Sustainable Synthesis of Microwave Assisted IONPs by Using Spinacia Oleracea: Enhances Resistance Against Fungal Wilt Infection by Inducing ROS and Modulating Defense System in Tomato Plants

Background: Changing climate enhances the survival of pests and pathogens which eventually affects the crop yield and reduces its economic value. To attain the sustainable food security, novel approaches should be employed. Nanobased agri-chemicals provide a distinctive mechanism to increase productivity and manage phytopathogens with minimum environmental distress. In-vitro and greenhouse studies were conducted to evaluate the potential of green synthesized iron oxide nanoparticles (IONPs) in suppressing the wilt infection caused by Fusarium oxysporum f.sp. lycospersici and to improve tomato growth (Solanum lycopersicum) and fruit quality. Results: Various microwave powers (100 W- 1000 W) were used to modulate the properties of green synthesize IONPs by using spinach as a starting material. The IONPs stabilized with black coffee extract were substantively characterized by X-ray diffraction analysis, fourier transform infrared spectroscopy, dielectric and impedance spectroscopy, X-ray photoelectron spectroscopy, Scanning and transmission electron microscopy and magnetization-analysis. XRD revealed cubic magnetite (Fe 3 O 4 ) phase with superparamagnetic nature, detected at all microwave powers. Binding energies of Fe 2p3/2 (712.05 eV) and Fe 2p1/2 (723.9 eV) of Fe 3 O 4 NPs was conrmed by XPS analysis observed at microwave power of 1000 W. Uniform, spherical shaped particles with an average diameter of 4 nm were conrmed by SEM and TEM analysis. Signicant reduction in mycelial growth and spore germination was perceived after exposure to different treatments of IONPs. Malformed mycelium, DNA fragmentation, alternation in cell membrane and ROS production in F. oxysporum indicates antimicrobial anity of iron oxide NPs. The particles were applied both through root (before transplantation) and foliar application (after two weeks) to infected seedlings. The IONPs signicantly reduced the disease severity by an average of 47.8% resulting in increased plant growth variables at exposure to 10 µg/mL of IONPs. Analysis of photosynthetic pigment, phenolic compounds and antioxidant enzymes in root and shoots signies an increasing trend after exposure comparisons ±

exposure to different treatments of IONPs. Malformed mycelium, DNA fragmentation, alternation in cell membrane and ROS production in F. oxysporum indicates antimicrobial a nity of iron oxide NPs. The particles were applied both through root (before transplantation) and foliar application (after two weeks) to infected seedlings. The IONPs signi cantly reduced the disease severity by an average of 47.8% resulting in increased plant growth variables at exposure to 10 µg/mL of IONPs. Analysis of photosynthetic pigment, phenolic compounds and antioxidant enzymes in root and shoots signi es an increasing trend after exposure to various concentrations of IONPs. Correspondingly, lycopene, vitamin C, total avonoids and protein content was substantially improved in tomato fruits after treatment of IONPs.
Conclusion: These ndings of current investigation suggests that IONPs owned antifungal properties to subdue Fusarium wilt disease by boosting plant growth and fruit quality.

