NA plates using cork borer. 50 μL (500 µg mL⁻¹) of each crude extract was dispensed in the wells. A negative control was prepared using the respective solvent used to dissolve the test sample. The plates were incubated for 24 h at 35 ± 2 °C, and inhibition zone of the bacterial growth was measured in “mm”. Each test was performed in triplicate.

2.5 Antifungal assay of basidiomycete extracts using potato dextrose agar plates

The antifungal activity of the n-hexane, chloroform, methanol and water extracts of the seven basidiomycetes was assessed against four phytopathogens (Colletotrichum gloeosporioides, Colletotrichum capsici, Curvularia lunata and Botrytis cinerea) using potato dextrose agar (PDA) plates. An aliquot of each crude extract was added to the PDA to obtain a final concentration of 500 µg mL⁻¹. Crude extract-containing PDA plates were inoculated with a 5-mm agar plug containing the respective pathogen fungus that had been grown on PDA (without extract) for 5 days. The plates were incubated at 25 ± 2 °C, and the radial growth of the C. gloeosporioides, C. capsici, C. lunata and B. cinerea phytopathogens was measured after one week. Each test was performed in triplicate.

2.6 Antimicrobial assays of G. applanatum fractions

All the G. applanatum fractions were subjected to antimicrobial assays at a final concentration of 200 µg mL⁻¹ against C. capsici and S. aureus as described above.

2.7 Column chromatography of G. applanatum methanol extract

The G. applanatum 70% methanol extract was dried in a rotary evaporator using a vacuum pump (v-700; BUCHI, Rotavapor R-3) at 50 °C. The dried methanol extract was partitioned between n-butanol and H₂O (1:1, v/v). The n-butanol layer was filtered and concentrated, affording 12.09 g extract. An aliquot of the crude extract was chromatographed by C300 silica gel column chromatography (3 cm × 60 cm; AG Tokyo, Japan). The column was developed using a gradient solvent system starting with 100% chloroform and chloroform-methanol (8:2–5:5) as eluents to give six fractions.

2.8 Determination of bioactive compounds in G. applanatum fraction 3 using HPLC

Briefly, G applanatum fraction 3 was filtered and centrifuged at 15,000 rpm for 5 min. The supernatant was completely dried using a rotary evaporator, and the remnant was then dissolved in 1 mL MeOH. A volume of 60 μL of filtered fraction 3 was injected into HPLC (Shimadzu, Tokyo, Japan). The HPLC system includes a pump, a column oven, a diode array detector set to 280 nm, a data collection system (EZchrom Elite TM; Hitachi High-Technologies Corporation, Tokyo, Japan), a DGU-14A degasser and a SIL-10ADvp autosampler. Separation was conducted at 30 °C on Agilent® Eclipse XDB C-18 reversed-phase column (250 mm × 4.6 mm length, 5 μm particle size). Identification and quantitative analyses were conducted by comparison with authentic standards.

2.9 Biosynthesis of AgNPs

AgNPs were prepared by biological synthesis route using fraction 3 of the G. applanatum (BT.MBKUD-7) methanol extract as reducing and capping agent. A reaction mixture consisted of 10 mL of the G. applanatum fraction 3, 1 mL of 100 mM AgNO₃ and 89 mL SDW was subjected to various pHs (4, 5, 6, 7 and 8), temperatures (15, 20, 25, 30 and 35 °C) and incubation periods (2, 4, 6, 12 and 24 h). The color change in the incubated supernatant was observed as the indicator for the synthesis of AgNPs. The cell-free filtrate (without AgNO₃) was maintained as control under the same conditions.

2.10 Characterization of AgNPs

The presence of AgNPs was initially observed by the visual change in color. AgNP detection was further confirmed by UV–Vis spectrum of the tested sample recorded at 420 nm using spectra ranging from 300 to 600 nm at room temperature. The presence of possible interactions between protein and AgNPs, and their type of binding was analyzed using FTIR spectrum recorded in absorption mode with Perkin Elmer spectrometer (Spectrum 1000) along with KBr pellets. The crystal structure and composition of AgNPs were determined by a Shimadzu powder X-ray diffractometer (PXRD-7000) using Ag Kα radiation of λ = 1.541°A in the 2θ range of 20–80°. The size of the AgNPs was calculated using the Debye-Scherrer’s equation (Dubey et al., 2010).

