Inuence of Bio-Inspired Ag doped MoO3 Nanoparticles in the Seedling Growth and Inhibitory Action Against Microbial Organisms

Herein we report the hydrothermal synthesis, characterization and biological applications of h-MoO 3 and silver doped MoO 3 nanoparticles (NPs). The phase formation of the synthesized NPs was identified using X-ray diffraction studies and vibrational spectral studies. The average crystallite size of the NPs tends to decrease as the dopant concentration increases. The surface morphology and the elemental composition of the nanoparticles were observed from SEM and EDAX analysis. The crystallite nature was obtained from HRTEM images. The band gap energies obtained from UV-DRS spectra for h-MoO 3 (3.26 eV) were starting to decrease as the concentration of the dopant Ag increases (3.22-2.76eV). The antibacterial activity of the prepared nanoparticles was tested against some gram positive and gram negative bacterial strains viz., Staphylococcus aureus, Bacillus cereus and Citrobacter koseri and Pseudomonas aeruginosa respectively . Also their seed germination properties were studied on foxtail and finger millet seeds for a period of seven days.


Introduction
Agriculture is considered to be the most vital and stable part due to the production of raw materials for food and feed producing industries. In order to meet the demands of the growing population, there is a necessity to enhance the existing food production. The deterioration of natural resources viz., land, water and soil due to contamination claims the need for agricultural development to be economic, viable and eco-friendly [1]. In the existing situation, the agricultural field is prone to irregular climatic changes, contamination of resources by various hazardous pollutants like fertilizers and pesticides which leads to the elevation of food demands for the growing population [2]. Thus, it is known that there is a need for increased crop production as well as better food security.
No doubt that the sustainable growth of agriculture totally depends on the new and innovative techniques like nanotechnology. In agro-industrial sector, nanotechnology is commonly used in the production of herbicides, fertilizers, fungicides, pesticides as well as nano-sensor materials [3]. These advances can be used to overcome the future demands in agriculture thereby increasing quality and yield of crops thus reducing the environmental pollution due to chemicals and also protecting the vegetatation against environmental stresses [4]. The of NPs serve a better application in agricultural filed by reducing the nutrient losses to enhance their yields and minimizing the cost of production to obtain maximum output [5].
The nanoherbicides and nanopesticides used for the treatment of weeds and pests is observed to significantly increase the crop production. Various types of polymeric and inorganic nanoparticles are utilized as nanoherbicides. Nanomaterials having specific antimicrobial properties aid in the prevention of microbial invasions. Using nanotechnology researchers have designed a smart delivery system to release the nutrients to the targeted site in a slow and controlled manner in order to tackle the nutrient deficiency in plants.
Among the transition metal oxides, molybdenum trioxide (MoO3) has attracted the attention of the researchers greatly on account of its multifunctional structural, optical, electronic properties along with better intercalation chemistry with unique chemical, electrochemical, and catalytic properties. Molybdenum trioxide (MoO3), an n-type semiconductor material with a wide band gap of 2.8-3.6 eV [6]. There are three common phases of MoO3: a thermodynamically stable orthorhombic phase (α-MoO3) and two metastable phases: monoclinic (β-MoO3) and hexagonal (h-MoO3) [7]. The hexagonal phase of MoO3 is composed of the zigzag chains of MoO6 octahedrons connecting through the cispositions [8,9]. In addition, MoO3 was found to be an effective antimicrobial against different bacterial strains [10]. Antimicrobial activity of MoO3 is related to their acidic surface involving the formation of intermediate molybdic acid [11]. Also, Mo is responsible for the conversion of nitrogen into ammonia (NH3) and important for the metabolism of nitrogen and sulfur. However, the increased amount of Mo in plants leads to molybdenum toxicity, causing yellowish leaves, reduced growth in seedlings, and increased concentrations of anthocyanin [12].
Silver nanoparticles (AgNPs) are extensively used for their antimicrobial property against a wide range of phytopathogens [13]. Silver nanoparticles can constantly release the silver ions, which may be considered the mechanism behind their antimicrobial effect. Owing to the electrostatic attraction and greater affinity towards sulfur proteins, silver ions can stick to the cytoplasmic membrane of the cell and increase the permeability of the membrane leading to disruption of the bacterial cell [14]. In recent times, silver nanoparticles have been involved in enhancing seed germination, plant growth and improving photosynthetic quantum efficiency and also serve as antimicrobial agents in treating plant diseases. There are many reports showing that AgNPs with appropriate concentrations play a crucial role in enhancing seed germination and plant growth [15].
Owing to the extensive applications of h-MoO3 and Ag nanoparticles, in this study, we report the synthesis of Ag doped h-MoO3 nanoparticles and their characterization using several analytical techniques. The antibacterial studies of these nanoparticles against Staphylococcus aureus, Bacillus cereus, Citrobacter koseri and Pseudomonas aeruginosa strains have been evaluated and also their seed germination properties on the seeds of foxtail millet and finger millet seeds have been studied.

