Biosynthesis of silver nanoparticles employing Trichoderma harzianum with enzymatic stimulation for the control of Sclerotinia sclerotiorum

Biogenic synthesis of silver nanoparticles employing fungi offers advantages, including the formation of a capping from fungal biomolecules, which provides stability and can contribute to biological activity. In this work, silver nanoparticles were synthesized using Trichoderma harzianum cultivated with (AgNP-TS) and without enzymatic stimulation (AgNP-T) by the cell wall of Sclerotinia sclerotiorum. The nanoparticles were evaluated for the control of S. sclerotiorum. The specific activity of the T. harzianum hydrolytic enzymes were determined in the filtrates and nanoparticles. Cytotoxicity and genotoxicity were also evaluated. Both the nanoparticles exhibited inhibitory activity towards S. sclerotiorum, with no new sclerotia development, however AgNP-TS was more effective against mycelial growth. Both the filtrates and the nanoparticles showed specific enzymatic activity. Low levels of cytotoxicity and genotoxicity were observed. This study opens perspectives for further exploration of fungal biogenic nanoparticles, indicating their use for the control of S. sclerotiorum and other agricultural pests.

physicochemical characterization of the nanoparticles. After the synthesis, the nanoparticles and the corresponding T. harzianum filtrates were analyzed by UV-visible spectroscopy in the wavelength range between 200 and 800 nm, with resolution of 1 nm, using a Shimadzu Multispec 1501 spectrophotometer. The pH values of the nanoparticles and the filtrates were measured using a pH meter (HMMPB-210).
The hydrodynamic diameters and polydispersities of the nanoparticles were measured by dynamic light scattering (DLS) and the zeta potentials were determined by microelectrophoresis, using a ZS90 particle analyzer (Malvern Instruments). The analyses were performed in triplicate, at 25 °C, with a fixed angle of 90°. The sizes and concentrations (NPs.mL −1 ) of the nanoparticles were also determined by nanoparticle tracking analysis (NTA), using a NanoSight LM10 cell (Malvern Analytical) coupled to a camera and controlled with NanoSight v. 2.3 software. The nanoparticles were diluted 50-fold in ultrapure water and 5 measurements were performed for each sample.
Hydrolytic enzyme specific activity assays. The specific activities of the T. harzianum hydrolytic enzymes β-1,3-glucanase, N-acetylglucosaminidase (NAGase), chitinase, and acid protease were determined using microplate assays in which the enzyme sources were the T. harzianum filtrates obtained with and without stimulation, as well as the corresponding nanoparticles (AgNP-TS and AgNP-T). The protein concentrations were first determined using the method of Bradford 31 , with bovine serum albumin (0.125, 0.250, 0.500, and 1.000 mg. mL −1 ) as standard. The assays of the four enzymes employed the methodology described by Qualhato et al. 21 .

Biological activities of the nanoparticles towards Sclerotinia sclerotiorum and effects on
Trichoderma harzianum. For evaluation of the ability of the nanoparticles to control the mycelial growth and sclerotia development of S. sclerotiorum, Petri dishes were prepared with potato-dextrose agar culture medium containing AgNP-TS and AgNP-T at final concentrations of 3 × 10 9 NPs.mL −1 , in triplicate. Controls were prepared using potato-dextrose agar alone and inoculated with T. harzianum (127 μg.mL −1 ). After solidification of the agar, a viable sclerotium was placed in the center of each plate, followed by keeping at room temperature for 15 days, with a photoperiod of 12 h. At the end of the period, the mycelium growth halo was measured and the number of new sclerotia was counted.
Evaluation was also made of possible effects of the nanoparticles on T. harzianum, given the importance of ensuring the viability of this biological control agent for both the control of phytopathogens and the induction of plant growth and development. For this, plates were prepared with culture media containing the nanoparticles, as described above, which were inoculated with T. harzianum at 127 μg.mL −1 , in duplicate. The cultures were kept at ambient temperature for 15 days, in the dark, followed by growth analysis.
