Nanosilica Schiff-Base Copper(II) Complexes with Sustainable

Research Article pubs.acs.org/journal/ascecg

Nanosilica Schiff-Base Copper(II) Complexes with Sustainable Antimicrobial Activity against Bacteria and Reduced Risk of Harm to Plant and Environment Wenbing Zhang, Tianyu Shi, Guanglong Ding, Darunee Punyapitak, Juanli Zhu, Dong Guo, Zhaopeng Zhang, Jianqiang Li, and Yongsong Cao*

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College of Plant Protection, China Agricultural University, Beijing 100193, China ABSTRACT: The increasing frequency of using traditional copper-based bactericides has resulted in phytotoxicity to plants and heavy metal pollution to the environment. A novel formulation of copper bactericides with excellent environmental compatibility and high systemicity as well as safety and low toxicity is a preferred choice to improve excessive defects of traditional copper-based bactericides. Nanosilica Schiff-base copper(II) complexes (Silica-NMP-Cu) were prepared and evaluated in terms of their antimicrobial activity and toxicity. The results showed that, compared with Kocide, the SilicaNMP-Cu had excellent antimicrobial activity against four kinds of bacteria with reduced 54.31−64.75% amount of copper and could move upward and downward freely in the plant with the water transportation as well as increase disease resistance by increasing the concentration of salicylic acid in plants, which could reduce the frequency of using copper-based bactericide. Application of the cucumber safety assessment and Allium cepa chromoson aberration assay demonstrated that the Silica-NMPCu was able to reduce the phytotoxicity and the genotoxicity of the copper. The drug delivery system developed offers an efficient way to control bacteria, as well as reduce the risk of harm to the plant and the environment. KEYWORDS: Nanosilica, Schiff-base, Copper(II), Antimicrobial, Safety, Translocation, Genotoxicity

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pathogens.13,14 In addition, metal toxicity, salinity, drought, and temperature stresses can be alleviated by silica application.15 Bordeaux mixture, copper hydroxide, and diversiform formulations of copper-based bactericides have been extendedly applied in controlling bacterial diseases of plants depending on their broad spectrum, relatively inexpensive price, and fewer instrumental requirements for application than other bactericides.16,17 However, considering the lack of systemic action, copper-based bactericides need to be applied frequently for lasting activity to obtain effective bacteriostasis results without causing significant plant damage or yield loss simultaneously.18−20 The accumulation of copper has been affirmed to lead to heavy metal pollution of the environment because of nonbiodegradability, biological magnification, and long persistent in the environment.21,22 Excessive copper in the environment is toxic to organisms, resulting in disturbance of the ecosystem, and it always can lessen soil respiration, microbial biomass, and microbial numbers.23,24 Moreover, cooper exposure has already been proved to cause the fatty change in the liver and oxidative stress in teleost fish.25,26 Hence,

INTRODUCTION The emergence of nanoparticles (NPs) which enhance the drug bioavailability, reduce drug toxicity, and improve the controlled release of drug molecules presents opportunities for enlarging the applicable range of traditional bactericides.1−3 The creation of NPs for enhanced bactericidal efficacy in drug release areas highly benefits from their small size and high surface to volume ratio, which provides a direct method for the active ingredient to interact closely with microbial membranes and is not merely due to the simple release of active ingredient in solution.4,5 Recently, nanosilica particles with uniform structures and prominent surface properties are deemed to have excellent biocompatibility among numerous carrier materials that have been investigated for drug delivery.6−8 As a suitable carrier of the active ingredient, nanosilica has been widely applied in the fields of pharmaceuticals, pesticides, coatings, and so on.9−11 Silicon, which exists in the form of silica and silicate, does not act as an essential nutrient element in the process of plant growth, but it is significant in plant biology and physiology, particularly improves disease resistance, as well as mitigates other biotic and abiotic stresses.12 The deposition of silica in epidermal cell walls offers mechanical and protective advantages; accordingly, it can be a physical barrier to infection and reduction in the sensitivity to enzymatic degradation by © 2016 American Chemical Society