2.11 Assay for AgNP antioxidant activity

DPPH scavenging activity was measured using IC₅₀ values as previously described (Abdelrahman et al., 2014). DPPH solution was prepared in ethanol and added to a serial dilution of G. applanatum-synthesized AgNPs and G. applanatum fraction 3. The reaction mixture was kept for 30 min under dark conditions at room temperature. Thereafter, the absorbance was read at 520 nm. The effective concentration, having 50% radical inhibition activity (IC₅₀) expressed as µg mL⁻¹, was calculated from the graph of the free radical scavenging activity. L-Ascorbic acid was used as a positive control. All experiments were repeated in triplicate.

2.12 Assay for antibacterial activities of AgNPs

An aliquot of the aqueous AgNPs, AgNO₃, G. applanatum fraction 3 (at a final concentration of 50 µg mL⁻¹) and SDW was dispensed on sterilized filter paper disc. The discs were placed equidistantly around the margin of the NA plates containing S. aureus or E. coli, and then the plates were incubated for 24 h at 35 ± 2 °C. Zone of inhibition of the bacterial growth was measured in mm. Each test was performed in triplicate.

2.13 In vivo leaflet assay for AgNP antifungal activities

One-month-old tomato (Solanum lycopersicum) and strawberry (Fragaria × ananassa) leaves were treated with an aliquot of the aqueous AgNPs and G. applanatum fraction 3 at a final concentration of 0, 12.5, 25 and 50 µg mL⁻¹, respectively. Agar plugs (5 mm in diameter) containing phytopathogen B. cinerea or C. gloeosporioides, which were grown on PDA for 5 days, were placed on AgNPs-wetted tomato and strawberry leaves, respectively. The treated leaves were incubated in sterilized plastic box for 7 days at 25 ± 2 °C under dark conditions. The development of disease was evaluated and recorded on the basis of the diameter of the necrotic lesion.

2.14 Statistical analysis

Data represented as means ± standard errors (SEs) of three repeats. The statistical analysis was performed by analysis of variance (ANOVA) using SPSS 20.0 (SPSS, Inc. USA). Heatmap clustering and box plot analysis were performed using R statistical package version 3.2.2 (www.r-project.org).

3.1 G. applanatum is the most effective antibacterial and antifungal basidiomycete for AgNP synthesis among the seven investigated basidiomycetes

The present study deals with the “green synthesis” of AgNPs using G. applanatum extract as reducing and capping agent, followed by the morphological and structural characterizations of synthesized AgNPs, as well as evaluation of their antimicrobial and antioxidant properties. Recently, syntheses of NPs using different fungal species have been reported (Devi and Joshi, 2015; Govindappa et al., 2016; Quester et al., 2016). Eukaryotic microbes, such as fungi, are considered as an exceptional choice for synthesizing NPs because they produce a great amount of secreted enzymes and proteins responsible for the bio-reduction (Gade et al., 2013; Cilerdžić et al., 2014). In addition, stabilizing and capping properties of the agents produced by fungi offer important advantages in “green synthesis” of NPs, including AgNP (Gade et al., 2013; Tran et al., 2013; Quester et al., 2016).

