Evaluation of phosphorus-solubilizing and phosphorus-mineralizing bacteria for improved germination and early seedling growth of rice

Introduction

Rice (Oryza sativa L.) is one of the most crucial staple food crops and the main calorie source for over half the world's population. Rice is one of the most important crops for global food and nutritional security, covering about 165 million hectares and producing more than 780 million tonnes of rice annually (Sawargaonkar et al., 2026). India is the 2nd largest producer of rice after China and also grown in varieties of agro climates. As the world population grows and land suitable for rice cultivation decreases, it is becoming a real challenge to increase the productivity of rice by sustainable agriculture practices (Darko et al., 2025; Kumar et al., 2026). Hence, to achieve sustainable rice production, nutrient use efficiency should be improved, and environmental impacts reduced [24,7,17] . Phosphorus (P) is a key macronutrient for plant growth and involved in many of the metabolic and biochemical pathways of the plant, such as energy transfer, photosynthesis and respiration, nucleic acid synthesis, cell division, membrane formation and root development. The proper supply of available phosphorus during germination and establishment of seedlings is especially important because it influences stand, root architecture, nutrient acquisition and grain yield. Although total phosphorus is plentiful in most agricultural soils, the amount present in plant available forms is very small. Soluble orthophosphate ions are quickly bound by precipitation with calcium, in alkaline soils, and with iron and aluminum, in acid soils, and also adsorbed on soil colloids and thus unavailable for plant uptake. Additionally, a significant portion of soil P is present as organic P (e.g., phytates, phospholipids, nucleic acids, and phosphoesters) that is not readily available to plants. Rice farming is a practice in which the application of large amounts of chemical phosphate fertilizers is common, as a means of overcoming phosphorus deficiency. The use efficiency of applied phosphorus fertilizers is generally low, however, with only 10-25% being used by crop during the first growing season with the excess phosphate becoming soil bound (as insoluble fraction) or loss by run-off and erosion (Cordell et al., 2009). The use of chemical fertilizers is a nonrenewable resource, leads to a depletion in non-renewable phosphate rock, soil nutrient imbalance and eutrophication of water bodies, as well as raises production costs. This has led to a greater interest in environmentally friendly options which will increase the availability of phosphorus and enhance fertilizer use efficiency (Gurav et al.,2024, Vance et al., 2003) [6,14,27] . The phosphorus-solubilizing and phosphorus-mineralizing bacteria (PSMB) which are beneficial soil microorganisms that are able to convert unavailable inorganic and organic phosphorus into soluble phosphorus which can be readily taken up by the plant. Phosphorus-solubilizing bacteria produce low-molecular-weight organic acids which bring about a decrease in rhizosphere pH, chelate the cations on the insoluble phosphate minerals, and release soluble phosphate ions. Phosphorus-mineralizing bacteria produce extracellular enzymes (acid phosphatases, alkaline phosphatases, phytases and phosphodiesterases) which are responsible for breaking down organic compounds of phosphorus into inorganic phosphate (Pang et al., 2024, Silva et al., 2023). By these complementary processes, PSMB increase the availability of phosphorus, increase nutrient uptake, stimulate root development and support sustainable nutrient cycling in agricultural soils. In addition to phosphorus mobilization, many PSMB also have multiple plant growth promoting properties including the production of phytohormones, biological nitrogen fixation, siderophore production, potassium and zinc solubilization and suppression of phytopathogens, which further promote plant growth and development (Goswami et al., 2019) [20,25,11] . The initial growth of seedling starts as rapidly dividing cells, which demand energy and nucleic acid synthesis at a rapid pace, seed germination and early seedling growth is highly sensitive to the availability of phosphorus. Improved phosphorus nutrition during germination results in quicker radicle emergence, growth of root, shoot growth, formation of more biomass and greater vigour of seedlings (Backer et al., 2018, Khoso et al., 2024). Seed inoculation with efficient phosphorus-solubilizing and mineralizing bacteria has been reported to increase the germination percentage, decrease mean germination time, increase root and shoot growth and seedling biomass in several cereal crops including rice (Gupta et al., 2015) [3,16,13] . However, the efficiency of the phosphorus mobilizing bacteria is variable between individual isolates because of their ability to mobilize phosphate, their production of phosphatase enzymes, their rhizosphere colonization capacity and their compatibility with their host plants. Therefore, screening of bacterial isolates should be performed systematically to isolate better strains having good efficiency in phosphorus mobilization and plant growth promotion. Identifying highly efficient isolates capable of enhancing early plant growth is an important prerequisite for developing effective biofertilizer formulations aimed at improving phosphorus use efficiency and reducing dependence on chemical phosphate fertilizers. Therefore, the present investigation was undertaken to evaluate the effect phosphorus-solubilizing and phosphorus-mineralizing bacterial isolates on seed germination and seedling growth of rice.

