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THE DIRECT QUANTITATIVE DETERMINATION OF SPECIES AND PATHOTYPES OF POTATO CYST NEMATODES IN SOIL BY ELISA AND DNA PROBES

Objective


Potato cyst nematodes Globodera pallida and G rostochiensis, cause substantial losses of potato production each year. An accurate, rapid identification method is proposed which will utilize monoclonal antibodies that will differentially recognize the two species of potato cyst nematodes (PCN) and which can be used in an enzyme linked immunosorbent assay (ELISA) to quantify population densities of PCN.

Nematode populations and potato genotypes appropriate for selection of virulence have been assembled and candidate propagation systems have been compared. Conditions for using a closed container system have been overcome by appropriate sterilization procedures. Polyclonal antisera and monoclonal antibodies (MAbs) have been raised to the 2 species of potato cyst nematodes (PCN). 2 polyclonal antisera raised against juvenile homogenates are being used experimentally. So far, 3 fusions to give cell lines producing MAbs raised against juvenile homogenates or juvenile proteins have been performed. About 20 antibodies which recognise the 2 species of PCN differentially have so far been identified and 2 of these have been used successfully in quantification assays.
Potato cyst nematodes, Clobodera pallida and G. rostochiensis, cause substantial losses of potato production each year and considerable expenditure is made in control programmes to minimize direct losses and in quarantine programmes to avoid further spread of these pests to presently uncontaminated land. Both types of programme rely upon an ability to identify these nematode species accurately. In addition, integrated control programmes require an accurate quantification of the numbers of nematodes present in soils. Identification of species and quantification of their population densities is presently achieved by labour-intensive methods which are subject to considerable errors and take several days to complete. Furthermore, the pathotypes, or races, that occur within each species can only be identified by tests which take several weeks to complete.

This project seeks to produce monoclonal antibodies that will differentially recognize the two species of potato cyst nematodes and can be used in an ELISA to quantify their population densities. Pure pathotypes will also be selected as a basis for production of antibodies that may differentially recognize pathotypes. The further problem of detecting ability in G. pallida to overcome resistance in cultivars with partially-inherited resisttance will be addressed by first producing nematode populations with selected levels of virulence. RFLP analysis will be used in an attempt to characterize virulent populations, followed by attempts to produce DNA probes which recognize these populations.

Provision is made within the project for optimization of ELISA protocols, the possibility of designing assays robust enough for use in the field, and the assessment of the utility of the tests in a surbey situation, where they will be required to work against a wide range of nematode populations.

Coordinator

BBSRC Institute of Arable Crops Research
Address
Rothamsted Experimental Station
AL5 2JQ Harpenden
United Kingdom

Participants (3)

UNIVERSIDADE DE TRAS-OS-MONTES E ALTO DOURO
Portugal
Address
Quinta De Prados
5000-911 Vila Real
Universidade de Coimbra
Portugal
Address
Paco Das Esculas
3000 Coimbra
Zeneca Group plc
United Kingdom
Address
Jealott's Hill Research Station
RG12 6EY Bracknell