Background
In recent times innovative and advance technological approaches has been employed in agriculture sector to encounter the surging challenges of food-security and sustainable-production [1]. Stratagems based on "nanoparticle-technology" have releveled fascinating outputs due to their innate characteristics [2]. Novel practices in agriculture are dynamic factor of research to meet the needs for consistent food supply in exploding global population [3]. Nanotechnology, an emitting interdisciplinary eld of modern era, has imperative impact on people's lives by resolving most of the scienti c problems and has proven its essentiality almost in every eld including agriculture and allied industries [4]. Nanoscale materials due to their distinctive properties such as improved e cacy, lesser eco-toxicity and reduced-inputs provides auspicious alternative for crop protection that has numerous advantages over traditional approaches and products [5]. Among various classes of nanoparticles, metal-oxide nanomaterials are generally pondered as innocuous both for environment and humans, speci cally iron oxide nanoparticle (IONPs), are the most prominent and biocompatible, possess antimicrobial properties against pathogenic fungi and bacteria [6]. Now-a-days nanotechnology has a new function in agriculture by implicating suppression of pathogen infection (bacterial, viral, fungal) by improving plant nutrition and thus directly enhances nutritional value and crop yield [7]. Additionally, nanomaterials may affect plant cells and its developmental stages including seed-germination, root induction, cell-metabolism, growth-index and biomass plus alters redox level [8]. Iron is a pivotal micronutrient cognate with various physiological functions in plants but generally found as insoluble Fe +3 form, owing to low solubility of minerals having iron, one method to overcome iron-de ciency is the usage of nanoparticles either as iron or iron oxide NPs [9]. Iron oxide nanoparticles being reduced in size and having higher solubility than other complex molecules facilities plants with greater level of iron [10]. Therefore, these compounds provide optimum environment for plant enhancement and averting stress conditions by generating secondary metabolites [11]. Iron being key constituent of cell-redox reaction, behaves like a co-factor for various anti-oxidant enzymes such as catalase (CAT), superoxide-dismutase (SOD), and peroxidase (POD), acts as a scavenger of ROS [12]. Even though oxidative stress is induced in response to nanoparticles, triggers the defence mechanism in plants such as higher activities of antioxidant enzymes eventually scavenge for ROS [13]. Earlier reports indicated that during stress conditions, iron oxide nanoparticles elevated the antioxidant activity in various plants such as, Mentha piperita, [9] Triticum aestivum, [14,15] Solanum lycopersicum [10] and Dracocephalum moldavica [4].
Tomato (Solanum lycopersicum L.) being the most notable crop after potato from commercial and economic perspective, ranked sixth in world by Food and Agriculture organization in terms of total annual production [16]. Crop yield is potentially reduced to 20% by soil-borne diseases that is tough to cope, alone fungal pathogens are responsible for loss of millions per years by reducing economic return [17].
Fusarium-wilt, one of the most devastating fungal-diseases, diminishing the yield index and nutritive value of various crops, particularly in tomatoes, it is initiated by Fusarium oxysporum f.sp lycopersici, affected tomato growth equally under greenhouse and eld trials [18]. Although there are different traditional ways to overcome this disease such as use of resistant verities and application of fungicides, but these approaches are environmentally un-sustainable and cost ineffectual, [17] so there is a demand of time to nd more innovative and effective ways to cope with fungal pathogens. Although limited number of reports available on the role of iron oxide nanoparticles in diseases management. Alam et al. reported antifungal e cacy of green synthesized iron oxide nanoparticles against polyphagous pathogen Verticillium dahlia by preventing the proliferation of mycelium [19]. Fe 3 O 4 nanoparticles induces the expression of miR159c in yellow medick plant against powdery mildew [20]. In-vitro application of IONPs showed signi cant antifungal properties, inhibiting the spore germination in various phytopathogens such as, Aspergillus niger and Fusarium solani; [21] Rhizopus oryzae, [22] Trichothecium roseum, Cladosporium herbarum, Penicillium chrysogenum, Alternaria alternata and A. niger; [23] P. expansum, A. niger, A.alternata, P. chrysogenum, Mucor plumbeus, T. roseum and Rhizoctonia solani [24]. Earlier studies indicate the formation and accumulation of reactive-oxygen-species (ROS) in microbial cells impedes the multiplication ability of the pathogens depicts the underlying mechanism of metal nanoparticles, however it is commonly anticipated that antimicrobial activity relies on direct-interaction among nanoparticles and living cells [25]. magnesium oxide nanoparticles reveal antagonist activity against fungal species by acting directly on fungal cells [26]. Chitosan-coated IONPs showed signi cant antimicrobial activity against Escherichia coli and Bacillus subtilis by up-regulating the ROS level [27].
Tomatoes are an important part of human diet as they contain various bio-active compounds like totalphenols, lycopene, vitamin-C, carotenoids and total-avonoids, that acts as antioxidant compounds with speci c physio-chemical and biological properties, plays a bene cial role in human health [28]. As iron oxide nanoparticles are safe to be used for medical and food applications, permitted by food and drug administration (FDA) [29], making IONPs as a potential candidate to study their fungicidal activity and nutritional effects on plants.
In current investigation, it was hypothesized that green synthesized IONPs will be more e cient in suppressing fusarium wilt disease in infected tomato plants. We demonstrated the synthesis of iron oxide nanoparticles (IONPs) by utilizing spinach powder and BC (black coffee) extract at various microwave powers i.e., 100 W-1000 W. The ndings of this study indicate the improved e cacy of microwave assisted IONPs in subduing fungal growth and enhancing plant growth, both under in-vitro and in-vivo conditions. In addition, alternations in mycelia, ROS production, membrane integrity and DNA fragmentation were evaluated in fungal pathogen. Importantly, the effects of IONPs on plant germination, disease index, enzymatic and non-enzymatic antioxidant enzymes were also measured. These ndings provide signi cant information on e cient supply and activation of defensive pathways in tomato plants in response to metal oxide exposure and has a potential to replace the conventional chemicals, which are also toxic to the environment.

Materials And Methods
Materials, reagents and fungal culture All the reagents and chemicals were of analytic grade, obtained from Sigma-Aldrich, (St Louis, MO, USA) consumed without any further puri cation unless otherwise speci ed. Fresh spinach leaves (Spinacia oleracea), bought from local vegetable market of Lahore, Pakistan, were used as a starting material. Black coffee extract was prepared by dissolving 20 g of coffee powder to 100 mL of deionized water (DI) by adjusting pH at 5. Tomato seeds were obtained from Ayub Agriculture Research Institute (AARI), Faisalabad, Pakistan. "Fusarium oxysporum f. sp. lycopersici (IAGS-1322)" used in this investigation as a challenging tomato pathogen, issued by Fungal-Biotechnology-Lab, Department of Plant Pathology, Faculty of Agricultural Sciences, University of the Punjab, Lahore, that was previously isolated from infected tomato elds and was preferred, based on its virulence. Stock culture was grown and maintained on potato dextrose agar (PDA) slants at 4 °C for long term use. For solid cultures of F. oxysporum, stock cultures were sub-cultured onto Petri plates having PDA and left for 7 days in dark at 28 °C. Deionized water was used as a solvent.

Green synthesis of Iron oxide Nanoparticles (IONPs)
For the synthesis of IONPs, spinach (Spinacia oleracea) was used as a staring material. Initially, spinach leaves were washed with deionized water and further dried at room temperature (25 o C) for 2 h. Dried leaves were kept in mu e furnace at 500 o C for 2 h to get powdered leaves. Nano ball milling was performed for 4 h at 3000 rpm in pulverisette (Fritsch pulverisette-23 mini mill) to obtained ne nanopowder. Four grams (4 g) of nano milled powder was dissolved in 400 mL of DI water under vigorous stirring (solution A). Black coffee (BC) extract was added dropwise to solution 'A' to prepare 0.01 M solution (solution 'B') under continuous stirring. Solution 'B' was further subjected to various microwave powers i.e., 100 W-1000 W, for the formation of stabilized iron-oxide powder. On and off time of microwave was precisely controlled to overcome the spitting process due to localized heat generated in microwave oven. The resultant product was separated by centrifugation and dried in vacuum oven 80 °C for further characterization. Details regarding characterization instruments is discuss in Experiment S1.
The schematic is shown in Fig. 1.

