Synthesis and antifungal activity of copper nanoparticles against Fusarium oxysporum pathogen of plants

For many decades, Cu2+ ions have been used as fungicides in agriculture. However, the accumulation of Cu2+ ions in the soil ecosystem will be disadvantage for environment. Thus, the substitution of copper nanoparticles for Cu2+ ion is necessary to develop long-term efficient, eco-friendly, and cost-effective fungicides. In this study, the copper nanoparticles were synthesized by chemical reduction method of Cu2+ with reductive agent of NaHB4 in chitosan stabilizer. Characterizations of copper nanoparticles were determined by UV–vis spectra, Fourier transform infrared spectroscopy, X ray diffraction patterns and Transmission Electron Microscopy images. The antifungal efficacy of CuNPs was evaluated by testing against Fusarium oxysporum fungi at various concentrations from 20–100 ppm. The results revealed that copper nanoparticles samples inhibited significantly the growth of Fusarium oxysporum and the smaller diameter is, the higher antifungal efficacy is. The copper nanoparticles with 26.5 nm expressed an antifungal efficacy is higher than copper nanoparticles with 29 nm. The complete inhibition was observed at concentrations ≥ 80 ppm after 1 day and even 7 days of incubation for 2.0 CuNPs (26.5 nm) sample while fungi still survive on PDA plates containing 1.5 CuNPs (29 nm).


Introduction
Copper is an essential micronutrient required for maintaining effective health and nutrition in plants. It plays an important role in the synthesis of chlorophyll, plant pigments and metabolism of protein as well as carbohydrate [1]. The deficiency of Cu may lead to various diseases in crop plants leading to loss in yield. Its deficiency also affects the vegetative growth, formation of grains, seeds, fruits and reduction in lignifications of cell walls. Moreover, Cu and its compounds possess remarkable bactericidal and fungicidal activity, therefore they have been used to control the crop diseases. Bordeaux solution was known globally, as an effective fungicide includes copper sulfate and lime water. This mixture was invented by French scientists and developed into plant protector against disease as downy mildew infection onto the grape trees [2]. Copper sulfate is a key ingredient in most of the commercially available fungicides for farm and garden. At present, there has been the organic farming in search for the copper substitute in crop protection and in minimizing the amounts of applied Cu. However, there are no active substances, techniques, or methods that could serve as a complete substitute for Cu [3].
The use of agrochemicals against plant diseases has direct economic benefits to the minimization of losses in crops. However, the frequent use of pesticides in bulk has caused tremendous harm to humans and environment by accumulation of residues in produce and the soil [4]. Moreover, the use of this agent has resulted in disease Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. resistance to this pesticide among microorganisms [5]. Thus, the application of nanotechnology in agriculture has gained more attention by plant protection, promoting plant growth, nanosensors, antifungal and antibacterial activities. Specially, copper nanoparticles received more consideration recently because of their cost effectiveness, the copper nanoparticles (CuNPs) are much cheaper than that of silver or gold nanoparticles. The improvement in antimicrobial activity and absorbable property of CuNPs in plant as compared to copper salt is due to their unique property i.e. large surface area to volume ratio [6]. The CuNPs as antibacterial agent by the copper surfaces can be used to kill bacteria, yeasts, and viruses which are known as 'contact killing'. This killing activity takes place at a rate of at least 7-8 logs per hour and no micro-organism survives after prolonged incubation on copper surfaces [7]. The morphology and oxidative stress play important roles in the antibacterial and antifungal activities. Since nanoparicles have a very small size, this is an advantage to easily interact with cell membranes and penetrate inside leading to increasing permeability and disturbing respiration. The nanoparticles mediated increase in reactive oxygen species (ROS) which can cause damage to deoxyribonucleic acid DNA, proteins, membranes and interfering in nutrient absorption of bacterial cell [8].
Worldwide, over 19,000 fungi are known to cause diseases in crop plants. Pathogen fungi such as Phytophthora infestans caused blight with potatoes, Alternaria solani caused irregular dark brown spots on leaves of sweet potatoes leading to early blight, Xanthomonas oryzae is harmful to leaves of rice and death in plants [9]. Especially, Furasium sp. is a common fungal disease on many crops and it is one of the most devastating diseases spotted on banana, tomato, potato, dragon fruit, watermelon, chickpea plants and other cucurbits [10,11]. In Viet Nam, tropical fruit is an important production sector of agriculture for exportation to EU, American and China market [12]. Pathogens are major causes for yield losses and diminished crop quality, so nanotechnology offers new strategy of biotechnology and agriculture [10]. Saharan et al demonstrated significant antifungal activity of copper nanoparticles against plant pathogenic fungi, namely Alternaria alternata and Fusarium oxysporum on tomato plants [13]. CuNPs treatments exhibited growth promoting effect in terms of plant height, root length, root weight, nodule number, weight of nodule and number of pods per plant in both experiments of pot and field were enhanced [14]. Recently, the investigations showed that incorporation of CuNPs into natural polymers, namely cellulose, starch and chitosan matrices significantly enhanced its antimicrobial and antifungal activity [15][16][17][18]. The CuNPs-Chitosan can combine the properties of biopolymer and nanosized copper as well as enhancement synergic effect of components, therefore, their application can be more effective than separately. Also, chitosan is a suitable stabilizer for pathogenous therapy target on plant [15]. This is demonstrated by the antifungal activity of CuNPs-Chitosan against two sclerotium-forming plant pathogenic fungi of Sclerotium rolfsii and Rhizoctonia solani [16]. The efficacy of Cu-chitosan nanoparticles (NPs) to plant was also reported by other author who evaluated antibacterial activity against bacterial pustule disease of soybean [17].
In previous studies, we prepared metal nanoparticles by radiolysis reduction of metal ions (Me n+) to Me 0 atoms by irradiation effects and aggregation into Me nanoparticles at room temperature. However, the reduction of Cu 2+ ions to Cu 0 atoms and then aggregation of clusters into Cu nanoparticles at temperature ranging from 60°C to 85°C [18,19]. Thus, in this study, CuNPs were synthesized by chemical reduction. We described one-step synthesis to form colloidal CuNPs from an aqueous solution at 20 mM Cu 2+ ions precursor by reduction of sodium borohydride (NaBH 4 ) at 80°C. Chitosan is used as an agent using capping and stabilizing for cupric nanoparticles. Physical chemistry characterizations and antifungal activity of CuNPschitosan against Fusarium oxysporum were investigated.

