Presently, the term “green synthesis” or “phytonanotechnology” has been coined for nanoparticle (NP) synthesis which has many advantages including its biocompatibility, scalability, and applicability by utilizing water which acts as a reduction medium (Noruzi, 2015). It has been recommended that vitamins, proteins, organic acids, amino acids, and secondary metabolites act like capping and stabilizing agents that reduce metal salts of synthesized NPs by playing a key role (Duan et al., 2015). Green synthesis ascends as an incipient approach which has more advantages over physical and chemicals ways of synthesizing NPs. Biological processes implicate fungal, bacterial, and plant enzymes that involve convoluted procedures for sustaining cell cultures under aseptic conditions while significant production of NPs was achieved by employing plant extracts, encompassing simplicity and applicability with low energy consumption; however, chemically synthesized NPs comprise of toxic reagents that remain as residues along with particles and ultimately nurture toxicity problems within human system (Qayyum et al., 2017; Oves et al., 2018).

In the last few decades, extensive research has been done on the effects of NPs on ecosystems including organisms and plants. Being an eco-friendly and a multifactorial biogenic material, application of NPs is receiving attention due to their specific physiochemical properties, serves as an inexpensive approach for assembling of innovative functional materials used almost in every area of science and technology like medicine, engineering, environment, and agriculture (Austin et al., 2014; Majdalawieh et al., 2014; Siripattanakul-Ratpukdi and Furhacker, 2014; Prasad et al., 2016; Vishwakarma et al., 2017). Due to the interdynamic properties of NPs, there are high opportunities to explore the potential of NPs while the nano-elicitive behavior of these minute particles may rely on their nature and methods of synthesis (Jasim et al., 2017).

Silver (Ag)NPs arise as potential antimicrobial agents by demonstrating stout antiviral, bacterial, fungal, and inflammatory activities which highlighted their significance (Qayyum et al., 2017). To our best acquaintance, for the development of novel antimicrobial agents to manage agricultural diseases, manipulations in the synthesis of AgNPs such as structural properties and surface coating are some of the methods which are being employed for enhancing strong phytopathogenic activities (Jamshidi and Ghanati, 2016; Kumari et al., 2017). Some reports describe antifungal activity of AgNPs on conidial development and fungal hyphae as well as presenting strong inhibitory effects of biosynthesized AgNPs against certain phytopathogens in fields in greenhouse conditions (Mishra et al., 2014; Madbouly et al., 2017). In some earlier studies, nanomaterials have been employed to boost plant germination, improve soil fertility, and enhance deprivation of pesticide wastes (Ghrair et al., 2010; Khot et al., 2012; El-Temsah et al., 2014). Among the different types of metals, AgNPs have been used extensively against different fungal plant pathogens, and their suppressive effects on growth and development have been investigated. Elgorban et al. (2016) observed antifungal activity of AgNPs in cotton plants. Nano-silver has also been applied to suppress soilborne diseases. AgNPs from Artemisia absinthium extracts have shown high effect against agriculturally imperative pathogens from the genus Phytophthora. Its single applications of 10 and 100 μg/ml of nano-silver resulted in 78–95% plant survival, while 5% survival was observed for untreated control (Ali M. et al., 2015). Inhibitory effect of various formulations of nanoscale silver was studied against Colletotrichum gloeosporioides and Sclerotium cepivorum (Jung et al., 2010; Aguilar-Mendez et al., 2011). Mallaiah (2015) observed 75–55% disease reduction by AgNPs in commercially available ornamental flower of Crossandra spp. infected by Fusarium wilt in pot culture.

Formerly, an extensive research had been done on the utilization of different plants for the synthesis of AgNPs including aloe vera leaf extract (Medda et al., 2015), Azadirachta indica leaf extract (Ahmed et al., 2016), Protium serratum leaf extract (Mohanta et al., 2017), Mangifera indica inflorescence aqueous extract (Qayyum et al., 2017), Phoenix dactylifera root hair extract (Oves et al., 2018), and Caesalpinia ferrea seed extract (Soares et al., 2018). However, it is still required to explore commercially feasible, economically stable, and eco-friendly safe routes for synthesis of AgNPs by utilizing various plant materials (Chung et al., 2016).

