Bio-functionalized nickel-silica nanoparticles suppress bacterial leaf blight disease in rice (Oryza sativa L.)

Introduction Bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most devastative diseases that threatens rice plants worldwide. Biosynthesized nanoparticle (NP) composite compounds have attracted attention as environmentally safe materials that possess antibacterial activity that could be used in managing plant diseases. Methods During this study, a nanocomposite of two important elements, nickel and silicon, was biosynthesized using extraction of saffron stigmas (Crocus sativus L.). Characterization of obtained nickel-silicon dioxide (Ni-SiO2) nanocomposite was investigated using Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), Transmission/Scanning electron microscopy (TEM/SEM), and energy-dispersive spectrum (EDS). Antibacterial activities of the biosynthesized Ni-SiO2 nanocomposite against Xoo were tested by measuring bacterial growth, biofilm formation, and dead Xoo cells. Results and discussions The bacterial growth (OD600) and biofilm formation (OD570) of Xoo treated with distilled water (control) was found to be 1.21 and 1.11, respectively. Treatment with Ni-SiO2 NPs composite, respectively, reduced the growth and biofilm formation by 89.07% and 80.40% at 200 μg/ml. The impact of obtained Ni-SiO2 nanocomposite at a concentration of 200 μg/ml was assayed on infected rice plants. Treatment of rice seedlings with Ni-SiO2 NPs composite only had a plant height of 64.8 cm while seedlings treated with distilled water reached a height of 45.20 cm. Notably, Xoo-infected seedlings treated with Ni-SiO2 NPs composite had a plant height of 57.10 cm. Furthermore, Ni-SiO2 NPs composite sprayed on inoculated seedlings had a decrease in disease leaf area from 43.83% in non-treated infected seedlings to 13.06% in treated seedlings. The FTIR spectra of biosynthesized Ni-SiO2 nanocomposite using saffron stigma extract showed different bands at 3,406, 1,643, 1,103, 600, and 470 cm−1. No impurities were found in the synthesized composite. Spherically shaped NPs were observed by using TEM and SEM. EDS revealed that Ni-SiO2 nanoparticles (NPs) have 13.26% Ni, 29.62% Si, and 57.11% O. Xoo treated with 200 µg/ml of Ni-SiO2 NPs composite drastically increased the apoptosis of bacterial cells to 99.61% in comparison with 2.23% recorded for the control. Conclusions The application of Ni-SiO2 NPs significantly improved the vitality of rice plants and reduced the severity of BLB.


