Synergistic Action of Biosynthesized Silver Nanoparticles and Culture Supernatant of Bacillus amyloliquefacience against the Soft Rot Pathogen Dickeya dadantii

Nanomaterials are increasingly being used for crop growth, especially as a new paradigm for plant disease management. Among the other nanomaterials, silver nanoparticles (AgNPs) draw a great deal of attention because of their unique features and multiple usages. Rapid expansion in nanotechnology and utilization of AgNPs in a large range of areas resulted in the substantial release of these nanoparticles into the soil and water environment, causing concern for the safety of ecosystems and phytosanitary. In an attempt to find an effective control measure for sweet potato soft rot disease, the pathogen Dickeya dadantii was exposed to AgNPs, the cell-free culture supernatant (CFCS) of Bacillus amyloliquefaciens alone, and both in combination. AgNPs were synthesized using CFCS of Bacillus amyloliquefaciens strain A3. The green synthesized AgNPs exhibited a characteristic surface plasmon resonance peak at 410–420 nm. Electron microscopy and X-ray diffraction spectroscopy determined the nanocrystalline nature and 20–100 nm diameters of AgNPs. Release of metal Ag+ ion from biosynthesized AgNPs increases with time. AgNPs and CFCS of B. amyloliquefaciens alone exhibited antibacterial activity against the growth, biofilm formation, swimming motility, and virulence of strain A3. The antibacterial activities elevated with the elevation in AgNPs and CFCS concentration. Similar antibacterial activities against D. dadantii were obtained with AgNPs at 50 µg·mL−1, 50% CFCS alone, and the combination of AgNPs at 12 µg·mL−1 and 12% CFCS of B. amyloliquefaciens. In planta experiments indicated that all the treatments reduced D. dadantii infection and increased plant growth. These findings suggest that AgNPs along with CFCS of B. amyloliquefaciens can be applied to minimize this bacterial disease by controlling pathogen-contaminated sweet potato tuber with minimum Ag nano-pollutant in the environment.


Introduction
The application of nanoscale-size materials possessing no less than one dimension in the 1 to 100 nanometers range is a newly emerging area in nanoscience and technology. The nanomaterials display exceptional physical, chemical, and electrical properties for their extreme surface-to-volume ratio [1]. The extensive use of nanoparticles raises concern about their environmental impact, but the impact of NPs on ecological processes and organisms where D represents the median crystalline nanoparticles size, K (0.94) represents the Scherrer constant, λ (0.15406 nm) represents the wavelength of X-ray, β represents the full breadth at half largest of the X-ray diffraction peak, and θ represents the Bragg angle.
Transmission (TEM) electron microscopy and scanning (SEM) electron microscopy FTIR spectrometry revealed the functional groups in the CFCS of B. amyloliquefaciens A3 that cause the reduction of Ag + , stabilization, and AgNPs capping. The FTIR spectrum of the synthesized AgNPs exhibited absorption peaks at 3354, 2925, 1651, 1574, 1395, 1084, 975, 805, and 533 cm −1 (Figure 1b). The peak at 3354 cm −1 is for N-H stretching vibration; the peak at 2925 cm −1 corresponds to C-H stretching vibration and N-H stretching of the amide group; the peak at 1651 cm −1 corresponds to -C=O and C=C stretching vibrations; the band at 1574 cm −1 is attributed to C=C stretching vibration; the peak at 1395 cm −1 is attributed to CH 3 or C-H bending; the peak at 975 cm −1 corresponds to C=C bending and C-H vibration; and the peaks at 1084 cm −1 , 805 cm −1 , and 533 cm −1 are for C=O stretching, C-H bending, and C-C skeleton vibration, respectively. The existence of these functional groups confirmed the presence of protein in the CFCS of B. amyloliquefaciens and also indicated that these groups act as reducing and stabilizing agents in the synthesis process of AgNPs [43,44]. Earlier reports showed that free amine groups as well as cysteine residue in proteins can combine with nanoparticles and stabilize the AgNPs [45].
The X-ray diffraction patterns revealed the nanocrystalline nature of synthesized AgNPs. Based on the Bragg reflection peak at 2θ, the values of 27.86, 32.27, 46.22, 54.86, and 76.74 correspond to planes (101), (111), (200), (220), and (311), respectively (Figure 2a). The four reflection peaks are correlated with the crystallographic nature of the centering face and the cubic nature of AgNPs, which was confirmed with a comparison to the powder diffraction card of the Joint Committee on Powder Diffraction Standard File No. JCPDS 04-0783. The average particle diameter of AgNPs is 50.3 nm, which was calculated using the Debye-Scherrer equation: where D represents the median crystalline nanoparticles size, K (0.94) represents the Scherrer constant, λ (0.15406 nm) represents the wavelength of X-ray, β represents the full breadth at half largest of the X-ray diffraction peak, and θ represents the Bragg angle.

