Potato (Solanum tuberosum) as a New Host for Pentastiridius leporinus (Hemiptera: Cixiidae) and Candidatus Arsenophonus Phytopathogenicus

Simple Summary Pentastiridius leporinus is a planthopper that transmits two bacterial pathogens to sugar beet plants: Arsenophonus and Stolbur. These bacteria cause an economically important disease known as syndrome basses richesses (SBR), characterized by deformed yellow leaves and low beet yields. Having observed potato fields in Germany infested with planthoppers and showing signs of leaf yellowing, we set out to identify the planthoppers and any transmitted pathogens. We identified P. leporinus by comparisons of COI and COII partial sequences and detected both bacterial pathogens in planthoppers, as well as in beet and potato plants. We found that P. leporinus nymphs and adults can transmit the bacteria and that long, warm summers allow two generations of planthoppers to breed, probably increasing the population size and threat of disease transmission in the following year. We conclude that the planthopper P. leporinus has expanded its host range to potato, and that further studies are needed, to facilitate the development of control strategies that minimize losses in sugar beet and potato crops. Abstract Pentastiridius leporinus is a planthopper (Hemiptera: Cixiidae) that vectors two phloem-restricted bacterial pathogens to sugar beet (Beta vulgaris (L.)): the γ-proteobacterium Candidatus Arsenophonus phytopathogenicus and the stolbur phytoplasma Candidatus Phytoplasma solani. These bacteria cause an economically important disease known as syndrome basses richesses (SBR), characterized by yellowing, deformed leaves and low beet yields. Having observed potato fields in Germany infested with cixiid planthoppers and showing signs of leaf yellowing, we used morphological criteria and COI and COII as molecular markers, to identify the planthoppers (adults and nymphs) primarily as P. leporinus. We analyzed planthoppers, potato tubers, and sugar beet roots and detected both pathogens in all sample types, confirming that P. leporinus adults and nymphs can transmit the bacteria. This is the first time that P. leporinus has been shown to transmit Arsenophonus to potato plants. We also found that two generations of P. leporinus were produced in the warm summer of 2022, which will probably increase the pest population size (and thus the prevalence of SBR) in 2023. We conclude that P. leporinus has expanded its host range to potato, and can now utilize both host plants during its developmental cycle, a finding that will facilitate the development of more efficient control strategies.


Introduction
The planthopper Pentastiridius leporinus (Linnaeus) belongs to the family Cixiidae (Hemiptera: Fulgoromorpha), and has been known to colonize sugar beet plants were transferred individually to microcentrifuge tubes or Petri dishes for further analysis in the laboratory. Field-collected adults were transferred to a 2-week-old potted potato plant var. Jule (Solana, Windeby, Germany), for egg deposition and hatching of first-instar nymphs. Plants were kept in the greenhouse in single cages (Cavea PopUp size M net cage (Howitec, Bolsward, The Netherlands)). The temperature of the chamber was set to 22/16 • C day/night, with a 16 h photoperiod. Images of field-collected and freshly-hatched nymphs were captured using a VHX-5000 digital microscope (Keyence Deutschland, Neu-Isenburg, Germany), and measured using the VHX-5000 communication software. Potato tubers expressing stolbur-like symptoms, characterized by the formation of side shoots, wilting, and rubbery tubers, were collected from the same locations. In the Eich and Ibersheim locations, we also collected 15 sugar beet samples with SBR symptoms, characterized by yellowing leaves, deformed young leaves, and necrosis of the vascular tissue and root tips.

Identification of Nymphs and Adults
To confirm the species, adults were morphologically examined [23] and DNA was extracted from nymphs and adults representing each location using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). A single fragment of the partial cytochrome oxidase subunit I and II (COI and COII) genes was amplified, as previously described [8], using primer pair C1J2441/C2N3661 [24]. The amplicons were purified with Exonuclease and SAP (New England Biolabs, Ipswich, MA, USA), followed by sequencing (Eurofins Genomics, Cologne, Germany). In total, seventeen adults were analyzed: for potato, four from Eich, seven from Lampertheim and five from Ibersheim; and for sugar beet, one from Ibersheim. As for the nymphs, nine were analyzed, one from potato in Eich, two from potato in Ibersheim, four from potato in Lampertheim, one from sugar beet in Eich, and one from sugar beet in Ibersheim. COI/COII sequences from three P. leporinus nymphs, collected from potato fields, one adult, collected from a sugar beet field, and reference sequences FN179288-FN179291 from P. leporinus and Hyalesthes obsoletus, were compared [8]. COI and COII sequences were trimmed to 972 bp and aligned using MUSCLE [25], and a phylogenetic tree was created using the neighbor-joining method and Geneious v10.2 (Biomatters, Auckland, New Zealand). Bootstrap analysis was conducted using 10,000 replications. The COI/COII sequences were submitted to Genbank, under accession numbers with sequential numbering from OQ420381 to OQ420405.