Introduction
Rice (Oryza sativa L.) is the most consumed cereal crop worldwide.Food and Agriculture Organization considers rice as an important crop for food security in the world (Wang et al., 2023).One of the most serious diseases infecting rice plants is bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv.oryzae (Xoo).BLB is a dominant diseases among various rice varieties (Singh et al., 2015).Infection by Xoo reduces the efficiency of photosynthesis and metabolism of rice plants, which subsequently leads to yield loss of up to 80% (Yasmin et al., 2017;Ma et al., 2023).
Diverse management strategies have been applied to control plant diseases.Use of chemical bactericides could be effective in controlling BLB.However, because of the extensive application of traditional chemical bactericides and antibiotics, it may catalyze mutations and lead to durable resistant races of pathogenic bacteria (Russo et al., 2008;Xu et al., 2010).Marques et al. (2009) reported that the widespread occurrence of copper and streptomycin resistance in field isolates and its adaptation to bactericides have a negative impact on the chemical management of Xanthomonas campestris pv.viticola.In China, different studies have reported streptomycin resistance in various phytopathogens.Choi et al. (2015) concluded that more than 50% of tested field strains of Pseudomonas syringae pv.tabaci showed medium-to high-level resistance to streptomycin.The evolution of Xoo strains in overcoming single-gene-based resistance has been reported.For instance, Xa4, a single-based breeding gene for BLB management has been defeated by Xoo sub-population evolution (Shanti et al., 2010).
Use of nanoparticles (NPs) to combat plant diseases is one of the best tools to enhance pathogen suppression while maintaining an ecofriendly and safe method as it results in the bioreduction of metals to stable metallic NPs through a green route (Melo et al., 2018).While many NPs have existed, currently, they have not been widely applied in plant pathology.However, the recent use of nano-medicine against human pathogens has re-evolution plant disease management approach (Elmer et al., 2018).Recently, NPs of metallic oxides (single and composites) have gained momentum in phytopathology.
The antibacterial action of NPs against phytopathogens is confirmed by many studies (Abdallah et al., 2020).For instance, ZnO NPs are found to be efficient against different pathogenic bacteria including Xoo and fungi (Ogunyemi et al., 2019).In addition, Cai et al. (2018) reported the antibacterial action of magnesium oxide NPs against Ralstonia solanacearum.The physicochemical characteristics of NPs increase their interaction with bacteria and improves their antimicrobial activities (Aziz et al., 2016;Rudramurthy et al., 2016;Ihtisham et al., 2021).NPs bind to the pathogen's cell wall causing deformation of cell membranes due to high-energy transfer and, subsequently, lead to the death of the pathogen (Pereira et al., 2022).In bacteria, metal NPs (MNPs) increase cell membrane permeability and cell destruction.Among known NPs, nickel has gained wide interest as an antifungal and antibacterial element.Nickel-based NPs have been used for controlling several plant pathogenic fungi (Ahmed et al., 2016;Markowicz, 2023).Jeyaraj Pandian et al. (2016) and Mirhosseini et al. (2018) reported a high growth inhibition against Gram-negative bacteria and Candida species (C.albicans and C. tropicalis) by using nickel oxide (NiO) NPs.
To improve NP properties and increase their efficacy, the synthesis of nanocomposites was recently tested for that purpose (Prakasham et al., 2010;Baig et al., 2021).Omanovic-Miklicǎnin et al. (2020) explained that the synthesis of nanocomposites consists of an assemblage of two different natural materials, which introduces material with greater performance characteristics than that of the original components separately.Lattuada and Hatton (2011) and Tao et al. (2008) reported that nanocomposites include the interaction between their different materials, and these interactive nanocomposites usually possess distinct properties that are not expressed in their individual elemental components.One of the most popular inert support materials is silica (Liou, 2004;Adam and Andas, 2007).The potential changes in the characterization of NiO NPs and their efficacy as a bactericide, when combined with supported component such as silica, and the role it could play in management of BLB are not known.Therefore, this study will investigate the potential use of such a nanocomposite in suppressing Xoo resulting in decreased disease severity.
MNPs are synthesized by many physiochemical methods such as co-precipitation, sol-gel, microemulsion, hydrothermal reaction, electrospray synthesis, and laser ablation.Biogenic methods such as using plant extracts can also be used for the synthesis of MNPs.Biosynthesis of NPs via plant extracts is economical, eco-friendly, and non-hazardous (Dubey et al., 2010;Alharbi et al., 2022).The ability of various plants (pomegranate, rose, banana, hibiscus, geranium leaves, cinnamomum, aloe, and basil) for the synthesis of NPs has been studied (Sathishkumar et al., 2009;Ahmad et al., 2010;Philip, 2010).Saffron (Crocus sativus), a bulbous perennial belonging to the iris family (Iridaceae) (Siddiqui et al., 2018), has been successfully used in the biosynthesis of several MNPs (Abootorabi et al., 2016;Bagherzade et al., 2017).The aqueous extract of saffron stigmas has OH groups from variety of phenolic compounds.These OH groups improve saffron's capability for the biosynthesis of NPs, as it reacts with metal ions and plays a role for the reduction of metal raw materials to MNPs (Khan and Rizvi, 2014).Therefore, this study aims to biosynthesize nickel-silicon NP composite using extract of saffron stigmas, to characterize the obtained composite, and to investigate the antibacterial action of obtained biosynthesized nickel and silica NPs against Xoo and its impact on rice plants challenged with Xoo.

Extraction of aqueous saffron
Aqueous saffron extraction was carried out in accordance to the method of Amin et al. (2017).A gram of dried saffron stigmas was added to 100 ml of deionized water in a beaker and then placed in a water bath for 4 h at 60°C.The extract was filtered twice using filter paper Whatman no.1 that was used directly for the synthesis of Ni-SiO 2 NP composite.