Metal Ag Release
The metal release data indicated that biosynthesized AgNPs significantly release Ag + ions in ddH20 during every time interval (Figure 4). After 8 h, a quick initial release of Ag + ions from the biosynthesized AgNPs was observed, which then attained a persistent value. Biosynthesized AgNPs released the most Ag + (2.25) after 24 h. Previous studies found that there is a correlation between the suppression of disease and the ion release profile [49]. Moreover, components of different media have a specific function in releasing metal ions from nanoparticles. Transmission (TEM) electron microscopy and scanning (SEM) electron microscopy were used to confirm the AgNP sizes that were synthesized from the CFCS of B. amyloliquefaciens. The TEM images ( Figure 2b) showed that most of the AgNPs had uniform size, were mono-dispersed, and about 20-100 nm in diameter with spherical shape and an average mean size of 48.5 nm, which were confirmed with the SEM images ( Figure 3a). These results are consistent with previous studies [35,43].

Metal Ag Release
The metal release data indicated that biosynthesized AgNPs significantly release Ag + ions in ddH20 during every time interval (Figure 4). After 8 h, a quick initial release of Ag + ions from the biosynthesized AgNPs was observed, which then attained a persistent value.  Energy dispersive spectroscopy further confirmed the dominance of metal silver (Ag 0 ) in the synthesized AgNPs. The typical absorption peak of the Ag element was exhibited by AgNPs at 3 KeV (Figure 3b), which confirmed that nano-sized silver was in a pure form, as earlier reports have shown [35,38,41,[46][47][48]. Spectroscopy showed 82.32 wt% Ag 0 along with 17.07 and 0.61 wt% of Cl and Al, respectively.

Metal Ag Release
The metal release data indicated that biosynthesized AgNPs significantly release Ag + ions in ddH 2 0 during every time interval (Figure 4). After 8 h, a quick initial release of Ag + ions from the biosynthesized AgNPs was observed, which then attained a persistent value. Biosynthesized AgNPs released the most Ag + (2.25) after 24 h. Previous studies found that there is a correlation between the suppression of disease and the ion release profile [49]. Moreover, components of different media have a specific function in releasing metal ions from nanoparticles.

Metal Ag Release
The metal release data indicated that biosynthesized AgNPs significantly release Ag + ions in ddH20 during every time interval (Figure 4). After 8 h, a quick initial release of Ag + ions from the biosynthesized AgNPs was observed, which then attained a persistent value. Biosynthesized AgNPs released the most Ag + (2.25) after 24 h. Previous studies found that there is a correlation between the suppression of disease and the ion release profile [49]. Moreover, components of different media have a specific function in releasing metal ions from nanoparticles.

