Effect of silver nanoparticles and *Bacillus cereus* LPR2 on the growth of *Zea mays*

The study addresses the need for sustainable strategies to increase crop productivity amid rising global food demand and environmental concerns related to chemical fertilizers. Maize (Zea mays) is a major cereal crop with growing global demand but suffers significant yield losses (approximately 12–40%) due to pests and fungal diseases such as Fusarium graminearum, Stenocarpella maydis, and Macrophomina phaseolina. Plant Growth Promoting Rhizobacteria (PGPR) offer an eco-friendly alternative by enhancing nutrient uptake and producing phytohormones, solubilizing minerals, producing siderophores, and antagonizing phytopathogens. Nanotechnology, particularly silver nanoparticles (AgNPs), has emerged as another eco-friendly tool with antimicrobial properties against plant pathogens and potential to improve plant growth. The research evaluates Bacillus cereus LPR2 (a PGPR isolated from spinach rhizosphere) and phytomediated AgNPs synthesized from Tagetes erecta for their individual and combined effects on maize seed germination and early growth, and for biocontrol against M. phaseolina.

Prior work has established diverse PGPR (e.g., Azotobacter, Azospirillum, Bacillus, Enterobacter, Klebsiella, Pseudomonas, Serratia, Variovorax) as beneficial for plant growth, stress tolerance, and biocontrol via mechanisms such as N2 fixation, phytohormone production (IAA, cytokinin, gibberellins), and mineral solubilization. Bacillus spp. are noted for resilience under adverse conditions. Nanotechnology-derived materials, including AgNPs, exhibit high surface-to-volume ratios and unique properties conducive to agriculture (fabrication, storage, packaging, and controlled nutrient delivery). AgNPs have broad antimicrobial activity and can be synthesized via green (plant-mediated) methods, which are cost-effective and eco-friendly. Interactions between nanoparticles and PGPR can be positive or negative depending on concentrations and contexts. Prior studies reported enhanced plant growth and pathogen suppression with PGPR–nanoparticle combinations (e.g., wheat with TiO2 nanoparticles) and variable effects of AgNPs across crops, sometimes inhibiting PGPR efficacy when combined.

• Isolation and identification of rhizospheric bacteria: Healthy spinach plants were collected from fields in Chunni Kalan (Punjab, India). Rhizospheric soil was serially diluted; 0.1 ml of 10^-1 dilution was spread on Nutrient Agar and incubated at 28 °C for 24–36 h. Distinct colonies were purified and stored at 4 °C. Isolates were preliminarily identified as Bacillus spp. based on morphological and biochemical traits.
• Molecular characterization: Genomic DNA of isolate LPR2 was extracted and its 16S rRNA gene amplified and sequenced. The sequence was submitted to NCBI to obtain an accession number and analyzed via BLAST for taxonomic identification.
• Biosynthesis of AgNPs from Tagetes erecta: Step 1: Prepare water extracts by boiling 100 mg finely cut leaves/flowers in 20 ml double distilled water (DDW) for 5–7 min with stirring. Centrifuge 10 ml of extract at 5000 rpm for 3–4 min to remove debris. Step 2: Prepare 30 mM AgNO3 stock (510 mg in 100 ml DDW); dilute to 3 mM working solution. Step 3: Green synthesis: Mix 10 ml plant extract with 90 ml of 3 mM AgNO3 under constant stirring. Observe color change to reddish-brown within 25–30 min indicating AgNP formation. Incubate 24 h at room temperature. Step 4: Isolation: Centrifuge at 20,000 rpm for 20 min; discard supernatant. Repeat thrice. Resuspend pellet in 5 ml DDW.
• Characterization of AgNPs: UV-Vis spectroscopy: Record spectra from 360–660 nm to detect surface plasmon resonance. HRTEM and EDX: Using FEI TECNAI G2 F20 (200 keV), obtain images to assess particle size/shape; perform EDX to confirm elemental composition.
• AgNP preparation for treatments: Prepare 200 ppm AgNP solution by diluting stock; further dilute to 25 ppm for 3 h seed soak. Apply 50 ppm AgNP foliar spray 6 days after sowing.
• PGPR attribute assays: IAA production: Grow culture in nutrient broth with 0.1 g/l tryptophan; after centrifugation, mix supernatant with O-phosphoric acid and Salkowski’s reagent; pink color indicates IAA. Phosphate solubilization: Spot isolates on Pikovskaya’s agar; incubate at 30 °C for 2 days; halo zone indicates solubilization. HCN production: Grow on glycine-supplemented agar with picric acid/Na2CO3-soaked filter paper lid; incubate 30 °C for 48–72 h; yellow-to-brown color change indicates HCN. Ammonia production: Inoculate peptone water; incubate 30 °C for 72 h; add Nessler’s reagent; yellow-brown precipitate indicates ammonia.
• Antagonistic activity against Macrophomina phaseolina: Dual culture assay on agar; place 5 mm pathogen plug in center; spot bacterial isolate 2 cm away. Incubate at 28 °C for 3–7 days. Calculate inhibition zone (%) = [(C – T)/C] × 100, where C = pathogen radial growth in control, T = growth in test.
• Seed bacterization: Surface-sterilize maize seeds (NaOCl 2–3%, ethanol 70%), rinse thoroughly. Grow B. cereus LPR2 in nutrient broth (48 h, 28 °C, shaker). Prepare 1% CMC slurry mixed with bacterial culture to coat seeds for 1 h; treatments included LPR2 alone, AgNPs (25 ppm soak 3 h; 50 ppm spray at day 6), and LPR2 + AgNPs.
• Pot assay: Sterilize garden soil with formalin; fill pots; sow 4 seeds per pot. Maintain moisture with sterile water. Uproot plants after 10 days to measure seed germination, shoot/root length, and fresh/dry weights of shoots and roots. Treatments: T1 LPR2; T2 AgNPs; T3 LPR2 + AgNPs; T4 control (1% CMC only).
• Statistics: Data analyzed by ANOVA; significance assessed at CD 1% and 5%.

