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.


Introduction
The syndrome 'basses richesses' (SBR) is a fast-spreading sugar beet (Beta vulgaris) disease leading to up to 5% absolute sugar content loss and severe yield reduction of the taproot [1][2][3]. Since the first report in 1991, a fast spread of SBR occurred in eastern France, based on mitochondrial cytochrome oxidase I gene (COI) sequences for taxonomic insect identification. The COI gene was used for identification of planthoppers in the genera Reptalus or Hyalesthes using species-specific primers [27,29], for sequence analysis of several cixiid species including P. leporinus, R. quinquecostatus, and H. obsoletus [16,27,29] or for phylogenetic analysis of cixiid and delphacid planthoppers including P. leporinus, Reptalus cuspidatus, and H. obsoletus [34]. The aim of this study was to establish a species-specific, inexpensive and time-saving PCR detection for P. leporinus eggs, immature stages and both male and female adults allowing differentiation from two other closely related species (R. quinquecostatus and H. obsoletus). In addition, sequence analysis showed that the designed primers enable differentiation of P. leporinus from all other Auchenorrhyncha species that have been described from sugar beet fields, including morphologically and taxonomically close as well as distantly related species. Furthermore, two common sources of insect collections (sweep netting with direct preservation or sticky trap collection) and two methods of template DNA preparation were evaluated. The evolutionary relationships based on the P. leporinus partial sequence of the COI gene confirmed the relationship between closely and distantly related Auchenorrhyncha species.

Planthopper Collection and Morphological Identification
Closely related cixiids (adult P. leporinus, R. quinquecostatus, and H. obsoletus) were field-collected with sweep nets or yellow sticky traps during summer 2020 from several locations in Germany (Baden-Württemberg, Rhineland-Palatinate, and Saxony). Morphological identification of the sweep net collected insects was carried out within 24 h after collection. Sticky traps 10 cm × 25 cm ('Gelbe Insekten-Leimtafeln', Aeroxon Insect Control GmbH, Waiblingen, Germany) were collected after seven days and transferred into polypropylene cards ('office discount Sichthüllen DIN A4 glasklar glatt 0,12 mm', office discount GmbH, Neufahrn bei München, Germany). Sticky trap collected specimens were stored on the traps for 14-18 months (long term) at room temperature (15-25 °C) before morphological identification was carried out.
The most common Auchenorrhyncha species reported from German sugar beet fields (species are provided in Section 2.4) were collected during summer 2020 and stored on sticky traps for 1-2 weeks (short term) before morphological identification was performed [17]. The aim of this study was to establish a species-specific, inexpensive and time-saving PCR detection for P. leporinus eggs, immature stages and both male and female adults allowing differentiation from two other closely related species (R. quinquecostatus and H. obsoletus). In addition, sequence analysis showed that the designed primers enable differentiation of P. leporinus from all other Auchenorrhyncha species that have been described from sugar beet fields, including morphologically and taxonomically close as well as distantly related species. Furthermore, two common sources of insect collections (sweep netting with direct preservation or sticky trap collection) and two methods of template DNA preparation were evaluated. The evolutionary relationships based on the P. leporinus partial sequence of the COI gene confirmed the relationship between closely and distantly related Auchenorrhyncha species.

Planthopper Collection and Morphological Identification
Closely related cixiids (adult P. leporinus, R. quinquecostatus, and H. obsoletus) were fieldcollected with sweep nets or yellow sticky traps during summer 2020 from several locations in Germany (Baden-Württemberg, Rhineland-Palatinate, and Saxony). Morphological identification of the sweep net collected insects was carried out within 24 h after collection. Sticky traps 10 cm × 25 cm ('Gelbe Insekten-Leimtafeln', Aeroxon Insect Control GmbH, Waiblingen, Germany) were collected after seven days and transferred into polypropylene cards ('office discount Sichthüllen DIN A4 glasklar glatt 0,12 mm', office discount GmbH, Neufahrn bei München, Germany). Sticky trap collected specimens were stored on the traps for 14-18 months (long term) at room temperature (15-25 • C) before morphological identification was carried out.
The most common Auchenorrhyncha species reported from German sugar beet fields (species are provided in Section 2.4) were collected during summer 2020 and stored on sticky traps for 1-2 weeks (short term) before morphological identification was performed [17].
Morphological identification of planthoppers was carried out with a stereomicroscope according to the taxonomic key of Biedermann & Niedringhaus [30]. Family and genus of individual female adult specimens were identified by observation of wings, pronotum, mesonotum, postnotum, and tarsus. Furthermore, the genital structures of male adults were evaluated to allow morphological identification at the species level. Hereafter, sweep net collected specimens were preserved in 96% ethanol and at −20 • C and sticky trap collected specimens with glue attached were preserved in 60% or 70% ethanol at room temperature until further use. Additionally, P. leporinus eggs and all five nymphal instars were obtained from a rearing on sugar beet [13]. Developmental stages of nymphs were determined under a stereomicroscope according to the key of Pfitzer et al. [13], before specimens were preserved in 96% ethanol at −20 • C until further use.

