Glyphosate (N-phosphonomethylglycine) is a herbicide that is used worldwidely. Its common trade name is Roundup. Its non-targeted species action makes it most popular herbicide. It was developed by Monsanto company. The primary target for glyphosate is the enzyme EPSPS (5-enolpyruvoylshikimate 3-phosphate synthase). When glyphosate binds to EPSPS it forms a very stable complex that essentially permanently disables the enzyme and hence affect the metabolic activity of the plant and results in its death. Finding Glyphosate degrading microorganisms from soil is an interesting topic since glyphosate is non- targeted in its toxicity. Microorganisms were isolated from soil samples, which were then identified by molecular method. Isolation of DNA, its amplification using 16s rRNA gene and its sequencing are the major steps involved. Bioinformatics tool helps to identify the microorganisms. Two microorganisms identified are Pseudomonas sp. and Achromobacter sp. In the phylogenetic analysis also the two organisms are grouped as separate clads. In these, strain 1 showed highest growth in the Glyphosate containing medium than strain 2. These results show that the bacterial strain may possess potential to be used in bioremediation of glyphosate-contaminated environments.
Table of Contents
1. Introduction
1.1 Objectives
1.2 Scope of the study
2. Review of literature
3. Hypothesis
4. Materials and Methods
4.1 Study area
4.2 Soil sample collection
4.3 Isolation of microorganisms from soil
4.3.1 Pure culture preparation
4.4 Morphological and biochemical tests
4.4.1 Gram staining
4.4.2 Colony morphology
4.4.3 Catalase test
4.4.4 Motility test
4.4.5 Mannitol salt agar test
4.4.6 Urease test
4.4.7 IMViC test
4.5 Molecular identification and isolation of DNA
4.6 Quantification of DNA
4.7 Agarose gel electrophoresis
4.8 PCR amplification using 16s rRNA
4.9 Data sequencing
4.10 Data analysis
4.11 Glyphosate degradation
4.12 Comparitive study for Glyphosate tolerance
4.13 Statistical analysis
5. Results and discussion
5.1 Isolation of microorganism from soil
5.2 Pure culture preparation, morphological and biochemical identification
5.3 Bacterial DNA isolation
5.4 PCR qmplification using 16s rRNA and sequencing
5.5 Sequence analysis
5.6 Molecular phylogentic analysis using maximum likelihood method
5.7 Evaluation of Glyphosate degredation capacity
5.8 Evaluation of Glyphosate tolerence capacity
6. Conclusions
Research Objectives and Themes
This research aims to isolate and identify glyphosate-resistant bacteria from agricultural soil samples in Kerala to explore their potential use in bioremediation. By applying molecular identification techniques and assessing the degradation capacities of the isolated bacterial strains, the study seeks to characterize organisms that can utilize glyphosate as a source of phosphorous, thereby offering an environmentally friendly method to restore contaminated agricultural soils.
- Isolation of glyphosate-resistant microorganisms from soil samples.
- Molecular identification of bacterial strains using 16s rRNA gene sequencing and BLAST analysis.
- Assessment of the glyphosate degradation capacity of identified strains.
- Evaluation of the pesticide tolerance limits of the isolated bacteria.
- Phylogenetic analysis to understand the diversity of the resistant strains.
Excerpt from the Book
4.3 Isolation of microorganisms from soil
The soil used for the enrichment and isolation of pesticide tolerantbacteria was obtained. 1gm of soil sample was put into a 150ml flask containing 30ml of sterile liquid Mineral Salt Medium (MSM) with10 microliter of glyphosate diluted in 1ml of distilled water. All the above flasks were incubated at 28 ± 2°C for 7days on static conditions (1st enrichment). From every flask, 5ml was re-inoculated to the flask with same medium composition aseptically and further incubated at 28 ± 2°C for 7 days on static conditions.
Summary of Chapters
1. Introduction: Outlines the global problem of soil pollution by pesticides and emphasizes the need for bioremediation strategies.
2. Review of literature: Provides background on the characteristics of glyphosate, its mechanism of action, and existing research on microbial degradation.
3. Hypothesis: States the premise that glyphosate-resistant bacteria exist in Kerala's agricultural soils and possess varying degradation capabilities.
4. Materials and Methods: Details the methodologies used for sampling, microbial isolation, biochemical characterization, DNA extraction, and statistical analysis.
5. Results and discussion: Presents the findings regarding the isolated bacteria, their biochemical profiles, molecular identification via BLAST, and their degradation and tolerance performance.
6. Conclusions: Summarizes the study's findings on the identified strains (Pseudomonas sp. and Achromobacter sp.) and their potential for future bioremediation applications.
Keywords
Glyphosate, Bioremediation, Pesticide Resistance, Pseudomonas, Achromobacter, 16s rRNA, DNA Isolation, PCR, Phylogenetic Analysis, Soil Microbiology, Degradation Capacity, Kerala, Roundup, Bioinformatics, BLAST.
Frequently Asked Questions
What is the primary focus of this research?
This work focuses on the isolation and molecular characterization of glyphosate-resistant bacteria from agricultural soil samples collected in Kerala, India.
What are the central themes of the research?
The central themes include soil pollution by pesticides, the effectiveness of bioremediation, microbial isolation techniques, and the molecular identification of resistant bacterial strains.
What is the primary objective of this study?
The primary objectives are to identify microorganisms resistant to glyphosate-containing pesticides and to assess their individual capacity to degrade glyphosate as a phosphorous source.
Which scientific methods were employed?
The researchers used microbial enrichment, morphological and biochemical tests (like Gram staining and IMViC tests), and molecular methods including DNA extraction, 16s rRNA PCR amplification, and sequence analysis using BLAST and MEGA7 software.
What does the main body of the work cover?
The main body covers the experimental design, the specific protocols for soil sampling and microbial identification, and the results of the laboratory experiments regarding glyphosate degradation and tolerance thresholds.
Which keywords characterize this work?
Key terms include Glyphosate, Bioremediation, Pseudomonas, Achromobacter, 16s rRNA, and PCR, among others.
Which two bacterial strains were specifically identified?
The research identified two distinct bacterial strains: Pseudomonas sp. and Achromobacter sp.
How did the researchers verify the tolerance of the bacteria?
The bacteria were tested on nutrient agar media supplemented with increasing concentrations of pesticide, ranging from 100µL to 2000µL, to observe their growth patterns and limitations.
What was the key conclusion regarding the bacterial strains' potential?
The researchers concluded that both strains showed significant potential for use in the bioremediation of glyphosate-contaminated environments, with strain S1 showing generally superior performance.
- Arbeit zitieren
- Dr. Prem Jose Vazhacharickal (Autor:in), Sajeshkumar N.K (Autor:in), Jiby John Mathew (Autor:in), C K Anjana (Autor:in), 2016, Isolation and molecular characterization of Glyphosate resistant bacteria from agricultural soils in Kerala, München, GRIN Verlag, https://www.grin.com/document/355561