Materials
All of the chemicals were analytical grade and were used as purchased without further purification. Copper (II) sulfate pentahydrate salt CuSO 4 .5H 2 O was 98.0% pure (Merck). Sodium borohydride (NaBH 4 , Reagent Plus 99.0%) from Sigma-Aldrich was used as the main reducing agent while ascorbic acid (99.7%, Prolabo) was used as the antioxidant of colloidal copper. Chitosan stabilizer with a deacetylation degree about 80% and Mw = 1.06 ×10 5 was prepared as reported previously [20]. Aqueous solutions were made in distilled water.

Synthesis of copper nanoparticles
In this work, the metallic copper nanostructures were obtained from the redox reaction between Cu 2+ and sodium borohydride in the presence of ascorbic acid and chitosan as capping agent. Copper sulfate solution as the precursor was prepared by dissolving 10 ml of CuSO 4 .5H 2 O (0.1 M) in 90 ml of chitosan solution at 1.5 and 2.0% (w/v, g ml −1 ) with lactic acid solvent. And then adding 0.5 ml of ascorbic acid -C 6 H 8 O 6 (0.01M) under vigorous stirring at 80°C for 15 min. Next, a 0.5 ml volume of NaBH 4 (4 mM) reductant was slowly dropped into the above mixture until the colour changed from bluish to dark purple, indicating the formation of CuNPs, namely 1.5 CuNPs and 2.0 CuNPs according to 1.5% and 2% initial chitosan concentration. The complete reaction will obtain by stirring solution continuously for 20 min.

