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Nano-silica 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 ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.6b01867 • Publication Date (Web): 23 Nov 2016 Downloaded from http://pubs.acs.org on November 29, 2016
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Nano-silica 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, Yongsong Cao*
College of Plant Protection, China Agricultural University, Beijing, China
*Corresponding author: NO.2 Yuanmingyuan West Road, China Agricultural University, Beijing, China, 100193 Telephone number: 86-10-62734302 (O), 86-10-62734302 (FAX) Email:
[email protected],
[email protected] 1
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Abstract The increasing frequency of using traditional copper-based bactericides has resulted in phytotoxicity to plants and heavy metal pollution to 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. Nano-silica 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 Silica-NMP-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 plant, 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-NMP-Cu was able to reduce the phytotoxicity and the genotoxicity of the copper. The drug delivery system developed offer an efficient way to control bacteria, as well as reduce the risk of harm to the plant and environment. Keywords: Nano-silica; Schiff-base; Copper (II); Antimicrobial; Safety; Translocation; Genotoxicity
Synopsis: :Based on nano-silica, new copper complexes with sustainable antimicrobial activity, environmental compatibility, high systemicity and low genotoxicity can be developed.
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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 applicable range of traditional bactericide.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 simply release of active ingredient in solution.4,
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Recently, nano-silica particles with uniform structures and prominent surface properties are deemed to be excellent biocompatibility among numerous carrier materials that have been investigated for drug delivery.6-8 As a suitable carrier of the active ingredient, nano-silica 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 pathogens.13, 14 In addition, metal toxicity, salinity, drought and temperature stresses can be alleviated by silica application.14 Bordeaux mixture, copper hydroxide, and diversiform formulations of copper-based bactericides have been extendedly applied in controlling bacterial diseases of plant depending on their broad spectrum, relatively inexpensive price and fewer instrumental requirement for application than other bactericides.16, 17 However, being lack of systemic action, copper-based bactericides need to 3
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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 non-biodegradability, biological magnification and long persistent in the environment of copper.21, 22 Excessive copper in the environment is toxic to organisms, resulting in disturbance of the ecosystem, and always can lessen soil respiration, microbial biomass, and microbial numbers.23,
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Moreover, cooper
exposure has already been proved to cause the fatty change in the liver and oxidative stress in teleost fish.25, 26 Hence, 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-base have been pervasively applied as biochemical, analytical and antimicrobial reagents in the past few years,27-29 especially act as an important role in medicinal chemistry due to their superiority of antibacterial effect.30 Owing to the properties of the metal center or the coordinate ligands or cooperation, the copper (II) complexes of Schiff-base have been shown to have stronger antibacterial activity, such as anticancer activity.31 Therefore, designing and synthesizing copper (II) complexes with Schiff-base ligand can be an excellent choice for the purpose of expanding the application range, reducing toxicity and environmental pollution of traditional copper-based bactericides. Herein, in order to develop a safe drug delivery system with less toxicity to non-target organisms and the environment, we prepared a novel antibacterial complex using nano-silica 4
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covalently bounded with Schiff-base and subsequently linked to the copper (II) by coordinating bond, which could combine the superiorities of nano-silica with the advantages of Schiff-base and copper. Nano-silica Schiff-base was prepared from nano-silica and N-methyl pyrrolidone (NMP) through a covalent reaction using 3-aminopropyltriethoxysilane (APTES) as a cross-linking agent. Following, the prepared nano-silica Schiff-base was further reacted with copper (II) via coordination bond, resulting in novel nano-silica 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 non-target plant were also evaluated. Meanwhile, the antimicrobial mechanism against bacteria, the translocation in plant and the genotoxic effects of the Silica-NMP-Cu were assessed. EXPERIMENTAL 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), tetraethyl orthosilicate (TEOS) were 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
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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 nano-silica Nano-silica was prepared according to the modified Stöber method described in the literature.32 In brief, 4.5 mL ammonia solution (0.9 mol L-1) and 32.2 mL ethanol were mixed in a three-necked flask equipped with a temperature control magnetic stirrer (SZCL-3A, Gongyi City Yuhua Instrument Co., Ltd, China) and continuously stirred at 600 rpm, and then 3.3 ml TEOS solution of ethanol (16 mmol L-1) was added dropwise at speed of 1.7 mL min-1 to a stirred mixture at 28-30 oC 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 amino-modified nano-silica to NMP was obtained via the covalent bonds between amine groups of nano-silica and carbonyl group of NMP. Firstly, 1 g of amino-modified nano-silica 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 nano-silica, was added to the mixture. The reactive mixture was stirred at 78 oC for 2 h. The predetermined amount of CuCl2·H2O as equal molar quantities with NMP was then added to the above suspension at 78 oC for 15 min under constant stirring at 600 rpm. Finally, the suspension was centrifuged and washed twice with deionized water and ethanol successively. After
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centrifugation and dried at 60 oC in oven, the Silica-NMP-Cu was obtained. The formation mechanism of Silica-NMP-Cu schematically presents in Scheme 1.
