A small-scale procedure for extracting nucleic acids from woody plants infected with various phytopathogens for PCR assay

Abstract

The complexity of most nucleic acid extraction procedures limits the number of samples that can be easily processed for analysis by polymerase chain reaction (PCR). A simple, small-scale procedure was developed which can be carried out entirely in 1.5-ml microfuge tubes whereby the container and contents are frozen with liquid nitrogen, tissue is pulverized, and targeted nucleic acids are extracted. DNA of bacterial and phytoplasmal plant pathogens was extracted in hot CTAB buffer followed by chloroform clarification. Following centrifugation, the DNA in the aqueous fraction was precipitated with isopropanol and resuspended in water. RNA originating from viruses and viroids was extracted from triturated tissue using STE buffer and phenol. The nucleic acid fraction was purified using CF-11 cellulose. All purified preparations were used as PCR or RT-PCR templates to detect DNA or RNA, respectively. These procedures were used to detect Xylella fastidiosa, peach yellow leaf roll phytoplasma, sour cherry green ring mottle virus, and peach latent mosaic viroid by agarose gel electrophoresis.