Biosynthesis of high antibacterial silver chloride nanoparticles against Ralstonia solanacearum using spent mushroom substrate extract

In this study, a green and highly efficient method was proposed to synthesize nano-silver chloride (nano-AgCl) using spent mushroom substrate (SMS) extract as a cheap reactant. Nanoparticles were characterized by a series of techniques like x-ray diffraction (XRD), energy dispersive spectroscopy (EDS), scanning electron microscopy (SEM), and transmission electron microscopy (TEM), which showed the formation of near-spherical silver chloride nanoparticles with an average size of about 8.30 nm. Notably, the synthesized nano-silver chloride has a more prominent antibacterial effect against Ralstonia solanacearum (EC50 = 5.18 mg L−1) than non-nano-sized silver chloride particles, nano-silver chloride synthesized by chemical method, and commercial pesticides. In-depth, the study of the mechanism revealed that nano-silver chloride could cause cell membrane disruption, DNA damage and intracellular generation of reactive oxygen species (·OH, ·O2− and 1O2), leading to peroxidation damage in Ralstonia solanacearum (R. solanacearum). Moreover, the reaction between nano-silver chloride and bacteria could be driven by intermolecular forces instead of electrostatic interactions. Our study provides a new approach to synthesizing nano-silver chloride as a highly efficient antibacterial agent and broadens the utilization of agricultural waste spent mushroom substrate.


Introduction
Nanomaterials ranging in size from 1-100 nm have extensive applications in medicine, food, agriculture, chemical industry, environment, and other fields due to their excellent interface effect and quantum size [1,2].Currently, nanomaterials have been extensively applied in sustainable agriculture.It is considered a promising alternative or supplement to traditional chemical pesticides and biopesticides, which are problematic for their low efficiency, high environmental toxicity, and susceptibility to pathogen resistance [3].Most previous studies have focused on traditional nano-antibacterial agents such as nano-ZnO, nano-TiO 2 and nano-Ag [4], while few have focused on nano-AgCl.In previous studies, AgCl nanoparticles are widely used as a photographic material, catalyst, ionic semiconductor, and reference electrode due to their high stability and optical activity [5][6][7].Recently, more studies indicated that nano-AgCl exhibited high antibacterial activity against Escherichia coli and Staphylococcus aureus with a low minimum inhibitory concentration (MIC) [5,8].However, the anti-pathogenic ability and mechanism of nano-AgCl have yet to be comprehensively investigated.In recent years, most synthesis methods of nano-AgCl have been established, such as electrochemical deposition [9], surfactantassisted synthesis [10], ultrasound [11], electrospinning [12] and reverse microemulsion method [13].Among these, electrochemical deposition and surfactant-assisted synthesis of nanoparticles are pure and structurally controllable, but suitable electrochemical reactions and surfactants are significant problems.Nanoparticles prepared by ultrasound and electrospinning with good controllability exhibit high particle size accuracy and excellent dispersibility.However, they are limited by the high energy consumption and complex operation.
And the reverse microemulsion method involves complex procedures and high criteria for selecting the reaction system [13].Nanomaterials synthesized by most of these methods are relatively pure but expensive and lead to the production of toxic by-products.Thus, a green and simple one-step formation method of nano-AgCl is urgently needed.
Biosynthesis, which is environmentally friendly, refers to using microbes or plants for intracellular or extracellular synthesis.Nano-AgCl synthesized by microorganism extracts such as Rumex acetosa, yeast, and Bacillus subtilis exhibited prominent antioxidant activity against different pathogens [4,14].Likewise, nano-AgCl with antibacterial activity can be synthesized by plant extracts such as Sargassum plagiophyllum and needles of Azadirachta indica plant fruit [15,16].However, those biosynthesis methods are challenging to achieve industrially because of the high cost and limited sources.Therefore, synthesizing nano-AgCl with a lower cost should be further explored.Agricultural wastes with low cost and renewable characteristics include farm residues, livestock and poultry manure, agricultural processing waste, human manure, household waste, etc. [17].Agricultural wastes could be extensively used as resource materials in many engineering applications, such as feedstocks for cement production, composting, and raw materials for developing low-dielectric glass ceramics [18][19][20].As an agricultural waste, the amount of spent mushroom substrate (SMS) produced is about five times that of mushrooms during the growth of mushrooms [21], which causes severe water environmental pollution due to its low efficiency.Previous reports showed that SMS contains rich polysaccharides, lignin, flavonoids, etc., which are macromolecular or reducing substances that can reduce metal ions, enabling the formation of nanoparticles [22][23][24].The spent mushroom substrate is usually composed of mushroom mycelium, inorganic salts, large quantities of hemicelluellins, cellulose and lignin unused after mushroom culture, and extracellular lignin modifying enzymes secreted during substrate colonization [22,25], which suggests that it may contain inorganic anions such as acid ions.Moreover, mycelium metabolites (proteins, polysaccharides, enzymes, etc.) may play a role in surface-mediated and catalytic synthesis, helping improve the biocompatibility and performance of nanoparticles [25,26].Hence, agricultural waste synthesising nanomaterials efficiently holds excellent promises because of low cost and environmental friendliness.
So far, various prevention strategies have been developed to reduce bacterial wilt disease, such as selecting resistant varieties, applying chemical and biological controls.Even though these efforts help control this disease, an efficient and environmentally friendly control measure for most of the affected host crops is still needed because most methods require much labour and are time-consuming.Recently, nanomaterials have been applied to control and manage agricultural diseases.For example, it was reported that nano-TiO 2 as an efficient nanopesticide exhibited a strong antimicrobial effect against R. solanacearum and had no significant effect on tomato growth [27].Thus, we should take full advantage of nanomaterials' physical and chemical properties to resist pathogens in agriculture.Herein, we employed SMS to synthesize nano-AgCl as a high antibacterial agent and explored its antibacterial mechanisms by microscopic visualization, DNA injury and EPR analysis (as illustrated in figure 1).Our study provides new insights into the synthesis of nano-AgCl using SMS extract and develops an eco-friendly and highly efficient nanopesticide for tomato bacterial control.