Characterizations of copper nanoparticles
The UV-vis spectra of the CuNPs were recorded on a Jasco V-630 spectrophotometer in the range from 200-600 nm, after CuNPs colloid was diluted 5 times. The Transmission Electron Micrography (TEM) images of CuNPs were taken on a JEOL, JEM-2100 electron microscope at an accelerated voltage of 120 kV. After specimens of CuNPs colloid were deposited on carbon-coated copper grids and dried 50°C for 30 min. Fourier transform infrared spectroscopy (FT-IR) spectra were performed on a Shimadzu FT-IR 8400 s instrument scanning from 500-4000 cm −1 . The solid CuNPs were separated from colloid by an ultra centrifugation at 40,000 RPM, and then dried at 80°C. X ray diffraction (XRD) patterns of crystalline CuNPs were measured on a Shimadzu 5A diffractomer with CuKα radiation (25 kV and 25 mA), scanning at a speed of 0.5°/min from 5 to 80°(2θ). The crystalline cupric nanoparticle has been estimated by using Debey-Scherrer formula: , β is the full width at half maximum (FWHM), θ is the diffraction angel (Bragg) and D is the particle diameter size [21].

In vitro antifungal activity assay
Fusarium oxysporum (F oxysporum) fungal samples were isolated from infected tomatoes, and then were grown on potato D-glucose agar (PDA) media for further experimentation. This media includes 1000 ml distilled water, 200 g l −1 potato extract, 20 g l −1 agar, and 20 g/l glucose. Colloidal CuNPs at concentrations of 0, 20, 40, 60, 80 and 100 ppm were introduced in PDA media which was steamed at 121°C for 15 min. Next, the mixture was shaken and then poured on sterilized petri dishes (90×15 mm). Media containing CuNPs was incubated at room temperature for 48 h. And then each Petri dish was inoculated at the center with a mycelial disc (6 mm diameter) taken at the periphery of F oxysporum colony grown on PDA. After incubation at 28°C for 1, 2, 3, 4, 5, 6 and 7 days, the radial growth of fungal mycelium was recorded. Radial inhibition was determined when the mycelial growth in the control plate reached the edge of the petri dish. The formula was used for calculation of the growth inhibition efficacy (%) which described by Margarita as follows: Where d 0 is the diameter of mycelial growth in the control plates and d a is the diameter of mycelial growth in the plates treated with CuNPs, respectively [16].

Statistical analysis
All fungal growth assays were carried out in triplicate and the data are presented as the mean standard deviation. Two-Factor analysis of variance (ANOVA) was performed to evaluate the interactions between both factors of concentration and culture period on inhibition efficacy of fungal growth for samples and the controls (without CuNPs).

