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Nanobrass CuZn nanoparticles as foliar spray non phytotoxic fungicides Orestis Antonoglou, Julietta Moustaka, Ioannis Dimosthenis Adamakis, Ilektra Sperdouli, Anastasia A. Pantazaki, Michael Moustakas, and Catherine Dendrinou-Samara ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b17017 • Publication Date (Web): 09 Jan 2018 Downloaded from http://pubs.acs.org on January 12, 2018

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ACS Applied Materials & Interfaces

Nanobrass CuZn nanoparticles as foliar spray non phytotoxic fungicides Orestis Antonogloua, Julietta Moustakab,c, Ioannis-Dimosthenis S. Adamakisb, Ilektra Sperdoulib, Anastasia A. Pantazakid, Michael Moustakasb,e,* , Catherine Dendrinou-Samaraa* a

Lab. of Inorganic Chemistry, Department of Chemistry, Aristotle University of Thessaloniki, 54124

Thessaloniki, Greece b c

Department of Botany, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece

Lab. of Molecular Entomology, Department of Biology, University of Crete, Voutes University

Campus, 70013 Heraklion, Crete, Greece d

Lab. of Biochemistry, Department of Chemistry, Aristotle University of Thessaloniki, 54124

Thessaloniki, Greece e

Division of Botany, Department of Biology, Faculty of Science, Istanbul University, 34134 Istanbul,

Turkey

*

Corresponding authors

E-mail address: [email protected] (C. D.-S.) E-mail address: [email protected] (M. M.)

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Keywords: Bimetallic nanoparticles Saccharomyces cerevisiae Hydrogen peroxide Lycopersicon esculentum Non-photochemical quenching Photoprotection Reactive oxygen species Redox state

ABSTRACT Inorganic nanoparticles (NPs) have been proposed as alternative fertilizers to suppress plant disease and increase crop yield. However, phytotoxicity of NPs remains a key factor for their massive employment in agricultural applications. In order to investigate new effective, non phytotoxic and inexpensive fungicides, in the present study CuZn bimetallic nanoparticles (BNPs) have been synthesized as antifungals, while assessment of photosystem II (PSII) efficiency by chlorophyll fluorescence imaging analysis is utilized as an effective and non-invasive phytotoxicity evaluation method. Thus, biocompatible coated, non-oxide contaminated CuZn BNPs of 20 nm crystallite size and 250 nm hydrodynamic diameter have been prepared by a microwave-assisted synthesis. BNPs' antifungal activity against Saccharomyces cerevisiae was found enhanced compared to monometallic Cu NPs. Reactive oxygen species (ROS) formation and photosystem II (PSII) functionality at low light (LL), and high light (HL) intensity were determined on tomato plants sprayed with 15 and 30 mg L-1 of BNPs for the evaluation of their phytotoxicity. Tomato leaves sprayed with 15 mg L-1 of BNPs displayed no significant difference in PSII functionality at LL, while exposure to 30 mg L-1 of BNPs for up to 90 min resulted in a reduced plastoquinone (PQ) pool that gave rise to H2O2 accumulation, initiating signaling networks and regulating acclimation responses. After 3 h of exposure to 30 mg L-1 of BNPs, PSII functionality at LL was similar to control, indicating non phytotoxic effects. Meanwhile, exposure of tomato leaves either enhanced (15 mg L-1), or did not have any significant effect (30 mg L1

) on PSII functionality at HL, attributed to the absence of semiconducting oxide phases and

photochemical toxicity-reducing modifications. The use of chlorophyll fluorescence imaging analysis is recommended as a tool to monitor NPs behavior on plants.

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1. Introduction During the last decade, the remarkable progress in nanotechnology has extended the application of nanomaterials and nanoparticles (NPs) in the agriculture sector 1. The NPs’ utilities in agriculture are several, but the most evident seem to be their use as fertilizers to enhance plant growth and yield and as pesticides for pest and disease management to improve plant protection 2, 3. In medical microbiological studies, inorganic metal-based NPs have been reported to exhibit antimicrobial activity 4, encouraging their use as alternative tools of disinfection in agriculture (nanoagroparticles). Some of the most extensively examined nanoagroparticles are silver, copper, zinc and titanium based NPs 5. Specificity, biocompatibility, solubility, stability and steady antimicrobial action are the desirable characteristics that should arise out of rational design 2. Still, the bioavailability and toxicity of nanoagroparticles remain key factors for their massive employment 6, 7. Most NPs cause phytotoxicity at some concentrations affecting the productivity of crops, degrading their quality and lowering the germination rate of seeds 8. To understand the possible benefits of applying nanotechnology to agriculture, the first step should be to analyze the effects of NPs in plants 9. In most studies contacted for the assessments of NPs’ phytotoxicity the used tests were germination inhibition, root elongation index, and shoot or root biomass decrease Stampoulis et al.

11

10-12

. However,

concluded that phytotoxicity tests such as root elongation and seed germination are

not appropriate to evaluate NPs toxicity to plant species since they are not sensitive enough. Meanwhile, the use of chlorophyll a fluorescence measurements appears to be a promising tool to analyze the effects of NPs on plants and monitoring plant-NPs interactions

13, 14

. Measurements of

chlorophyll a fluorescence emissions by photosystem II (PSII) is a well-known and widely used method to determine and detect the impact of stress on plants and probe the function of the photosynthetic apparatus

15-17

. The method of chlorophyll fluorescence imaging also permits the

visualization of the spatiotemporal variations of the abiotic stress effects on leaf photosynthesis, and it provides a non-invasive evaluation of PSII efficiency

18-20

. The disturbance of photosynthesis at the

molecular level is associated with low electron transport through PSII and/or with structural injury to PSII and the light-harvesting complexes 21. By measuring photochemical fluorescence quenching it is possible to measure the fraction of open or closed PSII reaction centers that is the redox state of the plastoquinone (PQ) pool. Recently it has been documented that toxicity effects of NPs’ are generated by lipid peroxidation, protein and DNA damage, due to oxidative stress generated by reactive oxygen species (ROS)

8, 22

.

