Rapid and specific detection of Pentastiridius leporinus by recombinase polymerase amplification assay.

Pentastiridius leporinus (Hemiptera: Cixiidae) is the main vector of an emerging and fast spreading sugar beet disease, the syndrome 'basses richesses' (SBR), in different European countries. The disease is caused by the γ-3-proteobacterium 'Candidatus Arsenophonus phytopathogenicus' and the phytoplasma 'Candidatus Phytoplasma solani' which are exclusively transmitted by planthoppers and can lead to a significant loss of sugar content and yield. Monitoring of this insect vector is important for disease management. However, the morphological identification is time consuming and challenging as two additional cixiid species Reptalus quinquecostatus and Hyalesthes obsoletus with a very close morphology have been reported in sugar beet fields. Further, identification of females and nymphs of P. leporinus at species level based on taxonomic key is not possible. In this study, an isothermal nucleic acid amplification based on recombinase polymerase amplification (RPA) was developed to specifically detect P. leporinus. In addition, real-time RPA was developed to detect both adults (male and female) and nymph stages using pure or crude nucleic acid extracts. The sensitivity of the real-time RPA for detection of P. leporinus was comparable to real-time PCR, but a shorter time (< 7 min) was required. This is a first report for real-time RPA application for P. leporinus detection using crude nucleic acid templates which can be applied for fast and specific detection of this vector in the field.

Effects of succession crops and soil tillage on suppressing the syndrome 'basses richesses' vector Pentastiridius leporinus in sugar beet.

BACKGROUND: Pentastiridius leporinus (Hemiptera: Cixiidae) is the most important vector of syndrome 'basses richesses' (SBR), a new disease that leads to severe economic losses in sugar beet. In this study, different soil tillage methods (ploughing and cultivator) and crops (winter wheat, spring wheat, maize and bare soil) following SBR-infested sugar beet were tested as potential management options in field trials. In the laboratory, the survival and development of first and third instar nymphs on wheat and maize was studied to further assess their suitability as host plants. RESULTS: In five out of seven field sites, reduced soil tillage had no effect on adult planthopper emergence compared to ploughing. In two sites, reduced tillage resulted in higher emergence rates. In nearly all field sites, up to 98.9% fewer emerging adults were detected in bare soil and maize, when compared to winter wheat. Under laboratory conditions, the lowest survival rate was found in first instar nymphs feeding on maize seedlings (4.2%), while 66.7% survived on wheat, over a period of 300 days. In contrast, 73.3% and 70% of third instar nymphs survived on wheat and maize over a period of 150 days. CONCLUSION: Soil tillage had little effect against Pentastiridius leporinus. Maize is a poor host for first instars but a suitable resource for third instar nymphs, the stage which encounters maize under field conditions. Hence, reductions in planthopper emergence in the field were likely caused by starvation due to the long host-free period between sugar beet harvest and the sowing of maize. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Molecular Detection of Pentastiridius leporinus, the Main Vector of the Syndrome 'Basses Richesses' in Sugar Beet.

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.

Life history traits and a method for continuous mass rearing of the planthopper Pentastiridius leporinus, a vector of the causal agent of syndrome 'basses richesses' in sugar beet.

BACKGROUND: The planthopper Pentastiridius leporinus (Hemiptera: Cixiidae) is the main vector of the γ-3 proteobacterium 'Candidatus Arsenophonus phytopathogenicus' which causes the syndrome 'basses richesses' (SBR) in sugar beet. SBR is a new and fast-spreading disease in Central Europe that leads to high yield losses. To date, the development of management strategies has been hampered by insufficient knowledge about general life history traits of the planthopper and, most importantly, the year-round availability of insects reared under controlled conditions. Rearing of P. leporinus has been considered challenging and to date no protocol exists. RESULTS: Here we describe a method for mass rearing P. leporinus on sugar beet from egg to adult that has produced five generations and over 20 000 individuals between June 2020 and March 2022. An alternative host such as wheat is not necessary for completing the life cycle. No-choice experiments showed that P. leporinus lays 139.1 ± 132.9 eggs on sugar beet, whereas no oviposition was observed on its nymphal host wheat. Head capsule width was identified as a trait that unequivocally distinguished the five nymphal instars. Developmental time from first instar to adult was 193.6 ± 35.8 days for males and 193.5 ± 59.2 days for females. Infection rates of adults were tested with a nested polymerase chain reaction. The results demonstrated that 70%-80% of reared planthoppers across all generations carried the SBR proteobacterium. CONCLUSION: The mass-rearing protocol and life history data will help overcome an important bottleneck in SBR research and enhance efforts in developing integrated pest management tools. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.