Biosynthesis of Silver Nanoparticles and Their Biocontrol Potentials Against Aspergillus Niger and Fusarium Udum

Silver nanoparticles can be biosynthesized from bacteria, fungi, and plant extracts but due to their ability to synthesize nanoparticles in varying sizes and shapes at ease, bacterial has drawn interest. Bacterial based biosynthesis is effective, inexpensive, and simple thus, Pseudomonas uorescence cell ltrates were used to synthesize silver nanoparticles in the present study. The chromatic shifts (yellow to brown) in the media after overnight incubation and the absorption of UV-Vis spectra at 420 nm conrmed the biosynthesis of AgNP’s. Besides that, the SPR analysis of AgNP’s showed a 400–500 nm band width, supporting the formation of silver nanoparticles and their small size with a uniform shape. AgNP’s transmission electron microscopy (TEM) images conrmed their shape as quasi spherical, mean size as 30 nm and anisotropy. From the Zeta potential analysis (-42.7 mV at pH = 7 with a single peak), highly repulsive nature of nanoparticles was conrmed. On the other hand, bio-fabricated silver nanoparticles were tested for antifungal activity against Fusarium udum and Aspergillus niger under in vitro conditions. At 150 ppm concentration of AgNP’s, Fusarium udum and Aspergillus niger were inhibited up to 100 and 80.50 %, respectively. In conclusion, synthesis of nanoparticle with aqueous Pseudomonas uorescence extract is simple and environmentally benign.


Introduction
Nanotechnology was introduced by Physicist and Nobel laureate, Professor Richard Feynman in 1959.

Synthesis and application of nanomaterials in different elds of investigation is called as
Nanotechnology (Duran et al. 2005). Nanoparticles act as fundamental building blocks for nanotechnology. Self-assembly of atoms or molecules at nanoscale was rst reported by Drexler, (1986) since then, rapid and extensive research has happened across the globe. Designing, synthesis and working with the molecular systems below 100 nm is popularly communicated as nanotechnology.
Nanotechnology is a rapidly growing eld with interdisciplinary applications especially in biology. Owing to their applicability, demand is increasing at an overwhelming rate concurrently manifested production is also recorded. Nanoparticles are synthesized from silver, gold, platinum, and palladium (noble metals) among these silver nanoparticles are most explored (Roy and Barik, 2010). Conventionally, nanomaterials are synthesized using either chemical or physical methods which include sol process, micelle, chemical precipitation, hydrothermal method, pyrolysis, and chemical vapour deposition. Often, these chemical and physical methods of preparing nanoparticles requires high-end toxic chemicals and consumes huge quantities of energy which are being re ected in their cost and restrict their avenues of applications, biology in particular, due to toxicity.
The future of nanotechnology relies on the development of e cient and eco -friendly nanomaterial synthesis protocols among a variety of over a range of biological compositions, proportions, shapes and elevated monodispersity. Biosynthesis of nanoparticles is an attractive opportunity to advance green nanotechnology, which has the opportunity to attain multiple applications, particularly in biology and agriculture. Bansal et al. (2011) stated that biosynthesis of nanoparticles is a kind of bottom-up method that combines the mechanism of reduction or oxidation. Silver, inhibits broad range of microorganisms by altering the composition of cell membrane and function (Mc Donnell et al., 1999), and interferes with the production of ATP by altering the expression of associated proteins (Yamanaka et al., 2005). Silver binds with the thiol groups of proteins and deactivate them (Davies and Etris, 1997).
Pulses exposed to several biotic and abiotic stresses because, in India they were poorly managed. Diseases like collar rot (Aspergillus niger) and wilt (Fusarium udum) reduces the yields of groundnut and pigeon pea by 50 (Ghewande et al. 2002) and 30-100 % respectively. The disease incidence of Fusarium and Aspergillus has been increasing year after year and most of the released cultivars became susceptible to these diseases, due to increased virulence in pathogen. However, the e cacy of commercial existed fungicides is low as most of organisms are resistant. Seed dressing with fungicides was found promising (Gangopadhyay et al., 1996) however, development of resistance was a big concern now a days and besides that damage of soil micro ora need to be addressed for environmental stability.
Therefore, there is a need to develop new fungicide formulations with enhanced e ciency against an array of disease-causing pathogens in agriculture using the emerging technologies like nanotechnology.
Several microorganisms have been utilized to synthesize AgNPs through either intracellular or extracellular (Klaus et al., 1999;Nair and Pradeep, 2002;Mukherjee et al., 2001;and Ahmad et al., 2003) mechanisms. For instance, Ag containing nanocrystals of different compositions were synthesized by Pseudomonas stutzeri AG259 bacterium (Klaus et al., 1999). In Fusarium oxysporum fungus, the reduction of Ag + ions were attributed to an enzymatic process involving NADH -dependent reductase (Ahmad et al., 2003). Pseudomonas uorescens produces extracellular enzymes and metabolites which are essential for the production of silver nanoparticles.