Synthesis of CuNPs by chemical reduction
It is noteworthy that the chemical reduction of metal salt precursors is one of the most convenient and promising synthetic approaches to obtain metallic nanoparticles given its simplicity, feasibility, and relatively inexpensive cost [22]. The rate of growth of the nanoparticles depends upon various variables, including the concentration of metal ions, the type of reductant, pH and temperature [23]. Sodium borohydride (NaBH 4 ) is perhaps one of the strongest reductant in small scale synthesis of metal nanoparticles and it is widely adopted in laboratories for its versatility in many chemical syntheses. It has been used in a broad variety of operative conditions and it proved to be useful for its fast reaction kinetics of reduction [24]. In this study, CuNPs were prepared by simple reduction of copper salts-CuSO 4 by NaBH 4 agent in chitosan stabilizer at 80°C and antioxidant of ascorbic acid. This process was modified from reduction of Cu 2+ by NaBH 4 in other surfactant, namely cetyltrimethyl ammonium bromide (CTAB) [25]. The mechanism for reduction of Cu 2+ ions to zero valent Cu atoms can be proposed as follows: Continuous reduction of the Cu 2+ causes aggregation of clusters into Cu nanoparticles. The formation of CuNPs colloid can be visually observed by change in the color of mixture from blue to dark purple (figure 1). The binding of Cu clusters by chitosan is achieved through the chelation of both amine and hydroxyl groups with Cu 2+ ions and then Cu atoms, thereby making it an excellent support for nucleation of Cu nanoparticles. Besides, the other amine groups of chitosan in acidic medium are protonated to form -NH 3 + ions, so that the metal nanoparticles as CuNPs can be stabilized by chitosan through electrostatics repulsions as well as chelate steric hindrance [26]. The use of biopolymer as capping and reducing agents represents an environmentally friendly alternative to hazardous organic solvents [27].
During reduction of Cu 2+ to Cu°and aggregation to CuNPs, the oxidation to Cu 2 O or CuO easily occurs, thus the ascorbic acid was added to the initial mixture for enhancement of reduction and antioxidation, due to its ability to scavenge free radicals and reactive oxygen molecules [28]. The absence of ascorbic acid in synthesis of CuNPs may lead on formation of mixture including Cu nanoparticles, Cu 2 O and CuO in reduction process of Cu 2+ by NaBH 4 . The supplement of the ascorbic acid in the above relative system with sufficient quantity leads to formation the pure CuNPs, non-oxidation, even the experimental conditions without deoxygenation or inert gas [29]. Reactions can be expressed as follows: The UV-vis absorption spectral analysis was carried out to examine the copper nanoparticles in colloid (figure 2). The absorption bands for Cu nanoparticles have been reported to be in the range of 500-600 nm [28,30]. The effect of different chitosan concentrations on the optical properties of CuNPs in suspension of CuNPs-Chitosan is determined by measuring UV-vis spectra. The results depicted a blue shift from 575.5 nm to 574 nm with respective increases in absorbance intensity for colloidal CuNPs in 1.5% and 2% chitosan. This means that the size of CuNPs decreased and a better dispersion of CuNPs with increasing stabilizer concentration. The chitosan concentration was maintained at 1.5 and 2% solution as this provided sufficient chitosan for stabilization of CuNPs and also allowed for an acceptable solution viscosity at room temperature. If the concentration of chitosan under 1.5% can cause coalescence and precipitation.

Characterizations of CuNPs
FTIR analysis was conducted to determine the molecular interactions between chitosan and the synthesized CuNPs. FTIR analysis shows the presence of bands at 3400-3500 cm −1 associated with stretching the hydrophilic groups had strong intensity as -OH and -NHgroups. The absorption peaks at 1500-1600 cm −1 are assigned to bending vibrations of adsorbed water groups -NH 2 in chitosan molecules ( figure 3(a)). After reduction of Cu 2+ -chitosan to Cu nanoparticles-chitosan by NaBH 4 solution, FTIR spectrum appeared the sharp peaks at 619.50 cm −1 and 791.24 cm −1 due to stretching Cu-O in the presence of CuNPs-chitosan ( figure 3(b)). The same results were also reported by other authors [27,28]. Figure 4(a) shows the diffraction peaks of CuNPs in the range of 20-80°, the peaks appear about 25°can be attributed to the structure of chitosan [31]. The Bragg reflections at 2θ = 43.3°, 52.1°and 74.2°were attributed to the (111), (200) and (220) planes of crystalline copper, respectively. This result is in agreement with the XDR peak positions of Cu were also indexed previously [32,33]. The full width at half-maximum (FWHM) of diffraction peak (111) around 43°in figure 4(b) is employed to determine the average crystalline size using Debye-Scherrer as formula (1). The crystalline sizes of CuNPs were calculated to be about 29 nm and 26.5 nm corresponding to samples of 1.5 CuNPs ( figure 4(b1)) and 2.0 CuNPs ( figure 4(b2)). Once nuclei are formed, they tend to aggregate in order to decrease the total surface energy. The use of chitosan as surfactant in this work avoiding aggregation of nanoparticles by steric repulsion between individuals. Thus, the size of CuNPs is smaller at the chitosan concentration is higher when other parameters are unchanged. The TEM images provide the detailed morphological insights on the shape and dimension of nanoparticles [34]. As the images showed in figure 5 the synthesized CuNPs shape is almost pherical and the particle distribution is relatively homogeneous, there was no agglomeration.

