Nanotechnology is one of the most promising methods for plant protection, it helps to develop alternative management strategies for efficient disease management at the field level. In addition, to suppressing disease and increasing crop productivity, it has a significant role in reducing pesticide contamination in the environment. Because of their functionalization properties and broad spectrum, NP-antimicrobial agents are a promising byproduct of the development of nanotechnology. NP-antimicrobial agents can eliminate the limitations that come with traditional antimicrobial agents (Hemeg 2017). The high surface area, high reactivity, and distinctive particle shape of metallic nanoparticles make them special in terms of physicochemical characteristics also, antibacterial properties, making them an ideal approach to overcoming bacterial resistance (Wang et al. 2017).
The ability to use nanomaterials for R. solanacearum suppression in vitro and crop protection in vivo required determining the effective concentration of bulks in vitro to prepare the appropriate suggested nanoparticle dosage. Results compared the efficiencies of bulk CuSO4 and bulk MgSO4 currently used against R. solanacearum, by comparing the CFU numbers of the various treatments to the control, the rates of cell viability were examined (Cui et al. 2016), the results showed that less inhibition was found for lower concentrations of 3 mg/mL of both bulk CuSO4 and bulk MgSO4. (Cai et al. 2018) mentioned that the doses of bulk MgO had minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of 500 and 600 mg/mL, respectively.
Ultraviolet (UV) visible spectroscopy revealed that CuO-NPs showed a surface plasmon absorption band at obtained peak at 220 nm with an absorption value around 0.2. According to (Mahapatra et al. 2008) the CuO-NPs optical absorption spectrum is from 270- 330 nm wavelength. While (Rajeshkumar and Rinitha 2018) reported that UV-visible spectra of CuO-NPs are from 200-300 nm wavelength range. CuO-NPs recorded nanoparticles in high-resolution transmission electron microscopy HR-TEM images are very small (3.3 nm and 6.41 nm), smooth, spherical, loose, and freely distributed homogeneously in the matrix. These findings support (Kumar 2014), said the CuO-NPs had spheres with particle sizes between 5.0 and 8.0 nm and (Shende 2016) who showed that TEM analysis of CuO-NPs was as rod-shaped, cylindrical, and elliptical particles, with an average copper nanoparticle size of 25 nm and a total concentration of 300.29 particles per frame, 13.53 particles per milliliter. However, (Chen et al. 2019) demonstrated that, with a clear crystal lattice structure of 2.29 nm, the obtained CuO-NPs' typical morphology as measured by TEM revealed irregular spherical particles and agglomerations of various sizes. Additionally, the synthesized CuO-NPs in the HRTEM image had a mean diameter of 20.20 nm, a scale from 12.91 to 33.45 nm, and were mono-dispersed spherical and rounded forms. (El-Batal et al. 2020). CuO-NPs are more stable because of their higher surface charge, which is expressed as a zeta potential of -39.5 mv. The zeta potential value of CuO-NPs, in contrast, was reported by (Shende 2016) to be -14.9 mV. FTIR spectroscopy of CuO-NPs in the present study displayed strong absorption bands at 3440.7, 1636, 78 and 432.11cm−1 explaining that different chemical compounds encircled metal nanoparticles which match well with the outcomes noted by (Pandiyarajan et al. 2013) confirmed that FTIR spectra of copper nanoparticles display three distinct bands that correspond to the vibrations of the Cu-O bond. The existence of a metal-oxygen connection was