Abundance of P. leporinus in 2022
The abundance of P. leporinus was evaluated using transparent, sticky "cloak" traps (PAL, CSALAMON Group, Budapest, Hungary), 25 × 35 cm. The traps were set up at one location in Hesse (coordinates 49.758758, 8.577285). The site was a commercial sugar beet field, 13.2 km away from the examined potato field in Eich (Table 1). Three traps were placed at distances of 10, 30, and 50 m from the edge of the field, on a pole at a height of 10 cm above the sugar beet vegetation. The sampling period started in May and ended in September, after two continuous weeks without catches. The traps were replaced weekly and the number of adults was determined. To evaluate the ability of P. leporinus nymphs to develop on potato plants, the head capsule width and dorsal length of 100 field-collected nymphs were measured, using a VHX-5000 digital microscope.

Transmission Assays with Nymphs
The ability of P. leporinus nymphs to transmit Arsenophonus and stolbur to uninfected potato plants, was tested by releasing five field-collected nymphs (third to fifth instar) onto twelve potato plants and three sugar beet plants, followed by feeding for an IAP of 66 days. The control group consisted of three potato plants without nymphs. Field-collected P. leporinus nymphs were placed directly onto 3-week-old sugar beet plants (KWS Annarosa, Einbeck, Germany) and 5-day-old potato plants (Colomba HZPC, Joure, The Netherlands), growing in Göttinger 3 L pots (Lamprecht-Verpackungen, Göttingen, Germany), filled with Frühstorfer potting soil LD 80 (Heinrichs, Ingelheim, Germany) and covered with expanded clay (Floragard, Oldenburg, Germany). Plants were kept in single cage, in a Cavea PopUp size M net cage (Howitec, Bolsward, The Netherlands), and watered moderately twice weekly. The temperature in the greenhouse chamber was set to 22/16 • C day/night, with a 16 h photoperiod.
After 66 days, nymphs were collected from plants and pooled by plant origin for DNA extraction, as described above. Potato plants were divided into leaves, upper stem, midsection, lower stem, tubers, and roots. Sugar beet plants were divided into leaves, leaf stems, upper root, midsection of root, root tip, and tender roots. DNA extracts from plants were prepared from 0.1 g samples according to a modified CTAB procedure [26], followed by qRT-PCR (Section 2.6).

Comparison of Arsenophonus Isolated from Potato Tubers, Beet Roots, and P. leporinus
Nested PCR with primer pairs Fra5/rP1 and Alb1/Oliv1, was carried out to amplify the Arsenophonus 16S ITS sequence [3]. The products were separated by 2% agarose gel electrophoresis and stained with 0.1 µL/ml SYBR-Safe (Thermo Fisher Scientific, Waltham, MA, USA), followed by visualization under UV light. The products were then cleaned up and sequenced as described above. Multiple sequences were aligned with MUSCLE, using Geneious v10.2, as described above. The Arsenophonus sequences were submitted to Genbank, under accession numbers with sequential numbering from OQ411083 to OQ411090.

Analysis of Arsenophonus and Stolbur Infections
Ninety-one individual insects, from three locations, were analyzed: thirty-six adults and thirty-six nymphs from potato fields, as well as nine adults and ten nymphs from sugar beet fields. Following DNA extraction, as described above, TaqMan qRT-PCR was performed using Luna Universal qRT-PCR Master Mix (New England Biolabs, Frankfurt, Germany), 25 ng of template DNA, and primer pairs KL437/438 and KL464/465, for the detection of Arsenophonus and stolbur, respectively [7]. The number of target copies was determined using absolute standard curves. Samples with a CT value > 35 were considered negative, which is equivalent to 22 copies for Arsenophonus and 14 copies for stolbur.
To assess the abundance of infected potato and sugar beet plants in two locations, 30 symptomatic sugar beet root samples and 45 symptomatic potato tuber samples were collected, from three locations, and tested for Arsenophonus and stolbur. DNA extracts were prepared from 0.1 g tuber or sugar beet root samples, using a modified CTAB method [26] followed by qRT-PCR, as described above.