Among the microorganisms, the basidiomycetes produce a wide range of biologically active metabolites that have scarcely been investigated. A broad spectrum of natural products with biological activity produced by basidiomycetes includes antimicrobial, antifungal and antiviral compounds (Anke, 1989), as well as plant growth regulators (Jogaiah et al., 2016). Although various investigations reported the useful bioactive compounds from basidiomycetes, not much attention has been paid on the “green synthesis” of NPs using this microbial origin. Hence, in this study, we were interested in examining whether basidiomycetes could effectively synthesize AgNPs with antioxidant and antimicrobial activities, which can be then used in plant protection against various pathogens as well as in medical industry. In order to determine the most effective fungal basidiomycete species among the seven basidiomycetes examined, we first discriminated these basidiomycetes based on their antibacterial and antifungal activities using the gradient solvent extraction system of n-hexane, chloroform, methanol and water. The obtained results were further subjected to heatmap clustering and box plot analysis (Fig. 1). The heatmap clustering results revealed a distinctive separation between the seven basidiomycete species, and G. applanatum extracts had the highest antibacterial and antifungal activities against the tested pathogens in comparison with that of other basidiomycetes (Fig. 1a, b). Additionally, box plot analysis of different G. applanatum solvent extracts indicated that the methanol extract exhibited the most significant antibacterial and antifungal activities against the tested pathogens as compared with other solvent extractions (Fig. 1c, d). G. applanatum methanol extract displayed the maximum bacterial growth inhibition with 18.50 mm inhibition zone against S. aureus, and the highest fungal growth inhibition of 75.49% against C. capsici (Fig. 1a and b). Therefore, G. applanatum methanol extract was further used for the “green synthesis” of AgNPs.

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Fig. 1 Antibacterial and antifungal properties of several basidiomycete extracts (500 µg mL⁻¹) using the gradient solvent system of n-hexane, chloroform, methanol and water. (a) Heatmap clustering of antibacterial activities of different solvent extracts from the Polyphorus sp. (BT.MBKUD-1), Termitomyces microcarpus (BT.MBKUD-2), Pleurotus florida (BT.MBKUD-3), Ganoderma lucidum (BT.MBKUD-4), G. applanatum (BT.MBKUD-7) and two unidentified basidiomycete species (BT.MBKUD-5 and BT.MBKUD-6). (b) Heatmap clustering of antifungal activities of different solvent extracts from seven basidiomycete species. (c) Box plot analysis of G. applanatum BT.MBKUD-7 antibacterial activities. (d) Box plot analysis of G. applanatum BT.MBKUD-7 antifungal activities. The heatmap color scale showed the antimicrobial range. Box plots labeled by the same letter(s) are not significantly different according to a Tukey’s honestly significant difference post hoc test.

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To optimize the efficacy and concentration of the G. applanatum methanol extract with regard to its antimicrobial properties, G. applanatum methanol extract was entirely freeze-dried and subjected to a column chromatography, resulting in 6 fractions. The obtained fractions were assessed for their antimicrobial activities at a final concentration of 200 µg mL⁻¹ (Fig. S1). Fraction 3 displayed a significantly higher antimicrobial activity in comparison with other tested fractions (Fig. S1), exhibiting 41.02% fungal growth inhibition against C. capsici and 10.13 mm inhibition zone against S. aureus. These data suggested that G. applanatum fraction 3 might contain the highest concentration of potential bioactive compounds that may induce the antimicrobial properties of AgNPs. To gain an insight into the nature of these key bioactive compounds in fraction 3, phytochemical analysis using HPLC-UV was carried out (Fig. S2 and Table 1). The HPLC results showed high phenolic contents in fraction 3, including protocatechuic acid, catechin and apigenin (Table 1). In support of our result, a recent finding reported that a G. applanatum strain has high phenolic contents, and these phenolic compounds are key components for the biological activities of G. applanatum (Zengin et al., 2015). The important role of phenolic compounds derived from plant extracts as potent antimicrobial and antioxidant agents has been well-established, which was attributed to the presence of a hydroxyl group bonded to the aromatic hydrocarbon ring (Abdelrahman et al., 2015; Fatrcova-Sramková et al., 2016; Tohma et al., 2016).

