Surveying cereal-infecting geminiviruses in Germany--diagnostics and direct sequencing using rolling circle amplification.

Geminiviruses have spread in German cereal crops during the last few years. In order to identify and classify them, we have compared conventional techniques (enzyme-linked immunosorbent assays, polymerase chain reaction, bacterial cloning, and sequencing) with a newly developed method which uses rolling circle amplification (RCA), restriction fragment length polymorphism (RFLP), and direct sequencing without a bacterial cloning step. Whereas immunological methods utilising polyclonal antibodies were reliable for detection of geminiviruses, they did not discriminate between different German mastrevirus species, in contrast to RCA/RFLP. Direct sequencing gave high fidelity results with the same quality as conventional cloning and sequencing but with significantly reduced effort and costs. Based on a survey of field-derived cereal samples and on DNA sequences of distinct virus isolates we propose two new mastrevirus species, to be named Barley dwarf virus (BDV), and Oat dwarf virus (ODV) according to sequence differences and host range studies. The results show the applicability of RCA-based techniques for field studies and the possibility of sequencing a geminiviral genome without cloning. This approach will accelerate genomics studies of all viruses with small circular DNA genomes. In addition, RCA proves to be a reliable technique allowing for the detection of new geminivirus species as it does not depend on the knowledge of specific primers.