Easy and Efficient DNA Extraction from Woody Plants for the Detection of Phytoplasmas by Polymerase Chain Reaction

Abstract

A simple and efficient procedure for the extraction of high-quality DNA from phytoplasma-infected woody and herbaceous plants for polymerase chain reaction (PCR) detection is described. This procedure does not require phenol, chloroform, or alcohol for the precipitation of nucleic acids. Herbaceous and woody plant material are extracted in an identical manner with no additional purification or enrichment steps required. The method utilizes commercially available microspin-column matrices, and the extraction of total DNA can be achieved in less than 1 h. The method has been used to successfully purify phytoplasma DNA from whole leaves, leaf petioles and midribs, roots, and dormant wood from a diverse selection of plant material. The phytoplasmas detected by PCR include pear decline, western X-disease, peach yellow leaf roll, peach rosette, apple proliferation, Australian grapevine yellows, and Vaccinium witches'-broom.

Impact of metagenomic DNA extraction procedures on the identifiable endophytic bacterial diversity in Sorghum bicolor (L. Moench)

“…Seven plant mDNA extraction protocols were tested; two classical protocols (SDS-or CTAB-based) and five commercial kits (MoBio PowerPlant Pro® DNA Isolation Kit, Qiagen DNeasy R Plant Mini Kit, Fermentas GeneJet Plant Genomic DNA Purification Kit, MoBio PowerSoil™ DNA Purification Kit and MoBio UltraClean® Soil DNA Isolation Kit). These protocols have previously been used to study endophytic bacterial communities (Drabkova et al, 2002;Green et al, 1999;Krechel et al, 2002;West et al, 2010). All kit-based DNA extrac-tion protocols were performed according to the manufacturer's instruc-tions, with the exception that starting plant material quantities were always 0.1 g or 0.3 g, and the final elution was performed in 50 μL buffer for normalization purposes.…”

Detection and molecular characterisation of a group 16SrIX phytoplasma infecting citrus (Citrus sinensis and C. limon), coffee (Coffea arabica), periwinkle (Catharanthus roseus), and tabebuia (Tabebuia heterophylla) in Puerto Rico

“…As described by Green et al (1999), the total DNA extraction from petioles and leaf midribs was carried out using a modified DNeasy Plant Mini Kit protocol (Qiagen, MD). Finally, the DNA was eluted in 100 μl of Qiagen's AE buffer (pre-warmed to 65°C) and stored at −20°C until required.…”

Detection of PCR inhibition in food and feed with a synthetic plasmid

“…The isolation protocol thus needs to be further optimised, e.g. by incorporating an additional purification step based on solid-phase extraction (SPE) (Green et al 1999;Ganopoulos et al 2011).…”