Antibacterial and Antifungal Studies of Biosynthesized Silver Nanoparticles against Plant Parasitic Nematode Meloidogyne incognita, Plant Pathogens Ralstonia solanacearum and Fusarium oxysporum

The possibility of using silver nanoparticles (AgNPs) to enhance the plants growth, crop production, and control of plant diseases is currently being researched. One of the most effective approaches for the production of AgNPs is green synthesis. Herein, we report a green and phytogenic synthesis of AgNPs by using aqueous extract of strawberry waste (solid waste after fruit juice extraction) as a novel bioresource, which is a non-hazardous and inexpensive that can act as a reducing, capping, and stabilizing agent. Successful biosynthesis of AgNPs was monitored by UV-visible spectroscopy showing a surface plasmon resonance (SPR) peak at ~415 nm. The X-ray diffraction studies confirm the face-centered cubic crystalline AgNPs. The scanning electron microscopy (SEM) and transmission electron microscopy (TEM) techniques confirm the rectangular shape with an average size of ~55 nm. The antibacterial and antifungal efficacy and inhibitory impact of the biosynthesized AgNPs were tested against nematode, Meloidogyne incognita, plant pathogenic bacterium, Ralstonia solanacearum and fungus, Fusarium oxysporum. These results confirm that biosynthesized AgNPs can significantly control these plant pathogens.


Introduction
Currently, around 1300 nanomaterials, with widespread potential applications, are currently available [1]. Silver nanoparticles (AgNPs) are a promising candidate in nanomedicine [2,3] and other AgNPs combinations, like those with biomaterials [4] to reduce microbial contamination. Globally in 2010, it is estimated that 260,000-309,000 metric tons of nanoparticles were produced [5], and worldwide consumption of nanomaterials was approximately 225,060-585,000 metric tons in 2014 to 2019 [6]. In nanomaterial science, green synthesis of nanomaterials has gained extensive attention as a sustainable, reliable, and eco-friendly protocol [7,8]. Plant extract can be used for synthesis of AgNPs. Plant extract of R. officinalis, U. dioica, and V. vitis-idaea were used for AgNPs synthesis [9]. Extract of alga Parachlorella kessleri can be used for AgNPs synthesis and they have antimicrobial potential [10,11]. Green synthesis of nanomaterials is an important alternative tool to reduce the destructive effects associated with the traditional methods of synthesis for nanoparticles commonly utilized in laboratory and industry settings [12][13][14][15]. The possibility of using nanomaterials to enhance the plants growth, crop production, and control of plant diseases is currently being researched all over the world with renewed vigor [16]. In recent years, nanotechnology has emerged as one of the most important fields of modern sciences [17,18]. Taniguchi in 1974 used the term nanotechnology to define the knowledge that deals with synthesis and application of nanosized particles (1-100 nm) of any material [19]. Nanotechnology has been increasingly applied to the development of antimicrobial materials for the management of pathogenic bacteria affecting agricultural crops, human beings, and animals. Significant development in nanomaterials synthesis, such as polymeric, carbon-based, and metallic NPs has attracted researcher attention for applications in managing plant diseases caused by pathogens [20]. AgNPs show the antimicrobial activity and gain lot of attention because of their unique antimicrobial, physiochemical, catalytic, and optical properties, as well as nanopharmaceuticals [21][22][23][24]. AgNPs show the inhibitory activity against many plant pathogenic fungi [25]. The plants, or plant extracts, which act as reducing and capping agents for NPs synthesis, are more advantageous, simple, and less prodigal over other biological primary sources [26]. Plant-mediated synthesis of NPs is preferred because it is a cost-effective, practical, environmentally friendly, and safe one-step method [27][28][29].
As agriculture is currently facing major challenges, due to climate change and high human population growth, it is difficult to meet the food demand so nanomaterials appear to be a useful tool for improving production and yields. Nanomaterials can be used to improve agricultural production by increasing the plant growth parameters.
Every year high crop loss occurs due to plant diseases caused by various pests and pathogens. R. solanacearum is a Gram-negative bacterium which causes wilt to more than 150 plant species of 33 different plant families. It is responsible for great economic losses Worldwide [30]. F. oxysporum fungus represents a complex species that includes many important plant and human pathogens [31]. Disease caused by Fusarium spp., especially by Fusarium wilt (FW) in many crop plants, is one of the most intensively studied plant diseases. Root knot nematode (M. incognita) also causes root knot disease in many important crops. Currently, various plant disease management approaches mainly relies on toxic fungicides and pesticides that are potentially noxious to environment and human health. The latest approach in management of plant diseases is the use of NPs, which may be very fruitful. NPss can be used as alternatives to pesticides, as nanopesticides [32].
In order to fully utilize the advantage of nanotechnology in plant disease protection and management, it becomes essential to analyze the effect of nanosized particles on microbes and also their application in synthesizing fungicides and pesticides. By keeping it in our mind, in this article we report a green, handy, and environmentally friendly approach for the biosynthesis of AgNPs, using aqueous extract of strawberry waste (solid waste after fruit juice extraction) as a novel bioresource, which is non-hazardous and inexpensive. Additionally, we analyzed the impact of biosynthesized AgNPs on root knot nematode M. incognita, bacterium R. solanacearum, and fungus F. oxysporum.