established by (Kumar 2014). (Awwad et al. 2015) demonstrated that the presence of amines N-H of amide as well as hydrogen-linked O-H groups of phenols and alcohols can be used to explain a peak at 3444 cm-1. In the synthesized CuO-NPs, this peak moved to the lower field at 3398 cm-1, the shifting of these bands strongly suggests that the protein of Malva sylvestris leaf extract's carboxylic acids and CuO-NPs work in concert to disperse, stabilize, and cap CuO-NPs. (Shende 2016) indicated that the FTIR spectrum had different chemical compounds, including terpenoids, alcohols, ketones, esters, aldehydes, and carboxylic acids, surrounding CuO-NPs. The synthesized CuO-NPs were recognized by their X-ray diffraction pattern as h k l (0 0 2), (1 1 -1), (1 1 1), (2 0 0), and (2 0 -2) which matches well with those reported by (Kumar 2014) XRD results were used to identify a pure copper oxide phase. The diffraction data are well coordinated with the JCPDS card (Card No. 89-5899) and implies a monoclinic structure; no impurity peak is visible. (Chen et al. 2019) conducted that the two peaks that serve as markers, located at 2q values of 35.49 and 38.72, which were given to the (111, 002) and (1-1 1), the X-ray diffraction pattern of the synthesized CuO nanoparticles obtained indicated the overwhelming presence of CuO in the produced powder using crystallographic planes. In contrast, Bragg's reflections were used to characterize the XRD estimate findings of the produced CuO NPs at (110), (002), (111), (202), (020), (202), (113), (311), (113) and (004), respectively (El-Batal et al. 2020). Ultraviolet (UV) visible spectroscopy of MgO-NPs showed a surface plasmon an absorption band at obtained peak between 200 – 300 nm with absorption value around 0.6. The maximum surface plasmon resonance (SPR) for MgO-NPs was detected at 260nm by (Essien et al. 2019). According to (Umetsu et al. 2019), the spectrum of MgO-NPs exhibits a broad absorption peak between 260-330 nm. The particle form of MgO-NPs was revealed by high-resolution transmission electron microscopy HR-TEM pictures to be quite tiny (3.7 nm and 6.79 nm), smooth, spherical, loose, and freely distributed homogeneously in the matrix. Also, (Cai et al. 2018) indicated that the MgO-NPs had a particle size range of 17–5 nm and were spherical. (Suresh et al. 2018) have previously employed TEM to assess the stability and particle size distribution of the MgO-NPs in various suspensions. The zeta potential of - 43.8 mv in this study indicates that MgO-NPs are more stable. (Cai et al. 2018) stated that the Zeta potential of nanoparticles considerably influences their antibacterial activity compared to their particle size. The FTIR spectroscopy of MgO-NPs displays strong absorption bands at 3436, 2079.31, 1637, 64, and 669.05 cm−1. The peaks explained that the multitude of distinct vibrations in the fingerprint region makes things difficult. FTIR spectrum of magnesium nanoparticles suggested that various chemical compounds encircled magnesium nanoparticles. Also, FTIR spectrum of magnesium nanoparticles analysis by (Imani and Safaei 2019) concluded that magnesium nanoparticles were surrounded by several organic compounds. The diffraction pattern (X-ray) of the as-prepared MgO-NPs, in the current study illustrated that MgO is single-phase with a crystalline structure indexed to the (2 2 0) and (2 0 0) diffraction planes. Previous studies recorded by (Imani and Safaei 2019; Umetsu et al. 2019) mentioned that as-prepared MgO-NPs displayed distinctive reflection peaks in their diffraction pattern that were indexed to the (1 1 1), (2 0 0), (2 2 0), (3 1 1), and (2 2 2) diffraction planes.