Materials
All the chemicals used in this research work are of analytical grade. Ammonium heptamolybdate tetrahydrate (AHM) was purchased from Sigma Aldrich and silver nitrate from Loba Chemie. Deionised water was used throughout the entire research work.

Synthesis of MoO3 and silver doped MoO3 nanoparticles
For the synthesis of MoO3 nanoparticles, 2.43 g of AHM ([NH4]6Mo7.O24.4H2O]) was dissolved in 10 mL of deionised water. After stirring for 15 mins, 10 mL of Con. HNO3 was added slowly in the aqueous solution containing AHM. The reaction mixture was then transferred to Teflon lined stainless steel autoclave (100 mL) and heated at 90 o C for 9 hours.
The system was then allowed to cool naturally to room temperature. The obtained precipitate was collected by centrifugation and washed several times with deionised water and ethanol.
Finally the powder was dried in vacuum oven at 70 o C for 12 hours. Similar procedure was followed for synthesizing Ag doped MoO3. During the reaction, silver nitrate was added along with AHM. The synthesis was carried out for three different weight percentages (1%, 3% & 5%) of silver nitrate along with AHM. The synthesized nanoparticles MoO3, 1% Ag doped MoO3, 3% Ag doped MoO3 and 5% Ag doped MoO3 which is mentioned as M, 1AM, 3AM and 5AM respectively. The formation of molybdenum trioxide from the precursor can be described by the following endothermic reaction, (NH4)6Mo7O244H2O 7MoO3(s) + 6NH3 (g) + 7H2O(g)

Characterization techniques
X-ray diffraction patterns of the synthesized samples were recorded on X'pert PRO powder X-ray diffractometer in 2θ range of 20°-60° at a scan rate of 2° min -1 using Cu-

Antimicrobial studies
Antimicrobial activity of the nanoparticles was carried out against four different bacterial strains i.e. Staphylococcus aureus, Bacillus cereus, Citrobacter koseri and Pseudomonas aeruginosa employing well diffusion method. It was performed by properly sterilizing the Mueller Hinton media of agar. After solidification, the wells were cut using a cork borer. The bacterial pathogens to be tested were swabbed onto the surface of Mueller Hinton agar plates. The cut pieces of thin film of samples were placed on the well. The plates were incubated at 37 o C for 24 hours, and then the zone of inhibition was measured in millimeters. Each antibacterial assay was performed in triplicate and mean values were reported.

Seed Germination
The experimental treatments involved four samples M, 1AM, 3AM and 5AM NPs for observing the germination of foxtail millet (Setaria italica (L.) P.Beauv.) and finger millet (Eleusine coracana (L.) Gaertn.) seeds. Healthy seeds of uniform size were selected to minimize the errors and were rinsed using water to clean them from chemicals and surface impurities. After rinsing the seeds in running tap water, they were incubated in a 5% (v/v) detergent solution for 5 min. Then, seeds were then immersed in 70% (v/v) ethanol solution (1 min) and finally in NaCl (1.5% (v/v), 10 min). Finally, the seeds were washed twice thoroughly in sterile distilled water. The experiment was performed under normal laboratory conditions in natural light. Germination tests were done in petri dishes containing a Whatman No-1 filter paper moistened with 5 ml of suspension or distilled water and each plate was loaded with fifteen seeds with 5ml suspension of NPs with specific concentration. The distilled water was utilized in control sample for the reference. The petri dishes were kept in a dark place and the number of seeds germinated and other relevant parameters were recorded on daily for a period of 7 days.

X-Ray Diffraction studies
The have been indexed and matched well with standard data card (JCPDS-21-0569). It was found that the diffraction peaks were ascribed to a metastable hexagonal phase of MoO3 [16]. The absence of any other diffraction peaks indicates the purity of the MoO3.
X-ray diffraction information reveals the microstructural details and different structural parameters. The lattice constants "a," "b," and "c" for h-MoO3 can be determined from the inter-planar spacing values of {h, k, l} planes with the Miller indices h, k, l by using the following equation [17]: where, dhkl is the inter-planar spacing of the {h, k, l} plane. The calculated values of lattice parameters for preferential orientation were given in table 1. The values are in good agreement with standard results for hexagonal phase and previously reported values [18].
The crystallite size (D) can be determined from Debye-Scherrer's equation [19]: where D,is the crystallite size, the wavelength of X-ray radiation, λ = 1.54056 Å for CuKα, the shape factor and constant (K = 0.90), βhkl is the instrumental corrected full-width at halfmaximum height (FWHM) of the diffraction peak (in radians) located at 2θ and θhkl the Bragg angle (in degrees), respectively. The calculated values of crystallite sizes for the nanoparticles are provided in table 1. From the XRD pattern, it is observed that the crystallite size of MoO3 was also found to decrease slightly with Ag doping.