Materials and methods
2.1.Preparation of SMS extract and nanomaterials synthesis R. solanacearum was kindly provided by the culture collection of the Plant Pathogenic Bacteriology Laboratory at the Fujian Agriculture and Forestry University.The SMS of Pleurotus geesteranus came from China National Engineering Research Center of JUNCAO Technology, Fujian Agriculture and Forestry University.SMS was removed from the polyethylene packaging and dried in an oven at 60 °C for 3 to 5 days.After that, SMS was finely milled to pass through a 60-mesh screen (0.42 mm) and stored at room temperature.Then, 30 g SMS powder was suspended in 180 ml double distilled water (ddH 2 O) and mixed in a shaker for 1 h.Subsequently, the mixture was autoclaved (121 °C) for 20 min and centrifuged to collect the supernatant.The supernatant was filtered by qualitative filter paper and two filter units (0.45 μm and 0.22 μm) in turn for further use.

Biological synthesis
Silver nitrate (AgNO 3 ) was dissolved in ddH 2 O to prepare a final concentration of 0.08 mol/L silver nitrate solution and then mixed with SMS extract obtained by hot water extraction in a volume ratio of 1.5:1.The mixture was stirred using a magnetic stirrer at 40 °C for 2 h.Subsequently, the reaction liquid was centrifuged and filtered to get precipitates, and it was dried overnight in an oven at 55 °C.

Chemical synthesis
AgCl particles were synthesized by direct precipitation with Ethylenediaminetetraacetic acid (EDTA) as a complexing agent.Firstly, 10 mmol L −1 ethylenediamine tetraacetic acid disodium (EDTA 2 Na) dissolved in deionized water was ultrasonically washed until completely dissolved.Then AgNO 3 was dissolved in deionized water to prepare a final concentration of 10 mmol L −1 and poured into the above EDTA 2 Na to mix.After adjusting the pH of the mixed solution with sodium hydroxide (NaOH) at a concentration of 0.1 mol / L, the solution was placed in a 25 °C thermostatic water bath for magnetic stirring for 2 h.Then, sodium chloride (NaCl) solution with a concentration of 10 mmol / L was dropped dropwise to obtain AgCl suspension.The obtained AgCl suspension was centrifuged and washed three times with distilled water and anhydrous ethanol, and the product was dried in an oven at 55 °C for 10 h [28].Both dried samples were ground into powder for subsequent use, respectively.