Material And Methods
Isolation of the pathogens A total of twenty Aspergillus niger and Fusarium udum infected plant samples of groundnut and pigeon pea were collected. The collected samples were utilized for the isolation of respective pathogens by tissue segment method. In brief, the infected portions were cut into 1 cm bits with sterile surgical blades and the surfaces were sterilized by dipping in 0.1 percent (%) sodium hypochlorite (NaOCl) for 30 seconds followed by 3 washings in sterile distilled water. These cuttings were placed on the sterilized PDA plates and incubated at 28±2 0 C in the BOD incubator for 5-7 days (Rangaswamy et al., 1999). Pure culture of Pseudomonas uorescens was obtained from the department of Agricultural Microbiology and Bioenergy, Hyderabad, India.
Bio-fabrication of silver nanoparticles from Pseudomonas uorescens A three-day old Pseudomonas uorescens pure culture was inoculated into 100 ml of King's B Broth and incubated at 28±2 0 C in an orbital shaker (120 rpm). The biomass was harvested after 3 Days of inoculation through sterilized Whatman No.1 lter paper. After the harvesting of biomass, silver nanoparticles were synthesized using the culture ltrate. Approximately, 2 g of culture ltrate of Pseudomonas uorescens was treated with the 50 ml of 1 mM AgNO 3 aqueous solution (odourless, boiling point 440 °C, molar mass 169.87 g/mol, density 4.35 g/cm 3 ) for the synthesis of silver nanoparticles. The whole mixture was incubated at room temperature for 72 h, change in media colour from colour less to brown colour con rms the formation of silver nanoparticles through reduction of silver ionic forms Ag + to Ag 0 (Adrian et al., 2020).

Ultraviolet-Visible (UV-Vis) spectroscopy analysis of nanoparticles
The reduction of silver (Ag + ) ions in the reaction mixture was monitored using UV-Vis spectrophotometer at 24 h. The Localized Surface Plasmon Resonance (LSPR) of AgNP's in the reaction mixture was Dispersion, shape and size of bio-fabricated silver nanoparticles was analysed by using Transmission Electron Microscopy (JEOL (JEM-1010)), with an accelerating voltage of 80 kV (Adrian et al., 2020). A drop of aqueous AgNP's was dried on the carbon-coated copper TEM grids and kept under vacuum in desiccators before loading them onto a specimen holder. The particle size distribution of silver nanoparticles was evaluated using ImageJ 1.45 software.

Silver nanoparticles characteristics
The aqueous suspension of silver nanoparticles ltered through a 0.22 μm syringe driven lter unit. Later, the ltrate was used for evaluating the size based on the principle of Dynamic Light Scattering (DLS) technique made in a Nanoparticle SZ-100 series compact scattering spectrometer (Mahmoodreza et al., 2019). The surface zeta potentials of nano particles measured by using Laser zeta meter (Malvern zeta seizer 2000, Malvern). In brief, 5 ml of liquid samples (nanoparticles) were diluted with 50 ml of DDW (double distil water) using NaCl as suspending electrolyte solution (2 x10 -2 M NaCl) and the samples were shaken for 30 minutes. After shaking, zeta potential and pH was recorded and of the metallic particles was measured. A zeta potential values were used to determine the surface potential of the silver nanoparticles. In each case, an average of three separate measurements was reported and the stability of NPs were measured when the values of zeta potential ranged from higher than +30 mV to lower than -30 mV (Akman et al., 2011).

