Isolation and Molecular Identification of Native Plant Growth-promoting Bacillus Strains from the Soil around Tehran Province

The escalating consumption of chemical fertilizers has imposed severe environmental and economic burdens on the society. Studies have shown the detrimental effects of these chemical agents on human health, necessitating a shift towards alternative fertilization practices. Research has focused on harnessing the potential of naturally occurring soil microorganisms as a sustainable and environmentally friendly solution to enhance plant growth. Biofertilizers, soil amendments enriched with beneficial microorganisms such as fungi, protozoa, actinomycetes, or algae, collectively known as plant growth-promoting bacteria (PGPB), represent a promising alternative to chemical fertilizers. Studies have shown that Bacillus-based biofertilizers can enhance nutrient uptake and regulate phytohormone metabolism within plants. Moreover, Bacillus strains exhibit remarkable resilience under environmental stress and secrete enzymes that disrupt the cell walls of pathogenic bacteria, viruses, fungi, and nematodes, thereby protecting plants from disease and promoting healthy growth and yield. In light of the aforementioned benefits, this study aims to isolate and molecularly identify native Bacillus strains with plant growth-promoting properties. The potential of these strains for incorporation into biofertilizers derived from Iranian native strains will be investigated.

In this study, rhizospheric and rhizoplane Bacillus strains from Iranian agricultural soils with Nitrogen-fixing ability were isolated and characterized for their plant growth-promoting properties. Strains with higher growth capacity were screened by 5% overnight culture in 100 ml NFB medium completed with 0.5% yeast extract. The strains were streaked onto Pikovskaya agar containing insoluble tricalcium phosphate. Quantitative analysis of phosphate solubilization was performed by culturing the strains in the mentioned liquid medium.  For the screening of (IAA) producer strains, the overnight culture of bacteria inoculated to a 250 ml Erlenmeyer-containing nutrient broth fortified with L-tryptophan. Then 2 ml of the supernatant was mixed with 2 ml Salkowski reagent. The optical absorbance was measured at 535 nm and the produced IAA was measured by standard curve graph. Production of ammonia was assayed by inoculation e of strains into peptone water. Nessler’s reagent was added to the medium. The development of yellow color showed the production of ammonia by the strain. The ability of HCN production was assayed by overnight culture of strain inoculated in nutrient agar slant fortified with glycine. The color change of the paper filter soaked in 0.5% picric acid and 2% sodium carbonate from yellow to brown indicated the production of HCN. Evaluation of antifungal activity against aspergillus niger, verticillium dahlia, and fusarium graminearum was performed on potato agar (MPA) plates, and calculated according to the following formula I= [(C-T)/C]*100. Quantitative measurement of ACC-deaminase activity of bacillus isolates was carried out using a modified NFB-ACC medium. siderophore production from cultural variables analyzed by CAS agar plate in reference to the change of color of CAS medium when microorganisms were grown in CAS agar plates. Phylogenetic relationships among the strains were established using 16S rDNA gene sequencing.  GraphPad Prism 6 software was used for statistical analysis. Data analysis was performed using one-way ANOVA and t-test and statistically significant was considered as p<0.05.

In the intricate realm of soil microbiology, the rhizosphere and rhizoplane stand out as vibrant microbial hotspots, teeming with life and playing pivotal roles in plant health and nutrition. The rhizosphere, the narrow zone of soil surrounding plant roots, is a dynamic environment enriched in root exudates, serving as a nutrient-rich haven for a diverse array of microorganisms. Within this intricate ecosystem, nitrogen-fixing bacteria emerge as crucial players, converting atmospheric nitrogen into a form usable by plants, thereby supporting their growth and productivity. Nitrogen-fixing growth-promoting bacteria isolated from the rhizosphere play a crucial role in regulating soil nitrogen availability for plants. Among the nitrogen-fixing bacteria that inhabit the rhizosphere, Bacillus strains have garnered attention for their exceptional ability to fix nitrogen and promote plant growth. These beneficial microbes possess a suite of plant growth-promoting traits, making them valuable assets for sustainable agricultural practices.

This study focused on isolating nitrogen-fixing bacteria. Over 80% of the isolated nitrogen-fixing bacilli were obtained from the rhizoplane, likely due to the high concentration of root exudates and microbial accumulation in this region. Despite previous studies indicating higher bacterial diversity in the rhizosphere, our findings suggest a distinct microbial niche within the rhizoplane supporting nitrogen-fixing bacteria. To select Bacillus strains with the highest plant growth-promoting potential, we screened them based on their growth ability, resulting in the selection of five promising strains. Phosphorus, a critical nutrient for plant growth, is often limiting. Bacillus strain S3 demonstrated the highest phosphate dissolution capacity (278.3 µg/mL). Bacilli secrete organic acids and produce siderophores that chelate iron bound to mineralized phosphates, making them accessible to plants. Moreover, studies have shown that bacilli can convert insoluble inorganic and organic phosphorus compounds into soluble forms via ACC deaminase enzyme production. Most strains exhibited ACC deaminase activity, enabling them to utilize 1-aminocyclopropane-1 as a nitrogen source. Siderophores, known for their biocontrol potential against plant pathogens, were produced by all strains except S3. Indole-3-acetic acid, a key plant hormone that promotes plant growth and germination, was produced in significant amounts by Bacillus strain S3. IAA production varies among species depending on nutrient availability. Bacillus indirectly enhances root growth by producing ammonia, a nitrogen source. Bacillus strains S3 and S5 exhibited the highest ammonia production, corroborating previous studies. Additionally, all strains demonstrated over 70% growth inhibition against fungal strains. Collectively, the obtained results underscore the promising potential of the native Bacillus strains isolated in this study for biofertilizer development. Their diverse plant growth-promoting traits, including nitrogen fixation, phosphate solubilization, ACC deaminase activity, siderophore production, IAA synthesis, ammonia production, and antifungal activity, make them valuable candidates for sustainable agricultural practices.