Molecular Detection of Pentastiridius leporinus, the Main Vector of the Syndrome ‘Basses Richesses’ in Sugar Beet

Abstract

Monitoring of Pentastiridius leporinus (Hemiptera: Auchenorrhyncha: Cixiidae), representing the main vector of the syndrome ‘basses richesses’ (SBR) disease in sugar beet is based on morphological identification. However, two other cixiid species, Reptalus quinquecostatus and Hyalesthes obsoletus with similar external characters are known to appear in sugar beet fields and are challenging to be distinguished from P. leporinus. We present a PCR-based method for species-specific detection of both male and female P. leporinus, directly after sweep net collection or after up to 18 months long term storage on sticky traps. Two methods of DNA template preparation, based on a commercial extraction kit or on simple grinding in phosphate-buffered saline (PBS) were compared. The latter method was also established for eggs and all five nymphal instars of P. leporinus from a rearing. Furthermore, in silico primer analysis showed that all Auchenorrhyncha species including far related species reported from sugar beet fields can be differentiated from P. leporinus. This was PCR-confirmed for the most common Auchenorrhyncha species from different German sugar beet fields. Sequence analysis of the P. leporinus mitochondrial cytochrome oxidase I gene (COI) amplicon showed a close relationship to COI from P. beieri but separated from the Reptalus and Hyalesthes species which are grouped into the same family Cixiidae. We present a sensitive, cost- and time-saving PCR-based method for reliable and specific detection of eggs and all nymphal instars, as well as male and female P. leporinus, after different methods of planthopper collection and template DNA template preparation that can be used in large scale monitoring assays.

Multiplex PCR for Discriminating Host Plant Associations of Hyalesthes obsoletus (Hemiptera: Cixiidae), a Key Vector and Driver of ‘Ca. Phytoplasma solani’ Epidemiology

“…Comparing the COI haplotype diversity identified in our study with data on previously (partially) COI-genotyped H. obsoletus specimens [10,48,62,71,72] allowed us to identify geographic ranges for some of them that were distinct from our own. The H1 haplotype, which was found in Montenegro and Greece (Table 1), is also present in Northern Italy (FN179291 and GU552999), Southeastern France (isolate Ho-Ss5, LT841328), and Germany (ON210854), whereas the H2 haplotype from Serbia, Romania, North Macedonia, and Montenegro is also found in South Russia, Rostov Oblast (GU553002), and confirmed for Romania (GU553001).…”

“…Hence, the method of multiplex PCR identification of H. obsoletus host plant associations presented here could not be used to discriminate H. obsoletus sensu lato from its morphologically distinct congeners (a total of 32 species excluding H. obsoletus [81]). In contrast to methods previously developed for molecular identification and differentiation of morphologically distinct cixiid planthoppers, including H. obsoletus [10,11,48], the current method could not be used for the differentiation of H. obsoletus sensu lato, i.e., host plant associations, from other Hyalesthes species that may co-occur together, such as H. luteipes. However, the latter species has distinctive morphological features compared to H. obsoletus sensu lato, and thus this is not a true limitation but rather a notion that specimens should be examined through a stereo microscope and identified by external morphological characteristics to the genus level prior to the molecular identification of host associations.…”

“…In addition, connections between haplotypes were validated by performing TCS network and median-joining (MJ) calculation [70], keeping the parameter e = 0 using the software PopART version 1.7 (http://popart.otago.ac.nz) (accessed on 6 February 2023). Finally, the H. obsoletus COI gene haplotypes were compared to a previously published reference sequence to establish their relationship [10,38,48,62,71,72]. -H18).…”