Inhibitory effect of Iron-oxide nanoparticles on fungal mycelial growth
In vitro antifungal activity of Iron-oxide nanoparticles on mycelial growth of F. oxysporum was evaluated by following agar dilution protocol. Twenty milliliter of PDA was poured into sterilized Petri dishes and measured amount of IONPs from stock solution was added to get the required concentrations. The nal concentrations in the growth media were 0.01, 0.5, 1.5, 2.5, 5, 7.5, 10, 12.5 and 15 µg of IONPs per mL. PDA plates without IONPs were used as a negative control while plates with fungicide treatment serves as positive control. Afterwards, fungal disc (4mm in diameter) was incised from the fringe of a seven-day old culture of F. oxysporum and inoculated aseptically at the center of each PDA solid media plate. Later, plates were sealed and incubated at 28 °C until the growth in the control plates extends to periphery. Each treatment was performed in triplicates. Photographs were captured and mycelial radial growth was estimated after 7 days. Percentage inhibition of radial growth by different concentrations of iron-oxide nanoparticles in comparison to control was calculated using equation (1): Where, R 1 : mean radial growth in control group, R 2 : mean radial growth in treatment group [30]. The protocol for determining suppressive activity of IONPs on fungal spore germination is discussed in Experiment S2.
Evaluating morphological alterations in fungal mycelia by SEM The morphological and ultrastructural changes in fungal hypha after treatment with IONPs were examined by Field-emission-scanning-electron-microscope (FE-SEM). In brief, control and nanoparticles treated F. oxysporum (10 and 15 µg/mL) mycelium, were scratched out and xed for 4 h with glutaraldehyde (2.5%) at 4 °C, post-xed with aqueous-osmium-tetroxide (1%), later the xed sample was washed for several times with phosphate-buffer-saline (pH:7, 0.5 M). Afterwards, the hyphal samples were exsiccated with ethanol in form of gradient-series (30%: 50%: 70%: 80%: 90% and 100%), twenty min per series. Subsequently, hyphal sections were left overnight in isoamyl-acetate. Finally, the sections were exposed to analytical level of dry CO 2 , conductively coated by gold sputter and observed under a FE-SEM (S-4800, Hitachi-Japan).
Evaluating Reactive oxygen species (ROS) production . For visual detection of ROS production in fungal mycelium was observed by confocal-laser scanning-uorescence microscope (CLSM, Zeiss LSM 7) with emission at 525 nm and excitation at 488 nm. The quanti cation of uorescence intensity was carried out by using Image J software [31].

Evaluating plasma membrane integrity
Integrity of plasma membrane of F. oxysporum after treatment with IONPs were explored by propidium iodide (PI: membrane-impermeable dye) according to the earlier protocol described by Wei et al. [32] with minor modi cations. Fungal mycelia were collected by following the above protocol. The suspension was not treated with nanoparticles considered as control. Subsequently, collected mycelia were stained with 10 µg/mL od PI for 15 min at 30 °C under dark conditions. After centrifugation at 5000 rpm for 5 min at 4°C , mycelia were rinsed twice with 0.5 M PBS (pH:7) to eradicate residual-dyes and re-suspended in the same buffer. Samples were observed by confocal-laser scanning-uorescence microscope (CLSM, Zeiss LSM 7) with emission at 617 nm and excitation at 536 nm, while results were analyzed in percentage with reference to the control. The quanti cation of uorescence intensity was carried out by using Image J software.
Next, the lysates were incubated with RNase (0.03 mg/L) and proteinase K (0.2 mg/mL) solution at 55 °C for 1 hour. After incubation period, the concoction was centrifuged for ten minutes at 10,000 rpm. The nuclear-DNA of F. oxysporum was then isolated by using phenol-chloroform mixture. The aqueous phase was then transferred to new tube and puri ed with isopropanol and placed at -20 °C for 20 min. The DNA samples were rinsed thrice with 70% ethanol and resuspended in TE buffer. DNA samples were run on a 1% agarose gel in 1x TAE buffer at 100 volts for 60 min stained with ethidium bromide (1mg/mL). The gel photographs were captured by using UV-transilluminator (Witeg, Germany).
Evaluating the impact of IONPs under green-house assay A tomato variety (Rio-Grande) susceptible to wilt disease caused by F. oxysporum were grown in small plastic pots under controlled conditions (temp: 30-28 ±1°C; humidity 85-90%) for 25-30 days, until 3-5 leaves were developed. The fungal pathogen i.e., Fusarium oxysporum was grown on potato dextrose broth (PDB) to develop conidial suspension, while inoculated concentration was adjusted to be 1x 10 6 spore/mL. Speci cally, one week before seedling transplantation, each plastic pot lled with sterilize soil, received 30 mL of spore suspension in a separate trial. Nanoparticle's treatment was performed by root dipping method [33] by keeping tomato plant roots in various concentrations of IONPs (0.1-15 µg/mL) at room temperature. Afterwards, uniform tomato seedlings were shifted to infected soil, three plants per pot. Positive and negative controls treated with fungicide (Nativo) and sterilized water. Foliar applications of nanoparticles sprays with interim of ve days were performed after two weeks of transplanting. Each treatment was adjusted by three replicates. The Fusarium wilt disease incidence (DI) and percent disease severity (PDS) was evaluated after 25 days of post inoculation. The disease severity for FW (Fusarium wilt) on tomato plants was determined using the following 0-6 indexing scale with minor modi cations [34].Disease severity scale: (1= Immune: symptomless, 2= Resistant: Initiation of wilting symptoms, 5% leaves showing yellowing and wilting, 3= Moderately Resistant: 6-10% wilting and yellowing of leaves, 4= Moderately susceptible: 11-12% leaves show symptoms, 5= Susceptible: 21-50% leaves showing wilting with yellow brown bicolouration, and 6= Highly susceptible: > 50% leaves show infection, dying and drying of plant. Percentage disease severity was calculated by using the following equation (2):

