Materials and chemicals
The chemicals used in this study, unless otherwise stated, were purchased from Sigma-Aldrich (St. Louis, MO, USA). The chemicals were analytical grade, and demineralized water was produced using an Aqual 25 reverse osmosis apparatus (Aqual, Česká, Czech Republic) and further treated with a Millipore System (Millipore System Inc., Billerica, MA, USA) to obtain ultrapure water with a corresponding resistivity of 18.20 MΩ cm (at 25 °C). All experiments used this ultrapure water unless otherwise stated. The pH values were evaluated using a pH meter (WTW inoLab, Weilheim, Germany) with a WTW SenTix pH electrode. For the determination of metals using atomic absorption spectroscopy, demineralized water obtained with a Millipore Milli-Q system (Millipore, Bedford, MA, USA) was used.
Synthesis on nanomaterials
Preparation of graphene oxide (GO)
GO was prepared by the chemical oxidation of 5.0 g graphite flakes (Sigma–Aldrich, 100 mesh, ≥ 75% min) in a mixture of concentrated H2SO4 (670 mL) and 30.0 g KMnO4 according to the modified Hummer’s method [51, 52]. The reaction mixture was stirred vigorously. After 10 days, the oxidation of graphite was terminated by the addition of H2O2 solution (250 mL, 30 wt% in H2O, Penta, Prague, Czech Republic). The formed GO was washed 3 times with 1 M HCl (37 wt% in H2O, Penta) and several times with ultrapure water (total volume used 60 L) until a constant pH value (3–4) was achieved.
Synthesis of the rGO-Cu-Ag nanocomposite
Solutions of AgNO3 (25.0 mL, 10 mM) and Cu(CH3COO)2 (25.0 mL, 10 mM) were added dropwise to a solution of GO (1.0 mL, 5.0 mg mL−1) under vigorous stirring. Then, the reducing agent Na[BH4] (40 mg) was slowly added to the reaction mixture, and the resulting mixture was stirred vigorously for 24 h at room temperature. The prepared nanocomposite was washed three times with 50.0 mL ultrapure water. The centrifuged nanocomposite (10 min, 6500 rcf) (Universal 320, Hettich, Tuttlingen, Germany) was filled up to a final volume 10.0 mL. Synthesis of rGO-Ag and rGO-Cu nanocomposites
For the synthesis of rGO-Ag, solutions of AgNO3 (50.0 mL, 10 mM) were added dropwise to a solution of GO (1.0 mL, 5.0 mg mL−1) under vigorous stirring. Then, the reducing agent Na[BH4] (40 mg) was slowly added to the reaction mixture, and the resulting mixture was stirred vigorously for 24 h at room temperature. The prepared nanocomposite was washed three times with 50.0 mL ultrapure water. The centrifuged nanocomposites (10 min, 6500 rcf) (Universal 320, Hettich, Tuttlingen, Germany) was filled up to a final volume 10.0 mL. The procedure for the synthesis of rGO-Cu was the same as that for rGO-Ag, and only AgNO3 was replaced with Cu(CH3COO)2.
Characterization of the rGO-Cu-Ag nanocomposite
Scanning electron microscopy and energy-dispersive X-ray spectroscopy (EDS)
The morphologies of the samples were determined using scanning electron microscopy (SEM). The dispersed samples were diluted 1:20 with ultrapure water and then applied to silicon wafers from Siegert Wafer company (Siegert Wafer GmbH, Aachen, Germany) and allowed to dry at room temperature (20–25 °C). Images of the samples were obtained using a MAIA 3 SEM (TESCAN Ltd, Brno, Czech Republic). An In-Beam SE detector with an accelerating voltage of 5 kV, a working distance of 3 mm and 50,000-fold magnification was used. Full frame capture was performed in UH Resolution mode and accumulation of image with image shift correction enabled, and it took approximately 0.5 min with the ∼ 0,32 µs/pixel dwell time. The spot size was set at 2.4 nm.