Materials and Methods

Plant Material and Bacterial Isolates

Nine bacterial isolates exhibiting phosphorus-solubilizing and phosphorus-mineralizing abilities were evaluated using the rice variety Improved Samba Mahsuri (ISM). The isolates were previously characterized and had exhibited inorganic phosphate-solubilizing efficiency and organic phosphorusmineralizing potential, along with other beneficial plant growth-promoting traits. The bacterial cultures were maintained on nutrient agar slants and glycerol stocks and periodically sub-cultured to maintain viability. Fresh bacterial cultures were grown in nutrient broth at 28 ± 2°C for 24-48 h under continuous shaking at 150 rpm. The cultures were harvested by centrifugation at 8,000 rpm for 10 min, washed twice with sterile phosphate-buffered saline (PBS), and resuspended in sterile PBS. The bacterial suspension was adjusted to an optical density of OD₆₀₀ = 1.0, corresponding to approximately 1 × 10⁸ CFU mL⁻¹, and used for seed inoculation (Pole et al., 2026) [22] .

Seed Surface Sterilization and Bacterial Inoculation

Rice seeds were surface sterilized by immersion in 70% ethanol for 1 min, followed by treatment with 0.1% mercuric chloride (HgCl₂) for 1 min. The sterilized seeds were rinsed six times with sterile distilled water to eliminate residual sterilizing agents. The seeds were then soaked separately in each bacterial suspension (approximately 1 × 10⁸ CFU mL⁻¹) for 24 h at 28°C under aseptic conditions ((Venkat et al., 2026).). Seeds soaked in sterile phosphate-buffered saline served as the uninoculated control. After inoculation, seeds from each treatment were transferred onto into small pots. Seed growth parameters observations were taken at 7,14 and 21 days after sowing [28] .

Experimental Design

The experiment was conducted as a Completely Randomized Design (CRD) consisting of ten treatments, including one uninoculated control and nine bacterial inoculation treatments. Each treatment was replicated three times. The treatments comprised individual inoculation with phosphorus-solubilizing and phosphorus-mineralizing bacterial isolate, while uninoculated seeds served as the control. All experimental units were maintained under identical environmental conditions throughout the study.

Germination Attributes

Seed germination was recorded daily until it fully germinates. Daily germination counts were used to determine germination percentage (GP) and mean germination time (MGT). Seedling growth was evaluated at 7, 14, and 21 days after inoculation (DAI). At each sampling interval, five randomly selected seedlings from each replication were carefully removed from the pots. Root length and shoot length were measured using a graduated ruler and expressed in centimetres. Fresh weights of roots, shoots, and seeds were immediately determined using an analytical balance. The plant samples were subsequently dried in a hot-air oven at 65 ± 2°C until constant weight was attained, after which root dry weight, shoot dry weight, and seed dry weight were recorded. These measurements were used to assess the influence of bacterial inoculation on early seedling growth, biomass accumulation, and seedling establishment over time.

Statistical Analysis

The experiment was designed in a completely randomized design (CRD) with three replications. Analysis of variance (ANOVA) was used to analyze the data and critical difference (CD) was used to compare the treatment means at 5% level of significance (Gomez and Gomez 1984) [10] .

