Sensitive detection of potato spindle tuber viroid using RT-PCR and identification of a viroid variant naturally infecting pepino plants

Abstract

A reverse-transcription polymerase chain reaction (RT-PCR) method was developed to detect full length potato spindle tuber viroid (PSTVd) using primer pair 5'-CCCTGAAGCGCTCCTCCGAG-3' (complementary to PSTVd nucleotides 69-88) and 5'ATCCCCGGGGAAACCTGGAGCGAAC-3' (homologous to PSTVd nucleotides 89-113). A full-length amplified PSTVd cDNA was detected using RT-PCR in GeneReleaser-treated nucleic acid extracts of potato tuber tissues taken from tuber periderm, cortical parenchyma, external phloem, xylem ring, perimedullary zone containing internal phloem, perimedullary starch-storage parenchyma or pith regions. The viroid was also detected using RT-PCR from total nucleic acid or GeneReleaser-treated sap extracts of true potato seed (TPS), and pollen. PSTVd detection from total nucleic acid extracts of TPS can be achieved without further treatment with GeneReleaser or after further purification. GeneReleaser-treated sap extracts of as little as five pollen grains were sufficient to yield an amplified full-length PSTVd cDNA by this method. A one-step short version of RT-PCR was shown to detect PSTVd from infected tomato leaf nucleic acids. The RT-PCR protocol was successful in detecting a naturally occurring variant of PSTVd from pepino (Solanum muricatum) plants that consists of 357 nucleotides and differs from the prototype 359 nucleotide PSTVd isolate in five positions.