A SMALL-SCALE PROCEDURE FOR EXTRACTING NUCLEIC-ACIDS FROM WOODY-PLANTS INFECTED WITH VARIOUS PHYTOPATHOGENS FOR PCR ASSAY

The complexity of most nucleic acid extraction procedures limits the n umber of samples that can be easily processed for analysis by polymera se chain reaction (PCR). A simple, small-scale procedure was developed which can be carried out entirely in 1.5-ml microfuge tubes whereby t he container and contents are frozen with liquid nitrogen, tissue is p ulverized, and targeted nucleic acids are extracted. DNA of bacterial and phytoplasmal plant pathogens was extracted in hot CTAB buffer foll owed by chloroform clarification. Following centrifugation, the DNA in the aqueous fraction was precipitated with isopropanol and resuspende d in water. RNA originating from viruses and viroids was extracted fro m triturated tissue using STE buffer and phenol. The nucleic acid frac tion was purified using CF-11 cellulose. All purified preparations wer e 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. (C) 1998 Elsevier Science B.V. All rights reserved.