Antibacterial activities of the CuO-NPs and MgO-NPs prepared in this study were investigated and the result noticed that CuO-NPs exhibited the highest antibacterial activity of 19.3 mm on TZC media and are more powerful than gentamycin. In this finding, antibacterial studies of CuO-NPs to a group of bacterial s, when compared to the positive control, S. flexneri and B. subtilis are the most sensitive to copper oxide nanoparticles (Penicillin G). The least sensitive was Salmonella typhimurium. (Pandiyarajan et al. 2013). Similar to this, (Awwad et al. 2015) noted that Shigella and Listeria inhibitory zones by CuO-NPs have radial diameters of 15 and 18 mm, respectively. According to (Cai et al. 2017), MgO-NPs offer a broad spectrum of direct toxicity against many pathogens. Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria. Additionally, these results are in line with other investigations that demonstrated the toxicity of these nanoparticles to phytopathogens, according to (Vanathi et al. 2016), CuO nanoparticles may show antifungal effectiveness against the following decreased phytopathogens: A. fumigatus > A. niger > F. oxysporum > A. flavus. Additionally, CuO-NPs-streptomycin has shown greater biofilm inhibition rates of about 90.99%, 84.23%, and 83.42% regarding C. michiganensis subsp. sepedonicus, Ralstonia solanacearum, and Dickeya solani, respectively (El-Batal et al. 2020). Concerning the fungi Aspergillus niger and Penicillium oxalicum, MgO-NPs show a wide range of toxicity (Sierra-Fernandez et al. 2017).The current data revealed that the bulks had weaker antibacterial activity than the nanoparticles at the same concentration against R. solanacearum. These results in agreement with (Mondal and Mani 2012) revealed that at the same concentration, the nanoparticles' antibacterial activity was superior to that of the bulk material. They reported that Cu nanoparticle was discovered to prevent the growth of the bacterial pathogen Xanthomonas axonopodis pv. punicae that causes bacterial blight of pomegranate at 0.2 ppm, which is >10,000-fold lower concentration than the typically used copper oxychloride (Cu2 (OH) 3Cl), similarly, (Cai et al. 2018) stated that compared to MgO-NPs, which ranged in concentration from 50 to 250 mg/mL, the comparable doses of bulk MgO were 500 and 600 mg/mL.
The MIC and MBC were tested to find out whether NPs might stop or eradicate the bacterial plant pathogens R. solanacearum growth. It was crucial to evaluate how these materials might be used in agriculture for crop protection. Using the microdilution method, the MIC and MBC were calculated. The findings showed that the MIC value of CuO-NPs against R. solanacearum was lower than that of MgO-NPs, and that at these concentrations, nearly all R. solanacearum cells were eradicated, indicating higher antibacterial activity. MICs were confirmed by numerous researchers (Mahapatra et al. 2008) demonstrated that Shigella, Salmonella paratyphi, Pseudomonas aeruginosa, and Klebsiella pneumoniae bacteria are all susceptible to the CuO nanoparticle dispersion (80-100 nm). The nanoparticles maintained their antibacterial activity until dilution of 1:128 demonstrated the zone of inhibition that was noticeable following treatment with nanoparticle suspension. Also, (Kumar 2014) demonstrated that CuO nanoparticle was tested against K. pneumonia and MIC was found to be 0.55µg\mL. While (Chen et al. 2019) showed that after being exposed to 250 mg mL-1 CuO-NPs, all R. solanacearum died. (Cai et al. 2018) mentioned that R. solanacearum's growth was suppressed by MgO-NPs at concentrations of 50 to 250 mg/mL, and the (MIC) and (MBC) values were 200 and 250 mg/mL, respectively. However, in this research, lipid analysis was conducted to clarify how cell material leakage affects respiratory function and ultimately results in cell death, the results revealed that the amounts of total lipids and neutral lipids of R. solanasearum decreased, while phospholipids increased in all treatments at MIC concentrations. CuO-NPs treatment gave the highest efficacy as the total lipids and neutral lipids decreased. These results by (Roy Choudhury et al. 2012) reported that synthesized sulphur nanoparticles (SNPs) were tested for their impact on enzymes and the overall lipid content of Aspergillus niger. The treated fungal isolates' total lipid content was dramatically reduced by SNPs, and the expression of numerous desaturase enzymes was significantly reduced. Ions produced by nanoparticles are thought to cause protein denaturation by attaching to and rupturing the negatively charged bacterial cell wall (Azam et al. 2012; Xin et al. 2019). According to (le Ouay and Stellacci 2015), the creation of ROS via lipid peroxidation, which causes cell material leakage and inhibits respiratory function, most likely leads in membrane damage. Cell death is the result of this respiratory activity. Additionally, the earlier literature highlighted that inorganic metal nanoparticles (such ZnO, Ag, TiO2, and Cu) are being used more and more as antibacterial, due to the buildup of reactive oxygen species (ROS), which could cause biological components including proteins, lipids, and even nucleic acids to be damaged. (Hemeg 2017).