Tomato (Solanum lycopersicum L.) is the most imperious vegetable harvest on earth that ranked second after potato (Jensen et al., 2010; FAOSTAT, 2014). Tomatoes are cultivated in different areas of Pakistan, with 11.05 tons per hectare (GOP, 2013). F. oxysporum f. sp. lycopersici, responsible for wilt, is an unavoidable infection of the grown tomatoes. Being a soilborne disease, it is only controlled by using resistant varieties and soil fumigants, but both have their own constraints. The impact of these pesticides on our ecosystem has already raised many questions on their utilization. Therefore, there is an essential need to adopt alternative approaches for the management of pathogens. An adequate approach that is being actively investigated involves NPs, such as metal oxides, which are used to control soilborne fungi (Shenashen et al., 2017).

The current study reports green synthesis of AgNPs by using the aqueous extract of M. azedarach and evaluates its potential application as an antifungal agent against F. oxysporum causing tomato wilt without intrusion of any supplementary physical and chemical steps. Silver has been chosen because of its antimicrobial activities either in NP or ionic form as well as lesser toxicity to mammalian cells (Ali K. et al., 2015). Moreover, the efficacy of Melia leaf extract (MLE)-AgNPs against Fusarium in tomato roots was evaluated under laboratory and greenhouse conditions by comparing it with a commercially available fungicide. The effects of MLE-AgNPs on tomato growth parameters and antioxidant enzymes were studied. Green synthesis of metal NPs by using plant material proficiently proved to be an eco-friendly and cost-effective approach.