During the light reactions of photosynthetic process, singlet oxygen (1O2) can be produced by PSII, if the chlorophyll triplet state transfers excitation energy to O2 23. Under excess light conditions or stress conditions, PSI acceptor site can directly reduce oxygen leading to the formation of superoxide ions (O2•-) that are rapidly dismutated to hydrogen peroxide (H2O2) by superoxide dismutase (SOD) and then converted to water by ascorbate peroxidase (APX) 24. An imbalance between ROS production and their detoxification results in oxidative damage

25

, and thus during various stress conditions,



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chloroplasts are the main targets of ROS-linked damage 26. The response of plants to this imbalance, before damage occurs in their cellular structures, is crucial for maintaining high rates of photosynthesis and also for their survival

25, 27

studied by estimating ROS

. Consequently, the toxicological impacts of NPS on plants can also be

8, 28

. However, ROS are not only harmful species causing oxidative stress

but are also important messengers involved in redox signaling, initiating signaling networks and regulating defense and acclimation responses to stress conditions ROS in the regulation of diverse stress responses in plants

30, 31

14, 29

. H2O2 play a major role among

.

Metallic and/or oxide Cu or ZnO NPs, have been previously evaluated by us and others as antimicrobial/nanoagrochemical agents against bacterial and fungal strains, as well as fertilizers 2, 3, 5, 32, 33

with a view to a lower formulated product and active component dose application than the

commercial agrochemicals 34. Moreover, phytotoxicity assessments point towards their occasional use with no deleterious consequences on plants. Recently, there is an emerging research on the collective properties of the most promising inorganic antimicrobial agents, either in a mixed ionic form or as bimetallic nanomaterials

35-38

. Different metals can be more effective in fighting off pathogenic

microorganisms in combination than as distinct units while the intrinsic efficacy of copper alloys, such as brass (Cu-Zn alloy), as advanced healthcare "touch surfaces" to eliminate an extensive variety of microorganisms that endanger public health is well projected

39

. However, synthesis of CuZn

nanoparticles known as nanobrass is challenging and is scarcely reported before

40, 41

, while to our

knowledge antimicrobial/agrochemical evaluation of nanobrass remains absent. In continuation of our efforts 33, 34, 38, 42, in the present work CuZn bimetallic nanoparticles (BNPs) were effectively synthesized through a microwave assistant polyol process where nitrate salts have been used as precursors and triethylene glycol has been employed in a triple role (surfactant/solvent/reductive agent). Microwave assisted synthesis has been selected as a fast, inexpensive and green mass-production approach. The BNPs were characterized through X-ray diffraction (XRD) and thermogravimetric analysis (TGA) while their primary particle size, morphology and composition has been determined by transmission electron microscopy (TEM), scanning electron microscope (SEM) and by energy dispersive X-ray spectrometry (EDX). The hydrodynamic size of BNPs in water was measured by dynamic light scattering (DLS) and their ionic release were determined by flame atomic absorption spectrophotometry (FAAS). The antifungal activity of the synthesized BNPs against S. cerevisiae was measured by the means of optical density while fungal inhibition and intracellular ROS production have been evaluated. Phytotoxicity effects were determined on tomato plants sprayed with two different concentrations of BNPs. ROS formation, measured through H2O2 accumulation, and PSII functionality, measured by chlorophyll fluorescence imaging analysis, at low and high light intensities, after 30 min, 90 min and 3 h exposure, were evaluated for any detrimental effects.


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2. Materials and methods

2.1. Synthesis of CuZn BNPs A microwave assisted polyol process (MW-PP) was selected for the synthesis of CuZn BNPs. For that purpose, a commercial microwave accelerated reaction system, Model MARS 6-240/50-CEM, was utilized. This system runs at a maximum frequency of 2450 MHz and a power of 1800 W where, unlike domestic analogues, additional aspects, namely complete automation and regulation of pressure, power, temperature and time, are on hands. The reaction was carried out in a double-walled vessel consisting of an inner Teflon container liner where temperature and pressure sensors are connected and an outer composite sleeve of high strength polymer. Zn(NO3)2·4H2O (2.0 mmol) and Cu(NO3)2·3H2O (2.0 mmol) were mixed and dissolved in 40mL of triethylene glycol (TrEG), followed by the transfer to the autoclave. The reaction was carried out at 245 °C with a hold time of 30 min and a ramp time heating step (from 25 °C to 245 °C) set at 15 min. After MW-PP, cooling of the autoclave till room temperature takes place for ~ 30 min, followed by centrifugation at 5000 rpm, where supernatants were disposed, and a grey-black precipitate was acquired and washed three times with ethanol, for the removal of unreacted precursors. All the reagents were of analytical grade and were used without any further purification. The utilized products were the following: Copper (II) nitrate trihydrate, Cu(NO3)2·3H2O (Merck, ≥ 99.5%, M = 241.60 g/mol), zinc (II) nitrate tetrahydrate, Zn(NO3)2·4H2O (Merck, ≥ 99.5%, M = 261.44 g/mol) and TrEG (Merck, ≥ 99%, M = 150.17 g/mol).

