DefinePK

Research Objective

To isolate and identify Pseudomonas fluorescens and Bacillus subtilis from the rhizospheric soil of Triticum aestivum and to assess their biocontrol activity against Fusarium oxysporum and Botrytis cinerea.

Methodology

Soil samples were collected from the rhizospheric soil of Triticum aestivum. Serial dilution was performed, and bacterial isolates were cultured on nutrient agar plates. Isolation and identification involved Gram staining, microscopic analysis, and biochemical tests (VP test, Methyl red test, Citrate test, Catalase test, Indole test). Molecular identification was conducted using DNA extraction, PCR amplification of the 16S rRNA gene, gel electrophoresis, and sequencing, followed by NCBI BLAST analysis. Antifungal activity was tested on potato dextrose agar (PDA) + 0.5% yeast extract plates by inoculating fungi in the center and bacteria on the sides.

Methodology Flowchart

                        graph TD
    A[Soil Sampling from Wheat Rhizosphere] --> B[Serial Dilution];
    B --> C[Bacterial Isolation on Nutrient Agar];
    C --> D[Initial Screening];
    D --> E[Gram Staining & Microscopic Analysis];
    E --> F[Biochemical Tests];
    F --> G[DNA Extraction];
    G --> H[PCR Amplification of 16S rRNA];
    H --> I[Gel Electrophoresis & Sequencing];
    I --> J[NCBI BLAST Analysis for Identification];
    J --> K[Antifungal Activity Testing];
    K --> L[Data Analysis & Conclusion];                    

Discussion

The study confirmed the presence of Bacillus subtilis and Pseudomonas fluorescens in wheat rhizosphere and their potential as biocontrol agents. The findings align with previous research demonstrating the antagonistic capabilities of these bacteria against fungal plant pathogens. The mechanisms of action, such as the production of enzymes and antimicrobial compounds, are discussed in relation to their biocontrol efficacy.

Key Findings

Twenty bacterial strains were isolated from the wheat rhizosphere. Sixteen were Gram-positive rods, and four were Gram-negative rods. Biochemical and molecular tests identified Bacillus subtilis (99% sequence identity) and Pseudomonas fluorescens (94% sequence identity). Bacillus subtilis showed significant biocontrol activity against both Fusarium oxysporum and Botrytis cinerea. Pseudomonas fluorescens demonstrated activity against Fusarium oxysporum but not Botrytis cinerea.

Conclusion

Bacillus subtilis and Pseudomonas fluorescens were successfully isolated and identified from the rhizospheric soil of Triticum aestivum. Bacillus subtilis proved to be an effective biocontrol agent against Fusarium oxysporum and Botrytis cinerea, while Pseudomonas fluorescens showed promise against Fusarium oxysporum. These bacteria have the potential to be used as biofertilizers and biopesticides.

Fact Check

1. Bacterial Identification: The study identified Bacillus subtilis with 99% sequence identity and Pseudomonas fluorescens with 94% sequence identity using NCBI BLAST analysis.
2. Biocontrol Efficacy: Bacillus subtilis demonstrated biocontrol activity against both Fusarium oxysporum and Botrytis cinerea, while Pseudomonas fluorescens was effective only against Fusarium oxysporum.
3. Gram Staining Results: Out of 20 isolated bacterial strains, 16 were Gram-positive, and 4 were Gram-negative.

Mind Map