Kit-based, low-toxicity method for extracting and purifying fungal DNA from ectomycorrhizal roots

摘要

Ectomycorrhizal fungi are ecologically and economically important because when they form associations with roots of species such as pines, oaks, and eucalypts, they may increase nutrient and water uptake (15). Functional and taxonomic diversity among the ectomycorrhizal fungi is great and thus, in many studies, it is important to identify individual fungal species. During the past few years, researchers have perfected DNA-based methods for the identification of ectomycorrhizal fungi. In most cases, this involves the extraction and purification of DNA from colonized roots, amplification of fungal genomic DNA, and either analysis of restriction fragments or sequencing (1, 3-5,7,9,10). The extraction of DNA from ectomycorrhizal roots, as commonly published, involves some combination of manual grinding, freezing and thawing, and incubation at elevated temperatures, while purification may involve chloroform or phenol/chloroform extraction of impurities and precipitation of DNA by alcohol (3,5,7,10). The process may take hours to complete, and it requires the use of a fume hood to vent chloroform and phenol vapors. We desired a simple, inexpensive, rapid, and less hazardous method for extracting and purifying readily amplifiable fungal DNA from ectomycorrhizal roots, and a kit-based method seemed ideal. However, as far as we are aware, there are no kits specifically designed to extract and purify fungal DNA from ectomycorrhizal roots. Here, we report on the successful use of a commercially available kit to do this without manual grinding, specialized shaking apparatus, or phenol/chloroform.