2.2. Characterization of CuZn BNPs The crystal structure of the synthesized BNPs was investigated through X-ray diffraction (XRD) performed on a Philips PW 1820 diffractometer at a scanning rate of 0.050/3s, in the 2θ range from 10 to 90 °, with monochromatized Cu Kα radiation (λ = 1.5406 nm). Thermogravimetric analysis (TGA) was employed to calculate the wt % percentage of the organic coating of the BNPs using SETA-RAM SetSys-1200 and carried out in the range from room temperature to 900oC at a heating rate of 10o C min-1 under an N2 atmosphere. Powder morphology of CuZn BNPs was determined by a JEOL 840A scanning electron microscope (SEM) coupled with energy dispersive X-ray spectrometry (EDX) spectra for estimating the inorganic composition. Primary particle size and morphology was determined by conventional transmission electron microscopy (TEM) images obtained with JEOL JEM 1010 microscope operating at 100 kV. For TEM observations we used suspensions of the nanoparticles deposited onto carboncoated copper grids. The hydrodynamic size of CuZn BNPs was determined by dynamic light scattering (DLS) measurements, carried out at 25 °C utilizing a Nano ZS Malvern apparatus. Ionic release estimation



was determined by flame atomic absorption spectrophotometry (FAAS), using a Perkin Elmer instrument model AAnalyst 800.

2.3. Evaluation of CuZn BNPs for antifungal activity The antifungal activity of the BNPs was evaluated as previously described by us 38, using the minimal medium salts broth (MMS) as a cultivation media and pH adjustments through monitoring in removed aliquots. Cultures consisting of 25 mL MMS, 5 mg of yeast S. cerevisiae dry mass and 15, 30, 50 and 100 mg L-1 of BNPs, respectively, as well as reference cultures were used. Pre-cultures were grown at 30 oC until the exponential phase. Fungal growth was estimated based on the absorbance at 600 nm, by using a spectrophotometer (Thermo Electron Corporation, Helios γ, USA). The absorbance values of the reference cultures were considered as the 100% of fungal growth. The MIC in mg L-1 is defined as the minimum concentration that inhibits the 50% of the fungal growth (IC50 value). 2.4. Estimation of reactive oxygen species in the fungal cultures Extracellularly and/or intracellularly ROS produced during the incubation of S. cerevisiae cultures with BNPs were calculated by the nitroblue tetrazolium (NBT) reduction protocol as previously described by Giannousi et al.

. 200 µL of fungal cells suspension (with an OD600 = 1.1

42

were incubated with two BNPs concentrations (15 and 30 mg L-1) and 500 µL of 1 g L-1 NBT at 30 °C for two different incubation times, 1 h and 3 h. Then, 100 µL of 0.1M HCl were added and the tubes were centrifuged at 1500 g for 10 min. The OD at 575 nm of the supernatants was measured for the extracellular ROS. The pellets were treated with 600 µL DMSO and were placed in a sonication bath to extract the reduced NBT. Lastly, 500 µL MMS was added and the reduced NBT from cells was measured as OD at 575 nm. Extracellular and intracellular ROS produced by cultures grown in the absence of BNPs for 1 h and 3 h were included in the study as a control.

2.5. Plant material and growth conditions Tomato (Lycopersicon esculentum Mill. cv. Meteor) plants 20 cm in height (Fig. S1 in supplemental information) were purchased from the market and transferred to a growth chamber with a 10-h photoperiod, 20 ± 1/18 ± 1 °C day/night temperature, photosynthetic photon flux density (PPFD) 130 ± 20 µmol quanta m-2 s-1 and relative humidity 50 ± 5/60 ± 5% day/night.

2.6. Exposure of tomato plants to CuZn BNPs Tomato (Lycopersicon esculentum Mill. cv. Meteor) plants were spray treated with two CuZn BNPs concentrations (15 and 30 mg L-1) under low light (LL) conditions, while control plants were sprayed with distilled water.


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2.7. Chlorophyll fluorescence analysis Chlorophyll fluorescence analysis was performed at room temperature in tomato leaves, control and exposed to BNPs, using an imaging-PAM fluorometer (Walz, Effeltrich, Germany), as described by Sperdouli and Moustakas 43. All samples were dark adapted for 15 min before measurement. Eleven areas of interest (AOI) were selected in each leaf. First Fo (minimum chlorophyll a fluorescence in the dark) and Fm (maximum chlorophyll a fluorescence in the dark) values were measured with darkadapted samples. Fm was obtained with a saturating pulse of 6000 µmol photons m-2 s-1 (470 nm, 800 ms) followed by application of actinic light (AL) with blue LED wavelength (470 nm) to assess steadystate photosynthesis (Fs). A low light intensity of AL 140 µmol photons m–2 s–1 (LL, low light), selected to match that of the growth conditions and a high light intensity of AL 900 µmol photons m–2 s–1 (HL, high light) were selected. The illumination time for each light intensity was 5 min with repetitive measurements of Fo΄ (minimum chlorophyll a fluorescence in the light) and Fm΄ (maximum chlorophyll a fluorescence in the light) every 20 s. The measured parameters were the maximum quantum efficiency of PSII [Fv/Fm = (Fm – Fo)/Fv], the effective quantum yield of photochemical energy conversion in PSII [ΦPSII = (Fm΄ - Fs)/Fm], the yield of regulated non-photochemical energy loss in PSII [ΦNPQ = 1 - ΦPSII – 1/{NPQ + 1 + qL (Fm/Fo - 1)}], the quantum yield of non-regulated energy loss in PSII [ΦNO = 1/{NPQ + 1 + qL (Fm/Fo - 1)}], and the redox state of the plastoquinone (PQ) [qP = (Fm΄ Fs)/(Fm΄ - Fo΄)], calculated all according to Kramer et al. 44. The non-photochemical quenching (NPQ), that estimates heat dissipation of excitation energy, was calculated as (Fm - Fm΄)/Fm΄, while the electron transport rate (ETR) was calculated as described previously 43. Color-coded images of Fv/Fm, ΦPSII, ΦNPQ, ΦNO, and qP after 5 min illumination with 140 µmol photons m–2 s–1 are also presented.

