IMPROVED RNA EXTRACTION FROM WOODY-PLANTS FOR THE DETECTION OF VIRAL PATHOGENS BY REVERSE TRANSCRIPTION-POLYMERASE CHAIN-REACTION

An efficient procedure for the extraction of high-quality RNA from woo dy plants without the use of phenol, organic solvents, or alcohol prec ipitation is described. The method employs commercially available spin -column matrices and mitigates the inhibitory effects of plant polysac charides and polyphenolic compounds commonly observed on subsequent po lymerase chain reaction amplification when conventional extraction met hods are applied to woody plant species. The method described has been successfully used in the development of highly sensitive reverse tran scription-polymerase chain reaction (RT-PCR) techniques for the detect ion of a number of viruses in their woody hosts. The viruses detected included apple stem grooving capillovirus (ASGV), apple stem pitting v irus, Prunus necrotic ringspot ilarvirus (PNRSV), grapevine fanleaf an d Arabis mosaic nepoviruses, and grapevine leafroll-associated closter ovirus type 3. The method described was equally effective for the extr action of viral RNA from either budwood, leaves, or flower blossoms as determined by the equivalent RT-PCR detection of ASGV and PNRSV from these tissues. Detection of viral RNA in samples of total plant RNA pr epared using this method was found to be as sensitive as was previousl y described for the immunocapture RT-PCR technique.