Biosynthesis of Ni-SiO 2 NP composite
To synthesize Ni-SiO 2 composite, a 100-ml solution of each element (1 mM bulk NiO and 1 mM bulk SiO 2 ) was prepared separately by adding previously prepared 100 ml of aqueous saffron extract to each one and then stirred at 180 Revolution Per Minute (rpm) for 4 h at 60°C.Then, Ni-SiO 2 composite was prepared by mixing the previously prepared solutions of NiO and SiO 2 using a ratio of 1:1 (v/v).The new mixture was swirled for 4 h at 60°C.The final solution was divided into 50-ml tubes and centrifuged (10,000 rpm/20 min).The pellets were retrievd and washed gently using ddH 2 O. Obtained pellets were lyophilized for 8 h.

Characterization of Ni-SiO 2 composite
To evaluate the formation of Ni-SiO2 NPs in the obtained powder, Fourier transform infrared spectroscopy (FTIR) analysis was done by employing spectrometer (Vector 22, Bruker, Germany) at the range of 500-4,000 cm −1 region at a resolution of 4 cm −1 .X-ray diffraction (XRD) was adopted to test the purity of obtained particles, and the mean crystallite size from XRD was calculated adopting the Scherrer equation (Jeffery, 1957).Transmission electron microscopy (TEM) was employed to observe morphology of NPs using (JEM-1230, JEOL, Akishima, Japan).Obtained NP powder was scanned by scanning electron microscopy (SEM) using (TM-1000, Hitachi, Japan).The SEM microscope was connected to energy-dispersive spectrum (EDS) to be assured of the presence of the elements.

In vitro inhibitory effect of Ni-SiO 2 NP composite and determination of minimum inhibition of concentration
Xoo strain GZ 0005 used for this investigation was collected from the Institute of Biotechnology, College of Agriculture and Biotechnology, Zhejiang University, China.The virulence of Xoo was tested and confirmed before the study.The antibacterial activity of Ni-SiO 2 NP composite against Xoo was evaluated by using the agar well diffusion assay as explained by Monteiro et al. (2013).An overnight 100 μl of Xoo culture (approximately 1 × 10 8 Colony forming unit (CFU)/ml) was added to 5 ml of Nutrient Agar (NA) medium, and, then, 50 μl each of previously prepared concentration (Ni-SiO 2 NP composite at 50, 100, and 200 mg/ml) was poured into 6-mm-diameter agar wells.Five replications were done for this assay; each replication was typified by a plate consisting of a well for each of the three concentrations.The plates were incubated at 30°C for 48 h.The clearance zone around the well was scaled after 48 h.The experiment was repeated following the same condition.
The minimum inhibition of concentration (MIC) of Ni-SiO 2 NP composite against Xoo was investigated as explained by Wiegand et al. (2008).In detail, 100 μl of an overnight culture of Xoo (approximately 1 × 10 8 CFU/ml) was poured into sterile tubes containing 5 ml of nutrient broth.Ni-SiO 2 NP composite was added to each tube, and the concentrations were adjusted to 50, 100, and 200 μg/ml each in respective tube.The tubes were kept in 30°C with shaking at approximately 180 rpm.After 48 h of incubation, MIC was measured using a UV spectrophotometer by the optical density at 600 nm (OD 600 ).The investigation was repeated twice.

Effect of Ni-SiO 2 NP composite on biofilm formation of Xoo
The ability of Ni-SiO 2 NP composite to inhibit Xoo biofilm was measured as described by Merritt et al. (2005).A 100 ml of overnight Xoo culture (1 × 10 8 CFU/ml) was added to Nutrient Broth (NB) medium containing Ni-SiO 2 NP composite to get a final concentration of 50, 100, and 200 mg/ml.The mixture was kept static in a 30°C incubator for 48 h to develop a biofilm in a 96-well plate.To stain the attached biofilm, crystal violet (CV) was added to the wells after discarding the supernatant.CH 3 COOH (33%) was used in solubilizing the CV attached to the biofilm and measured at OD 570 .