To check the elemental compositions of the generated nanocomposites, energy-dispersive X-ray spectroscopy (EDS) analysis was performed using an EDX detector made on a MIRA 2 SEM (TESCAN Ltd, Brno, Czech Republic). An Everhart–Thornley scintillation detector was used with an accelerating voltage of 15 kV and a work distance of 15 mm. The power of the detector was set so that the input signal was approximately 19,000–21,000 cts. At this setting, the output signal was approximately 15,000–16,000 cts, and the detector deadtime fluctuated between 19 and 21%. The time for each analysis was 10 min. The spot size was 40 nm.
Transmission electron microscopy (TEM) analysis and EDS
The samples were studied by an FEI Talos F200X HRTEM operated at 200 kV with a maximum beam current of 1.0 nA. The lower amount of beam current was chosen so as not to damage the GO in the samples. The microscope was equipped with a Super-X EDS system with four silicon drift detectors (SDDs) enabling element mapping. The samples were prepared on Au grid coated with a holey carbon film.
Determination of the Cu and Ag concentrations in rGO-Cu-Ag by atomic absorption spectrometry (AAS)
For the atomic absorption spectrometry (AAS) analysis, 0.1 mL of rGO-Cu-Ag, rGO-Ag, and rGO-Cu samples were decomposed with reaction mixtures containing 5.0 mL of suprapure HNO3 (70%, Merck, Germany) and 5.0 mL of Milli-Q water at 210 °C for 35 min (15 min operating temperature, 20 min holding time) using an Ethos ONE microwave extractor (Milestone, Sorisole, Italy). Determination of the Cu and Ag contents in the samples was performed using a 240 FS AA atomic absorption spectrometer (Agilent Technologies, Santa Clara, CA, USA) with flame atomization acetylene–air flame (oxygen flow 13.5 L min−1 and acetylene 2.0 L min−1). Standard solutions of Cu and Ag (1000 mg L−1, Merck, Darmstadt, Germany) were used to prepare the calibration solutions, which were acidified with 1 wt% concentrated suprapure HNO3. All solutions were prepared using demineralized water obtained with a Millipore Milli-Q system (Millipore, Bedford, MA, USA). The wavelength for Cu was 324.8 nm and for Ag was 328.1 nm.
Attenuated total reflectance Fourier transform infrared spectroscopy (ATR FTIR)
Fourier transform infrared spectroscopy (FTIR) spectra were collected using an INVENIO R FTIR spectrometer equipped with a single-reflection diamond ATR accessory—A225/Q Platinum ATR module (Bruker Optic Inc., Billerica, MA, USA). A fixed load was applied to each small amount of sample to ensure full contact of the solid with the diamond surface. Solid samples were directly analysed in lyophilized form. Before each measurement, background spectra were collected. Spectra were recorded at 25 °C from 4000 to 400 cm−1 at a resolution of 2 cm−1. Each spectrum was acquired by merging 128 interferograms. Bruker OPUS 8.1 (Bruker Optic Inc., Billerica, MA, USA) software was used for the spectra recording, and JDXview v0.2 software written by Norbert Haider, Vienna, Austria was used for further spectral evaluation [53].
X-ray powder diffraction (XRPD) sample preparation
A thin layer of a corresponding sample was deposited on the surface of a Si zero-background sample holder by evaporating water from the suspension. All the as-prepared samples on a zero-background sample holder were then placed into the sample holders for XRPD analysis.
X-ray powder diffraction–conventional Bragg–Brentano reflection geometry
Diffraction patterns were collected with a PANalytical X'Pert PRO diffractometer (Malvern Panalytical, Malvern, Worcestershire, United Kingdom) equipped with a conventional X-ray tube (Cu Kα radiation, 40 kV, 30 mA) and a linear position sensitive detector PIXcel with an anti-scatter shield. A programmable divergence slit set to a fixed value of 0.25°, Soller slit of 0.04 rad and mask of 15 mm were used in the primary beam. A programmable anti-scatter slit set to a fixed value of 0.25°, a Soller slit of 0.04 rad and a Ni beta-filter were used for the diffracted beam. Data were collected in the range of 5–90° 2θ with a step of 0.0131° and 500 s per step, producing a scan of approximately 3 h 46 min.