Using TEM, the morphological structure of NPs treated R. solanacearum cells was examined. The findings demonstrated that the bacteria were fragmented into several segments. The harmful behaviors observed in this experiment were mostly in line with earlier studies that have looked at the complex processes by which metal nanoparticles harm bacteria and how bacteria and nanoparticles interact. These processes may be explained by higher surface area of adhesins, which comprise a large variety of bacterial surface glycoprotein receptors (Jeevanandam and Klabunde 2002). According to one theory, the interaction between pathogens and nanoparticles is caused by the electrostatic attraction of positively charged nanoparticles to negatively charged cell membranes (Raffi et al. 2010). However, the activity of (negatively charged) nanoparticles is not explained by this process. Mechanisms postulate that pathogens' cell membranes or cell walls are immediately destroyed by toxic ions created by nanoparticles that attach to proteins with Sulphur in them (Dong et al. 2010). In this regard intermolecular cross-linking is brought about by the binding of Cu nanoparticles to nucleic acids they interact with the amino acids sulfhydryl group and/or carboxyl group, damaging the protein and impairing biological activities (Chatterjee et al. 2014). When proteins are oxidized, reactive oxygen species (ROS) are formed., which interferes with electron transport and causes damage to proteins (Imada et al. 2016). ROS is a naturally occurring byproduct of an organism's metabolism. A high level of ROS production may result in oxidative stress. The antioxidant defenses of the cells, such as the glutathione/glutathione disulfide (GSH/GSSG) ratio, guard against its formation. For the first time, (Cai et al. 2018) investigated that MgO nanoparticles have good antibacterial actions against R. solanacearum as rod-shaped bactericides that can be used both in vitro and in vivo. They discovered that DNA damage caused by reactive oxygen species (ROS) production contributed significantly to the antibacterial effect. According to the present study, CuO and MgO nanoparticles' effective surface charge density (zeta potential of - 39.5 and – 43.8 mV) is related to their antibacterial effectiveness, providing them a higher binding affinity for the bacterial reduction of Cu (II) to Cu(I) and Mg (II) to Mg (I) that produces poisonous H2O2, causing the cell viability to be destroyed (Chen et al. 2019).
Studying how humic acid and compost tea treated with NPs affected the growth of R. solanacearum, the results demonstrate that the viable counts of bacteria isolated from the infected soil samples indicated that the highest numbers of bacteria were present in compost tea than humic acid samples as compared with, the highest numbers of bacteria found in control (CFU. g. dry wt.). On the other hand, a lot of authors have criticized the contribution of compost to disease resistance (Termorshuizen et al. 2006; EL-Desuki 2010). According to (JianHua et al. 2012), the mechanism underlying the suppressive effects of composts on R. solanacearum-caused potato wilt was thought to involve changes in the physical and chemical qualities, enzyme activity, and quantity of microorganisms. To determine whether NPs, a chemical that is allegedly an antibacterial field agent, could control bacterial wilt disease in comparison to bulks and commercial bactericides, trials on potted plants were conducted in vivo when the greenhouse is set against the unaffected control. The greenhouse experiment administration of NPs significantly lessened the bacterial wilt disease destructiveness. Without nanoparticle therapy, potato tubers were almost completely dead, treatment with NPs considerably lessened the severity of the illness, resulting in a wilt index of 28.8% for CuO-NPs treatment and 30.6% wilt index for MgO-NPs compared with 57% and 60.5% for bulk CuSO4 and bulk MgSO4, respectively, in comparison with a 100% wilt index in the infected control and 44.4% wilt index in Rhizo-N. In particular, (Elmer and White 2016) noted that, whether in a soilless medium or in soil, during the vegetative stage, foliar spraying of CuO-NPs to tomato and eggplant plants at higher concentrations seemed to significantly slow the spread of the Fusarium wilt disease. (Chen et al. 2019) observed CuO-NP exposure at doses of 50, 125, and 250 mg/mL reduced the disease indices of tobacco seedlings by 74.2%, 62.2%, and 38.1%, respectively, which displayed various disease symptoms. By developing systemic resistance, (Imada et al. 2016) found that employing MgO-NPs at a