Identification and Morphometric Analysis of P. leporinus on Potato Plants
A recent increase in wilting syndromes in potato fields in Southwest Germany, was accompanied by the observation of hitherto unknown planthoppers in those affected fields. We identified the species as P. leporinus, based on morphological traits in potatoes (Solanum tuberosum var. Juventa). We found symptomatic potatoes colonized by nymphs, filamentous residues reminiscent of the characteristic wax structures of egg clutches, and hatching P. leporinus adults (Figure 1).

Identification and Morphometric Analysis of P. leporinus on Potato Plants
A recent increase in wilting syndromes in potato fields in Southwest Germany, was accompanied by the observation of hitherto unknown planthoppers in those affected fields. We identified the species as P. leporinus, based on morphological traits in potatoes (Solanum tuberosum var. Juventa). We found symptomatic potatoes colonized by nymphs, filamentous residues reminiscent of the characteristic wax structures of egg clutches, and hatching P. leporinus adults ( Figure 1).  Morphometric analysis was carried out on 100 of the field-collected nymphs (Table S1).

Molecular Identification of P. leporinus
We analyzed 23 individual nymphs and adults from potato and sugar beet fields, all of which (except one nymph) were identified as P. leporinus. The one exception, collected from sugar beet, was identified as Cixius sp., another planthopper in the family Cixiidae. Evolutionary relationships among the planthoppers and some reference specimens from other years and locations, were checked on the basis of COI and COII sequence similarity, resulting in the phylogenic tree shown in Figure 2. We analyzed 23 individual nymphs and adults from potato and sugar beet f of which (except one nymph) were identified as P. leporinus. The one exception, from sugar beet, was identified as Cixius sp., another planthopper in the family Evolutionary relationships among the planthoppers and some reference specim other years and locations, were checked on the basis of COI and COII sequence si resulting in the phylogenic tree shown in Figure 2. Based on COI and COII sequences, the planthoppers found in potato and su showed a closer genetic relationship to P. leporinus than the two known referen France and Russia. The nearest related species (P. beieri), as well as the more related H. obsoletus and Cixius sp. specimens, showed clear sequence differences.

Second Generation of Adults in Bickenbach, 2022
In 2022, 1336 P. leporinus adults were caught in a sugar beet field in Bickenbac using transparent, sticky traps ( Figure 3). The flight period lasted from calendar (mid-May) until calendar week 39 (end of September). At this location, 92.4% of tured individuals were males. Based on COI and COII sequences, the planthoppers found in potato and sugar beet showed a closer genetic relationship to P. leporinus than the two known references from France and Russia. The nearest related species (P. beieri), as well as the more distantly related H. obsoletus and Cixius sp. specimens, showed clear sequence differences.

Second Generation of Adults in Bickenbach, 2022
In 2022, 1336 P. leporinus adults were caught in a sugar beet field in Bickenbach, Hesse using transparent, sticky traps ( Figure 3). The flight period lasted from calendar week 20 (mid-May) until calendar week 39 (end of September). At this location, 92.4% of the captured individuals were males.
Surprisingly, two peaks of abundance were observed. The first occurred at the beginning of July (calendar week 27), followed by a decline until the end of July (calendar week 30), at which point the mean capture rate per trap fell to a low of 0.3 insects. Afterwards, the number of captured adults rose again, reaching a second peak at the end of August (calendar week 35). One insect was trapped at the end of September (calendar week 39). Insects 2023, 14, x FOR PEER REVIEW 7 of 13 Surprisingly, two peaks of abundance were observed. The first occurred at the beginning of July (calendar week 27), followed by a decline until the end of July (calendar week 30), at which point the mean capture rate per trap fell to a low of 0.3 insects. Afterwards, the number of captured adults rose again, reaching a second peak at the end of August (calendar week 35). One insect was trapped at the end of September (calendar week 39).