Received: August 5, 2016 Published: November 23, 2016 502

DOI: 10.1021/acssuschemeng.6b01867 ACS Sustainable Chem. Eng. 2017, 5, 502−509

Research Article

ACS Sustainable Chemistry & Engineering Scheme 1. Schematic Diagram of the Possible Formation Mechanism of Silica-NMP-Cu

analytical chemicals purchased from Sinopharm Chemical Reagent Beijing Co., Ltd., Beijing, China. Carbol fuchsin was obtained from Sigma-Aldrich (St. Louis, USA). Deionized water was applied for all reactions and treatment processes. 77% Kocide wettable powder was supplied by DuPont, USA. Acetonitrile and methanol were HPLC grade and purchased from J.T. Baker, USA. Acidovorax avenae subsp. citrulli (Aac), Clavibacter michiganense subsp. michiganense (Cmm), Pseudomonas syringe pv Lachryrnans (Psl), and Xanthomonas canpestris pv campestris (Xcc) strains were provided by Seed Pathology & Fungicide Pharmacology Lab., China Agriculture University. Preparation of Silica-NMP-Cu. Synthesis of Amino-Modified Nanosilica. Nanosilica was prepared according to the modified Stöber method described in the literature.32 In brief, 4.5 mL of ammonia solution (0.9 mol L−1) and 32.2 mL of ethanol were mixed in a threenecked flask equipped with a temperature controlled magnetic stirrer (SZCL-3A, Gongyi City Yuhua Instrument Co., Ltd., China) and continuously stirred at 600 rpm, and then 3.3 mL of TEOS solution of ethanol (16 mmol L−1) was added dropwise at a speed of 1.7 mL min−1 to a stirred mixture at 28−30 °C for 10 min under constant stirring. Finally, 0.8 mL of APTES was added to the flask and the reaction mixture was stirred at 50 °C for a further 2 h. The production was further filtered and washed three times with distilled water, centrifuged (6000 rpm), and dried at 60 °C.33 Synthesis of Silica-NMP-Cu. The chemical attachment of aminomodified nanosilica to NMP was obtained via the covalent bonds between the amine groups of nanosilica and the carbonyl group of NMP. First, 1 g of amino-modified nanosilica was added to 100 mL of ethanol and the mixture was continuously stirred so that it is entirely diffused. NMP, whose molar quantities were equal to that of nitrogen in nanosilica, was added to the mixture. The reactive mixture was stirred at 78 °C for 2 h. The predetermined amount of CuCl2·H2O as equal molar quantities with NMP was then added to the above suspension at 78 °C for 15 min under constant stirring at 600 rpm. Finally, the suspension was centrifuged and washed twice with deionized water and ethanol successively. After centrifugation and drying at 60 °C in an oven, the Silica-NMP-Cu was obtained. The formation mechanism of Silica-NMP-Cu is schematically presented in Scheme 1. Characterization. Fourier Transform Infrared Spectroscopy (FTIR). FT-IR spectra, recorded on a Jasco FT-IR 5300 spectrophotometer, were used to identify the different functional groups present in the samples. Samples were prepared as KBr pellets and scanned against a blank KBr pellet background at wavenumbers ranging from 4000 to 450 cm−1 with a resolution of 4.0 cm−1. Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) Observation. The morphology and structures of products were investigated using scanning electron microscopy (SEM,

