SIMPLE AND RAPID PREPARATION OF INFECTED-PLANT TISSUE-EXTRACTS FOR PCR AMPLIFICATION OF VIRUS, VIROID, AND MLO NUCLEIC-ACIDS

A rapid, simple method for preparing plant tissues infected with virus es, viroids, or MLOs using a commercial product known as Gene Releaser (TM) is described. The Gene Releaser (TM) polymeric matrix method pro duced plant extracts suitable for PCR amplification without the use of organic solvents, ethanol precipitation, or additional nucleic acid p urification techniques. Modification of maceration methods and/or extr action buffers resulted in the PCR amplification of potato spindle tub er, apple scar skin, and dapple apple viroids, as well as, genomic seg ments of plum pox potyvirus, grapevine virus B, grapevine leafroll-ass ociated virus III, and elm yellows MLO. These pathogens were amplified from tissue of woody and herbaceous hosts such as peach, apricot, app le, grapevine, elm, periwinkle and potato. The application of this pro duct for use with intractable tissue avoids lengthy and laborious extr action procedures. In our hands, about 20 samples could be prepared fo r PCR or RT-PCR in 1-2 h versus 1-3 days.