Copper nanoparticles hold promise in the effective management of maize diseases without impairing environmental health

A novel method of management of maize pathogens in vitro and in vivo using newly synthesized copper nanoparticles (CuNPs) has been documented in this study. CuNPs have been synthesized using CuSO4 as a precursor, NaBH4 and ascorbic acid as a reducing agent, and polyethylene glycol 8000 (PEG-8000) as a stabilizing agent. Characterization of CuNPs using a Transmission Electron Microscope (TEM) confirmed the nanoparticles’ size range of 35–70 nm. Fourier transform infrared spectroscopy (FTIR) revealed the association of alcohol groups and allyl halides group with CuNPs. The synthesized CuNPs exhibited significant inhibition at 20 ppm of three pathogenic fungi namely Macrophomina phaseolina, Bipolaris maydis, and Fusarium verticillioides, and at 50 ppm against Rhizoctonia solani. Bactericidal property of CuNPs was evidenced against Erwinia carotovora and Ralstonia solanacearum at 30 ppm. Evaluation of CuNPs in vivo against two diseases viz., maydis leaf blight (MLB) and banded leaf and sheath blight (BLSB) culminated in a reduction in percent disease index (PDI). Seed treatment together with foliar spray @ 300 ppm of CuNPs resulted in a significant reduction of MLB. However, BLSB disease was reduced relatively less at the same aforesaid concentration nevertheless, it was evinced best in controlling BLSB disease. CuNPs were found inimical against beneficial fungi and bacteria. However, a positive effect was observed on soil enzyme activities namely dehydrogenase, urease, and alkaline phosphatase and maize seedling characters viz., shoot length, root length, number of roots per seedlings, fresh and dry weight.


Introduction
Maize (Zea mays L.), popularly known as the queen of cereals, is an important Kharif crop with astonishingly high genetic potential, cultivated in different agro-climatic conditions covering approximately 150 mha in about 160 countries (Parihar et al., 2011). The United States, China, and Brazil are the top three maize-producing countries approximately accounting for 563 of the 717 million metric tons/year (Ranum et al., 2014). Maize is regarded for its nutritive quality, containing approximately 72% starch, 10% protein, and 4% fat, providing an energy density of 365 Vol:. (1234567890) Kcal/100 g. In addition, maize can be processed into various edible and industrial products such as starch, oil, alcohol, beverages, fuel, and ethanol (Ranum et al., 2014). Therefore, maize has a propitious industrial significance. However, plant pathogens pose a major constraint in its production, and a quest to overcome the problem has been endeavored for decades. With the constant tireless effort, many effective chemicals (fungicides, antibiotics, etc.) have been developed and introduced which can effectively manage various diseases rendered by phytopathogens. However, such conventional methods to manage phytopathogens have affected both environment and the farmer's economy due to its high toxicity and the liability of the applied fungicides getting wasted due to wind or surface runoff. It has been estimated that approx. 5.6 billion pounds of pesticides are used worldwide (Alavanja, 2009) and globally $38 billion is spent on pesticides each year (Pan-Germany, 2012). Its inimical effect on target organisms and the environment can be attributed to its high toxicity, non-biodegradable nature, and long residual activity (Aktar et al., 2009). Apparently, 80-90% of applied fungicides are wasted in the environment (Stephenson, 2003;Ghormade et al., 2011). Although the introduction of systemic fungicides took a major turn in the management of diseases, unfortunately, it has led to the development of resistance against fungicides because of their specific site of action. Therefore, the development of alternative antifungal agents like nano fungicides is of utmost need. Recently, nanoscience has emerged as an exciting yet unfathomed field having significant utility and application in agriculture as well. Nanotechnology can make disease management sustainable and eco-friendly by reducing toxicity and increasing the efficacy and shelf life of an antifungal agent. Concerning plant protection, the nanoparticles (Usually in the range of 10-100 nm) alone can be used directly as antifungal agents (Bramhanwade et al., 2016;Zain et al., 2014;Viet et al., 2016;Kanhed et al., 2014;Mondal & Mani, 2009) or as a nanocarrier of fungicides by entrapping or encapsulating which would result in controlled release of active ingredients increasing the efficacy (Mody et al., 2014). Metallic nanoparticles such as copper, silver, ferrous, titanium dioxide, zinc oxide, gold nanoparticles, etc. are widely being used as promising protectants. CuNPs can be exploited astoundingly to manage plant pathogens due to their remarkable antimicrobial properties. Moreover, nanoparticles are proven to show a synergistic effect when combined with biocontrol agents, generally recognized as safe substances (GRASS), biopolymers or essential oil, etc. (Beyki et al., 2014). Employing such a strategy would hold down pesticide usage by its high efficacy and durability and also would delay the development of fungal resistance. Nanoparticles possess the potential to prevent the development of multidrug resistance in bacteria by hindering quorum sensing, preventing bacterial efflux pumps activity, biofilm formation, etc. (Baptista et al., 2018), and the development of fungicide resistance in fungi due to its multiple sites of action. However, there are certain limitations of nanoparticles generally associated with the synthesis which involves the use and generation of toxic chemicals (Guilger-Casagrande & Lima, 2019). Moreover, nanoparticles bearing positive charges may result in cytotoxicity (Kutawa et al., 2021).
Copper was known to man since time immemorial as an essential element. Copper has drawn attention for its antimicrobial properties and various other advantages in several fields. Taking into account the myriad advantages of the copper compound, currently, they are of special interest to many for the synthesis of CuNPs. Several methods have been adopted for the synthesis of CuNPs such as chemical, physical, and biological methods. Chemical methods involve reducing agents, for instance, CuNPs synthesis by sodium hypophosphite as a reducing agent in ethylene glycol under microwave irradiation (Zhu et al., 2005), by ascorbic acid in the presence of chitosan using microwave heating (Zain et al., 2014), polyol method (Park et al., 2007), use of hydrazine and chitosan as reducing agent and stabilizer (Usman et al., 2013), etc.
In the present investigation, CuNPs synthesized by the chemical method were evaluated in vitro and in vivo. The CuNPs were evaluated for antimicrobial activities against five different fungi and two different bacteria. Under the net house conditions, the efficacy against two maize diseases namely MLB (Maydis leaf blight) and BLSB (Banded leaf sheath blight) were studied. The detrimental effect of synthesized CuNPs was ascertained against three different biocontrol fungi and two beneficial bacteria. An attempt was made to understand the influence of CuNPs on soil microbiome by analyzing soil enzyme activities. Further, the CuNPs were also evaluated for their effect on maize seed germination, and other seedling characteristics to get an insight into its phytotoxic traits.