Assessment of silver nanoparticles biocontrol potential against Aspergillus niger and Fusarium udum
To produce a nal concentration of 10, 30, 50, 100 and 150 ppm of AgNP's and AgNO 3 , 40 ml of modi ed PDA medium (Potatoes-400 g, Dextrose-40 g, Agar-40 g, distilled water-1000 ml) was combined with AgNP's (dried culture ltrate) and AgNO 3 solutions respectively. Approximately, 20 ml of this medium was poured in 90 mm diameter petri plate, control was maintained without AgNP's and AgNO 3 respectively.
Mycelial discs (7 mm) of 24 h old pathogens were inoculated at the centre of the plates and incubated at 28±2 o C until full growth was observed in control. In each treatment, ve replications were maintained.
The percent inhibition of radial growth of the test pathogens Aspergillus niger and Fusarium udum were calculated by using the following formula. Aspergillus niger (collar rot) affected seeds with blackish testa and rotted internal tissue and also mature plants with wilting and rotting symptoms at just below the ground level were collected. The affected portion turned dark, shrunken and shredded, and later covered by black spores of the pathogen. The pathogen was identi ed as Aspergillus niger based on microscopic examination (Figure 1). The fungus displayed upright and tiny conidiophores, nishing with globose swelling, holding phialides radiating from the entire surface. The conidia were found to be single celled, light to dark brown in colour, globose shaped and produced basipetally (Figure 1a and b). Mycelium was hyaline branched and septate.
The wilt (Fusarium udum) pathogen isolated from the infected plant roots of pigeon pea had slender hyaline hyphae with abundant branching, typically with small aerial growth ( Figure 2). Chlamydospores were globose and smooth walled. Macroconidia were straight to falcate, thin walled, septate but predominantly 3-septate. Microconidia fusiform to reniform, septate (Figure 2a and b), formation of micro and macroconidia was observed under compound microscope.