Characterization of nano-AgCl
The synthesized nanomaterials were identified using x-ray Diffraction (XRD) (Bruker, Germany) with a copper target at a 2θ angle of 20 ∼ 80°, and the average size of nano-AgCl is calculated by Scherrer equation.Further, the nanomaterials were measured via energy dispersive spectroscopy (EDS) (Hitachi, Japan).Then, the sample was characterized using UV-visible spectroscopy (UV-vis) recorded by UV1800PC (AUCY Scientific, China) under the 190 nm to 600 nm region.The Fourier transform infrared spectroscopy (FT-IR) measurements were recorded in the wave number range of 500-4,000 cm −1 using an FTIR-1500 Fourier Transform infrared spectrometer (Josvok, China).The sample processing steps are as follows: 1-2 mg sample and 200 mg pure KBr were ground and placed in a mould, and the mixture was pressed mechanically into sheets and placed in an oven to dry.Both sample and KBr should be dried and ground to a particle size of less than 2 μm to avoid scattering light.Subsequently, scanning electron microscope (SEM) (Hitachi, Tokyo, Japan, 200 kV) and transmission electron microscope (TEM) (JEM-1200EX, 120 kV) were used to characterize the general morphology and size distribution of nano-AgCl.After dispersing by ultrasound, the size and surface charge of nanoparticles were measured by Zeta Sizer Nano S (Malvern, United Kingdom).

2.3.
Inhibitory activity of nano-AgCl against agricultural pathogen R. solanacearum R. solanacearum was cultured in Nutrient Broth (NB) liquid medium (NaCl 3 g L −1 , bacterial peptone 5 g/l, yeast extract 2 g/l, sucrose 10 g L −1 , beef extract 6 g /L , p H 7.0-7.2) at 30 °C at 200 rpm for 24 h.Then, the bacteria were centrifuged at 8000 rpm for 3 min and the supernatant was decanted to collect the bacteria.The inhibition of R. solanacearum treated with nano-AgCl solution for 24 h was measured gradients (0-30.0mg/l).Subsequently, the 100 μl diluted cell suspensions treated with different concentrations of nano-AgCl were spread on NB solid medium and incubated for 48 h.All treatments were repeated three times, and the corresponding antibacterial rate was determined.The inhibition rate formula is as follows [29] N 1 is the number of bacterial colonies growing on the plate after being treated with ddH 2 O (blank control), and N 2 is the number of bacterial colonies growing on the plate after being treated with nano-AgCl of different concentrations.

Antibacterial efficacy comparison
The bacteria cultured in the NB liquid medium were centrifuged at 8000 rpm for 3 min, and washed once with ddH 2 O and phosphate buffer saline (PBS), respectively.Then the bacteria were treated with ddH 2 O, 1250 m g / L 2.5% zhongshengmycin, 7500 mg /L 20 % thiodiazole copper, 600 mg /L 20 % kasugamycin, 30 mg L −1 non nano-sized AgCl nanoparticles and 30 mg L −1 AgCl nanoparticles for 24 h, among the concentration of commercial pesticides referred to the pharmaceutical product description for field use concentration.After treatment of the above six kinds of reagents, the bacterial suspensions diluted by 10 6 times were spread on NB solid medium, and the number of bacterial colonies was counted after 48 h.The inhibition rate was calculated according to the formula in section 2.3.

Toxicological mechanism analysis 2.5.1. Live/Death assays and DNA damage
The solution in a microfuge tube containing equal volumes of SYTO ® 9 and propidium iodide (PI) was mixed thoroughly.Then, the mixture was applied to stain ddH 2 O-treated bacteria and 30.0 mg/l nano-AgCl-treated bacteria, and they were incubated at room temperature in the dark for 30 min.Soon, treated bacteria were centrifuged at 8000 rpm for 3 min and washed three times with sterile water.Subsequently 5 μl of the stained bacterial suspension trapped between a slide and an 18 mm square coverslip was observed in a confocal laser scanning microscope (FV1000 CLSM, OLYMPUS, Japan).The excitation/emission maxima of two dyes are about 480/500 nm for SYTO ® 9 and 490/635 nm for PI [30]. 2 ml of bacteria solution treated with ddH 2 O, 10 mg L −1 and 20 mg L −1 nano-AgCl for 24 h were centrifuged at 12000 rpm for 2 min to obtain the bacteria.Further the DNA of bacteria was extracted with TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit.DNA damage was analyzed in agarose gel by dissolving 0.20 g agarose in 20 ml of 1 × TAE buffer solution, accompanied by boiling and cooling.Then DNA electrophoresis was performed using DYY-6C agarose gel electrophoresis (Beijing, Liuyi, 150 V).