Scheme 1 Schematic diagram of the possible formation mechanism of Silica-NMP-Cu
Characterization Fourier transform infrared spectroscopy (FT-IR) FT-IR spectra, recorded on Jasco FT-IR 5300 spectrophotometer, were used to identify the different functional groups present in the samples. Samples were prepared as KBr pellet and scanned against a blank KBr pellet background at wave numbers ranging from 4000 to 450 cm-1 with a resolution of 4.0 cm-1. Scanning electron microscopy (SEM) and transmission electron microscope (TEM) observation The morphology and structures of products were investigated using scanning electron microscopy (SEM, S4800, HITACH, Japan) and transmission electron microscope (TEM, Tecnai G2 F30 S-TWIN, USA). X-ray diffraction (XRD) analysis
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The crystal structural analyses of products were carried out by using a Rigaku D/Max-RB X-ray diffractometer with Cu-Kα incident source at the scanning rate of 10o/min in 2θ range from 10o-100o. Antimicrobial activity of Silica-NMP-Cu The antimicrobial activity 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 of 10 mL lysogeny broth (LB) medium, which containing 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 oC. The growth of each culture was monitored by measuring its optical density at 600 nm (OD600) 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. The antimicrobial mechanism of Silica-NMP-Cu The antimicrobial mechanism was researched by 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 1 cm quartz colorimetric utensil, and then 0, 10, 20, 30, 40 µL of Silica-NMP-Cu suspension or CuCl2·H2O solution with concentration of 300 mg L-1 were dropwise added respectively by micropipettor, the mixtures were shaken 5 min and then determined the fluorescence intensities at range of 305-500 nm after each adding. The 8
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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 controlled the temperature at 25 oC. 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 treated with water, nano-silica at concentration of 12.5, 25, 50 and 100 mg L-1, and Kocide at concentration of 12.5, 25, 50 and 100 mg L-1 as 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 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 leaf tissue was homogenized in 10 mL 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 consisting 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 excitation wavelength of 310 nm and emission wavelength of 430 nm. The analysis was executed with injection volume of 20 µL and flow rate of 1.0 mL min-1. The translocation of Silica-NMP-Cu in plant The compound (FMN-Silica-NMP-Cu) was prepared by combining the FMN with complexes (Silica-NMP-Cu) via hydrogen bond and using laser scanning confocal microscope (LSCM, 9
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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 FMN-Silica-NMP-Cu at the three-leaf stage, and sampled after 2 h, 4 h, and 6 h treatment, respectively. Safety assessment of Silica-NMP-Cu to plant Experiments were conducted in the greenhouse of China Agricultural University, located in the Haidian district of Beijing. Average day/night temperature was about 25/16 oC, 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 fertilised weekly to optimise 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 7 days intervals. The experiments were arranged in a randomised 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 growth increase recorded as plant height and fresh weight increase of cucumber seedlings. After removing the dead tissue, plant heights and fresh weights of whole seedlings from the trays were determined and recorded, and then calculated the rate of increase. Allium cepa chromosome aberration assays The genotoxicity of copper was evaluated by Allium cepa assays based on the method by Rank 10
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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 treatment, the roots were collected and fixed using the methanol/acetic acid (3:1, v/v). After fixing, the samples were immersed in 1 M HCl at 60 oC 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 cover slip to squash and spread the cells. Under an optical microscope, about 500 cells was 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. RESULTS AND DISCUSSION Structural characterization of Silica-NMP-Cu The results of FT-IR The FT-IR results (Figure 1) show the infrared spectra of nano-silica, amino-modified silica, and Silica-NMP-Cu. The nano-silica (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 cm-1 and 471 cm-1 are symmetrical stretching vibration band and bend vibration band always attributed to the Si-O-Si groups. Compared with nano-silica, the amino-modified nano-silica (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 nano-silica. The spectrum of the Silica-NMP-Cu (Figure 1c) exhibits 11
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a much stronger absorption band of double-bonded carbonic acid (C=N) at about 1622 cm-1, in contrast with amino-modified nano-silica. 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.