preparing a novel formulation of copper bactericides with appropriate persistent period, excellent environmental compatibility, and well high systemicity as well as safety and low toxicity is a preferred method to improve excessive defects of traditional copper-based bactericides. Considered as great potential coordination compounds, transition metal complexes of Schiff-bases have been pervasively applied as biochemical, analytical, and antimicrobial reagents in the past few years,27−29 especially acting in an important role in medicinal chemistry due to their superiority of the antibacterial effect.30 Owing to the properties of the metal center or the coordinate ligands or cooperation, the copper(II) complexes of a Schiff-base have been shown to have stronger antibacterial activity, such as anticancer activity.31 Therefore, designing and synthesizing copper(II) complexes with a Schiff-base ligand can be an excellent choice for the purpose of expanding the application range and reducing toxicity and environmental pollution of traditional copper-based bactericides. Herein, in order to develop a safe drug delivery system with less toxicity to nontarget organisms and the environment, we prepared a novel antibacterial complex using nanosilica covalently bounded with a Schiff-base and subsequently linked to the copper(II) by a coordinating bond, which could combine the superiorities of nanosilica with the advantages of a Schiffbase and copper. A nanosilica Schiff-base was prepared from nanosilica and N-methyl pyrrolidone (NMP) through a covalent reaction using 3-aminopropyltriethoxysilane (APTES) as a cross-linking agent. Following, the prepared nanosilica Schiff-base was further reacted with copper(II) via a coordination bond, resulting in novel nanosilica Schiff-base copper complexes (Silica-NMP-Cu). The preparation conditions and the structural features of production were evaluated and characterized, as well as the effect of complexes on target bacteria and nontarget plants were also evaluated. Meanwhile, the antimicrobial mechanism against bacteria, the translocation in plants, and the genotoxic effects of the Silica-NMP-Cu were assessed.

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EXPERIMENTAL SECTION

Materials. Acetate, acetic acid, 3-aminopropyltriethoxysilane (APTES), ammonium hydroxide, bovine serum albumin (BSA), copper(II) chloride dihydrate (CuCl2·H2O), ethanol (99.9%), ether, flavin mononucleotide (FMN), hydrochloric acid (HCl), N-methylpyrrolidone (NMP), and tetraethyl orthosilicate (TEOS) were 503


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ACS Sustainable Chemistry & Engineering

temperature was about 25/16 °C, and humidity was at 60−80% during experiments. Cucumber seeds (Cucumis sativus L. cv. Zhongnong 12, Cucumis sativus L. cv. Zhongnong No.6, and Cucumis sativus L. cv. Biyu) were sown in trays (5000 cm2) carrying 50 flower pots filled with soil, and each pot contained 1−2 seeds. Cucumber seedlings were watered and fertilized weekly to optimize their growth. Silica-NMP-Cu application dosages were 37.5, 100, and 300 mg L−1, and treated with Kocide (37.5, 100, and 300 mg L−1), CuCl2·H2O (25, 50, and 100 mg L−1), and water as control. Each tray was sprayed with 25.0 mL of suspension. The suspension was totally sprayed 3 times with a microaerosol sprayer at the three-leaf stage of cucumber, and each time was a 7 days intervals. The experiments were arranged in a randomized complete-block design with four replications. The fresh weights and plant heights were determined 7 days after treatment (DAT). Twenty seedlings from each tray were selected, washed, and air-dried. Safety assessment was evaluated by a growth increase recorded as a plant height and fresh weight increase of cucumber seedlings. After removing the dead tissue, the plant heights and fresh weights of whole seedlings from the trays were determined and recorded, and then the rate of increase was calculated. Allium cepa Chromosome Aberration Assays. The genotoxicity of copper was evaluated by Allium cepa assays based on the method by Rank and Nielsen.35 Allium cepa seeds were germinated in water until the roots had reached a length of about 2 cm; then they were subjected to different copper concentrations of Kocide (1.5, 3, 6 mg L−1) and Silica-NMP-Cu (1.5, 3, 6 mg L−1) for periods of 24 h. Water was used as the control. After 24 h of treatment, the roots were collected and fixed using methanol/acetic acid (3:1, v/v). After fixing, the samples were immersed in 1 M HCl at 60 °C for 9 min and washed thrice in distilled water. The meristematic region of the root tip was cut and placed on a slide together with coloring by addition of 100 μL of carbol fuchsin, and then gently covered with a coverslip to squash and spread the cells. Under an optical microscope, about 500 cells were collected and observed on each slide to determine the mitotic index, which was considered the proportion of cells in division, and the chromosomal aberration index, which was considered the proportion of divisions with chromosomal aberrations. Each treatment was performed in triplicate.