The green synthesis of NPs has currently established an environment-friendly and safest method involving a wide range of biological entities including fungi, bacteria, and different plant parts. UV-vis spectroscopy is an important tool to inspect the formation and steadiness of metal NPs as absorption band is detected in UV-vis range due to the SPR of metal electrons in NP solution which ultimately gives information regarding the shape and size of the NPs (Link and El-Sayed, 2003; Noginov et al., 2006). The change in color regulates the synthesis of AgNPs from the aqueous extract of M. azedarach (Elumalai et al., 2010). The reduction of silver metal ions A⁺ into AgNPs is due to the presence of active molecules in the leaf extract (Ahmad et al., 2003). Most probably, the speedy rate of reaction by using microwave irradiations is due to constant and rapid heating of the medium, which gives homogeneous nucleation and synthesis of NPs (Ali K. et al., 2015). Integration of microwave chemistry with biosynthetic methods significantly enhances the synthesis of NPs without interrupting green reaction conditions (Joseph and Mathew, 2015). Our results support the observation of Francis et al. (2018), who recently reported on the formation of stable AgNPs from the leaf extract of Elephantopus scaber by using domestic oven irradiation. Active biomolecules present in the aqueous extract of M. azedarach interceded the formation of AgNPs (Tripathy et al., 2010; Sukirtha et al., 2011). There are evidences which state that gallic acid results in rapid reduction of silver nitrate into AgNPs which is due to hydrolysis of tannic acid into gallic acid and glucose under a slightly acidic and basic environment (Martinez-Castanon et al., 2008). Our results are in accordance to Ali K. et al. (2015) who synthesized AgNPs after 30 s of microwave irradiation. The UV-vis spectral analysis predicts that NPs are evenly distributed and somehow more spherical in shape. Small, uniform-sized NPs can be yielded by microwave synthesis in relatively lesser time, and that is the main attraction of this method. The rapid utilization of the initial materials minimizes the chances of agglomeration during microwave-assisted methods and results in narrow-sized distributed NPs (Nadagouda et al., 2011). Increasing pH of the reaction mixture results in an increased reduction of silver nitrate (AgNO₃) by aqueous MLE. This may be attributed to the fact that at higher pH, more H⁺ ions are available, which results in faster reduction of AgNO₃ and oxidation of the metabolites (Jayapriya and Lalitha, 2013). Parameters such as temperature and pH can be used to modify the size and shape of AgNPs, exclusive of any additional stabilizers, as cellular proteins themselves act as stabilizing agents (Aziz et al., 2019). Disparity in the metal salt concentrations and biological material influences the NP synthesis (Pimprikar et al., 2009). It is therefore suggested that microwave-assisted green synthesis by using MLEs acts as both capping and reducing agent and has been subjected for one pot green synthesis of MLE-AgNPs at rapid rate due to improved product yield, consuming less energy, reducing reaction time, comparatively smaller size, as it has been reported previously (Nadagouda et al., 2011; Ali K. et al., 2015). FTIR confirms the absorption of characteristic frequencies of IR radiations of different functional groups (Kalainila et al., 2014). The results of the current investigation are in accordance to previous studies (Raghunandan et al., 2011; Ali K. et al., 2015) that suggested that the peak from 1,607 to 1,636 cm^–1 is due to C=C aromatic ring stretching vibrations. However, Satyavani et al. (2011) proposed that the peaks at 1,620–1,636 cm^–1 represent carbonyl group (C=O) present in polyphenols such as theaflavin, epicatechin gallate, and catechin gallate. The major sources for these vibration bands are various phytochemicals, flavonoids, and alkaloids that are copiously present in the leaf. Biogenic AgNPs are capped by amine and amide group; in addition to, extracts are responsible for reduction of Ag⁺ to Ag⁰, involving the formation of transitional complexes with phenolic OH groups, FTIR insinuates that the presence of peptides on the surface of biogenic AgNPs plays a pivotal role in the synthesis and capping of NPs (Aziz et al., 2015). Few unassigned peaks in XRD pattern indicate the presence of some bio-organic proteins/compounds in the leaf extracts that crystallize on the surface of the AgNPs (Jemal et al., 2017). Ramanibai and Velayutham (2015) performed XRD for AgNPs synthesized from M. azedarach, and the calculated particle size was 22 nm, which is very close to our study. Size variability depends on many factors such as pH, temperature, and type of plant extracts (Musarrat et al., 2010). Previous reports showed various size ranges (0.5–350 nm) for AgNPs synthesized from different leaf extracts (Ahmed et al., 2016). Generally, it is common that due to SPR, silver nano-crystals showed a typical absorption peak at 3 keV in EDX (Magudapatty et al., 2001). Some other minor peaks in graph may be possible due to the presence of biomolecules in plant material bound to AgNPs (Reddy et al., 2015). The SEM results of the current study are in accordance with the work of Mehmood et al. (2017), who synthesized predominantly spherical shaped AgNPs from M. azedarach extract. The various sizes of the particles may be related with different shapes and may be due to aggregation of the smaller ones.

Dynamic light scattering is used to investigate polydispersity or monodispersity and quantitative size distribution of NPs in the colloidal solution. Sample preparations are mainly involved in variation of results (Zia et al., 2016). DLS results of the present study revealed larger particle size and polydispersity in comparison to TEM analysis which mainly included the size of capping agent that lids the surface of AgNPs (Bhakya et al., 2016). Zeta potential is an important parameter indicating the long-term stability and state of NP in dispersion. In accordance to previous literature, highly stable NPs have zeta potential values that range from greater than +25 mV or less than −25 mV. Due to aggregation of interparticle attractions, dispersions showed low zeta potential values; however, it also depends on solution pH and electrolyte concentration (Sukirtha et al., 2012). The zeta potential (−22.3 mV) for MLE-AgNPs in the current study indicates high stability which could be attributed to the presence of organic coating formed around the NPs.

