Introduction

The soil layer around plant roots, known as the rhizosphere, is critical to plant growth and development.^{1, 2} Rhizobacteria are bacterial communities that live in this region, and some of them, known as plant growth-promoting rhizobacteria (PGPR), have a favourable impact on plant growth. These PGPR promote plant growth directly or indirectly.³ These helpful rhizobacteria are classified into various genera, including Bacillus, Azotobacter, and Pseudomonas.

Bacillus is a genus of gram-positive bacteria commonly found in soil, with species such as Bacillus subtilis. ⁴ being diazotrophic. Extensive research has been conducted on the ability of Bacillus and Paenibacillus species to promote plant growth. ^{5, 6} It is well established that B. cereus, B. subtilis, and B. megaterium can serve as effective biocontrol agents against plant diseases. Additionally, antifungal peptides produced by species of Bacillus have the ability to regulate fungal growth.⁷

Paenibacillus and Bacillus are widely found in the rhizosphere. Very little information is available about this genus, but it is impossible that only a single species will dominate and outnumber other species in most soils.⁸ Many PGPR possess great potential as inoculants, and they can be used for agricultural purposes and environmental protection. These PGPR help to maintain the sustainability of agroecosystems. However, currently, PGPR are not effectively used in agriculture despite many positive reports in laboratory conditions. The use of a consortium of PGPR with different plant growth-boosting features can have a greater favourable impact on plant growth than using a single strain of PGPR. Inoculation of plants with mixed strains was found to be more effective than using a single strain.⁹ As a result, the purpose of this study was to determine the influence of a Bacillus sp. consortium on plant growth promotion.

Materials and Methods

Isoaltion and Characterization of Bacillus sp

Bacillus sp. from the rhizospheric zone were isolated as previously described.¹⁰ Colonies isolated were examined for their cell shape, gram reaction, and ability to form endospores.¹¹ Bacillus species were identified as previously described. ¹² Different biochemical tests were performed, such as arginine hydrolysis, catalase test, lipid hydrolysis, casein hydrolysis, gelatin liquefaction, starch hydrolysis, and urease test.

Figure 1: Urease Test                   Click here to view Figure

Figure 2: Starch Hydrolysis Test  Click here to view Figure

Figure 3: Arginine hydrolysis Test Click here to view Figure

Determination of plant growth-promoting traits of Bacillus sp.

Isolated Bacillus species were investigated for their diverse plant growth-promoting characteristics, including as IAA production ¹³, gibberellic acid production ¹⁴, phosphate solubilisation, siderophore production ¹⁵, and HCN production.¹⁶

Antifungal Activity

The antifungal properties of Bacillus sp. was assessed as described.¹⁷ Fusarium oxysporum was cultured on plates containing PDA (potato dextrose agar) and was inoculated at the center of the plates. A bacterial culture of 24 hours old was streaked parallel 3-4 cm away from the disc on both sides. The incubation of these plates was done at 30^oC for 96 hours. 

Plant growth promotion experiment

The highest producers of IAA, GA, HCN, and Phosphate solubilizers were employed for the growth promotion experiment in selected plants, including those isolates that show the highest antifungal activity. A Plant growth promotion experiment was conducted as previously described.¹⁸  Wheat seeds were dipped in 0.02% sodium hypochlorite for two to three minutes and were then washed thrice with sterilized distilled water. Carboxymethylcellulose (CMC) 1% solution was used as an adhesive. The seeds were treated with a broth that contained 1 ×10⁹ CFU/ml. Seeds were treated with each bacterial culture for 30 min and dried overnight. The four treated seeds were sown in soil containing pot and the length of the root, the shoot and the dry weight of the root and the shoot was noted on the eighth day, the fifteenth day and the twenty fifth day of treatment.

Plant growth promotion experiment using consortium of microorganisms

A plant growth promotion experiment using a consortium was conducted as previously described.¹⁹ The highest producers of IAA, GA, HCN, and Phosphate solubilizers were selected for the plant growth promotion experiment, including those isolates that show the highest antifungal activity. Wheat seeds were soaked for 2-3 minutes in 0.02% sodium hypochlorite, then washed three times with sterilised distilled water. The glue was made of 1% CMC (carboxymethylcellulose). Seeds were treated with broth containing 1 ×10⁹ CFU/ml. The consortium had a 1:1 (v/v) ratio of each broth culture. Seeds were treated with each consortium culture for 30 minutes and then dried overnight. The four treated seeds were sown in soil containing a pot, and the length and dry weight of the root and shoot were measured on the eighth, fifteenth, and twenty-fifth days of treatment.