2.8. Imaging of hydrogen peroxide production The production of hydrogen peroxide in tomato control plants (sprayed with distilled water) and CuZn BNPs sprayed leaves was evaluated by 2', 7'-dichlorofluorescein diacetate (DCF-DA, Sigma) as described previously 14, 31.

2.9. Statistical analyses Three replicates were conducted for each dose during the antifungal screening (evaluation of antifungal activity and estimation of ROS in the fungal cultures) and a one-way ANOVA statistical analysis was employed by treating each dose as a different level. This provides the SD values for each dose as well as the P values of statistical comparison with the controls of the experiment. Additionally, a nonlinear curve fit-growth/sigmoidal-dose response was applied to estimate IC50 values along with SD error. The paired t - test at a level of P < 0.05 was applied to analyze differences between chlorophyll fluorescence parameters 14, 45.



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3. Results and Discussion 3.1. Characterization of BNPs and synthetic aspects Microwave assisted manufacture of inorganic materials is a mass-production ‘green’ synthetic route that is fast, cheap, simple and energy efficient 46. Energy transport from microwaves to materials is reported to happen via resonance or relaxation, leading to swift heating. In our study, a microwaveassisted polyol process has been employed for the synthesis of CuZn BNPs (nanobrass) via the reaction of Zn and Cu nitrate salts with a biocompatible polyol member, triethylene glycol (TrEG). The principal of polyol process is using high boiling polyalcohols in the triple role of solvent, capping agent and reducing agent 47. The utilization of this process results in an effective control over the physicochemical characteristics of the NPs through size, shape, structure and surface chemistry known as 4S’s while eliminating the appearance of by-products. Moreover, NPs with a significant amount of biocompatible organic surface coating that provides high hydrophilicity are produced in that manner. The crystal structure of the BNPs was investigated through X-ray diffraction (XRD) at room temperature. The observed peaks at 42.4 o, 49.78 o and 73.47 o are attributed to the planes (111) (200) (220) of α-brass fcc phase (JCPDS no. 50-1333 and no. 65-6567), respectively, and are distinctively shifted from the fcc metallic copper pattern (JCPDS no. 04-0836), as reported elsewhere for brass BNPs 48

, and shown in Fig.1. Moreover, by using MDI’s Jade software for a whole peak fit and lattice

calculation and based on the main crystallographic plane (111) of the fcc structure, the lattice parameter, a, was found 3.6294 Å which is noticeably distorted compared to the value of metallic copper (3.615 Å). Brass is classified as a substitutional alloy and the observed distortion of the lattice parameter stems from the substitution of copper atoms by zinc ones, as zinc has a slightly higher atomic radius (1.34 instead of 1.28 Å). It has been shown previously that brass materials can adapt many crystal structures, namely α, β or γ forms, where copper rich or zinc rich compositions can coexist due to the high miscibility of the two metals

40, 41, 48, 49

. Regarding the peak at 35.84 o, it can be assigned to either

metallic hexagonal Zn (JCPDS no. 04-0831) or γ-brass (JCPDS no. 5-6566). However, as it can be seen in Fig. 1, the majority of the peaks of the hexagonal Zn pattern are absent and in so the peak at 35.84 o is attributed to the (222) plane of the zinc-rich γ-brass (JCPDS no. 5-6566). By utilizing the easy quantitative option of MDI’s Jade, the % wt crystal composition is calculated at 76% α-brass and 24% γ-brass. The peak at 22.89 o correlates to the partial formation of graphitic carbon via dehydrogenation oxidation reactions and part-transformation of the polyols during the synthesis and is in agreement with previous results of our group for CuFe BNPs 38. The as formed graphitic carbon enhances the reductive atmosphere of the synthesis and is involved in the formation mechanism of the bimetallic NPs

38

. By

obtaining the full width at half-maximum (FWHM) of the main peak and by applying the Scherrer equation, crystallite size was calculated at 20 nm. In so, the absence of oxide phases ZnO, Cu2O, CuO and precursor reflections was attained by setting the synthetic reaction temperature (245 °C) close to the boiling point of TrEG (285 °C) that favors the formation of pure bimetallic NPs 50.


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Figure 1. The X-ray diffraction (XRD) patterns of the synthesized brass BNPs at 10-85 degrees and at 40-52 degrees (inset). Fig. 2 illustrates the SEM image of the synthesized CuZn BNPs. Seven spectrum areas were chosen for the estimation of the inorganic composition of BNPs by EDX. Results are presented in Table 1, where spectrum 7 provides a whole image elemental composition of Cu0.56Zn0.44. By combining the easy quantitative results by XRD and the elemental composition given by SEM, the resulted composition of the as prepared BNPs is given as α-Cu47Zn29/γ-Cu9Zn15. Composition correlates well with copper-rich and zinc-rich areas presented in the SEM image (spectrums 2-5), as well as with the maximum miscibility of Zn in the α-brass fcc phase, which is 38.4 % wt. Thermogravimetric analysis (TGA) was employed to calculate % wt of the organic coating of the BNPs. At Fig. S2 (supplemental information) the observed weight loss corresponds to the gradual decomposition of TrEG form the surface of BNPs. The cumulative organic content derived by the end point of the curve is 32 % wt.