Transmission electron microscope images also depict the AgNP distribution (Nagati et al., 2012). Coating with capping agent signifying the presence of biomolecules in the leaf extract like flavonoids, proteins, and polyphenols indicates the stabilization of NPs and prevents agglomeration even within aggregates (Pugazhendhi et al., 2015). This type of inherent capping agents allows perfect dispersion of NPs in bio reduced colloidal solution (Kathiresan et al., 2010).

The present study specifies that various concentrations of AgNPs from lower to higher significantly inhibited the radial growth of F. oxysporum and found to be more efficient which are in accordance with previous studies (e.g., Wani et al., 2012). Different studies reported antifungal activities of the AgNPs (Vivek et al., 2011). Various researchers worked on the synthesis of NPs from different plant extracts to explore their antifungal potential. Phull et al. (2016) studied antifungal activity of AgNPs synthesized from Bergenia ciliata against various fungi, and their results indicated that the NPs were more effective as compared to the B. ciliata extract alone. The bio-fabricated AgNPs at different concentrations accounted for complete inhibition of conidial germination of Bipolaris sorokiniana, spot blotch pathogen of wheat, whereas 100% conidial germination was observed in the absence of these NPs (Mishra et al., 2014). Balashanmugam et al. (2016) attempted to synthesize efficient and stable biocompatible AgNPs from the aqueous leaf extract of Cassia roxburghii which exhibited higher antifungal activity against plant pathogenic fungi including F. oxysporum, Curvularia sp., and Rhizoctonia solani as compared to the conventional antifungal drug, so their findings suggest that these phyto-synthesized NPs have potential to be used as an effective growth inhibitor. AgNPs exhibited antifungal activity by inactivating the sulfhydryl groups in the fungal cell wall by forming insoluble compounds, while disruption of membrane-bound lipids and enzymes resulted in lysis of the cell (Duran et al., 2005). AgNPs expressed antifungal activity by destructing the membrane integrity of the pathogen (Elgorban et al., 2016). In a former study, Krishnaraj et al. (2012) stated that AgNPs exhibited better antifungal activity against various phytopathogenic fungi. Some other authors also reported that the antimicrobial process may involve the creation of pores by binding AgNPs to the external proteins, intrusive with replication of DNA or forming ROS (Duran et al., 2016; Ottoni et al., 2017). Biogenic nanoparticles coupled with their intrinsic properties are suitable for antimicrobial activities. The synergistic effect of green synthesized AgNPs combining with fungicides was explored against three pathologically reported fungal strains, and this showed significant resistance against infectious agents (Aziz et al., 2016). Elamawi et al. (2018) evaluated the antifungal activity by estimating 90% reduction of colony-forming phytopathogenic fungi including Fusarium moniliforme, F. verticillioides, Penicillium brevicompactum, Pyricularia oryzae, and Helminthosporium oryzae by using AgNPs.

The SEM is extensively used to illustrate the direct interactions between NPs and biological materials (Chen et al., 2014). Disruption of fungal hyphae and cell wall of F. oxysporum at different concentrations of MLE-AgNPs induced toxic effects on fungal cell wall which can be attributed to the disorder in the biosynthesis of chitin. The fungal cell wall has complex dynamics and primarily composed of chitin, glycoproteins, mannans, and glucans. Disturbance of chitin synthesis results in outcomes in the form of wall disintegration and deformed and osmotically unstable fungal cells (Bowman and Free, 2006). It has been insinuated that silver ions are emancipated to the growth media from MLE-AgNPs, which attach on the surface of the cell wall by diffusing into fungal cells (Pariona et al., 2019). SEM monographs in the current study clearly show the deformation of mycelium by the action of nanomaterials; it clearly indicates morphological changes in cell such as surface damage and inflammation as well as loss of cell wall integrity. Similarly, Ouda (2014) studied some fungi and observed a detrimental effect of silver and copper NPs on fungal hyphae. Oxidative stress induced by AgNPs on fungal cells was also suggested as one of the key mechanisms for enhancing the cytotoxic effect. The formation and stable dispersion of the NPs affect their antimicrobial activity; however, during the interaction of biological cells and NPs, excretion of silver and increased residence time enhance the activity (Stark, 2011; Li et al., 2013). Eventually, the NPs inhibited the spore germination, leading to hyphal mutilation and impeding sporulation.