Antibacterial Activity of the Combination of CFCS and AgNPs against D. dadantii
AgNPs (50 µL), CFCS (50%), and the combination of AgNPs (12 µL) and CFCS (12%) exhibit a similar extended of inhibition of growth and swimming motility against D. dadantii strain A3 and tissue maceration in sweet potato. The inhibition of in vivo tissue maceration using tuber slices was in general agreement with the result of in vitro bacterial growth, biofilm formation, and swimming motility.
In sweet potato tubers, D. dadantii created a maceration zone by degrading plant cell walls in the sweet potato slice. One day after inoculation, the maceration zone was about 32 mm (Table 4). The combination of AgNPs (12 µL) and CFCS (12%) significantly inhibited (65%) tissue maceration, whereas AgNPs (50 µL) and CFCS (50%) alone showed a similar extent of inhibition.
TEM also revealed the morphological changes in D. dadantii cells after treatment with the combination of AgNPs (12 µL) and CFCS (12%). After 4 h of treatment, most D. dadantii cells were dead, as indicated by distorted cells with disintegrated cytoplasm and cell walls (Figure 5c,d). On the other hand, D. dadantii cells grown without treatment had intact cell walls with dense cytoplasm filled in the cell (Figure 5a,b).
TEM also revealed the morphological changes in D. dadantii cells after treatment with the combination of AgNPs (12 µL) and CFCS (12%). After 4 h of treatment, most D. dadantii cells were dead, as indicated by distorted cells with disintegrated cytoplasm and cell walls (Figure 5c,d). On the other hand, D. dadantii cells grown without treatment had intact cell walls with dense cytoplasm filled in the cell (Figure 5a,b). During the in planta experiment, D. dadantii was inoculated into sweet potato seed tubers, and due to the infection, the tubers became rotten. D. dadantii was then recovered from the rotten inoculated tubers. Seed tubers treated with water germinated and became small seedlings 21 days post-inoculation. On the other hand, seed tubers treated with AgNPs (50 µg·mL −1 ) from the CFCS of B. amyloliquefaciens A3, the CFCS of B. During the in planta experiment, D. dadantii was inoculated into sweet potato seed tubers, and due to the infection, the tubers became rotten. D. dadantii was then recovered from the rotten inoculated tubers. Seed tubers treated with water germinated and became small seedlings 21 days post-inoculation. On the other hand, seed tubers treated with Ag-NPs (50 µg·mL −1 ) from the CFCS of B. amyloliquefaciens A3, the CFCS of B. amyloliquefaciens A3 (50%), and the combination of AgNPs (12 µg·mL −1 ) and CFCS (12%) germinated and became seedlings with a greater height than the seedlings grown from the water-treated seed tubers (Table 5). The fresh weight and dry weight of seedlings grown from sweet potato seed tubers treated with AgNPs (50 µg·mL −1 ) from the CFCS of B. amyloliquefaciens A3, the CFCS of B. amyloliquefaciens A3 (50%), and the combination of AgNPs (12 µg·mL −1 ) and CFCS (12%) were higher than the fresh and dry weight of the seedlings grown from water-treated seed tubers (Table 5). Table 5. Seedling height, fresh and dry weight of seedlings raised from sweet potato seed tubers treated with water, D. dadantii, D. dadantii with the CFCS of B. amyloliquefaciens A3 (50%), AgNPs (50 µg·mL −1 ) from the CFCS of B. amyloliquefaciens A3, or the combination of AgNPs (12 µg·mL −1 ) and CFCS (12%).
AgNPs may transport Ag + more efficiently to the bacterial membrane and cytoplasm [50]. Ag + release from the 20-80 nm silver nanoparticle and subsequent pene- tration to the bacterial cell membrane might be the primary cause for the antibacterial action of AgNPs [50][51][52][53]. The CFCS of B. amyloliquefaciens-derived AgNPs were about 20-100 nm in width and inhibited the growth, biofilm formation, and swimming motility of D. dadantii through attachment to surfaces of bacterial cells, which may release toxic Ag + and damage the cell membrane. After penetration into the D. dadantii cells, AgNPs may generate oxidative stress and interact with the sulfur present in the membrane proteins and the phosphorous remaining in the DNA to disrupt the respiratory chain and cell division, which lead to cell death [53,54]. The antimicrobial actions of AgNPs are mainly due to the release of Ag + , and the release of Ag + even in very low concentrations can account for the biological response [50].
On the other hand, Bacillus spp. are known to be active against a large range of phytopathogenic bacteria through competition with phytopathogens and antibiosis by producing antibiotics and metabolites. As a biocontrol agent, the main attribute of Bacillus spp. is their various secondary metabolites and capability to produce a wide range of structurally various antimicrobial substances [15]. In comparison with other isolates, 8% of the whole genome of plant-associated B. amyloliquefaciens strain FZB42 is involved in the production of secondary metabolites such as bacteriocins, antimicrobial peptides and lipopeptides, polyketides, and siderophore with microbial activities [55,56]. A higher concentration of surfactin was observed in the CFCS of B. amyloliquefaciens strain A3 among the effective strain against D. dadantii [37]. Surfactins belonging to lipopeptides with strong antibacterial activities [14,57] may play a key role in the inhibition of growth and biofilm formation of D. dadantii. Previous studies showed that silver nanoparticles synthesized with the incubation of the CFCS of Bacillus amyloliquefaciens strain A3 also exhibited antibacterial activities [35,38]. Selection of Bacillus amyloliquefaciens strain A3 can be used for the production of CFCS and the synthesis of AgNPs, which avoids the screening of bacteria and can be used as a reference strain for the synthesis of AgNPs.
In conclusion, we provide a simple, environmentally safe, and economically viable control strategy to combat the bacterial pathogen Dickeya dadantii using a combination of AgNPs and CFCS. Dickeya dadantii-infected sweet potato can be treated with the combination of AgNPs (12 µg) and CFCS (12%) to produce a healthy seed crop. This can minimize the labor required to produce a large amount of CFCS and reduce the chance of releasing Ag + from AgNPs. The combination of AgNPs and CFCS acts as a Dickeya dadantii inhibitor and growth promoter for the plant. The promising potency of the combination of AgNPs and cell-free culture supernatant can draw immense research interest in the production of green-synthesized nano pesticides.