Template DNA Preparation
Detailed information about experimental samples is provided in Supplementary  Table S1. Insect DNA templates were obtained either by using 'DNeasy Blood & Tissue Kit' (QIAGEN GmbH, Hilden, Germany) according to the manufacturer's instructions or simply by crushing the insects in phosphate-buffered saline (PBS) as described by Priti et al. [35] with slight modifications. Individual insects were transferred into 1.5 mL microcentrifuge tubes with 60 µL (Stictocephala bisonia adults: 120 µL) or 30 µL (eggs and nymphs) PBS (pH 7.4), then crushed with a sterile micropestle and incubated at 100 • C for 10 min. Additionally, the tubes were centrifuged for 10 min with 13,500× g at room temperature. The supernatant (template DNA concentrations are provided in Section 2.3) was used as a PCR template. DNA quality and quantity were analyzed with a spectrophotometer ('DeNovix DS-11 , DeNovix Inc., Wilmington, DE, USA). To avoid DNA contamination between samples, we used a single undamaged insect for DNA preparation. Furthermore, to avoid DNA degradation, DNA extracts by means of DNeasy Blood & Tissue Kit were diluted in AE buffer and PBS extracted DNA were used in a short time, within a week.

Primer Design and PCR Conditions
COI sequences of P. leporinus, R. quinquecostatus, and H. obsoletus were obtained from the NCBI database (National Center for Biotechnology Information, U.S. National Library of Medicine, Rockville Pike, MD, USA) and multiple-aligned with the software BioEdit 7.2 [36] for primer design. COI sequences of P. leporinus were also compared to each two additional representative taxonomically close Reptalus and Hyalesthes species (R. melanochaetus, R. panzeri, H. luteipes, and H. scotti) for species-specific primer design. The specific P. leporinus fw1 and rv1 primers were designed to have no miss-match with the COI gene of P. leporinus but show miss-match with the COI gene of the closely related species.
Furthermore, the specificity of the designed primers was tested In silico on all Auchenorrhyncha species reported to occur in sugar beet fields [5,11,17] for which COI sequences were available at the NCBI database. A list of primers (Table 1) is provided. A~1000 bp fragment of the COI gene was amplified with primers Ron and Calvin [37] and used as a control for DNA quality. Another PCR protocol was used for amplification of a~1000 bp fragment of the COI region from R. quinquecostatus and H. obsoletus with primers UEA3 and UEA8 according to Lunt et al. [38]. PCR reactions were carried out in a mixture with a final volume of 20 µL, consisting of 10 µL DreamTaq PCR Master Mix (2X), 0.5 µM of each primer and the same (UEA3 and UEA8) or double (Ron and Calvin) template DNA concentrations compared to species-specific PCR (described above). Thermocycling conditions consisted of 95 • C for 2 min, 35 cycles at 95 • C for 30 s, 51 • C (Ron and Calvin) or 54 • C (UEA3 and UEA8) for 30 s and 72 • C for 75 s and a final 72 • C step for 10 min.
PCR products were separated on 1 % agarose gels and stained with 'Gelred' (Biotium, Landing Pkwy, CA, USA) next to a 'GeneRuler 1 kb DNA ladder' (Thermo Fisher Scientific, Waltham, MA, USA). PCR products were sequenced (Microsynth Seqlab GmbH, Göttingen, Germany) and the data were used in phylogenetic analysis. Furthermore, COI sequences were aligned to sequences from the NCBI database to support morphological determination (see Section 2.1).

