Evaluation of Plant Growth Promoting Traits of Silicate Solubilizing Bacillus Tequilensis SKSSB09 in Zea mays L.

Maize (Zea mays L.) is a pivotal and extensively cultivated crop globally. In developed nations, it is primarily used as animal feed and raw material for agricultural processing industries.
       
In developing countries, it provides food, assuring nutritional security for humans and livestock (Prasanna et al., 2021; Lelago et al., 2025). Maize tends to show resilience to various adverse weather and agroecological conditions (Semugenze et al., 2024; Bhaumik et al., 2025). With its exceptional significance, maize is widely acclaimed as the crown of cereals. Maize is a complete source of nutrients for starch, calcium, fibre, essential minerals and vitamins (Adinarayana et al., 2024; Rouf Shah et al., 2016.
       
In recent decades, due to adverse abiotic conditions viz. drought, water logging, salinity and heavy metal toxicity, the world has noticed a drastic decline in productivity, threatening the security of food. Among all the abiotic stress, drought created devastating losses leading to sharp decline in the productivity (Sukumar et al., 2023). Silicon is one of the major minerals on the earth which occupies 31% of the soil and absorbed by plants in the form of monosilicic acid. Silicon has determined benefits in securing plants from lethal injuries of biological and environmental stresses (Karuganti et al., 2023).
       
The silicate solubilizing bacteria group is responsible for transforming insoluble silicates into soluble silica, enhancing soil fertility and plant nutrition. These silicate solubilizing bacteria secrete the silicase enzyme and organic acids responsible for silicate breakdown into soluble silica (Cruz et al., 2022; Jain 2025). In this scenario, silicate solubilizing bacteria was much studied due to its ability to enhance drought alleviation in Zea mays L. and other crops. These silicate solubilizing bacteria has the ability to secrete organic acids, ACC deaminase and plant growth-promoting molecules which induce systemic acquired resistance to the plants in fighting against biotic and abiotic stresses.
       
A major auxin family hormone, indole acetic acid (IAA) is produced by plant beneficial microbial inoculants. This IAA regulates the wide range of plant bodily functions including germination, cell division, vegetative growth, secondary metabolite biosynthesis and stress response, highlighting its importance in plant development and adaptation. Some of the silicate solubilizing bacteria like Bacillus tequilensis has multipotential abilities viz. solubilizing the complex silicates, secretion of IAA and showing antagonistic activity against plant pathogens (Sukumar et al., 2023; Karuganti et al., 2023).
       
Globally, plant beneficial microbial inoculants gained significant importance due to their plant growth promoting attributes and the ability to induce resistance in plants. Although individual microbial inoculants are widely available in the global market, their utility is limited as they typically offer only a single benefit. Microbial consortia-based formulations gained importance due to their multiple plant growth promoting abilities (Lugtenberg and Kamilova, 2009).
       
This research focuses on the plant growth-promoting effects of silicate solubilizing Bacillus tequilensis SKSSB09 in Zea mays L. The study aims to test plant growth promoting efficiency of Bacillus tequilensis individually and in combination with the Paenibacillus azotofixans BFPA09 (nitrogen fixing bacteria), Bacillus megaterium KLBM01 (phosphate solubilizing bacteria), Bacillus mucilaginous BFBM06 (potash solubilizing bacteria) and synthetic fertilizers.

Sourcing of microbial cultures
 
The microbial cultures used in this study were sourced from Biofac Inputs Private Limited in Hyderabad and included Bacillus tequilensis SKSSB09 (silicate solubilizing), Paenibacillus azotofixans BFPA09 (nitrogen-fixing), Bacillus megaterium KLBM01 (phosphate solubilizing) and Bacillus mucilaginous BFBM06 (potash solubilizing). These cultures were revived, subcultured on soybean casein digest agar and stored at 4oC for long-term use.
 
