Strategies for the detection of potential beet necrotic yellow vein virus genome recombinations which might arise as a result of growing A type coat protein gene-expressing sugarbeets in soil containing B type virus.

We have searched for beet necrotic yellow vein virus (BNYVV) populations with a recombined genome which could possibly arise when transgenic sugarbeets expressing the coat protein gene of A type BNYVV are grown in soil containing Polymyxa betae carrying B type BNYVV, in soil samples from previous field release experiments and in a greenhouse model experiment. In order to accelerate the potential evolution of virus populations with recombined genomes in the model experiment, eight successive crops of sugarbeet plantlets were grown in the same soil samples over a period of 3 years. For the sensitive detection of recombined BNYVV genomes, we used nested PCRs with sense primers that are preferentially extended on the A type BNYVV sequence in the region of the coat protein gene and antisense primers which are preferentially extended on the B type BNYVV sequence in a region downstream of the coat protein gene which is not present in the transgene. Controls with mixtures of sap from plants which were singly infected with A or with B type BNYVV only revealed that, unless proper precautions are taken, PCR-mediated recombination artifacts may readily be produced. A method was developed that is able to detect A type/B type recombinant RNA molecules up to dilutions of one to a million in pure B type RNA molecules. Inspite of this high sensitivity we failed to detect any BNYVV with a recombined genome in the transgenic plants of the model experiment or at the sites of the previous field release experiments.