Detection of the Quarantine Species Thrips palmi by Loop-Mediated Isothermal Amplification

Abstract

Thrips palmi (from the order Thysanoptera) is a serious insect pest of various crops, including vegetables, fruits and ornamental plants, causing significant economic losses. Its presence constitutes a double threat; not only does T. palmi feed on the plants, it is also a vector for several plant viruses. T. palmi originated in Asia, but has spread to North and Central America, Africa, Oceania and the Caribbean in recent decades. This species has been sporadically noted in Europe and is under quarantine regulation in the European Union. For non-specialists its larval stages are indistinguishable morphologically from another widespread and serious insect pest Frankliniella occidentalis (a non-quarantine species in the European Union) as well as other frequently occurring thrips. In this study, we have developed a loop-mediated isothermal amplification protocol to amplify rDNA regions of T. palmi. The results were consistent whether isolated DNA or crushed insects were used as template, indicating that the DNA isolation step could be omitted. The described method is species-specific and sensitive and provides a rapid diagnostic tool to detect T. palmi in the field.

PCR-RFLP method to distinguish Frankliniella occidentalis, Frankliniella intonsa, Frankliniella pallida and Frankliniella tenuicornis

“…Sequences of 18S-ITS1-5,8S-ITS2-28S rDNA region from a number of thrips species (F. occidentalis, F. intonsa, F. pallida, F. tenuicornis, Thrips palmi, T. tabaci, T. major, T. trehernei, T. sambuci, T. simplex, T. roepkei), obtained during our previous study (Przybylska et al 2015) and F. schultzei sequence downloaded from NCBI databases (National Center for Biotechnology Information) were aligned using BioEdit software (Hall 1999) and regions conserved only for Frankliniella species were chosen for designing forward and reverse primers amplifying products of different lengths. Next, this Frankliniella species sequences alignment was used to indicate restriction enzymes sites which would allow for differentiation between F. occidentalis, F. intonsa, F. pallida and F. tenuicornis.…”

“…Also several protocols were developed for quarantine thrips species (e.g. Przybylska et al 2015). In this study PCR-RFLP (Polymerase Chain Reaction − Restriction Fragment Length Polymorphism) was used which allows for the amplification of a conserved region of DNA sequence using PCR followed by the digestion of the amplified fragment with appropriate restriction enzymes which enables the detection of genetic variation between identified species.…”

“…There are two other protocols for distinguishing thrips species using PCR-RFLP assay (Brunner et al 2002;Mainali et al 2008) but not for all four species analyzed in this study. Other protocols for thrips detection are based on real-time PCR (Huang et al 2010), PCR (Zhang et al 2012) or Loop-mediated Isothermal Amplification (LAMP) (Przybylska et al 2015).…”

“…There are some protocols for thrips species based on mtCOI (Brunner et al 2002;Zhang et al 2012) but there are some reports about several intraspecific variants of this region in the case of thrips (Rebijith et al 2012) which might result in an ambiguity of detection with this marker. We chose the ITS1 rDNA region because sequences analysis done in our previous study (Przybylska et al 2015) demonstrated its interspecific variations between Frankliniella and Thrips species and the stability within the studied species. Therefore we found appropriate fragments which made it possible to design universal primers and the amplification of DNA from all four analyzed species.…”

Loop‐mediated isothermal amplification (LAMP) for the identification of invasive Aedes mosquito species

“…Over the last decade, numerous LAMP assays have been developed for genetic analyses, not only mainly for the diagnosis of pathogens of medical, veterinary or agricultural importance (Bühlmann et al, 2013;Mansour et al, 2015;Li et al, 2017), but also for identifying food constituents (Focke et al, 2013) and other applications. With regard to insects, few LAMP assays have been reported for the identification of pest species (Huang et al, 2009;Hsieh et al, 2012;Fekrat et al, 2015;Przybylska et al, 2015;Ide et al, 2016;Blaser et al, 2018;Sabahi et al, 2018). The LAMP-based identification of mosquitoes has so far been developed only for the differentiation of Anopheles gambiae s.s. Giles, 1902and Anopholes arabiensis Patton, 1905(Bonizzoni et al, 2009.…”