Statistical analysis
Statistical-analysis was achieved by using GraphPad-Prism (8.4.3) software (GraphPad-software, CA, USA). One-way-analysis-of-variance (ANOVA) or students t-test was employed for comparing means and difference between group mean, by using Tukey's multiple comparison test. The outcomes were presented as the mean ± standard-deviation (SD) for three or more replicates. Asterisks (* P< 0.05; **P< 0.01; ***P<0.001) represents signi cant differences.

Results And Discussion
Structural analysis of green synthesized IONPs XRD patterns of iron oxide nanoparticles (IONPs) synthesized by using spinach leaves and (black coffee) BC extract, subjected to various microwave powers i.e., 100 W to 1000 W ( IONPs. The phenolic-OH as well as ortho-dihydroxyphenyl groups present in the chemical structure of tannins are responsible for complex formation with iron. These groups also participate in redox reactions [35]. Therefore, production of Fe 3 O 4 nanoparticles is mainly governed by the tannins present in BC extract. Also, the use of microwave radiation makes it possible to synthesize pure phase Fe 3 O 4 nanoparticles without any further heat treatment. Microwave energy is converted into heat energy which is highly depends upon the nature of utilizing solvent. The parameter which determines the capability of material for this conversion is termed as "tangent loss" (tan δ). Usually for microwave radiations having typical frequency of 2.45 GHz, solvents with higher value of tan δ are preferable choice as they can absorb excellent amount of radiations thus convert it into heat energy [36]. Due to higher tangent loss of polyphenols (i.e., constituents of BC, tannin), high heat will be produced in microwave oven thus leading towards the phase pure Fe 3 O 4 nanoparticles even without further heat treatment.
Where, k is shape factor taken as 0.9, λ is the wavelength of Cu ka source, β is full width at half maximum (FWHM), θ is the diffraction angle of highest intensity peak. Variation in crystallite size was observed with the variation in various extracts used during microwave powers. Black coffee along with microwave heating played a critical role not only in achieving Fe 3 O 4 phase of iron oxide ( Fig. 2) but also in achieving a small value of crystallite size ( Figure S1). XRD analysis also revealed that synthesized nanoparticles using 100 W -200 W power possess amorphous behavior while for higher microwave powers crystalline nature of iron oxide nanoparticles along with phase stability was observed (Fig. 2). Major factors in uencing crystallite size are lowest surface energy, grain boundary energy and diffusion of surface atoms. Microwave energy was observed to be effective to tune these parameters thus leading to formation of stabilized and pure phased Fe 3 O 4 nanoparticles. Highest crystallite size i.e., ~15 nm along with lowest dislocation value was observed after using microwave power of 1000 W ( Figure S1).