Application to Adult and Immature Specimens
The specificity of P. leporinus fw1 and rv1 primers was tested on both male and female adults of P. leporinus, R. quinquecostatus, and H. obsoletus using the two template preparation methods. Furthermore, these primers were also tested for detection of eggs and all nymphal instars of P. leporinus after PBS template preparation. For these assays, we had no access to R. quinquecostatus and H. obsoletus immature specimens, so only adults were used as the negative control.

Evolutionary Relationships
The amplified part of the COI (341 bp in size) of P. leporinus was sequenced and applied for BLAST search. Ten representative entries from Pentastiridius spp., Reptalus spp., and Hyalesthes spp. were selected to test their phylogenetic relationship using the neighbor-joining method [39]. Catonia carolina (family: Achilidae) and Tettigometra virescens (family: Tettigometridae) were used as outgroups. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches [40]. Furthermore, evolutionary divergence between sequences was estimated and the number of base substitutions per site from between the sequences is shown. The evolutionary distances were computed using the Maximum composite likelihood method [41] and are in the units of the number of base substitutions per site. All ambiguous positions were removed for each sequence pair (pairwise deletion option). Evolutionary analysis was conducted using MEGA X [42]. Similarly, the part of the COI gene (ca. 1000 bp depending on the species) amplified from P. leporinus, R. quinquecostatus, and H. obsoletus using universal primers, was sequenced, and used in a BLAST search. Each one representative COI sequence from the NCBI database per Auchenorrhyncha family and subfamily reported from sugar beet fields [17] was aligned and used for phylogenetic analysis, including another taxonomically close family Delphacidae [43]. This was to show whether the amplified COI sequence is helpful to group these closely and far related species.

Species-Specific Primer Design
In silico analysis was conducted to test the specificity of the newly designed P. leporinus fw1 and rv1 primers towards the COI gene of various species within the genera Pentastiridius, Reptalus, and Hyalesthes available from the NCBI database. No mismatches to the primers were observed for the different P. leporinus sequences (FN179289, FN179288, Figure 2A). However, one to four mismatches to the forward and three to nine mismatches to the reverse primer were observed in the sequences of each three Reptalus and Hyalesthes species, respectively. Each sequence displayed at least one mismatch at the 3 ends of both primers and the mismatches accumulated at the 3 ends (Figure 2A). The primer positions on P. leporinus COI are displayed in Figure 2B. In silico a 341 bp PCR product was obtained.