Propidium iodide staining of the mycelium indicated the extent of cell death and suggested that MLE-AgNPs have the ability for causing irretrievable damage and disintegration of cellular membranes by altering the membrane permeability. SEM observations are confirmed by these findings. Fungicidal effect of AgNPs against F. oxysporum was evidenced by expressing a high level of red fluorescence of PI intensity that was directly proportional to the exposed dose of MLE-AgNPs. As an example, the mycelium of F. oxysporum showed cell death and loss of membrane integrity after uptake of PI upon treatment with lawsone (Dananjaya et al., 2017). Similarly, DCFH-DA fluorescence specifies intracellular ROS production in fungal mycelium. ROS is naturally available in the cells as highly reactive signaling molecules leading to cell death by inducing oxidative stress responses in fungi and bacteria and thus serves as an indicator for depicting the physiological status of the cell (Dupre-Crochet et al., 2013; Dayem et al., 2017). These ROS are a set of ephemeral reactive-oxidants, encompassing hydrogen peroxide (H₂O₂), hydroxyl-radical (–OH), singlet oxygen, superoxide radical, etc. (Liu et al., 2011). Chen et al. (2016) proposed that ROS induces inhibition and morphological damage to fungal spores when GO-AgNPs comes in contact with fungal mycelium, which also confirms current observations.

Elmer and White (2016) proposed that NPs have a great potential to play an important role in agriculture sector while their studies on the effect of different types of NPs on tomatoes and eggplants both under greenhouse and field conditions suggested that plant weight and yield are improved even when they are grown in disease-infested soils. The effects of AgNPs on disease reduction and growth parameters are more pronounced at higher concentrations. Similarly, Kim et al. (2012) stated that fungal inhibition amplifies with increased concentration of AgNPs. This phenomenon arises due to higher density of AgNPs solution which has a property to saturate, adhere to the fungal hyphae, and disengage the pathogen invasion. Elmer and White (2016) studied the effect of foliar treated micronutrient NPs in disease-infested soil under greenhouse and field on tomatoes and eggplants and found that NPs performed well and affected plant yield and weight positively. Micronutrients have reduced mobility in plants especially in neutral soils, yet they play a key role in root health, so NPs have potential of micronutrients to sustain roots in disease-infected soils, but it also depends on application efficacy; therefore, NPs have a great potential to be a part of agriculture sector (Elmer and White, 2016). Madbouly et al. (2017) investigated in vivo and in vitro effects of biosynthesized AgNPs against Fusarium wilt, and their findings suggested that these NPs have antifungal potency to be used as synthetic fungicides as they enhanced growth parameters especially the roots when treated with various concentrations and reduced disease severity by 90% by minimizing the number of wilted seedlings. It was also proposed that coating of seedlings with AgNPs may provoke plant growth and prevent the intrusion of phytopathogens (Abdelmalek and Salaheldin, 2016). The antifungal potency of AgNPs provides a shielding effect around the roots and acts as a barrier to invasive fungi by preventing colonization and development of wilt symptoms. Incubation time also affected the treatment of seedlings with AgNPs, as longer times allowed complete saturation of seedling roots and hence play a key role in controlling the wilt disease (Madbouly, 2018).