Materials and synthesis
For the synthesis of CuNPs, chemicals viz., copper sulphate (CuSO 4 ), sodium borohydride (NaBH 4 ), ascorbic acid (C 6 H 8 O 6 ), and polyethylene glycol 8000 (PEG-8000) of analytical grade were used. The CuNPs were synthesized by employing a modified standardized protocol (Kathad & Gajera, 2014). A volume of 50 ml of CuSO 4 solution of 0.1 M was poured in a 250 ml capacity flat bottom round flask. Ten ml of 0.01 M PEG 8000 was added to it. The solution was stirred briskly using a magnetic stirrer for 30 min. After 30 min of constant stirring, 0.02 M ascorbic acids (20 ml) were added gradually to the mixture using a dropping funnel. The mixture was allowed to stir for 10 min followed by the addition of 0.01 M NaBH 4 (40 ml) by using a dropping funnel.
The reaction mixture was then stirred for two more hours by heating at 50 °C (Fig. 1).

Characterization of copper nanoparticles
The synthesized CuNPs were subjected to transmission electron microscopy (TEM, Jeol 1011 100 kV, Japan) for morphological studies. The sample was prepared on a 400-mesh carbon-coated copper grid. Before placing on the carbon grid, the liquid sample was sonicated for 40 min at room temperature, and then one drop of the sample was placed on the grid using a micro-pipette. After 2-3 sec, the grid was stained with 2% uranyl acetate and the sample was allowed to dry for 1 h followed by the observation of the sample under the electron microscope. Fourier transform infrared (FTIR) spectroscopy (Nicolet 6700 FTIR System, USA) was carried out to determine the functional groups associated with the synthesized CuNPs. The sample was prepared by adding 100 mg of spectral-grade KBr which was further pressed under the pressure of 6000 kg cm −2 for about 2 min which yielded a translucent KBr pellet. The pellet obtained was used for FTIR analysis. The sample's spectra were collected at a resolution and  (Nene & Thapliyal, 1979) was adopted to evaluate the efficacy of synthesized CuNPs. The in vitro assay was repeated twice against all the fungi and bacteria under study. Different concentrations of CuNPs (20 to 1000 ppm) were added to the media to ascertain their efficacy. Conventional fungicides (Carbendazim 50% WP, Hexaconazole 5% EC, Manocozeb 45% WP, Copper oxychloride) were also used as a negative control as per the recommendation. A cut-out disc (4-5 mm) of actively growing fungus mycelium (4-7 days old) was placed at the centre on the solidified PDA of previously labelled Petri plates. The plates were incubated at 28 ± 2 °C in a BOD incubator. The radial growth of the test fungus was measured in all the treatments when complete growth was attained in the untreated control plates. For an antibacterial test of the synthesized CuNPs, cultures of Erwinia carotovora and Ralstonia solanacearum were obtained from the Bacteriology Laboratory of ICAR-IARI. The growth inhibitory activity of CuNPs against these bacteria was analyzed quantitatively in nutrient broth. The optical density (OD) value /CFU count (CFU ml −1 ) of the nutrient broth amended with CuNPs with different concentrations (20 to 100 ppm) was recorded after incubation for 48 h using BioPhotometer (Mondal et al., 2010).
In vivo ( Half-filled conical flasks with cleaned and soaked seeds were sterilized for consecutive two days at 15 lbs. for 30 min. Mycelia disc from the actively growing culture plate was cut and dropped in sterilized sorghum flasks. The flask was incubated at 28 °C for 8 days with intermittent shaking at two days intervals for uniform growth. Fully grown fungus on seeds was air-dried at room temperature for 5 days and then ground to powder. Fresh sorghum seed ground powder was mixed with fungus-laid seed powder in a 1:1 ratio. Rhizoctonia solani f.sp. sasakii inoculums were prepared using barley seeds. Flask was quarter filled with overnight soaked seeds and autoclaved two times at 121 °C (15 lbs) for 30 min for two consecutive days. Fresh mycelia discs were put mixed with sterilized barley seeds and incubated for 10 days with daily shaking of the flask (Ahuja & Payak, 1978).
MLB was inoculated in 30 days old plant (stage 5 of maize growth stages, FAO Manual, 1971) of CM-500 susceptible varieties by the whorl inoculation method of Payak and Sharma (1983). About 5 g inoculums powder was spread on the leaves of the central whorl in the evening hours. BLSB was inoculated with 15 days old barley grain culture of Rhizoctonia solani f.sp. sasakii on 40 days old plant of CM 501. Each plant was inoculated by inserting 3-4 barley grains in between the stalk and sheath of the second or third internodes from the soil surface. Sufficient humidity of the experimental plot was 1 3 Vol.: (0123456789) maintained using overhead water sprinklers two times a day from the next day of inoculation.
Evaluation of the efficacy of CuNPs on disease management was determined by a periodic recording of disease data. MLB disease was recorded twice at 20 (15 days after CuNPs spray) and 30 days (25 days after CuNPs spray) days after inoculation (DAI) using a 1-9 scale adopted in the All India Coordinated Maize Improvement Project (AICMIP), 2016. The scale had been modified from the rating scales of Balint-Kurti et al. (2006), and Mitiku et al. (2014). The Percent disease index (PDI) i.e. severity was calculated by applying the formula described by McKinney (1923).
In the case of BLSB, the length of the infected area in each inoculated plant was documented on 20 and 30 DAI. Disease severity was recorded by using a modified 1-5 rating scale based on the area affected (Payak & Sharma, 1983). The PDI was calculated by using the following formula.