S4800, HITACH, Japan) and transmission electron microscopy (TEM, Tecnai G2 F30 S-TWIN, USA). X-ray Diffraction (XRD) Analysis. The crystal structural analyses of products were carried out by using a Rigaku D/Max-RB X-ray diffractometer with a Cu Kα incident source at the scanning rate of 10°/min in the 2θ range from 10° to 100°. Antimicrobial Activity of Silica-NMP-Cu. The antimicrobial activities of Silica-NMP-Cu against Psl, Aac, Xcc, and Cmm were determined by the turbidimetric method. An aliquot (100 μL) of the bacterial suspension (108 CFU mL−1) was cultured into a conical flask (35 mL) containing 10 mL of lysogeny broth (LB) medium, which contained series concentrations of Silica-NMP-Cu (0, 12.5, 25, 50, 100, 300 mg L−1), 77% Kocide wettable powder (0, 12.5, 25, 50, 100, 300 mg L−1), and CuCl2·H2O (0, 6.25, 12.5, 25, 50, 100 mg L−1) as positive control. The mixtures were inoculated at 120 r min−1 for 8− 10 h under 28−30 °C. The growth of each culture was monitored by measuring its optical density at 600 nm (OD 600 ) through spectrophotometric colorimetry, and the same operation was conducted in three replicates. The half maximal effective concentration (EC50) values were calculated by the linear regression of the probit % of inhibition of the number of bacteria as a function of the log of inhibitor concentrations. Antimicrobial Mechanism of Silica-NMP-Cu. The antimicrobial mechanism was researched by a static fluorescence quenching experiment performed via interacting BSA with Silica-NMP-Cu and CuCl2·H2O. Specifically, 500 μL of BSA solution with concentration of 10−7 mol L−1 was added in a 1 cm quartz colorimetric utensil, and then 0, 10, 20, 30, and 40 μL of Silica-NMP-Cu suspension or CuCl2·H2O solution with concentration of 300 mg L−1 was dropwise added respectively by micropipettor, the mixtures were shaken 5 min, and then the fluorescence intensities were determined in the range 305− 500 nm after each addition. The fluorescence intensities were recorded using an F-4500 fluorescence spectrophotometer (HITACHI, Japan) with an excitation wavelength (λex) of 283 nm and excitation and emission slit widths of 5 nm at a scanning speed of 300 nm min−1, and the temperature was controlled at 25 °C. The antimicrobial mechanism was also researched by determining the change of salicylic acid content in cucumber (Cucumis sativus L. cv. Zhongnong 12) after sprayed Silica-NMP-Cu. The application dosages were 12.5, 25, 50, and 100 mg L−1, and samples were treated with water, nanosilica at the concentrations 12.5, 25, 50, and 100 mg L−1, and Kocide at the concentrations 12.5, 25, 50, and 100 mg L−1 as a control. The suspension was sprayed with a microaerosol sprayer at the three-leaf stage of cucumber, and sampled after 1 h, 1 d, 2 d, 3 d, and 4 d of treatment, respectively. The detection of salicylic acid content was performed on leaf extracts. Cucumber leaves from treatment and control plants were ground after freezing with liquid nitrogen, and then 0.5 g of leaf tissue was homogenized in 10 mL of extract (water:methanol:ether:acetate = 20:16:16:1, w/w) and extracted for 12 h; after centrifugation at 15000 rpm for 10 min, the supernatant was collected and used for analysis. The supernatant was filtered through a 0.22 μm filter membrane and injected into the HPLC system which consisted of two LC-20ATvp pumps and an RF-20AXL fluorescence detector (Shimadzu, Japan). The mobile phase was composed of methanol as eluent A, and water with acetate-sodium/ acetic acid buffer (pH = 3.2) as eluent B. The samples were determined with an excitation wavelength of 310 nm and an emission wavelength of 430 nm. The analysis was executed with an injection volume of 20 μL and a flow rate of 1.0 mL min−1. Translocation of Silica-NMP-Cu in Plants. The compound (FMN-Silica-NMP-Cu) was prepared by combining the FMN with complexes (Silica-NMP-Cu) via a hydrogen bond and using laser scanning confocal microscopy (LSCM, Olympus FluoView FV1000, Japan) to observe the location of them in the plant.34 The cucumber was sprayed in upper or lower positions with 100 mg L−1 of FMNSilica-NMP-Cu at the three-leaf stage, and sampled after 2, 4, and 6 h of treatment, respectively. Safety Assessment of Silica-NMP-Cu to Plants. Experiments were conducted in the greenhouse of China Agricultural University, located in the Haidian district of Beijing. Average day/night