Bacteria
The pathogenic strain CZ1501 of D. dadantii was isolated from infected sweet potato stems cultivated in Hangzhou of Zhejiang province, China, and the biocontrol strain B. amyloliquefaciens strain A3 isolated from rice seeds [58] were used in this study. Both bacteria were grown in nutrient agar or broth medium consisting of 10 g of tryptone, 2.5 g of glucose, 5 g of NaCl, and 3 g of beef extract with or without 15 g of agar per liter, respectively, at pH 7.0. 3 mM AgNO 3 were added together and used as the control. AgNPs synthesis was observed when the solution changed from a light yellow to dark brown color. After adding 2 mL of the dark brown solution with 2 mL of Milli-Q water, the UV-Visible spectra were observed at 1 nm resolution in the range of 200-750 nm using a Simadzu UV-2550 spectrometer (Shimadzu, Kyoto, Japan). AgNPs were obtained from the dark brown solution with centrifugation at 27,200× g for 10 min followed by washing two times with Milli-Q water, after which they were freeze-dried with Alpha 1-2 LD plus (GmbH, Marburg, Germany). The freeze-dried AgNPs were weighed, and a stock solution of 50 mg.mL −1 was prepared by dissolving AgNPs with Milli-Q water.

Characterization of AgNPs Synthesized with CFCS with B. amyloliquefaciens
The functional groups for the biosynthesis and stabilization of AgNPs were confirmed using Fourier transforms infrared (FTIR) spectroscopy. In detail, 1 mg of freeze-dried AgNP powder was mixed with 300 mg KBr, and thin pellets were made using a hydraulic pellet press. FTIR spectrum was documented with an AVATAR 370 FTIR spectrometer (Thermo Nicolet, Madison, WI, USA) in the range between 500 and 4000 cm −1 with a 4 cm −1 resolution. FTIR spectra were interpreted using the table of Bruker infrared (Bruker Optics Inc., Billerica, MA, USA) and the absorption table of Libre Texts infrared spectroscopy (https://chem.libretexts.prg/) accessed on 8 June 2018.
To observe the AgNP size and morphology, SEM and TEM were used. One drop of the AgNPs solution was applied onto the carbon-coated copper grid and then dried under a mercury lamp. The SU8010 field emission scanning electron microscope (Hitachi, Tokyo, Japan) and a JEM-1230 transmission electron microscope (JEOL, Tokyo, Japan) were used to observe the grid carried in AgNPs. An X-Max N energy dispersive spectrometer (Oxford Instruments, Oxford, UK) was used to detect the silver element in AgNPs at 20 keV.
The X-ray diffraction pattern was used to understand the crystallographic structure of AgNPs. A coated film of freeze-dried AgNP powder was kept on the glass slide and analyzed using a D8 Advance Diffractometer (Bruker, Karlsruhe, Germany) maintaining an operational condition of 40 kV and 40 mA with a Cu-Kα radiation ranging from 20 • to 80 • at the 2θ angle.

