Identification and management of virulence genes in potato cyst nematodes

Abstract

Efficient and accurate diagnostic assays are essential for the design and evaluation of control measures of the potato cyst nematodesGlobodera rostochiensis andG. pallida by means of resistance. The hybridoma technology and the polymerase chain reaction (PCR) offer in potential various possibilities to design such diagnostic tests for routine purposes. We set out to devise a refined advisory system based on biochemical assays by using the following stepwise approach. In the early 80's a research program was started to develop an immunoassay to differentiate the two sibling species of potato cyst nematodes. Species specific monoclonal antibodies were raised against nematode proteins which are thermostable, abundant and homologous, and which enable reliable species identification using single eggs. The second step to improve the management of virulence genes is aimed at discriminating groups of populations within a species (‘virulence groups’ or ‘pathotypes’). The concept is that the number of initial populations introduced from South America is limited and that numerous Dutch populations (‘secondary founders’) are closely related by descent. Biochemical characters revealed by two-dimensional gel electrophoresis (2-DGE) of polypeptides, PCR in combination with restriction enzyme digests and RAPD (Random amplified polymorphic DNA) will be used to delineate groups of populations. The final diagnostic assay will be based on PCR. One of the challenges will be to devise a manageable number of primers to recognize all distinct groups. The third research line is aimed at developing a PCR assay based on the virulence genes themselves. Genetic studies showed that virulence inG. rostochiensis towards the H1 resistance gene is inherited at a single locus and is recessive to avirulence. To identify molecular markers linked to the virulence gene, 300 virulent lines were selected via backcrossing the F1 (Aa) with the virulent (aa) parent line. Molecular differences between the parent lines were obtained by 2-DGE, RFLP's (restriction fragment length polymorphisms) and RAPD. Especially RAPD proved to be a valuable technique to construct a linkage map. Screening 80 primers (10-mer) resolved more than 120 markers. RAPD will eventually lead to flanking DNA sequences, which will be used to isolate and characterize the virulence gene. Sequence information of the virulence gene inG. rostochiensis for the H1 resistance gene can be used to devise primers for a PCR assay and may also provide a starting point to isolate other virulence genes.