The reaction mixture consisted of the G. applanatum fraction 3 and AgNO₃ was subjected to different conditions of pH, temperature and incubation period in order to optimize the uniformity and size of AgNPs. The reduction of AgNO₃ to AgNPs was visually detected by the change in color of the mixture from pale yellow to dark brown, which indicated the formation of AgNPs. UV–Vis absorbance spectra of the synthesized AgNPs at different pHs, temperatures and incubation time points were then recorded (Fig. 2). Strong and broad surface plasmon resonance (SPR) peak was observed at 420 nm at pH 6–7, which demonstrated the synthesis of AgNPs (Fig. 2). Previous studies suggested that an SPR peak between 410 and 450 nm is characteristic for AgNPs, and might be attributed to spherical NPs (Rafiuddin, 2012; Jyoti et al., 2015). The main absorption band for AgNPs was shifted to higher unresolved absorption wavelength for AgNPs formed at other pH values, indicating that the synthesized AgNPs at these pHs are not spherical, and may exhibit different sizes and shapes (Fig. 2a). Likewise, the time-course analysis of AgNP biosynthesis process by G. applanatum fraction 3 clearly demonstrated the rapid biosynthesis of AgNPs as evidenced by the absorbance intensity at 2 h, and the increase in the incubation time accelerated the AgNP biosynthesis process with maximum absorbance intensity being detected at 6 and 12 h (Fig. 2b). The effect of temperature on the rate of AgNP biosynthesis by G. applanatum fraction 3 showed a characteristic absorbance of the synthesized AgNPs at 420 nm with maximum absorbance intensity being observed at 25 and 30 °C (Fig. 2c).

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Fig. 2 UV–Vis spectra of silver nanoparticles synthesized by Ganoderma applanatum fraction 3 at (a) different pHs, (b) incubation times (hours) and (c) temperatures.

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3.2 Characterization of the synthesized AgNPs by FTIR and X-ray diffraction

FTIR is a valuable tool to investigate the core-shell morphology of the synthesized AgNPs, and also provides crucial information about the chemical bonds and molecular structures responsible for the bio-reduction of Ag⁺ and capping/stabilization of the AgNPs (Jyoti et al., 2015). The observed bands were compared with standard values to identify the functional groups. FTIR spectra showed absorption bands at 3620.82, 3420.75, 1600.81, 1384.41, 1048.84 and 472.08 cm⁻¹ (Fig. 3a), indicating the presence of capping agent with the NPs. The bands from 3620.82 to 3420.75 cm⁻¹ correspond to O—H stretching, indicating the presence of alcohol and phenol. The band at 1600.81 cm⁻¹ in the spectra corresponds to N ⚌ O and C ⚌ O stretches, indicating the presence of amide and nitro groups. The bands from 1384.41 to 1048.84 cm⁻¹ correspond to the C—O and N ⚌ O stretches, indicating the presence of nitro and ester groups, respectively. These functional groups have an important role in the stability/capping and biological activities of AgNPs (Jyoti et al., 2015; Prakash et al., 2013). The FTIR data indicated that the synthesized AgNPs were enclosed by G. applanatum proteins and amino acids, which may be responsible for the stability of the AgNPs, and that the structure of proteins derived from G. applanatum was not affected by the interactions with synthesized AgNPs.

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Fig. 3 The core-shell morphology and crystalline pattern of Ganoderma applanatum-synthesized silver nanoparticles (AgNPs). (a) Fourier transform infrared spectroscopy and (b) X-ray diffraction of AgNPs synthesized by G. applanatum fraction 3. The infrared spectroscopy wave numbers and the Bragg reflections with 2θ values are shown within each corresponding peak.

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3.3 Characterization of synthesized AgNPs by XRD, SEM and TEM

The crystalline pattern of AgNPs was further verified by X-ray crystallography, and the XRD of the synthesized AgNPs was recorded (Fig. 3b). The diffracted intensities were recorded from 20° to 80°. The Bragg reflections with 2θ values of 39.01°, 46.57°, 65.21° and 78.13° corresponded to the (1 1 1), (2 0 0), (2 2 0) and (3 1 1) sets of lattice planes indicated the bands for face-centered cubic structure of Ag and its crystalline nature (Prakash et al., 2013). The calculated crystallite size of the AgNPs ranged from 20 to 25 nm.