Results and Discussion

Germination Percentage and Mean Germination Time

The bacterial isolates significantly influenced germination percentage (GP) and mean germination time (MGT) of rice seeds compared with the uninoculated control (Fig. 1, Fig. 2). Among the evaluated treatments, Isolate The enhanced germination observed in inoculated seeds demonstrates the beneficial influence of plant growthpromoting bacteria on early seed germination and seedling establishment. The highest performance of Isolate 5, followed by Isolates 2 and 9, suggests that these isolates possess strong plant growth-promoting characteristics that facilitate rapid germination and vigorous seedling emergence (Basu et al., 2021). Enhanced germination may be attributed to the production of phytohormones such as indole-3-acetic acid (IAA), gibberellins, and cytokinins, which stimulate embryo growth, activate hydrolytic enzymes responsible for mobilization of seed reserves, and accelerate radicle emergence (Abd El-Mageed et al., 2022). In addition, beneficial bacteria improve nutrient availability in the vicinity of germinating seeds and release metabolites that enhance seed metabolic activity, resulting in faster and more uniform germination [4,1] . The reduction in mean germination time further indicates synchronized and rapid seedling emergence, which is advantageous for achieving better crop establishment under field conditions. Early and uniform germination enables seedlings to utilize available resources more efficiently and improves their competitiveness during the initial stages of growth (Lotfi et al., 2025) [19] . Plant growth-promoting bacteria are also known to produce ACC deaminase, which reduces stress-induced ethylene levels, thereby promoting faster germination and enhancing seedling establishment (Gupta et al., 2022). These physiological and biochemical mechanisms likely contributed to the increased performance of Isolates 5, 2, and 9. The present findings are consistent with earlier reports demonstrating that PGPB enhance seed germination through phytohormone production, nutrient mobilization, and improved seed metabolic activity (Harahap et al., 2023). Previous studies have shown that phosphate-solubilizing and rhizosphere-associated bacteria significantly increase germination percentage while reducing mean germination time, ultimately leading to improved seedling vigor and crop establishment (Rawat et al., 2022). The enhanced germination observed in the present study was also reflected in the subsequent improvements in seedling growth and biomass accumulation, confirming the sustained beneficial effects of bacterial inoculation during early plant development [12,15,23] .

Effect of Phosphorous solubilizing and mineralizing bacterial inoculation on growth parameters of rice seedlings at 7 days after sowing

Phosphorus-solubilizing and mineralizing bacterial inoculation significantly enhanced the early growth and biomass accumulation of rice seedlings at 7 days after sowing (DAS) (Table 1). Significant variation was observed among the bacterial isolates for all growth parameters. Isolate 5 produced the greatest root length (8.83 cm) and shoot length (12.40 cm), followed by Isolate 9 (8.50 and 12.00 cm, respectively) and Isolate 2 (8.23 and 11.80 cm, respectively). Moderate improvements were recorded with Isolates 8 and 7, whereas Isolates 1, 3, 4, and 6 exhibited comparatively lower responses. The uninoculated control consistently recorded the lowest root and shoot growth. A similar trend was observed for biomass accumulation. Isolate 5 recorded the highest seed fresh weight (0.340 g), root fresh weight (0.098 g), and shoot fresh weight (0.242 g), followed by Isolate 9 and Isolate 2. Likewise, maximum seedling dry weight (0.106 g), root dry weight (0.021 g), and shoot dry weight (0.085 g) were recorded in Isolate 5, whereas the control produced the lowest fresh and dry biomass. Overall, Isolate 5 exhibited the greatest plant growth-promoting potential during the initial stages of seedling establishment. The enhancement in seedling growth and biomass following bacterial inoculation demonstrates the effectiveness of phosphorus-solubilizing and mineralizing bacteria in promoting early rice seedling establishment. The enhanced performance of Isolate 5, followed by Isolates 9 and 2, may be attributed to increased phosphorus availability resulting from phosphate solubilization and mineralization, coupled with the production of plant growth-promoting metabolites such as indole-3-acetic acid (IAA), gibberellins, and cytokinins. Enhanced phosphorus nutrition during early growth stimulates root proliferation, thereby improving water and nutrient acquisition and ultimately increasing biomass accumulation (Yavdav et al., 2014, Awlachew and Mengistie, G. Y. ( 2022)). The present findings are in agreement with previous studies reporting that inoculation with phosphate-solubilizing bacteria enhances early seedling growth by improving phosphorus availability, stimulating root development, and increasing biomass accumulation (Phringpaen et al., 2023) [30,2] .