Live/dead assays to infer the cell membrane integrity
Fluorescence emitted from propidium iodide (PI) of dead bacterial cells after incubation with the NPs was measured by flow cytometer (Kumar et al., 2011).Xoo culture (1 × 10 8 CFU/ ml) was centrifuged (5,000 rpm/5 min), and Ni-SiO2 NP (200 μg/ ml) composite was added to the obtained pellets for 4 h.PI was added in the dark for 30 min to stain the chromatin of bacterial cells.Subsequently, the dead cell ratio of Xoo cells was measured by flow cytometry (FC) (Gallios Beckman Coulter, Germany).

Effect of Ni-SiO 2 composite on rice seedlings infected with Xoo
The experiment was conducted as complete randomized blocks.Five replications were used per treatment.Three rice seedlings (cv.II You 023 Oryza sativa L.) in each replicate were sown in small pots filled with sterile soil and kept in the growth chamber under 28 ± 2°C , 80% relative humidity with a photoperiod of 16-h light and 8-h dark.This experiment consisted of four treatments that include the following: 1.In the first treatment, 3-week-old rice seedlings were sprayed with a suspension of Ni-SiO 2 NP composite (200 μg/ml); after 48 h, the rice seedlings were inoculated with Xoo strain GZ 0005 culture (1 × 10 8 CFU/ml) via leaf clipping.2. In the second test treatment, 3-week-old rice seedlings were sprayed with distilled water; after 48 h, the rice seedlings were inoculated with Xoo strain GZ 0005 culture (1 × 10 8 CFU/ml) via leaf clipping.3.In the third test treatment, 3-week-old rice seedlings were sprayed with a suspension of Ni-SiO 2 NP composite (200 μg/ml), and no Xoo inoculation was applied.4. The fourth treatment, 3-week-old rice seedlings were sprayed with distilled water, and no Xoo inoculation was applied.
The experiment was carried out at 11:00 a.m. to ensure that the stomata had opened.Diseased leaf area, plant height, and fresh and dry biomass weight were recorded 1 month after application of Ni-SiO 2 NP composite on rice plants.The percentage of diseased leaf area (DLA%) was calculated as follows: DLA % = Total lesion area of the test sample= Total leaf area of the test sample Â 100 %

Statistical analysis
Data were subjected to analysis of variance using SAS, 2003 software (SAS Institute, Cary, NC, USA).The general linear model procedure was used to check the significant differences among the main treatments.Individual comparisons between mean values were performed using Duncan's method (P ≤ 0.05).Simple linear regression (SLR) analysis was performed to better understand the relationship between concentrations of Ni-SiO 2 NP composite and inhibition zone, bacterial growth inhibition, and biofilm formation inhibition.The fitted regression model was stated as a regression equation, coefficient of determination (R 2 ), R 2 adj , and p-value as determined by the F-test (P ≤ 0.05).

Characterization of Ni-Si O 2 NP composite
The FTIR spectra of biosynthesized Ni-SiO 2 NP composite revealed various bands at 3,406, 1,643, 1,103, 800, and 470 cm −1 (Figure 1A).The band at 3,406 cm −1 was assigned to hydroxl stretch hydrogen bonds, the band at 1,643 cm −1 was related to C=C stretch, and the band at 1,103 cm −1 indicates C-O stretches.The peaks at 800 and 470 cm −1 were attributed to the symmetric vibration of Si atoms.XRD pattern showed no impurities in tested samples.The sharpest diffraction peaks were recorded at 2q around 43°, which can be indexed as (202) for nickel, and at 2q around 20°, representing (101) for silica (Figure 1B).Spherically shaped NPs were observed by using TEM (Figure 1C) and SEM (Figure 1D).Data from the EDS of Ni (Figure 2A), Si (Figure 2B), O (Figure 2C), and Ni-SiO 2 NPs (Figure 2D) revealed that Ni-SiO 2 NP composite has 13.26% Ni, 29.62% Si, and 57.11% O (Figure 2E).