SEM and TEM studies provide further insight into the morphology and particle size distribution profile of the AgNPs (Fig. 4). The SEM micrograph recorded at different magnifications indicated the AgNP aggregates (Fig. 4a, b). The SEM micrograph showed the spherical AgNPs in the size ranged between 20 and 25 nm Additionally, TEM images provided further details of the morphology of the synthesized AgNPs (Fig. 4c, d). The spherical nature of the AgNPs was also verified by TEM analysis with an average diameter between 20 and 25 nm. Thus, both SEM and TEM results strengthened the XRD crystalline size of the synthesized AgNPs.

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Fig. 4 Scanning electron microscopic (SEM) and transmission electron microscopic (TEM) images of silver nanoparticles (AgNPs) synthesized by Ganoderma applanatum. (a) and (b) SEM images at different magnifications. (c) and (d) TEM micrographs showed the spherical AgNPs at different magnifications.

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3.4 Antioxidant activity of the synthesized AgNPs

The antioxidant activity of the G. applanatum-synthesized AgNPs and G. applanatum fraction 3 was evaluated using the DPPH IC₅₀ scavenging assay, with ascorbic acid being a positive standard. The maximum DPPH scavenging activity was recorded with ascorbic acid (IC₅₀, 37.53 µg mL⁻¹) and AgNPs (IC₅₀, 98.73 µg mL⁻¹), whereas the lowest scavenging activity was recorded for the G. applanatum fraction 3 (IC₅₀, 118.62 µg mL⁻¹) (Table 2). Our data were in line with the results of previous studies that have reported the antioxidant activities of AgNPs synthesized using the crude extracts derived from Chenopodium murale, Bergenia ciliate and Malus domestica (Abdel-Aziz et al., 2014; Nagaich et al., 2016; Phull et al., 2016). It is well-established that the antioxidant activity is majorly attributed to the phenolic contents of the living organism (Abdelrahman et al., 2016). The FTIR data demonstrated the presence of phenol groups (Fig. 3a) in G. applanatum-synthesized AgNPs which may contribute to their DPPH scavenging activity.

3.5 In vitro antibacterial activities of the synthesized AgNPs

Next, the antibacterial activities of G. applanatum-synthesized AgNPs were evaluated against two clinical bacterial pathogens (S. aureus and E. coli) using agar disc diffusion method (Fig. 5a, b). At the final concentration of 50 µg mL⁻¹, the AgNPs exhibited higher antibacterial activities against S. aureus (16.33 mm) and E. coli (13.33 mm) in comparison with the crude G. applanatum fraction 3 (7.4 and 6.8 mm, respectively) and AgNO₃ (1.83 and 0.85 mm, respectively) (Fig. 5c, d). The differential antibacterial activities between AgNPs and AgNO₃ clearly demonstrate the importance of G. applanatum extract in the improvement of the antibacterial properties of the synthesized AgNPs. In support of our findings, antibacterial activities of AgNPs, which were synthesized from Mimusops elengi leaf extract and the marine bacterium Ochrobactrum anhtropi, against various clinical pathogens were also reported (Zhou et al., 2012; Prakash et al., 2013; Thomas et al., 2014; Le Ouay and Stellacci, 2015). The antibacterial effect of AgNPs could be explained on the basis of small-sized AgNPs synthesized by G. applanatum and functional capping groups that provide a better interaction with the bacterial cell membranes (Zhou et al., 2012; Thomas et al., 2014; Le Ouay and Stellacci, 2015). In addition, AgNP interaction with bacterial cell membrane will facilitate the Ag⁺ penetration and interactions with cellular DNA and proteins, resulting in loss of cell viability and cell death (Rafiuddin, 2012; Le Ouay and Stellacci, 2015).

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Fig. 5 Antibacterial activities of silver nanoparticles (AgNPs) synthesized by Ganoderma applanatum fraction 3. (a) and (b) Images showing antibacterial activities of AgNPs at a final concentration of 50 µg mL⁻¹ against Staphylococcus aureus and Escherichia coli, respectively. (c) and (d) The diameter of inhibition zone in S. aureus and E. coli, respectively. Values are means ± standard errors (n = 3). Significance levels between control versus AgNPs, control versus G. applanatum fraction 3 (F3), and control versus AgNO₃ (50 µg mL⁻¹) are indicated by asterisks (*P < .05, ^**P < .01 and ^***P < .001) according to an independent Student’s t-test.

