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Comparison of Methods for Isolating Entomopathogenic Fungi from Soil Samples
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Two efficient methods for isolation of high-quality genomic DNA from entomopathogenic fungi

María G Serna-Domínguez1, Gilda Y Andrade-Michel1, Hugo C Arredondo-Bernal1

  • 1Centro Nacional de Referencia de Control Biológico, Km 1.5 Carretera Tecomán-Estación FFCC, Col. Tepeyac, C.P. 28110 Tecomán, Colima, Mexico.

Journal of Microbiological Methods
|March 30, 2018
PubMed

Insights

Optimizing DNA extraction from entomopathogenic fungi (EPF) is crucial. Liquid nitrogen homogenization with DNeasy® Plant Mini Kit or SDS buffer yielded high-quality genomic DNA, outperforming other methods for downstream molecular applications.

Area of Science:

  • Mycology
  • Molecular Biology
  • Biotechnology

Background:

  • Entomopathogenic fungi (EPF) possess complex cell walls and secondary metabolites that hinder DNA extraction.
  • Existing commercial and conventional DNA isolation methods are not universally optimal for all fungal species.
  • Efficient genomic DNA extraction is vital for molecular studies and applications involving EPF.

Purpose of the Study:

  • To compare the efficacy of three commercial and three conventional DNA extraction methods for four EPF species.
  • To evaluate the impact of different cell lysis procedures (liquid nitrogen homogenization, bead-beating) on DNA yield, purity, and integrity.
  • To identify the optimal DNA extraction protocol for high-quality genomic DNA suitable for PCR-based analyses.

Main Methods:

  • Evaluated three commercial kits (Qiagen DNeasy®, Promega Wizard®, Axygen™ Multisource) and three conventional buffers (SDS, CTAB/PVPP, CTAB/β-mercaptoethanol).
  • Compared three cell lysis techniques: liquid nitrogen homogenization, tungsten-carbide bead-beating, and stainless-steel bead-beating.
  • Tested protocols on four EPF species: Beauveria bassiana, Hirsutella citriformis, Isaria javanica, and Metarhizium anisopliae.
  • Assessed DNA quality using PCR amplification of translation elongation factor 1-α (TEF) and molecular markers (ISSR, AFLP).

Main Results:

  • Liquid nitrogen homogenization combined with either the DNeasy® Plant Mini Kit (QN) or SDS buffer (SN) significantly improved DNA yield.
  • The QN and SN methods produced genomic DNA with good purity (~1.8 A260/230) and high integrity (>20,000 bp).
  • These lysis and buffer combinations outperformed bead-beating methods and other tested protocols.
  • Purified DNA from SN and QN protocols yielded reliable and reproducible results in PCR-based analyses.

Conclusions:

  • Liquid nitrogen homogenization coupled with DNeasy® Plant Mini Kit or SDS buffer provides a robust method for high-quality genomic DNA extraction from EPF.
  • The optimized SN and QN protocols ensure DNA suitable for various downstream molecular applications, despite inter-species variations.
  • This study offers valuable insights for selecting efficient DNA extraction strategies in entomopathogenic fungi research.
Keywords:
Bead-beatingDNA extractionEntomopathogenic fungiHigh-quality DNALiquid nitrogenMolecular markers

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