Biology of Pentastiridius leporinus and approaches to control the main vector of the syndrome ‘basses richesses’ in sugar beet

Pentastiridius leporinus (Hemiptera: Cixiidae) is the main vector of Syndrome ‘basses richesses’ (SBR), a fast spreading sugar beet (Beta vulgaris) disease in Central Europe. The disease is caused by two procaryotic phloem limited bacterial pathogens, the γ 3 proteobacterium ‘Candidatus Arsenophonus phytopathogenicus’ (here: SBR proteobacterium), and the stolbur phytoplasma (16SrXII A subgroup) ‘Candidatus Phytoplasma solani’ (here: SBR phytoplasma). SBR infections in sugar beet can lead to maximum 5 % losses in absolute sugar content and more than 25 % yield reduction in the taproots. P. leporinus has adapted from its natural host reed (Phragmites australis) to sugar beet in crop rotation with cereals such as winter wheat (Triticum aestivum) or spring barley (Hordeum vulgare). However, the developmental stages of P. leporinus have not been characterized and no rearing protocol for this planthopper is available. In addition, agronomical measures for sustainable P. leporinus control are lacking. Further, other species of Auchenorrhyncha were reported from sugar beet fields and species such as Hyalesthes obsoletus and Reptalus quinquecostatus (Hemiptera: Cixiidae) are morphologically closely related to P. leporinus, can be hardly distinguished and only male adults can be identified to the species level using morphological keys. Thus, a reliable molecular method is required for species identification of all P. leporinus developmental stages. To establish a P. leporinus rearing, suitable conditions such as temperature, humidity and light were optimized. The established rearing allowed continuous production of ca. 20,000 specimens and up to five generations exclusively on sugar beet in 21 months without dormancy. In a no choice experiment, median survival time for adults was 24 days and females laid 139.1 ± 132.9 eggs on sugar beet. In contrast, the females rapidly died (6 days median survival time) and did not oviposit on wheat plants. Development time of second to fifth instars increased with the age and first instar was most vulnerable (25.7 % mortality), compared to the other instars (3 13.4 % mortality). Finally, development time from first instar to adult was remarkably faster under controlled (males: 193.6 ± 35.8 days, females: 193.5 ± 59.2 days) compared to field conditions (ca. one year). The fifth instar nymphs were successfully (68.7 %) emerged as adults within 140 days after being transferred to potted sugar beets with a top layer of expanded clay. Further, in the first three generations of the rearing, 70 80 % of adult planthoppers (n=10) carried SBR proteobacterium according to polymerase chain reaction (PCR). In contrast, SBR phytoplasma was not detected. Thus, SBR proteobacterium infected P. leporinus can be cultivated using this protocol. To study agronomical measures for P. leporinus control, adult P. leporinus emergence was quantified after variations in soil tillage (tillage by plough or cultivator) and succession crops (spring wheat, spring barley, maize, and bare soil were compared to winter wheat) following SBR infested sugar beet in field experiments. In five of seven field trial sites, adult numbers from winter wheat did not differ between tillage by plough or cultivator and in two sites, adult P. leporinus emergence was significantly (p<0.05) lower after ploughing. Further, adult numbers were significantly reduced (up to 98.9 %) in bare soil and in the latest sown spring crop maize, in all three and four analysed field trial sites, respectively. Under controlled environmental conditions, survival and development of P. leporinus first and third instar nymphs were tested on wheat, maize, and barley (only tested for first instar) seedlings. The survival rates in first instar were 4.2 % on maize, 29.2 % on barley seedlings and 66.7 % on wheat over 300 days. Furthermore, 4.2 %, 29.2 %, and 62.5 % of first instar nymphs developed into adults on maize, barley, and wheat, respectively. In third instar nymphs, similar survival rates were observed for maize (70 %) and wheat (73.3 %) over 150 days. More, 56.7 % and 50 % of third instar nymphs developed into adults on maize and wheat, respectively. It is assumed that the effects observed in maize were caused by starvation of nymphs due the long host free period after sugar beet harvest. To establish a PCR based molecular method for reliable, time and cost saving identification of all P. leporinus development stages, species specific primers were designed on highly conserved parts of mitochondrial cytochrome oxidase I gene (COI) which had mismatches on the 3’ ends when compared to sequences of R. quinquecostatus and H. obsoletus. PCR method was established using two methods of template DNA preparation including a commercial kit or a simple, time and cost saving insect crushing in phosphate buffered saline (PBS). The established PCR based method allowed species specific detection of P. leporinus male and female adults and differentiation from R. quinquecostatus and H. obsoletus, and also other common Auchenorrhyncha species that were collected from German sugar beet fields. Further, all immature stages of P. leporinus (eggs and all five nymphal instars) were specifically detected. In addition, phylogenetic analysis based on the specifically amplified COI fragment showed that P. leporinus differs from the other close planthopper species including R. quinquecostatus and H. obsoletus. In conclusion, this study provides basic knowledge on the biology of P. leporinus and the mass rearing protocol enables year round availability and accelerated research on the planthopper. Furthermore, agronomic measures for vector control were analysed on multiple field trial sites and potential further steps for research are discussed. The established molecular method can be used for monitoring of all life stages of P. leporinus in sugar beet and other host plants without additional sequencing steps.

2023-08-22