Zeta potential and SEM analysis
In order to remove the interference of the culture medium, the bacteria were redispersed and washed thoroughly with sterile water three times in advance and then centrifuged to obtain the bacteria.Subsequently, the incubated bacteria were dispersed in deionized water to detect the surface charge via a Zeta potential analyzer with high sensitivity.The measurement was repeated three times and averaged to obtain the Zeta potential.In addition, the bacterial solution treated with ddH 2 O and 30.0 mg/l nano-AgCl for 24 h, respectively, was centrifuged at 8000 rpm for 3 min and then washed three times with PBS.After that, the bacteria were fixed with a 2.5% glutaraldehyde solution at 4 °C for 12 h, and further immobilized with 1% osmotic acid, and dehydrated in acetone.Two hours later, the bacteria were observed by scanning electron microscopy.

The determination of Reactive oxygen species (ROS)
The production of ROS in bacteria was measured by the sensitive dye 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA).The bacteria treated with ddH 2 O and 30.0 mg/l nano-AgCl were cultured at 30 °C for 24 h and then centrifuged at 8000 rpm for 3 min.Further, the bacteria treated with ddH 2 O, nano-AgCl, and Rosup (positive control) were added with 10 μmol/L DCFH-DA and incubated at 37 °C with shaking at 200 rpm for 30 min.Subsequently, these samples were centrifuged at 8000 rpm for 3 min and washed three times with ddH 2 O.Then, 5 μl for each sample was prepared for observation under a laser confocal microscope (excitation wavelength 448 nm/emission wavelength 525 nm).

Electron paramagnetic resonance (EPR) assays
In-depth, the ROS produced by the samples were measured using EPR spectrometer (Bruker, Germany) with a 100 W mercury lamp as a light source (at the wavelength of 265-800 nm) at room temperature.5, 5-dimethylpyrroline 1-oxide (DMPO) and 4-Oxo-2,2,6, 6-tetramethyl-1-piperidinyl (TEMP) as two spin trapping agents were added to R. solanacearum, nano-AgCl, and nano-AgCl-treated bacterial suspensions, respectively.Herein, the concentration of two trapping agents is 50 mmol L −1 and nano-AgCl is 30 mg L −1 .The EPR spectra data was recorded during irradiation to detect the type of ROS produced.

