Multimodal Impact of Bacillus subtilis-derived Zinc oxide and Copper Oxide Nanoparticles on Citrus Biochemistry, Horticultural Attributes and Greening Disease Management

Multimodal Impact of Bacillus subtilis-derived Zinc oxide and Copper Oxide Nanoparticles on Citrus Biochemistry, Horticultural Attributes and Greening Disease Management | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Multimodal Impact of Bacillus subtilis-derived Zinc oxide and Copper Oxide Nanoparticles on Citrus Biochemistry, Horticultural Attributes and Greening Disease Management Asad Ullah, Muhammad Atiq, Azeem Akram, Owais Iqbal, Muhammad Usman, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7379214/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Present research work explored the multimodal function of Bacillus subtilis -mediated zinc oxide and copper oxide nanoparticles towards citrus greening disease, with their impact on the kinnow biochemical response. UV-Vis spectroscopy confirmed nanoparticle formation with surface plasmon resonance peaks at 380 nm (ZnONPs) and 280 nm (CuONPs), while particle sizes averaged 60 nm and 85 nm, respectively. Zeta potentials were − 35 mV (ZnONPs) and − 20 mV (CuONPs), indicating stable colloidal dispersions. XRD analysis confirmed their FCC crystalline nature, and FTIR spectra revealed plant-derived functional groups (O–H, N–H, C = O, C–O) involved in nanoparticle capping. SEM imaging showed irregular spherical ZnONPs (45–55 nm) and rod-shaped CuONPs (70–80 nm). Greenhouse assays showed significant disease suppression, with ZnONPs + CuONPs reducing incidence to 16.95%, outperforming ZnONPs (19.61%) and CuONPs (28.76%). Concentration- and time-dependent reductions under ZnONPs + CuONPs reached 12.84% at 0.75% and 12.06% at 21 days. Field trials confirmed similar trends (25.90% incidence under ZnONPs + CuONPs). Biochemically, ZnONPs at 0.75% enhanced SOD (6.06 µg/g FW), POD (5.98), CAT (5.04), TPC (5.38), TSP (5.85), TSS (6.15), proline (4.99 µmol/g FW), H₂O₂ (4.73), and MDA (5.43). Agronomic traits were also improved at 100 mg/L ZnONPs: juice pH (3.50), juice content (46.46%), vitamin C (16.03 mg/100 mL), fruit weight (213.53 g), diameter (72.29 mm), peel thickness (9.29 mm), and yield (258.03 kg/tree). These findings underscore the promise of biogenic ZnONPs and CuONPs as sustainable, multifunctional tools for integrated disease management and crop health improvement. Biological sciences/Biochemistry Biological sciences/Biological techniques Biological sciences/Biotechnology Earth and environmental sciences/Environmental sciences Physical sciences/Nanoscience and technology Biological sciences/Plant sciences Citrus reticulata HLB Candidatus Liberibacter asiaticus ZnONPs CuONPs Bacillus subtilis biochemical markers Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Citrus is well known for its rich profile of bioactive compounds, mainly grown in tropical and subtropical regions and belongs to the Rutaceae family 1 2 . This fruit is popular for its refreshing taste, a natural source of valuable metabolites with both nutritional and medicinal benefits 3 . This fruit crop is highly vulnerable to many diseases, but among these, huanglongbing has emerged as one of the most devastating, as it drastically reduces yields and shortens the life span of citrus trees 4 . HLB is caused by Candidatus Liberibacter , a gram-negative bacterium that colonizes the phloem of infected trees and it spreads primarily through contaminated grafting material and by insect vectors, most notably the Asian citrus psyllid ( Diaphorina citri ), which picks up the bacterium during phloem feeding and then transfers it to healthy trees as it continues feeding 5 . Although there is no evidence of seed transmission, grafting and psyllids are efficient routes for the pathogen. In Asia, Diaphorina citri is the major vector, while in some other parts of the world, Trioza erytreae also plays a role in its spread 6 . Environmental conditions, particularly warm and humid weather with temperatures between 16°C and 33°C, supports the development of the disease, which is characterized by blotchy vein yellowing, small and misshapen fruits with uneven coloring, thick rinds, excessive acidity, low sugar content, a bitter taste and premature fruit drop, collectively resulting in severe economic losses 7 . Traditionally, farmers have relied on chemical pesticides and antibiotics because they work quickly and are relatively inexpensive, but their overuse led to environmental pollution, risks to human health, damage to crops and the emergence of resistant strains of pathogens 8 . These drawbacks have prompted researchers to explore safer and more sustainable solutions, such as biological control using beneficial bacteria like Lysobacter antibioticus , Pseudomonas , and Bacillus species 9 . In recent decades, nanotechnology has gained significant weight in the field of plant protection 10 . Nanoparticles, due to their small size ranges between 1–100 nm have unique physical properties like a large surface area to volume ratio which makes them highly reactive and ensure bioavailability of essential nutrients to plant system with enhanced efficacy to combat disease 11 12 13 . It has been explored that these physical properties of NPs also facilitate deep penetration into plant tissue, perform efficiently at lower doses, which makes them a cost-effective alternative option to conventional chemotherapy 14 15 16 . Multiple approaches (like physical, chemical and biological) are being used for the synthesis of NPs, among these, the chemical method is common, relies on hazardous reagents and generates toxic waste, posing risks to human health and the environment 17 18 . On the other hand, physical methods require expensive equipment and a lot of energy to carry out the synthetic process 19 , conversely, biological synthesis offers a safer and eco-friendly route due to the usage of bioresources (like bacteria, fungi, algae and yeast) for biosynthesis of nanoparticles 20 . Microbial synthesis is a promising avenue because the bioactive metabolites of microorganisms act as natural reducing and stabilizing agents, eliminating the need for toxic reagents and precursors commonly used in traditional routes 21 . Biogenic approach tends to produce more stable, biodegradable and less toxic NPs, with minimal impact on the environment and non-target microflora 22 . Many prior studies have witnessed that nanoparticles activate the host antioxidant defense responses by triggering systemic acquired resistance against a variety of phytopathogens 23 . Among several microbial resources investigated, Bacillus subtilis stands out as the most potent bio-reducing agent for bio fabrication of NPs 24 . So, a recent research effort was made to explore the impact of a microbial nanofactory, Bacillus subtilis-derived Zinc oxide and copper oxide nanoparticles, on the biochemistry and horticultural attributes of kinnow for the management of citrus greening disease. Methodology Iodine Kit Test to Pre-Screen Symptomatic Samples Citrus greening disease was diagnosed using the indirect iodine test described by 25 based on the presence of characteristic splotchy leaf mottling. Symptomatic and asymptomatic leaves were collected and processed at the Molecular Phytobacteriology Laboratory, University of Agriculture, Faisalabad. For this purpose, leaf tissue (3 g) was homogenized with 3 mL of distilled water, the extract was filtered into a sachet, and iodine-litmus paper, followed by iodine solution, was applied. A blue or purple color change indicated infection, whereas the absence of color change confirmed healthy samples. Bacillus subtilis -based Synthesis of ZnONPs and CuONPs Zinc oxide and copper oxide nanoparticles were synthesized following the protocol of 26 with minor modifications. Bacillus subtilis , obtained from the Institute of Soil and Environmental Sciences, University of Agriculture Faisalabad, was suspended aerobically in nitrate medium and incubated on a rotary shaker at 120 rpm for 24 h at 28°C. After incubation, the culture was centrifuged at 6,000 rpm for 10 min at 6°C to collect the cell-free supernatant. The supernatant was inoculated into 250 mL Erlenmeyer flasks containing 100 mL of sterile nutrient broth and incubated on a rotary shaker at 200 rpm for 48 h at room temperature. The culture was then centrifuged at 12,000 rpm for 10 min to separate the biomass, which was resuspended in 100 mL of 1 mM zinc nitrate (Zn(NO₃)₂) or 1 mM copper sulfate (CuSO₄) aqueous solution in 250 mL flasks for ZnO and CuO nanoparticle synthesis, respectively. The reaction mixtures were incubated on a rotary shaker (200 rpm) at room temperature for 24 h in light to facilitate the reduction of metal ions to nanoparticles. Heat-killed bacterial samples with metal salts served as controls. The synthesized nanoparticles were characterized using UV–visible spectroscopy (UV-Vis), Fourier-transform infrared spectroscopy (FTIR), particle size analysis (PSA), zeta potential, and scanning electron microscopy (SEM). Evaluation of Bacillus subtilis -derived ZnONPs and CuONPs against citrus greening disease under Greenhouse conditions In the greenhouse trial, antibacterial efficacy of Bacillus subtilis -based zinc oxide and copper oxide