XRD Analysis of the Synthesized AgNPs
An X-ray diffraction pattern of the synthesized AgNPs was recorded at 10-80 • 2θ range, as shown in Figure 1a. It was carried out to confirm the formation, structure, and crystalline nature of the synthesized AgNPs. The XRD pattern shows the prominent diffraction peaks at 38.4 • , 46.3 • , 64.7 • , and 78.2 • , which correspond to the (111), (200), (220), and (311) planes of metallic silver, respectively [21,23]. These peaks are well attributed to the standard JCPDS data of the silver with face-centered cubic (fcc) crystal lattice structure (JCPDS No. 00-004-0783) [21,23]. This confirms the polycrystalline nature of the synthesized AgNPs. The average crystallite size was calculated using Scherrer's formula and found to be~55 nm [21,23].

UV-Vis Spectroscopic Analysis of the Synthesized AgNPs
UV-Vis absorption spectroscopic analysis was performed to confirm the biosynthesis of AgNPs. It was observed that a colorless solution of AgNO 3 starts changing to wine red color after 30 min, due to the formation of AgNPs. The UV-vis spectrum of the synthesized AgNPs is shown in Figure 1b. It showed an absorption band at~415 nm, which is a characteristic band of surface plasma resonance (SPR) of AgNPs [21,23].

SEM of the Biosynthesized AgNPs
SEM analysis at different magnification of the biosynthesized AgNPs was carried out to investigate their morphology and size. SEM (Figure 2A,B) showed biosynthesized AgNPs with an almost rectangular shape. AgNPs aliquot prepared the coverslip and fixed the coverslip on an aluminum stub observed by SEM, as previously described by [33]. A sample of bacteria for SEM analysis was prepared by centrifugation of a broth culture of bacteria. Pellet was isolated and washed with phosphate buffer. 0.25% gluteraldehyde was added and left for some time. Samples were dehydrated by an ethanol series. Samples were added on SEM stub using two-sided tape.

TEM and SAED Studies of the Biosynthesized AgNPs
TEM analysis was performed to study and confirm the morphology and size of the synthesized AgNPs. TEM analysis ( Figure 3A,B) illustrates aggregates with an average size of 300 nm, consisting of AgNPs about 55 nm in size. The X-ray diffraction pattern of the synthesized AgNPs cannot provide information about particle size. AgNPs are in nanoscale, having the rectangular shape with average size of 55-70 nm. The selected area electron diffraction (SAED) pattern ( Figure 3B) shows well-resolved lattice fringes, diffraction cycles, and concentric rings correspondingly indexed to the (111), (200), (220), and (311) planes of the "fcc" silver phase, which is the characteristic crystal planes of elemental Ag 0 of highly crystalline nature of the biosynthesized AgNPs. The SAED patterns are in good agreement with the attributed XRD result, which also suggests the similar reflections of the synthesized AgNPs.

FTIR Analysis of the Biosynthesized AgNPs
FTIR spectroscopic analysis was performed to ascertain the involvement of possible phytochemicals and bio molecules used in the synthesis. Figure 4 shows the FTIR spectrum of obtained samples manifesting prominent transmittance peaks located at 3307 cm −1 (NH), 1733 cm −1 carbonyl group (C=O), and 1628 cm −1 (stretching -C=C-) attributed to aromatic ring stretching vibrations, respectively. These peaks suggested the presence of flavonoids and other phenolics in the extract [23]. The presence of flavonoids and other phenolics in the aqueous extract of strawberry waste could be responsible for the bio reduction of Ag + ions and formation of AgNPs. The FTIR spectra (Figure 4) of AgNPs show stretching frequency bands at 3307 cm −1 , 1628 cm −1 , 1228 cm −1 , 1035 cm −1 , and 553 cm −1 . Consequently, the occurrence of these peaks in the FTIR spectrum of AgNPs evidently indicates the dual role of the strawberry waste (peel) extract, both as a green reducing agent and also as a stabilizing agent.