Silver nanoparticles biosynthesis and speci cations
The majority of metal nanoparticles have optical properties that are proportional to the size and form. The bio-fabrication of silver nanoparticles (AgNPs) has been veri ed by means of a notable transition in media colour from yellow to brown after 24 hours of incubation (Figure 3), signalling the biotransformation of Ag + to Ag 0 by reductive enzymes (Ahmad et al., 2003;Saifuddin et al., 2009). However, we point out that in the present research, over colour production was observed, which may be attributed to the difference in the existence of the organism and the size and shape of metal particles. In addition, the variation in relative behaviour in the reduction of silver nitrate ions to mental nanoparticles due to the existence of the proteins formed may be also be the explanation for the shift in colour. Surface plasmon vibration gives silver nanoparticles a peculiar brownish yellow colour. Light evokes the free electrons in silver nanoparticles as observed under UV-Vis spectroscopy, and transmits to a higher energy level, but the electron is unstable in an excited state and returns to the level of base energy and a photon is emitted simultaneously (Thangaraju et al., 2012). Correspondingly, the spectrum of silver nanoparticles demonstrated highest surface plasmon resonance at 420 nm. The existane of silver nanoparticles in an aqueous solution is usually veri ed by sharp SRP's in the 350-600 nm range (Sastry et al., 1998;Henglein, 1993). Figure 4 reveals that the SPR is in the 400-500 nm band, suggesting that bio-fabricated silver nanoparticles are smaller size and identical in form. As per Mie theory, only a single SPR band is expected in the absorption spectra of spherical nanoparticles whereas, the number of peaks increases as anisotropy increases (Raut Rajesh et al., 2009). We also recorded a single peak in the present study that indicates the silver nanoparticles are spherical in nature. This nding was the primary con rmation of size, form and distribution, further veri ed by TEM analysis.
The size and form of individual bio-fabricated silver nanoparticles is demonstrated by TEM micrographs with good visibility of lattice space ( Figure 5). With an average size of 30 nm, they are quasi spherical in form and anisotropic in nature. Moreover, without major agglomeration and morphological variation, they are all well scattered. These observations are in accordance with the nding of the small sized nanoparticles of distinct shapes found by Abdel-Raouf et al., (2018). The particles are strongly monodispersed, in good alignment with the high zeta potential reported (-42.7mV).
The particle size distribution of bio-fabricated silver nanoparticles has been evaluated with Dynamic Light Scattering (DLS). Based on the results, the mean size of bio-fabricated silver nanoparticles was approximately 66.0 nm ( Figure 6) and the range of nanoparticles was 32 to 115 nm, based on the ndings. As predicted, due to the variations in measurements by the devices, the DLS calculated value is marginally larger than TEM calculated value i.e., TEM calculates the number based on size distribution of the physical dimension without capping agent, while the DLS measures the hydrodynamic diameter that is the particle diameter as well as the ions or molecules that are connected and travel along with them in silver nanoparticles in solution. Hence, DLS measurements are always greater than the TEM analysis (Huang et al., 2007).
To obtain additional insights into the stability of the bio-fabricated silver nanoparticles, zeta potential analysis will typically be performed. The magnitude of zeta potential provides a hint a hint of the potential stability of the colloid, considering stable particles of more than +30 mV or more than -30 mV (Melendrez et al., 2010). In this respect, the zeta potential value of bio-fabricated silver nanoparticles was -42.7 mV at pH=7 with a single peak (Figure 7), whereas, the relative high zeta potential suggests that particles are highly scattered due to heavy repulsion between synthesized nanoparticles. If every hydrosol has a strong negative or positive zeta potential, colloidal particles will appear to repel each other and the particles will not tend to agglomerate. It is thus shown that, because of its high negative charge, AGNP's are stable in nature.
Antifungal potentials of silver nanoparticles Silver nanoparticles have recently received considerable interest in the eld of phytopathogenic fungi control due to the increased resistance to fungicides and antibiotics. The capacity of silver nanoparticles to regulate phytopathogens must be investigated thoroughly. The bio-fabricated silver in the current research have demonstrated excellent in vitro inhibition of phytopathogenic fungi. The suspension of silver nanoparticles and AgNO 3 has been used to investigate the antifungal activity against Aspergillus niger and Fusarium udum on PDA. The effect of silver nanoparticles was compared with that of silver nitrate at concentrations of (AgNO 3 ) at 10, 30, 50, 100 and 150 ppm respectively. With increased concentration, the percent inhibition was improved and highest was recorded at 150 ppm. The percent inhibition of Aspergillus niger by silver nanoparticles is in the range of 45 to 80.55 % while, AgNO 3 supressed the pathogen 55.5 to 87.19 percent range. The percent inhibition of Aspergillus niger and Fusarium udum was registered as 87. 19 and 74.8 percent, respectively, at 150 ppm of AgNO 3 concentration (Figure 8 and 9). Whereas the percent inhibition of Aspergillus niger and Fusarium udum was reported as 80.50 and 100 percent, respectively, at 150 ppm of AgNP's concentration. As compared with silver nanoparticles, Aspergillus niger was greatly inhibited by AgNO 3 . The antifungal activity of silver nanoparticles varies with the type of fungus and the size of silver nanoparticles that are closely associated with the pit formation in fungal cell wall (Shafaghat, 2015). However, against Fusarium udum, silver nanoparticles demonstrated superior antifungal e cacy. Interestingly, the same concentration (150 ppm) of silver nitrate and bio-fabricated silver nanoparticles displayed superior antifungal activity against various species, including Aspergillus niger, and Fusarium udum. In general, important inhibitory action against Aspergillus niger and Fusarium udum was induced by the silver nanoparticles was studied. By selectively invading the cell membranes, silver nanoparticles interrupt the membrane potential of fungus (Kim et al., 2009a), and conidial germination (Lamsal et al., 2011).

Conclusions
Silver nanoparticles biosynthesis was accomplished using culture ltrates of P. uorescence and aqueous solution of 1 mM AgNO 3 . Later, using UV-Vis spectroscopy, they were characterized and validated by SPR and TEM study. It was observed that the nanoparticles produced by the isolate had an average size of 30 nm. The study of transmission electron microscopy (TEM) indicated a strong dispersion among the biosynthesized silver nanoparticles. An in vitro analysis of silver nanoparticles revealed a clear antifungal effect against Fusarium udum and Aspergillus niger, and the diameter of inhibition zone differed depending on the fungal species. The ndings recommend that silver nanoparticles from P. uorescence culture ltrate can be used as biopesticide agents.

Statistical analysis
Experiments were conducted in Completely Randomized Design (CRD). Data was analysed following the statistical methods outlined by Gomez and Gomez (1984).