Molecular Docking of Biosynthesized Zinc Oxide Nanoparticles to Screen Their Impact on Fungal Pathogen of Carrot Plant

Zinc plays a key role in plants growth and application of Zinc can, therefore, contribute to crop yield improvement. Nowadays, nanoparticles have received high attention because to their novel properties. The current work is done with an aim to investigate the biosynthesis of zinc oxide nanoparticles (ZnO NPs) and effect on fungus Rhizoctonia solani and on carrot crop. Use of nanoparticles as a nano-fertilizer requires an understanding of nanoparticles impact on crop plants We have used seed coat of almond for the synthesis of zinc oxide nanoparticles (ZnO NPs) characterized by EDS, FTIR, SEM and TEM. Spray with 50ppm and 100 ppm caused signicant increase in plant growth parameter of carrot plants. It has been reported that the synthesized ZnO NPs demonstrated an inhibitory activity against plant pathogenic fungi R. solani. Antifungal eciency of ZnONPs was further explained with help of Molecular docking analysis. Conrmation of the least binding energy was used to predict binding site of receptor with NPs to know mechanistic approach. ZnONPs are likely to interact with the pathogens by mechanical enfolding which may be one of the major toxicity actions against R. solani by ZnONPs.


Introduction
In the past years, toxic effect of pesticides has been reported on human health and environment. Pesticide in food causes diseases like cancer in humans. For example pesticide DDT (dichlorodiphenyltrichloroethane) used in agriculture causes cancer or other diseases in humans (Cohn et al., 2007). Alternate to these pesticides is required. Nanomaterials (NPs) have received high attention due to their unique properties and bene cial in agriculture and other sectors. Globally in 2010 it is estimated that 260,000-309,000 metric tons of nanoparticles were produced (Yadav et al., 2014)  Nanomaterials have been reported to absorb 15-20 times higher than their bulk particles (Srivastav et al. 2016). ZnO NPs levels in environment were approximately 3.1-31 µg/kg in soil and 76-760 µg/L in water (Ghosh et al., 2016). Zinc oxide nanoparticles (ZnO NPs) are capable to enhance agriculture production and crop yield (Sabir et. al. 2014). Nanoscale treatment of zinc oxide promotes stem and root growth in peanuts (Prasad et al. 2012 Awwad 2020). In this study an attempt is made to green synthesis of zinc oxide nanoparticles were done and their effect were examined on the growth of carrot plant and screen their effect on plant pathogenic fungus Rhizoctonia solani. In addition, molecular docking was performed to understand interactions between receptor of R. solani and biosynthesized nanoparticles and estimating the binding a nities.

Materials And Methods
Green synthesis of zinc nanoparticles Zinc nitrate GR used as such (purchased from Merck, India). 100 mL, 1 mM solution of zinc nitrate was prepared in an Erlenmeyer ask. Take 10 ml extract of seed coat of almond. After that 0.67gm of zinc acetate was added in 50ml of water after constant heating and stirring for 12 hours add 2 plates of NaOH in 25 ml of distilled water add in the reaction mixture and further heating with stirring the sample was centrifuged and then dried in oven. Zn + to Zn 0 respectively. The UV-Visible spectroscopy that comes in the range of 272 nm con rms the formation of ZnONPs respectively.

Plant culture
Carrot seeds were sown in pots separately after seed surface sterilization by 0.1% sodium hypochlorite for 5-7 minutes and washed with distilled water. After ten days of seed germination plants grown in each pot were sprayed with 50 ppm and 100 ppm ZnO NPs. Each treatment was replicated ve times and pots were arranged on a greenhouse bench at 26 ± 5°C and watered regularly. The experiment was terminated 50 days. Plant growth parameters were analysed and total chlorophyll and carotenoids content of carrot leaf also estimated.

Fungus culture
Rhizoctonia solani fungus was cultured on potato dextrose agar (PDA) medium at 25°C. Fungal mycelium after culture was treated with synthesized ZnO NPs. SEM and confocal analyses were done to analyze the effect of ZnO NPs.