Species-Specific Primer Design
In silico analysis was conducted to test the specificity of the newly designed P. leporinus fw1 and rv1 primers towards the COI gene of various species within the genera Pentastiridius, Reptalus, and Hyalesthes available from the NCBI database. No mismatches to the primers were observed for the different P. leporinus sequences (FN179289, FN179288, Figure 2A). However, one to four mismatches to the forward and three to nine mismatches to the reverse primer were observed in the sequences of each three Reptalus and Hyalesthes species, respectively. Each sequence displayed at least one mismatch at the 3′ ends of both primers and the mismatches accumulated at the 3′ ends ( Figure 2A). The primer positions on P. leporinus COI are displayed in Figure 2B. In silico a 341 bp PCR product was obtained. Alignment of the specific primers to the COI gene of various Auchenorrhyncha genera or species, reported from sugar beet fields, showed 2 to 14 mismatches to the fw1 primer and 3 to 23 mismatches to the rv1 primer (Supplementary Figure S1). Most of the mismatches occurred at the primers 3′ end. Two exceptions (J. obscurella and N. campestris), Alignment of the specific primers to the COI gene of various Auchenorrhyncha genera or species, reported from sugar beet fields, showed 2 to 14 mismatches to the fw1 primer and 3 to 23 mismatches to the rv1 primer (Supplementary Figure S1). Most of the mismatches occurred at the primers 3 end. Two exceptions (J. obscurella and N. campestris), where the mismatches to P. leporinus fw1 primer were not located at the 3 ends, were observed. However, seven and ten mismatches, respectively, were observed for these two species to P. leporinus rv1 primer and at least two of the mismatches were located at the 3 ends. Therefore, distantly related Auchenorrhyncha species from sugar beet fields may not be detected with these specific primers.
Furthermore, the universal COI primer pairs Ron/Calvin and UEA3/UEA8 were aligned to the P. leporinus, R. quinquecostatus, and H. obsoletus COI sequences. Ron and Calvin primers were used for molecular detection of cixiids according to Urban et al. [44] and UEA3 and UEA8 primers were designed for general COI amplification of hemipteran insects [38]. The numbers and positions of mismatches are shown in Figure 3A,B. Primer positions on the COI sequences are represented in Figure 3C. Ron and Calvin primers each had a maximum of one mismatch with P. leporinus, R. quinquecostatus, and H. obsoletus COI. UEA8 primer had three mismatches with P. leporinus COI (one mismatch on the next-to-last Insects 2022, 13, 992 7 of 15 nucleotide at the 3 end, Figure 3B) which is expected to interfere with PCR amplification ( Figure 3C). Furthermore, the universal COI primer pairs Ron/Calvin and UEA3/UEA8 aligned to the P. leporinus, R. quinquecostatus, and H. obsoletus COI sequences. Ro Calvin primers were used for molecular detection of cixiids according to Urban et a and UEA3 and UEA8 primers were designed for general COI amplification of hemip insects [38]. The numbers and positions of mismatches are shown in Figure 3A,B. P positions on the COI sequences are represented in Figure 3C. Ron and Calvin primer had a maximum of one mismatch with P. leporinus, R. quinquecostatus, and H. obs COI. UEA8 primer had three mismatches with P. leporinus COI (one mismatch on the to-last nucleotide at the 3′ end, Figure 3B) which is expected to interfere with PCR a fication ( Figure 3C). leporinus, R. quinquecostatus, and H. obsoletus COI schematic maps. Arrows represent the locat the primers on the COI gene and show that Ron and UEA3 as well as Calvin and UEA8 partly lapped. The green color shows the fragment that was amplified and sequenced in this study fo species. The blue color shows the available sequence from the NCBI database. Figure 3. Alignment of the universal primers (Ron/Calvin and UEA3/UEA8) to P. leporinus, R. quinquecostatus, and H. obsoletus COI sequences and primer location within the COI gene. Alignment of (A) Ron/Calvin and (B) UEA3/UEA8 primers to the COI gene of P. leporinus, R. quinquecostatus, and H. obsoletus. Dots mark identical nucleotides in the primers and the analyzed sequences. Asterisks mark the positions of conserved nucleotides within primer sequences. Nucleotide mismatches between primers and analyzed sequences including numbers are indicated for each sequence. (C) P. leporinus, R. quinquecostatus, and H. obsoletus COI schematic maps. Arrows represent the locations of the primers on the COI gene and show that Ron and UEA3 as well as Calvin and UEA8 partly overlapped. The green color shows the fragment that was amplified and sequenced in this study for each species. The blue color shows the available sequence from the NCBI database.

PCR Validation on Adult Planthoppers
The specificity of P. leporinus fw1 and rv1 primers was tested on DNA templates, prepared with a DNeasy Blood & Tissue Kit, from male and female adult P. leporinus, R. quinquecostatus, and H. obsoletus. In the specific P. leporinus PCR, 100% of the P. leporinus specimens and no unspecific sample were detected ( Figure 4). However, in the general PCR using universal primers (Ron and Calvin), for both sweep net and sticky trap collected specimens, all samples were detected. Notably, 25% of sticky trap collected insects produced only weak bands. Furthermore, in the general COI PCR using UEA3 and UEA8 primers, no DNA amplification was observed for P. leporinus specimens but 100% of the R. quinquecostatus and H. obsoletus specimens produced amplicons. However, 50% of the PCR products obtained from sticky trap collected insects were rather weak (Supplementary Figure S2). PCR using universal primers (Ron and Calvin), for both sweep net and sticky trap collected specimens, all samples were detected. Notably, 25% of sticky trap collected insects produced only weak bands. Furthermore, in the general COI PCR using UEA3 and UEA8 primers, no DNA amplification was observed for P. leporinus specimens but 100% of the R. quinquecostatus and H. obsoletus specimens produced amplicons. However, 50% of the PCR products obtained from sticky trap collected insects were rather weak (Supplementary Figure S2). Amplification of COI fragments from PBS extracts is shown in Figure 5. PBS extracts had a lower quality, compared with DNeasy Blood & Tissue Kit DNA extracts (data not shown). A part of the COI was amplified from 75% of the sweep net and 100% of the sticky trap collected specimens in the general COI PCR with Ron and Calvin primers, however 25% of the sticky trap collected samples produced weak bands. In specific P. leporinus PCR, 100% of P. leporinus specimens and none of the other samples were detected. In the general COI PCR with UEA3 and UEA8 primers, DNA from none of P. leporinus and 75% (sweep net collected) or 100% (sticky trap collected) of R. quinquecostatus and H. obsoletus samples were amplified. However, most of the sticky trap collected samples produced rather weak bands. The obtained COI sequences in this study from P. leporinus, R. quin- Amplification of COI fragments from PBS extracts is shown in Figure 5. PBS extracts had a lower quality, compared with DNeasy Blood & Tissue Kit DNA extracts (data not shown). A part of the COI was amplified from 75% of the sweep net and 100% of the sticky trap collected specimens in the general COI PCR with Ron and Calvin primers, however 25% of the sticky trap collected samples produced weak bands. In specific P. leporinus PCR, 100% of P. leporinus specimens and none of the other samples were detected. In the general COI PCR with UEA3 and UEA8 primers, DNA from none of P. leporinus and 75% (sweep net collected) or 100% (sticky trap collected) of R. quinquecostatus and H. obsoletus samples were amplified. However, most of the sticky trap collected samples produced rather weak bands. The obtained COI sequences in this study from P. leporinus, R. quinquecostatus, and H. obsoletus using universal primers were aligned and the consensus sequences were submitted to the NCBI database (accession numbers ON094072, ON094073, and ON210854).

