TABLE CONTENTS

comprise about 15-20% of the population. Quantification using real time PCR corresponded
to that using microscopic examination (r 2 ≥ 0.95) without concerns for variation in DNA
quantity of nematodes at different stages. The effect of DNA quantities in lesion nematode at
different stages and the stage structures in field samples on quantification will be discussed.
The method we developed should be efficient and cost effective particularly for large
numbers of samples for diagnostic services and research plots.
Reliability and Limits of Published Molecular Tests for the Specific
Identification of Potato Cysts Nematodes (PCN) of Quarantine Concern
Anthoine, G. (1), A.M. Chappé (1), D. Fouville (2), E. Henriquez Flores (3), D. Mugniéry (2)
& E. Grenier (2)
(1) Laboratoire National de la Protection des Végétaux – Unité de nématologie – Domaine de la Motte – BP
35327 – F-35653 LE RHEU cedex – France; (2) INRA, Agrocampus Rennes, Université Rennes 1, UMR1099
BiO3P, F-35653 LE RHEU – France; (3) Gobierno de Chile, Servicio Agricola y Ganadero, Laboratorio y
Estacion Cuarentenaria Agricola, Ruta 68, km 22, Santiago, Chile.
In the context of international trade and international or regional quarantine requirements,
diagnostic laboratories need reliable identification tools. In the European Union Globodera
pallida and G. rostochiensis are the only Globodera species considered as quarantine pests
(EU 2000-29 directive). For national survey or import controls, identification is often based
on morphological and molecular tests. But which the reliability for the result whether we
choose one tool or another and when faced to the wide diversity observed in South-America
where Globodera originated from? The specificity of nine published molecular tests (PCR,
PCR-RFLP) was evaluated against a set of G. pallida, G. rostochiensis, G. tabacum, ‘G.
mexicana’, and G. artemisiae populations, carefully chosen to represent the widest genetic
diversity known (Europe, The Americas). It appears that all tests are not reliable when
compared to published results. Overall, G. pallida populations were correctly identified, even
if some RFLP patterns were lacking (South American populations). But when considering G.
rostochiensis populations, several tests gave false negative or positive results. Lack of
amplification or observed at a wrong size respectively explain false negatives (especially for
one Bolivian population) and positives (confusion with other species). Most of the G.
tabacum populations were often confused with PCN and the ‘G. mexicana’ populations
usually identified as G. pallida. These results highlight the need of both a deep validation of
molecular tools before use for official analysis and further scientific investigations regarding
the taxonomic status of particular populations (G. ‘mexicana’ and some G. sp populations).
At present, common operating procedures for detection and identification of regulated
nematodes are discussed at a regional or international level. Thus we propose that the
development of new molecular tools should include an obligatory validation process on a
defined species set and the definition of limit of use before adoption as a reference test.
5 th International Congress of Nematology, 2008 125