Antifungal activity of CuNPs on mycelial growth
The antifungal test was carried out with samples of 1.5 CuNPs (figure 6, series A) and 2.0 CuNPs (figure 6, series B) corresponding to the diameter of CuNPs to be about 29 nm and 26.5 nm. The antifungal activity of the synthesized CuNPs was evaluated by measuring the mycelia radial growth for all treatments. Figure 6 shows the growth inhibition of F oxysporum fungus on the plates which were cultured with CuNPs at the various concentrations of 20, 40, 60, 80 and 100 ppm, after incubation periods of 2 days, 4 days and 7 days.  In most cases, the diameters of mycelium in samples which were supplemented CuNPs being smaller than the control samples without CuNPs (ĐC), especially it was expressed clearly at concentrations 40 ppm for 2.0 CuNPs (series B) and 60 ppm for 1.5 CuNPs (series A). The quantitative results in table 1 show that the more the CuNPs concentration increases, the more the antifungal efficacy (E, %) according to formula (2) increases. The growth inhibition of F oxysporum almost completely i.e. the E value obtained 100% for plates which were treated with 2.0 CuNPs sample at 80 ppm and 100 ppm even prolongation of incubation to 7 days (series B). At this conditions, the mycelial growth on the plates contain 1.5 CuNPs still continue although it is insignificant (E = 95%). The difference in antifungal efficacy between two samples is also depicted at other concentrations in figure 7. The toxicity of copper nanoparticles depends on the combination of several factors such as concentration, length of exposure, humidity, and temperature [35]. It has been reported that copper interacts with microorganisms in various ways including cell membrane permeabilization, membrane lipid peroxidation, protein alteration, and denaturation of nucleic acids, ultimately leading to cell death [16]. The result of analysis of variance (ANOVA) revealed that both factors of concentration and incubation period had significant effects on the antifungal efficacy, p < 0.05 (table 2). The value of F rows > F crit confimed that the concentration effects significantly on the antifungal efficacy. The quantification of fungicide is an important factor in agriculture by relation to the antifungal efficacy as well as the accumulation of residues in the soil.
In this study, the antifungal efficacy can be explained by a 'contact killing' mechanism of cupric nanoparticles was published by many authors [36,37]. It was proposed that the copper provided a surface for killing bacteria, fungi, and viruses with extensive cell membrane damage, by cell vacuole enlargement and disappearances. No live microorganisms were recovered from the copper surfaces, after prolonged exposure. The nanoparticles mediated increase in reactive oxygen species (ROS) level, however the phytopathogenic fungi usually have stronger resistance to ROS attack than bacteria or viruses, because of their much larger size and thicker cell wall/membrane. Thus, effective alternatives to chemical fungicides are difficult to develop [38]. The combination between CuNPs and chitosan results in synergic effect on antifungal activity of components. Direct activity of chitosan against viruses, bacteria and fungi has been reported on the inactivation of replication, which leads to the stoppage of multiplication and spread [39]. Chitosan, when applied to plant tissues, it acts as physical barrier around pathogen penetration sites, preventing the pathogen from spreading and invading other healthy tissues. Besides, chitosans as chelators for minerals and metals, it can chelate nutrients and minerals (i.e., Fe, Cu), preventing pathogens from accessing them thus reducing fungal spoilage [40]. Antifungal efficacy of the 2.0 CuNPs sample is higher than 1.5 CuNPs sample by it has both smaller size and higher chitosan concentration.
These results indicate that Cu-NPs-Chitosan strongly suppressed F oxysporum under in vitro condition. The tests in vivo for antifungal activity on plants will be investigated further in future.

Conclusion
Copper nanoparticles -chitosan colloid was synthesized via the chemical method using reduction agent of sodium borohydride and copper sulfate as precursor. Different analytical techniques such as UV-vis spectra and TEM images confirmed the formation of copper nanoparticles in chitosan. The results of the in vitro test showed that CuNPs significantly inhibited the growth of F oxysporum pathogen for tropical plant according to the size of CuNPs. The initial experiments demonstrated CuNPs can be used as nano-based fungicides in a simple and cost-effective preparation manner. Of course, further in vivo tests for antifungal efficacy as well as the impact of CuNPs on the environment and other characterizations will be investigated in future.