Dielectric analysis of green synthesized IONPs
Short ranged electrical conduction of material is dependent on the dielectric properties which it exhibits. Charged particles in the material experience a displacement as a result of applied electric eld and gets pile up at the interfaces and hence creation of dipoles takes place. Frequency dependent dielectric constant of iron oxide nanoparticles (IONPs) was obtained by impedance analyzer using Eq. (6), whereas the tangent loss was calculated using Eq. (7) [38].
Where, 'C' represents capacitance, 'd' is for specimen's thickness, 'A' is for area, 'ρ' is for resistivity and permittivity of free space is represented by 'ε o' .
Variation in dielectric constant and tangent loss as function of frequency at room temperature was shown in Fig. 3A, B. Different types of polarization i.e., ionic, electronic, orientational and dipole are accompanied with dielectric constant under the effect of an alternating electric eld. At low frequency space charge polarization is dominant whereas, at high frequency ionic and electronic polarizations dominate in polycrystalline material. When an external electric eld is applied dispersion occurs in space charges and these space charges require some time to align themselves in the direction of applied eld. At low frequency space charges get enough time for their arrangement as per applied eld. However, at high frequency values charges do not get enough time to orient themselves in the direction of the applied eld, thus, resulting in low dielectric values (Fig. 3A). Dielectric constant almost remained same for samples synthesized with low microwave powers i.e., 100W-200W. Small values of dielectric constant at low microwave power is due to amorphous behaviour of iron oxide nanoparticles as observed in XRD patterns (Fig. 2). Whereas increase in dielectric constant was observed with the increase in microwave power from 300W-1000W. High value of dielectric constant is attributed to the presence of both Fe 3+ and Fe 2+ cations in Fe 3 O 4 phase of iron oxide. Heterogeneity in Fe 3 O 4 structure arises because of the existence of Fe 2+ cations that gives high polarization, leading to higher value of dielectric constant [39,40]. High dielectric constant can be employed in agricultural eld i.e., plant pathology for investigating activities of pathogenic microbes including fungi.
Tangent loss for as synthesized nanoparticles is shown in Fig. 3B. The plot of tangent loss shows normal dispersion behaviour due to space charge polarization. Relatively, higher values of tangent loss are observed for nanoparticles prepared at low microwave power i.e., 100 W -200 W whereas, low values of tangent loss are observed for high microwave powers. Comparison of dielectric constant as well as tangent loss with respect to varying microwave powers at log f =5 and log f =1.3 ( Figure S2).
Conductivity is categorized in two regions, i.e., low frequency region and high frequency region. At low frequency region conductivity is known as dc-like conductivity because of free charge carriers. Whereas, at high frequency it is known as ac conductivity because of bound charge carriers. Figure S3A shows variation in conductivity as a function of frequency for all of the samples prepared using various microwave powers i.e., from 100 W-1000 W. Small values of conductivity, because of the hopping mechanism of charge carriers, are observed even at high frequency values. Comparison of conductivity values at log f =5 and log f =7.3 by using various microwave powers is exhibited in Fig. S3B.
Impedance is a property that offered an opposition to the ow of electric current. Conduction in nanomaterials can be best comprehended on the base of complex impedance spectroscopy which is an effective technique to differentiate the resistive and conducting elements in the circuit. Complex impedance (Z*) is presented in Eq. (9) [38] while real (Z') and imaginary (Z'') impedance are given in Eq. (10) and (11) '' = The variation of real and imaginary impedance in iron oxide nanoparticles with respect to frequency is depicted in Fig. 4A,B. Two regions are observed in Z' plots (Fig. 4A). First is the region of low frequencies in which there is a slight decrease in Z'. Second is the region in which Z' decreases monotonically and becomes constant at higher frequencies. This behavior is connected with conductivity of charge carriers observed in Fig. 4A where conductivity increases at high frequencies [41,42]. Z'' (Fig. 4B) shows different relaxation peaks at different values of frequency for changes in microwave powers. Information regarding grains and interface effects in poly crystalline materials can be realized on the basis of these relaxation peaks which demonstrate different relaxation mechanism happening in nanoparticles [42].
Relaxation peaks at different microwave powers spread in different zones of frequency explains the distinguished participation of grains and grain boundaries. To obtain the exact contribution of grains and grain boundaries towards the conduction process Cole-Cole plots i.e., plots between Z'' and Z' were studied (Fig. 4C). Generally, Cole-Cole plot contain of three semi circles. First semi-circle in high frequency regime represents the effect of grains resistance, second semicircle in the middle range of frequency correspond to grain boundary resistance and the third one in low frequency range represents the resistance offered by grain-to-grain boundary interface. These plots provide information about electrical characteristics of material (grains and grain boundaries) [43].
FTIR analysis of green synthesized IONPs FTIR spectra of green synthesized IONPs using BC extract and dried using various microwave powers is depicted in Fig. 5A,B. Peak appeared at ~ 560 cm -1 was assigned to characteristic band of Fe-O that also in well agreement with previously reported literature [44]. The band at 1068 cm -1 was appeared due to C-N stretching. While, peaks appearing at 1561 cm -1 and 1650 cm -1 were ascribed to C=C stretching vibrational bands due to presence of aromatic rings / phenolic groups present in BC extract. Presence of phenolic feature indicates the capping effect on the surface of IONPs. Absorption band appeared at 2352 cm -1 corresponds to atmospheric CO 2 [45].

