Green synthesis of Copper Nanoparticles Using Rosemary Extract to Reduce Postharvest Decays Caused by Botrytis Cinerea in Tomato

INTRODUCTION
Tomato (Solanum lycopersicum L.) is one of the most popular vegetable crops with a world production of more than 164 million tons per year. It is the seventh most important crop in the world after maize, rice, wheat, potatoes, soybeans and cassava. Based on FAO data in 2019, tomato production in Iran was about 5/25 million tons. Tomato is one of the very perishable fruits and it has a relatively short storage life. Depending on humidity and temperature, its quality immediately decreases and becomes unacceptable, so that post-harvest losses are on average between 24 to 40 percent in developing countries and between 2-20 percent in developed countries. Therefore, the decrease of post-harvest losses is one of the important programs to improve shelf life and food security in the world. Growth of fungal pathogens is the main cause of fruit rots during postharvest. Several species of fungi are responsible for this fruit rots. Botrytis cinerea is a fungal pathogen that leads to gray mold disease in more than 235 plant species, such as tomato, grape, strawberry, apple, and pear. It is difficult to control fungal growth of B.cinerea because this fungus has showed resistance to conventional fungicides. To overcome this resistance, it is important to explore novel antifungal agents, which may replace current control strategies. Many studies have shown bactericidal effects and anti-fungal activities of various metal nanoparticles, including silver, zinc, gold and copper. Among the metal nanoparticles, copper nanoparticles are one of the most extensively studied nanoparticles that can be synthesized using chemical, physical and biological approaches. Physical and chemical synthesis methods are the most common scheme for the synthesis of copper nanoparticles but these methods require toxic solvents, high-energy consumption and stabilizing agents which can cause harmful effects on health and environment. In contrast, biological synthesis methods are low cost, environmentally safe and are less toxic chemicals in the synthesis process. Microorganism cell or plant extracts are considered a reducing agent in the biological synthesis of copper nanoparticles. In plant extract, some metabolites, including terpenoids, polyphenols, sugars, alkaloids, phenolic acids, and proteins, play an important role in the bio-reduction. Many studies have shown that various plant extracts can be utilized to synthesis copper nanoparticles. In recent years, nanoparticle materials have received increasing attention due to their unique physical and chemical properties which differ significantly from their conventional counterparts [1-13].
Rosemary (Rosmarinus officinalis L.) is an evergreen shrub with fragrant needle-like leaves that are used in the medicine, cosmetic and food industries. The rosemary leaf extract is one of the major bioactive compounds which have been reported to possess many biological activities such as antibacterial, antioxidant, antifungal, etc [14].
In the present study, leaf extract of rosemary was used for the biosynthesis of copper nanoparticles and was investigated by advanced characterization techniques. Also, antifungal activity against gray mold disease was investigated by growing Botrytis cinerea on agar plates supplemented with different concentrations of copper nanoparticles. Finally, concentrations of copper nanoparticles that can inhibit tomato fruit rots during postharvest were determined in vivo condition.

MATERIALS AND METHODS
Plant material collection and extract preparation
Rosemary (Rosmarinus officinalis L.) plant was collected from the local habitat in Markazi province, Arak, Iran. Leaves were washed with distilled water, dried at the shadow and finely powdered by the electric grinder. 300 g of powdered leaves were added to 1000 ml distilled water with the concentration of 300 g/L and boiled in at 70 °C for 1 hour. The resultant extract was filtered through sterile Whatman No. 1 filter paper and storage at 4 °C for further use (Fig. 1).

Biosynthesis of copper nanoparticles 
Copper (II) sulfate (CuSO4) and sodium borohydride pellets (NaBH4) was purchased from Merck (Germany). To synthesis copper nanoparticles, 24.9 g of Copper (II) sulfate was dissolved in 1000 ml rosemary extract. The solution was put on the heater and heating with mixing continued until the solution color changes to bluish-green. The product was cooled at room temperature. NaBH4 (2 M) was added drop-wise while stirring. A black crystalline precipitate of copper oxide was obtained, which is washed repeatedly with distilled water, filtered and dried in an oven at 90 °C to obtain the copper nanoparticles powder (Fig. 1).

Characterization of synthesized nanoparticles
The solutions were characterized by absorption spectroscopy in the UV-Vis Spectrophotometer. The morphology and size of produced nanoparticles were characterized by Scanning electron Microscopy. The formation of copper nanoparticles using X-ray diffraction (XRD) with a Ni filter and Cu Kα radiation (λ =0.15406 nm), on the PANalytical X’pert PRO powder X-Ray diffractometer. Also, dynamic light scattering (DLS) was used to determine the average size of synthesized copper nanoparticles.

In vitro antifungal assay
Pathogenic fungus of Botrytis cinerea was obtained from Iranian Biological Resource Center and it was cultured on PDA (Potato Dextrose Agar) at 25 ◦C in the dark. Antifungal tests were performed by the agar dilution method with some modifications [15]. The autoclaved PDA media with copper nanoparticles solution at concentrations of 0, 3, 5, 7, 11 and 15 mmol/l were poured into the Petri dishes (8 cm diameter). The fungi were inoculated after the PDA media solidified. A disc (1.5 cm) of mycelia material taken from the edge of 7-day-old fungal cultures was placed in the center of each Petri dish. The Petri dish with the inoculums was then incubated at 25 ◦C. The diameter of developed colonies was measured when fungal mycelium covered one plate in the control treatment [16]. Inhibition percentage in colony diameter was calculated using the following formula:

Where: Δd0 and Δd are the average diameters of fungal colonies in the control and treatment.