Experiments on Ag Release
The metal Ag release from the biosynthesized AgNPs was calculated in sterile ddH 2 0 according to the previously described method [59] with a few modifications. In brief, suspensions were prepared using 25 µg·mL −1 AgNPs in ddH 2 0 with three replications, and the pH was adjusted to 7. The suspensions were incubated at 26 ± 2 • C under continuous shaking at 150 rpm. After 2, 4, 8, 16, and 24 h time intervals, 1 mL aliquot was taken from each suspension and centrifuged at 13,000× g to discard NPs. The acquired supernatants were digested by applying HClO 4 :HNO 3 (1:3), and the Ag content was analyzed using inductively coupled plasma-mass spectrometry (ICP-MS; Optima 8000DV, Pekin-Elmer, Waltham, MA, USA). The quality of the investigative data was ensured by running a standard suspension containing 100 µg·mL −1 continually in each of the 5 samples.

Antibacterial Assay
D. dadantii CZ1501 was grown in nutrient broth at 30 • C with shaking at 200 rpm to the mid-exponential phase, and the concentration of bacterial cells was adjusted to about 5 × 10 8 CFU·mL −1 before use.
Antibacterial activities of AgNPs synthesized with the CFCS of B. amyloliquefaciens strain A3, the CFCS of B. amyloliquefaciens strain A3, and the combination of AgNPs with CFCS were determined in the nutrient broth. Briefly, 5 mL of nutrient broth was inoculated with 100 µL D. dadantii suspensions and used as the control. AgNPs from B. amyloliquefaciens strain A3 were adjusted to concentrations of 12, 25, and 50 µg·mL −1 by adding nutrient broth. Furthermore, 100 µL D. dadantii suspensions were inoculated into 5 mL of nutrient broth containing AgNPs. D. dadantii suspensions (100 µL), NA broth (2.4 mL), the CFCS of B. amyloliquefaciens A3 (0.6 mL or 1.25 mL or 2.5 mL), and sterile distilled water (1.9 mL or 1.25 mL or 0 mL) to a total volume of 5 mL were added into a glass tube to obtain final concentrations of 12, 25, and 50% CFCS. D. dadantii suspensions (100 µL), NA broth (2.4 mL), the CFCS of A3 (0.6 mL or 1.25 mL or 2.5 mL), and sterile distilled water (1.9 mL or 1.25 mL or 0 mL) were mixed, and AgNPs were added with nutrient broth consisting of CFCS to obtain concentrations of 12, 25, and 50 µg·mL −1 . The samples were then incubated at 30 • C and 200 rpm for 24 h. After that, the optical density of the samples was measured at 600 nm (OD 600 ) using a SpectraMax spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). This experiment was repeated three times with three replications of each treatment.
The effect of 50 µg·mL −1 synthesized AgNPs from B. amyloliquefaciens strain A3, 50% of the CFCS of B. amyloliquefaciens strain A3, and the combination of AgNPs (12 µg·mL −1 ) and CFCS (12%) on the swimming motility of D. dadantii strain CZ1501 was determined with semi-solid nutrient agar [0.3% (w/v)]. A plate without AgNPs and CFCS served as the control. AgNPs were added to semisolid nutrient agar [0.3%, w/v)] to a concentration of 50 µg·mL −1 . Equal amounts of the CFCF of B. amyloliquefaciens strain A3 and semisolid nutrient agar [0.6% (w/v)] were mixed to obtain a 50% CFCS concentration. AgNPs and CFCS were added to the semi-solid nutrient agar to obtain a final concentration of 12 µL AgNPs and 12% of CFCS. In the center of each swimming plate, a suspension (5 µL) of D. dadantii was spotted and incubated for 48 h at 30 • C. The diameter of D. dadantii colonies was measured. The experiment was repeated three times with three replicates of each treatment.
The inhibition of biofilm formed by D. dadantii strain CZ1501 using AgNPs (50 µg·mL −1 ) from B. amyloliquefaciens strain A3, CFCS of B. amyloliquefaciens strain A3 (50%), the combination of AgNPs (12 µg·mL −1 ) and CFCS (12%) was determined using the method of crystal violet staining, which was quantified by measuring the absorbance at 590 nm (OD 590 ) using a 96-well microtiter plate. AgNPs were added with nutrient broth to obtain a concentration of 50 µg·mL −1 . AgNPs were added with nutrient broth consisting of 12% CFCS to a concentration of 12 µg·mL −1 . An amount of 100 µL D. dadantii suspensions was mixed with 100 µL of the CFCS of B. amyloliquefaciens A3 or nutrient broth composed of 50 µg·mL −1 AgNPs (100 µL) or nutrient broth containing 12 µg·mL −1 AgNPs along with 12% CFCS (100 µL). The mixtures were independently added to the wells of a 96-well microplate. Furthermore, a 100 µL of D. dadantii suspension added with 100 µL of distilled water was used as the control, whereas 100 µL of nutrient broth with 100 µL of distilled water was used as the blank. The plates were then incubated without shaking at 30 • C for 24 h. The liquid from each well was discarded and gently washed three times using distilled water followed by 1 h of air drying. In addition, 200 µL crystal violet (1%, w/v) was used to stain the bacterial cells that attached to the wells. A thorough washing was performed after incubation for 30 min at room temperature using distilled water. The crystal violet dye in the bacterial cells was dissolved by adding 33% (v/v) of 200 µL acetic acid in each well. The OD value at 590 nm was measured using SpectraMax spectrophotometer (Molecular Device, Sunnyvale, CA, USA). The experiment was repeated three times with six replicates of each treatment.
The effect of AgNPs (50 µg·mL −1 ) from B. amyloliquefaciens strain A3, the CFCS of B. amyloliquefaciens strain A3 (50%), and the combination of AgNPs (12 µg·mL −1 ) and CFCS (12%) on in vivo antagonistic actions against D. dadantii was assayed with tuber slices of sweet potato. After surface sterilization with 70% (v/v) ethanol, the sweet potato tubers were rinsed with sterile distilled water, and 10 mm thick slices were prepared. The tuber slices were kept in a solution containing 50 µg·mL −1 AgNPs or 50% CFCS or 12 µg·mL −1 AgNPs along with 12% CFCS for 1 h, and after that, they were air-dried for 1 h in Petri dishes. Then, 5 µL of D. dadantii cell suspension was spotted by puncturing at the center of the slice. The slices were incubated at 30 • C for 24 h. The diameter of the maceration zones created by D. dadantii around the punctures was measured. The experiment was repeated three times with three replicates of each treatment.

