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

SIMPLE SUMMARY: Pentastiridius leporinus is the main vector of a new and fast spreading disease, the syndrome ‘basses richesses’ (SBR) in sugar beet. SBR causes high sugar content and yield losses in Central Europe. Monitoring of this insect vector based on morphological identification is challenging as two other cixiid species Reptalus quinquecostatus and Hyalesthes obsoletus with similar external characters are known to additionally appear in sugar beet fields. In this study, a PCR-based method is provided for simple and reliable detection of P. leporinus collected via sweep nets and sticky traps. This method also detects eggs and all nymphal stages and differentiates this vector from the most common Auchenorrhyncha species occurring in sugar beet fields. Furthermore, the phylogenetic relationship of these morphologically close cixiid species was investigated based on the mitochondrial cytochrome oxidase I gene (COI). 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.