A serological approach to the identification of potato cyst nematodes

Inglés. The potato cyst nematode species G.rostochiensis and G.pallida are a threat to the cultivation of potatoes. Their presence in the soil embodies a potential financial loss to the farmer either because of harvest reduction, or because of rejection of seed potatoes, and other crops with adhering soil, for certification. Together with crop rotation and the use of nematicides, resistant potato varieties are essential for the control of these parasites. A reliable, simple and quick screening test to characterize and monitor field infestations of potato cyst nematodes according to species should improve possibilities for control, by means of cultivating resistant potato varieties. Both parasites are hard to distinguish, and considerable attention has been paid to the identification of these nematode species by methods other than those based on morphological characteristics. Such methods have mostly been based on biochemical entities, but have not proven suitable for routine applications. In this thesis, the outlines are presented of an immunoassay, which is based on species-differentiating monoclonal antibodies, and which can be used for routine purposes.To raise species-differentiating (monoclonal) antibodies, species-specific antigens must be available. In chapter II the purification of two major groups of heat-stable proteins from homogenates of eggs of G.rostochiensis and G.pallida is described. SDS-polyacrylamide gel electrophoresis revealed two bands specific for G.rostochiensis with apparent molecular weights of 20.7 kD and 18.0 kD, and three bands specific for G.pallida with apparent molecular weights of 21.0 kD, 20.5 kD and 17.0 kD. Two-dimensional gel electrophoresis revealed that actually four thermostable proteins are present in either nematode species, of which two (present in the 17.0 kD and 18.0 kD band respectively) have the same apparent molecular weight but differ in iso-electric point. Conventional antisera made against proteins of either one of the Globodera species were shown to exhibit a strong cross-reactivity with these species-specific proteins.Monoclonal antibodies (McAbs) hold the promise of better differentiating reagents since they recognize a single epitope with a possible reduction in the extent of cross-reactivity. The development of such antibodies using thermostable proteins of either nematode species as an antigen source is described in chapter IV. The antibodies produced by three of the hybridomas (WGP 1, WGP 2 and WGP 3) bind preferentially to thermostable protein antigens of G.pallida while two other hybridomas (WGR 11 and WGR 12) produce antibodies which prefer binding to proteins of G.rostochiensis . Most of the hybridomas which were isolated, however, produce antibodies which bind to thermostable proteins from both potato cyst nematode species. To quantitate the differences in affinity, binding constants were determined (chapter IV) according to the method as described in chapter III. In immunoblotting experiments, it was demonstrated that all G.pallida specific, and some aspecific, McAbs bind to the same two proteins in a mixture of four thermostable proteins from either G.pallida alone, or G.rostochiensis and G.pallida . Besides their very similar physico-chemical characteristics this can be interpreted as another indication that these proteins are homologous. In addition, the reactivity with thermostable proteins from other commonly occurring cyst nematodes was also investigated. Three categories of McAbs could be distinguished: i) WGP 2 and WGP 3, which only bind to thermostable proteins from G.pallida , ii) WGP 1, which binds to proteins of both G.pallida and G.rostochiensis , and iii) all other McAbs, which bind to thermostable proteins of potato cyst, and other cyst nematodes. In chapter V, the topological relationship between the antigenic sites on the G.rostochiensis and G.pallida antigens as defined by six of these McAbs was determined with a competition ELISA.From the McAbs so far characterized, a deliberate choice was made with respect to the development of a routine test for the identification of the potato cyst nematodes G.rostochiensis and G.pallida . In chapter VI, the reactivity of these McAbs in a direct ELISA was predicted and verified with the use of the formerly established binding constants.Thus, the McAbs obtained and characterized in this study establish a basis for the serological identification of the potato cyst nematode species G.rostochiensis and G.pallida by an immunoassay. In chapter VII, the implications of such an assay for the reduction of nematicide applications, and the utilization of resistant potato varieties is discussed.

Holandés; flamenco. M.b.v. de biotechnologische techniek van zuivere antistoffen is een routinetoets ontwikkeld voor de soortherkenning van aardappelcysteaaltjes, waartoe een aantal eiwitten zijn geisoleerd die soortspecifiek zijn voor aaltjes