Figure 2. Scanning electron microscope (SEM) image of the synthesized CuZn BNPs with the seven spectrum areas used for the calculation of the elemental composition by energy dispersive X-ray spectrometry (EDX). TEM images of the BNPs (Fig. 3) reveal the formation of nano-clusters with a size of approximately 250 nm. Additionally, by zooming in the edge of the clusters (Fig. 3 inset) BNPs with a size of roughly 20 nm can be spotted. DLS measurements of the aqueous suspensions of the BNPs (Fig. S3, supplemental information) provided a hydrodynamic diameter estimation of 239 nm with a polydispersity index (PdI) of 0.272 (mean size provided by intensity and numbers measurements). The formation of nano-clusters (Fig. 3) is in good agreement with the data derived from the DLS measurements (Fig. S3). The amphiphilic nature of TrEG, along with its polar nature as a solvent and the lipophilicity of CuZn BNPs leads to a cluster formation. TrEG, which amounts to 32 % wt of the BNPs (Fig. S2), constitutes the clustering agent, encapsulating the as-formed hydrophobic brass nanoparticles during the synthesis. Furthermore, the hydrodynamic diameter of 41 nm provided by the DLS numbers measurement (Fig. S3) could be linked to the encapsulated CuZn BNPs inside the clusters (Fig. 3 inset). This observation is supported by crystallite size data received by XRD (Fig. 1).

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Figure 3. Transmission electron microscopy (TEM) images of the synthesized CuZn BNPs. In the inset NPs of roughly 20 nm can be spotted. The stability of the CuZn BNPs in water was investigated through leaching tests by measuring the ionic release of 30 mg L-1 aqueous suspensions of BNPs after 24h of incubation, using FAAS. The amount of leached ions was found 1.78 mg L-1 for Cu and 2.19 mg L-1 for Zn. The relatively low amount of leached ions ensures the stability of the aqueous suspensions, while the higher values found for zinc compared to copper are due to dezincification, a common phenomenon for brass materials.



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Table 1. Elemental composition percentages of the synthesized CuZn BNPs calculated by EDX for all spectrum areas shown in SEM image (Fig. 2). Spectrum

Cu

Zn

Spectrum 1

50.88 %

49.12 %

Spectrum 2

48.82 %

51.18 %

Spectrum 3

47.59 %

52.41 %

Spectrum 4

52.76 %

47.24 %

Spectrum 5

53.49 %

46.51%

Spectrum 6

50.88 %

49.12%

Spectrum 7

55.95 %

44.05 %

Mean

51.48 %

48.52 %

STD

2.84

2.84

Max.

55.95 %

52.41 %

Min.

47.59 %

44.05 %

3.2. Evaluation of CuZn BNPs for antifungal activity The antifungal activity of CuZn BNPs against the fungal strain S. cerevisiae is estimated in terms of growth inhibition by measuring the optical density of fungal cultures at 600 nm, 5 h and 24 h after the injection of various suspensions of BNPs (15, 30, 50 and 100 mg L-1) at the logarithmic growth phase of the cultures (Fig. 4). Cultures grown in the absence of BNPs are also included in the study as cultures of reference (control). S. cerevisiae is one of the most extensively examined eukaryotic organisms as a microbiological model. Fungal growth inhibition is observed in a concentration-dependent manner. The minimal inhibitory concentration (MIC) values (IC50) which inhibits the 50% of fungal growth, calculated after 5 h and 24 h of incubation, are 27.1 ± 1.89 mg L-1 and 36.9 ± 1.63 mg L-1, respectively. Fungal growth in the absence of BNPs accounts for 100% fungal growth. A nonlinear curve fit-growth/sigmoidal-dose response was applied to estimate the IC50 and values are mean of three replicates along with SD error. All values were found statistically significant, P ≤ 0.01. The IC50 values calculated for CuZn BNPs (Fig. 4) are significantly lower than values obtained in a similar manner for synthesized monometallic Cu NPs, 63 ± 1.72 mg L-1, previously reported by our group

. The co-inclusion of zinc appears to enhance the antifungal activity of the

38, 42

BNPs. As the IC50 values were found below 50 and 100 mg L-1 BNPs, these concentrations are not included in the remaining experiments.


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Figure 4. Saccharomyces cerevisiae growth inhibition (%) and IC50 values estimated by optical density measurements 5 (a) and 24 h (b) after the incubation of fungal cultures with CuZn BNPs. Values are mean of three replicates along with SD error bars. All values were found statistically significant, P ≤ 0.01(**).

3.3 Estimation of reactive oxygen species in the fungal cultures The nitroblue tetrazolium (NBT) assay was utilized to calculate the extracellular and intracellular superoxide radicals (O2•−) produced during the incubation of fungal cultures with CuZn BNPs as previously described by Giannousi et al.

42

. NBT is able to bypass the cell membrane and enter the

•−

fungal cells. In the presence of O2 , NBT gets reduced to its formazan blue-derivative that can be quantified by measuring the absorbance of the solution at 575 nm. Thus, higher measured concentrations of the formazan blue-derivative translate to higher production of O2•−. Two different ACS Paragon Plus13 Environment


concentrations of BNPs (15 and 30 mg L-1) were evaluated at two different incubation times, 1 h and 3 h, and the results are given in Fig. 5. Values are mean of three replicates along with SD (P ≤ 0.01).