Transmission Assay
The detection of Arsenophonus and stolbur in nymphs and plants, by qRT-PCR, is summarized in Table 2. All groups of nymphs tested positive for Arsenophonus, but only one of fifteen groups tested positive for stolbur. Three of twelve potato plants tested positive for Arsenophonus, whereas stolbur was not detected in any potato plants. In the first potato plant, Arsenophonus was detected in the tubers with 45 copies. In the second plant, 141 copies were found in the roots. The third potato plant was more severely infected, with 85,635 copies in the lower stem, 5341 in the roots, 172 copies in the leaves, and 93 in the tubers. Arsenophonus and stolbur were both detected in one of the three sugar beet plants, which contained 23 copies of Arsenophonus and 1416 copies of stolbur in the root tip. Neither pathogen was found in the control group.

Similarity of Arsenophonus in all Organisms
The Arsenophonus qRT-PCR results were verified by nested PCR followed by agarose gel electrophoresis [3]. Infected potato tubers showed the same characteristic banding pattern as infected sugar beet and planthopper samples ( Figure S1). No bands were observed in the negative control. Two Arsenophonus amplicons from planthoppers, one from sugar beet, and four from potato plants were sequenced, revealing 100% identity

Transmission Assay
The detection of Arsenophonus and stolbur in nymphs and plants, by qRT-PCR, is summarized in Table 2. All groups of nymphs tested positive for Arsenophonus, but only one of fifteen groups tested positive for stolbur. Three of twelve potato plants tested positive for Arsenophonus, whereas stolbur was not detected in any potato plants. In the first potato plant, Arsenophonus was detected in the tubers with 45 copies. In the second plant, 141 copies were found in the roots. The third potato plant was more severely infected, with 85,635 copies in the lower stem, 5341 in the roots, 172 copies in the leaves, and 93 in the tubers. Arsenophonus and stolbur were both detected in one of the three sugar beet plants, which contained 23 copies of Arsenophonus and 1416 copies of stolbur in the root tip. Neither pathogen was found in the control group.

Similarity of Arsenophonus in All Organisms
The Arsenophonus qRT-PCR results were verified by nested PCR followed by agarose gel electrophoresis [3]. Infected potato tubers showed the same characteristic banding pattern as infected sugar beet and planthopper samples ( Figure S1). No bands were observed in the negative control. Two Arsenophonus amplicons from planthoppers, one from sugar beet, and four from potato plants were sequenced, revealing 100% identity with the Arsenophonus 16S ITS sequence DQ834353.1 (positions 80-419 bp) submitted by [3].

Prevalence of Arsenophonus and Stolbur in P. leporinus and Plant Material
We tested 91 P. leporinus adults and nymphs for Arsenophonus and stolbur, 69.2% of which were positive for Arsenophonus and 2.2% of which were positive for stolbur. Each adult positive for stolbur was also positive for Arsenophonus (Figure 4). The prevalence of Arsenophonus in both plant species was high: 95% of the sugar beet roots and 100% of the potato tuber samples were positive for this species. In addition, stolbur was detected in 20% of the potato tubers and 45% of the beet root samples ( Figure 5).
We tested 91 P. leporinus adults and nymphs for Arsenophonus and stolbur, 69.2% of which were positive for Arsenophonus and 2.2% of which were positive for stolbur. Each adult positive for stolbur was also positive for Arsenophonus (Figure 4). The prevalence of Arsenophonus in both plant species was high: 95% of the sugar beet roots and 100% of the potato tuber samples were positive for this species. In addition, stolbur was detected in 20% of the potato tubers and 45% of the beet root samples ( Figure  5).  We analyzed forty-five potato tuber samples in total. Among the fifteen samples from Eich, all were positive only for Arsenophonus, except a single uninfected sample. Among the fifteen samples from Ibersheim, seven were positive only for Arsenophonus, seven were positive for both pathogens and one was positive only for stolbur. Among the fifteen samples from Lampertheim, fourteen were positive for Arsenophonus alone and one sample was positive for both pathogens. We analyzed thirty sugar beet samples in total, all of which were positive for Arsenophonus or a double infection. Among the fifteen samples from Eich, seven were positive for Arsenophonus and eight were positive for both pathogens. Among the fifteen samples from Ibersheim, nine were positive for Arsenophonus and six were positive for both pathogens.