nanoparticles individually and in integration was tested against citrus greening disease. One-year-old citrus plants of uniform size and development, previously confirmed to be infected with CGD, were maintained under controlled greenhouse conditions (25–30°C, 60–70% relative humidity, 12 h photoperiod supplemented with artificial light when necessary). Treatments consisted of ZnONPs and CuONPs at concentrations of 0.25%, 0.50% and 75%, either alone or combined, along with an untreated control. The experiment was laid out in a Completely Randomized Design with five replicates per treatment. For trunk injection, 100 mL, 75 mL and 50 mL of nanoparticle solution were injected per plant for the 0.25%, 0.50% and 75% concentrations, respectively, using a Cypress Tree Injector (Model CTP-01). Solutions were prepared by dissolving 0.25 g, 0.50 g and 0.75 g of nanoparticles in 100 mL of water to achieve the respective concentrations. Disease severity and physiological responses were assessed at 7, 14 and 21 days after treatment. Assessment of Bacillus subtilis-mediated ZnONPs and CuONPs against citrus greening disease under field conditions A field-scale experiment was performed to investigate the efficacy of Bacillus subtilis -based zinc oxide and copper oxide nanoparticles, applied individually and in combination, against citrus greening disease in Citrus reticulata (Kinnow). The trial was established in a citrus plantation using 5-year-old trees (2.0–2.5 m height, 2 m canopy width, 4.5 m spacing) that had been confirmed as infected with citrus greening disease using iodine test kits. Treatments included ZnONPs and CuONPs at 0.25%, 0.50% and 0.75%, either alone or combined, along with an untreated control, arranged in a Randomized Complete Block Design (RCBD) with three replicates per treatment. Trunk injection volumes were adjusted to deliver the required nanoparticle concentrations: 200 mL, 150 mL and 100 mL for 0.25%, 0.50% and 0.75% treatments, respectively. Solutions were prepared by dissolving 0.25 g, 0.50 g and 75 g of nanoparticles in 100 mL of water, resulting in a total dose of 1 g, 1.13 g and 1 g per tree, respectively. Injections were carried out using a Cypress Tree Injector (Model CTP-01) to ensure precise delivery into the vascular system 25 . Disease incidence and severity were assessed at 7, 14 and 21 days after treatment. Sample Preparation for Biochemical Analysis Leaves from the Kinnow were collected from nanoparticles (ZnONPs and CuONPs) treated and untreated plants under stress conditions induced by the pathogen. The leaves were cut into small pieces and 0.5 g of the leaf sample was ground in a pestle and mortar with KH₂PO₄ buffer. The samples were centrifuged (Horizon 6 Flex) at 12,000 rpm for 5 minutes and the supernatant was collected for biochemical analysis. Estimation of superoxide dismutase, peroxidase catalase and hydrogen peroxide The levels of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and hydrogen peroxide (H 2 O 2 ) concentration were estimated in treated and untreated kinnow leaves under HLB stress. For SOD activity, a reaction mixture containing 100 µL enzyme extract, 100 µL Nitroblue Tetrazolium (NBT), 200 µL Triton X-500, 200 µL potassium phosphate buffer (pH 5), 200 µL methionine and 800 µL distilled water was exposed to UV light for 15 minutes to activate riboflavin and generate superoxide anions. After adding 100 µL riboflavin, absorbance was measured at 560 nm 27 . POD activity was quantified by using a mixture of 800 µL potassium phosphate buffer (pH 5), 100 µL enzyme extract, 20 mM guaiacol, and 100 µL of 40 mM H 2 O 2 and absorbance was recorded at 470 nm 28 (Hameed et al. , 2021). For CAT activity, a reaction mixture of 100 µL enzyme extract and 100 µL of 5.9 mM H 2 O 2 was used, and absorbance was measured at 240 nm 28 . H 2 O 2 concentration was determined by grinding 50 mg fresh leaf sample in trichloroacetic acid buffer and centrifuged at 12,000 rpm for 15 minutes at 4°C. The supernatant was mixed with potassium phosphate buffer (pH 7) and potassium iodide. The reaction mixture was incubated for 5 minutes, and absorbance was measured at 390 nm. H 2 O 2 concentration was expressed as µmol/g fresh weight 29 . Quantification of Proline, Total soluble sugars, Total Soluble Proteins, Total Phenolic and Malondialdehyde Contents For proline estimation, leaf samples were homogenized in 3% sulfosalicylic acid and a reaction mixture of proline, glacial acetic acid, and acidic ninhydrin (1:1:1 ratio) was prepared. After heating at 100°C for one hour, the mixture was cooled in an ice bath and extracted with toluene. The optical density of the upper toluene phase was measured at 520 nm 30 . To quantify total soluble sugars, 100 mg of leaf sample was hydrolyzed with 5 mL of 2.5 N HCl at 100°C for three hours. After neutralization and centrifugation, 0.5 µL of the supernatant was treated with Anthrone reagent and heated and absorbance was measured at 630 nm 31 . Total soluble proteins were quantified using the Bradford method 32 (Bradford, 1976). Leaf enzyme extract (40 µL) was prepared in potassium phosphate buffer (pH 5), vortexed (V-3, ELMI), and centrifuged. Bradford reagent (160 µL) was added to the supernatant and incubated for 5 minutes. Absorbance was measured at 595 nm using a spectrophotometer (Hitachi U-2001, model 121-003). For total phenolic contents determination, 1 g of leaf sample was extracted, centrifuged, and the supernatant was mixed with F-C reagent and Na 2 CO 3 . After one hour, absorbance was recorded at 765 nm 28 . MDA content was estimated by grinding 0.5 g of leaf with liquid nitrogen and extracting with 1% TCA. After centrifugation, the supernatant was mixed with TBA, heated at 95°C for 30 minutes, and absorbance was measured at 532 nm 33 . Impact of biogenic ZnONPs on fruit quality and productivity parameters of kinnow trees The field experiment was laid out in an RCBD, with foliar applications of biogenic ZnONPs at 0.25%,0.50% and 0.75% applied thrice from March to November. Kinnow fruit quality and productivity parameters (Juice content, juice pH, vitamin C, fruit weight, fruit diameters, peel thickness and total yield) were assessed. Juice content (%) was determined from 10 fruits/tree via formula: (Juice weight ÷ Fruit weight) × 100 34 . Kinnow Juice pH was measured with a calibrated pH meter and vitamin C was estimated using a spectrophotometer (Hitachi U-2001, model 121-003) 35 34 . Average fruit diameters, fruit weight and peel thickness were calculated from 10 collected fruits/tree and yield was expressed as kg/tree 34 . Data Analysis Data from greenhouse and field experiments were analyzed by using CRD and RCBD, respectively. The statistical analysis was performed via PROC MIXED procedure in the Statistical Analysis System (SAS) (Version 9.4). Results Characterization of Bacillus subtilis -based nanoparticles Optical and surface characterization of ZnONPs and CuONPs UV–Visible spectroscopy verified the successful biosynthesis of nanoparticles with ZnONPs exhibiting a characteristic surface plasmon resonance peak at 380 nm and an optical density of 1.12 (Fig. 1 A). CuONPs displayed a peak at 280 nm with an optical density of 0.99 (Fig. 1 B). Particle size analysis revealed that ZnONPs had a distinct peak at 60 nm, with a distribution range of 55–65 nm (Fig. 1 C), whereas CuONPs displayed a peak at 85 nm, ranging from 80–90 nm (Fig. 1 D). Zeta potential measurements indicated negative surface charges for both nanoparticle types, suggesting good colloidal stability. ZnONPs exhibited a peak zeta potential of − 35 mV (range: − 45 to − 25 mV) (Fig. 1 E), while CuONPs had a peak at − 20 mV (range: − 30 to − 10 mV) (Fig. 1 F). Structural and morphological characterization of ZnONPs and CuONPs XRD analysis confirmed the crystalline nature of both nanoparticles. ZnONPs exhibited characteristic peaks at 31.6°, 34.2°, 34.3°, 47.4°, and 56.4°, corresponding to the (100), (002), (101), (102), and (110) planes of the FCC zinc oxide structure (Fig. 2 A), while CuONPs showed peaks at 32.4°, 38.7°, 48.6°, and 61.6°, indexed to the (110), (111), (200), (202), and (113) planes of FCC copper oxide (Fig. 2 B). FTIR spectra indicated the presence of plant-derived functional groups involved in nanoparticle synthesis. ZnONPs (Fig. 2 C) exhibited peaks at 3386 cm⁻¹ (O–H/N–H), 1648 cm⁻¹ (C = O, amide I), 1539 cm⁻¹ (C = C, N–H, flavonoid-related), 1393 cm⁻¹ (C–O of polyols), and 601 cm⁻¹ (C–H bending or Zn–O interaction), while CuONPs (Fig. 2 D) showed peaks at 3400 cm⁻¹ (O–H/N–H), 1630 cm⁻¹ (C–H stretching), 1410 cm⁻¹ (C = O), 1039 cm⁻¹ (C–H bending), and 1071 cm⁻¹ (C–O), confirming the involvement of phenolics, flavonoids, and proteins. SEM imaging revealed that ZnONPs were Irregular spherical-shaped particles with slight agglomeration, measuring 45–55nm (average 50 nm) (Fig. 2 E), whereas CuONPs appeared as rod-shaped structures with uniform morphology, sized 70–80 nm (average 75 nm) (Fig. 2 F). Management of citrus greening under greenhouse and field conditions using Bacillus subtilis- mediated ZnONPs and CuONPs In greenhouse experiment, ZnONPs CuONPs demonstrated the lowest disease incidence of citrus greening (16.95%), followed by ZnONPs (19.61%) and CuONPs (28.76%) compared to the control (Fig. 3 A). The interaction