Rhazya Stricta Assisted Green Synthesis of Multifunctional Carbon Coated Copper Oxide Nanosheets for Photocatalysis of Dyes and Antibacterial Candidate Against Solanaceous Pathogens

The studies of metal oxides in environmental remediation of chemical and biological pollutants are gaining huge importance. Herein, we report the facile synthesis of multi-functional copper oxide nanosheets (CuO NS) using an aqueous extract of Rhazya stricta. The phytochemical investigation of R. stricta indicated the presence of saponins, tannins, and reducing sugars responsible for the reduction and stabilization of CuO NS. The formation of CuO NS was conrmed by x-ray diffraction and UV-visible spectrophotometer with specic Surface Plasmon Resonance at 294 nm. Further characterization of the CuO NS was done by FE-SEM, FTIR, and XRD. The obtained CuO NS were poly-dispersed with an average size of 20 nm. Interestingly these particles were aligned together in the form of 3D cubical sheets layered above each other via self-assembly. The as-synthesized CuO NS shows enhanced antibacterial potential (17.63 mm, mean inhibition zone) as compared to the known antibiotics (11.51 mm, mean inhibition zone) against the wilt causing bacteria of Solanaceous crop, including Ralstonia solanacearum and Clavibacter michiganensis. Furthermore, the appreciable photocatalytic potential of CuO NS has been observed, causing 83% degradation of methylene blue (MB) upon solar irradiation. The synthesis methodology is devoid of any toxic waste and/or by-products and could be used to produce eco-friendly CuO nanomaterial for industrial uses.


Introduction
Metal oxide nanostructures have drawn considerable interest due to their enhanced photocatalytic properties, low cost and wide range of biological and industrial applications 12 . Besides having excellent antimicrobial properties, metal oxide nanoparticles could be used for drug delivery, having increased resistance against multidrug resistance (MDR) pathogens. Metal nanostructures such as silver (Ag), gold (Au), and Iron (Fe) have been widely studied for their bioactivity and applications in various consumer products 345 . These can be synthesized using different methods, including chemical 6 , electrochemical 7 , sol-gel 8 and condensation 9 , etc. but with certain limitations such as the generation of hazardous waste, use of toxic chemicals and solvents, the di culty in optimizing the extent of scaling up synthetic processes, and utilization of high energy 10 . The recent development in nanotechnology combined with green chemistry, led to the development of environmentally friendly, non-toxic, and cost effective procedures for the fabrication of nanomaterials 11 . Due to the large surface to volume ratio, the nanomaterials are highly reactive and results in extremely bene cial properties, including mechanical, biochemical, biotechnology, optics, catalysis, and medicines 12 .
Recently copper oxide nanostructures have gained attention due to their low cost in comparison to the existing metal nanoparticles such as Ag and Au. They are also considered almost ten times cheaper than their other counterparts. Copper oxide nanostructures are believed to have high sensitivity against both Gram-negative & Gram-positive microorganisms. They have a high potential as an external microbial agent and in the form of biocidal lm over medical devices 13 . However, very limited studies are available for their use in agriculture for the control of pathogens.
Wilting of Solanaceous crops caused by bacteria, including Ralstonia solanacearum and Clavibacter michiganensis is of major concern for the growers 14 . Both of these bacteria spread rapidly on their outbreak and cause severe damage by clogging the vascular system through extracellular polysaccharides 1516 . These pathogenic bacteria are generally controlled by using commercially available antibiotics that are expensive and pose threats to the environment. Furthermore, rapid industrialization and excess use of dyes are posing a severe threat to the water quality and biotic ecology. These organic pollutants are highly mutagenic and carcinogenic to human life. Dyes are poorly degradable pollutant and their complex chemical structure make them di cult to remove from water 17 . Therefore, it is of utmost importance to have eco-friendly products for the control of these pathogens and for the removal of both biological and chemical pollutants.
To the best of our knowledge, there has not been any report on the single-step one-pot synthesis procedure carbon-coated CuO-NS using an aqueous extract of R stricta. Herein, we report a one-pot, environmentally friendly method for the fabrication of multi-functional CuO NS for photocatalytic degradation of methylene blue (MB) and their possible use as a bio-control agent against wilt causing bacterial pathogens of Solanaceous crops to enhance quality and agricultural productivity.

