Quantitative detection of cereal geminiviruses

Abstract

AbstractThe Geminiviruses _Wheat dwarf virus_ (WDV) and _Barley dwarf virus_ (BDV) cause symptoms such as dwarfing, stunting and yellowing in infected cereals and can lead to reduced yield and even death of the plant. Both viruses are transmitted by the leafhopper _Psammotettix alienus_ Dahlb. in a persistent non propagative manner. WDV is reported in Germany since the early 1990s. In the future an increase of infections is predicted due to global warming which leads to prolonged active periods of the vector in autumn. Furthermore infected plants can hibernate and serve as source of infection in spring.For the first time quantitative PCR assays were developed which allow the sensitive detection and quantification of WDV and BDV both in plants and vectors. Both viruses are detected with primers located in conserved regions of the viral genome. Taqman® Hydrolysis probes were designed for differentiating detection of WDV and BDV. The absolute quantification of viral copies is done by a standard dilution series of cloned viral DNA. The sensitivity of the method was compared to DAS-ELISA. For this purpose, sap of heavily infected plants was diluted with sap of healthy plants. With qPCR, virus was detectable in dilutions of 10-8 whereas the ELISA reaches the limit of detection at dilutions of 10-4.Field plants were analyzed to investigate how many viral copies can be found under natural infection conditions in late spring. ELISA and rating of symptoms were performed in parallel. It could be shown that viral copy number does not correlate with severity of disease symptoms.Winter barley cultivar ‘Rubina’ was inoculated with viruliferous _P. alienus_ under greenhouse conditions to monitor spatial development of infection. Virus titre increased continuously over 4 weeks. The virus was unequally distributed in the plant; the highest amounts were detected in the youngest leaf.Body and head of _P. alienus_ were analyzed separately to quantify virus content. The viruliferous leafhoppers were kept on plants for 7 days to infect them. It became evident that virus content of the vector does not correlate with its ability to infect plants.These highly sensitive and specific PCR assays allow a wide range of applications, e.g. investigations of the development of virus infection in the plant, vector transmission studies and comparisons of virus content of different cultivars for resistance breeding purposes.