between treatment and concentration (T×C) revealed that the lowest disease incidence was recorded with ZnONPs CuONPs (20.67%, 17.34%, 12.84%), followed by ZnONPs (22.89%, 20.11%, 15.84%) and CuONPs (33.50%, 28.89%, 23.89%) at 0.25%, 0.50%, and 0.75%, respectively (Fig. 3 B). Similarly, the treatment and time (T×D) interaction showed that ZnONPs + CuONPs exhibited the least disease incidence (22.84%, 15.95%, 12.06%), followed by ZnONPs (26.34%, 19.50%, 13.00%) and CuONPs (34.22%, 28.56%, 23.50%) after 7, 14, and 21 days, respectively (Fig. 3 C). In field conditions, a similar trend was observed. ZnONPs + CuONPs resulted in the lowest disease incidence (25.90%), followed by ZnONPs (29.85%) and CuONPs (32.06%) compared to the control (Fig. 3 D). The T×C interaction revealed reduced disease incidence with ZnONPs + CuONPs (29.59%, 26.31%, 21.81%), followed by ZnONPs (33.59%, 30.26%, 25.70%) and CuONPs (35.81%, 33.03%, 27.33%) across increasing concentrations (Fig. 3 E). Similarly, the T×D interaction confirmed that ZnONPs + CuONPs (31.87%, 24.81%, 21.03%) outperformed ZnONPs (35.76%, 28.81%, 24.98%) and CuONPs (39.26%, 32.42%, 24.50%) after 7, 14, and 21 days, respectively (Fig. 3 F). ZnONPs and CuONPs on the amount of biochemicals under HLB-induced stress Biochemical assay revealed that ZnONPs showed the highest SOD level (5.30 µg/g FW), followed by CuONPs (4.18 µg/g FW), whereas the control exhibited the lowest level (Fig. 4 ). A consistent increase in SOD was observed with rising concentrations. At 0.25%, SOD levels were 4.48 (ZnONPs), 3.83 (CuONPs), and 2.59 (control); at 0.50%, 5.35, 4.14, and 2.69; and at 0.75%, 6.06, 4.55, and 2.87, respectively (Fig. 5 ). POD level followed a similar trend. ZnONPs exhibited the highest POD (5.31 µg/g FW), followed by CuONPs (4.14 µg/g FW), and the control (2.38 µg/g FW) (Fig. 4 ). Increasing concentrations led to enhanced POD levels: at 0.25%, values were 4.62, 3.85, and 2.38; at 0.50%, 5.33, 4.06, and 2.44; and at 0.75%, 5.98, 4.50, and 2.60 (Fig. 5 ). CAT level was also significantly enhanced under ZnONPs (4.36 µg/g FW) compared to CuONPs (3.16 µg/g FW) and the control (2.53 µg/g FW) (Fig. 4 ). At 0.25%, CAT values were 3.70, 2.80, and 2.53; at 0.50%, 4.32, 3.12, and 2.56; and at 0.75%, 5.04, 3.56, and 2.76 (Fig. 5 ). TSS increased across concentrations: 4.72, 3.96, and 3.62 at 0.25%; 5.44, 4.23, and 3.74 at 0.50%; and 6.15, 4.54, and 3.85 at 0.75% (Fig. 5 ). TSP content was highest under ZnONPs (5.16 µg/g FW), followed by CuONPs (3.96 µg/g FW) and control (3.44 µg/g FW) (Fig. 4 ). TSP increased with concentration: 4.46, 3.72, and 3.37 at 0.25%; 5.16, 3.86, and 2.44 at 0.50%; and 5.85, 4.31, and 3.55 at 0.75% (Fig. 5 ). ZnONPs application also enhanced TPC levels (4.70 µg/g FW), compared to CuONPs (3.57 µg/g FW) and control (2.89 µg/g FW) (Fig. 4 ). At 0.25%, TPC values were 4.03, 3.27, and 2.81; at 0.50%, 4.69, 3.52, and 2.88; and at 0.75%, 5.38, 3.93, and 2.96 (Fig. 5 ). ZnONPs-treated plants also showed the highest H₂O₂ accumulation (4.02 µg/g FW), followed by CuONPs (2.87 µg/g FW) and control (1.99 µg/g FW) (Fig. 4 ). Concentration-wise responses were as follows: at 0.25%, 3.33, 2.59, and 1.90; at 0.50%, 4.01, 2.81, and 1.97; and at 0.75%, 4.73, 3.22, and 2.09 (Fig. 5 ). ZnONPs also resulted in the highest TSS content (5.44 µg/g FW), compared to CuONPs (4.24 µg/g FW) and the control (3.73 µg/g FW) (Fig. 4 ). Proline content was also level under ZnONPs (4.27 µmol/g FW), followed by CuONPs (3.13µmol/g FW) and control (2.47 µmol/g FW) (Fig. 4 ). Proline levels increased with concentrations: at 0.25%, 3.54, 2.80, and 2.43; at 0.50%, 4.28, 3.07, and 2.44; and at 0.75%, 4.99, 3.52, and 2.55 (Fig. 5 ). MDA content peaked in ZnONPs (4.70 µmol/g FW), followed by CuONPs (3.50 µmol/g FW) and control (2.79 µmol/g FW) (Fig. 4 ). Values increased with concentration: 3.98, 3.22, and 2.68 at 0.25%; 4.68, 3.46, and 2.79 at 0.50%; and 5.43, 3.81, and 2.90 at 0.75% (Fig. 5 ). Impact of Bacillus subtilis- based ZnONPs on horticultural attributes of Kinnow ZnONPs significantly improved juice pH, juice content, vitamin C, fruit weight, diameter, peel thickness, and yield per tree compared to the control (Fig. 6 ). At 0.25%, values were: juice pH (2.50), juice content (35.59%), vitamin C (9.97 mg/100 mL), fruit weight (198.53 g), diameter (70.29 mm), peel thickness (5.19 mm), and yield (246.93 kg). At 0.50%: pH (3.00), juice content (42.56%), vitamin C (12.93 mg/100 mL), weight (203.70 g), diameter (71.29 mm), peel thickness (7.69 mm), yield (253.03 kg). Maximum values were achieved at 0.75%: juice pH (3.50), juice content (46.46%), vitamin C (16.03 mg/100 mL), weight (213.53 g), diameter (72.29 mm), peel thickness (9.29 mm), and yield (258.03 kg), as shown in Fig. 7 . Discussion The beneficial role of bacteria in nanoparticle synthesis has been extensively explored due to their ability to produce stable and functional nanomaterials through biological reduction 36 . In the present work, successful biosynthesis of copper oxide and zinc oxide nanoparticles was initially confirmed via UV–visible spectroscopy, displaying characteristic absorption peaks for CuONPs and ZnONPs. Zeta analysis showed a narrow size distribution, and zeta potential measurements indicated a negative surface charge, reflecting good electrostatic stability. X-ray diffraction confirmed the crystalline nature of the particles, with CuONPs exhibiting a monoclinic phase and ZnONPs a hexagonal wurtzite structure. Scanning electron microscopy revealed that CuONPs were slightly irregular spherical to oval, whereas ZnONPs were more uniformly spherical; minor aggregation occurred due to biomolecules from bacterial synthesis 37 . Greenhouse and field experiments evaluated the efficacy of these nanoparticles against citrus greening caused by Candidatus Liberibacter . In greenhouse trials (0.25%, 0.50% and 0.75%), ZnONPs, CuONPs, and their combination significantly reduced disease incidence compared with untreated controls, with the combination showing the greatest effect. Field trials showed the same general trends as the greenhouse experiments, although the level of disease suppression was lower. This reduction was mainly linked to fluctuating temperatures, rainfall, differences in soil composition and the natural competition from other microbes. In contrast, the superior results observed in greenhouse conditions were largely due to the controlled environment, which minimized UV breakdown of nanoparticles, leaching, and excessive humidity factors that allowed the nanoparticles to stay in contact with the pathogen for longer 38 . This is in agreement with prior studies showing that metallic NPs possess strong antimicrobial activity against a wide range of plant pathogens such as Fusarium, Alternaria , Penicillium , Phoma and Rhizoctonia species 39 40 . NPs efficacy is directly linked to their large surface area and nanoscale reactivity, which enable close contact with bacterial cells, to penetrate cell walls, disrupt membrane structure, alter permeability and release reactive metal ions that bind to proteins and nucleic acids 41 . From nanoparticle surfaces ions such as Cu²⁺ and Zn²⁺ release inside plant tissues, along with the local surface plasmon resonance effect which lead to the generation of reactive oxygen species 42 . These ROS (H₂O₂, O₂⁻ and ˙OH) damage DNA, proteins and lipids, ultimately leading to oxidative stress and microbial cell death 43 . An additional copper oxide layer forms on microbial cell walls in the case of CuONPs, further weakening membrane integrity and interfering with vital enzymatic pathways 44 45 . Integrated application of ZnONPs and CuONPs exerted synergistic effect which boosted ROS production and metal ion release, leading to more severe disruption of Candidatus Liberibacter cellular functions, both in greenhouse and in field conditions, as indicated by 46 . It is scientifically proven that plants respond to biotic stress through complex defense mechanisms involving enzymatic and non-enzymatic metabolites that mitigate oxidative damage and maintain cellular homeostasis 47 . Nanoparticles, due to their nanoscale size, surface activity and ability to penetrate plant tissues, have emerged as effective tools for modulating plant defense responses 48 . Biofabricated NPs, particularly zinc and copper, regulated reactive oxygen species levels and activated antioxidant defense responses 49 . In the present work, biosynthesized zinc oxide and copper nanoparticles were tested individually at 0.25%, 0.50% and 0.75% under greenhouse and field conditions for their impact on kinnow biochemistry under Candidatus Liberibacter -induced stress. Both NPs significantly upregulated defense-related enzyme activities and elevated the concentrations of hydrogen peroxide, malondialdehyde, proline, total soluble proteins, total phenolics and total soluble sugars in infected plants compared to control. Comparatively, ZnONPs consistently produced stronger responses than CuONPs across all biochemical markers. The elevation of H₂O₂ and MDA levels reflects a pronounced oxidative burst and lipid peroxidation, while higher proline levels indicate improved osmotic adjustment and stress signaling. This superior biological function of ZnONPs may be attributed to zinc’s critical role as a