Statistical analysis
All data were expressed as mean ± standard deviation (SD).The results were analyzed by Origin 2018 and Image J, followed by SPSS V26 via one-way analysis of variance (ANOVA) and Duncan's test.A P-value of <0.05 was considered statistically significant.
Further, the EDS of the sample powder showed strong signals at 0.28 keV and 2.8 keV (Cl), 3.0 keV (Ag) (figure 3(a)), which conformed to the characteristic peaks of silver chloride nanocrystallites.However, other impurity peaks might be caused by the preparation method and the deposition of complex components in the SMS on the surface of nano-AgCl [26,32].According to the UV analysis, the synthesis nanoparticles have two absorption peaks at 200 nm and 255 nm (figure 3(b)).Previous studies reported that the absorption of nanosilver chloride is 200-350 nm, while nano-silver absorbs at 390-550 nm [4,33,34].The blue shift of absorption peak may be due to the following reasons: (i) SMS is rich in phytochemicals such as polysaccharides and flavonoids [35].The polysaccharide plays a role as a nano-bioreactor, in which the silver chloride is precipitated in the form of nanoparticles [34].The results combined with EDS also showed the presence of impurity atoms such as carbon and oxygen.These compounds mostly contain UV-absorbing functional groups such as hydroxyl and ketone.Therefore, the absorption may be due to the adhesion of polysaccharides, complexes and other impurities on the surface of the nanoparticles [5,36,37].(ii) The average size of nanoparticles synthesized in our study is only 8.30 nm.Reports showed that the surface plasmon resonance (SPR) absorption band is largely affected by the shape and size of the nanoparticle and the dielectric constant of the surrounding medium [36].Some researchers reported a blue shift of SPR peak, and the particle size decreased with the increase of plant extract [38].Also, the high sensitivity of SPR to changes in the surrounding medium could be the reason for the wide variations of absorption bands.iii) The amount of synthesized nano-silver chloride is related to the silver and chloride ions concentration.When chloride ions are consumed in the reaction system, the remaining silver ions also affect the position of the UV absorption peak [34,36].In addition, FT-IR analysis represented the occurrence of O-H stretching vibration (3421.09cm −1 ), C=C stretching vibration (1596.59cm −1 ), and C-O phenolic compounds (1039.37 cm −1 ) [24, 39] (figure 3(c)).The presence of these groups may be responsible for the shift of the UV-vis absorption peak of nano-AgCl.Among them, the -OH group with reducibility and polarity might be the cause of the biological conversion of Ag ions into silver chloride nanoparticles by avoiding the direct reduction of silver ions to silver nanoparticles first, and helping the nanoparticles disperse in the water phase [40].
Moreover, SEM and TEM observations demonstrated that the synthesized nano-AgCl powder had a nearspherical shape, and its size distribution was 4.28 ± 2.80 nm (figures 3(d)-(f)).The wide dispersity of AgCl particle size might be due to process parameters (reactant concentration, temperature, etc.) and uneven distribution of complex substances such as proteins, amino acids and carbohydrates in the SMS extract.The uneven distribution of these substances in the solution may lead to different reaction processes, causing the different sizes of synthesized nanoparticles [26,41].The polydispersity of nanoparticle size could lead to different numbers of nanoparticles penetrating cells, resulting in poor reproducibility of the antibacterial effect of nano-AgCl.However, the size of the nano-powder dispersed in water was found to be approximately 1100.51 nm, suggesting that there was a particular agglomeration phenomenon.It is worth noting that nano-AgCl in this study was characterized by smaller particle size and more uniform shape distribution compared with the other methods to synthesize nano-AgCl.For instance, Gopinath V et al and Aletayeb P et al produced near-spherical nano-AgCl with a particle size range of 10-19 nm via Cissus quadrangularis extract and Nostoc sp [5,42].Moreover, nano-AgCl with a particle size of 80 nm was synthesized using a chemical method [43].Furthermore, the small size effect and surface effect may be better for future applications.
Due to their rich composition, SMS has been used in many fields, such as feed additives, plant fertilizers, energy supply, pollutant treatment, secondary culture fermentation, and plant disease control [35].Currently, technologies such as Py-GC/MS analysis [35] and pyrolysis [44] are applied to analyze the composition of complex SMS supernatants.SMS is rich in organic matter and inorganic salts, in which organic matter contains biological macromolecules such as polysaccharides, lignin, proteins, etc.These substances that act as reducing or capping agents can induce silver nitrate to obtain nano-AgCl.At present, studies have reported that a variety of polysaccharides can stabilize Ag + to inhibit the production of large-size silver chloride [45], and we speculate that the Cl -in the synthesis of nano-AgCl might come from the inorganic salts and mycelium metabolites produced during the growth of mushrooms [22,25].In addition, due to the presence of reducing substances such as phenolic acids and flavonoids in the SMS [23,24,39], some silver ions could also be reduced to silver nanoparticles.However, the primary generation of nano-AgCl may be related to the amount of each substance And the further formation mechanism remains to be explored.