Effect of CuNPs on beneficial fungi and bacteria
The inimical effect of CuNPs was ascertained against three beneficial and benign fungi namely, Trichoderma virens, Paecilomyces lilacinus, and Chaetomium globosum, and two bacteria namely, Pseudomonas putida and Bacillus subtilis. The poisoned food technique was adopted to evaluate its effect on fungi in the concentration range of 20 to 1000 ppm whereas the effect on beneficial bacteria was analyzed in vitro quantitatively in nutrient broth amended with CuNPs at 10 to 100 ppm concentrations (Mondal & Mani, 2012).

Determination of the effect of CuNPs on soil enzyme activities
The experiment was carried out in earthen pots filled with one kg of soil collected from the maize field. Suspensions of chemically synthesized CuNPs and conventional copper oxychloride (Blitox) at 200 ppm and 400 ppm were poured into the potted field soil. Plain water was poured into the untreated control soil. The soil samples from the treated pots were collected periodically on the 0th, 15th, and 30th day for the determination of activities. Each treatment was comprised of three replications. The collected soil samples were sieved and used for the analysis of the activities of three soil enzymes.
Assay of alkaline phosphatase activity The estimation of alkaline phosphatase activity was performed by the method prescribed by Tabatabai and Bremner (1969). The chemicals acquired for the preparation of reagents were analytically pure. The reagents were prepared as per the prescription. Air-dried soil sample, 0.1 g was put in a test tube; to it, toluene (0.2 ml) was added, followed by the addition of 4 ml of modified universal buffer (pH 11) and 1 ml of 0.025 M p-nitrophenyl phosphate (PNP) solution. The test tubes were incubated at 37 °C for an hour with constant shaking. After the incubation, 1 ml of CaCl 2 (0.5 M) and 4 ml of NaOH (0.5 M) were added to the mixture followed by filtration of soil suspension through Whatman filter paper No. 1. Simultaneously, a blank was also prepared in the same way except for the addition of the soil sample. The optical density (OD value) of the filtrate was measured by a spectrophotometer (Thermo fisher scientific, EVO 300 PC) at 440 nm wavelength. The phosphatase activity in each sample in terms of concentration of p-nitrophenyl was computed and expressed as a mole of p-nitrophenol released per gram of dry soil per hour (μg PNPP/g soil/h). where, x concentration of p-nitrophenol (μg ml −1 filtrate) V total volume of soil solution dw dry weight of 1 g moist soil sw weight of soil sample t incubation time

Assay of dehydrogenase activity
The 2-3-5-triphenyl tetrazolium chloride (TTC) reduction technique (Casida, 1977) was used to estimate the dehydrogenase activity under the influence of the CuNPs treatment. The reagents used in the experiment were analytically pure. Six grams of soil sample collected from the pots were taken in a glass screw tube of 30 ml capacity. A pinch of calcium carbonate (CaCO 3 ) was added to the soil sample. Then 1 ml of freshly prepared 3% TTC was added followed by vortexing of the soil sample. To ensure the submergence of the sample, 2 ml of distilled water was poured followed by incubation at 28-30 °C for 24 h. Then 10 ml methanol was added to tubes and was left undisturbed at room temperature for 30 min to allow it to change color. The appearance of red/orange color reveals the enzyme activity. Whatman filter paper no. 1 was used to filter the suspension. A spectrophotometer (Thermo Fisher Scientific, EVO 300 PC) was used at 485 nm using methanol as a blank to read the optical density of the filtrate. Dehydrogenase activity per gram of dry soil was expressed as microgram formazan per gram of dry soil per hour (μg TPF released/g of soil/day).
where 10 = methanol added and 6 = soil taken in g.