In Vitro Antifungal and Antibacterial Activity of AgNPs
Until now, only a few articles including (at the same publication) results of antinematode, antifungal, and antibacterial studies against plant pathogens using AgNPs have been reported [23,[34][35][36][37][38][39][40][41]. Therefore, in this work, we screened the biosynthesized AgNPs against the plant pathogenic bacterium, R. solanacearum, and fungus, F. oxysporum. As a result, we found a strong inhibition zone around the paper disc dipped in 100 µg/mL AgNPs, placed in nutrient agar medium inoculated with R. solanacearum and the inhibition zone is absent around the control disc (without biosynthesized AgNPs) (Figure 5b,c). The presence of the inhibition zone, induced (Figure 5c) by biosynthesized AgNPs, proves their antibacterial activity. The SEM analysis of the treated R. solanacearum was used to observe the morphological changes on the surface of the bacteria treated with AgNPs. Figure 5d shows the bacterial cell disruption after treating with the synthesized AgNPs. Khan et al. reported the similar antibacterial activities of the AgNPs [23]. This study confirms that AgNPs exhibited antimicrobial activities by damaging the cell membranes. Similarly, biosynthesized AgNPs were tested against the plant pathogenic fungi, F. oxysporum (Figure 6a-e). In vitro studies show that AgNPs inhibited the mycelial growth of the fungus, F. oxysporum. The growth of F. oxysporum was considerably reduced to 40-50%.  Figure 6c shows fungal mycelium in potato dextrose medium (control without AgNPs) and Figure 6d shows fungal mycelium treated with 100 µg/mL AgNPs. Similar work has been reported by Khan et al., as well as Ghazy et al., in which antifungal activities of the AgNPs were observed [23,35]. The SEM analysis (Figure 6e) of the treated F. oxysporum was used to observe the morphological changes on the surface of the mycelium treated with AgNPs. Disruption of fungal mycelium has been observed at several places. Khan et al. has reported antifungal effects of AgNPs against plant pathogenic fungi and has observed a similar fatal effect on fungal mycelium [23]. This confirms that the biosynthesized AgNPs can be used as an antifungal agent to minimize their negative impacts on crop plants.
The results of the present study showed that AgNPs have antibacterial and antifungal activity in vitro against R. solanacearum and F. oxysporum, respectively. This finding is consistent with previous studies, which showed that AgNPs are effective for killing phytopathogens (including fungi, Gram-positive bacteria, and Gram-negative bacteria) (Table 1) [23,[35][36][37][38][39][40][41]. Not all of them have carried out a basic characterization of the biogenic AgNPs obtained, which makes it difficult to explain the mechanism by which AgNPs interact with these microorganisms. Mishra et al. synthesized AgNPs and found that AgNPs can manage spot blotch disease in wheat [38]. Gupta and Chauhan reported fungicidal properties of AgNPs against Alternaria brassicicola fungus that causes Black Spot of cauliflower and radish [42]. Khan et al. synthesized AgNPs from corn and reported antibacterial and antifungal activity [35]. Ocsoy et al. has shown the effect of DNA-Directed AgNPs on Graphene Oxide (GO) as an antibacterial against bacteria, Xanthomonas perforans. Bacterial spot, caused by X. perforans, is a major disease of tomatoes, leading to a reduction in production by 10-50%. DNA-directed AgNPs grown on GO effectively decrease X. perforans cell viability in culture and on plants. At 16 ppm, these composites show excellent antibacterial capability. Application of these composites at 100 ppm on tomato transplants in a greenhouse experiment significantly reduced the severity of bacterial spot disease, compared to untreated plants [20].
AgNPs release silver ions inside the bacterial cells and enhance their bactericidal activity [43]. The impact of AgNPs on the antibacterial and antifungal activity depends on the size and shape of the synthesized NPs. From this study, it was observed that the biosynthesized AgNPs exhibit antibacterial and antifungal activities by damaging the cell membranes.