structural and catalytic cofactor in antioxidant enzymes, its contribution to redox homeostasis and the smaller particle size and higher bioavailability of ZnONPs, which facilitate efficient uptake and priming of plant defense pathways. Comparatively, ZnONPs proved more efficacious than CuONPs in boosting stress-responsive pathways, indicating their pivotal role in HLB mitigation, as evidenced by 50 , where ZnONPs upmodulated antioxidant responses under biotic stress. Conclusion The combined application of ZnONPs and CuONPs resulted in superior disease suppression under both greenhouse and field conditions. Both nanoparticles significantly influenced the plant's biochemical responses under stress, with ZnONPs showing a comparatively greater impact. Furthermore, ZnONPs alone markedly improved key fruit quality and productivity parameters. The biosynthesized ZnONPs and CuONPs are recommended for citrus greening management and overall crop performance improvement. Declarations Declaration of planting materials We authors declared that all plant-related procedures were carried out in accordance with relevant institutional, national, and international guidelines and regulations. Acknowledgement: We authors are thankful to Scientists of Plant Bacteriology laboratory for the successful completion of this research Funding We thank United Arab Emirates University for providing a postdoctoral grant on climate action to (Qurban Ali-#12S140). Data availability The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request. Declaration of generative AI and AI-assisted technologies in the writing process During the preparation of this work the author(s) used ChatGPT in order to improve language clarity and grammar of the manuscript. 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15:51:01","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":42699,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7379214/v1/c381498f0692fd1cb3203138.png"},{"id":95277254,"identity":"111e706a-0512-45bc-8629-5cbd9fe6d92f","added_by":"auto","created_at":"2025-11-06 08:35:48","extension":"png","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":33981,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-7379214/v1/17b498f0df08770a5f5d9ff8.png"},{"id":95313690,"identity":"af2cf120-4833-4af0-93d0-9664160329e5","added_by":"auto","created_at":"2025-11-06 15:51:52","extension":"xml","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":127018,"visible":true,"origin":"","legend":"","description":"","filename":"5d6b0611435e4c4ab7a04b72e5acdaf01structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7379214/v1/805cbea908c3e21186cd7caf.xml"},{"id":95277247,"identity":"3697aea6-74db-486c-bc71-2d7a34f40b75","added_by":"auto","created_at":"2025-11-06 08:35:48","extension":"html","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":139394,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7379214/v1/460dd953cd5df8c2cc62db1a.html"},{"id":95277224,"identity":"8f952601-5274-4032-9072-79dfc8b8c74d","added_by":"auto","created_at":"2025-11-06 08:35:47","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":100646,"visible":true,"origin":"","legend":"\u003cp\u003eOptical and surface characterization of \u003cem\u003eBacillus subtilis\u003c/em\u003e-synthesized ZnONPs and CuONPs: UV–Vis spectra (A, B), particle size distribution (C, D), and zeta potential analysis (E, F).\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7379214/v1/ccdf28b14dd51164bab2543f.jpg"},{"id":95277232,"identity":"1880f961-d4ab-401d-bcec-d9069edc3d5d","added_by":"auto","created_at":"2025-11-06 08:35:47","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":146901,"visible":true,"origin":"","legend":"\u003cp\u003eStructural and morphological characterization of ZnONPs and CuONPs: XRD patterns confirming crystallinity (A, B), FTIR spectra showing functional groups (C, D), and SEM images depicting particle morphology (E, F).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7379214/v1/43928579bf5fc849ac237e58.jpg"},{"id":95313181,"identity":"de592f42-33b2-40a1-b9af-9e7552274503","added_by":"auto","created_at":"2025-11-06 15:51:02","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":116981,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eImpact of Bacillus subtillus-based nanoparticles on citrus greening management under Greenhouse (A–C) and Field (D–F) conditions.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7379214/v1/7a2ec512bba71e3aeff282de.jpg"},{"id":95277222,"identity":"7260dcbc-4fb9-4b5b-b77d-9a178b89b8af","added_by":"auto","created_at":"2025-11-06 08:35:47","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":132476,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of \u003cem\u003eBacillus subtilis-\u003c/em\u003ebased NPs on amount of biochemicals in treated and untreated kinnow leaves under HLB-induced stress\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7379214/v1/7fc0cae23a59e5b980e38e66.jpg"},{"id":95313457,"identity":"9ffa6316-9276-47c6-8435-fe97013b935e","added_by":"auto","created_at":"2025-11-06 15:51:27","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":142876,"visible":true,"origin":"","legend":"\u003cp\u003eDose-dependent Impact of \u003cem\u003eBacillus subtilis-\u003c/em\u003ebased NPs on amount of biochemicals in treated and untreated kinnow leaves under HLB-induced stress\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7379214/v1/2adf1c19d2298194b5725339.jpg"},{"id":95277225,"identity":"975d9a55-b383-4927-849c-b7978a5e2f63","added_by":"auto","created_at":"2025-11-06 08:35:47","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":103709,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of \u003cem\u003eBacillus subtilis-based\u003c/em\u003e ZnONPs on horticultural attributes of Kinnow\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7379214/v1/dd2b873e84ad96fbaa602d42.jpg"},{"id":95277229,"identity":"083d3d01-1d7a-4f5a-a17e-f81731d45741","added_by":"auto","created_at":"2025-11-06 08:35:47","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":116993,"visible":true,"origin":"","legend":"\u003cp\u003eDose-dependent impact of \u003cem\u003eBacillus subtilis-\u003c/em\u003emediated ZnONPs on fruit quality and productivity parameters in kinnow trees under biotic stress\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7379214/v1/ecf6dbfde4fc47361f526445.jpg"},{"id":95313897,"identity":"bed4105e-5a40-47b5-ae63-2dfbb4d3101d","added_by":"auto","created_at":"2025-11-06 15:52:12","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":115520,"visible":true,"origin":"","legend":"\u003cp\u003eGeneralized antibacterial mode of action of ZnONPs via cellular penetration, biomolecular disruption, and reactive oxygen species (ROS) generation.\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7379214/v1/bfa01f6094774e997d0db07b.jpg"},{"id":95313450,"identity":"14be9b51-fc29-49c2-af4e-a3135970bc96","added_by":"auto","created_at":"2025-11-06 15:51:27","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":92901,"visible":true,"origin":"","legend":"\u003cp\u003eGeneralized antibacterial mode of action of CuONPs via cellular penetration, biomolecular disruption, and reactive oxygen species (ROS) generation.\u003c/p\u003e","description":"","filename":"9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7379214/v1/2a8364befa616cd602763195.jpg"},{"id":96903649,"identity":"5f806d34-a30e-46dc-b599-a4de980134cf","added_by":"auto","created_at":"2025-11-27 11:53:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2292967,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7379214/v1/ef4ecad6-9518-44e0-8221-6f6525aca478.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Multimodal Impact of Bacillus subtilis-derived Zinc oxide and Copper Oxide Nanoparticles on Citrus Biochemistry, Horticultural Attributes and Greening Disease Management","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCitrus is well known for its rich profile of bioactive compounds, mainly grown in tropical and subtropical regions and belongs to the Rutaceae family \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. This fruit is popular for its refreshing taste, a natural source of valuable metabolites with both nutritional and medicinal benefits \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. This fruit crop is highly vulnerable to many diseases, but among these, huanglongbing has emerged as one of the most devastating, as it drastically reduces yields and shortens the life span of citrus trees \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. HLB is caused by \u003cem\u003eCandidatus Liberibacter\u003c/em\u003e, a gram-negative bacterium that colonizes the phloem of infected trees and it spreads primarily through contaminated grafting material and by insect vectors, most notably the Asian citrus psyllid (\u003cem\u003eDiaphorina citri\u003c/em\u003e), which picks up the bacterium during phloem feeding and then transfers it to healthy trees as it continues feeding \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Although there is no evidence of seed transmission, grafting and psyllids are efficient routes for the pathogen. In Asia, \u003cem\u003eDiaphorina citri\u003c/em\u003e is the major vector, while in some other parts of the world, \u003cem\u003eTrioza