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

Abstract

Three randomly amplified polymorphic DNA (RAPD) markers, OPA-12420, OPB-061200 and OPA-01700, 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 régions de séquences caractéristiques (SCAR) par une technique PCR - Trois marqueurs d'ADN polymorphique amplifiée au hasard (RAPD) OPA-12420, OPB-O61200 et OPA-OI700, respectivement spécifiques des espèces de nématodes Meloidogyne arenaria, M. incognita et M. javanica, ont été identifiés. Après le séquençage de ces produits RAPD-PCR, les amorces les plus longues de 18 à 23 nucléotides ont été choisies pour compléter les séquences terminales d'ADN des fragments d'ADN. Cela a conduit à trois paires d'amorces spécifiques de l'espèce, utilisées pour amplifier les régions des séquences caractéristiques (SCAR). Les lots d'amorces SCAR mis au point ont été utilisés avec succès lors d'essais directs, rapides et surs pour identifier M. incognita, M. javanica et M. arenia. Les marqueurs peuvent être amplifiés à partir de l'ADN des masses d'oeufs, des juvéniles de deuxième stade ou des femelles. Cette technique d'identification spécifique est donc indépendante des différents états de développement du nématode. De plus la technique SCAR-PCR a été appliquée avec succès à l'ADN extrait du matériel végétal infesté. Cette méthode présente des potentialités d'amélioration permettant d'envisager des tests pratiques d'identification de routine, facilitant ainsi le contrôle de ces parasites économiquement importants.

Response of heat-stable tomato genotypes to Mi-1 virulent root-knot nematode populations

“…M. javanica was identified by Fjav/Rjav primer produced 670 bp (Figure 1). Our finding showed accordance with previous studies (Zijlstra et al, 2000;Randig et al, 2002, Devran & Söğüt, 2009. Therefore, molecular results indicated that root-knot nematode species used in this study become pure culture.…”

“…Pure culture isolates of M. incognita and M. javanica were confirmed by PCR analysis using the species-specific primers inc14F/inc14R (Randin et al, 2002) and Fjav/Rjav (Zijlstra et al, 2000). The presence of Mi-1 gene in tested plants was checked by Mi23 marker (Seah et al, 2007).…”

Identification of the economically important plant parasitic nematodes in vineyards areas of Izmir and Manisa provinces by morphological and molecular techniques

“…Bu primerlerin, pozitif olan örneklerde 420 bp'da bant oluşturdukları saptanmıştır (Şekil 3a). Zijlstra et al (2000), dört farklı M. arenaria popülasyonu kullanarak yaptıkları çalışmada, tüm örneklerde 420 bp spesifik DNA bandını elde etmişlerdir. Benzer diğer bir çalışmada Kök-ur nematodlarının moleküler olarak tek bireyden tanımlanması için teşhis anahtarlarının oluşturulması amacıyla üç farklı M. arenaria popülasyonu kullanılmış ve bu örneklerde 420 bp DNA bandı elde edilmiştir (Adam et al, 2007).…”

Natural occurrence of fungal egg parasites of root-knot nematodes, Meloidogyne spp. in organic and integrated vegetable production systems in Spain

“…Meloidogyne species were identified according to the morphology of the perineal pattern of the females, and by SCAR-PCR markers (Zijlstra et al 2000).…”