Assay of urease activity
The urease activity was determined by the method of McGarity and Myers (1967). Five grams of soil were taken in a tube and 2.5 ml of urea solution was added to it, subsequently incubating at 37 °C for 2 h. Then 50 ml potassium chloride (KCl) solution was added to the sample by giving brisk stirring for 30 min. The filtrate was collected by centrifuging the samples at 10000 rpm for 10 minutes. One ml filtrate was taken in a vial and 9 ml distilled water was added to it. Then 5 ml volume of sodium salicylate (C 7 H 5 NaO 3 )/NaOH was added followed by the addition of 2 ml sodium dichloroisocyanurate (C 3 Cl 2 N 3 NaO 3 ). The mixture was kept at room temperature for 30 min. Finally, the optical density value was taken at 690 nm wavelength using a spectrophotometer (Thermo Electron Corporation, 400 L/4). Concentration and activity were calculated by the following formula.

Effect of CuNPs on plant characters of maize seedlings
To determine the effect of CuNPs on seed viability, percent germination was considered as an indicator (Karimi et al., 2012). The experiment was conducted under lab conditions two times. A total of sixty healthy seeds of two different varieties viz., CM-500 and CM-501 were used for each treatment. A complete randomized design (CRD) was followed for three different treatments (viz., control, 100 ppm, and 300 ppm) with five replications in each. Seeds were soaked in 100 and 300 ppm solutions of CuNPs, and in distilled water (untreated control) for one night. The treated seeds were placed on the absorbent cotton pad by maintaining adequate space between them. The whole setup was incubated at 28 °C for 6 days. On the 6th day, germination data were recorded and the percent germination was calculated. Further, the length of the shoot, as well as the roots, were measured using a scale, and the numbers of roots were counted and recorded treatmentwise. To determine the effect of CuNPs on the biomass of seedlings, fresh weight and dry weight were measured. Fresh weight (in mg) was recorded on the 6th day of whole seedlings by using a weighing microbalance (Sartorius). The fresh seedling samples were wrapped in aluminum foil, labeled treatment-wise, and ovendried consecutively for 3 days at 50 °C. After the complete drying of the samples, dry weight (in mg) was recorded.

Statistical analysis
The lab experiments were conducted by adopting a complete randomized design (CRD) and the net house (in vivo) experiments were conducted in a randomized block design (RBD). A total of three replications were taken for all the experiments except for the study of the effect of CuNPs on plant characteristics of maize seedlings, where 5 replications were taken. The experiments in-vitro and in-vivo were repeated twice. The statistical analysis of the data (converted by angular transformation) generated in the experiments was performed by following the procedure of SAS 9.4 (SAS Institute, 2003, Cary, NC). The Activity ( g NH4 − N∕g FW∕h) = Concentration in ppm× 52.5 Phytoparasitica (2023) 51:593-619 598 significant difference between the treatments' mean was determined after analysis of variance (ANOVA) (Gomez & Gomez, 1984) followed by the least significant difference test (LSD) (P ≤ 0.5).

Synthesis and characterization of copper nanoparticles
The color change indicated the formation of copper nanoparticles. The addition of ascorbic acid (C 6 H 8 O 6 ) led to the color change to light green confirming the size reduction. The addition of sodium borohydride (NaBH 4 ) led to a change in color from greenishyellow to slightly yellow and transition to red which eventually turned into a brick red/brown color indicating further reduction (Fig. 1). The PEG-8000 conditioned a reaction medium and stabilized the synthesized CuNPs. The C 6 H 8 O 6 and NaBH 4 possibly could have reduced the Cu 2+ to Cu + and further to Cu 0 . TEM analysis measured CuNPs of size 35-70 nm on a carbon grid that was spherical and appeared agglomerated (Fig. 2).
FTIR revealed prominent peaks at 3440, 2924, 1643, 1097, and 678.72 cm −1 (Fig. 3). The peaks at 3440 cm −1 correspond to the O-H stretching of the alcohol group. The peaks at 2924 and 1643 cm −1 overlap with C-H stretching, and N-H bending, respectively. The peaks at 1097 and 678.72 cm −1 can be assigned to the C-O stretching of alcohol group; and the C-Cl stretching of allyl halides (Sharon et al., 2018).
In vitro fungicidal efficacy of synthesized CuNPs against maize pathogens CuNPs rendered complete radial growth inhibition (100%) at 300 ppm against R. solani f.sp. sasakii, B. maydis, and F. verticillioides (Table 1, Fig. 4A, B, and C). However, 1000 ppm of commercial fungicide; carbendazim for R. solani f.sp. sasakii, and F. verticillioides, and 2000 ppm of Mancozeb for B. maydis is recommended. Concerning the concentration of CuNPs, significant inhibition was observed right from 50 ppm onwards in the case of R. solani f.sp. sasakii (34.20%), from 20 ppm in the case of F. verticillioides (13.34%) and B. maydis (39.34%). The CuNPs were also found highly effective against Macrophomina phaseolina in all the tested concentrations (Table 2, Fig. 4D). In the case of S. roflsii, significant inhibition (14.93%) was seen from 300 ppm and complete inhibition was achieved at 1000 ppm of CuNPs which was at par with commercial counterpart Hexaconazole at   Fig. 4E). It was also observed that with the increase in the concentration of CuNPs, the radial growth of all five fungal pathogens was consistently reduced.