erytreae\u003c/em\u003e also plays a role in its spread \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Environmental conditions, particularly warm and humid weather with temperatures between 16\u0026deg;C and 33\u0026deg;C, supports the development of the disease, which is characterized by blotchy vein yellowing, small and misshapen fruits with uneven coloring, thick rinds, excessive acidity, low sugar content, a bitter taste and premature fruit drop, collectively resulting in severe economic losses \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Traditionally, farmers have relied on chemical pesticides and antibiotics because they work quickly and are relatively inexpensive, but their overuse led to environmental pollution, risks to human health, damage to crops and the emergence of resistant strains of pathogens \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. These drawbacks have prompted researchers to explore safer and more sustainable solutions, such as biological control using beneficial bacteria like \u003cem\u003eLysobacter antibioticus\u003c/em\u003e, \u003cem\u003ePseudomonas\u003c/em\u003e, and \u003cem\u003eBacillus\u003c/em\u003e species \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. In recent decades, nanotechnology has gained significant weight in the field of plant protection \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Nanoparticles, due to their small size ranges between 1\u0026ndash;100 nm have unique physical properties like a large surface area to volume ratio which makes them highly reactive and ensure bioavailability of essential nutrients to plant system with enhanced efficacy to combat disease \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. It has been explored that these physical properties of NPs also facilitate deep penetration into plant tissue, perform efficiently at lower doses, which makes them a cost-effective alternative option to conventional chemotherapy \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Multiple approaches (like physical, chemical and biological) are being used for the synthesis of NPs, among these, the chemical method is common, relies on hazardous reagents and generates toxic waste, posing risks to human health and the environment \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. On the other hand, physical methods require expensive equipment and a lot of energy to carry out the synthetic process \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, conversely, biological synthesis offers a safer and eco-friendly route due to the usage of bioresources (like bacteria, fungi, algae and yeast) for biosynthesis of nanoparticles \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Microbial synthesis is a promising avenue because the bioactive metabolites of microorganisms act as natural reducing and stabilizing agents, eliminating the need for toxic reagents and precursors commonly used in traditional routes \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Biogenic approach tends to produce more stable, biodegradable and less toxic NPs, with minimal impact on the environment and non-target microflora \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eMany prior studies have witnessed that nanoparticles activate the host antioxidant defense responses by triggering systemic acquired resistance against a variety of phytopathogens \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Among several microbial resources investigated, \u003cem\u003eBacillus subtilis\u003c/em\u003e stands out as the most potent bio-reducing agent for bio fabrication of NPs \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. So, a recent research effort was made to explore the impact of \u003cem\u003ea\u003c/em\u003e microbial nanofactory, \u003cem\u003eBacillus subtilis-derived\u003c/em\u003e Zinc oxide and copper oxide nanoparticles, on the biochemistry and horticultural attributes of kinnow for the management of citrus greening disease.\u003c/p\u003e"},{"header":"Methodology","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eIodine Kit Test to Pre-Screen Symptomatic Samples\u003c/h2\u003e\u003cp\u003eCitrus greening disease was diagnosed using the indirect iodine test described by \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e based on the presence of characteristic splotchy leaf mottling. Symptomatic and asymptomatic leaves were collected and processed at the Molecular Phytobacteriology Laboratory, University of Agriculture, Faisalabad. For this purpose, leaf tissue (3 g) was homogenized with 3 mL of distilled water, the extract was filtered into a sachet, and iodine-litmus paper, followed by iodine solution, was applied. A blue or purple color change indicated infection, whereas the absence of color change confirmed healthy samples.\u003c/p\u003e\u003cp\u003e\u003cb\u003eBacillus subtilis\u003c/b\u003e\u003cb\u003e-based Synthesis of ZnONPs and CuONPs\u003c/b\u003e\u003c/p\u003e\u003cp\u003eZinc oxide and copper oxide nanoparticles were synthesized following the protocol of \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e with minor modifications. \u003cem\u003eBacillus subtilis\u003c/em\u003e, obtained from the Institute of Soil and Environmental Sciences, University of Agriculture Faisalabad, was suspended aerobically in nitrate medium and incubated on a rotary shaker at 120 rpm for 24 h at 28\u0026deg;C. After incubation, the culture was centrifuged at 6,000 rpm for 10 min at 6\u0026deg;C to collect the cell-free supernatant. The supernatant was inoculated into 250 mL Erlenmeyer flasks containing 100 mL of sterile nutrient broth and incubated on a rotary shaker at 200 rpm for 48 h at room temperature. The culture was then centrifuged at 12,000 rpm for 10 min to separate the biomass, which was resuspended in 100 mL of 1 mM zinc nitrate (Zn(NO₃)₂) or 1 mM copper sulfate (CuSO₄) aqueous solution in 250 mL flasks for ZnO and CuO nanoparticle synthesis, respectively. The reaction mixtures were incubated on a rotary shaker (200 rpm) at room temperature for 24 h in light to facilitate the reduction of metal ions to nanoparticles. Heat-killed bacterial samples with metal salts served as controls. The synthesized nanoparticles were characterized using UV\u0026ndash;visible spectroscopy (UV-Vis), Fourier-transform infrared spectroscopy (FTIR), particle size analysis (PSA), zeta potential, and scanning electron microscopy (SEM).\u003c/p\u003e\u003cp\u003e\u003cb\u003eEvaluation of\u003c/b\u003e \u003cb\u003eBacillus subtilis\u003c/b\u003e\u003cb\u003e-derived ZnONPs and CuONPs against citrus greening disease under Greenhouse conditions\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn the greenhouse trial, antibacterial efficacy of \u003cem\u003eBacillus subtilis\u003c/em\u003e-based zinc oxide and copper oxide nanoparticles individually and in integration was tested against citrus greening disease. One-year-old citrus plants of uniform size and development, previously confirmed to be infected with CGD, were maintained under controlled greenhouse conditions (25\u0026ndash;30\u0026deg;C, 60\u0026ndash;70% relative humidity, 12 h photoperiod supplemented with artificial light when necessary). Treatments consisted of ZnONPs and CuONPs at concentrations of 0.25%, 0.50% and 75%, either alone or combined, along with an untreated control. The experiment was laid out in a Completely Randomized Design with five replicates per treatment. For trunk injection, 100 mL, 75 mL and 50 mL of nanoparticle solution were injected per plant for the 0.25%, 0.50% and 75% concentrations, respectively, using a Cypress Tree Injector (Model CTP-01). Solutions were prepared by dissolving 0.25 g, 0.50 g and 0.75 g of nanoparticles in 100 mL of water to achieve the respective concentrations. Disease severity and physiological responses were assessed at 7, 14 and 21 days after treatment.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAssessment of\u003c/b\u003e \u003cb\u003eBacillus subtilis-mediated\u003c/b\u003e \u003cb\u003eZnONPs and CuONPs against citrus greening disease under field conditions\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA field-scale experiment was performed to investigate the efficacy of \u003cem\u003eBacillus subtilis\u003c/em\u003e-based zinc oxide and copper oxide nanoparticles, applied individually and in combination, against citrus greening disease in \u003cem\u003eCitrus reticulata\u003c/em\u003e (Kinnow). The trial was established in a citrus plantation using 5-year-old trees (2.0\u0026ndash;2.5 m height, 2 m canopy width, 4.5 m spacing) that had been confirmed as infected with citrus greening disease using iodine test kits. Treatments included ZnONPs and CuONPs at 0.25%, 0.50% and 0.75%, either alone or combined, along with an untreated control, arranged in a Randomized Complete Block Design (RCBD) with three replicates per treatment. Trunk injection volumes were adjusted to deliver the required nanoparticle concentrations: 200 mL, 150 mL and 100 mL for 0.25%, 0.50% and 0.75% treatments, respectively. Solutions were prepared by dissolving 0.25 g, 0.50 g and 75 g of nanoparticles in 100 mL of water, resulting in a total dose of 1 g, 1.13 g and 1 g per tree, respectively. Injections were carried out using a Cypress Tree Injector (Model CTP-01) to ensure precise delivery into the vascular system \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Disease incidence and severity were assessed at 7, 14 and 21 days after treatment.