Inhibitory effect of nano-AgCl on R. solanacearum and antibacterial efficacy comparison
The concentration of nanomaterials showed a positive correlation with its antibacterial effect, namely higher concentrations resulting in increased inhibition of R. solanacearum.The inhibitory rate could exceed 90% when the nanoparticle concentration was 30.0 mg/l and the EC 50 value was 5.18 mg L −1 , which showed that nano-AgCl had a strong ability to inhibit the growth of agricultural pathogens (figures 4(a) and (b)).The increased Ag ions released by nano-AgCl might be driven by the intracellular and extracellular concentration difference to enter the cell readily, leading to a better antibacterial effect.Moreover, the extent of cell membrane damage and change in membrane permeability could be influenced by the concentration of Ag ions capable of penetrating the cell, followed by inducing damage at the molecular level, such as peroxidation and DNA damage [4,46].
Moreover, the commonly used anti-bacterial wilt pesticides were applied to compare the antibacterial effect of nano-AgCl in this study.The results showed that the level of inhibition rates of various antibacterial agents were Kasugamycin ≈ AgCl NPs > Thiodiazole copper > Non nano-sized AgCl particles > Zhongshengmycin (figures 4(c) and (d)).Both kasugamycin and nano-AgCl, along with the inhibition rates of over 90%, showed prominent antibacterial effects against R. solanacearum.Next, compared with nano-AgCl synthesized by chemical method, the nanoparticles synthesized by this method have stronger activity against R. solanacearum (figures 4(e) and (f)).The biosynthetic nanoparticles with smaller size could be more conducive to entering into the organism to play an antibacterial role.And raw materials of biosynthesis can provide extremely rich reaction precursors and catalysts, realizing self-assembly or hierarchical self-assembly of nanomaterials with good biocompatibility [26,31,47], which can avoid the problems of toxicity in traditional chemical synthesis.Hence, it is evident that biosynthetic nano-AgCl possesses a significant advantage in its antibacterial efficacy.
Compared with commercial pesticides and non-nano-sized AgCl, it was found that synthetic nano-AgCl has significant antibacterial properties at low doses, which helps to improve drug resistance and pesticide In terms of antibacterial mechanism, zhongshengmycin and kasugamycin mainly inhibit the protein synthesis of bacteria [48,49].And thiodiazole copper acts through thiazole groups and copper ions, the two types of ions can bind with proteins on the cell membrane and some intracellular enzymes, leading to the dysfunction of bacteria [50].It has been reported that silver ions can cause damage to cell membranes and DNA and destroy the microbial electron transport system, respiratory system and material transport system [46], thereby inhibiting cell growth and reducing ATP synthesis.

Antibacterial mechanism of nano-AgCl
The toxicity and antimicrobial properties of nanomaterials rely on their particle size, surface, shape, composition, solubility and some other factors [29].The antimicrobial properties of the same nanomaterials synthesized by different biological methods differ.In order to further determine the antibacterial mechanism of nano-AgCl, we applied fluorescence and electron microscopy to observe the bacteria treated with ddH 2 O and nano-AgCl (30 mg L −1 ), respectively, to evaluate the activity of bacterial cell membranes.SYTO ® 9 is highly permeable and can penetrate both living and dead cells, but PI does not readily pass through cell membranes and can only stain dead cells.In addition, due to the high molecular weight of PI, the green fluorescence caused by SYTO ® 9 is inhibited, and the red fluorescence is enhanced under a CLSM observation [51].As a result, living cells show green fluorescence, while dead cells show red fluorescence.In this study, the bacteria treated with ddH 2 O showed strong green fluorescence under a fluorescence microscope, which indicated the integrity and vitality of cell membrane.However, the bacteria treated with 30 mg L −1 nano-AgCl showed both green and red fluorescence, suggesting that the bacteria died and the cell membrane was destroyed (figures 5(a) and (b)).DNA damage may also significantly affect the synthesis of some enzymes within cells.Gel electrophoresis demonstrated that the DNA band of bacteria treated with nano-AgCl were narrower and more blurred than that treated with ddH 2 O, and it implied that nano-AgCl could cause a missing fragment or an open double strand of DNA (figure 5(c)).All these findings indicate that nano-AgCl has a strong inhibitory effect on bacterial growth.
By SEM observation, it was found that bacteria treated with ddH 2 O were relatively round and full (figures 5(d) and (e)).In contrast, the cells treated with nano-AgCl showed abnormal phenomena such as flat, twisted, irregular, etc. (figures 5(f) and (g)).It was speculated that the nanomaterials may change the permeability of the cell membrane, thus resulting in the seepage of some substances and affecting the survival and growth of bacterial cells [52].Furthermore, the Zeta potential of R. solanacearum and nano-AgCl was measured, and it was found that both bacteria and nano-AgCl carried negative charges (figure 5(h)).Based on the result of Zeta potential, we speculated that the combination of nano-AgCl and bacteria could be driven by intermolecular forces [24,53] or attraction between silver ions (released by nano-AgCl) and cell membranes (with a negative charge), leading to severe damage to cell intact and bacterial DNA.
DCFH-DA is a fluorescent probe extensively used to detect ROS content.It can enter cells and was hydrolyzed into dichlorodihydro-fluorescein (DCFH) under the action of intracellular enzymes, and DCFH combines with the generated ROS to produce 2′,7′-dichlorofluorescein (DCF).DCFH-DA does not induce fluorescence, while DCF exhibits green fluorescence [54].Here, it was observed by CLSM that the bacteria treated with ddH 2 O showed no fluorescence, while the bacteria treated with nano-AgCl and Rosup showed green fluorescence (figure 6(a)), indicating that both induced intracellular oxidative stress reactions and produced ROS.The production of ROS may be a significant cause of DNA damage, cell wall rupture and membrane permeability change [4].Further, the species of ROS was detected via EPR spectrum based on the transition of unpaired electrons in a magnetic field, and it was found that the R. solanacearum without nano-AgCl treatment and nano-AgCl did not produce ROS.In contrast, the tested R. solanacearum exposed to nano-AgCl showed significant production of ROS (•OH, 1 O 2 and •O 2-) (figure 6(b)), revealing that nano-AgCl could cause an increase in the production of ROS within bacterial cells.The result was also consistent with the above findings.
In the present study, through more profound mechanism exploration, we found that nano-AgCl can result in cell membrane damage and morphological abnormalities, bacterial DNA disruption, and ROS generation.In more severe cases, it can even cause cell death.And we speculated that nano-AgCl could contact with bacteria through the release of Ag + , triggering the occurrence of cellular stress reaction and leading to peroxidation damage.In-depth, this might impact ribosomal subunit protein and the activity of respiratory chain enzymes for bacterial cell growth [55,56].Moreover, it was reported that metallic nanomaterials interacting with functional groups such as SH in cell wall proteins can affect the function of the cell wall [57].