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3.6 In vivo antifungal activities of the synthesized AgNPs

In the next line of our study, we were curious whether the synthesized AgNPs could provide protection to plants against phytopathogens. Leaflet assay was carried out using one-month-old tomato and strawberry leaves treated with different concentrations of AgNPs and G. applantum fraction 3 extract (0, 12.5, 25 and 50 µg mL⁻¹). The treated-tomato and -strawberry leaves were further inoculated with 5-mm PDA plug of B. cinerea and C. gloeosporioides, respectively (Figs. 6 and 7). The antifungal activities of synthesized AgNPs were significantly increased with the increasing concentrations of AgNPs (Figs. 6c and 7c). The maximum fungal growth inhibition was observed at 50 µg mL⁻¹ AgNPs, whereas tomato and strawberry leaves exhibited a full necrotic lesion and subsequent leaf wilting without AgNP treatment. The observed results showed the capability of AgNPs to prevent the fungal spreading in the inoculated-tomato and -strawberry leaves without affecting the leaf morphological status. In support of our findings, in vitro antifungal activities of AgNPs synthesized from turnip leaf extract and sodium citrate solution, against wood-degrading fungal pathogens (e.g. Gloeophyllum abietinum, G. trabeum, Chaetomium globosum and Phanerochaete sordida) and cotton pathogen Rhizoctonia solani, respectively, were reported in previous studies (Narayanan and Park, 2015; Elgorban et al., 2016; Haghighi Pak et al., 2016). However, the in vivo antifungal activity of AgNPs has rarely been examined. Thus, the finding of our in vivo antifungal assay provides strong support for the development of agrochemical NP agents with the cost-effective and environmentally acceptable platform. Further studies are required to determine the efficacy of AgNPs against various phytopathogens in different cropping systems under controls and field conditions for their broad and efficient applications in agriculture. The hypothetical antimicrobial mechanism of AgNPs has been summarized in Fig. 8. Taken together, it can be concluded that the “green synthesis” of AgNPs using G. applanatum extract is a cost-effective and environmentally acceptable platform that excludes the hazards resulted from the use of toxic reducing/capping agents. Moreover, this process could be simply scaled up for the industrial applications to maximize the yield of the AgNPs.

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Fig. 6 Antifungal activities of silver nanoparticles (AgNPs) synthesized by Ganoderma applanatum fraction 3 (F3). (a) and (b) In vivo antifungal leaflet assay of one-month-old tomato leaves treated with different concentrations of F3 or AgNPs (0, 12.5, 25 and 50 µg mL⁻¹), and then challenge-inoculated with Botrytis cinerea. (c) Fungal growth inhibition percentage of B. cinerea. Values are means ± standard errors (n = 3). Significance difference between F3 and AgNPs at each concentration is indicated by asterisks (*P < .05, ^**P < .01 and ^***P < .001) according to an analysis of variance test.

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Fig. 7 Antifungal activities of silver nanoparticles (AgNPs) synthesized by Ganoderma applanatum fraction 3 (F3). (a) and (b) In vivo antifungal leaflet assay of one-month-old strawberry leaves treated with different concentrations of F3 or AgNPs (0, 12.5, 25 and 50 µg mL⁻¹), and then challenge-inoculated with Colletotrichum gloeosporioides. (c) Fungal growth inhibition percentage of C. gloeosporioides. Values are means ± standard errors (n = 3). Significance difference between F3 and AgNPs at each concentration is indicated by asterisks (*P < .05, ^**P < .01 and ^***P < .001) according to an analysis of variance test.

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Fig. 8 Potential antibacterial mechanisms of silver nanoparticles synthesized by Ganoderma applanatum fraction 3 (GaF3). ROS, reactive oxygen species; F_O, a component of ATP synthase; α and β, membrane subunits of ATP synthase; SU I-IV, subunits of the cytochrome bo₃ ubiquinol oxidase.

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