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSample Preparation for Biochemical Analysis\u003c/h3\u003e\n\u003cp\u003eLeaves from the Kinnow were collected from nanoparticles (ZnONPs and CuONPs) treated and untreated plants under stress conditions induced by the pathogen. The leaves were cut into small pieces and 0.5 g of the leaf sample was ground in a pestle and mortar with KH₂PO₄ buffer. The samples were centrifuged (Horizon 6 Flex) at 12,000 rpm for 5 minutes and the supernatant was collected for biochemical analysis.\u003c/p\u003e\n\u003ch3\u003eEstimation of superoxide dismutase, peroxidase catalase and hydrogen peroxide\u003c/h3\u003e\n\u003cp\u003eThe levels of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) concentration were estimated in treated and untreated kinnow leaves under HLB stress. For SOD activity, a reaction mixture containing 100 \u0026micro;L enzyme extract, 100 \u0026micro;L Nitroblue Tetrazolium (NBT), 200 \u0026micro;L Triton X-500, 200 \u0026micro;L potassium phosphate buffer (pH 5), 200 \u0026micro;L methionine and 800 \u0026micro;L distilled water was exposed to UV light for 15 minutes to activate riboflavin and generate superoxide anions. After adding 100 \u0026micro;L riboflavin, absorbance was measured at 560 nm \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. POD activity was quantified by using a mixture of 800 \u0026micro;L potassium phosphate buffer (pH 5), 100 \u0026micro;L enzyme extract, 20 mM guaiacol, and 100 \u0026micro;L of 40 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and absorbance was recorded at 470 nm \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e(Hameed \u003cem\u003eet al.\u003c/em\u003e, 2021). For CAT activity, a reaction mixture of 100 \u0026micro;L enzyme extract and 100 \u0026micro;L of 5.9 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was used, and absorbance was measured at 240 nm \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration was determined by grinding 50 mg fresh leaf sample in trichloroacetic acid buffer and centrifuged at 12,000 rpm for 15 minutes at 4\u0026deg;C. The supernatant was mixed with potassium phosphate buffer (pH 7) and potassium iodide. The reaction mixture was incubated for 5 minutes, and absorbance was measured at 390 nm. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration was expressed as \u0026micro;mol/g fresh weight \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eQuantification of Proline, Total soluble sugars, Total Soluble Proteins, Total Phenolic and Malondialdehyde Contents\u003c/h3\u003e\n\u003cp\u003eFor proline estimation, leaf samples were homogenized in 3% sulfosalicylic acid and a reaction mixture of proline, glacial acetic acid, and acidic ninhydrin (1:1:1 ratio) was prepared. After heating at 100\u0026deg;C for one hour, the mixture was cooled in an ice bath and extracted with toluene. The optical density of the upper toluene phase was measured at 520 nm \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. To quantify total soluble sugars, 100 mg of leaf sample was hydrolyzed with 5 mL of 2.5 N HCl at 100\u0026deg;C for three hours. After neutralization and centrifugation, 0.5 \u0026micro;L of the supernatant was treated with Anthrone reagent and heated and absorbance was measured at 630 nm \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Total soluble proteins were quantified using the Bradford method \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e(Bradford, 1976). Leaf enzyme extract (40 \u0026micro;L) was prepared in potassium phosphate buffer (pH 5), vortexed (V-3, ELMI), and centrifuged. Bradford reagent (160 \u0026micro;L) was added to the supernatant and incubated for 5 minutes. Absorbance was measured at 595 nm using a spectrophotometer (Hitachi U-2001, model 121-003). For total phenolic contents determination, 1 g of leaf sample was extracted, centrifuged, and the supernatant was mixed with F-C reagent and Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e. After one hour, absorbance was recorded at 765 nm \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. MDA content was estimated by grinding 0.5 g of leaf with liquid nitrogen and extracting with 1% TCA. After centrifugation, the supernatant was mixed with TBA, heated at 95\u0026deg;C for 30 minutes, and absorbance was measured at 532 nm \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eImpact of biogenic ZnONPs on fruit quality and productivity parameters of kinnow trees\u003c/h3\u003e\n\u003cp\u003eThe field experiment was laid out in an RCBD, with foliar applications of biogenic ZnONPs at 0.25%,0.50% and 0.75% applied thrice from March to November. Kinnow fruit quality and productivity parameters (Juice content, juice pH, vitamin C, fruit weight, fruit diameters, peel thickness and total yield) were assessed. Juice content (%) was determined from 10 fruits/tree via formula: (Juice weight\u0026thinsp;\u0026divide;\u0026thinsp;Fruit weight) \u0026times; 100 \u003csup\u003e34\u003c/sup\u003e. Kinnow Juice pH was measured with a calibrated pH meter and vitamin C was estimated using a spectrophotometer (Hitachi U-2001, model 121-003) \u003csup\u003e35 34\u003c/sup\u003e. Average fruit diameters, fruit weight and peel thickness were calculated from 10 collected fruits/tree and yield was expressed as kg/tree \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eData Analysis\u003c/h2\u003e\u003cp\u003eData from greenhouse and field experiments were analyzed by using CRD and RCBD, respectively. The statistical analysis was performed via PROC MIXED procedure in the Statistical Analysis System (SAS) (Version 9.4).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eCharacterization of\u003c/b\u003e \u003cb\u003eBacillus subtilis\u003c/b\u003e\u003cb\u003e-based nanoparticles\u003c/b\u003e\u003c/p\u003e\n\u003ch3\u003eOptical and surface characterization of ZnONPs and CuONPs\u003c/h3\u003e\n\u003cp\u003eUV\u0026ndash;Visible spectroscopy verified the successful biosynthesis of nanoparticles with ZnONPs exhibiting a characteristic surface plasmon resonance peak at 380 nm and an optical density of 1.12 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). CuONPs displayed a peak at 280 nm with an optical density of 0.99 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Particle size analysis revealed that ZnONPs had a distinct peak at 60 nm, with a distribution range of 55\u0026ndash;65 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), whereas CuONPs displayed a peak at 85 nm, ranging from 80\u0026ndash;90 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Zeta potential measurements indicated negative surface charges for both nanoparticle types, suggesting good colloidal stability. ZnONPs exhibited a peak zeta potential of \u0026minus;\u0026thinsp;35 mV (range: \u0026minus;\u0026thinsp;45 to \u0026minus;\u0026thinsp;25 mV) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), while CuONPs had a peak at \u0026minus;\u0026thinsp;20 mV (range: \u0026minus;\u0026thinsp;30 to \u0026minus;\u0026thinsp;10 mV) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eStructural and morphological characterization of ZnONPs and CuONPs\u003c/h2\u003e\u003cp\u003eXRD analysis confirmed the crystalline nature of both nanoparticles. ZnONPs exhibited characteristic peaks at 31.6\u0026deg;, 34.2\u0026deg;, 34.3\u0026deg;, 47.4\u0026deg;, and 56.4\u0026deg;, corresponding to the (100), (002), (101), (102), and (110) planes of the FCC zinc oxide structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), while CuONPs showed peaks at 32.4\u0026deg;, 38.7\u0026deg;, 48.6\u0026deg;, and 61.6\u0026deg;, indexed to the (110), (111), (200), (202), and (113) planes of FCC copper oxide (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). FTIR spectra indicated the presence of plant-derived functional groups involved in nanoparticle synthesis. ZnONPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) exhibited peaks at 3386 cm⁻\u0026sup1; (O\u0026ndash;H/N\u0026ndash;H), 1648 cm⁻\u0026sup1; (C\u0026thinsp;=\u0026thinsp;O, amide I), 1539 cm⁻\u0026sup1; (C\u0026thinsp;=\u0026thinsp;C, N\u0026ndash;H, flavonoid-related), 1393 cm⁻\u0026sup1; (C\u0026ndash;O of polyols), and 601 cm⁻\u0026sup1; (C\u0026ndash;H bending or Zn\u0026ndash;O interaction), while CuONPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) showed peaks at 3400 cm⁻\u0026sup1; (O\u0026ndash;H/N\u0026ndash;H), 1630 cm⁻\u0026sup1; (C\u0026ndash;H stretching), 1410 cm⁻\u0026sup1; (C\u0026thinsp;=\u0026thinsp;O), 1039 cm⁻\u0026sup1; (C\u0026ndash;H bending), and 1071 cm⁻\u0026sup1; (C\u0026ndash;O), confirming the involvement of phenolics, flavonoids, and proteins. SEM imaging revealed that ZnONPs were Irregular spherical-shaped particles with slight agglomeration, measuring 45\u0026ndash;55nm (average 50 nm) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), whereas CuONPs appeared as rod-shaped structures with uniform morphology, sized 70\u0026ndash;80 nm (average 75 nm) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eManagement of citrus greening under greenhouse and