Molecular detection and multilocus sequence analysis of Candidatus Phytoplasma solani-related strains infecting potato and sugar beet plants in Southern Germany

doi:10.21203/rs.3.rs-4001387/v1

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Molecular detection and multilocus sequence analysis of Candidatus Phytoplasma solani-related strains infecting potato and sugar beet plants in Southern Germany

https://doi.org/10.21203/rs.3.rs-4001387/v1

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Syndrome ‘basses richesses’ (SBR) disease in sugar beet caused by two phloem-limited pathogens, 'Candidatus Arsenophonus phytopathogenicus' and 'Candidatus Phytoplasma solani' is a fastspreading disease in Central Europe. The planthopper vector, Pentastiridius leporinus (Cixiidae), has recently expanded its host range to potato in Germany. However, the genetic diversity of 'Ca P. solani' in potato and possible association to SBR phytoplasma is unknown. In this study we charachterized ‘Ca. P. solani’ infecting sugar beet and potato plants in close distance fields in Southern Germany. Initially, the pathogen was detected in the potato tubers (18.7%) and sugar beet roots (60%) using TaqMan Real-Time PCR. Then, the sequences of 16S rRNA and other informative genes (stamp, vmp1, tuf and secY) were analysed in a number of infected potato and sugar beet plants. The phytoplasma strain infecting sugar beet in Southern Germany was classified into 16SrXII-P subgroup, a novel subgroup recently reported from sugar beet in Eastern Germany. While, the potato related strains were close to 16SrXII-A, which is a common subgroup for potato stolbur reported in Europe. The multilocus sequence analysis (MLSA) of non-ribosomal genes of the phytoplasma strains showed that the potato strain is clearly different from the sugar beet associated strain in this region. The presence and prevalence of 16SrXII-P in sugar beet in Southern and Eastern Germany suggests that this subgroup is dominant in sugar beet in Germany. In addition, this study elucidates for the first time, the genetic diversity of ‘Ca. P. solani’ strains in potato in Germany with a possible different source rather than sugar beet. Further investigation is required to investigate genetic variation of ‘Ca. P. solani’ in all sugar beet and potato-growing regions including weeds host in Central Europe to better understand the epidemiology of both sugar beet SBR and potato stolbur disease.

Phytoplasma

Pentastiridius leporinus

sugar beet SBR

Stolbur

Syndrome ‘basses richesses’ (SBR) in sugar beet (Beta vulgaris) is a fast-spreading disease in Central Europe (France, Switzerland and Germany) and causing up to 5% absolute sugar content loss with severe yield reduction of the taproot (Ćurčić et al., 2021amétey et al., 2007a, Bressan et al., 2008a, Gatineau et al., 2002). Symptoms of the SBR disease include asymmetric and lancet-shaped leaf deformations, yellowing of older leaves, and necrosis of the vascular tissue in roots (Bressan et al., 2008a). The causal agents of SBR are the phytoplasma from the stolbur group (16SrXII group) ‘Candidatus Phytoplasma solani’ and the γ-3-proteobacterium ‘Candidatus Arsenophonus phytopathogenicus’ (hereafter called: Arsenophonus) which are exclusively transmitted by planthoppers (Bressan et al., 2008amétey et al., 2007b). Pentastiridius leporinus (Hemiptera: Cixiidae) is the main vector for SBR disease in sugar beet fields (Sémétey et al., 2007a, Bressan et al., 2008a, Gatineau et al., 2002). This is due to the high population densities, infection rates and the ability to transmit both pathogens and vertical transmission of the SBR proteobacterium to their offspring (Bressan et al., 2008amétey et al., 2007a, Mahillon et al., 2022, Pfitzer et al., 2020). This planthopper has been host-shifted from natural host plant reed (Phragmites australis) to sugar beet and other crops including barley (Hordeum vulgare) and winter wheat (Triticum aestivum) during crop rotations (Bressan et al., 2009a). Recently, P. leporinus has expanded its host range to potato in Germany and both pathogens were detected in this crop (Behrmann et al., 2023).

Potato (Solanum tuberosum L.) plants are affected by phytoplasma diseases including ‘stolbur’, ‘purple top’ and ‘witches broom’ (Klein, 2001). Stolbur disease (yellow type disease) is characterized by a significant yield loss in crops including potato and sugar beet. Symptoms of stolbur in potato plants include aerial tubers, shortened internodes, upward rolling and purplish or red discoloration of the top leaves, early senescence and finally, plant wilt and death. In addition, the suitability of tubers for fried potato processing is affected by the increased sucrose content in infected tubers (Lindner et al., 2011).