Composition of biosynthesized Ni and SiO 2 NPs enhances their antibacterial activity against Xoo
The ability of Ni, Si, and Ni-SiO 2 NPs to inhibit Xoo bacteria was investigated by using plate assay technique (Figures 3A-C, respectively).Three concentrations (50, 100, and 200 μg/ml) of each NP were tested.In general, all the tested NPs had a dose-dependent antibacterial action against Xoo that significantly inhibited its growth in vitro (Figure 3D).It is worth mentioning that the Ni-SiO 2 NP composite was the most efficient NPs suppressing Xoo growth (Figures 3C, D).The three tested concentrations of Ni-SiO 2 NP composite (50, 100, and 200 μg/ml) produced inhibition zones of 2.1, 2.4, and 2.9 cm, respectively, compared with 0.9, 1.3, and 1.5 cm for SiO 2 and 0.8, 1.1, and 1.2 cm for NiO NPs (Figure 3D).The MIC of the Ni-SiO 2 NPs was 200 mg/ml, in which Xoo growth was inhibited by 89.07%, whereas using 50 and 100 mg/ml resulted in 21.50% and 54.37% inhibition, respectively.

Ni-SiO 2 NP composite inhibits bacterial growth of Xoo in nutrient broth
Furthermore, because of the superiority of Ni-SiO 2 NP composite over NiO and SiO 2 NPs, the focus was placed on it throughout the rest of this study.Briefly, in nutrient broth, Ni-SiO 2 NP composite significantly inhibited the growth of Xoo in a dosedependent manner as revealed by OD 600 (Figure 4A).In other words, the inhibition extents increased from 50< 100< 200 μg/ml.In agreement with these findings, SLR showed strong negative correlation (y = 1.1876 − 0.0055x, R 2 = 0.9833, R 2 adj = 0.9750, and P = 0.0084) between Xoo bacterial growth (OD 600 ) and Ni-SiO 2 NP concentrations (μg/ml) (Figure 4B).

Ni-SiO 2 NP composite inhibits biofilm formation of Xoo
Likewise, Ni-SiO 2 NP composite significantly hindered biofilm development of Xoo cells in a dose-dependent manner because the higher concentrations showed lower biofilm formation, and vice versa, as indicated by optical density at 570 nm (OD 570 ) (Figure 4C).Antibiofilm activity of 25.91%, 61.06%, and 80.40% were detected as a result of using Ni-SiO 2 NPs of 50, 100, and 200 μg/ml, respectively.In addition, SLR showed a strong negative correlation (y = 1.0382 − 0.0045x, R 2 = 0.9246, R 2 adj = 0.8869, and P = 0.0384) between biofilm formation (OD 570 ) and Ni-SiO 2 concentrations (μg/ml) (Figure 4D).In panels (B) and (D), the linear fit regression line is presented as a dashed line, whereas the 95% confidence intervals are light blue-shaded and edged by dotted lines).Regression equations, R2, R2 adj , and P-value based on the F-test (P< 0.05) were also obtained and presented within the graph.Abdallah et al. 10.3389/fpls.2023.1216782Frontiers in Plant Science frontiersin.org

Ni-SiO 2 NP composite causes cell injury or death to Xoo
Moreover, cell damage/apoptosis of Xoo cells was assessed using FC and PI-based method.Briefly, incubation of Xoo with Ni-SiO 2 NP composite (200 μg/ml) drastically increased the apoptosis of the bacterial cells to 99.61% (Figure 4E) compared with 2.23% for the mock control (distilled water; Figure 4F).Together, in addition to the inhibition of bacterial growth, our findings proved that Ni-SiO 2 NP composite might cause cell puncture or death to Xoo when it was amended with a concentration of 200 μg/ml.