Magnetic response of green synthesized IONPs
Superparamagnetic response of green synthesized IONPs at various microwave powers i.e., 100 W -1000 W, was detected through M-H loops (Fig. 6). However, high saturation magnetization (~21.72 emu/g) was observed for IONPs synthesized at 1000 W (Fig. 6J). Superparamagnetic behavior arises when size of single domain becomes so small that thermal energy can easily overcome anisotropy energy barrier. As the particle size is decreased number of surface spins contributing to magnetization increases [46]. Such behavior of IONPs makes it potential candidate for agricultural applications in terms of targeted delivery of nutrients and controlled release of pesticides in plant. Variation in saturation magnetization (Ms) was observed by using various microwave powers ( Figure S4).
X-ray photoelectron spectroscopic (XPS) analysis of green synthesized IONPs XPS analysis of IONPs was performed by using spinach as a precursor along with black coffee (BC) extract at various microwave powers (100 W-1000 W) with interval of 100 W (Fig. 7 A, B). It can be observed that binding energy peaks of Fe 2p 3/2 and Fe 2p 1/2 at 710.9 eV and 724.5 eV are due to magnetite (Fe 3 O 4 ), respectively (Fig. 7A) [47]. Splitted spin-orbit peak of Fe 2p are wide due to less chemical shift between Fe 2+ & Fe 3+ [48]. Figure. 7b represents the spectra of O1s core level. Peak present at 529.9 eV is due to existence of O -2 species and 531.7 eV is due to existence of OHspecies present on iron oxide surface. Other peak at 532.9 eV is associated with adsorbed H 2 O molecules [49].
Surface morphology and size distribution of green synthesized IONPs The bene ts of using microwave-assisted approach in contrast to chemical-methods includes in-core heating of materials in a rapid way and its speci c chemical bonds gives discerning absorbance, which results as nano size particles, having uniform size and shape, depicted from microscopic images (SEM and TEM). The morphology and particle size distribution of green synthesized iron oxide nanoparticles (IONPs) at 1000 W power were revealed by SEM and TEM analysis ( Figure S5). Microscopic images indicate uniform, spherical, polydisperse, less-aggregated crystalline nature of IONPs ( Figure S5 A, C) which endorses the purity of the sample without any other phases. Figure S5B showed the particles size distribution of IONPs by SEM, was in the range of 3-23 nm with an average diameter of 4.99 ± 0.17 nm. Correspondingly, the mean particle size of 4.08 ± 0.19 nm was determined by TEM, varying from 1-15 nm, conferring to Gaussian t of particle size distribution ( Figure S5D). These results display the narrow size distribution of green synthesized IONPs, which are somehow coherent with previous studies [50][51][52][53].
IONPs as inducer of antifungal activity-inhibiting fungal growth and spore germination We investigated the in-vitro antifungal activity of biosynthesized iron oxide nanoparticles (IONPs) synthesized at various microwave powers (100 W-1000 W) as shown in Table S1. After initial screening, the IONPs at 1000W was selected and used for next experiments which also justify the phenomena of highest magnetic saturation at this microwave power. The emergence of resistance in plant fungal pathogens against agrochemicals leads towards development of more e cient antifungal agents which must be eco-friendly. Therefore, antifungal potential of nanoparticles is bene cial in agriculture sector as they emerged as "innovative-generation fungicides" [54]. Figure 8 shows the effect of INOPs on fungal mycelial growth and spore germination at various tested concentrations (0.01 µg/mL -15 µg/mL) against F. oxysporum, the causal agent of tomato wilt.
IONPs exhibited strong inhibitory effect on radial growth of fungal mycelia on PDA medium as compared with control treatment (Fig. 8A). It is evident from the results that antifungal activity of iron oxide nanoparticles signi cantly increases in a dosage-dependent form. After seven days of post inoculation, 15 µg/mL of IONPs strikingly minimized F. oxysporum growth by 90.84 ± 0.56%, in contrast to the control treatment. For lower concentrations, ranges from 0.01-1.5 µg/mL, displayed minimum growth inhibition i.e., below 50% whereas, fungicide exposure yielded 89.77 1.24%, relative to the growth rate in control group (Fig. 8B). Former studies indicated the antimicrobial activity of IONPs, and their nding suggests that this activity increases gradually from lower to higher concentration [55,[22][23].
The sporicidal activity of IONPs on spore's germination of F. oxysporum was illustrated in Fig. 8C,D. Disruption of fungal membrane indicates the inhibiting action of nanoparticles as observed in this study is owing to the biocidal action of nanoparticles, furthermore, smaller size NPs retain large surface area gets readily attached and absorbed to disassemble the microbial cell membrane, leads to deterioration of intracellular organelles, eventually results in death of microorganism [56,57]. After six hours of incubation with various concentrations of iron oxide nanoparticles, the microscopic images revealed that spore suspension of F. oxysporum displayed a sharp decline in spore germination rate in contrast to the untreated control samples (Fig. 8D). In fungal life cycle, spore germination and maturation are vital phases for successful plant colonization, but once the germination is subdued after treated with graphene oxide, spore cannot develop into mature mycelium to initiate the infection cycle [35]. It can be clearly indicated from the results that germination rate of spores was gradually reduced in response to the increasing concentrations. A statistically signi cant reduction in germination rate was observed at concentrations ranges from 1.5 -15 µg/mL, while minimum germination rate was up to 5.38 ± 1.38% at 15 µg/mL in comparison with the control group (88.58 ± 0.69%). Analogously, in case of fungicide, signi cant inhibition to spore germination was determined to be 18.56 ± 0.86% respectively (Fig. 8C).
Devi et al. worked on two fungal species (A. niger and M. piriformis) proposed that greater surface interaction among iron oxide nanoparticles and fungal membranes played a signi cant role in antifungal activity [58]. Analogously, Saleem et al. also exhibited antifungal potency of green synthesized iron-oxide nanoparticles against A. avus and F. oxysporum, their nding suggests that IONPs have potential to be used for biological applications [59].
IONPs induce changes in cell-wall morphology, viability and ROS production in F. oxysporum The SEM micrographs revealed malformed mycelium after treatment with iron oxide nanoparticles can be attributed to distortion of chitin synthesis and cell envelop; that shields the leakage of cellular component into extracellular environment [19]. SEM visualization indicates the detrimental effects of iron-oxide NPs on F. oxysporum. The IONPs treated mycelia revealed some eccentric morphological characteristics as compared to the control (Fig. 9A). In control, cylindrical shaped mycelium has healthy smooth turgid surface with clear conidiation (Fig. 9A-a). Whereas, after contact with IONPs, the remarkable structural changes were induced in fungal hyphae as observed in Fig. 9A-b,c. At 10 µg/mL, hypha became deformed showing irregular shrinkage with minute granules on surface ( Fig. 9A-b). The impairment was intensi ed at 15 µg/mL, the hyphae become recessed, slender and stacked together including rifts or blebs (Fig. 9A-c). Earlier investigations suggested that consecutive interactions occurred due to magnetic nanoparticles which stimulated microbial toxicity, such as discharging of metal-ions, affecting protein synchronization and cellular-homeostasis; lipid-peroxidation and nucleic acid impairment by accumulation of reactive oxygen species (ROS) and mutilation of cell integrity by membrane- The antifungal property of IONPs was further veri ed by using uorescent dyes i.e., Propidium iodide (PI) and 2',7'-dichloro-dihydro-uorescein-diacetate (H 2 DCFDA). The uorescence intensity of fungal hyphae was signi cantly enhanced in dose-dependent manner on the treatment of IONPs in contrast to the untreated control. Alike outcome was perceived for IONPs bound with amphotericin B, by a reaction between aldehyde and amine groups against candida strains [61]. Propidium iodide is a DNA-uorescentprobe that invades disrupted plasma membrane of cell, emitting red color uorescence from stained nucleus [62]. The effect of IONPs on membrane integrity of F. oxysporum in control and treated fungal mycelial samples were depicted in Fig. 9B. As pragmatic from the RFP (red uorescence protein) images a slight red-uorescence was detected in the control hyphae whereas, stronger uorescence was observed in treated ones. After 15 mins of exposure, the PI uorescence intensity in treated groups (5,10 and 15 µg/mL) were 2.21, 3.09 and 3.69 folds, signi cantly higher than the control group (Fig. 9D). The GFP (Green uorescence protein) micrographs of control and IONPs treated fungal mycelia of ROS accumulation were illustrated in Fig. 9C . By comparing the uorescence intensities as shown above speci es that the fungal-nano interactions were relatively stronger after treatment with IONPs that eventually increases the variation in free-energy content, resulting more ROS generation. In line up with current investigation, Arakha et al. found that chitosan-coated IONPs in culture media has ability to enhance ROS production by altering the interaction pattern among bio-nano interface, plays a critical role for antimicrobial a nity of iron oxide NPs [27].
IONPs as inducer of DNA-fragmentation in F. oxysporum DNA-fragmentation is another biochemical key feature of apoptosis (programmed-cell-death) [67]. The inter-nucleosomal cleavage of genomic DNA was studied in F. oxysporum subjected to various concentrations of IONPs. DNA was isolated from fungal cells and analyzed by agarose gel electrophoresis. The electro-phoretogram is presented in Figure S6. As it is clear from the gel image that typical DNA ladder were formed in control group however "DNA-laddering" of non-chronological DNA fragments were found in IONPs treated group. Moreover, it can be observed that DNA-cleavage increases in IONPs treated samples in concentration-dependent manner. Gel containing DNAs of F. oxysporum also indicates single high molecular-DNA-bands in lanes 1, 2 and 3, treated with 0.01, 0.5 and 1.5 µg/mL IONPs. However, the intensities of these DNA-bands were less than the control group (lane 10).
Additionally, the smeared-DNA in all lanes is less than 1Kb and apparently appears weaker than the . ROS is reported to be implicate in DNA mutilation by IONPs effecting DNA bases such as pyrimidine and purine, contributing to reduce the bio lm formation in bacterial cell [29].

