Multiplex Polymerase Chain Reaction Identification of Single Individuals of the Longidorid Nematodes Xiphinema index, X. diversicaudatum, X. vuittenezi, and X. italiae Using Specific Primers from Ribosomal Genes

Abstract

The species X. index, X. diversicaudatum, X. vuittenezi, and X. italiae are established (E) or putative (P) vectors of Grapevine fanleaf virus (GFLV) (E), Arabis mosaic virus (E), Grapevine chrome mosaic virus (P), and GFLV (P) nepoviruses of grapevine, respectively. All four species are very closely related taxonomically and their low field densities make them difficult to identify from morphological and morphometrical diagnostic characters when only single or few individuals are detected. To improve diagnostic accuracy, a simple method was developed. The internal transcribed spacer 1 (ITS1) region spanning the 18S and 5.8S ribosomal genes was sequenced in one population of each species using two conserved primers from these genes. The ITS1 fragments were 1,132 bp (X. vuittenezi), 1,153 bp (X. index), 1,175 bp (X. diversicaudatum), and 1,190 bp (X. italiae), i.e., a difference of over 5% between the extremes. The sequence variability made it possible to design species-specific internal sense primers that amplified, in combination with the same antisense ITS1 primer, a single signature fragment (340 bp for X. index, 414 bp for X. italiae, 591 bp for X. vuittenezi, and 813 bp for X. diversicaudatum). Tests with DNA from a single adult or juvenile nematode confirmed the specificity of the primers from diverse isolates or populations. The primers were successfully used in a multiplex test for the reliable detection of two to four mixed species, each represented by a single individual. This multiplex-based diagnostic tool will be particularly useful for successful nematode management practices in vineyards.

Morphological characterisation and diagnostics of Xiphinema non-americanum group species (Nematoda: Longidoridae) from Romania using mutiplex PCR

“…9). Single fragments of approximately 590, 340, 800 and 410 bp were amplified for X. vuittenezi (lanes 2-3, Wang et al (2003). The PCR products for both studied populations of X. vuittenezi and X. italiae were of the same size (data not shown for Adamclisi and Bîrlad populations of X. italiae).…”

“…Amplification was carried out in a 25-μl reaction mixture containing the 2.5 μl lysis buffer (nematode lysate as PCR template), 1x Platinum Taq DNA polymerase buffer (Invitrogen), 1.5 mM MgCl 2 (Invitrogen), 0.2 mM each of dATP, dCTP, dGTP, and dTTP (Sigma 10mM), 0.8 pmol each primer, and 0.5 units of Platinum Taq DNA polymerase (Invitrogen). The following primers A-ITS1, I27, D24, V18, ITA26 were used (Wang et al, 2003). Amplifications were performed in a thermal cycler (Master cycler Pro SEppendorf), with the following cycling conditions: 95 °C for 3 min followed by 39 cycles at 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min 30 s, and ending with 1 cycle at 72 °C for 5 min and storage at 4 °C.…”

“…Xiphinema italiae, X. index and X. vuittenezi have been recorded from grapevine in different regions of Romania ..... (Romaşcu, 1971;Romaşcu & Zinca, 1974); X. diversicaudatum was recorded also from the rhizosphere of strawberry, cherry, peach and plum trees (Romaşcu, 1981), however the data on their morphology and variability are limited and developmental stages were not described. The purposes of this work are 1) to provide data on the morphology of adults and juvenile stages of these species from Romania; 2) to test the multiplex polymerase chain reaction using species-specific primers (Wang et al, 2003) and 3) to study the genetic variability of two X. vuittenezi populations which have shown differences in their morphology.…”

“…Xiphinema diversicaudatum, X. index, X. vuittenezi and X. italiae were recovered from vineyards and cherry fruit trees; adults and juvenile stages were described and analysed and the morphology/variability discussed. Multiplex PCR diagnostic test using species-specific primers designed by Wang et al (2003) yielded amplification products with expected lengths for all screened populations of these four species. Two ribosomal markers (D2-D3 28 LSU rDNA and ITS) were sequenced and ITS RFLP patterns were obtained from two X. vuittenezi populations, which have shown some morphological differences.…”

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 414 bp'da bant oluşturdukları saptanmıştır (Şekil 7 b). Bu bulgular, Xiphinema cinsi nematodların moleküler olarak tanımlanması amacıyla yürütülen çalışmada elde edilen sonuçlarla uyumluluk göstermiştir (Wang et al, 2003). …”