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Functional and Statistical Validation of Nitrogen-Fixing Plant Growth–Promoting Bacteria for Ethylene Regulation and Seed Germination Under Osmotic Stress

This study validates the functional and statistical efficacy of selected nitrogen-fixing bacteria, particularly *Lactobacillus pasteurii* DSM 23907 and *Bacillus rugosus*, in mitigating osmotic stress and significantly enhancing seed germination and seedling vigor through mechanisms including ammonia production, ACC deaminase activity, and nitrogenase function.

Original authors: Gottumukkala Hiranmayee, Sarada Prasanna Mallick, Chinthalapudi Aravind, Gandu Harini, Koppula Akshay, Palnati Purna Mahesh, Kari Sree Ranga Mangala Deepika, Golamari Siva Reddy

Published 2026-08-10

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CC BY 4.0

Original authors: Gottumukkala Hiranmayee, Sarada Prasanna Mallick, Chinthalapudi Aravind, Gandu Harini, Koppula Akshay, Palnati Purna Mahesh, Kari Sree Ranga Mangala Deepika, Golamari Siva Reddy

Original paper licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). ✨ This is an AI-generated explanation of the paper below. It is not written or endorsed by the authors. For technical accuracy, refer to the original paper. Read full disclaimer

Imagine the soil as a bustling city where plants are the residents and tiny, invisible microbes are the helpful neighbors. Some of these neighbors are like master chefs who can turn the air we breathe—specifically nitrogen gas, which is useless to plants on its own—into a delicious, nutritious meal called ammonia. This process is called "Biological Nitrogen Fixation," and it's the plant world's way of making its own fertilizer. But life in this soil city isn't always easy. Sometimes, the weather gets too hot and dry, creating "osmotic stress," which is just a fancy way of saying the water is so scarce or salty that plants can't drink it up, causing them to wilt and struggle to grow.

Enter the "Plant Growth-Promoting Bacteria" (PGPB). Think of these as the ultimate plant bodyguards. They don't just bring food; they also have a special trick to lower a plant's stress levels. Plants produce a chemical called ethylene when they are scared or stressed (like during a drought), which acts like a panic button that can actually stop them from growing. Some bacteria carry a tool called "ACC deaminase," which acts like a stress-relief counselor, breaking down that panic button so the plant can keep growing even when the going gets tough. Scientists are always on the hunt for the best of these bacterial neighbors to help farmers grow food without relying so heavily on chemical fertilizers, especially as the climate gets drier.

This study is like a tryout camp for four specific bacterial candidates: Corynebacterium accolens, Bacillus rugosus, Lactobacillus pasteurii DSM 23907, and Cytobacillus firmus. The researchers wanted to see which of these microbes were the true champions at two things: making nitrogen food and calming plant stress. They put the bacteria through a series of tests, starting with checking how much ammonia they could produce. The results showed that Corynebacterium accolens, Bacillus rugosus, and Lactobacillus pasteurii were the heavy hitters, turning out a lot of ammonia, while the others were a bit weaker.

Next, the team tested their "stress-relief" skills by measuring ACC deaminase activity. This is the enzyme that lowers the plant's panic levels. Here, Lactobacillus pasteurii DSM 23907 was the clear star, showing the highest activity at 1.487 ± 0.050 µmol α-KB mg⁻¹ protein h⁻¹, followed closely by Bacillus rugosus at 1.050 ± 0.044. The other two bacteria had significantly lower activity. To double-check their nitrogen-making powers, the scientists used a test called the Acetylene Reduction Assay, which measures how much ethylene gas the bacteria produce as a sign of nitrogen-fixing work. Again, Lactobacillus pasteurii DSM 23907 took the crown with the highest activity of 38.46 ± 3.59 nmol/OD/hr, significantly outperforming the others.

The real drama, however, happened in the seed germination lab. The researchers took okra seeds and put them in a "drought simulator" using a substance called Polyethylene Glycol (PEG) to create water stress. As expected, the stressed seeds without help were in trouble: their germination rates dropped to about 40% or even 34%, they took longer to sprout, and their seedlings were weak and short. But when the researchers added the bacteria, the story changed completely. The seeds inoculated with Bacillus rugosus or Lactobacillus pasteurii bounced back, with germination rates soaring back up to around 76–77%. Not only did they sprout more, but they also grew faster and stronger. The "Seedling Vigor Index," a score that combines how many seeds sprouted with how big they got, more than doubled for the bacteria-treated seeds compared to the stressed ones that got no help.

The data suggests that while all four bacteria had some good traits, Lactobacillus pasteurii DSM 23907 was the most consistent all-rounder, followed by Bacillus rugosus. These two showed they could not only fix nitrogen but also effectively shield seeds from the harsh effects of drought. The study concludes that these bacteria are promising candidates for becoming "biofertilizers"—natural helpers that could help farmers grow crops in dry conditions without needing as many chemical inputs. However, the authors note that these results are from lab experiments, and more testing in greenhouses and real fields is needed to confirm they work just as well in the messy, unpredictable real world.