Figure 5. Intracellular O2•− generated and detected by the NBT assay 1 h and 3 h after the incubation of fungal cultures with CuZn BNPs (15 and 30 mg L-1). Values are mean of three replicates along with SD error bars. All values were found statistically significant, P ≤ 0.01(**). No significant extracellular O2•− were detected for both tested concentrations of BNPs and both incubation times suggesting that CuZn BNPs enter the fungal cells. The intracellular O2•− calculated for cultures exposed to 15 and 30 mg L-1 of CuZn BNPs for 1 h did not differ compared to controls. However, in cultures incubated with the same concentrations of BNPs but for 3 h, a concentrationdependent increase was observed. This increased intracellular production of O2•− is expected, as intracellular production of ROS is already established as a possible antimicrobial mechanism for metallic NPs 4.

3.4. Chlorophyll fluorescence analysis 3.4.1. Allocation of the absorbed light energy in PSII after exposure to CuZn BNPs The allocation of absorbed light energy in PSII was estimated by measuring the quantum yield of photochemical energy conversion (ΦPSII), the quantum yield of regulated non-photochemical energy loss (ΦNPQ), and the quantum yield of non-regulated non-photochemical energy loss (ΦNO), in control and tomato leaves sprayed with CuZn BNPs. The allocation of absorbed light energy in PSII in tomato leaves exposed to 15 mg L-1 CuZn BNPs


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for 30 min, 90 min, and 3h did not differ compared to controls (Fig. 6a, c, e) at the low light (LL) intensity (140 µmol photons m–2 s–1), but in tomato leaves exposed to 30 mg L-1 CuZn BNPs for 30 min, and 90 min, ΦPSII decreased significantly (Fig. 6a), while there was a significant increase in ΦNPQ (Fig. 6c). This significant increase in ΦNPQ resulted in an unchanged ΦNO compared to control tomato leaves (Fig. 6e). The allocation of absorbed light energy in PSII in tomato leaves exposed to 30 mg L-1 CuZn BNPs for 3 h at LL did not differ compared to controls (Fig. 6a, c, e). At high light (HL) intensity (900 µmol photons m–2 s–1), the allocation of absorbed light energy in PSII in tomato leaves exposed to either 15 or 30 mg L-1 CuZn BNPs at all exposure durations, did not differ compared to control ones (Fig. 6b, d, f), with the exception of a significant increase of ΦPSII in tomato leaves exposed to 15 mg L-1 CuZn BNPs for 90 min (Fig. 6b).

Figure 6. Changes in the allocation of the absorbed light energy in PSII of tomato leaves exposed to distilled water (control), or 15 and 30 mg L-1CuZn BNPs concentrations for 30 min, 1 h and 30 min, and 3 h. The illumination time was 5 min, and the actinic light (AL) 140 µmol photons m–2 s–1 (low light, LL), or 900 µmol photons m–2 s–1 (high light, HL). The quantum efficiency of PSII



photochemistry (ΦPSΙΙ), at low LL (a), and HL (b), the quantum yield for dissipation by downregulation in PSII (ΦNPQ) at low LL (c), and HL (d), and the quantum yield of non-regulated energy dissipated in PSII (ΦNO) at low LL (e), and HL (f). Error bars on columns are standard deviations based on five leaves (each with eleven areas of interest, AOI) from five plants. Columns with same letters are statistically not different results (P < 0.05).

3.4.2. Non-photochemical quenching and electron transport rate in response to CuZn BNPs The non-photochemical quenching that reflects heat dissipation of excitation energy in the antenna system (NPQ), did not differ compared to control ones at LL in tomato leaves exposed to 15 mg L-1 CuZn BNPs at all exposure durations, but in tomato leaves exposed to 30 mg L-1 CuZn BNPs for 30 min, and 90 min, NPQ increased significantly (Fig. 7a). In tomato leaves exposed to 30 mg L-1 CuZn BNPs for 3h at LL, NPQ did not differ compared to controls (Fig. 7a). At HL intensity in tomato leaves exposed either to 15 or 30 mg L-1 CuZn BNPs, at all exposure durations, NPQ did not differ compared to control ones (Fig. 7b). The relative PSII electron transport rate (ETR) did not differ compared to control ones at LL in tomato leaves exposed to 15 mg L-1 CuZn BNPs at all exposure durations (Fig. 7c), but in tomato leaves exposed to 30 mg L-1 CuZn BNPs for 30 min, and 90 min, ETR decreased significantly (Fig. 7c). In tomato leaves exposed to 30 mg L-1 CuZn BNPs for 3h at LL, ETR did not differ compared to controls (Fig. 7c). At HL intensity, ETR in tomato leaves exposed to 15 mg L-1 CuZn BNPs for 30 min, and 3 h, or at all exposure durations to 30 mg L-1 CuZn BNPs, did not differ compared to control ones (Fig. 7d), while in tomato leaves exposed to 15 mg L-1 CuZn BNPs for 90 min, ETR increased significantly compared to control ones (Fig. 7d).

3.4.3. The redox state of PSII after exposure to CuZn BNPs The redox state of the PQ pool (qP), did not differ compared to control ones at LL in tomato leaves exposed to 15 mg L-1 CuZn BNPs at all exposure durations (Fig. 7e), but it was significantly less oxidized in tomato leaves exposed to 30 mg L-1 CuZn BNPs for 30 min, and 90 min, (Fig. 7e). In tomato leaves exposed to 30 mg L-1 CuZn BNPs for 3 h at LL, the redox state of PQ pool did not differ compared to controls (Fig. 7e). At HL intensity in tomato leaves exposed either to 15 or 30 mg L-1 CuZn BNPs, at all exposure durations, qP, did not differ compared to control ones, with the exception of tomato leaves exposed to 15 mg L-1 CuZn BNPs for 90 min, and 3 h, when it was significantly more oxidized compared to control ones (Fig. 7f).