Nine 16Sr groups of phytoplasmas including 16SrI, 16SrII, 16SrIII, 16SrV, 16SrVI, 16SrVIII, 16SrXII, 16SrXIII and 16SrXVIII have been reported in potato. 16SrXII-A (Ca. P. solani’) is the most commonly identified in potato plants in Europe (Belgium, Romania, Serbia, Greece, Montenegro, Turkey and Russia) (Holeva et al., 2014, Çağlar & Şimşek, 2022, Girsova et al., 2016, Ember et al., 2011, Jović et al., 2011, Mitrović et al., 2015). ´Bois noir´ is another economic and studied stolbur diseases affecting grapevines (Quaglino et al., 2013). The pathogenic agent ‘Ca. P. solani’ is transmitted (mainly?) by Hyalesthes obsoletus (Cixiidae) which is polyphagous and can complete its life cycle on a few plant species (Sforza et al., 1998, Maixner, 1994). Further, Stolbur causes rubbery tap root disease (RTD) in sugar beet in Southeast Europe (Ćurčić et al., 2021b) which is also transmitted by Reptalus quinquecostatus (Dufour) (Kosovac et al., 2023). Symptoms include yellowing and necrosis of the oldest leaves, wilting and rubbery root, decline of the aboveground and root rotting in some cases (Kosovac et al., 2023, Ćurčić et al., 2021b).

Multigene genotyping techniques have been developed to distinguish different phytoplasma strains. Since the routinely used 16SrDNA sequences often is not sufficient for strain discrimination and epidemiological investigations (Ray, 2013), additional molecular marker such as tuf (translation elongation factor Tu), secY (translocation protein SecY), vmp1 (variable membrane protein) and stamp (stolbur phytoplasma antigenic membrane protein) genes have been used to distinguish various strains of ‘Ca. P. solani’ (Quaglino et al., 2013, Fabre et al., 2011, Passera et al., 2020). Using this technique for identification of ‘Ca. P. solani’ strains, 80, 59 and 33 genetic variants based on the sequences of vmp1, stamp and secY, respectively, have been reported (Pierro et al., 2018). For example, the most common multilocus genotype for the causal agent of sugar beet RTD in Serbia and the Pannonian Plain is dSTOLg (tuf-d/STOL/V2-TA) (Ćurčić et al., 2021a). In the same report, a single stolbur phytoplasma (strain Z187; Bickenbach; Gemany) in sugar beet was also assigned in defined in tuf-b1/Z187/V4 group.

In addition, analysis of a tuf gene sequences show a link between ‘Ca. P. solani’ Tuf-types (a, b1, and b2) and various ecological systems (Pierro et al., 2018, Cvrković et al., 2014). This housekeeping gene encodes the elongation factor Tu and contains two main types based on an RFLP assay. Tuf-a type infects stinging nettle (Urtica dioica), Tuf -b (later called Tuf -b1) is linked to field bindweed (Convolvulus arvensis) (Johannesen et al., 2012) and Tuf-c was detected in H. obsoletus and hedge bindweed (Calystegia sepium) as an alternate host plant for grapevine yellows associated phytoplasmas in a limited area of Germany (Langer & Maixner, 2004). In Germany, Tuf-a strains of ‘Ca. P. solani’ spread through an epidemic cycle including U. dioica as main herbaceous host and Tuf-b phytoplasmas predominantly via a cycle with C. arvensis (Langer and Maixner 2004, Johannesen et al. 2012). In addition, two new Tuf groups, Tufab (Kosovac et al., 2016, Atanasova et al., 2015) and Tuf-b2 (Jamshidi et al., 2019) (Plavec et al., 2015), were reported in association with stinging nettle and grapevine, respectively.

SecY gene encodes an integral membrane protein that forms a heterotrimeric channel-like structure with SecG and SecE in the SecY secretion pathway system (Mori & Ito, 2001, Lee et al., 2006). The SecYEG translocon is conserved and plays an essential role in the bacterial protein transport regulation and translocation across the cytoplasmic membrane (Ma et al., 2019). Phylogenetic analysis based on the secY gene enables finer differentiation of phytoplasma strains as compared with 16S rRNA (Lee et al., 2010).