Application of Ni-SiO 2 NP composite improves plant growth and reduces disease severity of BLB in rice
A notable improvement in rice growth was observed when Ni-SiO 2 NP composite was applied at a concentration of 200 μg/ml as a foliar application on healthy and Xoo-infected rice plants under greenhouse conditions (Figure 5A).Interestingly, Ni-SiO 2 NP composite application notably increased the leaf length of treated rice plants (Figures 5B, C); however, it reduced the total diseased leaf area in Xoo-infected rice plants (Figure 5D).Accordingly, the disease leaf area decreased from 43.83% in non-treated control plants to 13.06% when Ni-SiO 2 NP composite was applied to infected plants (Figure 5D).Moreover, the amendment with Ni-SiO 2 NPs significantly increased the height of non-infected rice plants to 64.8 cm in comparison with 45.2 cm of plants amended with only water (Figure 5E).Likewise, treating Xoo-infected rice plants with Ni-SiO 2 NP composite significantly increased plant height to 57.1 cm compared with non-treated infected rice plants, which appeared short with an average plant height just below 20 cm.Similarly, the application of Ni-SiO 2 NPs produced almost the same pattern in terms of root length (Figure 5F).In addition to improving rice growth, using Ni-SiO 2 NP composite showed a positive effect on biomass.Briefly, application of Ni-SiO 2 NPs significantly increased both fresh (Figure 5G) and dry (Figure 5H) weight of treated healthy and Xoo-infected rice plants compared with non-treated ones.Abdallah et al. 10.3389/fpls.2023.1216782Frontiers in Plant Science frontiersin.org

