Development of species‐specific primers for rapid diagnosis of Tetranychus urticae, T. kanzawai, T. phaselus and T. truncatus (Acari: Tetranychidae)

Abstract

Species diagnosis is of the utmost importance to both pest management and plant quarantine services. Because of difficulties in the morphological diagnosis of spider mites, molecular techniques are of great value to rapidly and accurately diagnose closely related species. We examined four species of genus Tetranychus (the green and red forms of T. urticae, and T. kanzawai, T. phaselus and T. truncatus), which are found in Korea and are of significance to plant quarantine services. DNA samples isolated from a single egg, larva or adult weighed 64–188 ng. We designed species‐specific primers by performing sequence alignment for 107 sequences of the ribosomal internal transcribed spacer 2 (ITS2) region, which we obtained from GenBank, and sequences generated in this study. Specific nucleotides of each species were selected for designing primers specific for each species. Each species‐specific primer pair, when used to perform PCR analyses, detected only the species from which it originated. However, a T. urticae‐specific primer pair did not discriminate between the green and red forms of this species. These species‐specific primers can be applied in practice for the rapid and accurate diagnosis of spider mite species in plant quarantine and in agricultural fields.

Species identification of spider mites (Tetranychidae: Tetranychinae): a review of methods

“…Osakabe, 1987 Gotoh, 1992Gotoh et al, 2007Arabuli et al, 2019Kajiwara et al, 2016Ben-David et al, 2007Hinomoto et al, 2007Matsuda et al, 2012Matsuda et al, 2013Arabuli et al, 2019Osakabe et al, 2008Arimoto et al, 2013Hurtado et al, 2008Ovalle et al, 2020Khaing et al, 2015Shim et al, 2016Zélé et al, 2018Sinaie et al, 2018Li et al, 2015 as Oligonychus and Amphitetranychus). However, some closely related species (e. g., from genus Tetranychus) may be indistinguishable from each other by their barcode se quences, as it has been shown for T. kanzawai, T. parakanzawai, and T. ezoensis having similar ITS sequences (Matsuda et al, 2013); and T. urticae and T. turkestani having similar COI gene sequences (Hinomoto et al, 2007;Konoplev et al, 2017).…”

“…Another approach for rapid mite identification has been selection of species-specific primers. The DNA regions where such primers are defined should be common for the species being determined, but the sequences themselves should differ between species of the same genus (Shim et al, 2016). In this case, both whole pairs of species-specific primers can be used, as well as a combination of one universal primer and the second primer being unique for a particular species.…”

Incomplete reproductive barriers and genomic differentiation impact the spread of resistance mutations between green‐ and red‐colour morphs of a cosmopolitan mite pest

“…Despite intensive work, evidence of genetic differentiation between the colour morphs is inconclusive. Previous molecular studies are restricted to examining one or a few loci, such as mitochondrial DNA, ribosomal DNA internal transcribed spacer regions (ITS) or microsatellites, which have generated conflicting evidence of both strong genetic differentiation and of genetic similarity between the morphs, in addition to revealing a putative history of introgression with closely related species, such as Tetranychus turkestani (Bailly et al, 2004; Auger et al, 2013; Ben‐David et al, 2007; Hinomoto et al, 2007; Lee et al, 1999; Li, 2010; Navajas et al, 2001; Navajas & Boursot, 2003; Noge et al, 2005; Ros & Breeuwer, 2007; Shim et al, 2016; Toda et al, 2000; Vargas et al, 2005; Xie et al, 2006). Both T. turkestani and T. urticae are polyphagous species that can co‐occur along their large geographical distribution (Ben‐David et al, 2007; Migeon & Dorkeld, 2023; Saba, 1975a, 1975b).…”

Development and use of a single real‐time PCR assay to identify the three spider mite species Tetranychus urticae, Tetranychus lambi and Tetranychus ludeni (Acari: Tetranychidae)

“…Species‐specific primers of ITS2 have been used to identify T. urticae , Tetranychus kanzawai , Tetranychus phaselus and T etranychus truncatus in Korea (Shim et al . 2016), and most recently multiplex PCR has been developed to detect spider species (Sinaie et al . 2018; Zele et al .…”