Collection of plant material
The experiment and collection of plant material is done in accordance with the relevant national and international guidelines. 18 Arial parts of Rhazya stricta (Wild), were collected with permission from local forest o cers in the month of April 2020 from district Karak, Khyber Pakhtunkhwa, Pakistan, located at 33°7'12N 71°5'41E ( Figure 1). The plant samples were stored in a paper bag and transferred to the laboratory within 48 hours and identi ed by Dr. Muhammad Ishfaq Khan. A specimen of the sample was deposited in a special herbarium for weeds and medicinal plants (voucher No. MIK-5/20-397), Department of weed science, The University of Agriculture Peshawar. The collected samples were rinsed with distilled water and air-dried under shade for 14 days. The dried samples were chopped into small pieces using a sterile scissor, and the sample was boiled at 60°C distilled water for 20 min (10% weight to volume ratio) on the heating mantle. The solution was cooled to room temperature and vacuum ltered to obtain the aqueous extract concentrate (brown color) and stored at 4°C for further use.

Biosynthesis of CuO NS
For the synthesis of CuO NS, 60 mL of freshly prepared aqueous extract of Rhazya stricta was added to 80 mL of 0.5 M aqueous copper sulfate pentahydrate (CuSO 4 .5H 2 O) solution at room temperature. A prominent change in color from blue to green was observed as soon as both solutions come in contact, which indicates the reduction of ionic copper. The obtained green mixture upon heating at 80 °C for 12 hours turned to the brown mixture. The reaction mixture was then allowed to cool at room temperature, and the brown suspended particles were separated through centrifugation (10000 RPM for 10 min). The precipitates were thoroughly washed with distilled water and absolute ethanol to remove unreacted reagents and biomolecules. The obtained solid material was dried under N 2 ow and stored at 4 °C for further characterization.

UV-visible spectral analysis
The bio-reduction of Cu 2+ ion solution through an aqueous extract of Rhazya stricta was monitored using Optima Sp3000+ (Japan) split beam UV-vis spectrometer for its maximum absorption v/s wavelength range against the aqueous extract of Rhazya stricta as a blank. Upon completion, the reaction mixture was centrifuged at 10,000 rpm for 10 minutes to eliminate any uncoordinated bio-molecules. The obtained solid material was re-suspended in double-distilled de-ionized water and scanned from 200-1100 nm wavelength.

Fourier transform infrared spectroscopy (FT-IR) analysis
The Fourier Transform Infrared spectra were recorded under identical conditions in the range 400-4000 cm -1 region using FTIR Spectrometer (SHIMADZHU; Japan).

X-ray diffraction analysis
The phase identity, crystalline structure, and crystallite size were determined from the XRD data using Cu-kα radiation source. The CuO NS powder was coated on a glass substrate and submitted for their crystal structure analysis. The results were recorded as a graph with 2θ vs. intensity at the x-axis and y-axis, respectively.

Field Emission Scanning electron microscopy (FE-SEM)
The surface morphology and size distribution of as-synthesized CuO NS were characterized using TESCAN MAIA3 Field Emission Scanning Electron Microscopy (FE-SEM). A drop of an aqueous solution of Cu NS obtained after puri cation via repeated centrifugation was placed on a Silicon (Si) substrate and let to dry. The samples were then characterized at an accelerated voltage of 5.0 kV.

Antibacterial Assays
Synthesized CuO NS were tested for inhibition against the wilt-causing bacterial pathogens of Solanaceous crops, including Ralstonia solanacearum and Clavibacter michiganensis. The antibacterial assay was carried out by the disc diffusion method 21 . Both bacterial strains were developed using nutrient broth at 38°C for 24 hours and then streaked over Potato Dextrose Agar's surface using sterile cotton swabs. The sterile paper disk (5 mm) was adsorbed with 10 µL of the reaction mixture, and the disc was placed on the surface of the plate. As a control 10 µL plant extract, 10 µL 0.5 M aqueous CuSO 4 solution, and a positive control 10 µL of streptomycin (0.5 M in distilled water) were separately adsorbed on sterile paper discs (5 mm). It was then placed on a prepared lawn of bacterial cultures on PDA to assess their effect on pathogens. The plates were incubated at 38°C for 24 hours, and bacterial growth inhibition was observed as clear halos (zones) around the discs.

Photocatalytic property
The photocatalytic potential of biogenic CuO NS was carried out at the end of May under sunlight irradiation.
To 100 mL (10 µg mL -1 ) aqueous methylene blue solution in a transparent ask, 10 mg of synthesized CuO NS were added and allowed to stir for 2 hours and 20 minutes under direct sunlight. A control experiment (in the absence of CuO NS) was also maintained simultaneously. After every 20 min of reaction duration, 5 mL sample aliquot was collected and centrifuged (10000 rpm) for 6 minutes to remove suspended CuO NS. The supernatant was collected and examined by a UV-Vis spectrophotometer at λ max 664 nm for the estimation of available unreacted MB. The following formula was used to determine the percent photo-degradation e ciency (PD%) of MB.
Where PD% denote percent photodegradation, C 0 and C t represents the absorbance of MB at time 0 min and t min Statistical analysis The data was obtained in triplicates. The antibacterial activity is presented as a mean zone of inhibition (mm) ±S.E, the different letters in columns are statistically signi cant at P< 0.05