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Figure 7. Changes in the chlorophyll fluorescence parameters in tomato leaves exposed to distilled water (control), or 15 and 30 mg L-1CuZn BNPs concentrations for 30 min, 1 h and 30 min, and 3 h. The illumination time was 5 min, and the actinic light (AL) 140 µmol photons m–2 s–1 (low light, LL), or 900 µmol photons m–2 s–1 (high light, HL). The non-photochemical fluorescence quenching (NPQ) at low LL (a), and HL (b), the relative PSII electron transport rate (ETR) at low LL (c), and HL (d), and the reduction state of the plastoquinone pool (qp) at low LL (e), and HL (f). Error bars on columns are standard deviations based on five leaves (each with eleven areas of interest, AOI) from five plants. Columns with same letters are statistically not different results (P < 0.05).

3.4.4. Spatiotemporal variation of PSII responses to CuZn BNPs Spatiotemporal heterogeneity was observed in the color-coded images of chlorophyll fluorescence parameters in tomato leaves exposed to CuZn BNPs mainly after LL treatment, and not after HL treatment. Thus, color-coded images of the parameters Fv/Fm, ΦPSII, ΦNPQ, ΦNO, and qP are presented after 5 min illumination with 140 µmol photons m–2 s–1 (Fig. 8).



Figure 8. Representative chlorophyll fluorescence images, after 5 min illumination at 140 µmol photons m-2 sec-1, of the potential (maximal) quantum yield of PSII (Fv/Fm), the actual (effective) quantum yield


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of PSII photochemistry (ΦPSΙΙ), the quantum yield for dissipation by downregulation in PSII (ΦNPQ), the quantum yield of non-regulated energy loss in PSII (ΦNO), and the reduction state of the plastoquinone pool (qp) of tomato leaves exposed to distilled water (control), or 15 and 30 mg L-1CuZn BNPs concentrations for 30 min, 1 h and 30 min, and 3 h. Vertical scale bar at the right side of the images indicates the value of false color that ranges from black (pixel values 0.0) to purple (1.0). In each image the circles represent the areas of interest (AOI), which are accompanied by values of the selected fluorescence parameter. The average value of each photosynthetic parameter is presented in the figure. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Among the chlorophyll fluorescence parameters examined, the highest spatiotemporal heterogeneity was observed in the redox state of the PQ pool (qP), in tomato leaves exposed to 30 mg L-1 CuZn BNPs (Fig. 8). Spatiotemporal heterogeneity was also observed in the quantum yield of photochemical energy conversion (ΦPSII), and the yield of regulated non-photochemical energy loss in PSII (ΦNPQ), in tomato leaves exposed to 30 mg L-1 CuZn BNPs. The highest heterogeneity compared to control was observed after 90 min exposure to 30 mg L-1 CuZn BNPs (Fig. 8).

3.5. Hydrogen peroxide production in response to CuZn BNPs To investigate H2O2 production in vivo, tomato control and CuZn BNPs sprayed leaves were incubated in darkness with 2', 7'-dichlorofluorescein diacetate (DCF-DA). The H2O2 real-time staining patterns showed almost no effect of 15 mg L-1 CuZn BNPs (Fig. 9c, e, g) on H2O2 production in tomato leaves compared to control ones (Fig. 9a,b), while after 30 min, and 90 min exposure to 30 mg L-1 CuZn BNPs (Fig. 9d, f) increased H2O2 production was detected, mainly in the leaf veins. However, after 3h exposure to 30 mg L-1 CuZn BNPs, H2O2 production was undetectable (Fig. 9h). In control leaves H2O2 production could be detected only in leaf hairs (Fig. 9b).

3.6. Phytotoxicity evaluation. When light energy is more than what can be used to drive photosynthesis, it results in the production of ROS that causes photodamage to the photosynthetic machinery, primarily PSII, resulting in a decrease in photosynthetic activity 25, 51. Extra light energy can lead to the generation of the excited triplet chlorophyll (3Chl*) if the singlet-excited chlorophyll a (1Chl*) is not de-excited 51. In PSII, the triplet state of chlorophyll (3Chl*) can transfer excitation energy to the chemically inert triplet ground state of O2 that leads to the formation of singlet oxygen (1O2) 23, 29. In the electron transport chain, a high reduced PQ pool will increase the chances for O2 to receive electrons from PSI and become overreduced, forming superoxide radicals (O2•−) 25.



Figure 9. Representative patterns of H2O2 production in tomato leaves exposed to distilled water (control), or 15 and 30 mg L-1CuZn BNPs concentrations for 30 min, 1 h and 30 min, and 3 h. H2O2 in controls (a) and (b); H2O2 after 30 min exposure to 15 (c) and 30 (d) mg L-1 CuZn BNPs; H2O2 after 1 h and 30 min exposure to 15 (e) and 30 (f) mg L-1 CuZn BNPs; H2O2 after 3 h exposure to 15 (g) and 30 (h) mg L-1 CuZn BNPs. Scale bare: 200 µm. Increased H2O2 content is indicated by light green color.