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RESULTS AND DISCUSSION

Structural Characterization of Silica-NMP-Cu. Results of FT-IR. The FT-IR results (Figure 1) show the infrared spectra of nanosilica, amino-modified silica, and Silica-NMPCu. The nanosilica (Figure 1a) shows a broad band at about 1075 cm−1, assigned to antisymmetrical stretching vibration modes of Si−O−Si groups, and the peaks toward 793 and 471 cm−1 are the symmetrical stretching vibration band and the bend vibration band always attributed to the Si−O−Si groups.

Figure 1. FT-IR spectra of nanosilica (a), amino-modified silica (b), and Silica-NMP-Cu (c). 504


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ACS Sustainable Chemistry & Engineering Compared with nanosilica, the amino-modified nanosilica (Figure 1b) shows the characteristic vibration bands of the amine (−NH2) at 3300 cm−1 and the absorption band of the methylene group (−CH2−) at 2980 cm−1. This indicated APTES had been successfully attached onto the surface of nanosilica. The spectrum of the Silica-NMP-Cu (Figure 1c) exhibits a much stronger absorption band of double-bonded carbonic acid (CN) at about 1622 cm−1, in contrast with amino-modified nanosilica. Besides, the absorption band of the methyl (−CH3) at 1384 cm−1 is attributed to the NMP, demonstrating that the Silica-NMP-Cu has been successfully achieved. XRD Analysis of Silica-NMP-Cu. The XRD results are presented in Figure 2. Similar patterns of broad peaks ranging

Figure 3. SEM images of nanosilica (a) and Silica-NMP-Cu (b); TEM images of nanosilica (c) and Silica-NMP-Cu (d).

7.65, 22.26, and 10.17 mg L−1 for Xcc, and 6.23, 8.95, and 3.84 mg L−1 for Cmm, respectively. According to results, CuCl2· 2H2O displays better antimicrobial efficacy depending on its ion form particularity, but it was not considered to be used directly due to its phytotoxic to plants. Compared with the Kocide, the Silica-NMP-Cu reduces 64.75%, 59.67%, 54.31%, and 57.09% of copper concentration for four kinds of bacteria with the same antibacterial effects, which indicates that SilicaNMP-Cu could greatly reduce the dosage of pesticide and replace the traditional copper agents in the agricultural area. Antimicrobial Mechanism. The effects of CuCl2·2H2O and Silica-NMP-Cu on the fluorescence intensity of bovine serum albumin (BSA) are presented in Figure 4. It is obvious that BSA has a strong fluorescence emission peaked at about 350 nm, and significantly decreases with the additive volume of CuCl2·2H2O varying from 10 to 40 μL. The fluorescence quenching occurs due to the interactions of copper ions with the sulfydryl (-SH), amino (−NH2), and carboxyl (−COOH) of the BSA. When BSA is dropwise added with 10, 20, 30, or 40 μL of Silica-NMP-Cu, a remarkable intrinsic fluorescence decrease of BSA is observed which shows the consistent trend of CuCl2·2H2O. Therefore, Silica-NMP-Cu can also combine with the sulfydryl (-SH), amino (−NH2), and carboxyl (−COOH) groups of the bacterial protein, which causes plant diseases, and then restrains bacteria growth. Table 2 shows that the concentration of salicylic acid (SA) in cucumber leaves is obviously proportional to the concentration of treatments, but is inversely proportional to the time. Compared with CuCl2·2H2O, the high level concentration of SA is present for a significantly lasting time under treatments of nanosilica and Silica-NMP-Cu. SA accumulation is essential for expression of multiple modes of plant disease resistance which can be increased by silica;12,37 improvement of the disease resistance by Silica-NMP-Cu indicates that Silica-NMP-Cu has exactly the same advantage as silica. Combining the superiorities of CuCl2·2H2O and silica, Silica-NMP-Cu shows efficient antimicrobial activity and well improvement of plant disease resistance, which reflects that the antimicrobial mechanism of Silica-NMP-Cu depends on the antimicrobial activity of the copper and plant disease resistance of nanosilica. Translocation of Silica-NMP-Cu in Plants. Figure 5 shows that FMN-Silica-NMP-Cu can be found in the cell wall, vascular, stomatal cell, and microtubule of upper leaves after spraying 4 h in the lower leaves of cucumber. The fluorescence