Results And Discussion
The detection of phytochemical in aqueous extract of Rhazya stricta revealed the presence of various groups of bioactive natural products (Table 1). These natural products, especially polyphenols, are believed to be responsible for the reduction of metal ions and the formation of metallic nanoparticles 22 .  (Figure 2-A). This change in the color is due to the excitation of surface Plasmon vibration of CuO NS, which generated a highintensity peak appearing at 294 nm and a minor peak at 314 could be from the different size/shape of CuO NS ( Figure 2-B Inset) 23 . We opined that this is also attributed to the n →π* transition of C=O bonds 24 . The peak appearing at 226 nm, 244 nm and 258 nm (Fig 2-B inset) in the low-frequency region are the characteristic peaks of graphene nanosheets corresponding to the π conjugation network or π → π* transition 25  0.34 nm. The slightly higher d spacing value suggests the presence of some oxygen functional groups between the carbon layers. Besides this, the broadened peak corresponding to the (002) plane is believed to be due to the disordered carbon structure indicating the formation of graphene sheets as observed by Ashish and Sundara 34 , which might be due to the self-assembly of carbon atoms.

Fourier transform infrared spectroscopy (FT-IR) analysis
Various bio-molecules having different functional groups were responsible for reducing CuSO 4 and then stabilizing CuO NS derived from bioactive molecules. Various functional groups were found to be attached to nanoparticles' surface during synthesis. FTIR is one of the most important techniques used to identify functional groups attached to nanomaterials' surface. Figure 4 shows a sharp band appearing at 630 cm -1 , the characteristics band of pure monoclinic CuO reported earlier 35 . The peak appearing at 1040 cm -1 is attributed to the C-H stretching vibrations. A sharp absorption band at 1600 cm -1 is believed to be due to the selfassembled disordered carbon sheets. The broad peak at 3297 cm -1 is believed to be due to OH groups' existence on the surface of CuO NS. A sharp absorption band at 2930 cm -1 is because of the CH and CH 2 groups. This suggests that the hydroxyl groups might have worked as reducing and stabilizing agents for the fabrication and consequently prevented the accumulation of pure CuO NS. The appearance of bands at 1600 cm -1 and that of the 2930 cm -1 is believed to predict graphene sheets interacting with CuO NS 36 . Chemical linkages on the surface of CuO NS suggest that the hydroxyl and carbonyl groups might have reacted as reducing and stabilizing agents for the fabrication of CuO material and consequently prevented the accumulation of CuO NS.

Field Emission Electron Microscopy of CuO NS
The eld emission scanning electron microscopy (FE-SEM) was used to analyze the morphology of asprepared CuO nanosheets, as shown in Figure 5. It can be observed that CuO crystallizes in the form of smaller nanoparticles, which are polydispersed and aligned together in the proper sequence. These nanoparticles, upon diffusion, lead to the formation of nanosheets. The sheet's width appears to be less than 20 nm, which leads to highly reactive edges and corners.
Antibacterial potential of CuO NS against wilt causing pathogens The antibacterial properties of CuO NS were evaluated against the wilt-causing bacterial pathogens of Solanaceous crops. Two bacterial strains, including Clavibactermichiganensis (gram +ve) and Ralstoniasolanacearum (gram -ve), were tested using the disk diffusion method. In the present study, the synthesized CuO NS showed signi cant zones of inhibitions against both bacterial strains. The Cu ions are known to disrupt various biochemical processes 37 Table   2 shows the biosynthesized CuO NS possessed higher antibacterial activity against both R. solanacearum and C. michiganensis with an inhibition zone measured as 17.30 mm and 17.97 mm, respectively (p<0.05).
Signi cantly improved antibacterial activity of the CuO NS against both tested bacterial strains was recorded compared to standard antibiotics (p<0.05).
Interestingly both selected bacteria started to develop resistance after 18 hours against Streptomycin, and clear hallow around disk became turbid after 24 hours. In contrast, selected bacteria did not show any growth around CuO NS, and clear hallow remained persistent after 24 hours ( Figure 6). The aqueous extract of Rhazya stricta (blank) showed no activity against both R. solanacearum and C. michiganensis. The dense growth of both bacterial isolates was seen (Figure 6, blank)

Conclusion
The successful one-pot eco-friendly synthesis of Copper NS was achieved using an aqueous extract of Rhazya stricta in 24 hours under mild conditions. Regular self-assembly of synthesized CuO NS was recorded in the form of three-dimensional cubical sheets with signi cant photocatalytic degradation e ciency against methylene blue. The nanosheets show a prominent bactericidal potential against wilt causing bacterial pathogens of Solanaceous plants. The Cu liberated in the eld as a by-product could also be used as a micronutrient by the crops.