Isolation of pathogens
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 identified as Aspergillus niger based on microscopic examination (Figure 1). The fungus displayed upright and tiny conidiophores, finishing 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 specifications
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 verified 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 Ag0 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 verified 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 finding was the primary confirmation of size, form and distribution, further verified 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 finding 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 findings. 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 field 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 AgNO3 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 (AgNO3) 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, AgNO3 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 AgNO3 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 AgNO3. 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 efficacy. 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).