Viability/cytotoxicity and genotoxicity assays using the nanoparticles. The cytotoxic effects of the nanoparticles were evaluated using the V79 (chinese hamster lung fibroblast), 3T3 (albino Swiss mouse embryo fibroblast), and HaCat (human keratinocyte) cell lines. The techniques employed were the tetrazolium reduction assay (MTT test for mitochondrial activity), image cytometry (cell viability, apoptosis, and necrosis), and the trypan blue exclusion test (cell viability). For the MTT assay, the cells were previously cultured in Dulbecco's modified eagle medium (DMEM), plated in 96-well plates (5 × 10 4 cells per well), and exposed for 24 h to the nanoparticles at concentrations from 0.1 × 10 9 to 3.5 × 10 9 NPs.mL −1 . The wells were than washed with phosphate buffer saline (PBS), MTT solution (5 mg.mL −1 ) was added, and the plates were left for 3 h. Finally, the samples were fixed with dimethylsulphoxide (DMSO) and were read using a microplate reader at 540 nm. Image cytometry analyses of cell viability, apoptosis, and necrosis were performed using an apoptosis kit with Annexin V Alexa Fluor TM 488 and propidium iodide (Invitrogen). The cells were exposed for 1 h to the nanoparticles at a concentration of 3 × 10 9 NPs.mL −1 and were then prepared as specified by the manufacturer. The readings were obtained using a Tali TM image cytometer (Invitrogen). For the trypan blue exclusion assay, the cells were exposed for 1 h to the nanoparticles at 3 × 10 9 NPs.mL −1 , followed by staining with trypan blue and counting the cells using an optical microscope, considering cells stained blue to be dead. The positive controls were cells exposed to 0.5 M hydrogen peroxide, while the negative controls were cells kept in culture medium without any exposure.
The genotoxic potentials of the nanoparticles were determined using the Allium cepa assay, as described by Lima et al. 32 . The roots were exposed for 24 h to AgNP-TS and AgNP-T at concentrations of 1 × 10 10 and 3 × 10 9 NPs.mL −1 , followed by fixing in ethanol:acetic acid (3:1). For preparation of the slides (in triplicate), the roots were hydrolyzed in 1 M HCl, at 60 °C, and stained with Schiff 's reagent. The meristematic regions were cut, stained with acetic carmine, and crushed under cover slips. The cells were observed under an optical microscope, with counting of the cells in division and those that presented chromosomal alterations, hence obtaining the mitotic index (MI) and chromosomal alteration index (AI) values.
The comet assays to evaluate the effects of AgNP-TS and AgNP-T were performed using an adaptation of the methodology described by Singh et al. 33 . The previously cultured cells were exposed for 1 h to the nanoparticles at concentrations of 3 × 10 9 NP.mL −1 , followed by mixing with agarose, application to pre-gelatinized slides, and keeping for 1 h in lysis solution. Following neutralization, the slides were kept for 20 min in electrophoresis buffer and were subsequently submitted to electrophoresis for 20 min at 22 V and 300 mA. The slides were then fixed, stained with silver, and visual scoring was performed under an optical microscope.
Statistical analyses. Statistical treatment of the data employed one-way analysis of variance (ANOVA) followed by Tukey's test, with a significance level of p < 0.05. These analyses were performed with GraphPad Prism 7.0 software.

Results and Discussion
physicochemical characterization of the nanoparticles. Silver nanoparticles were successfully synthesized using the filtrates from T. harzianum cultivated in the presence and absence of the cell wall of S. sclerotiorum, which resulted in the nanoparticles AgNP-TS and AgNP-T. The filtrates showed color change from light yellow to reddish-brown 72 h after addition of AgNO 3 , due to the surface plasmon resonance of the silver 34 . The synthesis was confirmed by UV-visible spectroscopy, with peaks obtained at 409 and 413 nm for AgNP-TS and AgNP-T, respectively (Fig. 1A), indicating the presence of elemental silver 35,36 . Analysis of the filtrates used in the synthesis revealed peaks at 221 and 215 nm for the samples obtained with and without stimulation by the cell wall of S. sclerotiorum, respectively. These absorbance peaks observed for the filtrates were also present in www.nature.com/scientificreports www.nature.com/scientificreports/ the spectra for the nanoparticles, reflecting the presence of filtrate proteins in their compositions. In previous studies, Phanjom and Ahmed 37 reported peaks at 210 and 260 nm for filtrate of the fungus Aspergillus oryzae used for the synthesis of silver nanoparticles, which were attributed to amides and amino acid residues, respectively. Durán et al. 38 used Fusarium oxysporum to synthesize silver nanoparticles, with peaks at 265 nm attributed to aromatic amino acids of proteins released into the filtrate, which contributed to silver nitrate reduction and stabilization of the nanoparticles. Ballotin et al. 39 found two main UV-vis bands for silver nanoparticles synthesized using Aspergillus tubingensis, with one at 440 nm, confirming formation of the silver nanoparticles, and another at 280 nm, attributed to aromatic amino acids such as tryptophan, tyrosine, and phenylalanine residues, which composed the protein capping of the nanoparticles. The presence of peaks corresponding to proteins and amino acid residues in both the filtrates and the capped nanoparticles confirmed the release of these compounds from the fungal biomass dispersed in water, indicating that they were involved in the process of reduction of the metal ions, leading to the formation and stabilization of the nanoparticles.