Figure 2. XRD patterns of the nanosilica (a), Silica-NMP (b), and Silica-NMP-Cu (c).

from 15 to 30° in 2θ angles of nanosilica (Figure 2a) and SilicaNMP (Figure 2b) confirm the amorphous nature of the silica, and are identical with those reported in literature.36 The XRD pattern of the Silica-NMP-Cu (Figure 2c) shows peaks corresponding to copper. The XRD peaks of the crystal plane for Cu(100) appearing at 32.21° (2θ), for Cu(111) appearing at 39.65° (2θ), and for Cu(200) appearing at 50.00° (2θ) determine that copper ions have been successfully attached to the Silica-NMP. Meanwhile, the XRD pattern of Silica-NMPCu with peak ranging from 15 to 30° in 2θ is the characteristic peak of nanosilica, which demonstrates that the Silica-NMP-Cu has been achieved. Results of SEM and TEM Observation. The SEM and TEM were used to further examine the particle size, structure, and morphology of samples. It is observed clearly from the SEM images (Figure 3a and 3b) that the particles are spheres with regular shape and homogeneous particle size, which is about 300 nm; meanwhile, the surface of nanosilica (Figure 3a) is smoother than that of Silica-NMP-Cu (Figure 3b). In contrast with the nanosilica (Figure 3c), the TEM image of Silica-NMPCu (Figure 3d) shows that the surface is attached by a branching structure, which indicates that the copper(II) has been successfully attached on nanosilica via a coordinate bond of a combination between copper ions and a Schiff-base. Antimicrobial Activity. The antimicrobial activities of CuCl2·2H2O, Kocide, and Silica-NMP-Cu against Psl, Aac, Xcc, and Cmm were determined by the turbidimetric method, and the toxicity tests are summarized in Table 1. When obtaining the same inhibitory effect, the copper concentrations of CuCl2· 2H2O, Kocide, and Silica-NMP-Cu were 16.54, 58.83, and 20.74 mg L−1 for Psl, 11.86, 29.41, and 11.86 mg L−1 for Aac, 505


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ACS Sustainable Chemistry & Engineering Table 1. EC50 Values of CuCl2·2H2O, Kocide, and Silica-NMP-Cu against Psl, Aac, Xcc, and Cmm Treatment

Percentage of copper (%)

Bacterial

CuCl2·2H2O

37.4

Kocide

50.5

Silica-NMP-Cu

16.9

Psl Aac Xcc Cmm Psl Aac Xcc Cmm Psl Aac Xcc Cmm

Toxicity equations y y y y y y y y y y y y

= = = = = = = = = = = =

1.273x 3.423x 5.266x 3.702x 2.399x 3.267x 3.106x 2.621x 2.394x 2.820x 2.755x 2.616x

