Evaluation and optimization of lysis method for microbial DNA extraction from epiphytic phyllosphere samples

Area of Science:

• Microbiology
• Molecular Biology
• Plant Science

Background:

• Analyzing epiphytic phyllosphere microbiomes is challenging due to plant-derived biomolecule interference.
• Sequencing-based techniques require high-quality DNA extraction for accurate microbial community analysis.
• Both mechanical and enzymatic lysis methods are commonly used for DNA extraction from phyllosphere samples.

Purpose of the Study:

• To evaluate the impact of mechanical versus enzymatic lysis on DNA extraction yield, purity, integrity, and microbial 16S rRNA gene copy number.
• To assess the effect of these lysis methods on bacterial community composition, diversity, and reproducibility using 16S rRNA gene amplicon sequencing.

Main Methods:

• Comparison of DNA extraction using mechanical and enzymatic lysis methods.
• Assessment of DNA yield, purity, and integrity (DIN).
• 16S rRNA gene amplicon sequencing to analyze bacterial community composition and diversity.

Main Results:

• Enzymatic lysis yielded significantly more DNA but with suboptimal quality and inconsistent community composition.
• Mechanical lysis resulted in lower DNA yield but higher purity, integrity, and reproducible microbial community profiles.
• Mechanical disruption demonstrated superior reproducibility across different extraction conditions.

Conclusions:

• Mechanical DNA disruption is more suitable for epiphytic phyllosphere samples than enzymatic disruption.
• Mechanical lysis ensures more reliable and reproducible microbiome analysis in phyllosphere studies.
• Optimized DNA extraction is crucial for accurate characterization of plant-associated microbial communities.