Figure 1 FT-IR spectra of nano-silica (a), amino-modified silica (b), and Silica-NMP-Cu (c)
The XRD analysis of Silica-NMP-Cu The XRD results are presented in Figure 2. Similar patterns of broad peaks ranging from 15 to 30o in 2θ angles of nano-silica (Figure 2a) and Silica-NMP (Figure 2b) confirm the amorphous nature of the silica, which 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 crystal plane for Cu (100) appearing at 32.21o (2θ), Cu (111) appearing at 39.65o (2θ) and Cu (200) appearing at 50.00o (2θ) determine that copper ions have been successfully attached to the Silica-NMP. Meanwhile, the XRD pattern of Silica-NMP-Cu with peak ranging from 15 to 30o in 2θ is the characteristic peak of nano-silica, which demonstrates that the Silica-NMP-Cu has been achieved.
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Figure 2 XRD patterns of the nano-silica (a), Silica-NMP (b) and Silica-NMP-Cu (c)
The 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 sphere with regular shape and homogeneous particle size which is about 300 nm, meanwhile, the surface of nano-silica (Figure 3a) is smoother than that of Silica-NMP-Cu (Figure 3b). On contrast with the nano-silica (Figure 3c), the TEM image of Silica-NMP-Cu (Figure 3d) shows that the surface is attached by branching structure, which indicates that the copper (II) has been successfully attached on nano-silica via coordinate bond of combination between copper ions and Schiff-base.
Figure 3 SEM images of nano-silica (a) and Silica-NMP-Cu (b), TEM images of nano-silica (c) 13
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and Silica-NMP-Cu (d)
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, 7.65, 22.26, 10.17 mg L-1 for Xcc, 6.23, 8.95 and 3.84 mg L-1 for Cmm, respectively. According to results, the 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 same antibacterial effects, which indicates that Silica-NMP-Cu could greatly reduce the dosage of pesticide and replace the traditional copper agents in the agricultural area.
Table 1 The EC50 values of CuCl2·2H2O, Kocide and Silica-NMP-Cu against Psl, Aac, Xcc and Cmm Percentage of Treatment
Toxicity
Correlation
EC50 (mg
Concentration of
equations
(r2)
L-1)
copper (mg L-1)
Psl
y =1.273x+2.905
0.9742
44.23
16.54
Aac
y =3.423x-0.139
0.9738
31.72
11.86
Xcc
y=5.266x-1.903
0.9491
20.46
7.65
Cmm
y=3.702x+0.476
0.9215
16.67
6.23
Psl
y=2.399x+0.043
0.9881
116.49
58.83
Bacterial copper (%)
CuCl2·2H2O
Kocide
37.4
50.5
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Silica-NMP-C
Aac
y=3.267x-0.767
0.9692
58.24
29.41
Xcc
y=3.106x-0.107
0.9083
44.08
22.26
Cmm
y=2.621x+1.728
0.8185
17.72
8.95
Psl
y =2.394x-0.001
0.9706
122.74
20.74
Aac
y=2.820x-0.206
0.9664
70.16
11.86
Xcc
y=2.755x+0.098
0.8729
60.16
10.17
Cmm
y=2.616x+1.453
0.8115
22.70
3.84
16.9 u
The antimicrobial mechanism The effects of CuCl2·2H2O and Silica-NMP-Cu on 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 varied from 10 to 40 µL. The fluorescence quenching occurs due to the interactions between copper ions with the sulfydryl (-SH), amino (-NH2) and carboxyl (-COOH) of the BSA. When BSA is dropwise added with 10, 20, 30, 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 sulfydryl (-SH), amino (-NH2) and carboxyl (-COOH) groups of bacterial protein which causes plant diseases, and then restrains bacteria growth.