Impact of IONPs on tomato growth-parameters
All concentrations of Iron oxide nanoparticles (0.01-15 µg/mL) signi cantly enhanced the growth parameters (length (root and shoot), biomass (fresh and dry weight) and plant height of tomato plants infected with Fusarium wilt under pot bioassay (Fig. 10: Figure S7)  Figure S7B). Furthermore, the average root and shoot length under 5-15 µg/mL were greatly analogous to the control treatment, predominantly for 12.5 µg/mL showed an increase of 76.3% (roots) and 79.05% (shoots) respectively. Moreover, statistically signi cant difference was found for plant biomass (fresh and dry weight) in contrast to the control treatment ( Figure S7C). The fresh and dry weight was superior with 12 µg/mL treatment surpassing the control by 60.9% and 67.1%. While fungicide treatments followed the same trend for fresh and dry weight by showing an increase of 43.8% and 47.7%, with respect to the control. Earlier studies reported increase in seed germination, plantbiomass, seedling growth/vigor and yield after application of iron oxide nanoparticles [70][71][72][73][74]. Treatment of tomato seeds with Fe 3 O 4 nanoparticles had no side effects on plant growth and development [75]. Similarly, our results veri ed the previous research by observing the enhancement in growth parameters of tomato plants treated with various concentrations of IONPs.

Impact of IONPs on disease attributes of tomato wilt
Virulence of many pathogens relies on iron procurement so; occasionally microbial infections can be avoided by using iron-chelating products that inhibit pathogen's ability to approach iron [76]. Pot bioassays were performed to evaluate whether IONPs, considered as an agricultural antifungal agent, have potential to control Fusarium wilt disease (Fig. 10D,E). As shown in Figure S8A,B treating infected tomato seedlings with IONPs reduced the severity and incidence of Fusarium wilt caused by F. oxysporum. Furthermore, a clear positive correlation exists between the concentrations of NPs and disease-index. The disease in control plants were speci cally severe after 25 days of post inoculation, and the disease severity came up to 96.67% , however the disease severity of tomato seedlings exposed to IONPs at concentrations of 10, 12.5 and 15 µg/mL were reduced to 47.78%, 43.33% and 45% respectively. Though, with fungicide treatment a decline of 55% in disease severity was achieved ( Figure  S8A). The corresponding disease incidence in IONPs treated plants was 46.6%, 33.3% and 46.7% in contrast to the 100% control ( Figure. S8B). Plants activate a toxic oxidative-burst by increasing iron levels to minimize pathogen virulence; roots mutualistic interactions also encounter phyto-diseases via iron uptake as well as competition for iron acquisition induces a systemic resistance that signal components in roots for iron-uptake [77]. There has no work been done so far on the application of iron oxide nanoparticles to combat plant diseases under eld conditions, so recent work is considered as novel, and the results clearly indicates that these NPs has potential to become a part of disease management.