Formulation of the microbial consortium
 
For each bacterial culture, 100 mL of soybean casein digest broth was autoclaved and cooled. A loopful of each bacterial culture was inoculated and incubated at 120 rpm for 24 to 48 h on an orbital shaker. Microbial samples were collected and centrifuged using the REMI C-24 BL. The cell pellet was used to determine the total microbial count using the spread plate technique, expressed as CFU/g. 1 g of each cell biomass was suspended in 1 L of double distilled water and mixed homogeneously using the magnetic stirrer (REMI- 5MLH) for 15 min. The microbial cells were encapsulated with 1% of glycerol to increase the viability of the cells. The total microbial count of the formulated microbial consortium was as assessed on N-free malate agar with composition of malic acid (5 g/L), yeast extract (0.1 g/L), potassium hydroxide (4 g/L), KH2PO4 (0.5 g/L), MgSO₄ (0.1 g/L), NaCl (0.02 g/L), FeSO₄ (0.05 g/L), MnSO₄ (0.01 g/L), Na₂MoO₄ (0.002 g/L), bromothymol blue (0.5% alcoholic solution) of 2 mL/L, adjusted to pH 7.0. Pikovask’s agar (composition: glucose 10 g/L, aluminium sulphate 0.5 g/L, NaCl 0.3 g/L, KCl 0.3 g/L, MgSO₄ 0.3 g/L, FeSO₄ 0.03 g/L, MnSO₄ 0.03 g/L, tricalcium phosphate 3 g/L and pH adjusted to 6.8) and Aleksandrov’s agar (composition: MgSO₄ 0.5 g/L, CaCO₃ 0.1 g/L, dextrose 5 g/L, FeCl₃ 0.005 g/L, Ca₃PO₄ 2 g/L and agar 20 g/L) to check the compatibility and presented as CFU/ml (Raju et al., 2020).
 
In-planta experimental design for studying the plant growth attributes
 
Maize seeds (Rasi RMH 3499) were surface sterilized with 0.1% HgCl2 and rinsed with sterile water. The seeds were then sown in seedling trays with double-sterilized coco peat and after 10 days, transplanted into 8 Kg pots with a sterilized pot mix (40% garden soil, 20% coco peat, 20% vermicompost and 20% vermiculite). Treatments were applied based on the experimental design, with four replications per treatment (Table 1). Irrigation was daily and data were collected twice per week. The trial continued until cob harvesting, recording shoot dry weight, shoot length, root dry weight, root length and yield. Dry weights were determined by drying plant parts at 80^oC for 24 h and one-factor ANOVA was used for data analysis (Raju et al., 2020; Karuganti  et al., 2023).


 
Estimation of N, P, K in leaves
 
Older leaves from the base of plants were collected before harvesting (130 days) and dried in a hot air oven at 63^oC for 48 h to ensure complete desiccation. Total nitrogen content was determined using the Kjeldahl apparatus. For phosphorus (P) and potassium (K) analysis, the samples were incinerated at 400^oC for 2 h, then treated with magnesium nitrate and vaporized. After moistening with 1N nitric acid and drying, the ash was dissolved in 0.1N HNO₃ and aliquots were analysed for P and K using a Perkin-Elmer flame photometer (Bennet et al., 1962).
 
Estimation of pigments
 
Pigment extraction was performed on 1 g fresh leaf samples collected before harvesting (130 days), cut into 0.5 cm pieces and immersed in 10 mL of 80% acetone, then incubated at 4^oC for 24 h. Chlorophyll a, chlorophyll b and carotenoid contents were measured spectrophotometrically at 663, 645 and 480 nm using a UV Visible spectro-photometer 117 TS (Saleem et al., 2021).
 

Chlorophylla (mg/g) = (12.7 x Absorbance at 663 - 2.69 x Absorbance at 645] x (Volume of extract in mL/1000) x Fresh weight of the sample

 
 

Chlorophyll b (mg/g) = (22.9 x Absorbance at 645- 4.68 x Absorbance at 663] x (Volume of extract in mL/1000) x Fresh weight of the sample
 

India has experienced a notable increase in maize production, marked by a 35% rise in cultivated area and a 48% improvement in yield. However, productivity faces challenges from abiotic and biotic stresses, worsened by climate change, particularly drought (Yadav et al., 2016; Song et al., 2020). Microbial inoculants, such as Bacillus tequilensis, are essential for sustainable agriculture as they enhance nutrient availability and plant growth. Bacillus tequilensis helps alleviate drought stress by solubilizing silicates and producing indole-3-acetic acid (IAA).
       