field conditions using\u003c/b\u003e \u003cb\u003eBacillus subtilis-\u003c/b\u003e\u003cb\u003emediated ZnONPs and CuONPs\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn greenhouse experiment, ZnONPs CuONPs demonstrated the lowest disease incidence of citrus greening (16.95%), followed by ZnONPs (19.61%) and CuONPs (28.76%) compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The interaction between treatment and concentration (T\u0026times;C) revealed that the lowest disease incidence was recorded with ZnONPs CuONPs (20.67%, 17.34%, 12.84%), followed by ZnONPs (22.89%, 20.11%, 15.84%) and CuONPs (33.50%, 28.89%, 23.89%) at 0.25%, 0.50%, and 0.75%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Similarly, the treatment and time (T\u0026times;D) interaction showed that ZnONPs\u0026thinsp;+\u0026thinsp;CuONPs exhibited the least disease incidence (22.84%, 15.95%, 12.06%), followed by ZnONPs (26.34%, 19.50%, 13.00%) and CuONPs (34.22%, 28.56%, 23.50%) after 7, 14, and 21 days, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). In field conditions, a similar trend was observed. ZnONPs\u0026thinsp;+\u0026thinsp;CuONPs resulted in the lowest disease incidence (25.90%), followed by ZnONPs (29.85%) and CuONPs (32.06%) compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). The T\u0026times;C interaction revealed reduced disease incidence with ZnONPs\u0026thinsp;+\u0026thinsp;CuONPs (29.59%, 26.31%, 21.81%), followed by ZnONPs (33.59%, 30.26%, 25.70%) and CuONPs (35.81%, 33.03%, 27.33%) across increasing concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Similarly, the T\u0026times;D interaction confirmed that ZnONPs\u0026thinsp;+\u0026thinsp;CuONPs (31.87%, 24.81%, 21.03%) outperformed ZnONPs (35.76%, 28.81%, 24.98%) and CuONPs (39.26%, 32.42%, 24.50%) after 7, 14, and 21 days, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eZnONPs and CuONPs on the amount of biochemicals under HLB-induced stress\u003c/h2\u003e\u003cp\u003eBiochemical assay revealed that ZnONPs showed the highest SOD level (5.30 \u0026micro;g/g FW), followed by CuONPs (4.18 \u0026micro;g/g FW), whereas the control exhibited the lowest level (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). A consistent increase in SOD was observed with rising concentrations. At 0.25%, SOD levels were 4.48 (ZnONPs), 3.83 (CuONPs), and 2.59 (control); at 0.50%, 5.35, 4.14, and 2.69; and at 0.75%, 6.06, 4.55, and 2.87, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). POD level followed a similar trend. ZnONPs exhibited the highest POD (5.31 \u0026micro;g/g FW), followed by CuONPs (4.14 \u0026micro;g/g FW), and the control (2.38 \u0026micro;g/g FW) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Increasing concentrations led to enhanced POD levels: at 0.25%, values were 4.62, 3.85, and 2.38; at 0.50%, 5.33, 4.06, and 2.44; and at 0.75%, 5.98, 4.50, and 2.60 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). CAT level was also significantly enhanced under ZnONPs (4.36 \u0026micro;g/g FW) compared to CuONPs (3.16 \u0026micro;g/g FW) and the control (2.53 \u0026micro;g/g FW) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). At 0.25%, CAT values were 3.70, 2.80, and 2.53; at 0.50%, 4.32, 3.12, and 2.56; and at 0.75%, 5.04, 3.56, and 2.76 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). TSS increased across concentrations: 4.72, 3.96, and 3.62 at 0.25%; 5.44, 4.23, and 3.74 at 0.50%; and 6.15, 4.54, and 3.85 at 0.75% (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). TSP content was highest under ZnONPs (5.16 \u0026micro;g/g FW), followed by CuONPs (3.96 \u0026micro;g/g FW) and control (3.44 \u0026micro;g/g FW) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). TSP increased with concentration: 4.46, 3.72, and 3.37 at 0.25%; 5.16, 3.86, and 2.44 at 0.50%; and 5.85, 4.31, and 3.55 at 0.75% (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). ZnONPs application also enhanced TPC levels (4.70 \u0026micro;g/g FW), compared to CuONPs (3.57 \u0026micro;g/g FW) and control (2.89 \u0026micro;g/g FW) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). At 0.25%, TPC values were 4.03, 3.27, and 2.81; at 0.50%, 4.69, 3.52, and 2.88; and at 0.75%, 5.38, 3.93, and 2.96 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). ZnONPs-treated plants also showed the highest H₂O₂ accumulation (4.02 \u0026micro;g/g FW), followed by CuONPs (2.87 \u0026micro;g/g FW) and control (1.99 \u0026micro;g/g FW) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Concentration-wise responses were as follows: at 0.25%, 3.33, 2.59, and 1.90; at 0.50%, 4.01, 2.81, and 1.97; and at 0.75%, 4.73, 3.22, and 2.09 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). ZnONPs also resulted in the highest TSS content (5.44 \u0026micro;g/g FW), compared to CuONPs (4.24 \u0026micro;g/g FW) and the control (3.73 \u0026micro;g/g FW) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Proline content was also level under ZnONPs (4.27 \u0026micro;mol/g FW), followed by CuONPs (3.13\u0026micro;mol/g FW) and control (2.47 \u0026micro;mol/g FW) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Proline levels increased with concentrations: at 0.25%, 3.54, 2.80, and 2.43; at 0.50%, 4.28, 3.07, and 2.44; and at 0.75%, 4.99, 3.52, and 2.55 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). MDA content peaked in ZnONPs (4.70 \u0026micro;mol/g FW), followed by CuONPs (3.50 \u0026micro;mol/g FW) and control (2.79 \u0026micro;mol/g FW) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Values increased with concentration: 3.98, 3.22, and 2.68 at 0.25%; 4.68, 3.46, and 2.79 at 0.50%; and 5.43, 3.81, and 2.90 at 0.75% (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eImpact of\u003c/b\u003e \u003cb\u003eBacillus subtilis-\u003c/b\u003e\u003cb\u003ebased ZnONPs on horticultural attributes of Kinnow\u003c/b\u003e\u003c/p\u003e\u003cp\u003eZnONPs significantly improved juice pH, juice content, vitamin C, fruit weight, diameter, peel thickness, and yield per tree compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). At 0.25%, values were: juice pH (2.50), juice content (35.59%), vitamin C (9.97 mg/100 mL), fruit weight (198.53 g), diameter (70.29 mm), peel thickness (5.19 mm), and yield (246.93 kg). At 0.50%: pH (3.00), juice content (42.56%), vitamin C (12.93 mg/100 mL), weight (203.70 g), diameter (71.29 mm), peel thickness (7.69 mm), yield (253.03 kg). Maximum values were achieved at 0.75%: juice pH (3.50), juice content (46.46%), vitamin C (16.03 mg/100 mL), weight (213.53 g), diameter (72.29 mm), peel thickness (9.29 mm), and yield (258.03 kg), as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe beneficial role of bacteria in nanoparticle synthesis has been extensively explored due to their ability to produce stable and functional nanomaterials through biological reduction \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. In the present work, successful biosynthesis of copper oxide and zinc oxide nanoparticles was initially confirmed via UV\u0026ndash;visible spectroscopy, displaying characteristic absorption peaks for CuONPs and ZnONPs. Zeta analysis showed a narrow size distribution, and zeta potential measurements indicated a negative surface charge, reflecting good electrostatic stability. X-ray diffraction confirmed the crystalline nature of the particles, with CuONPs exhibiting a monoclinic phase and ZnONPs a hexagonal wurtzite structure. Scanning electron microscopy revealed that CuONPs were slightly irregular spherical to oval, whereas ZnONPs were more uniformly spherical; minor aggregation occurred due to biomolecules from bacterial synthesis \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Greenhouse and field experiments evaluated the efficacy of these nanoparticles against citrus greening caused by \u003cem\u003eCandidatus Liberibacter\u003c/em\u003e. In greenhouse trials (0.25%, 0.50% and 0.75%), ZnONPs, CuONPs, and their combination significantly reduced disease incidence compared with untreated controls, with the combination showing the greatest effect. Field trials showed the same general trends as the greenhouse experiments, although the level of disease suppression was lower. This reduction was mainly linked to fluctuating temperatures, rainfall, differences in soil composition and the natural competition from other microbes. In contrast, the superior results observed in greenhouse conditions were largely due to the controlled environment, which minimized UV breakdown of nanoparticles, leaching, and excessive humidity factors that allowed the nanoparticles to stay in contact with the pathogen for longer \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. This is in agreement with prior studies showing that metallic NPs possess strong antimicrobial activity against a wide range of plant pathogens such as Fusarium, \u003cem\u003eAlternaria\u003c/em\u003e, \u003cem\u003ePenicillium\u003c/em\u003e, \u003cem\u003ePhoma\u003c/em\u003e and \u003cem\u003eRhizoctonia\u003c/em\u003e species \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e39\u003c/span\u003e \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. NPs efficacy is directly linked to their large surface area and nanoscale reactivity, which enable close contact with bacterial cells, to penetrate