Identification of Meloidogyne incognita, M. javanica and M. arenaria using sequence characterised amplified region (SCAR) based PCR assays

TLDR: Three randomly amplified polymorphic DNA markers species specific to the root-knot nematode species Meloidogyne arenaria, M. incognita and M. javanica respectively, were identified and the SCAR-PCR assay was successfully applied using DNA extracts from infested plant material.

Abstract: Three randomly amplified polymorphic DNA (RAPD) markers, OPA-12 420 , OPB-06 1200 and OPA-01 700 , species specific to the root-knot nematode species Meloidogyne arenaria , M. incognita and M. javanica respectively, were identified. After sequencing these RAPD-PCR products, longer primers of 18 to 23 nucleotides were designed to complement the terminal DNA sequences of the DNA fragments. This resulted in three pairs of species specific primers that were used to amplify the sequence characterised amplified regions (SCARs). The developed sets of SCAR primers were successfully used in straightforward, fast and reliable PCR assays to identify M. incognita , M. javanica and M. arenaria . The length variant SCAR markers can be amplified from DNA from egg masses, second stage juveniles and females. This species identification technique is therefore independent of the nematode's life cycle stage. Moreover the SCAR-PCR assay was successfully applied using DNA extracts from infested plant material. The method has potential to be optimised for routine practical diagnostic tests facilitating the control of these economically important pest organisms. Identification de Meloigyne incognita, M. javanica et M. arenaria au moyen de l'amplification de regions de sequences caracteristiques (SCAR) par une technique PCR - Trois marqueurs d'ADN polymorphique amplifiee au hasard (RAPD) OPA-12 420 , OPB-O6 1200 et OPA-OI 700 , respectivement specifiques des especes de nematodes Meloidogyne arenaria , M. incognita et M. javanica , ont ete identifies. Apres le sequencage de ces produits RAPD-PCR, les amorces les plus longues de 18 a 23 nucleotides ont ete choisies pour completer les sequences terminales d'ADN des fragments d'ADN. Cela a conduit a trois paires d'amorces specifiques de l'espece, utilisees pour amplifier les regions des sequences caracteristiques (SCAR). Les lots d'amorces SCAR mis au point ont ete utilises avec succes lors d'essais directs, rapides et surs pour identifier M. incognita , M. javanica et M. arenia . Les marqueurs peuvent etre amplifies a partir de l'ADN des masses d'oeufs, des juveniles de deuxieme stade ou des femelles. Cette technique d'identification specifique est donc independante des differents etats de developpement du nematode. De plus la technique SCAR-PCR a ete appliquee avec succes a l'ADN extrait du materiel vegetal infeste. Cette methode presente des potentialites d'amelioration permettant d'envisager des tests pratiques d'identification de routine, facilitant ainsi le controle de ces parasites economiquement importants.