Effect of AgNPs on Nematode M. Incognita
In vitro studies showed that synthesized AgNPs could inhibit the hatching of M. incognita from eggs. AgNPs alone reduces the hatching ( Table 2). Hatching of nematodes occurs highest in double distilled water at 24 and 48 h. Synthesized AgNPs show a toxic effect on nematodes and cause the death of hatched nematodes after 48 h of hatching. As the exposure time increases from 24 h to 48 h, activity of nematodes decreases. Lowest death of nematodes reported in double distilled water after 48 h. Previous studies also reported the toxic effect of biogenic AgNPs on nematodes [41]. Other works also reported toxic effect of the chemical synthetized AgNPs on M. incognita [44,45].

Reagents
AgNO 3 (GR93200), potato dextrose agar (PDA) and all other chemicals, reagents, and solvents used in this work were purchased from Merck India Ltd., Mumbai, India, and were used without any purification. Fungus, F. oxysporum (MTCC 1755) and bacteria R. solanacearum (BI0001) were used for screening the antifungal and antibacterial activity, respectively. They cultured on selected media for use.

Preparation of Aqueous Extract of Strawberry Waste
Strawberry waste (solid waste left after fruit juice extraction) was used to synthesize AgNPs on the basis of cost effectiveness, ease of availability, and environment friendliness. The waste material was cleaned with distilled water to remove contaminated contents and air-dried in sunlight. Material was crushed into a fine powder by a grinder and 20 mg fine powder was kept in a flask containing 200 mL of double distilled water and refluxed for 30 min. The extract was cooled down at room temperature and filtered with Whatman filter paper no.1 for further process.

Biosynthesis of AgNPs Using Aqueous Extract of Strawberry Waste
An aqueous solution of 1 mM AgNO 3 of 100 mL was prepared in an Erlenmeyer flask. A 10 mL of strawberry extract was added to 90 mL of this AgNO 3 aqueous solution and left at room temperature with continuous stirring until color changes were apparent. During this, reduction of Ag + to Ag 0 was observed by the color change of solution from colorless to wine red. AgNPs formation was further confirmed by UV-visible spectroscopy.

Bacterial Inoculum
Bacterium, R. solanacearum was cultured on nutrient agar medium. Nutrient agar plates were streaked separately with a pure colony of R. solanacearum and incubated at 30 • C for 24 h [23,46]. Antibacterial activity of biosynthesized AgNPs was screened against R. solanacearum. A small paper disc was dipped in a 100 µg/mL AgNPs solution and placed in media containing R. solanacearum and compared with control (paper disc without NPs). These plates were incubated at 35 • C for~24 h, after which, diameters of the inhibition zones were measured. Presence of inhibition zone is a sign of antibacterial activity. All the tests were run in triplicate.

Fungus Inoculum
Fungus, F. oxysporum, was grown in Petri dishes containing potato dextrose agar (PDA) medium at 25 • C for 15 days. For obtaining sufficient inoculum, F. oxysporum was cultured on Richard's liquid medium [47]. Antifungal activity was evaluated by poisoned food technique [48,49] by using biosynthesized AgNPs. The treated plates were compared with the control to calculate the percent inhibition of mycelia growth by using the formula given in [50]. Experiments were carried out in triplicate.

Root Knot Nematodes
Root-knot nematode, M. incognita, was isolated from infected roots and cultured ( Figure 7). AgNPs were used to screening their effect on M. incognita. To determine the nematicidal activity of prepared AgNPs, 2.5 mL (100 µg/mL) suspension was dissolved in 7.5 mL distilled water in Petri plates. Five egg masses of nematodes were placed on each Petri plate. One Petri plate had five egg masses and 10 mL distilled water was used as a control (without AgNPs). 100 µg/mL solutions were prepared by dissolving 0.1 mL solutions of synthesized AgNPs in one liter of distilled water. All the tests were run in triplicate.

Conclusions
This study accomplishes the biosynthesis of AgNPs using aqueous extract of strawberry waste. The biosynthesized AgNPs showed antinematode, antibacterial, and antifungal response against M. incognita, R. solanacearum, and F. oxysporum, respectively, owing to considerable antibacterial and antifungal activities. The biosynthesized AgNPs showed acute, potent antifungal effects on fungi tested in vitro, probably through rupture or fissure of membrane integrity. Hence, we can accede that the biosynthesized AgNPs can be used against plant pathogens for retrieval of agricultural production, food safety, and a range of other applications.