Detection of Immature Life Stages of P. leporinus
The COI was amplified from all immature P. leporinus specimens, including eggs and all five nymphal stages, using the universal Ron and Calvin primers and specific P. leporinus primers ( Figure 6). No DNA was amplified from immature specimens using UEA3 and UEA8 primers. In general, single, and clear bands with the expected product size were obtained for all specimens with specific primers. quecostatus, and H. obsoletus using universal primers were aligned and the consensus sequences were submitted to the NCBI database (accession numbers ON094072, ON094073, and ON210854).

Detection of Immature Life Stages of P. leporinus
The COI was amplified from all immature P. leporinus specimens, including eggs and all five nymphal stages, using the universal Ron and Calvin primers and specific P. leporinus primers ( Figure 6). No DNA was amplified from immature specimens using UEA3 and UEA8 primers. In general, single, and clear bands with the expected product size were obtained for all specimens with specific primers.

Detection of Distantly Related Species from Sugar Beet Fields
The specificity of P. leporinus primers was tested on the most common Auchenorrhyncha species from German sugar beet fields including closely and distantly related species. No DNA was amplified from other species besides P. leporinus with specific P. leporinus PCR (Supplementary Figures S2 and S3). In general COI PCR with Ron and Cal-

Detection of Distantly Related Species from Sugar Beet Fields
The specificity of P. leporinus primers was tested on the most common Auchenorrhyncha species from German sugar beet fields including closely and distantly related species. No DNA was amplified from other species besides P. leporinus with specific P. leporinus PCR (Supplementary Figures S2 and S3). In general COI PCR with Ron and Calvin primers, a part of the COI gene was amplified from P. leporinus, R. quinquecostatus, H. obsoletus, F. florii, J. pellucida, and J. obscurella specimens. The obtained COI sequences in this study of F. florii and Javesella sp. were aligned, and the consensus sequences were submitted to the NCBI database with the accession numbers OP090544, OP068197, and OP103664. In the general COI PCR with UEA3 and UEA8 primers, DNA from R. quinquecostatus, H. obsoletus and one P. alienus specimen was amplified.

Evolutionary Relationships
The phylogenetic relationship of morphologically closely related planthoppers was analyzed based on partial P. leporinus COI sequence amplified with specific primers and NCBI COI sequences of various species from the genera Pentastiridius, Reptalus, and Hyalesthes (Figure 7). The aim was to test whether the specifically amplified COI fragment is sufficient to differentiate those closely related species. Members of the three species clearly separated to different main branches of the phylogenetic tree, confirming morphological differences. Based on this analysis, two P. leporinus specimens from Russia (FN179288) and France (FN179289) were phylogenetically closest to the German collections and P. beieri was the closest species to P. leporinus in this study. Thus, intraspecific genetic distance to P. leporinus from Russia (0.0) and France (0.6) was lower than interspecific distance to P. beieri (5.1) (Supplementary Table S2). Therefore, the specifically amplified COI fragment was variable enough to differentiate Pentastiridius spp., Reptalus spp., and Hyalesthes spp. from each other.