Evaluation of X-ray patterns
Qualitative analysis was performed with the HighScorePlus software package (Malvern PANalytical, The Netherlands, version 5.1.0) together with the PDF-4 + database [54]. Line profile analysis was performed using routines implemented in the HighScorePlus software [55]. Diffraction lines were fitted using the Pseudo Voigt profile function with split width and shape. No background subtraction was performed. The calculated values of the integral breadths of the diffraction lines (Bobs) were then corrected for instrumental broadening (Bstd). The net values of the integral breadths (Bstruct) and the positions of the diffraction lines were then entered into the Scherrer formula [56] to obtain the appropriate crystallite size in the corresponding direction. K (crystal shape factor) corresponding to the cubic shape of particles (K = 1) was used. The correction for instrumental broadening was performed with the NIST SRM660a standard (LaB6) that was analysed with the same geometry, and the Bstd values were determined by the same procedure.
Raman spectroscopy
A Renishaw InVia Raman microscope (Gloucestershire, UK) was used to collect the Raman spectra. A laser beam with a wavelength of 633 nm was used to excite the molecules, and 0.75 mW of laser energy (5% of 15 mW) was used. The sample surfaces were investigated via a 50 × L objective. The time per spectrum was 5 s, and 32 repeats of spectra were collected and further analysed in Renishaw WiRE software version 5.2. When the spectra repeats were averaged and smoothed, the bands were identified after subtracting the baselines.
Atomic force microscopy (AFM)
A Bruker Dimension FastScan atomic force microscope (Bruker Nano Surface, Santa Barbara, CA, USA) operated in PeakForce tapping mode was used for GO and rGO-Cu-Ag characterization. Silicon nitride triangular cantilevers “SCANASYST-AIR” (Bruker Nano Surface) characterized by a spring constant of 0.671 N m−1 and a sensitivity of 91.67 nm V−1 equipped with a tetrahedral silicon tip with a nominal tip radius of 2 nm were used for imaging. Images were taken at 1000 × 1000 pixels with a PeakForce Tapping frequency of 2 kHz and an amplitude of 55 nm. The gain parameters were set automatically by the ScanAsyst algorithm. All images were collected under ambient conditions at 38% relative humidity and 22.5 °C with a scanning raster rate of 0.67 Hz.
X-ray photoelectron spectroscopy (XPS)
X-ray photoelectron spectroscopy (XPS, Kratos Axis Supra with monochromatic Al Kα X-ray radiation, emission current of 15 mA and hybrid lens mode, Manchester, UK) was used for the analysis of the surface of the rGO-Cu-Ag nanocomposite. High-resolution spectra were measured with a pass energy of 20 eV. The spectra were fitted using a combination of Gaussian–Lorentzian line shapes in CasaXPS software version 2.3.22. The Shirley algorithm was used to establish the background of the spectra.
