Development of a real‐time PCR method for the detection of the dagger nematodes Xiphinema index, X. diversicaudatum, X. vuittenezi and X. italiae, and for the quantification of X. index numbers

Abstract

The ectoparasitic dagger nematodes Xiphinema index and Xiphinema diversicaudatum, often at low numbers in the soil, are vectors of grapevine nepoviruses, which cause huge agronomical problems for the vineyard industry. This study reports a method, based on real‐time PCR, for the specific detection of these species and of the closely related non‐vector species Xiphinema vuittenezi and Xiphinema italiae. Specific primers and TaqMan probes were designed from the ribosomal DNA internal transcribed spacer 1 (ITS1), enabling the specific detection of single individuals of each of the X. index, X. diversicaudatum, X. italiae and X. vuittenezi species whatever the nematode population. The specificity of detection and absence of false positive reaction were confirmed in samples of each species mixed with the three other Xiphinema species or mixed with nematodes representative from other genera (non‐plant‐parasitic Dorylaimida, Longidorus sp., Meloidogyne spp., Globodera spp. and Pratylenchus sp.). The method was shown to be valid for the relative quantification of X. index numbers through its use, from crude nematode extracts of soil samples, in a greenhouse assay of grapevine accessions ranging from highly susceptible to resistant. As an alternative to time‐consuming microscopic identification and counting, this real‐time PCR method will provide a fast, sensitive and reliable diagnostic and relative quantification technique for X. index nematodes extracted from fields or controlled conditions.

Genetic diversity of Bursaphelenchus cocophilus in South America

“…Accurate and quick detection of this plant-parasitic nematode is very important to prevent the introduction and dispersal of the pest to new sites (Ye & Giblin-Davis, 2013;Van Ghelder et al, 2015). The design of speciesspecific primers is necessary for B. cocophilus, since there are no indisputable morphological diagnostic characters for this species in the described members of the genus.…”

“…The ITS-1/2 sequence can provide useful genetic markers for species, due to their low intraspecific variation (Gasser, 2001). The ITS-1/2 region was used for the design of species-specific primers in other species of nematodes such as Xiphinema, for conventional PCR (Oliveira et al, 2005) and real-time PCR (Van Ghelder et al, 2015); B. xylophilus nested PCRbased (Takeuchi et al, 2005) and real-time PCR (Cao et al, 2005;Ye & Giblin-Davis, 2013); Meloidogyne hapla, M. chitwoodi and M. fallax multiplex PCR (Zijlstra, 1997) and Pratylenchus coffeae (Saeki et al, 2003). Until now, B. cocophilus has been identified based only on morphological characters or by using molecular markers (28S, 18S and mtCOI) (Ye et al, 2007).…”

The feeding strategies of plant-parasitic nematodes

“…Beslenmelerini seçilen hücrelerde çok kısa bir sürede gerçekleştirmektedirler. Daha uzun sitilete sahip Belonolaimus, Trichodorus ve Xiphinema gibi nematod cinslerine bağlı türler, bitkilerin endodermal hücrelerinden (Şekil 4), nisbeten daha kısa sitilete sahip Tylenchorhynchus cinsine bağlı nematod türleri epidermal hücrelerden, Psilenchus, Tylenchus ve Atylenchus cinslerine bağlı nematod türleri ise yalnızca kılcal köklerden beslenmektedirler (Wyss, 1997;Bilgrami & Gaugler, 2004;Van Ghelder et al, 2015). Bu gruba ait nematodların konukçularında beslenmeleri sonucunda kök hücrelerinde nekrozlar, kök uçlarında gal benzeri yapılar (gall-likes welling) ve uzayan beslenme periyotları sonucunda ise hücre duvarlarının erimesi sonucu syncytia' ya benzer oluşumlara neden olabilmektedirler (Wyss, 1997).…”

“…Bu beslenme tipleri, nematodun bitkinin hangi dokusundan beslenebileceğini, bu dokularda nasıl zararlara neden olabileceğini göstermektedir. Bitki paraziti nematodların beslenme davranışları hakkındaki toprak üstü, toprak altı bitki dokularında beslenebilmeleri, hareketli ya da sabit özellikte olabilmeleri, beslenmeleri sonucu oluşturdukları özelleşmiş hücreler, beslenmelerinin ilerleyen dönemlerinde vücut yapılarında gözlemlenen farklılıklar, bazı bitki virüs hastalıklarına vektörlük yapabilmeleri gibi bilgiler, genel olarak bu canlıların cins düzeyinde sınıflandırılmasına imkân sunabilmektedir (Manzanilla-Lopez et al 2004;Van Ghelder et al, 2015).…”

A Rapid Diagnostic for Detection ofAphelenchoides besseyiandA. fujianensisBased on Real-Time PCR

“…(Koyama et al 2016;Oliveira et al 2017;Yan et al 2012Yan et al , 2013, Globodera pallida and G. rostochiensis (Nakhla et al 2010), Meloidogyne spp. (Sapkota et al 2016;Zhao et al 2010), Rotylenchulus reniformis (Sayler et al 2012), Xiphinema index (Van Ghelder et al 2015), and Bursaphelenchus xylophilus (Ye and Giblin-Davis 2013). However, few molecular assays for detection yet alone quantification of Aphelenchoides spp.…”