Pandey et al. (2014) observed that with increasing concentrations of AgNPs, the chlorophyll content also increased. The findings of the present work are in accordance to these results. Farghaly and Nafady (2015) noticed significant stimulation for Chl “b” and carotenoids in tomato plants for biosynthesized AgNPs while they noted stress effect by measuring reduced Chl “a” after 35 days of treatments. Similarly, Pak et al. (2017) proposed that carotenoid content increased in Dracocephalum moldavica with increasing concentrations of AgNPs that protect the plant against ROS. Thus, antioxidant properties of carotenoids provide a protective function against free radicals of metallic and metal NPs (Sakihama and Yamasaki, 2002). Elbeshehy et al. (2014) studied that systemic resistance was induced in Vicia faba against bean yellow mosaic virus (BYMV) by using biosynthesized AgNPs and results in increased concentration of photosynthetic pigments, whereas it was decreased in infected plants likewise. Postinfection treatment of AgNPs resulted in reduced percentage infection, disease severity, and virus concentration.

Different functions of plants such as defensive approach from biocidal and herbivory against fungal and bacterial pathogens as well as structural stability are related to phenolic activity (Heldt, 1997). Virus-infected broad bean leaves treated with AgNPs indicated maximum accumulation of phenolic contents in comparison to the infected leaves at different levels of treatments (Elbeshehy et al., 2014). From our study, it was clear that disease severity was reduced with increased activities of phenolics in tomato plants treated with different concentrations of AgNPs, so the infected roots of treated tomato seedlings were found to be more resistant as compared to the control ones.

Therefore, a noteworthy increase in the activities of phenolics, PO, PPO, and PAL at the higher concentration of MLE-AgNPs is usually regarded as tolerance indicator in tomato plants. So, these results suggest that biosynthesized AgNPs enhance the level of antioxidant enzymes which may act like a defensive mechanism against oxidative stress due to disease, whereas healthy growth of plant was associated with antioxidant defenses. Like current observations, Kumari et al. (2017) found that biosynthesized AgNPs are able to diminish the pathogenic population of Alternaria solani, the causative agent of early blight in tomato in a concentration-dependent manner and reported that pretreatment of particles on leaves increased the host resistance and prevents the infection by increasing the antioxidant and chlorophyll content. Plant resistance was enhanced by improved enzyme activities involved in phenyl-propenoid pathway viz: PAL, PO, and POO (Trotel-Aziz et al., 2008; Jourdan et al., 2009; Radjacommare et al., 2010; Akram and Anjum, 2011). The increased activity of PO, PPO, and PAL might be associated with cell wall strengthening and production of certain bioactive compounds including lignin, suberin, quinones, and melanin which act like a protective shield for the approaching pathogens by destroying their pectolytic enzymes (Kuzniak and Urbanek, 2000; Gomez-Vasquez et al., 2004; Fugate et al., 2016), while PPO plays a key role in initiation of defense resistance against plant diseases by catalyzing phenolic oxidation (Thipyapong and Stiffens, 1997). Pak et al. (2017) proposed that silver accumulated in root tissues after AgNP treatment mainly exists in the form of NPs which were highly stable and did not release ionic silver after inflowing the cells, so they showed least toxicity in comparison to ionic silver.

Nanotechnology plays a dynamic role in introducing multiple approaches for suppressing disease, enhancing disease diagnostics, and developing new measures for manipulation of plants and pathogens. Thus, biosynthesized MLE-AgNPs is an eco-friendly approach to control Fusarium wilt of tomatoes at various concentrations by suppressing the growth of F. oxysporum. MLE-AgNPs has shown strong potential to restrain the fungal population both in lab and field trials in dose-dependent manner. Application of NPs on pre-infected roots of tomato plants successfully reduced the wilt by increasing the resistance of the host plant and enhancing the growth parameters of tomato seedlings. Photosynthetic pigments and antioxidant enzymes increased with the various concentrations of MLE-AgNPs. So, this study provides a basis that biosynthesized NPs can be used as an alternative to conventional fungicides and become helpful in minimizing environmental pollution. Hence, they have the ability to replace the health hazards of chemical fungicides.

The datasets generated for this study are available on request to the corresponding author.

TA and HA designed the study. HA has worked as a research scholar for this project. SR and SN have provided technical guidance for various analyses. All the authors read and approved the final version of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.