+ 2.905 − 0.139 − 1.903 + 0.476 + 0.043 − 0.767 − 0.107 + 1.728 − 0.001 − 0.206 + 0.098 + 1.453

Correlation (r2)

EC50 (mg L−1)

Conc of copper (mg L−1)

0.9742 0.9738 0.9491 0.9215 0.9881 0.9692 0.9083 0.8185 0.9706 0.9664 0.8729 0.8115

44.23 31.72 20.46 16.67 116.49 58.24 44.08 17.72 122.74 70.16 60.16 22.70

16.54 11.86 7.65 6.23 58.83 29.41 22.26 8.95 20.74 11.86 10.17 3.84

Figure 5. Distribution of FMN-Silica-NMP-Cu in leaves: cell wall (a), vascular (b), stomatal cell, (c) and microtubule (d).

can also be observed in the lower leaves after 6 h of treatment in the upper leaves of cucumber. However, the fluorescence cannot be found in cucumber seedling tissues after Kocide treatment with the same procedure. The results indicate that the movement of Silica-NMP-Cu probably depends on water

Figure 4. Fluorescence spectra of BSA for the different addition volumes of CuCl2·2H2O (a) and Silica-NMP-Cu (b).

Table 2. Concentration of SA in Cucumber Leaves after Treatment of Water, CuCl2·2H2O, Nano-Silica, and Silica-NMP-Cu concentrations of SA (μg L−1) Treatment CK CuCl2·2H2O

Nanosilica

Silica-NMP-Cu

−1

Conc (mg L )

6h

1d

2d

3d

4d

0 12.5 25 50 100 12.5 25 50 100 12.5 25 50 100

45.67 287.63 404.00 539.25

51.34 210.31 369.00 487.94

49.23 139.06 227.48 276.95

44.37 107.84 158.68 198.34

50.45 78.75 117.33 157.26

182.28 275.79 345.37 543.42 174.53 253.75 351.06 521.77

176.15 256.21 325.82 430.29 162.38 238.47 338.77 418.79

153.26 239.52 300.75 314.56 151.35 227.61 309.42 307.41

133.21 183.39 279.35 226.91 139.77 203.79 281.93 248.35

110.78 157.01 256.04 180.94 128.63 185.34 278.15 195.45

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Figure 6. Effects of Silica-NMP-Cu (a) and Kocide (b) with concentration of 300 mg L−1, and of CuCl2·2H2O (c) with concentration of 50 mg L−1, and of the control group (d) on the seedling shape of Cucumis sativus L. cv. Zhongnong 12.

Table 3. Effects of Silica-NMP-Cu on the Plant Height and Fresh Weight of Cucumbera Plant height (cm) cucumber Cucumis sativus L. cv. Zhongnong 12

Cucumis sativus L. cv. Zhongnong No.6

Cucumis sativus L. cv. Biyu

a

Fresh weight (g)

Dosage (mg L−1)

Pretreatment

7 DAT

CK 37.5 100 300 CK 37.5 100 300 CK 37.5 100 300

24.32 23.57 24.21 24.55 27.85 25.66 26.28 28.1 28.51 29.96 30.64 31.32

35.85 33.24 34.12 32.91 44.63 43.02 44.35 45.16 45.22 46.43 47.15 46.93

Rate of increase (cm/d) Pretreatment 0.47aa 0.41a 0.41a 0.34a 0.60a 0.68a 0.69a 0.61a 0.59a 0.55a 0.54a 0.50a

16.85 16.66 17.28 17.14 20.51 19.96 20.64 21.32 18.51 19.96 20.64 21.32

7 DAT

Rate of increase (g/d)

31.56 30.23 30.91 31.01 46.61 45.42 46.86 48.28 46.87 47.74 48.52 48.93

0.87a 0.81a 0.79a 0.81a 1.27a 1.28a 1.27a 1.26a 1.53a 1.40a 1.35a 1.30a

Values marked with letters are significantly different according to the Duncan test (P < 0.05).