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Figure 4 Fluorescence spectra of BSA in the different adding volume of CuCl2·2H2O (a) and Silica-NMP-Cu (b)
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 presents significantly lasting time under treatments of nano-silica 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 same advantage of 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 antimicrobial activity of copper and plant disease resistance of nano-silica.
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Table 2 The concentration of SA in cucumber leaves after treatment of water, CuCl2·2H2O, nano-Silica and Silica-NMP-Cu The concentrations of SA (µg L-1)
Concentration Treatment
CK
CuCl2·2H2O
Nano-silica
Silica-NMP-Cu
(mg L-1)
6h
1d
2d
3d
4d
0
45.67
51.34
49.23
44.37
50.45
12.5
287.63
210.31
139.06
107.84
78.75
25
404.00
369.00
227.48
158.68
117.33
50
539.25
487.94
276.95
198.34
157.26
100
–
–
–
–
–
12.5
182.28
176.15
153.26
133.21
110.78
25
275.79
256.21
239.52
183.39
157.01
50
345.37
325.82
300.75
279.35
256.04
100
543.42
430.29
314.56
226.91
180.94
12.5
174.53
162.38
151.35
139.77
128.63
25
253.75
238.47
227.61
203.79
185.34
50
351.06
338.77
309.42
281.93
278.15
100
521.77
418.79
307.41
248.35
195.45
The translocation of Silica-NMP-Cu in plant 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 lower leaves of cucumber. The fluorescence can also be observed in the lower leaves after 6 h treatment in the upper leaves of cucumber. However, the fluorescence cannot be found in cucumber seedling tissues after Kocide treatment 17
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with the same procedure. The results indicate that the movement of Silica-NMP-Cu probably depends on water transportation of plant, especially the transpiration. Being lack of systemic action, traditional copper-based bactericides should be distributed uniformly and applied 2-3 times with one week interval to control plant disease.38 Due to 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.
Figure 5 The distribution of FMN-Silica-NMP-Cu in leaves: cell wall (a), vascular (b), stomatal cell (c) and microtubule (d)
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 control group (Figure 6d), cucumbers grow well without malformed, chlorotic and necrotic seedlings. However, under the treatment of Kocide (Figure 6b) at concentration of 300 mg L-1, the slight inter-veinal chlorosis on leaves of cucumber is appeared, and the leaves of cucumber exhibit serious chlorotic 18
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and curly appearance under treatment of CuCl2·2H2O with concentration of 50 mg L-1 (Figure 6c). Meanwhile, effects of Silica-NMP-Cu on 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 treatment have no obvious difference with the control group. Therefore, the Silica-NMP-Cu is safe for cucumber seedling growth without phytotoxicity.
Figure 6 Effects of Silica-NMP-Cu (a) and Kocide (b) with concentration of 300 mg L-1, CuCl2·2H2O (c) with concentration of 50 mg L-1, and 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 cucumber
cucumber
Dosage (mg L−1)
Plant height(cm) Pre-treatment 7 DAT
Rate of increase (cm/d)
Fresh weight(g) Pre-treatment 7 DAT
Rate of increase (g/d)
CK
24.32
35.85
0.47a*
16.85
31.56
0.87a
37.5
23.57
33.24
0.41a
16.66
30.23
0.81a
100
24.21
34.12
0.41a
17.28
30.91
0.79a
300
24.55
32.91
0.34a
17.14
31.01
0.81a
Cucumis sativus L. cv. Zhongnong 12
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CK
27.85
44.63
0.60a
20.51
46.61
1.27a
37.5
25.66
43.02
0.68a
19.96
45.42
1.28a
100
26.28
44.35
0.69a
20.64
46.86
1.27a
300
28.1
45.16
0.61a
21.32
48.28
1.26a
CK
28.51
45.22
0.59a
18.51
46.87
1.53a
Cucumis sativus
37.5
29.96
46.43
0.55a
19.96
47.74
1.40a
L. cv. Biyu
100
30.64
47.15
0.54a
20.64
48.52
1.35a
300
31.32
46.93
0.50a
21.32
48.93
1.30a
Cucumis sativus L. cv. Zhongnong No.6
* Values marked with letters are significantly different according to Duncan test (P