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(For interpretation to color, the reader is referred to the web version of this article). The quantum yield of photochemical energy conversion (ΦPSII) at LL decreased significantly in tomato leaves exposed to 30 mg L-1 CuZn BNPs for 30 min, and 90 min (Fig. 6a), but the significant increase in ΦNPQ (Fig. 6c) resulted in an unchanged ΦNO compared to control tomato leaves (Fig. 6e). ΦNO consists of chlorophyll fluorescence internal conversion, and intersystem crossing, which leads to the formation of 1O2 via 3Chl* 52, 53. Thus, despite the significant decrease of ΦPSII, the unchanged ΦNO suggests an almost unchanged 1O2 status. To control chloroplast ROS production effects, plants have developed several photoprotective mechanisms to counteract the harmful effects of excessive light energy absorption

25, 51

. At the

molecular level, the photoprotective mechanisms include NPQ of excessive energy as heat, scavenging of ROS by enzymatic and non-enzymatic antioxidant molecules, and modification of the photosynthetic machinery to enhance photon usage and reduce light absorption 51, 54. ΦPSΙΙ at LL decreased significantly in tomato leaves exposed to 30 mg L-1 CuZn BNPs for 30 min, and 90 min (Fig. 6a), and despite a significant increase in the photoprotective mechanisms of NPQ (Fig. 7a), the PQ pool became more reduced (Fig. 7e), with a simultaneous increase in H2O2 production (Fig. 9d, f). In terrestrial plants sufficient photoprotection can be achieved only if NPQ is regulated in such a way that PSII reaction centers remain open under given conditions 31, 55. Closed reaction centers or, in other words reduced PQ pool indicates excess excitation energy and thus an imbalance between energy supply and demand

56-58

.

Under such conditions, released H2O2 can be transmitted throughout the leaf veins to act as a longdistance signaling molecule

59

. The intracellular signaling transduction pathways concerning NPQ are

initiated by the redox state of the PQ pool

60

that controls photosynthetic gene expression

61

, also

comprising a mechanism of plant acclimation 60. This photosynthesis-derived H2O2 underlies the twin role of photosynthesis in energy fixation and gene regulation

62

and serves as a signaling molecule

triggering a stress-defense reaction in plant’s response to oxidative stress 27, 30. Zinc and Cu are important micronutrients required for plant growth and development 63. Soil Zn deficiency is a major problem and during crop developmental stages adequate Zn supply is recommended to improve productivity and the nutrient content in the edible parts 3. Currently, used fertilizers and metal-based pesticides are subject to leaching and precipitation by soil constituents 2, which can be avoided by the application of metallic NPs with foliar spray. Thus, the synthesized nanobrass BNPs can also be effective as plant micronutrients for additional enhancement of growth through nutritional benefits. The phytotoxicity of NPs released into the natural environment depends unavoidably on the light irradiation from the sun or artificial lighting 64. In most cases HL enhanced NPs’ phytotoxicity, usually by promoting the photo-induced generation of ROS, but this is mainly the case for semiconducting inorganic oxides, such as Cu2O, CuO, ZnO and TiO2 65-67. In contrast, light induced reduction of toxicity is reported for metallic silver (Ag) NPs, arising from photo-induced changes in their colloidal stability,



aggregation phenomena and lessening in their ionic release 68. Exposure of tomato leaves to CuZn BNPs did not have any negative effect on PSII functionality at HL, verifying the absence of semiconducting oxide phases from the BNPs (Fig. 1). Additionally, as ΦPSΙΙ at LL temporary decreased in tomato leaves exposed to 30 mg L-1 CuZn BNPs and no such effect was observed in HL, it is suggested that photoinduced physicochemical modifications reduced the negative effects of BNPs observed at LL 68.


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4. Conclusions Low cost effective antimicrobials are highly sought after. Copper-zinc alloys (brass) fit those perspectives amongst the already known antimicrobials of silver and copper which are expensive and less resistant, respectively. Despite the importance of this material, antimicrobial/agrochemical evaluation of nanobrass remains absent while synthesis of nanobrass is scarcely reported. The utilization of a microwave-assisted polyol process at a temperature close to the boiling point of triethylene glycol resulted in the effective isolation of non-oxide contaminated CuZn bimetallic nanoparticles (nanobrass). Biocompatible coated hydrophilic nanoclusters of 20 nm crystallite size, a hydrodynamic diameter of ~ 250 nm and no significant ionic leaching are produced in that manner. The antifungal activity of the as-produced bimetallic nanoparticles against S. cerevisiae was found enhanced compared to monometallic Cu nanoparticles, with IC50 value of 27.1 ± 1.89 mg L-1 calculated for CuZn. Meanwhile, the hydrophilicity of the resulted CuZn nanoparticles allows for application through foliar spray, avoiding leaching and precipitation by soil constituents. No phytotoxic effects were observed on photosystem II functionality (low and high light intensity) on tomato plants sprayed with CuZn nanoparticles’ concentrations ≤ their calculated IC50 value. Thus, the synthesized nanobrass can be effectively used to suppress fungal activity directly and also as plant micronutrients, with a view to increase crop growth and yield. Measurements of chlorophyll a fluorescence emissions by photosystem II proved to be a good indicator to probe the function of the photosynthetic machinery of tomato plants exposed to nanoparticles and chlorophyll fluorescence imaging analysis is recommended as a tool to monitor the spatiotemporal variations of nanoparticles’ effects on plants. Among the studied parameters, the reduction status of the plastoquinone pool displayed the highest spatiotemporal heterogeneity, being the most sensitive and suitable indicator to probe the function of the photosynthetic apparatus and determine the impact of stress on plants 69.

Supporting Information Additional data with the photograph of the sprayed tomato plants, thermogravimetric analysis (TGA) and dynamic light scattering (DLS) measurements of the bimetallic CuZn nanoparticles. This material is available free of charge via the Internet.

Conflict of interest The authors declare that there are no conflicts of interest

Acknowledgment This research was not funded by any external sources of funding to the authors. TEM images were recorded by Dr. Stefanos Mourdikoudis.



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thaliana to paraquat exposure. Pest. Biochem. Physiol. 2014, 111, 1–6

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