Witczak, Natasha

In Germany, maize (Zea mays L.) is cultivated on approximately 2.4 Mha, making it one of the most widely grown arable crops alongside wheat and barley (German Federal Statistical Office, 2025). Although maize has previously been identified as a host of Candidatus Phytoplasma solani (PHYPSO, 16SrXII-A), particularly in relation to maize redness (MR) disease detected in southeastern Europe (Jović et al., 2009), its role as a host of ‘Candidatus Arsenophonus phytopathogenicus’ (ARSEPH) has not been reported previously. The planthopper Pentastiridius leporinus (Hemiptera: Cixiidae) is the major vector for transmitting both pathogens in many crops in Germany (Rinklef et al., 2024). MR symptoms include patchy premature drying, reddish discoloration of leaves and midribs, poorly filled or aborted ears, and malformed kernels. In August and September 2025, maize plants showing MR-like symptoms were observed in different fields in Otzberg 49°48'25.3"N, 8°52'28.8"E (South Hesse), Bellheim 49°10′19.9N, 8°15′50.8E, Bockenheim 49°36'18.6"N, 8°11'30. E"O, Ibersheim 49°43′36.5″N, 8°24′34.6″E (Rhineland-Palatinate) and Ettling 48°48'50.0"N, 11°40'17.2"E (Baden-Württemberg). Samples from roots (25), leaves (10), ears (14) and kernels (9) of 14 selected symptomatic plants were collected for molecular analysis. DNA was extracted using a CTAB-based protocol. ARSEPH was detected by qPCR targeting the manA gene according to Zübert and Kube (2021). Positive results were obtained from ears of six plants from Ibersheim and Ettling, leaves of three plants from Bockenheim and Otzberg, kernels of two plants from Bellheim, and roots of one plant from Bockenheim. Four ARSEPH-positive samples (two ears and two kernels) were further analyzed using the Fra4/Fra5 PCR assay (Zreik et al. 1998). Sanger sequencing of the resulting amplicons followed by BLAST analysis confirmed all four sequences as ‘Candidatus Arsenophonus phytopathogenicus’ with 100% identity to reference sequence PP400342. The sequences have been deposited in GenBank under accession numbers PZ623476, PZ623478, PZ623483, and PZ626926. Furthermore, all samples were screened for PHYPSO using the qPCR assay described by Behrmann et al. (2022) with primers KL464/KL465. Seven ear samples from Ibersheim yielded positive qPCR signals. To further validate these results, DNA from qPCR-positive samples was subjected to nested PCR targeting the 16S rRNA gene using primer pairs P1/P7 (Schneider et al., 1995) and R16F2n/R16R2 (Gundersen and Lee, 1996), followed by Sanger sequencing and BLAST analysis against the NCBI GenBank database. However, repeated sequencing did not provide sufficient sequence identity to the reference strain STOL11 (GenBank accession number AF248959) to confirm the qPCR-positive samples as PHYPSO. To our knowledge, this is the first report of ARSEPH in maize in Germany. Its detection in maize extends the known host range of this phloem-limited bacterium. The high ARSEPH loads in ears and kernels detected in reproductive tissues are consistent with preferential accumulation in sink organs during grain filling. Whether maize contributes to pathogen persistence and transmission within the Pentastiridius leporinus or other potential vectors’ pathosystem remains to be investigated.

‘Candidatus Phytoplasma solani’ of the 16SrXII group is an emerging vector-borne pathogen in European crop production. The cixiid planthopper Hyalesthes obsoletus transmits 16SrXII-A stolbur phytoplasmas that are associated with diseases in grapevine, potato, and various weeds. While 16SrXII-P genomes transmitted by Pentastiridius leporinus are available, no genome of an H. obsoletus-transmissible 16SrXII-A phytoplasma has been reported from Germany. Here, we present insights into the phylogenetic position and pathogen–host interactions through the functional reconstruction of the complete 832,614 bp genome of the H. obsoletus transmissible ‘Ca. P. solani’ 16SrXII-A strain POT from a potato field. Phylogenetic analyses highlight the heterogeneity within the stolbur group using whole-genome alignment and a BUSCO-based core gene analysis approach. The POT chromosome shares highest average nucleotide identity with Italian bindweed-associated genomes and displays strong synteny with the c5 strain. Consistent with the typical phytoplasma architecture, the POT genome combines mobile-element-driven instability with a conserved core metabolism. Virulence factors include transposon-linked effectors but lack pathogenicity island organisation. POT further differs from other 16SrXII-group phytoplasmas through unique collagen-like proteins that could contribute to virulence. These findings provide a robust genomic framework that improves diagnostics, enables strain-level resolution and supports the assessment of breeding materials under stolbur phytoplasma pressure, thereby refining our understanding of stolbur phytoplasma diversity and highlighting the evolutionary divergence within the 16SrXII subgroup.