Discussion
Several species of planthoppers and psyllids cause serious damage to potato crops by direct feeding and/or by transmitting pathogens, especially phytoplasmas, bacteria, and viruses [27]. Many planthopper species associated with potato fields belong to the family Cixiidae, including H. obsoletus, Reptalus quinquecostatus (Dufour) and R. panzeri (Löw) [22]. Our study presents the first molecular evidence that potato plants are colonized by P. leporinus. We also confirmed adult feeding, egg laying, hatching, and nymphal feeding on potato plants and tubers. Our data therefore suggest that P. leporinus can move between sugar beet and potato host plants, as represented in an updated life cycle diagram ( Figure 6). To compare the suitability of sugar beet and potato as hosts, long-term life cycle trials are needed to understand the dynamics of P. leporinus developmental biology. To compare these host interactions, suitable protocols for some host plants under controlled conditions have already been established by [6].
We analyzed forty-five potato tuber samples in total. Among the fifteen samples from Eich, all were positive only for Arsenophonus, except a single uninfected sample. Among the fifteen samples from Ibersheim, seven were positive only for Arsenophonus, seven were positive for both pathogens and one was positive only for stolbur. Among the fifteen samples from Lampertheim, fourteen were positive for Arsenophonus alone and one sample was positive for both pathogens. We analyzed thirty sugar beet samples in total, all of which were positive for Arsenophonus or a double infection. Among the fifteen samples from Eich, seven were positive for Arsenophonus and eight were positive for both pathogens. Among the fifteen samples from Ibersheim, nine were positive for Arsenophonus and six were positive for both pathogens.