Discussion
NPs have been applied in the field of agriculture as highly effective bactericides, fungicides, and nano fertilizers due to their small size, large surface area, and high reaction (Elmer and White, 2018;Hossain et al., 2019;Ogunyemi et al., 2019).The synthesis of NPs produced a variety of morphology, sizes, and compositions that were determined by numerous physical, chemical, and biological techniques (Pagar et al., 2023).Our study aimed to biosynthesize Ni-SiO 2 NP composite with new properties that could contribute to the management of BLB by using extraction of saffron stigmas (Crocus sativus L.).
Studies of the infrared spectrum were conducted to explore the potential mechanism behind the formation of Ni-SiO 2 NP composite and information about the functional groups (Irshad et al., 2018;Petousis et al., 2020).The FTIR spectra of biosynthesized Ni-SiO 2 NP composite revealed various peaks that confirmed the presence of important bonds such as hydroxyl stretch, C=C stretch, C-H, and Si-O-Si bond (Majewski et al., 2013;Adel et al., 2022).Upon reviewing infrared spectrum results, plant extract of saffron stigmas could be responsible for the bio-reduction of Ni-SiO 2 NP composite.Moreover, silica bonds contributed to the stability of NiO NPs.Phytochemicals easily show the ability to synthesize nickel NPs (Singh et al., 2016;Shwetha et al., 2021).The obtained results show that there are no impurities revealed by the XRD pattern in biosynthesized Ni-SiO 2 composite.
Silica was able to improve the morphological characteristics of nickel NPs including particles size.Spherically shaped NPs were observed by using TEM and SEM.The size of obtained nickel-silica composite averaged between 12.6 and 27.8 nm.Our finding matches a study by Saha et al. (2015), which was able to synthesize Ni NPs of a size range of 10-30 nm in Ni-SiO 2 composite prepared by sol-gel route.The size of the produced Ni NPs was smaller in comparison with that of other synthesis protocol (Lajevardi et al., 2013).
Data collected from EDS revealed that Ni-Si-O NP composite has 13.26% Ni, 29.62% Si, and 57.11% O.As reported by Saha et al. (2015), one Si atom reacts with two O 2 atoms to form SiO 2 .Thus, 29.62% Si present in the composite combines with 57.11% O 2 to produce SiO 2 .The crystalline nature of synthesized Ni-SiO 2 NP composite was investigated by XRD technique.The wide spectrum range of 20°and 30°is attributed to the presence of an amorphous Si matrix.The formation of NiO is exempted from the phase analysis by XRD.
Ni-SiO 2 NP composite was able to inhibit Xoo growth and significantly increase the ratio of Xoo dead cells to 99.61% compared with 2.23% for control.Therefore, according to the obtained results, Ni-SiO 2 NP composite can be used as bactericides that have antimicrobial activity as documented by Ahmed et al. (2016) andJeyaraj Pandian et al. (2016).As NPs have positive or low negative charges, they are electrostatically attracted and adhered to the negatively charged cell membrane of bacteria (Zein El-Abdeen and Farroh, 2019).Subsequently, it caused irregular pit formations on the cell wall of the pathogenic bacteria that facilitate the entry of NPs into periplasmic space and inside bacterial cells (Ninganagouda et al., 2014;Wang et al., 2023).The high efficacy of Ni-SiO 2 NP composite against Xoo could be due to the size of the nickel NPs that have been reduced by silicon to range approximately from 10 to 30 nm, which allows nickel NPs to intensively enter the bacterial cell, resulting in ion accumulation that contributes to membrane porosity damaging the cytoplasm and cell structures.This destruction of cell structure caused the escape of the embedded cell contents, leading to bacterial cell death (Jeyaraj Pandian et al., 2016;Zhu et al., 2022).
The inhibition of biofilm formation, which was detected by using Ni-SiO 2 NP composite, confirms and matches that of the previous studies on metal oxide NPs (Lee et al., 2014).Le Ouay and Stellacci (2015) and Pellieux et al. (2000) documented that the inhibitory effect of NPs on bacteria is linked to the formation of Reactive Oxygen Species (ROS).ROS promotes oxidative stress in cells and induces DNA, protein, lipids, and cell damage (Piao et al., 2011;Zhang et al., 2018).In addition to the bactericidal effect of Ni-SiO 2 NP composite, it enhanced rice growth and significantly increased the height of the plant.It also showed a positive effect on rice seedlings' biomass fresh and dry weight.Mirzajani et al. (2014) and Syu et al. (2014) reported that rice treated with NPs enhanced root growth, which may be due to the interaction between NPs and ROS scavenging, hormone signaling pathways, and auxin.Tarafdar et al. (2014) and Zafar et al. (2016) stated that metal oxide NPs shows enhancement on shoot length of Pennisetum americanum and Brassica nigra.
This study proved that the application of Ni-SiO 2 NP composite significantly decreased the biofilm of Xoo, which subsequently decreased the virulence of the bacteria.The treatment with MgO and MnO 2 NPs at the primary stages of growth caused a promotion in rice seedlings growth and increased the photosynthetic parameters while reducing BLB expression (Ogunyemi et al., 2023).On the basis of this report, it can be inferred that, because NPs had a positive impact on photosynthesis, the plant yield will invariably be positively affected.Xu et al. (2021) reported that the application of titanium dioxide NPs on two different cultivars of rice (WYJ23 and YY2640) significantly increased the agronomic data and yield.Therefore, on the basis of reports of the positive impacts of NPs application, it indicates that the treatment of rice with NPs improves both the agronomic trait and yield of rice irrespective of the cultivar or NPs used.
This present work provides helpful and useful insights for using the Ni-SiO 2 NP composite as potent applications for antibacterial activities.Ni-SiO 2 NP composite, which is cheap, stable, and nontoxic, indicates a promising safe result that can be used not only in the management of plant diseases but also as a medical treatment for human diseases.Ni NPs were used for their antibacterial activity in the field of medicine and were found to be effective when used for targeting cancer cells (Sudhasree et al., 2014;Ezhilarasi et al., 2016).Hence, despite numerous reports about the antibacterial activity of individual NP elements against Xoo, there are few studies of the nanocomposites against this pathogen (Namburi et al., 2021;Chauhan et al., 2023).Therefore, the report of this study is novel, which helps to bridge the gap of the management of Xoo using Ni-SiO 2 NP composite.

