Sodium sulphite inhibition of potato and cherry polyphenolics in nucleic acid extraction for virus detection by RT-PCR.

Phenolic compounds from plant tissues inhibit reverse transcription-polymerase chain reaction (RT-PCR). Multiple-step protocols using several additives to inhibit polyphenolic compounds during nucleic acid extraction are common, but time consuming and laborious. The current research highlights that the inclusion of 0.65 to 0.70% of sodium sulphite in the extraction buffer minimizes the pigmentation of nucleic acid extracts and improves the RT-PCR detection of Potato virus Y (PVY) and Potato leafroll virus (PLRV) in potato (Solanum tuberosum) tubers and Prune dwarf virus (PDV) and Prunus necrotic ringspot virus (PNRSV) in leaves and bark in the sweet cherry (Prunus avium) tree. Substituting sodium sulphite in the nucleic acid extraction buffer eliminated the use of proteinase K during extraction. Reagents phosphate buffered saline (PBS)-Tween 20 and polyvinylpyrrolidone (PVP) were also no longer required during RT or PCR phase. The resultant nucleic acid extracts were suitable for both duplex and multiplex RT-PCR. This simple and less expensive nucleic acid extraction protocol has proved very effective for potato cv. Russet Norkotah, which contains a high amount of polyphenolics. Comparing commercially available RNA extraction kits (Catrimox and RNeasy), the sodium sulphite based extraction protocol yielded two to three times higher amounts of RNA, while maintaining comparable virus detection by RT-PCR. The sodium sulphite based extraction protocol was equally effective in potato tubers, and in leaves and bark from the cherry tree.

The infectivity of dimeric potato yellow mosaic geminivirus clones in different hosts.

Head-to-tail dimeric clones of both DNA A and DNA B of potato yellow mosaic geminivirus (PYMV) were constructed. These constructs were infectious when inoculated onto Nicotiana benthamiana plants either as DNA or by agroinoculation and were also infectious for tomato plants by agroinoculation. The dimers were not infectious for potato plants following inoculation by either method. Symptom induction required both DNA A and DNA B but agroinoculation with DNA A alone resulted in virus spread in 30% of the inoculated N. benthamiana plants. Leaf disc explants of N. benthamiana, tomato and potato could all be infected by agroinoculation indicating that the method of delivery of the DNA to intact potato plants was unsuitable for successful inoculation rather than an inherent inability of the virus to replicate/spread in potato per se. Neither whole plants nor leaf discs of sugar beet supported the replication of PYMV DNA.