of Silica-NMP-Cu on the growth at 7 DAT in the greenhouse were investigated, and the results are shown in Table 3. According to the statistical results, the plant height and fresh weight of the treatment have no obvious difference with the control group. Therefore, the Silica-NMP-Cu is safe for cucumber seedling growth without phytotoxicity. Allium cepa Chromosome Aberration Assays. Copper can cause damage to the DNA, with genotoxic and mutagenic effects which can lead to health problems that include possible hereditary impacts.39 The effects of Kocide and Silica-NMP-Cu suspension at the copper concentrations of 1.5, 3, and 6 mg L−1 on the cell division and chromosome behavior of A. cepa are presented in Figure 7. Compared with control, the mitotic index for the Kocide was obviously decreased as the concentration increased, and the differences between the Kocide at all concentrations and the control were statistically significant. The mitotic index of the Silica-NMP-Cu was significantly higher than that of the Kocide at same concentration, demonstrating that the complexes reduce the cytotoxicity of the copper (Figure 7A). The chromosomal

transportation of the plant, especially the transpiration. Because of the lack of systemic action, traditional copper-based bactericides should be distributed uniformly and applied 2−3 times with a 1 week interval to control plant disease.38 Due to the small particle size of about 300 nm, large surface area, and high adsorption capacity, Silica- NMP-Cu could move upward and downward freely in the plant with the water transportation. Therefore, Silica-NMP-Cu can obviously reduce the frequency of using pesticide. Safety Assessment to Cucumber. The results of safety assessment of Cucumis sativus L. cv. Zhongnong 12 show that there are no significant differences between the treatment of Silica-NMP-Cu (Figure 6a) and the control group (Figure 6d); cucumbers grow well without malformed, chlorotic, and necrotic seedlings. However, under the treatment of Kocide (Figure 6b) at the concentration 300 mg L−1, the slight interveinal chlorosis on the leaves of cucumber appeared, and the leaves of cucumber exhibit serious chlorotic and curly appearance under treatment of CuCl 2 ·2H 2O with the concentration 50 mg L−1 (Figure 6c). Meanwhile, the effects 507



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AUTHOR INFORMATION

Corresponding Author

*Telephone number: 86-10-62734302 (O), Fax: 86-1062734302, E-mail: [email protected], [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

This work was supported by the National Natural Science Foundation of China (31471799) and the National Department Public Benefit Research Foundation of China (201303031).

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Figure 7. Genotoxicity of the Silica-NMP-Cu against A. cepa.: (A) mitotic index; (B) chromosome aberration index. The statistical analysis was performed by comparison with (a) the control, and (b) the Kocide. Asterisk (*) indicates a statistically significant difference at the 0.05 level.

aberration indexes of the Kocide and Silica-NMP-Cu were higher than that of the control, and the index was increased with the raising of concentration (Figure 7B). Obviously, significantly higher index values were obtained for the treatments using the Kocide at all concentrations compared to the control and the Silica-NMP-Cu, while the index values between the Silica-NMP-Cu and the control were similar, indicating that the complexes could reduce the genotoxic effects by reducing the concentration of free copper ions.

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CONCLUSIONS In this study, we described a novel nanoscale drug delivery system which can be successfully used in agriculture. Based on the nanosilica, Silica-NMP-Cu, with excellent antibacterial activity, was prepared to improve the translocation and reduce the toxicity of copper to plants and the environment. The results showed that the Silica-NMP-Cu was remarkable on antibacterial activity to four kinds of bacteria and safety to cucumber growth, and could increase the concentration of SA to induce plant resistance as well as improve the translocation of copper in cucumber seedlings. Therefore, this complex model may expand the application range of traditional copperbased bactericides by offering an efficient way to control bacteria, as well as reduce the risk of harm to plants and the environment. 508


Research Article

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Nanosilica Schiff-Base Copper(II) Complexes with Sustainable

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