Quantitative detection of Potato virus Y and Potato leaf roll virus by real time PCR – a molecular approach with numerous applications in potato research

Abstract

Virus resistance research on potato has a high priority. In African potato growing countries the majority of seed tubers offered on rural markets are infected with potato viruses, such as Potato virus Y (PVY) and/or Potato leaf roll virus (PLRV) resulting in a catastrophic reduction of yields. By contrast, in industrial nations virus infection of potatoes is virtually irrelevant due to efficient seed certification schemes. Nevertheless, the high yield loss potential has led PVY and PLRV becoming two of the most important pathogens on the potato crop worldwide. Due to their high replication rate viruses evolve quickly and could overcome existing resistances. In the recent history, common PVY strains, were more and more replaced by recombinant strains, such as PVYN-Wi , PVYN:O or PVYNTN that are highly virulent and able to cause the potato tuber necrotic ring spot disease (PTNRD). Taken together, these challenges in potato research and production call for an increased knowledge about the epidemiology of PVY and PLRV strains. Furthermore, breeding programs have to focus on the introduction of new resistance genes and generation of varieties that are adapted to the climatic conditions of their growing regions. These efforts can be supported by reliable and sensitive virus detection methods. In this thesis the development and applications of reverse transcription real-time PCR (RTqPCR) detection assays are presented that are able to quantify the worldwide most important potato viruses PVY and PLRV and thus can contribute to several areas of potato research. It is demonstrated that the developed RT-qPCR assay for the quantification of PVY is highly sensitive and can be utilized for the direct testing even of freshly harvested tubers during seed certification. A similar sensitive assay was applied to evaluate Solanum species and progenies of somatic hybrids regarding their level of resistance to PVY. The accumulation of PVY differed in progenies of wild potato species that previously were uniformly classified as extreme resistant. Therefore, RT-qPCR may be an interesting tool for a resistance evaluation in breeding programs. Another application of RT-qPCR is the estimation of virulence of different PLRV isolates. A correlation between the PLRV titer in potato plants and virulence could not be assessed. However, a discriminating quantification of different PLRV RNA species allows further epidemiological studies. Finally, RT-qPCR was demonstrated to be a useful tool to evaluate the equivalence of genetically modified potatoes regarding their level of susceptibility to PVY. An increased susceptibility to PVY could be a reliable indicator for possible unintended effects caused by the genetic modification. It is shown that equivalence is difficult to approve if the non-transgenic comparator is non-equivalent and if the classification into equivalent or non-equivalent is dependent on environmental conditions.