3.3.5S
NEMATODE DIAGNOSTICS: IN SEARCH OF ANTIGENIC MARKERS

KG DAVIES

IACR-Rothamsted, Harpenden, Hertfordshire AL5 2JQ, UK

Background and objectives
Plant parasitic nematodes are important crop pests which are difficult to identify and quantify. The purpose of any diagnostic system is to identify the pests to a level whereby a management system can be applied. The tools available for the management of nematodes range from quarantine controls for plant health certification through crop rotation, resistant cultivars, trap cropping, biological control, the application of nematicides and soil sterilization. Each possible control measure requires the pest nematode to be identified as quickly and reliably as possible to a stringency compatible with the control techniques available. Identification of nematode pests traditionally has been done using morphology and host range, but more recently protein electrophoresis and now nucleic acid and immunological approaches are being developed and exploited for identification and quantification. At Rothamsted, research into immunological approaches to identify and quantify cyst and root-knot nematodes has been done and will be reviewed.

Materials and methods
Monoclonal antibodies have been raised to soluble protein homogenates extracted from cyst (Globodera and Heterodera spp.) and root-knot nematodes (Meloidogyne spp.) in either rats or mice using standard cell-fusion techniques. The products were screened by ELISA and potential diagnostic antigens characterized by electrophoresis and immunofluorescence. Where discrimination between species using antibodies proved problematic, a diagnostic protein, identified by electrophoresis, was purified using a method based on isoelectrofocusing, and antibodies raised to the purified protein.

Results and conclusions
Several fusions were performed and a small proportion of the antibodies raised could distinguish between different species, pathotypes and populations. However, only a very small proportion, less than 2%, were capable of differentiating the nematodes with the required stringency. For example, in the case of potato cyst nematodes, two monoclonal antibodies were identified as useful in differentiating Globodera pallida from G. rostochiensis by ELISA. The characterization of these antigens showed they recognized the same proteins as are currently used to identify these nematodes using isoelectric focusing. Immunolocalization studies showed the antigens to be associated with the anterior region of second-stage juveniles, notably the mouth and the amphids, and serendipitously they were only present in viable cysts. Although these antibodies did not cross-react with the majority of other plant parasitic nematodes tested, they were found to cross-react with Globodera tabacum. An immunoassay based on the homogenization of cysts has been developed which can differentiate G. pallida from G. rostochiensis. An immunoassay has also been successful in the identification and quantification of Heterodera avenae from processed soil samples containing up to 20% organic matter. The raising of antibodies to identify root-knot nematodes proved to be more problematic. Although monoclonal antibodies were identified which could differentiate some of the more important Meloidogyne spp. and differentiate them from cyst nematodes, early work could not differentiate the two most imortant ones, M. incognita and M. javanica. However, the raising of antibodies to an esterase enzyme, already used to differentiate one species of Meloidogyne from another, proved to be successful.

The withdrawal of the nematicide methyl bromide, and planned reductions in the use of other nematicides, focuses attention on alternative strategies for nematode control, of which crop rotation and the growth of resistant cultivars are going to become increasingly important. Therefore, the 'Holy Grail' of diagnostics will be the identification of markers which are predictive of host range. The work reported here with potato cyst nematodes shows that immunological techniques have the ability to fulfil such a role.

References
Davies KG, Curtis HC, Evans K, 1996. Pesticide Science 47, 81-87.
Curtis RHC, Al-Hinai MS, Diggines AER, Evans K, 1997. Nematologica 43, 199-213.