This study evaluates Bacillus tequilensis SKSSB09 alongside other microbial inoculants and synthetic fertilizers, utilizing cultures sourced from Biofac Inputs Private Limited in Hyderabad. A pot trial with seven treatments was conducted to assess the effectiveness of different plant growth-promoting rhizobacteria (PGPR) strains and synthetic fertilizers (Table 1).
 
Enhancement of shoot length and dry weight in maize
 
The combined application of NPK, SSB (Bacillus tequilensis, 1.0x10⁸  CFU/mL at 10 mL/L) and microbial consortia (Paenibacillus azotofixans, Bacillus mucilaginosus and Bacillus megaterium, 1.0x10⁸  CFU/mL at 10 mL/L) in T6 treatment significantly enhanced maize shoot length and dry weight. The values recorded before harvesting in T6 recorded the highest shoot length (219.95±3.96 cm), followed by T5 (205.13±2.28 cm), compared to the control (T7, 118.33±1.91 cm) (Fig 1). This improvement was attributed to phytohormone production, including indole acetic acid (IAA), atmospheric nitrogen fixation and phosphorus and potassium solubilization by the PGPR.


       
T6 had the highest shoot dry weight at 89.6±1.37 g, followed by T5 at 84.5 ± 0.98 g, compared to the control at 54.08±1.41 g (Fig 2). This increase in dry weight was linked to greater shoot length and enhanced nutrient availability, phytohormone synthesis and stress tolerance due to plant growth-promoting rhizobacteria (PGPR).

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The PGPR consortia showed greater plant growth-promoting abilities compared to individual treatments. This finding aligns with previous studies that reported improved plant growth from microbial inoculants, such as Azotobacter, Pseudomonas, Azospirillum and Serratia spp. (Gholami et al., 2008; Ali et al., 2017; Akther et al., 2024; Ghosh et al., 2018). Additionally, these results support earlier reports indicating that microbial inoculants, including Sinorhizobium spp., can enhance growth while reducing the need for chemical fertilizers without compromising yield (Zahir et al., 2006).
       
Moreover, silicate-solubilizing bacteria enhanced stress tolerance, including salinity resistance, by improving photosynthetic efficiency (Kubi et al., 2021). This research underscores the potential of microbial inoculants in sustainable agriculture to boost crop productivity while decreasing dependence on chemical fertilizers.
 
Effect of PGPR on root length and dry weight in maize
 
The T6 treatment, which combines NPK, SSB (Bacillus tequilensis) and microbial consortia (including Paenibacillus azotofixans, Bacillus mucilaginosus and Bacillus megaterium), showed the highest root proliferation at 61.55±1.17 cm. This result is significantly better than the control group (T7), which measured 37.4 cm (Fig 3). These findings align with previous research that demonstrated enhanced root lengths with Bacillus species (56.46 cm) and Pseudomonas species (29.33 cm) (Imran et al., 2020; Zahir et al., 2006).

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The increase in root length is due to phytohormones that stimulate cell elongation and division in root tissues, enhancing growth and nutrient uptake (Ahemad et al., 2014; Hayat et al., 2010). PGPR (Plant Growth-Promoting Rhizobacteria) likely improves nutrient cycling, soil structure and microbial communities, promoting root development (Bashan and de-Bashan, 2010).
       
In terms of root dry weight, treatment T6 showed the highest increase (17.05±0.68 g), followed by T5 (16.48±0.58 g), compared to the control (11.8±0.47 g) (Fig 4). This aligns with studies using microbial inoculants like Pseudomonas putida and Acinetobacter spp., which also enhanced root biomass (Kumar et al., 2023). The increased dry weight in T6 is linked to better nutrient acquisition and rhizosphere conditions improved by PGPR (Glick et al., 2014; Berg et al., 2009). The microbial consortia likely optimized soil health, contributing to stronger root growth (Lambers et al., 2009).