Technical Summary: Functional and Statistical Validation of Nitrogen-Fixing Plant Growth–Promoting Bacteria for Ethylene Regulation and Seed Germination Under Osmotic Stress

Problem Statement
Plants require nitrogen for essential physiological processes, yet atmospheric nitrogen (N₂) is inert and must be converted into bioavailable forms like ammonium. While biological nitrogen fixation (BNF) by diazotrophs is a natural solution, the efficiency of synthetic nitrogen fertilizers remains low (<50% uptake), leading to environmental degradation. Furthermore, abiotic stresses, particularly osmotic stress caused by drought and salinity, severely inhibit seed germination, delay germination timing, and stunt seedling growth by disrupting water potential and elevating ethylene levels. There is a need to identify and validate indigenous, non-symbiotic bacterial isolates that possess multifunctional traits—specifically nitrogen fixation and ethylene regulation—to serve as sustainable biofertilizers capable of mitigating these stresses.

Methodology
The study utilized four bacterial isolates previously obtained from the rhizosphere of plants in Tattiannaram, Telangana: Corynebacterium accolens, Bacillus rugosus, Lactobacillus pasteurii DSM 23907, and Cytobacillus firmus. The experimental workflow involved three primary phases:

Functional Screening:
- Ammonia Production: Isolates were cultured in peptone broth and tested with Nessler's reagent to detect ammonia, a proxy for nitrogen fixation potential.
- ACC Deaminase Activity: Isolates were grown in DF minimal medium with ACC as the sole nitrogen source. Enzymatic activity was quantified by measuring $\alpha$ -ketobutyrate production via colorimetric detection (DNPH reagent) after cell lysis.
- Nitrogenase Activity: The Acetylene Reduction Assay (ARA) was performed to measure ethylene production, serving as an indirect indicator of nitrogenase activity.
Seed Germination Assay under Osmotic Stress:
- Abelmoschus esculentus (okra) seeds were surface-sterilized and inoculated with bacterial suspensions ( $10^8$ CFU/ml).
- Germination was tested under controlled conditions using Polyethylene Glycol (PEG-6000) to simulate osmotic stress at potentials of -0.2 MPa (10% w/v) and -0.5 MPa (20% w/v).
- Controls included uninoculated seeds in water and uninoculated seeds in PEG solutions.
Statistical Analysis:
- Data were analyzed using one-way ANOVA to determine significant differences among treatments.
- Post-hoc pairwise comparisons were conducted using Tukey's HSD test ( $p < 0.05$ ).
- Heatmap analysis was employed to visualize trends across multiple growth parameters.

Key Results

Nitrogen Fixation Potential:
- Ammonia Production: C. accolens, B. rugosus, and L. pasteurii DSM 23907 exhibited strong color changes indicating high ammonia production, comparable to or exceeding that of the positive control (Azotobacter).
- ACC Deaminase Activity: The positive control exhibited the highest enzyme activity (1.62 ± 0.01 $\mu$ mol $\alpha$ -KB mg⁻¹ protein h⁻¹). Among the isolates, L. pasteurii DSM 23907 demonstrated the highest activity (1.487 ± 0.050 $\mu$ mol $\alpha$ -KB mg⁻¹ protein h⁻¹), followed by B. rugosus (1.050 ± 0.044). C. accolens and C. firmus showed significantly lower but statistically similar activities.
- Nitrogenase Activity (ARA): The positive control exhibited the highest specific activity (63.70 ± 13.90 nmol/OD/hr). Among the isolates, L. pasteurii DSM 23907 recorded the highest specific activity (38.46 ± 3.59 nmol/OD/hr), significantly outperforming B. rugosus (27.98 ± 2.08), C. firmus (24.09 ± 2.64), and C. accolens (20.49 ± 1.51). All isolates showed activity significantly lower than the positive control but distinct from the negative control.
Seed Germination and Stress Mitigation:
- Stress Impact: PEG-induced drought stress significantly reduced germination percentages (dropping to ~40% at -0.2 MPa and ~35% at -0.5 MPa), increased Mean Germination Time (MGT), and reduced root/shoot lengths and Seedling Vigor Index (SVI).
- Bacterial Inoculation Effects: Inoculation with B. rugosus and L. pasteurii DSM 23907 markedly alleviated stress.
  - Under -0.2 MPa stress, inoculation restored germination to ~76–81% and SVI to ~1390–1660.
  - Under -0.5 MPa stress, inoculation restored germination to ~77–79% and SVI to ~1400–1414.
- Comparative Performance: L. pasteurii DSM 23907 generally showed superior efficacy in promoting root/shoot development and SVI compared to B. rugosus. Inoculated seedlings under stress exhibited SVI values more than double those of uninoculated stressed seedlings.

Significance and Claims
The paper claims that the investigated isolates, particularly Lactobacillus pasteurii DSM 23907 and Bacillus rugosus, possess multifunctional plant growth-promoting traits. Specifically, they demonstrate:

Dual Functionality: The ability to fix nitrogen (via nitrogenase and ammonia production) and regulate plant stress hormones (via ACC deaminase activity).
Stress Alleviation: A proven capacity to mitigate the adverse effects of osmotic stress on seed germination and early seedling vigor in Abelmoschus esculentus.
Biofertilizer Potential: These isolates are identified as promising candidates for biofertilizer development to support sustainable agriculture, potentially reducing reliance on synthetic nitrogen inputs and enhancing crop resilience in water-limited conditions.

The authors conclude that while these strains show strong potential in functional assays and seed germination trials, further greenhouse and field-level validation is required to confirm their efficacy under natural environmental conditions. The study emphasizes that nitrogenase efficiency does not always correlate with cell density, highlighting the necessity of functional trait evaluation for specific strain selection.

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