Evolutionary Relationships
The phylogenetic relationship of morphologically closely related planthoppers was analyzed based on partial P. leporinus COI sequence amplified with specific primers and NCBI COI sequences of various species from the genera Pentastiridius, Reptalus, and Hyalesthes ( Figure 7). The aim was to test whether the specifically amplified COI fragment is sufficient to differentiate those closely related species. Members of the three species clearly separated to different main branches of the phylogenetic tree, confirming morphological differences. Based on this analysis, two P. leporinus specimens from Russia (FN179288) and France (FN179289) were phylogenetically closest to the German collections and P. beieri was the closest species to P. leporinus in this study. Thus, intraspecific genetic distance to P. leporinus from Russia (0.0) and France (0.6) was lower than interspecific distance to P. beieri (5.1) (Supplementary Table S2). Therefore, the specifically amplified COI fragment was variable enough to differentiate Pentastiridius spp., Reptalus spp., and Hyalesthes spp. from each other. Figure 7. Evolutionary relationships of selected members of Cixiidae based on the partial COI sequence amplified from P. leporinus using specific primers in this study and COI sequences from the NCBI database of closely related species from genus Pentastiridius and each three species from two taxonomically close genera Reptalus and Hyalesthes. The sequence obtained in this study is shown in bold. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The specifically amplified COI fragment is differentiating the closely related species. Catonia carolina from the Achilidae family and Tettigometra virescens from the Tettigometridae family were used as outgroups.
The phylogenetic relationship of closely and distantly related Auchenorrhyncha species reported from sugar beet fields based on the COI sequence amplified with universal primer pairs showed that P. leporinus, R. quinquecostatus, and H. obsoletus are closely related and grouped into Cixiidae (Supplementary Figure S4). This confirms the close mor- Figure 7. Evolutionary relationships of selected members of Cixiidae based on the partial COI sequence amplified from P. leporinus using specific primers in this study and COI sequences from the NCBI database of closely related species from genus Pentastiridius and each three species from two taxonomically close genera Reptalus and Hyalesthes. The sequence obtained in this study is shown in bold. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The specifically amplified COI fragment is differentiating the closely related species. Catonia carolina from the Achilidae family and Tettigometra virescens from the Tettigometridae family were used as outgroups.
The phylogenetic relationship of closely and distantly related Auchenorrhyncha species reported from sugar beet fields based on the COI sequence amplified with universal primer pairs showed that P. leporinus, R. quinquecostatus, and H. obsoletus are closely related and grouped into Cixiidae (Supplementary Figure S4). This confirms the close morphological features for these species. Additionally, these COI sequences were useful to clearly differentiate Cixiidae members from Delphacidae and all other representatives from different Auchenorrhyncha families and subfamilies reported in sugar beet fields.

