Method for the identification of Meloidogyne enterolobii by Real-Time PCR

Meloidogyne enterolobii is a tropical and subtropical root-knot nematode (RKN) posing a major threat to agricultural production systems worldwide. This nematode is a highly polyphagous species infecting a broad host range and is able to reproduce on crop genotypes carrying resistance genes to the major tropical RKN. These characteristics make the management of this nematode very difficult; therefore, its introduction and spread into the European Union territory would have severe economic and social consequences. M. enterolobii is a quarantine plant-parasitic nematode (Commission Implementing Regulation (EU) 2021/2285 Annex II: part A) included in the EPPO A2 list along with M. chitwoodi and M. fallax. These species must be controlled permanently throughout the European Union, as soon as they appear, regardless of the stage of development and/or the plants on which they are detected. Import controls have already detected M. enterolobii in soil, roots and tubers samples.
Regulatory requirements concerning this nematode mean that an official surveillance plan needs to be put in place for plant for planting, fruits and vegetables likely to host M. enterolobii. Until now, most of the identification of M. enterolobii was performed by morphological and molecular analyses, the latter using the conventional PCR test described by Wishart et al. (2002). As analytical methods are evolving in their performance, this work aimed to evaluate the performance of the real-time PCR identification approach developed by Kiewnick et al. (2015). The aim is to assess whether this test can be applied, regardless of the developmental stage of the nematodes (juveniles, females and males) (eggs were not taken into account in this study, as they are not included in the stages extracted from samples for identification) and whether the method is suitable and reliable for routine analysis.
So far, in the laboratory, Meloidogyne enterolobii has been identified by conventional PCR (Wishart et al. (2002). Both partners of the EURL, the French and Belgian National Reference Laboratories, took part in an EUPHRESCO project entitled "Development and validation of innovative diagnostic tools for detection and identification of Meloidogyne enterolobii in support of integrated plant protection strategies", in which TOPIC 4 gave rise to a Test Performance Study (TPS). The TPS aimed at assessing the robustness of a real-time PCR test developed by Kiewnick et al. (2015) (hereafter Kiewnick test) to detect M. enterolobii in nematode suspensions obtained from soils, roots or substrates. This conclusive validation was also the subject of a publication (Braun-Kiewnick et al. 2016).
Minor modifications were made to the test compared with the publication, for reasons of harmonisation with practices already in place in the laboratory and after checking that this had no impact on the performance of the test.