Impact of IONPs on photosynthetic pigments
The impacts of IONPs on the photosynthetic pigments were also accessed in this study by comparing diseased plants with treated ones (Figure S9A,B). Plant stress can also be indicated by changes in photosynthetic pigments [78]. Results indicated that various treatments of IONPs induces an increase in photosynthetic pigments. After exposure to 12.5 µg/mL IONPs, total chlorophyll and carotenoid contents in treated plants were signi cantly increased by 75.6% and 70.3% respectively in comparison to the control however, lower doses (0.01 and 0.5 µg/mL) showed decline up to 27.7% and 1.94% for chlorophyll content and 19.05 and 8.54% for carotenoid content respectively. Askary et al. reported the similar results with application of nano-iron fertilizer [9]. Compared to the control, increased pigment production was detected for fungicide treatment, with an elevation of 56.1% and 55.1% (p< 0.001) respectively. Iron plays a vital role in synthesis of chlorophyll; iron chlorosis reduces the level of photosynthetic pigments in plants effected the process of photosynthesis, while Fe-chlorosis is more frequent in photosystem-II analogous to photosystem-I [9]. The results demonstrated that higher doses of IONPs enhanced the synthesis of photosynthetic pigments in diseased plants by reducing chlorosis.

Impact of IONPs on phenolic content and antioxidant enzymatic activities
Plants endures stress environment by generating higher quantities of antioxidant enzymes, which enhances tolerance against oxidative burst [13]. Figure 11, 12: Figure  . SOD activity signi cantly enhanced in roots and shoots of infected tomato plants exposed to IONPs in parallel to the control treatment. The maximum activity, both in roots and shoots was recorded at 12.5 µg/mL, which showed 3.11 and 2.79folds higher than the control treatment, whereas its activity dropped 0.39 and 0.42 times at 15 µg/mL compared with the control (Fig. 11B). Rui

Impact of IONPs on growth and non-enzymatic compounds of tomato fruit
Fruit quality is imperative for merchandise, pulpy fruits are putrescible, certain biotic and abiotic agents are intricating in deteriorating the quality of product [93]. Table 1 indicates substantial improvement in fruit variables (average weight and number) and non-enzymatic compounds of tomato fruit exposed to various concentrations of IONPs (Fig. 10F,G). avonoid and Vitamin C content in tomato fruit after application of selenium and copper nanoparticles [102]. Proteins has a key role in fruit growth and quality, tomato ripening is associated with function of regulatory proteins involved in initiation of ethylene-biosynthesis [103][104]. Total protein content signi cantly increased at the highest concentrations of IONPs in comparison to the control, reaching up to 28.1%, 61.4%, 66.2%, 73.9% and 70.3 % with 5-15 µg/mL of IONPs respectively, the value also increased by 69.6% for fungicide treatment. However, the rest of the treatments didn't show signi cant difference from the control. Zaho et al. noticed increased protein content in cucumber fruit after application of nanoparticles [105].

Conclusion
Exploiting green-synthesized iron oxide nanoparticle, it was demonstrated that these nanoscale materials have a potential to become a part of diseases management system. Iron oxide nanoparticles (IONPs) nanoparticles were synthesized by mean of green approach using spinach as a starting material and black coffee as reducing / stabilizing agent. Microwave powers were varied form 100W-1000W in order to tune the properties of resulting product. XRD results showed cubic magnetite (Fe 3 O 4 ) phase of nanoparticles having superparamagnetic nature for all of the microwave powers. X-ray Photoelectron spectroscopy (XPS) results also con rmed the binding energies of Fe 2p3/2 (712.05 eV) and Fe 2p1/2 (723.9 eV) of Fe 3 O 4 NPs synthesized using microwave power of 1000W. FTIR analysis con rmed the presence of cubic magnetite (Fe 3 O 4 ) phase for all of the microwave powers. Iron oxide nanoparticles extorted a strong antifungal activity against F. oxysporum at highest treatments. Exposure to IONPs not only inhibited fungal growth under in-vitro test, but also manages the Fusarium wilt of Tomato in pot bioassay. ROS generation, mycelium deformation and DNA fragmentation are due to interaction among nanoparticles and fungal cells could be related to intrinsic mechanism of the iron-oxide nanoparticles. Biosynthesized IONPs positively affects plant growth parameters and fruit quality by reducing the disease index. These ndings clearly indicate that these nanomaterials have potential to repressed phytofungal-pathogens and paved a new pathway for nanoparticles to be used in agriculture in eco-friendly way.    with different concentrations of NPs viewed under light microscope (10x magni cation). Data represents as a mean ± SD (n=3) of three replicates indicating signi cant-difference (**p<0.01; *** p< 0.001) as compared to control by one-way-ANOVA (P< 0.05) and Tukey's-multiple comparing tests. dichloro-dihydro-uorescein diacetate (DCFH-DA) in dose-dependent manner. Graph denotes signi cantdifference (**P < 0.01; *** P < 0.001) in uorescence-intensity between different concentrations of nanoparticle and control group performed by one-way-ANOVA at P < 0.05 and Tukey multiple comparisons analysis. Each bar represents as a mean ± SD of three independent experiments. Figure 10