Conclusion
In conclusion, the use of saffron stigma extract in biosynthesizing Ni-SiO 2 NPs successfully produced a pure composite.The composite of nickel-silica particles have a small size range of 12.6-27.8nm.The composite had the ability to inhibit Xoo growth to the point where 89.07% of Xoo cells were killed when treated with Ni-SiO 2 NP composite (200 μg/ml).The obtained composite also showed that the bacterial anti-biofilm activity reached 80.40% and achieved 99.61% dead cells of Xoo.The application of Ni-SiO 2 NP composite significantly promoted the growth of rice plants challenged with Xoo compared with untreated plants.Ni-SiO 2 NP composite increased biomass fresh and dry weight.In general, Ni-SiO 2 NP composite is a promising effective tool for suppressing Xoo infection on rice plants.On the basis of the potent antibacterial activity of the synthesize nanocomposite recorded in this study, we hereby suggest future studies to be conducted on the mechanism of nanocomposite on ROS and phytohoromones and their effect on rice plants yield using different cultivars.
FIGURE 2 Energy dispersion spectrum (EDS) Ni-SiO 2 NP composite.(A) Ni Ka1, (B) Si Ka1, (C) O Ka1, (D) Ni-SiO 2 , and (E) composite.Energy-dispersive spectrum showing the predominance of Ni, Si, and O elements and the percentage of each element in the Ni-SiO 2 NP composite.
FIGURE 3 In vitro antibacterial action of NiO, SiO 2 , and Ni-SiO 2 NP composite against Xoo.(A-C) Antibacterial activity of different concentrations (50, 100, and 200 µg/ml) NiO, SiO 2 , and Ni-SiO 2 NP composite, respectively, against Xoo.(D) Diameters of the inhibition zones of Xoo after the treatment of NPs (50, 100, and 200 µg/ml).(E, G, I) Diameters of the inhibition zones of Xoo after the treatment with different concentrations of NiO, SiO 2 , and Ni-SiO 2 NP composite, respectively.Vertical bars represent the means ± standard deviation (means ± SD) of three biological replicates (n = 3).Different letters indicate statistically significant differences among treatments, whereas bars followed by the same letter(s) are not significantly different (P ≤ 0.05).(F,H, J) Simple linear regression between concentrations (µg/ml) NiO, SiO 2 , and Ni-SiO 2 NP composite, respectively, and the inhibition zones (cm).The linear fit regression line is presented as a dashed line, whereas the 95% confidence intervals are light blue-shaded and edged by dotted lines.Regression equations, R2, R2 adj , and p-value based on the F-test (P< 0.05) were also obtained and presented within the graph.
FIGURE 4 Antibacterial activity of Ni-SiO 2 NP composite against Xoo.(A) Bacterial growth of Xoo in nutrient broth containing different concentrations of Ni-SiO 2 NP composite (0, 50, 100, or 200 µg/ml) as indicated by optical density at 600 nm (OD 600 ).(B) Simple linear regression between concentrations of Ni-SiO 2 NP composite (µg/ml) and Xoo Bacterial growth (OD 600 ).(C) Biofilm formation of Xoo after the treatment with different concentrations of Ni-SiO 2 NP composite (0, 50, 100, or 200 µg ml −1 ) as indicated by optical density at 570 nm (OD 570 ).(D) Simple linear regression between concentrations of Ni-SiO 2 NP composite (µg/ml) and Xoo biofilm formation (OD 570 ).(E, F) Flow cytometry observations of Xoo cells after incubation with Ni-SiO 2 NP composite (200 mg/ml) or distilled water, respectively.In panels (A) and (C), bars represent the means ± standard deviation (means ± SD) of three biological replicates (n = 3).Different letters indicate statistically significant differences among treatments (P ≤ 0.05).In panels (B) and (D), the linear fit regression line is presented as a dashed line, whereas the 95% confidence intervals are light blue-shaded and edged by dotted lines).Regression equations, R2, R2 adj , and P-value based on the F-test (P< 0.05) were also obtained and presented within the graph.
FIGURE 5 Effect of application of Ni-SiO 2 NP composite on rice growth and disease severity of bacterial blight disease of rice caused by Xanthomonas oryzae pv.oryzae (A, B) Ni-SiO 2 -treated vs. non-treated healthy and Xoo-infected rice plants and leaves, respectively.(C) Leaf length (cm), (D) diseased leaf area (%), (E) shoot length (cm), (F) root length (cm), (G) plant fresh weight (g), and (H) plant dry weight (g) of Ni-SiO 2 treated vs. non-treated healthy and Xoo-infected rice plants.Bars represent the means ± standard deviation (means ± SD) of three biological replicates (n = 3).Different letters indicate statistically significant differences among treatments (P ≤ 0.05).Mock = Control.