In vitro bactericidal efficacy of synthesized CuNPs
Two phytopathogenic bacteria namely R. solanacearum and E. carotovora were taken into account  under the present investigation. Among the CuNPs treatments, the highest OD (3.20 and 2.18) and CFU (3.20 × 10 9 and 2.18 × 10 9 ) values were found at 10 ppm while the least OD (1.16 and 0.54) and CFU (1.16 × 10 9 and 0.54 × 10 9 ) at 100 ppm for E. carotovora and R. solanacearum, respectively (Suppl. Table 1). In the case of E. carotovora, a significant reduction in bacterial growth was recorded from 30 ppm (OD 2.54) onwards as compared to the positive control (OD 3.05) (Fig. 5A, Suppl. Fig. 1A). Similarly, the growth of R. solanacearum significantly reduced from the 30 ppm CuNPs (OD 1.98) onwards as compared to the control OD (2.34). The CuNPs treatments in the series, immediate to each other, did not reflect any significant difference (Fig. 5B, Suppl. Fig. 1B). However, 100 ppm of CuNPs was perceived at par with the antibiotic streptomycin sulphate at 200 ppm in the case of E. carotovora. In the case of R. solanacearum, although 100 ppm of CuNPs was evidenced to be at par with 200 ppm of streptomycin sulphate in restricting bacterial growth, it was manifold better than the copper oxychloride (Blitox) at 1000 ppm.
Efficacy evaluation of CuNPs in vivo for maize disease management The incidence of MLB disease was significantly less in treatments as compared to negative (inoculated) control in both seasons (Table 3). Among the treatments, the least incidence (20.37%) was recorded in T6 (Spray+ ST@ 300 ppm), followed by T2 (24.07%), T5 (23.45%), and T4 (23.15%), however, no significant difference between the treatments viz., T2, T4, T5, and T6 was observed. A maximum of 55.25% disease incidence was recorded in the negative control where no chemicals were applied, whereas the fungicide Mancozeb (2000 ppm) significantly restricted MLB incidence to 37.22%. Furthermore, significantly less disease incidence was evidenced when CuNPs were used as seed treatment and foliar spray at 300 ppm and seed treatment+spray at 100 and 300 ppm as compared to 2000 ppm of Mancozeb (Fig.6A). The overall MLB incidence recorded on 1st scoring at 20 DAI showed a slight increase after 10 days (2nd scoring) in treatments in both seasons.
In the case of BLSB disease (Table 4), treatments except for T3 (spray @100 ppm) exhibited significantly less disease incidence as compared to the negative control (71.05%) (Fig. 6B). Among the different treatments of CuNPs under study, T6 (Spray+ ST @ 300 ppm) performed best with the least PDI of 46.81% which was at par with Carbendazim spray (39.55%). Comparatively, seed treatments with CuNPs at the rate of 100 ppm (57.92%) and 300 ppm (55.33%) performed better than the spray with CuNPs at the rate of 100 ppm (67.05%) and 300 ppm (60.39%). Spraying with the lower dose of CuNPs (100 ppm) resulted in higher disease incidence, statistically at par with the negative control. In all the treatments, BLSB incidence was found to increase as evidenced by two times diseases scoring, 20 and 30 DAI.

Determination of the inimical effect of CuNPs against beneficial fungi
The growth of Trichoderma virens was significantly inhibited by the CuNPs from the concentration of 80 ppm onwards as compared to the positive control, and complete inhibition (100%) was observed at 300 ppm which confirmed the inimical effect of CuNPs on T. virens (Fig. 7A, Suppl. Fig. 2A, Supp. Table 2). In the case of Chaetomium globosum, significant inhibition in the growth was noticed right from 20 ppm (24.24%) (Fig. 7B, Suppl. Fig. 2B, Supp. Table 2). With the increase in the concentration of CuNPs, radial growth of C. globosum was reduced and thus 100% inhibition was observed at 300 ppm. In the case of Paecilomyces lilacinus, a reduction in the radial growth was observed from 100 ppm of CuNPs (Fig. 7C, Suppl. Fig. 2C, Suppl. Table 2). As compared to T. virens and C. globosum, P. lilacinus appeared to be slightly more tolerant since complete inhibition (100%) of growth was observed at higher concentrations (600 ppm) of CuNPs.