 
Effect of PGPR on grain weight in maize
 
T6 exhibited the highest average grain weight of 17.0±0.196 g, followed by T5 at 16.875±0.312 g, while the control (T7) showed a significantly lower weight of 8.725±0.582 g (Fig 5). The increase in grain weight is attributed to the combined effects of Bacillus tequilensis and PGPR on nutrient uptake and stress tolerance (Ojuederie and Babalola, 2023).


       
Bacillus tequilensis enhances silicon availability, improving cell wall strength, drought tolerance and photosynthetic efficiency, all contributing to increased grain weight (Ali et al., 2017). PGPR further boosts growth by enhancing nutrient uptake and producing phytohormones, promoting better grain filling. The dual inoculation of Bacillus tequilensis and PGPR enhances nutrient absorption and stress resilience, leading to increased grain weight, as supported by previous studies (Akther et al., 2024; Babaji et al., 2014).
 
Enhancement of nutrient uptake
 
Analysis of NPK content in pot trials revealed that T6 had the highest nitrogen (1.94%), phosphorus (18.19%) and potash (15.1%) content among all treatments, while the control recorded significantly lower levels (0.86% N, 8.1% P, 7.2% K) (Fig 6).


       
The increased NPK content in T6 is attributed to the synergistic action of PGPR and SSB. PGPR fixes atmospheric nitrogen, while SSB solubilizes silicate minerals, improving soil fertility. This combined effect enhances mineralization in the rhizosphere, increasing nitrogen availability and promoting chlorophyll production and growth. In general, during the harvesting period, plant nitrogen requirement will be less and the same results were reciprocated in the trial conducted.
       
Bacillus tequilensis, by producing organic acids and phosphatase enzymes, solubilizes phosphorus, silica and enhances root development. The higher silica availability improves drought tolerance, nutrient transport, enzyme activities and stress resilience in maize (Solomon et al., 2024; Kiran et al., 2022).
 
Effect of PGPR on chlorophyll and carotenoid content
 
The values recorded before the harvesting showed highest chlorophyll content in T6 (0.925 ± 0.038 mg/g), with T5 and T6 also demonstrating superior carotenoid content (0.974±0.037 and 0.974±0.041, respectively). In contrast, the control treatment had lower chlorophyll (0.649±0.027 mg/g) and carotenoid (0.683±0.029 mg/g) levels (Fig 7). These results align with prior studies reporting similar increases in chlorophyll and carotenoid content (Ali et al., 2017).


       

The enhanced photosynthetic pigments in this study are attributed to PGPR and Bacillus tequilensis, which improve chlorophyll content in maize leaves, thereby enhancing photosynthesis. The silicate-solubilizing bacteria and microbial consortia help to stabilize chlorophyll, reducing degradation and improving photosynthetic efficiency. Additionally, the increased carotenoid content, including lutein and zeaxanthin, boosts antioxidant activity, protecting against oxidative stress and enhancing overall plant health (Katagiri et al., 2002; Ali et al., 2017).

The results obtained revealed the plant growth-promoting abilities of the silicate solubilizing Bacillus tequilensis SKSSB09. The particular microbial strain also can induce drought mitigation in Zea mays L. as per the previous research reports. The Bacillus tequilensis strain showed synergism with the other microbial inoculants and synthetic fertilizers. Encouraging plant growth promotion results were obtained revealing the ability of the Bacillus tequilensis and microbial consortium in the nutrient uptake ability and plant growth. Further research focuses on field trials to evaluate the long-term benefits and scalability of these treatments in various agroecosystems.

We would like to express our deepest gratitude to the AADHAAR, R and D Lab, Biofac Inputs Private Limited, Hyderabad for their technical assistance and financial aid.
 
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The authors have no conflict of interest.