Results

A total of 34 Bacillus strains were isolated and identified from seven districts of Marathwada. These strains were characterized morphologically and biochemically. All isolates were gram-positive, rod-shaped, and motile, with the presence of endospores being a common characteristic. The isolates were tested for their ability to promote plant growth by producing siderophores, gibberellic acid, phosphate solubilization, IAA, and HCN. The strains producing the highest amounts of IAA, gibberellic acid, HCN, and those exhibiting the largest zones of phosphate solubilization and antifungal activity were selected for further investigation. Five Bacillus strains were chosen for the plant growth promotion experiment (Table 1). In this experiment, wheat seeds treated with Yb20 exhibited the longest root length after 15 days, while the highest shoot length of 16.55 cm was observed with Yb1 after 25 days of treatment (Table 3).  After 25 days of treatment, the B4 consortium (IAA producer + GA producer + phosphate solubilizer + HCN producer + antifungal activity) had the best results in terms of root length, shoot length, and dry root and shoot weight for wheat seeds. The B4 consortium’s root and shoot lengths were 11.80 cm and 14.63 cm, respectively (Table 5). The highest dry weight of the root was seen with consortia B3, while the highest dry weight of the shoot was obtained by B4 after 25 days. This study found that the use of Bacillus sp. consortia was more effective in boosting plant growth, in terms of root and shoot length and root and shoot dry weight, than the use of individual Bacillus.

Table 1: Isolates of Bacillus Selected for Plant Growth Promotion Experiment.

 Table 2: Consortium of Bacillus used for the experiment

 Table 3: Effect of   Bacillus   isolates on root length and shoot length of wheat seeds

Figure 4: Effect of Bacillus isolates on wheat seeds Click here to view Figure

Table 4: Effect of Bacillus isolates on dry root weight and dry shoot weight of wheat seeds. 

 Table 5: Effect of consortium of Bacillus   isolates on root length and shoot length of wheat seeds 

Figure 5: Effect of consortium of Bacillus sp on wheat seeds  Click here to view Figure

Table 6: Effect of consortium of Bacillus isolates on dry root weight and dry shoot weight of wheat seeds 

Discussion

PGPR exert beneficial effects on plants by different mechanisms such as the production of phytohormones, phosphate solubilization, nitrogen fixation, and biocontrol.²⁰  Bacillus is a common bacterium genus in the rhizosphere, and the plant growth-promoting properties of numerous of its strains have been well researched, revealing the mechanisms involved.²¹ Field investigations with microbial consortia of Pseudomonas, Azospirillum, and Bacillus on Withania somnifera showed significant increases in plant height and root length compared to individual strains.²² This suggests that combining soil microbial diversity to form consortia of two or more plant growth-promoting rhizobacteria (PGPR) can improve plant development.

The findings of this study highlight the significant potential of Bacillus spp. consortia for increasing wheat growth by improving nutrient uptake, stress tolerance, and overall plant health. The observed increases in root and shoot biomass, length, and yield metrics demonstrate the efficacy of these beneficial bacteria in promoting sustainable agriculture methods.²³ This technique, which takes advantage of the plant growth-promoting capabilities of Bacillus spp., offers a possible alternative to artificial fertilisers, contributing to environmentally friendly and resilient wheat agriculture. Additional study is required to optimise application methods and assess the long-term impacts on soil health and crop output.

Conclusion

For promoting plant growth, a consortium of microorganisms may be more effective than a single strain. Numerous Bacillus sp. isolates have several characteristics that promote plant development. These isolates can be applied to agriculture with success. Utilizing these bacteria for sustainable agriculture can be aided by research on their variety. 

Acknowledgement

The author would like to thank the Department of biotechnology, Yeshwant Mahavidyalaya, Nanded, Maharashtra for carrying out research work. 

Funding Sources

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Conflict of Interest

The authors do not have any conflict of interest.

Data Availability Statement

This statement does not apply to this article.

Ethics Statement

This research did not involve human participants, animal subjects, or any material that requires ethical approval.

Informed Consent Statement

This study did not involve human participants, and therefore, informed consent was not required.

Clinical Trial Registration

This research does not involve any clinical trials.

Permission to reproduce material from other sources

Not Applicable

Author Contributions

Aniruddha Ratnakar Apastambh: Methodology, Data Collection, Writing – Original Draft, Review

Mirza Mushtaq Vaseem Baig: Analysis, Editing, Supervision, Conceptualization. 

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