Molecular docking
Using Auto Dock method (Morris et al. 2009) molecular docking study was performed to know the preferred binding mode and binding sites of ZnONPs with pathogenic receptor. 3D protein structure of Rhizoctonia solani pathogenic receptor was retrieved from the PMDB database and allocated PMBD Id is PM0079487.The Crystallographic Information File (CIF) of ZnO was downloaded from the website of the materials project. The CIF of ZnO was converted and saved into PDB format and used as ligand for docking study. Before docking simulation, Gasteiger partial charges, adding Kolman charges, polar, nonpolar hydrogen atoms and Lamarckian genetic terminology were applied to ZnONPs and receptor. In this docking study AutoGrid produced a large grid map to cover the entire surface of the protein. Quality control and quality assurance The analytical grade of chemicals and reagents was used for overall analysis. The deionized water was used for reagent preparation and dilution. The chemicals and reagents were purchased from Merck, India. A total of three replicas of samples were investigated to eliminate the error during sample collection and preparation.

Results And Discussion
In the last decade several papers reported for biosynthesis of zinc oxide nanoparticles (ZnONPs) from plants (Rao, 2016;Choudhary, 2018). After reviewing previous literature we synthesized zinc oxide nanoparticles (ZnO NPs) from seed coat of almond. We carried out the biosynthesis of ZnONPs from the FTIR analysis FTIR spectroscopy analysis was performed to ascertain the involvement of possible plant bio-compound responsible for reduction of Zn + ions and capping and stabilization of bio-reduced ZnO NPs synthesized by using plant extract. Figure 2 shows the aqueous and synthesized ZnO NPs using almond seed coat extract where the absorption spectrum manifests prominent transmittance located at 3434 (NH), 1638, 1563, 1416 and 528 cm − 1 in the region (Fig. 2). The strong show to the -C C-stretches ( avanones) and broad peaks indicating the -N-H-stretches (amide group) and cyclic CH 2 stretches (aliphatic group).
The prominent band at 528 cm − 1 con rms the formation of ZnO NPs. The FTIR of ZnO NPs and exist band at 3434 (NH), 1638, 1563, 1416 and 528 cm − 1 , the occurrence of these peak in the FTIR spectrum of ZnO NPs evidently indicates the dual role as a green reducing agent and also as a stabilizing agent.

Antifungal activity
The antifungal activity of prepared ZnO NPs was investigated against fungus R. solani. It was evident from SEM analysis that ZnO NPs disturb the fungus mycelium. This was due to the binding of prepared ZnO NPs to the outer membrane of fungus shown in Confocal image (Fig. 7). ZnO NPs inhibit the growth of fungi by causing deformation in fungal hyphae (He et al. 2011). Khan and Siddiqui (2018) reported inhibitory effect of ZnO NPs on bacterium Ralstonia solanacearum, fungus Phomopsis vexans and plant parasitic nematode Meloidogyne incognita.

Molecular docking analysis
In order to understand the in vitro e ciency of ZnO NPs, the ligand protein model was used in the molecular docking study. Docking of ZnO NPs into a modeled receptor, endochitinase (PM0079487) was performed to know proper orientation of nanoparticles with in receptor including non covalent interactions between the active site of receptor and ZnO NPs leading to the design of new drugs for further biological research. Docking pose with binding energy (-8.60 kcal/mol) was considered a best model for describing interactions. The potential optimal combination between the ligand and the receptor protein is illustrated as can be seen in the Fig. 8a- (Fig. 8e).These interactions of the receptor with ZnONPs involve stabilizing the docked compound in the amino acid cavity of the receptor to disturb the proliferation of fungal mycelium. In vitro experimental study may be in good agreement with the binding interaction of ZnONPs with the receptor carried out by Molecular docking study. In addition, nanoparticles are thought to interact with the fungal mycelium by mechanical coating (Dharni et al. 2015), such in uence could be one of the main toxicity actions of ZnONPs against R. solani to prevent endochitinase of R. solani leads to inactivation of enzymes.

Conclusion
Overall conclusion is that the ZnO NPs exhibit broad spectrum biocidal activity towards different plant pathogenic bacteria and fungi. In this study, we have demonstrated the green synthesis of ZnO nanoparticles from plant part. Synthesized NPs improve the plant growth of carrot plants. Based on the current results it proved antifungal activity of synthesized ZnONPs. The binding interactions between nanoparticles and receptors were analyzed using a molecular docking study. It can be concluded that the ZnO nanoparticles constitute an effective antimicrobial agent against pathogenic microorganisms and can improve the plant growth.

Declarations Ethical Approval
Not applicable