The hydrodynamic diameters, polydispersity indices, and zeta potentials of the nanoparticles were determined by the DLS and microelectrophoresis techniques. The AgNP-TS and AgNP-T nanoparticles presented mean hydrodynamic diameters of 57.02 ± 1.75 and 81.84 ± 0.67 nm, respectively (Fig. 1B). The polydispersity and zeta potential values were 0.49 ± 0.01 and −18.70 ± 3.01 mV for AgNP-TS and 0.52 ± 0.00 and −18.30 ± 1.73 mV for AgNP-T, respectively. The difference in size between the nanoparticles synthesized using the filtrates of T. harzianum with and without stimulation by the S. sclerotiorum cell wall could have been due to the different culture medium compositions. Various factors have been reported to influence nanoparticle physicochemical characteristics, including the composition of the medium in which the reducing agent is cultivated 40 .
The NTA technique was also used to determine the sizes and concentrations of the nanoparticles (Fig. 1C), resulting in different concentrations for AgNP-TS and AgNP-T, with particle sizes of 88.0 ± 7.3 and 182.5 ± 6.9 nm, respectively. After characterization, the stock concentration of the nanoparticles was standardized at 1 × 10 10 NPs.mL −1 , in order to facilitate the activity and toxicity evaluations. Durán et al. 38 reported the presence of a capping around biogenic nanoparticles synthesized using the fungus Fusarium oxysporum, which avoided aggregation. In agreement with the previous report, the nanoparticles synthesized in the present work by the biogenic method presented a capping of proteins and other biomolecules derived from the organism used as the reducing agent (T. harzianum). This capping provided stability to the nanoparticles, so that they maintained their original size and did not aggregate 35 .
The pH of the medium in which the synthesis occurs (in this case, the filtrates) can affect the size and morphology of the nanoparticles. A higher pH leads to smaller nanoparticles with spherical morphology, while a lower pH results in larger nanoparticles in the forms of rods and prisms 41 . In the present case, both filtrates presented pH of 7.2, while no significant changes were observed after the synthesis, with near-neutral values of 7.2 and 7.3 for AgNP-TS and AgNP-T, respectively. It has been reported that for non-biogenic nanoparticles, the pH and size of newly synthesized nanoparticles influence their reactivity, with lower values of these parameters indicating greater possibility of dissolution and ionization, which could lead to increased toxic effects 42 .
Determination of hydrolytic enzyme specific activity. The assays used to determine the specific activities of the T. harzianum hydrolytic enzymes β-1,3-glucanase, NAGase, chitinase, and acid protease detected the activities of these enzymes in both the filtrates, as well as in the AgNP-TS and AgNP-T nanoparticles (Fig. 2). It was not possible to compare the activity levels of the filtrates and the corresponding nanoparticles, due to the concentration changes in the synthesis processes. However, it could be seen that the highest specific activity was obtained for NAGase, followed by β-1,3-glucanase for both the filtrates and the nanoparticles. The specific activity of chitinase and acid protease were also observed with lower values, especially for acid protease. Small differences were observed between the enzymatic activities for the filtrates obtained with and without stimulation by the cell wall of S. sclerotiorum, and between the activities for different nanoparticles. Higher specific activity of the NAGase and chitinase was observed for AgNP-TS in comparison with AgNP-T. Regarding to the filtrates, the exposure of T. harzianum to the cell wall of S. sclerotiorum stimulated the specific activity of the enzyme NAGase.