Discussion
DNA barcoding is a well-established method for insect species identification [31]. It is based on the COI sequence comparison with database sequences [31,32]. In addition, insect sequences from internal transcribed spacers (ITS) or 5.8S-ITS2 rDNA are used for species-specific detection [27,28,45]. Species-specific molecular detection methods are rapid and cost-saving compared to analysis of morphological traits and reduce the risk of misidentification [31,46]. In the presented study, species-specific primers were designed on highly conserved parts of the COI gene of the target species as the COI gene was variable enough to distinguish P. leporinus from all other Auchenorrhyncha species reported from sugar beet fields. Supporting our approach, several studies demonstrated that the COI gene was exclusively and successfully used for species-specific insect detection. For example, the COI gene was used for species-specific detection of Reptalus spp. [27], Hyalesthes spp. [29], Trissolcus japonicus [46], and Hishimonus spp. [47].
In this study, a specific PCR assay was established to detect the main vector of the SBR disease in sugar beet. The method can be applied to detect P. leporinus and discriminate this insect from other morphologically closely related cixiids including R. quinquecostatus and H. obsoletus [25]. Additionally, the In silico analysis demonstrated that other more distantly related Auchenorrhyncha species, reported from sugar beet fields, will not be detected due to missing target sequence similarity. Supporting the In silico analysis, P. leporinus was differentiated from the most common Auchenorrhyncha species reported from German sugar beet fields, including taxonomically distantly related species such as Empoasca spp., F. florii or C. placida.
Immature stages represent the longest time-period of the P. leporinus life cycle [8] and morphological description as well as taxonomic keys are missing to precisely discriminate P. leporinus immature stages from other cixiids. Molecular methods have been used to identify the immature stages of insects which also expands the monitoring period of insect vectors [29,31]. Similarly, Figure 6 shows that the developed protocol allows detection of all P. leporinus immature stages.
We provide a PCR method that reliably (100% detection rate of P. leporinus specimens) detects both male and female P. leporinus, either from sweep net or sticky trap collection, even if the insects were preserved in 96% ethanol at −20 • C within 24 h after sweep net collection or stored for a short (1-2 weeks) or long time (up to 18 months) on the sticky traps at room temperature before they were preserved in 60 or 70% ethanol. Sticky trap collected specimens were successfully detected without removing sticky trap glue from the insect bodies. Additionally, we established this method with a simple and time saving DNA preparation by grinding specimens in PBS. PBS extracts were successfully used for specific detection of all insect life stages including eggs, nymphs, and adults. Thus, this simple and cheap method is suitable for large scale monitoring assays. Furthermore, sequencing of PCR products is not required due to the species-specificity of this protocol.
The published universal primers (Ron and Calvin) allow the detection of P. leporinus only after sequencing the PCR products which is time consuming. In addition, the amplicons for some samples are low in concentration possibly due to the degeneracy of primers.
With the lower quality of template DNA in PBS extracts, this degeneracy resulted in weaker signals. Due to the 100% amplification rate of the analyzed P. leporinus samples with specific primers, the provided specific primers are more efficient and precise, compared to universal PCR with Ron and Calvin primers. The Ron primer was originally designed for general amplification of lepidopterans, dipterans, coleopterans, thysanopterans, hemipterans, and homopterans [48] and the Calvin primer was originally used to analyze species from the genera Enchenopa and Campylenchia within the family Membracidae [49]. Later, the primer pair Ron and Calvin was used for molecular detection of planthoppers from the infraorder Fulgoromorpha and the families Cixiidae and Delphacidae [44]. Amplification of delphacid DNA with Ron and Calvin primers was also demonstrated in the study of Argüello Caro et al. [37]. In our experiments, cixiid (P. leporinus, R. quinquecostatus, H. obsoletus) and delphacid (J. pellucida, J. obscurella) DNA was amplified. However, specimens of the families Cicadellidae (exception: F. florii) and Membracidae (S. bisonia), which belong to the infraorder Cicadomorpha were not detected. Therefore, the Ron and Calvin primer combination was no perfect choice to generally detect all Auchenorrhyncha species by sequencing.
Although UEA3 and UEA8 primers were designed for general COI amplification of hemipteran insects [38], due to mismatches, they never amplified P. leporinus in this study. This primer pair therefore may only be of use as a negative control for P. leporinus detection. Additionally, only one of three P. alienus specimens was amplified besides R. quinquecostatus and H. obsoletus and no other distantly related species, suggesting that this primer pair is not suitable for general COI amplification of Auchenorrhyncha species from sugar beet fields.
The evolutionary relationships of numerous cixiid species including Pentastiridius sp., R. quinquecostatus, and H. scotti have been extensively analyzed based on a large fragment (3652 bp in size) of COI, Cytochrome b, nuclear 18S rDNA and 28S rDNA genes [43]. Similarly, the COI gene (800 bp in size) was used for phylogenetic analysis of cixiids and delphacids including P. leporinus, R. cuspidatus, and H. obsoletus [16,34]. Therefore, the COI gene is a suitable gene for differentiation of these species. In our study, we confirmed that a partial COI fragment (341 bp) that was specifically amplified from P. leporinus in comparison to sequences from R. quinquecostatus and H. obsoletus can be sufficient to differentiate these morphologically close species.
In addition, phylogenetic analysis for these species based on the generally amplified COI fragments (~1000 bp) in comparison to representative species of all Auchenorrhyncha families and subfamilies reported from sugar beet fields confirmed the close morphological features for these three species and that the two close families Cixiidae and Delphacidae can be clearly separated ( Figure S4). In several studies, the close relationship between Cixiidae and Delphacidae has been reported [25,43,44] which supports the presented phylogenetic analysis based on the COI gene.
In conclusion, we provide here a sensitive, cost-and time-saving molecular method for reliable and specific detection of all immature stages as well as male and female P. leporinus, after different methods of planthopper collection and template DNA preparation. This technique has the potential to be used in large scale monitoring assays.