• Identification: Among five isolates (LPR1–LPR5), LPR2 exhibited superior PGPR traits (IAA, HCN, ammonia production, phosphate solubilization). 16S rRNA sequencing identified LPR2 as Bacillus cereus LPR2 (NCBI accession MH997647.1).
• AgNP characterization: UV-Vis spectra showed surface plasmon resonance near 420 nm, indicative of AgNP formation. HRTEM revealed predominantly oval particles ranging from 20–60 nm (average approximately 60 nm). EDX confirmed silver as the constituent element.
• Antagonism: LPR2 demonstrated the strongest antifungal activity against Macrophomina phaseolina, with a mean inhibition zone of 16.2 mm and 72% growth inhibition after 7 days, outperforming LPR3 (14.6 mm) and LPR5 (11.7 mm).
• Pot assay (10 days post-sowing): Seed germination: LPR2 = 87.5%; AgNPs = 87.5%; LPR2 + AgNPs = 75%; Control = 50%. Growth metrics (mean values; significance vs control): • Root length (cm): LPR2 12.567 (p≤0.01); AgNPs 10.767 (p≤0.01); LPR2+AgNPs 10.367 (p≤0.01); Control 8.901. • Shoot length (cm): LPR2 12.500 (p≤0.01); AgNPs 10.066 (p≤0.01); LPR2+AgNPs 9.133 (ns); Control 8.500. • Root fresh/dry weight (g): LPR2 1.1967* / 0.257**; AgNPs 1.067** / 0.167 (ns); LPR2+AgNPs 0.783 (ns) / 0.113 (ns); Control 0.623 / 0.085. • Shoot fresh/dry weight (g): LPR2 0.647** / 0.140**; AgNPs 0.520 (ns) / 0.090**; LPR2+AgNPs 0.460 (ns) / 0.073 (ns); Control 0.413 / 0.050.
• Overall, LPR2 alone yielded the greatest enhancement of germination and early growth, followed by AgNPs alone; the combination LPR2 + AgNPs did not outperform individual treatments.

The findings support the hypothesis that both PGPR (Bacillus cereus LPR2) and phytomediated AgNPs can individually promote maize seed germination and early vegetative growth, while LPR2 also provides strong biocontrol against M. phaseolina. The lack of synergistic effect in the combined LPR2 + AgNPs treatment aligns with reports that AgNPs can sometimes inhibit PGPR activity or exert variable effects depending on concentration, plant species, and context. Mechanistically, AgNPs may enhance germination by penetrating the seed coat and improving water uptake, while LPR2 promotes growth via phytohormone production (IAA), nutrient solubilization (phosphate), and antimicrobial metabolites (e.g., HCN), contributing to pathogen suppression. The results underscore the potential of deploying LPR2 as a biofertilizer/biocontrol agent and AgNPs as a growth stimulator, but also highlight the need to carefully manage nanoparticle–microbe interactions to avoid antagonism.

Bacillus cereus LPR2 exhibited multiple plant growth-promoting traits (IAA, HCN, ammonia production; phosphate solubilization) and strong antagonism against Macrophomina phaseolina. Green-synthesized AgNPs (20–60 nm) from Tagetes erecta enhanced maize germination and early growth compared to control. In pot assays, LPR2 alone provided the highest improvements in germination and biomass, followed by AgNPs; their combination did not yield additive benefits. These results indicate that B. cereus LPR2 and AgNPs can be applied individually as a bioinoculant and a growth stimulator, respectively, for maize. Future work should optimize dosing, timing, and formulations, evaluate longer growth periods and field conditions, and further investigate nanoparticle–PGPR interactions across crops and pathogens.

• Short experimental duration: growth parameters assessed only up to 10 days after sowing.
• Pot assay under controlled conditions; field variability not assessed.
• Limited replication for biomass measurements (means of 3 plants per set reported).
• Single PGPR strain (B. cereus LPR2) and single plant species (maize) examined.
• AgNPs evaluated at limited dosages (25 ppm seed soak; 50 ppm foliar spray) without a broader dose–response.
• Antagonistic testing performed against a single pathogen (Macrophomina phaseolina).