cell walls, disrupt membrane structure, alter permeability and release reactive metal ions that bind to proteins and nucleic acids \u003csup\u003e41\u003c/sup\u003e. From nanoparticle surfaces ions such as Cu\u0026sup2;⁺ and Zn\u0026sup2;⁺ release inside plant tissues, along with the local surface plasmon resonance effect which lead to the generation of reactive oxygen species \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. These ROS (H₂O₂, O₂⁻ and ˙OH) damage DNA, proteins and lipids, ultimately leading to oxidative stress and microbial cell death \u003csup\u003e43\u003c/sup\u003e. An additional copper oxide layer forms on microbial cell walls in the case of CuONPs, further weakening membrane integrity and interfering with vital enzymatic pathways \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e44\u003c/span\u003e \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Integrated application of ZnONPs and CuONPs exerted synergistic effect which boosted ROS production and metal ion release, leading to more severe disruption of \u003cem\u003eCandidatus Liberibacter\u003c/em\u003e cellular functions, both in greenhouse and in field conditions, as indicated by \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIt is scientifically proven that plants respond to biotic stress through complex defense mechanisms involving enzymatic and non-enzymatic metabolites that mitigate oxidative damage and maintain cellular homeostasis \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Nanoparticles, due to their nanoscale size, surface activity and ability to penetrate plant tissues, have emerged as effective tools for modulating plant defense responses \u003csup\u003e48\u003c/sup\u003e. Biofabricated NPs, particularly zinc and copper, regulated reactive oxygen species levels and activated antioxidant defense responses \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. In the present work, biosynthesized zinc oxide and copper nanoparticles were tested individually at 0.25%, 0.50% and 0.75% under greenhouse and field conditions for their impact on kinnow biochemistry under \u003cem\u003eCandidatus Liberibacter\u003c/em\u003e-induced stress. Both NPs significantly upregulated defense-related enzyme activities and elevated the concentrations of hydrogen peroxide, malondialdehyde, proline, total soluble proteins, total phenolics and total soluble sugars in infected plants compared to control. Comparatively, ZnONPs consistently produced stronger responses than CuONPs across all biochemical markers. The elevation of H₂O₂ and MDA levels reflects a pronounced oxidative burst and lipid peroxidation, while higher proline levels indicate improved osmotic adjustment and stress signaling. This superior biological function of ZnONPs may be attributed to zinc\u0026rsquo;s critical role as a structural and catalytic cofactor in antioxidant enzymes, its contribution to redox homeostasis and the smaller particle size and higher bioavailability of ZnONPs, which facilitate efficient uptake and priming of plant defense pathways. Comparatively, ZnONPs proved more efficacious than CuONPs in boosting stress-responsive pathways, indicating their pivotal role in HLB mitigation, as evidenced by \u003csup\u003e50\u003c/sup\u003e, where ZnONPs upmodulated antioxidant responses under biotic stress.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe combined application of ZnONPs and CuONPs resulted in superior disease suppression under both greenhouse and field conditions. Both nanoparticles significantly influenced the plant's biochemical responses under stress, with ZnONPs showing a comparatively greater impact. Furthermore, ZnONPs alone markedly improved key fruit quality and productivity parameters. The biosynthesized ZnONPs and CuONPs are recommended for citrus greening management and overall crop performance improvement.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDeclaration of planting materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe authors declared that all plant-related procedures were carried out in accordance with relevant institutional, national, and international guidelines and regulations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eWe authors are thankful to Scientists of Plant Bacteriology laboratory for the successful completion of this research\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank United Arab Emirates University for providing a postdoctoral grant on climate action to (Qurban Ali-#12S140).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of generative AI and AI-assisted technologies in the writing process\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring the preparation of this work the author(s) used ChatGPT in order to improve language clarity and grammar of the manuscript. After using this tool/service, the author(s) reviewed and edited the content as needed and take(s) full responsibility for the content of the published article.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAU. conducted research and Wrote intial manuscript.MA, NA, IA conceive idea and supervise research.AA. data analysisQA. Provide funding and edited manuscript.MU, AN. draw figures AH ,AW help and edited manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbobatta, W.F. 2019. Nutritional benefits of citrus fruits. \u003cem\u003eAmerican Journal of Biomedical Science and Research\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e: 303-306.\u003c/li\u003e\n\u003cli\u003eSiddhartha, A., H. Kaur, K. Bains and S. Kaur. 2025. 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Induction of tomato plant biochemical immune responses by the synthesized zinc oxide nanoparticles against wilt-induced \u003cem\u003eFusarium oxysporum\u003c/em\u003e. \u003cem\u003eInternational Microbiology\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e(2):435\u0026ndash;448.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Citrus reticulata, HLB, Candidatus Liberibacter asiaticus, ZnONPs, CuONPs, Bacillus subtilis, biochemical markers","lastPublishedDoi":"10.21203/rs.3.rs-7379214/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7379214/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePresent research work explored the multimodal function of \u003cem\u003eBacillus subtilis\u003c/em\u003e-mediated zinc oxide and copper oxide nanoparticles towards citrus greening disease, with their impact on the kinnow biochemical response. UV-Vis spectroscopy confirmed nanoparticle formation with surface plasmon resonance peaks at 380 nm (ZnONPs) and 280 nm (CuONPs), while particle sizes averaged 60 nm and 85 nm, respectively. Zeta potentials were \u0026minus;\u0026thinsp;35 mV (ZnONPs) and \u0026minus;\u0026thinsp;20 mV (CuONPs), indicating stable colloidal dispersions. XRD analysis confirmed their FCC crystalline nature, and FTIR spectra revealed plant-derived functional groups (O\u0026ndash;H, N\u0026ndash;H, C\u0026thinsp;=\u0026thinsp;O, C\u0026ndash;O) involved in nanoparticle capping. SEM imaging showed irregular spherical ZnONPs (45\u0026ndash;55 nm) and rod-shaped CuONPs (70\u0026ndash;80 nm). Greenhouse assays showed significant disease suppression, with ZnONPs\u0026thinsp;+\u0026thinsp;CuONPs reducing incidence to 16.95%, outperforming ZnONPs (19.61%) and CuONPs (28.76%). Concentration- and time-dependent reductions under ZnONPs\u0026thinsp;+\u0026thinsp;CuONPs reached 12.84% at 0.75% and 12.06% at 21 days. Field trials confirmed similar trends (25.90% incidence under ZnONPs\u0026thinsp;+\u0026thinsp;CuONPs). Biochemically, ZnONPs at 0.75% enhanced SOD (6.06 \u0026micro;g/g FW), POD (5.98), CAT (5.04), TPC (5.38), TSP (5.85), TSS (6.15), proline (4.99 \u0026micro;mol/g FW), H₂O₂ (4.73), and MDA (5.43). Agronomic traits were also improved at 100 mg/L ZnONPs: juice pH (3.50), juice content (46.46%), vitamin C (16.03 mg/100 mL), fruit weight (213.53 g), diameter (72.29 mm), peel thickness (9.29 mm), and yield (258.03 kg/tree). These findings underscore the promise of biogenic ZnONPs and CuONPs as sustainable, multifunctional tools for integrated disease management and crop health improvement.\u003c/p\u003e","manuscriptTitle":"Multimodal Impact of Bacillus subtilis-derived Zinc oxide and Copper Oxide Nanoparticles on Citrus Biochemistry, Horticultural Attributes and Greening Disease Management","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-06 08:35:43","doi":"10.21203/rs.3.rs-7379214/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d9e845d5-472c-4532-988f-d2c2ba0e1258","owner":[],"postedDate":"November 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":57405056,"name":"Biological sciences/Biochemistry"},{"id":57405057,"name":"Biological sciences/Biological techniques"},{"id":57405058,"name":"Biological sciences/Biotechnology"},{"id":57405059,"name":"Earth and environmental sciences/Environmental sciences"},{"id":57405060,"name":"Physical sciences/Nanoscience and technology"},{"id":57405061,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2025-11-27T11:53:25+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-06 08:35:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7379214","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7379214","identity":"rs-7379214","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}