Determination of the inimical effect of CuNPs against beneficial bacteria
CuNPs against B. subtilis exhibited a significant reduction in OD value from 20 ppm (0.52) onwards indicating a reduction in the growing number of the cell as compared to the control (0.98) (Suppl. Table 3). The concentrations of 10 and 20 ppm turned out to be statistically significant but beyond 20 ppm, when two immediate treatments were compared, no significant reduction in OD or growth of bacteria was observed. The effect of CuNPs at 50 ppm and 90 ppm was at par with the Blitox (1000 ppm) and Streptomycin sulphate (200 ppm), respectively (Fig.8A, Suppl. Fig. 3A, Suppl. Table 3). As compared to B. subtilis, the OD value for P. putida (Fig.8B, Suppl. Fig. 3B, Suppl. Table 3) was higher and a significant reduction in growth was observed from 30 ppm onwards. The 100 ppm concentration of CuNPs was more harmful to both the beneficial bacteria than the Streptomycin sulphate @200 ppm and blitox @1000 ppm.  The alkaline phosphatase activity was found high in both COC and CuNPs treated soil as compared to control ( Fig. 9C and D, Suppl. Table 5). Among the two chemicals, alkaline phosphatase activity in the soil treated with CuNPs was higher than the activity estimated for COC. In the case of COC at 200 ppm, the alkaline phosphatase activity was decreased from 466.80 to 445.19 μg pNPP/g soil/h on the 15th day which was elevated on the 30th day (490.28 μg pNPP/g soil/h). At a higher concentration of 400 ppm (COC), the enzyme activity increased from the base level of 497.79 (1st day) to 540.06 (15th day), and eventually decreased to 472.43 μg pNPP/g soil/h (30th day). In the case of CuNPs at 200 and 400 ppm, a significant sharp increase of alkaline phosphatase activity was seen on the 15th day (784.25 and 832.15 μg pNPP/g soil/h, respectively), however, the activity declined on the 30th day (772.04 and 713.81 μg pNPP/g soil/h respectively.). As compared to COC, CuNPs treatment resulted in significantly higher activity.
The activity of urease in terms of μg NH 4 -N/ g/ soil/h was found to be significantly high on the 15th day (3.931 μg NH 4 -N/ g/soil/h) in soil treated with CuNPs (400 ppm) which further reduced on the 30th day (3.669 μg NH 4 -N/ g/soil/h) ( Fig. 9E and F, Suppl. Table 6). The difference among the treatments remained insignificant in the case of COC treatments as well as in CuNPs treatment except for CuNPs treatment at 400 ppm. A comparison of the CuNPs and COC treatments did not show any significant difference in the urease activity.
The changes in enzyme activities observed in this study demonstrated that the soil microbial communities and their activities are influenced by metallic nanoparticles or possibly certain genes responsible for specific enzymatic functions are influenced.

Effect of CuNPs on plant characters of maize seedlings
Two maize varieties viz., CM-500 and CM-501 were evaluated after CuNPs treatment at 100 ppm and 300 ppm concentrations. The experiment was repeated twice. Both varieties exhibited 100% germination in both the experiments. In the case of CM-500 ( Fig. 10A and C) all the characters under consideration i.e. number of roots per seedlings, shoot length, root length, fresh weight, and dry weight exhibited a steady increase, which was statistically significant with the increase in the concentration of CuNPs upto 300 ppm (Table 5). Similarly, an increase in all the seedling's characters was perceived in CM 501 ( Fig. 10B and C).