concentration of up to 1% greatly reduced the incidence of disease in tomato plants against R. solanacearum. On the other side (Khan and Siddiqui 2018) demonstrated that ZnO-NPs in two concentrations were employed and sprayed, for the management of the disease complex of eggplant brought on by Phomopsis vexans, R. solanacearum, and Meloidogyne incognita, seed treatment, and soil inoculation are used. When 200 ZnO NPs were sprayed onto plants, the disease index was lowered to one. More significantly, plants exposed to NPs in the current study exhibited an increase in yield when compared to the control, with CuO-NPs having the strongest effect when compared to the others. As a result, it has been determined that all the NPs employed might increase yield in comparison to lesser values of the equivalent bulks. These results about the emergence of nanoparticle-based growth are consistent with earlier studies. When compared to the control sample, soybean seedlings treated with CuO-NPs at a concentration of 60 ppm showed a 177% increase in root biomass and a 140% rise in shoot biomass. A 200% increase in root biomass and a 177% increase in shoot biomass were seen in chickpea seedlings treated with 100 ppm (Adhikari 2012). Additionally, it is highlighted that Cu ions can act as a microelement and encourage plant development in small concentrations (Rajput et al. 2018). The current results are also consistent with other studies (Rathore and Tarafdar 2015), which indicated that MgO-NPs could increase native nutrient mobilization and soil health while also increasing wheat crop output. Mg metal nanoscale particles have been found in numerous investigations to promote plant growth (Cai et al. 2018).
In this study, the highest values of chlorophyll (a) and chlorophyll (b) were when CuO-NPs and MgO-NPs were applied to soil, respectively, compared to lower values of equivalent bulks. Treatment with NPs considerably increased chlorophyll content. According to certain studies, the nanoparticles improved root pore formation, nutrient uptake, and hydro-mineral flow (Castiglione et al. 2011). (Mahmood 2015) reports revealed that the plants absorption of nutrients and chlorophyll was both enhanced by the nanoparticles. (Khan and Siddiqui 2018) reported that when NPs were sprayed on the eggplant to treat them for the disease complex brought on by Phomopsis vexans, R. solanacearum, and Meloidogyne incognita, the biggest rise in chlorophyll, carotenoid levels, and plant growth was seen.
The present data demonstrated that NPs could increase values of peroxidase, polyphenol oxidase, and phenolic contents and the highest activity values of peroxidase and polyphenol oxidase were at CuO-NPs and MgO-NPs addition which almost in range of healthy plants values. While the lowest activity values were at the addition of bulk MgSO4, and bulk CuSO4. Under specific circumstances, polyphenols can serve as pro-oxidants and produce radicals as electrophiles (Sakihama et al. 2002). Rapid O2 production resulted from treating plant roots with NP. The interaction of NP with polyphenols or tomato plant extract also resulted in O2 production. These findings imply that an interaction between the roots' polyphenols and NP caused O2 to be produced in the roots. Thankfully, studies show that in tomato plants, MgO-NPs stimulate the signaling pathways for salicylic acid (SA), jasmonic acid (JA), and ethylene (ET), which results in systemic resistance to R. solanacearum (Imada et al. 2016). A few years ago, significant efforts have been made to clarify enzymes involved in defense mechanisms against R. solanacearum invasion and disease resistance. According to (Vanitha and Umesha 2008), Lipoxygenase (LOX) and polyphenol oxidase (PPO), two defensive enzymes, actively contribute to a plant's resistance to bacterial wilt and may limit the growth of R. solanacearum. When a bacterial pathogen attacks, tomato (Solanum lycopersicum) responds by activating various defense responses, including alterations in cellular metabolism, particularly in the phenylalanine ammonia-lyase (PAL), peroxidase (POX), and polyphenol oxidase enzyme activities (PPO). This research was conducted by (Mandal et al. 2013). Additionally, (Adss 2014) found that when a bacterial pathogen attack occurs, various defense systems are triggered in plants. Increased PO activity during incompatible contacts and phenol chemicals' strengthening of cell walls are correlated were also reported by (Rajeswari 2014) as well as (Panche et al. 2016) reported a connection between phenol strengthening of cell walls and increased PO activity during incompatible contacts.