FTIR spectral studies
The functional groups of the products with the best crystalline degree were identified by FTIR (Fig. 5)     Antibacterial activity was performed for 100 μg/mL dosage of the nanoparticles (Fig. 8a).
It is observed that the biocidal effect of h-MoO3 improves with increasing the concentration of Ag against Staphylococcus aureus, Citrobacter koseri and Pseudomonas aeruginosa. For Staphylococcus aureus, no significant activity is observed for h-MoO3 NPs.
But for the gram positive bacteria (Bacillus cereus), the zone of inhibition is found to be decreased from 12 mm to 07 mm for an increasing amount of dopant (Ag) concentration in h-MoO3 nanoparticles. The highest zone of inhibition was observed against Pseudomonas aeruginosa for 5AM and it is found to be 23 mm in diameter.
To explain the mechanism, it is necessary to understand the interaction between nanoparticle and bacterial cell wall which leads to the destruction of the cell wall [29]. Along with this, the size, shape, structure and composition of nanomaterials also play a crucial role in the bactericidal effect [30].
Silver present in the form of Ag + is more easily released and directly react with cell membranes when immersed in a practical biological system. But the Ag metal bound to the h-MoO3 particles must first undergo dissolution to form Ag + and then migrate into the bacterial cell [31]. It is known that, silver ions could easily interact with a number of electron donor groups like phosphates, thiols, hydroxyls, indoles and imidazoles thereby causing damage to the cell membrane and releasing the ROS, finally causing bacterial death [32].
It can be assumed that with decreasing particle size, the number of particles per unit volume increases resulting in increased surface area and increased generation of hydrogen peroxide [33]. The hydrogen peroxide, hydroxyl radicals, and superoxide allied to ROS group can harm the DNA and may cause the cell death. The histogram (Fig. 8b)

Seed Germination of Foxtail millet and finger millet seeds
The prepared h-MoO3 and Ag doped h-MoO3 NPs are used as fertilizer for improving the growth of foxtail and finger millet seeds at room temperature. Both the seeds were germinated for seven days and then various parameters were recorded such as root and shoot length, seed vigor index and germination percentage are presented in figure 10.

Representation of zone of inhibition (diameter in mm)
Antibacterial activity (100 µg/mL) The germination percentage and the seed vigor index are the indicators for the enhancement in germination rate of the seeds. The growth response of seeds in the presence of h-MoO3, Ag doped h-MoO3 and distilled water are analysed.
The germination percentage and the quality of germinated seeds can be identified by calculating the seed vigor index using the following equation [34].
. X 100 It is clear from the results that there is enhancement in germination rate of the foxtail millet (  by both the seeds, increased their percentage germination but the germination being prohibited for higher Ag concentration (5%) due to toxicity and the results are in consistent with literature [35].

Fig 10. (a,d) Germination percentage, (b,e) Root and shoot length, and (c,f) seed vigour index of foxtail millet and finger millet seeds respectively treated with MoO3 and Ag doped MoO3 nanoparticles
Many researchers have reported the impact produced by nanoparticles on seeds germination, but, the exact mechanism is still not well-understood. It is generally observed that the h-MoO3 and Ag doped h-MoO3 NPs can penetrate into the seed coat and activate the embryo so as to increase the uptake of water and other nutrients [36]. During the penetration of the h-MoO3 NPs more new pores are created which absorb the nutrients and thereby increasing the seed germination percentage and growth rate.
The enhanced growth rate of seeds may be due to uptake of water (for supplying energy to the seeds which is essential for germination) and nutrients by the treatment with nanoparticles [37]. The increase in the seed growth might be due to molybdenum, an important micronutrient for plant growth which is involved in several oxidation-reduction processes in plants. Accordingly, the NPs are proved to have positive effects in promotion of root and shoot formation, and also accumulation of plant biomass from seeds in most of the crops compared to the untreated one [38].
Silver based nanoparticles are commonly used in the food and agriculture application at specified concentrations due to their well-known antimicrobial activity and root regeneration property. The highest concentration tested for Ag doped NPs (higher concentrations induce oxidative stress in plant cells) was not found to be effective for plant growth but had a robust antimicrobial effect, which would be beneficial in during seed germination period. The silver doped NPs at moderate concentrations significantly enhance seed germination, germination %, seed vigour index, root length and shoot length of seeds.
Even higher concentrations of Ag doped NPs have led to a severe decrease in the growth parameters of seeds which were associated with accumulation of Ag in the stems and roots [39].

Conclusion
The seedling growth for both the seeds, but shows the maximum zone of inhibition against all the bacterial strains.

Declarations
The authors declare no conflict of interest. Raman spectra of MoO3 and Ag doped MoO3 nanoparticles