Conclusion
In summary, nano-AgCl with an average particle size of 8.30 nm and a negative charge was innovatively synthesized from SMS water extract, which is different from most of the traditional biosynthesis methods using microorganisms, plants or other reductive substances as raw materials to synthesize nanomaterials.Nanoparticles synthesized by this approach exhibited prominent antibacterial activity against R. solanacearum, whose EC 50 of 5.18 mg L -1 is smaller than other commercially available pesticides.Additionally, a more indepth investigation into the antibacterial mechanism of nano-AgCl revealed that it could cause DNA damage and ROS production, resulting in cell physiological activities blocked, bacterial deformation or even cell death.This work provides a green and facile method to synthesize high-antibacterial activity nano-AgCl and broadens the application of SMS and nanomaterials in agriculture.However, the complexity of biological resources used in biosynthesis might cause various impurities, such as silver nanoparticles.Moreover, it should be noted that biosynthesized nanomaterials bring problems of wide particle size distribution and uneven particle size.Therefore, further improving the purity and particle size uniformity of nanoparticles is needed to be studied in future.In addition, the safety assessment of biosynthesized nanomaterials should also be worthy focusing on continuously.Agriculture and Forestry University Construction Project for the Technological Innovation and Service System of the Tea Industry Chain (K1520005A03).

Figure 1 .
Figure 1.(a) Composition of spent mushroom substrate, (b) synthesis scheme of nano-AgCl, and (c) illustration of antibacterial mechanism of nano-AgCl on R. solanacearum.

Figure 4 .
Figure 4. Bacterial culture plates and inhibition rates, showing antibacterial activity of AgCl NPs at various concentrations (a and b); Comparison of the bacterial culture plates and inhibition rates of non-nano-sized AgCl particles, commercially available pesticides (c and d) and chemically synthesized nano-AgCl (e and f).(CK refers to the control group treated with the same volume of ddH 2 O).

Figure 5 .
Figure 5. Bacterial viability (a and b), DNA damage by nano-AgCl (c), and SEM images of R. solanacearum treated with ddH 2 O (d) and (e) and 30 mg l −1 nano-AgCl (f) and (g); Zeta potential of R. solanacearum and nano-AgCl (h).