Effect of Phosphorous solubilizing and mineralizing bacterial inoculation on growth parameters of rice seedlings at 14 days after sowing

The growth-promoting effects of bacterial inoculation became more pronounced by 14 DAS, resulting in significant improvements in seedling growth and biomass accumulation compared with the uninoculated control (Table 2). Among the tested isolates, Isolate 5 recorded the highest root length (12.82 cm) and shoot length (14.95 cm), followed by Isolate 9 (12.46 and 14.58 cm, respectively) and Isolate 2 (12.02 and 14.12 cm, respectively). Intermediate responses were observed with Isolates 6 and 8, whereas the remaining isolates produced comparatively lower growth. Fresh biomass production followed the same trend, with Isolate 5 recording the highest seed fresh weight (0.446 g), root fresh weight (0.131 g), and shoot fresh weight (0.312 g). Similarly, the highest seedling dry weight (0.139 g), root dry weight (0.027 g), and shoot dry weight (0.112 g) were obtained with Isolate 5, followed by Isolates 9 and 2. The control consistently recorded the lowest biomass values. The sustained improvement in seedling growth at 14 DAS suggests successful rhizosphere colonization and continued activity of the inoculated bacteria. Improved phosphorus availability, together with enhanced nutrient mobilization and phytohormone production, likely contributed to increased root development and photosynthetic capacity, resulting in greater fresh and dry matter accumulation (Dobrzyński et al., 2026). The consistent enhancement of Isolate 5, followed by Isolates 9 and 2, indicates that these isolates possess multiple plant growth-promoting traits that remained active throughout the early vegetative stage. Similar observations have been reported by Bhat et al., 2023 and Sun et al., 2024 [8, 5, 26] , who demonstrated that plant growth-promoting rhizobacteria improve nutrient acquisition, root architecture, and biomass production, leading to sustained enhancement of seedling growth during the early vegetative stage.

Effect of Phosphorous solubilizing and mineralizing bacterial inoculation on growth parameters of rice seedlings at 21 days after sowing

Significant improvements in seedling growth and biomass accumulation were maintained at 21 DAS following inoculation with phosphorus-solubilizing and mineralizing bacteria (Table 3). Among the evaluated isolates, Isolate 9 recorded the greatest root length (14.82 cm) and shoot length (19.72 cm), while Isolate 5 (14.18 and 18.04 cm, respectively) and Isolate 2 (14.34 and 17.18 cm, respectively) also produced substantially greater growth than the control. Despite the greater seedling elongation observed with Isolate 9, Isolate 5 produced the highest seed fresh weight (0.612 g), root fresh weight (0.180 g), and shoot fresh weight (0.425 g). It also recorded the maximum seedling dry weight (0.190 g), root dry weight (0.036 g), and shoot dry weight (0.154 g), followed closely by Isolates 9 and 2. The uninoculated control consistently exhibited the lowest fresh and dry biomass accumulation. The continued enhancement of growth and biomass up to 21 DAS indicates that the beneficial effects of bacterial inoculation were sustained throughout the seedling establishment period. Although Isolate 9 promoted greater root and shoot elongation, Isolate 5 consistently accumulated higher fresh and dry biomass, suggesting more efficient nutrient utilization and assimilate partitioning. The increased performance of Isolates 5, 9, and 2 demonstrates their potential as effective phosphorus-solubilizing and mineralizing bioinoculants capable of improving early rice seedling vigor and establishing a strong foundation for subsequent crop growth. The sustained improvement in seedling growth and biomass observed in the present study corroborates earlier reports indicating that phosphorussolubilizing bacteria promote root development, nutrient uptake, and dry matter accumulation, ultimately improving crop establishment and productivity (Elhaissoufi et al., 2023, Li et al., 2022 and Wang et al., 2022) [9,28,29] .

Conclusion

The present study demonstrated that seed inoculation with phosphorus-solubilizing and phosphorus-mineralizing bacteria significantly improved germination, seedling growth, and biomass accumulation in rice under pot conditions. Among the nine bacterial isolates evaluated, Isolate 5 consistently exhibited superior performance by recording the highest germination percentage, the shortest mean germination time, and the greatest fresh and dry biomass at 7, 14, and 21 DAS. Although Isolate 9 recorded the highest root and shoot lengths at 21 DAS, Isolate 5 maintained the highest overall biomass accumulation, indicating its greater efficiency in promoting early seedling vigor. Isolate 2 also performed consistently well across all growth parameters. The enhanced performance of the inoculated seedlings highlights the potential of phosphorussolubilizing and phosphorus-mineralizing bacteria with plant growth-promoting traits to stimulate early crop establishment. These beneficial microorganisms can serve as sustainable bioinoculants to reduce dependence on chemical phosphorus fertilizers while improving nutrient use efficiency and seedling development.

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