The effectivity of the biogenic nanoparticles for the control of S. sclerotiorum can be attributed to the specific activity of the hydrolytic enzymes present in the capping of the nanoparticles. In a previous study, Guilger et al. 10 evaluated the effects of commercial AgNPs, non-biogenic and uncapped, and these nanoparticles were not able to control fungal development.
Geraldine et al. 23 evaluated the specific activity of the hydrolytic enzymes NAGase, acid phosphatase, β-glucosidase, lipase, β-1,3-glucanase and proteases in the filtrates of different Trichoderma species and strains after exposure to S. sclerotiorum cell wall. All the species and strains showed enzymatic activity in different proportions and in a varied way, with the highest activities for NAGase, acid phosphatase, and protease.
Qualhato et al. 21 also reported the production and secretion of β-1,3-glucanase, NAGase, chitinase, acid phosphatase, acid proteases and alginate lyase in the filtrates of Trichoderma spp. grown in the presence of the cell wall of the pathogenic fungi Fusarium solani, Rhizoctonia solani and S. sclerotiorum. To our knowledge, no prior studies have examined the specific activity of fungal hydrolytic enzymes in samples of biogenic silver nanoparticles.

Biological activity of the nanoparticles against Sclerotinia sclerotiorum and effects on
Trichoderma harzianum. The biological activity results showed that the AgNP-TS and AgNP-T nanoparticles presented potential for the control of S. sclerotiorum, since mycelial growth was reduced and there was no formation of new sclerotia. Treatment using T. harzianum also resulted in inhibition of S. sclerotiorum. As shown in Fig. 3A, many new sclerotia were formed from the precursor sclerotium at the edges of the control plate (CTR), with a mean of 116.5 ± 7.7 sclerotia, while the AgNP-TS and AgNP-T plates showed no new sclerotia and reduced mycelial growth. The greatest mycelial inhibition was provided by AgNP-TS, which capping was derived from the filtrate obtained using stimulation to enhance the production of T. harzianum hydrolytic enzymes (Fig. 3B).
The superior control of mycelial growth achieved using AgNP-TS could have been related to the smaller nanoparticle size, given that previous work has found that a smaller nanoparticle size is associated with higher activity 43 . An additional consideration is that these nanoparticles were synthesized using filtrate from a culture in which there had been stimulation of the production of hydrolytic enzymes that could act in degradation of the cell walls of the phytopathogen. The better control of mycelial growth by AgNP-TS can be attributed to the higher activity of the enzymes NAGase and chitinase from these nanoparticles.
The inhibition of microorganisms by silver nanoparticles is due to the strong surface oxidative activity of the particles and the release of ions 44 . However, in the case of biogenic nanoparticles, there may also be a contribution of the stabilizing capping derived from the reducing organism, which deserves further investigation.
Previous studies have investigated the use of silver nanoparticles for controlling agricultural phytopathogens including S. sclerotiorum, with effective inhibitory activity observed in vitro [45][46][47][48] . However, the mechanism of action of nanoparticles is not yet understood 49 . Krishnaraj et al. 46 tested different concentrations of silver nanoparticles against the phytopathogenic fungi Alternaria alternata, Sclerotinia sclerotiorum, Macrophomina phaseolina, Rhizoctonia solani, Botrytis cinerea, and Curvularia lunata, with inhibitory effects observed against all these www.nature.com/scientificreports www.nature.com/scientificreports/ species. Despite such promising results, there have been no previous studies concerning the biogenic synthesis of nanoparticles with stimulation of the metabolism of the biological agent employed for the reduction and stabilization. This is important, since enzyme production by fungi is directly influenced by the conditions under which the organisms are cultivated 50 .
Trichoderma harzianum is widely used as a biological control agent therefore it is important to evaluate possible effects of nanomaterials against this fungus 51 . In our study the nanoparticles caused no alteration of T. harzianum growth (Fig. 3C), which was an important finding, indicating the possibility of its use in combination with the nanoparticles.
The use of nanotechnological products for the control of pests in agriculture is considered to be safer than employing traditional agrochemicals, since it helps to avoid excessive accumulation in the environment of chemicals that can cause residual toxicity 49 . An additional consideration is that the use of chemical fungicides can lead to the development of resistance by phytopathogens 52,53 . Nonetheless, it is possible that the release of new nanomaterials into the environment could become a problem for humans and other organisms 54 . For this reason, it is vital that their potential toxicities towards biological systems should be thoroughly investigated, in order to ensure the safe development of effective new nanomaterials 55 . The nanometric size of nanoparticles enables their internalization within the cells of living organisms, which might lead to serious consequences 56 .