Antibacterial testing
Antibacterial assay, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
The antibacterial activity of the rGO-Cu-Ag nanocomposite against X. euvesicatoria was evaluated by the determination of viable bacteria using the colony-forming unit (CFU) enumeration technique. Preliminary determination of MIC was carried out as an evaluation of the individual nanocomposites at final concentrations of 0.01, 0.1, and 5 µg mL−1. The effectiveness of the rGO-Cu-Ag nanocomposite was compared with those of the single-metal nanocomposites containing reduced graphene oxide with copper (rGO-Cu) or silver (rGO-Ag). For this purpose, X. euvesicatoria strain no. 2968 obtained from the National Collection of Plant Pathogenic Bacteria (NCPPB, London, UK) was cultured in Luria–Bertani (LB) broth (Sigma-Aldrich) at 28 °C overnight. The obtained bacterial suspension was adjusted to an optical density of 0.1 at 600 nm (OD600) (approx. 1 × 108 CFU mL−1) and then serially diluted in LB broth to a concentration of approx. 2 × 106 CFU mL−1. The nanocomposites were mixed with bacterial suspension and incubated for 24 h at 28 °C with continuous shaking at 110 rpm (ES–20, Biosan, Warren, Michigan, USA). For the nontreated control, ultrapure water was used instead of the nanocomposites. To determine the number of viable bacteria, the pour plate method was used. In detail, 100 µL samples from each mixture were diluted in a decimal series of 10−5–10−6. A volume 100 µL from each dilution was pipetted on the centre of a sterile Petri dish (90 mm diameter). Sterile, molten (44 to 46 °C) plate count agar (Himedia, Mumbai, India) was added and mixed with the sample by swirling the plate. The samples were cooled at room temperature until solidified and then inverted and incubated at 28 °C until bacterial colonies were visible on nontreated control plates. Subsequently, bacterial colonies formed in or on the plate were counted. The effect on bacterial growth was reported as a percent of the CFU number for the nontreated control. A similar assay was used to determine the minimum bactericidal concentration (MBC) of the rGO-Cu-Ag nanocomposite. The nanocomposite was used at concentrations of 6.25, 12.5, 25.0 and 50.0 µg mL−1. The MIC value was established by the lowest concentration of rGO-Cu-Ag nanocomposite that did not permit any visible growth of X. euvesicatoria in or on the plate count medium. This was done by observing post-incubated agar plates for the presence or absence of bacteria after 72 h.
Observation of morphological changes in bacteria
Fresh bacterial cultures were prepared in LB broth for 24 h at 28 °C. The bacterial suspension was diluted with sterile saline containing the nanocomposite and shaken at 120 rpm overnight. The final concentration of the nanocomposite in the solution was 0, 6.25, 12.5, 25.0, 50.0 and 500 µg mL−1. The solution was centrifuged at 7000 g for 15 min, and the pellet was washed 3 times in 10 mL of ultrapure water. For the evaluation of bacterial cell morphology in the presence of the tested nanocomposite, the washed pellet was dispersed in 2 mL of ultrapure water and diluted 100 times. Then, 5 µL of suspension was loaded on a silicon wafer and allowed to dry in laminar flow. Images of the samples were made using a SEM, MAIA 3, Brno, Czech Republic. The detector was an external SE detector with an accelerating voltage of 2 kV, and the work distance was 3 mm and was scanned by using analytical mode.
DNA analysis
The bacterial suspension was prepared as described above, mixed with fresh LB broth to OD600 = 0.1 corresponding to 1 × 108 CFU and cultivated overnight with the nanocomposite at concentrations of 6.25, 12.5, 25.0 and 50.0 µg mL−1. Sterile saline was used as a positive control instead of the nanocomposite. Samples were centrifuged in 2-mL tubes at 15,000 g for 5 min, and total DNA was extracted from the pellet using Nucleospin Tissue (Macherey Nagel, Düren, Germany) according to the manufacturer’s protocol. DNA samples were mixed with 5X Green GoTaq Reaction Buffer (Promega, Madison, WI, USA) and run on a 1.2% agarose gel (Thermo Fisher Scientific, Waltham, MA, USA) coloured by GelRed (Biotium, Fremont, CA, USA). Samples were visualized using a UV transilluminator MUV21—312 (Major Science, Saratoga, California, USA).