Transmission Electron Microscopy
The combined effect of AgNPs and CFCS on the structure of D. dadantii cells was determined using TEM. D. dadantii suspension alone or D. dadantii suspension containing a combination of AgNPs (12 µg·mL −1 ) and CFCS of B. amyloliquefaciens A3 (12%) were incubated at rotary shaking at 200 rpm and 30 • C for 4 h. The sample for TEM analysis was prepared followed by harvesting D. dadantii cells as described in our previous work [35], and observations were made using the JEM-1230 transmission electron microscope.

In Planta Assay
An in planta experiment was assayed to evaluate the efficacy of AgNPs (50 µg·mL −1 ) derived from the CFCS of B. amyloliquefaciens strain A3, the CFCS of B. amyloliquefaciens strain A3 (50%), and the combination of AgNPs (12 µg·mL −1 ) and CFCS (12%) for the growth promotion of sweet potato seedlings and the suppression of D. dadantii according to the method of [37] with slight modifications. In brief, after surface sterilization with 70% (v/v) ethanol, the sweet potato tubers were rinsed with sterile distilled water and immersed in D. dadantii suspension (1 × 10 7 CFU mL −1 ) or in sterile distilled water for 4 h. The seed tubers were air dried for 12 h and then immersed in sterile distilled water or AgNPs (50 µg·mL −1 ) from B. amyloliquefaciens strain A3, B. amyloliquefaciens strain A3-originated CFCS (50%), or the combination of AgNPs (12 µg·mL −1 ) and CFCS (12%) for 4 h. After treatment, all seed tubers were kept on three-layered moistened and sterilized filter papers in Petri dishes of 15 cm diameter. A single seed tuber was placed in a petri dish and was kept in a growth chamber for 21 days, maintaining 28 • C temperature, 80% humidity, and a 12/12-h light/dark photoperiod. After 21 days of incubation, seedling height was measured. The experiment was repeated three times with six replications of each treatment.

Statistical Analysis
Experimental data were analyzed using SPSS software version 16 (SPSS, Chicago, IL, USA) at a p < 0.05 level of significance. All data are presented as the mean of at least three values with their standard error for each independent experiment.

Data Availability Statement:
The data is contained within the manuscript.