Discussion
The chemical method of nanoparticle synthesis is considered a traditional method and is widely being used. Such method involves the use of certain chemical reagents as a reducing agent such as sodium borohydride (NaBH 4 ), potassium bitartrate (KC 4 H 5 O 6 ), sodium hypophosphite (NaPO 2 H 2 ) (Zhu et al., 2004), hydrazine (N 2 H 4 ) (Usman et al., 2013), glucose (C 6 H 12 O 6 ) (Suvarna et al., 2017), ascorbic acid (C 6 H 8 O 6 ) (Zain et al., 2014), diethylene glycol (Park et al., 2007). In addition, a stabilizing agent is used which prevents the agglomeration of synthesized nanoparticles by capping the nanoparticles. There are two important approaches to synthesis viz., down strategy and bottom-up strategy. In the former strategy, the precursor is converted to particles in the nano-size range whereas, in the latter approach, nanoparticles are synthesized by joining atom by atom (Zielonka & Klimek-Ochab, 2017). In the present investigation, NaBH 4 and the ascorbic acid act as a reducing agent, reducing the size of Cu precursor to CuNPs whereas PEG 8000 conditions a good reaction medium and stabilizes the formed CuNPs by capping those (Shameli et al.,    2012). Possibly, Cu 2+ under the influence of the reducing agent first gets converted to Cu + and then to Cu 0 resulting in the reduction of particle size. A similar result was obtained by Kaur et al. (2014) andSoomro et al. (2014) using NaBH 4 as a reducing agent which generated nanoparticles in size range from 40 to 80 nm and of 15× 14 nm size, respectively. The synthesis is influenced by factors such as concentrations of reactants, pH of the reaction medium, temperature, etc. Relation between the size of the nanoparticles and the concentration was shown by Zain et al. (2014) where they found that with the increase in the concentration of copper nitrate and silver nitrate, the size of the respective nanoparticles increased proportionately. The effect of temperature on the synthesis was reported by Rahimi et al. (2010). The temperature at 50 °C with reducing agent to the precursor ratio (R/P) 2 and 8 and at 60 °C with R/P 2 did not result in the formation. However, the temperature at 60 °C with R/P 4 and 6 and temperature at 75 °C with R/P as 4 led to the synthesis of copper nanoparticles without any precipitation. Although 85 °C with R/P 4 could also generate nanoparticles but resulted in precipitation. Traiwatcharanon et al. (2016) studied the effect of pH on particle size. They observed the surface plasmon resonance peak to be at >330 nm for acidic medium (pH 4 and 6 and) and at >420 nm for basic medium (pH 8 and 10 nm) which implies that the AgNPs size was smaller in the acidic medium. It was shown that the concentration of acid to base ratio would influence the size of nanoparticles by influencing the formation of nuclei (Ahmad & Sharma, 2012). Change in color/optical properties is the first and foremost indication of nanoparticle synthesis. In our study, with the reaction time and addition of reactants color changed from light yellow, dark yellow to red and eventually to the dark brick brown-red color. A similar observation was reported by Umer et al. (2012Umer et al. ( , 2014, Rahimi et al. (2010), Khalid et al. (2015), and Jain et al. (2015) where they perceived the final colour of the reaction mixture to be dark brown in color.
Copper was known to possess antimicrobial properties since the seventeenth century and hence the water was being stored in utensils made of copper. Moreover, Cu is an important micronutrient to plants. Taking into account the importance, copper was exploited in the present investigation. In the present study, five important maize fungal pathogens were tested. As less as 200 ppm of CuNPs rendered 100% inhibition in M. phaseolina and 300 ppm exhibited complete control of F. verticillioides, R. solani, f. sp. Sasakii, and B. maydis. However, to attain complete inhibition of S. rolfsii a concentration of 1000 ppm was required, although significant inhibition was apparent from 80 ppm. Pertaining to the findings of the present investigation, similar effectiveness of CuNPs was reported by Viet et al. (2016) at 450 ppm against Fusarium sp., Bramhanwade et al. (2016) against F. oxysporum and F. equiseti, and Kanhed et al. (2014) against Phoma destructiva, C. lunata, A. alternata, and F. oxysporum, Banik and Luque (2017, 800 ppm, respectively. The aforesaid findings perceived the effectiveness of CuNPs better than the commercial fungicides. In agreement with previous reports, the present investigation confirms the better effectiveness of CuNPs over commercial fungicides (Mancozeb, Carbendazim, Copper oxychloride, and Hexaconazole) used, exhibiting significant inhibition of the fungal pathogens at the concentration of CuNPs as low as 20 ppm. The enhanced fungicidal activity of CuNPs is due to their reduced size or high surface area to volume ratio. Moreover, its ability to disrupt enzymes by binding to sulfydryl amino and carboxyl groups of amino acids and by virtue of their small size, CuNPs even disrupt the DNA helix of the microbes (Shobha et al., 2014). Furthermore, CuNPs are also found to be affecting membrane integrity and membrane lipids (Santo et al., 2008). In our study, we have demonstrated the effectiveness of CuNPs against two bacteria namely, R. solanacearum and E. carotovora at a concentration of 30 and 20 ppm, respectively. The effectiveness against bacteria could be attributed to the ability of CuNPs to cross the bacterial cell wall, thereafter affecting the shape and functions of the cell membrane. It also affects bacterial DNA and enzymes, creates oxidative stress, and alters gene expression (Slavin et al., 2017). The bactericidal effect of CuNPs has been reported by Mani (2009, 2012) and Mondal et al. (2010) against Xanthomonas axonopodis pv. phaseoli, X. oryzae pv. oryzae and X. axonopodis pv. punicae, respectively at very low concentration (0.2 ppm) of CuNPs. Usman et al. (2013) also reported growth inhibition of several bacterial species (Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella choleraesuis, B. subtilis, and Candida albicans) at 30 ppm which supports the findings of the present study. A more precise mechanism of CuNPs against R. solanacearum had been elucidated by You et al. (2018) where they found bacterial cytomembrane was highly damaged due to absorption of CuNPs; moreover, several genes related to pathogenesis were down-regulated. However, counterproductive results against the beneficial microbes namely T. virens, C. globosum, B. subtilis, and P. putida were obtained. A similar result was reported by Ruparelia et al. (2008) against B. subtilis strain MTCC 441 growth inhibition at 20 μg/ml (20 ppm). The findings of the present investigation support the earlier report of Banik and Luque (2017), in which they observed an effective concentration of CuNPs at 200 mg/ml against Pseudomonas syringae.