Plant testing
Greenhouse experiment
Cultivars of pepper cv. Citrina and tomato cv. Mandat were used as plant material. Plants were grown in 280 mL pots containing standard substrate TS 4 (Klasmann-Deilmann GmbH, Geeste, Germany) and kept in greenhouse conditions at 22–26 °C and ≥ 70% relative humidity. For the experiment, plants at the four-leaf stage were used. According to the antibacterial assay, the rGO-Cu-Ag concentration of 50 µg mL−1 was selected as potentially effective for the plant treatment. To test the phytotoxicity of the nanocomposites on the plants, a concentration of 500 µg mL−1 was also included. The nanocomposites were applied to the plants by spraying (LaboPlast Spray Bottle, nozzle diameter 0.6 mm, Bürkle GmbH, Ban Bellingen, Germany), and after 24 h, the plants were sprayed using a 1 × 108 CFU bacterial suspension. After inoculation, the plants were covered by polyethylene bags for 48 h to increase the humidity. In the positive control, sterile saline was used instead of the nanocomposite. Plants in the negative control were sprayed with nanocomposites at a concentration of 500 µg mL−1, and sterile saline was used instead of bacteria. Negative control samples were used to evaluate the phytotoxicity of rGO-Cu-Ag. As another plant treatment, a 0.35% solution of the commercial product Kocide® 2000 (DuPont, Wilmington, DE, USA) was applied according to the manufacturer’s protocol. The experiment was carried out in two repetitions. In total, eight plants per treatment were used. The evaluation of disease symptoms was carried out on the seventh and fourteenth days after inoculation. The occurrence of bacterial spots was evaluated using a four-point scale: 0—healthy leaves without symptoms, 1—low symptom occurrence (1–3 dots per leaf), 2—1/3 of the leaf surface infected, 3—high symptom occurrence (more than 1/3 of the leaf surface infected). Based on symptom evaluation, the disease severity (DS) was calculated using the following formula [57]:
$$DS\left(\mathrm{\%}\right)=\frac{\sum \left(number \, of \, plants \, in \, a \, disease \, scale \, point\times disease \, scale \, point\right)}{\left(total \, number \, of \, plants \, \times maximum \, disease \, scale \, point\right)}\times 100.$$
Relative quantification of gene expression
Samples of tomato plants treated with 500 µg mL−1 rGO-Cu-Ag and inoculated with X. euvesicatoria (rGO-Cu-Ag + Xe), treated with 500 µg mL−1 rGO-Cu-Ag and noninoculated (rGO-Cu-Ag), nontreated and inoculated with X. euvesicatoria, and nontreated and noninoculated (NTC) were used for gene expression analysis. For each sample, four leaves of one plant were harvested 21 days after treatment. Plant samples were frozen at − 80 °C and ground to a fine powder using a mortar and pestle. An amount 100 mg of homogenized tissue were used for total RNA extraction using Spectrum Plant total RNA (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. Extracted RNA was quantified by a Modulus™ Single Tube Multimode Reader (Turner BioSystems, Sunnyvale, CA, USA) using a Quant-iT™ RiboGreen™ RNA Assay Kit (Invitrogen, Carlsbad, CA, USA), and the concentrations of all samples were adjusted to 75 ng µL−1. Reverse transcription was performed using random hexamer primers (Roche, Basel, Switzerland) and RevertAid Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA, USA). The cDNA was used as a template for the real-time PCR with the primer pairs and is shown in Additional file 1: Table. S1.
Betatubulin was used as a reference gene for the normalization of gene expression between the samples. The real-time PCR of a 20 µL volume consisted of 1 × HotSybr qPCR Kit (MCLab, San Francisco, CA, USA), 2 µL of prepared cDNA, 0.3 µM of each primer of the primer pair, and PCR grade water. The reactions for each sample were prepared in duplicate and run using a real-time PCR cycler RotorGene 3000 (Corbett Research, Sydney, Australia). The cycling conditions were 10 min at 95 °C, 40 cycles of 1 min at 95 °C, 1 min at 58 °C, and 1 min at 72 °C for Btub [58], PR1 [59], CAT [60] and TomQ’a [61]. The temperature profile for the qPCR of the PoP [62] and PRQb [61] genes was 10 min at 95 °C, 40 cycles of 15 s at 95 °C, 1 min at 60 °C, and 1 min at 72 °C. For relative quantification, the Livak and Schmittgen [63] method was used, and the analyses were performed using qbase + software (Biogazelle, Zwijnaarde, Belgium).
Statistical analysis
The obtained data were analysed by Statistica CZ software (StatSoft, Prague, Czech Republic). The data were subjected to analysis by single-factor ANOVA. Statistical differences (α = 0.05) were determined according to Duncan’s test. Gene expression data were subjected to analysis by the single-factor ANOVA test (p < 0.05), and the differences between the variants were then determined according to Tukey’s test using qbase + software (Biogazelle, Gent, Belgium).