In vivo antifungal assay
The antifungal activity of copper nanoparticles solution was evaluated in tomato (Solanum lycopersicum “Cherry tomato”) purchased in a local supermarket. Tomato fruits were properly disinfected with 0.5% (v/v) sodium hypochlorite solution for 2 min, then dipped in sterilized water for 2 min and left at room temperature until they dried completely. Subsequently, tomato fruits were treated with copper nanoparticles solution at four concentrations (0, 5, 15 and 25 mmol/l), and dried at room temperature. The tomato fruits were placed in plastic boxes to artificially inoculate by B.cinerea and maintained at 24 °C for 10 days. Disease incidences and symptoms of the treated tomato fruits were measured and observed 10 days after inoculation. All treatments were carried out with three replicates and three fruits for each treatment (Fig. 2). The rate of fruit rots was performed according to the method of Meena et al (2020) with slight modification [17].

Tomato fruits quality assay
The fruit firmness was measured on the two opposite sides of tomato fruit samples by using a penetrometer. Total Soluble Solids (TSS) and pH in the extracted fruit juice were measured according to the instructions mentioned in previous studies [18].

RESULTS AND DISCUSSION
UV–vis spectroscopy analysis
The UV–vis spectrum of synthesized Cu nanoparticles indicated a maximum absorbance at around 540 nm, which was specific for Cu nanoparticles (Fig. 3). Similarly, several studies have also showed the absorption spectrum between 540 and 560 nm due to the formation of Cu nanoparticles [19].

SEM images of synthesized copper nanoparticles 
SEM images of copper nanoparticles synthesized with rosemary extract were showed in Fig. 4 and this image indicates that the copper nanoparticles with average diameter size about 50 nm.

XRD pattern of the synthesized copper nanoparticles
The structure and formation of copper nanoparticles from the rosemary extract were analysised by X-ray diffraction and it showed the distinctive peaks indexed to the crystal planes of face-centered cubic silver (Fig. 5). The diffraction profile had an intense peak at 2θ of 43◦, 50◦ and 4◦, corresponding to (1 1 1),(2 0 0), (2 2 0), and (3 1 1) planes respectively. Our result is in conformity with the reports of the Joint Committee on Powder (JCPDS: 03-1015).

Dynamic light scattering
Dynamic light scattering (DLS) analysis determined the average particle size distribution profile of synthesized nanoparticles and the size of copper nanoparticles were in the range 50 nm hydrodynamic diameter (Fig. 6).

In vitro inhibitory effect of copper nanoparticles against B. cinerea
Our results showed that the copper nanoparticles have significant inhibitory effect on the pathogen growth of B. cinerea compared to the control. In vitro culture, the inhibitory rates of 0, 3, 7, 11 and 15 mmol/l copper nanoparticles were 45.71%, 74.78%, 85.43% and 100% at five day after inoculation, respectively (Fig. 7). Various studies have been shown to copper nanoparticles have the antifungal activity against plant infecting fungi and increasing concentration of copper nanoparticles can be enhanced antifungal effectiveness [20-21]. Similar to our results, copper nanoparticles were also found to be suitable for the control of the fungal pathogen Botrytis cinerea at 15 mg/L concentration [22].

In vivo effects of copper nanoparticles on microbial decay of tomato
The microbial decay was visually inspected in stored tomatoes up to 10 days considering the extent of microbial infection on fruit surface in the scale ranging from 1 to 4 (Fig. 8). Microbial decay was increased in control tomatoes after being stored for 10 days while copper nanoparticles (0.25 %) remarkably inhibited microbial decay (Table 1). Performance of copper nanoparticles on inhibition microbial decay was not only due to the antimicrobial nature of copper but also by its effect on preventing plant cell death by enhancing plant cell immunity [17].

Effects of copper nanoparticles on fruits quality of tomato
In vivo results showed that, the fruits firmness were significantly affected by increasing copper nanoparticles concentrations, but there was no significant change in dry weight percentage, pH, total soluble solids (TSS) and vitamin C, 10 days after storage (Table 1). 
Similar to our results, the application of Cu nanoparticles + chitosan increased the firmness of tomato fruits but it didn’t make differences in the total soluble solids [23]. Also, Abdel-Rahman et al (2020) reported that the tomato fruits treated with natural nanoparticles at different concentrations greatly reduced the development of black mold rot and significantly maintained fruit quality characteristics such as fruit firmness, titratable acidity and total soluble solids during cold storage [24].

CONCLUSION
In conclusion, copper nanoparticles with sizes of 70 nm were green synthesized by using the extract of Rosmarinus officinalis. Botrytis growth was completely blocked in vitro when copper nanoparticles with concentration of 15 mmol/l were added to the fungus culture medium. Also copper nanoparticles solution at 25 mmol/l were the most effective treatment against fungal decay in response to artificial inoculation of tomato fruits with B. cinerea and it can be useful in the control of gray mold and postharvest quality of tomato.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interests regarding the publication of this manuscript.