To ascertain the reliability of in-vitro results, experiments in in-vivo (Net house) conditions are imperative. The efficacy evaluation in-vivo was carried out twice to confirm the reliability of synthesized CuNPs. The severity of two diseases under study viz., MLB and BLSB with the treatment of CuNPs at 300 ppm (spray+seed treatment) were significantly reduced as compared to the treatment with respective commercial fungicides. Typical symptoms of both MLB and BLSB diseases started appearing at 3-4 DAI. The substantial decrease in PDI of both MLB and BLSB diseases could be due to the direct effect of CuNPs on the fungal pathogens as an antifungal agent, distressing the pathogen's physiology by various mechanisms. Another possible reason could be the activation of defense genes/mechanisms in maize plants after exposure to CuNPs. The results achieved in the present investigation are more or less in agreement with Choudhary et al. (2017), who observed the maize plants treated with Cu-chitosan NPs suffered from less disease, due to induction in defense response through higher antioxidants such as peroxidase and superoxide dismutase and activation of defense genes polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL) against Curvularia lunata, the incitant of Curvularia leaf spot (CLS) disease. A similar observation was recorded by Zhao et al. (2017), in which they observed an increase in phenolic compounds when maize plants were treated with Cu(OH) 2 nano-pesticide. The CuNPs in combination with chitosan-polyvinyl alcohol hydrogels (Cs-PVA) resulted in increased expression of defense genes in tomato plants under salt stress (Hernández-Hernández et al., 2018) and reactive oxygen species (ROS) as well as peroxidase activity in finger millet against Pyricularia grisea (Sathiyabama & Manikandan, 2016).
Soil enzyme activities provide an idea about soil fertility and productivity (Tiwari et al., 1989). It is also a measure of microbial biomass and microbial activity (Klose & Tabatabai, 1999) and hence it is a direct indicator of soil quality (Pascual et al., 2000). Owing to their definite significance in organic matter transformation and phosphorous cycle, three different enzymes were targeted; dehydrogenase, alkaline phosphatase, and urease. Dehydrogenase has an important role in transferring hydrogen or electron from substrate to acceptor during the initial stages of oxidations; hence, considered an adequate tool to assess microbial oxidative activity (Ross, 1971). Phosphatase activity is vital for the release of phosphorous from organically bound phosphorous (Nannipieri et al., 2011). Hydrolytic conversion of urea into CO 2 and NH 4 is carried out by the urease enzyme, hence acting as a regulator of nitrogen economy in soil (Swensen & Bakken, 1998). The decrease in the activity of two enzymes on the 30th day is possibly due to the exhaustion of nutrients in the soil. A similar trend in change in activities was also reported by Gopal et al. (2012), where they noted the shootup in the activities of dehydrogenase, alkaline phosphatase, and acidic phosphatase on the 30th day when treated with nano hexaconazole, but a gradual fall in activity was observed which reached to a minimum on 60th day. You et al. (2018) reported adverse effects of four metal oxide nanoparticles, i.e., zinc oxide (ZnO NPs), titanium dioxide (TiO 2 NPs), cerium dioxide (CeO 2 NPs), and magnetite (Fe 3 O 4 NPs) on the soil enzyme activities viz., invertase, urease, catalase, and phosphatase. McGee et al. (2017) also evaluated the effect of AgNPs, SiO 2 NPs, and Al 2 ONPs on soil enzyme activities of dehydrogenase and urease and observed a decrease in the activities. Contradicting earlier reports, the present study confirms no adverse effect of CuNPs on soil enzyme activities. However, better insight can be achieved about the effect of CuNPs on soil enzyme activities (microbial activities) by applying advanced approaches like meta-transcriptomics and metaproteomics.
The phytotoxicity of nanomaterial is under purview, therefore, to determine the effect of synthesized CuNPs on maize seed germination and seedling characters, a study under the lab conditions was carried out. Enhancing effect was observed in all the seedling characters taken into account after the CuNPs treatment which is possibly due to the role of copper as a micronutrient. It is well established that the concentration of copper in the range of 4 to 20 ppm (Landis & Steenis, 2000) is required for the normal development and physiological function of the plant. The significance of copper as a micronutrient is enormous as it acts as the main factor that activates the enzymes responsible for catalyzing reactions in the plant. Another function of copper in a plant is protein regulation by producing 'vitamin A' which ensures protein synthesis, mitochondrial respiration, activation of several enzymes like polyphenol oxidase (PPO), superoxide dismutase (SOD), amino oxidase, etc. role in oxidative stress response (Landis & Steenis, 2000;Pich & Scholz, 1996;Passam et al., 2007). An earlier report by Yasmeen et al. (2015) supports the present result, where they observed a significant increase in the percentage of seed germination of wheat seeds treated with copper (CuNPs), silver (AgNPs), and iron (FeNPs) nanoparticles. A similar result was obtained by Adhikari et al. (2012) when CuO nanoparticles were tested against the seeds of soybean and chickpea. Up to 200 ppm, no effect on germination was observed, but root development was inhibited at above 500 ppm of CuONPs. Also, the result of the present investigation is in impeccable agreement with the findings of Gautam et al. (2016) where they reported enhanced seed germination and seed vigor index (SVI) of soybean (Glycine max (L) Merr.) by 15% and 50.08%, respectively when treated with 200 ppm of CuO NPs However, the higher concentration drastically reduced the germination percentage as well as SVI. Consistent with earlier described results, 100-400 ppm of sulphur nanoparticles (SNPs) also reported enhancing the growth of Cucurbita pepo (summer squash) by increasing the number of leaves and branches stem girth and height of the plant. However, Salem et al. (2016) observed a slight reduction in growth at 400 ppm. Contradicting previous findings, Lin and Xing (2007) reported the inimical effect of five different types of nanoparticles viz., multi-walled carbon nano-tube, aluminium, alumina, zinc, and zinc oxide nanoparticles on root growth and seed germination of six plant species viz., cucumber, radish, lettuce, rape, ryegrass, and corn. They discern a harmful effect of 2000 ppm of Al 2 O 3 NPs on seed germination and root elongation of different plant species. On the other hand, ZnNPs and ZnONPs did inhibit seed germination at lower concentrations except for ryegrass and corn seeds, respectively. Hence the output of the  sasakii. Compared to other fungi, S. rolfsii appeared to be tolerant as its growth was checked at 1000 ppm. The growth of pathogenic bacteria was significantly reduced by CuNPs even at a concentration as low as 20 ppm. Economical and effective management of MLB and BLSB disease of maize was also possible using CuNPs which was manifold better than the commercial fungicides/bactericides without impairing the normal growth of the maize plant. Moreover, CuNPs proved to have a positive effect on maize seedlings' characters and exhibited no adverse effect on soil enzyme activities. The higher concentration (>50 ppm) of CuNPs exhibited an inimical effect on beneficial organisms which indicated standardizing the concentration of CuNPs compatible with such widely used biocontrol agents. Our results demonstrate the high potential of nanoparticles as protectants in managing phytopathogens.