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Updated: Mar 27, 2026
Isolation, Characterization, and Total DNA Extraction to Identify Endophytic Fungi in Mycoheterotrophic Plants
Published on: May 5, 2023
Direct DNA extraction method of an obligate parasitic fungus from infected plant tissue
1Key Laboratory of Agro-Biodiversity and Pest Management of Education Ministry of China, Yunnan Agricultural University, Kunming, Yunnan, China.
Abstract:
Powdery mildew and rust fungi are obligate parasites that cannot live without host organisms. They are difficult to culture in synthetic medium in the laboratory. Genomic DNA extraction is one of the basic molecular techniques used to study the genetic structure of populations. In this study, 2 different DNA extraction methods, Chelex-100 and cetyltrimethylammonium bromide (CTAB), were used to extract DNA from euonymus powdery mildew and Puccinia striiformis f. sp Tritici. Polymerase chain reaction was carried out with a race-specific-marker rDNA-internal transcribed spacer sequence. Both DNA extraction methods were compared and analyzed. The results showed that both Chelex-100 and CTAB were effective for extracting genomic DNA from infected plant tissue. However, less DNA was required for the Chelex-100 method than for the CTAB method, and the Chelex-100 method involved fewer steps, was simpler and safer, and did not require organic solvents compared to the CTAB method. DNA quality was evaluated by polymerase chain reaction, and the results showed that genomic DNA extracted using the Chelex-100 method was better than that using CTAB method, and was sufficient for studying the genetic structure of population.
Insights
Chelex-100 and cetyltrimethylammonium bromide (CTAB) methods effectively extract DNA from plant pathogens. Chelex-100 requires less DNA, is simpler, safer, and yields higher quality DNA for population genetic studies.
Area of Science:
- Plant Pathology
- Molecular Biology
- Genetics
Background:
- Powdery mildew and rust fungi are obligate parasites, making laboratory cultivation challenging.
- Genomic DNA extraction is crucial for studying fungal population genetics.
- Efficient DNA extraction methods are needed for these difficult-to-culture organisms.
Purpose of the Study:
- To compare the efficacy of Chelex-100 and cetyltrimethylammonium bromide (CTAB) methods for DNA extraction from plant pathogenic fungi.
- To evaluate DNA quality and suitability for downstream molecular applications, such as PCR.
- To identify the optimal DNA extraction method for population genetic studies of euonymus powdery mildew and Puccinia striiformis.
Main Methods:
- DNA was extracted from infected plant tissues using both Chelex-100 and CTAB methods.
- Polymerase chain reaction (PCR) was performed using a race-specific marker (rDNA-internal transcribed spacer sequence).
- Extracted DNA quality and yield were compared between the two methods.
Main Results:
- Both Chelex-100 and CTAB methods successfully extracted genomic DNA from infected plant tissues.
- Chelex-100 required significantly less starting material compared to CTAB.
- PCR amplification indicated that DNA extracted using Chelex-100 was of higher quality and more suitable for population genetic analysis than DNA extracted using CTAB.
Conclusions:
- Chelex-100 is a more efficient, simpler, safer, and cost-effective method for DNA extraction from powdery mildew and rust fungi compared to CTAB.
- The Chelex-100 method provides high-quality DNA suitable for population genetic studies.
- This method facilitates research on the genetic structure of fungal populations, aiding in disease management strategies.
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