Viability/cytotoxicity and genotoxicity evaluation of the nanoparticles. Evaluation of the cytotoxic effects of the nanoparticles using the MTT test, which indicates the mitochondrial activity of the cells, revealed differences in the viability percentages for the different cell lines. However, despite presenting different sensitivities, no IC50 values were observed for any of the samples, indicating low cytotoxicity of the nanoparticles in the range of exposure concentrations employed (Fig. 4A), including the concentration of interest for the control of S. sclerotiorum (3 × 10 9 NPs.mL −1 ). These results were consistent with the direct analyses of cytotoxicity using the image cytometry (Fig. 4B) and trypan blue (Fig. 4C) methods, although it is important to take the different exposure periods into consideration (24 h for the MTT test and 1 h for the image cytometry and trypan blue methods). In the image cytometry tests, the two types of nanoparticle caused similar levels of apoptosis and necrosis, with the HaCat cell line presenting viability equivalent to that of the control. The results of the trypan blue exclusion assays indicated that exposure to both types of nanoparticle led to significant decreases of the viabilities of the 3T3 and HaCat cells, while the V79 cells only showed decreased viability when exposed to AgNP-TS. In comparison of the two types of nanoparticle, AgNP-TS caused the lowest viabilities of the V79 and HaCat cells.
Silver nanoparticles can alter normal cellular functions, affect membrane integrity, and initiate programmed death processes 57 . This cytotoxicity may arise from physicochemical interactions between silver atoms and the functional groups of cellular proteins 58 . However, although these effects apply to silver nanoparticles in general, the nanoparticles obtained using biogenic synthesis present specific characteristics related to the organism used in the reduction process, with the cytotoxicity generally being lower than observed for commercial nanoparticles www.nature.com/scientificreports www.nature.com/scientificreports/ and solutions containing silver ions 59 . Skladanowski et al. 60 , using the MTT test, reported an absence of cytotoxicity in L929 mouse fibroblasts exposed to silver nanoparticles synthesized using Streptomyces sp. NH28.
The Allium cepa assay was used to investigate possible genotoxicity of the nanoparticles and mitotic index alteration. At the two exposure concentrations tested, both types of nanoparticle caused the mitotic index values to decrease, compared to the control (Fig. 5A). The alteration indices indicated that both AgNP-TS and AgNP-T caused increased chromosomal alterations, at the exposure concentrations, with no difference between the effects of the two nanoparticle types (Fig. 5B).
The results of the Allium cepa assay were consistent with those obtained in a similar trial using the plant species Drimia polyantha, where decreases of the mitotic index and the presence of chromosomal alterations were found following exposure to the highest concentrations of biogenic silver nanoparticles synthesized using the plant Getonia floribunda 61 . Similar results were obtained using the Allium cepa assay to evaluate the effects of low concentrations of silver nanoparticles synthesized using the plant Swertia chirata 62 . The toxic effects caused by silver nanoparticles may be due to the generation of reactive oxygen species and DNA damage, leading to apoptosis, as well as the release of Ag + ions, which depends on the rate of dissolution of the nanoparticles within the cells [63][64][65] .
Further evaluation of the genotoxicity of the nanoparticles was performed using comet assays to determine the extent of DNA damage in the V79, 3T3, and HaCat cell lines. Both types of nanoparticle caused increases of the damage indices for the three cell lines, compared to the control, with the V79 cells showing the greatest sensitivity to AgNP-TS (Fig. 5C).
conclusions Silver nanoparticles were successfully synthesized using the filtrates from T. harzianum cultivated in the presence and absence of the cell wall of S. sclerotiorum, which resulted in nanoparticles with different physicochemical characteristics. Both AgNP-TS and AgNP-T were able to control the growth of S. sclerotiorum, with inhibition of mycelial growth and prevention of the formation of new sclerotia. The most effective control of mycelial growth was achieved using AgNP-TS, which could be attributed to the smaller hydrodynamic diameter of the nanoparticles, as well as a possible effect of the biomolecules from the capping of the nanoparticles, which were derived from the filtrate obtained using stimulation of the enzymatic production of T. harzianum. The effect of the