Discussion
Several species of planthoppers and psyllids cause serious damage to potato crops by direct feeding and/or by transmitting pathogens, especially phytoplasmas, bacteria, and viruses [27]. Many planthopper species associated with potato fields belong to the family Cixiidae, including H. obsoletus, Reptalus quinquecostatus (Dufour) and R. panzeri (Löw) [22]. Our study presents the first molecular evidence that potato plants are colonized by P. leporinus. We also confirmed adult feeding, egg laying, hatching, and nymphal feeding on potato plants and tubers. Our data therefore suggest that P. leporinus can move between sugar beet and potato host plants, as represented in an updated life cycle diagram ( Figure 6). To compare the suitability of sugar beet and potato as hosts, long-term life cycle trials are needed to understand the dynamics of P. leporinus developmental biology. To compare these host interactions, suitable protocols for some host plants under controlled conditions have already been established by [6].  The identification of species was not possible based on morphological characteristics alone during the nymphal stages, so we sequenced COI amplicons to provide speciesdependent molecular markers. We found that the nymphs and adults on sugar beet and potato plants were 100% identical to the P. leporinus reference sequences, which differed from the H. obsoletus and Cixius sp. amplicons. However, further genomic analysis of planthoppers is necessary to identify potential differences between populations.
Cixiid species such as P. leporinus are typically univoltine-they produce a single generation per year [4]. However, the examination of sticky traps and soil samples provided convincing evidence that the hot and dry summer in 2022 enabled P. leporinus to produce a second generation of adults in September, which were able to lay eggs. Under field conditions in 2020 and 2021, there was no clear evidence of bivoltine development, whereas a second generation of adults was also evident, from the monitoring of sticky traps, in 2018 [28]. The ability to produce two generations in 2022 is likely to increase the number of adults in the region in 2023. This enhances the threat of economic damage caused by P. leporinus, in regions where potato and sugar beet are cultivated in the same rotation. Rising temperatures, caused by climate change, are predicted to reduce crop yields, by encouraging the growth of pest insect populations [29], and projections in staple cereals such as rice, maize, and wheat indicate a yield loss of up to 25% per degree Celsius increase. Our data show a real-time change in voltinism in P. leporinus, probably due to the increase in temperatures.
During our field study in September, we found one Cixius sp. nymph on sugar beet in Eich (Rhineland-Palatinate). Cixius wagneri (China) is known to transmit Candidatus Phlomobacter fragariae, which causes marginal chlorosis in strawberries [30]. In France, C. wagneri in heavily-infested sugar beets, were shown to carry and transmit Arsenophonus [10]. We found one Cixius sp. nymph on sugar beet, but no adults in the sticky traps. Further studies monitoring cixiid species in sugar beet and potato crops, should also include Cixius species as potential Arsenophonus vectors in Southwest Germany. The recent finding of stolbur in sugar beet [6], and the observed host shift of P. leporinus to potato, may also influence the disease pressure in this region. In Ibersheim, two H. obsoletus individuals were found on sticky traps in sugar beet crops (data not shown). Further studies are needed to clarify the population density of H. obsoletus and identity which bacteria are carried. Interactions between sugar beet, potato, and vine plants could lead to an outbreak of stolbur in Rhineland-Palatinate's vineyards and should therefore be investigated in a timely manner [15].
We conducted transmission assays to determine whether P. leporinus can vector Arsenophonus and/or stolbur to potato plants. Transmission from adults to sugar beet seedlings was previously reported [10,12], and was possible within an IAP of 3 days and occurred over a period of 33 days. Transmission assays from nymphs to sugar beet seedlings were also conducted previously for an IAP of 30 days, revealing a transmission frequency of 60%, based on PCR detection [12]. We provided quantitative data confirming that P. leporinus nymphs were able to transmit Arsenophonus to the stem, tender roots, and tubers of potato plants, after 66 days, as well as the first evidence of stolbur transmission from P. leporinus nymphs to sugar beet. Stolbur depends on horizontal transmission via insect vectors [31,32]. H. obsoletus was previously used to confirm the transmission of stolbur to sugar beet seedlings, because no stolbur-infected specimens of P. leporinus were available at that time [10].
We detected Arsenophonus in P. leporinus, as well as sugar beet root tips and potato tubers from the field. We found that 90% of all potato and sugar beet samples tested positive for Arsenophonus. The qRT-PCR results were confirmed by endpoint PCR, using the primer pairs Fra5/rP1 and Alb1/Oliv1 [3]. All potato, sugar beet, and planthopper samples showed the same band pattern. Furthermore, Arsenophonus 16S ITS sequences from different samples were identical, showing that all isolated strains shared a close genetic relationship. Further sequencing of these strains should be carried out, to determine if there is a genetic basis for the host shift from sugar beet to potato.
In our study, only rubbery potato tubers and yellowing sugar beets were collected for analysis. Stolbur was detected in 20% of potato samples, significantly lower than the prevalence of Arsenophonus. The symptoms of Arsenophonus in sugar beet and strawberry plants are convergent with those of phytoplasmas [5]. We conclude that stolburlike symptoms in potato can also be triggered by Arsenophonus, and 80% of the examined potato tubers were infected with Arsenophonus alone.
More studies examining the relationship between Arsenophonus, potato plants, and planthopper vectors are urgently needed, because we currently lack an in-depth characterization of symptoms and the quantification of damage caused by these pathogens, under controlled conditions. It is also unclear where, inside the plant, Arsenophonus localizes, accumulates, and propagates. The prevalence of both pathogens in different regions of Germany is still unknown and must be assessed to facilitate appropriate containment measures. Seed potatoes could potentially spread both diseases [33]. Potential control strategies could include population suppression in P. leporinus and the modification of crop rotations to avoid consecutive crops of potato and sugar beet.

Conclusions
Sugar beet and potato are important root crops, with a significant socioeconomic value in the Rhine Valley. In Rhineland-Palatinate, both crops together covered more than 23,000 ha (6.0% of total arable land) in 2022 [34]. In this study, we present evidence that the planthopper P. leporinus has expanded its host range to potato, which will help in the development of control strategies to prevent the loss of sugar beet and potato crops. Phylogenetic analysis suggested that the planthoppers found in potato and sugar beet from different locations in Southwest Germany had a common origin. Most of the potato plants with disease symptoms such as side shoots, wilting, and rubbery tubers were colonized by Arsenophonus, in the absence of stolbur. We confirmed the ability of P. leporinus nymphs to transmit Arsenophonus to potato and stolbur to sugar beet. Our field data also provide evidence that P. leporinus can produce a second generation per year, under favorable conditions. Supplementary Materials: The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/insects14030281/s1, Table S1: morphometric measurements; Figure S1: gel electrophoresis.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.