Stamp and vmp1 are also housekeeping genes that encode membrane proteins. These genes were used for phylogenetic analyses to better understand the phytoplasma population structures and dynamics (Quaglino et al., 2016, Murolo & Romanazzi, 2015). Vmp1 protein (57.8 kDa) contains a predicted C-terminal transmembrane domain that is most likely embedded in the phytoplasma membrane (Cimerman et al., 2009). Vmp1 involves in protein translation, secretion and maturation (Cimerman et al., 2009). Stamp encodes a protein with 157 amino acid length with a predicted signal peptide and also a C-terminal hydrophobic alpha-helix. This gene is more variable than the house-keeping gene secY (Fabre et al., 2011).

Investigating the genetic diversity of phytoplasma enables a better understanding their epidemiology and also monitoring and management of the phytoplasma associated disease. The presence of ‘Ca. P. solani’ strains (16SrXII-P subgroup) has been recently reported in sugar beet in Eastern Germany (Duduk et al., 2023). Further, potato stolbur was detected in potato fields (Behrmann et al., 2023), grapevine, U. dioica and C. arvensis in Germany (Ulrich, 2010, Jarausch et al., 2014) but not genetically characterized. There is no report on identification of potato strain/s and possible association to ‘Ca. P. solani’ infecting sugar beet in close distance. In this study, we investigated the genetic diversity of ‘Ca. P. solani’ infecting sugar beet and potato plants in Sothern Germany to shed light on the potential genetic variants of the pathogen in each crop and possible spread of ‘Ca. P. solani’ across potato and sugar beet by their common insect vector, P. leporinus. For this, initially the pathogen was detected in field-collected potato tubers and sugar beet roots using TaqMan Real-time qPCR. Based on the nested-PCR assay and sequencing for 16S rRNA and other informative genes (stamp, vmp1, tuf and secY), the genetic variation and their epidemiological relations were characterized. This study elucidates for the first time, the genetic variation of ‘Ca. P. solani’ strains in potato in Germany. Further, the phylogenetic relation of the potato strain and sugar beet strain in Southern Germany was analysed.

Sources of Nucleic Acids and Field Sampling

In September 2022, potato tubers from 11 fields in southern Germany (Meimsheim, Lauffen, and Massenbachhausen) and roots of a sugar beet in a field in the same region (Untereisesheim close to Heilbronn) were collected. Potato plants showed yellowing and mainly produced rubbery tubers. Sugar beet roots were collected from plants with typical SBR symptoms. The samples were cleaned and stored at 4°C until use.

Detection of SBR pathogens using TaqMan qPCR

Plant DNA was isolated from potato tubers and sugar beet roots. For this, 150 mg of plant tissues were grinded in liquid nitrogen and used for DNA extraction according to instruction of MagMAX kit (ThermoFischer Scientific) using KingFisher Duo Prime instrument (ThermoFischer Scientific). The extracted nucleic acids were measured with Nanodrop (Ds-11 Spectrophotometer; Denovix) and diluted with sterile water to a final concentration of about 20 ng/µl.

TaqMan Real-time PCR was performed using 20 ng of template DNA, and primer pair 16SrRNA-F/16SrRNA-R for the detection of ‘Ca P. solani’ (Christensen et al., 2004). The fluorogenic probes were synthesized by Eurofins Genomic (Germany). P2-Phy-16S probe was labelled with Hex (Hexachloro-fluorescein) reporter dye at 5’ end and the quenching dye, BHQ-1, at 3’ end and used for ‘Ca. P. solani’ detection. The final reaction volume of 20 µL contained 2x TaqMan qPCR master mix (ThermoFischer, Maxima probe qPCR), 0.9 µM of each primer and 0.4 µM probe; with cycling parameters 37°C for 2 min; 95°C for 3 min; 40 cycles of 95°C for 15 Sec, 60°C for 30 Sec. A similar reaction with specific primers and probe (P1-SBR-HSP20, Table S1) was used for detection of ‘Ca. A. phytopathogenicus’. Samples with a Cq value > 36.5 were considered negative, which is equivalent to 100 copies for Arsenophonus (Zübert & Kube, 2021). Samples with a Cq value > 35.1 were considered negative, which is equivalent to 10 copies for phytoplasma (Schneider et al., 2020). Table S1 shows the sequence details for primers and probes.

Amplification and sequencing of 16SrRNA, tuf, vmp1, stamp and secY genes

Initial phytoplasma identification was through nested-PCR assay and sequencing using phytoplasma universal primer pairs P1 /P7 and R16F2n/R2 primer pairs (Schneider et al., 1995, Deng & Hiruki, 1991) to amplify a fragment of ~ 1.2 kbp from the 16SrRNA gene in the phytoplasma positive samples.

Further characterization of ‘Ca. P. solani’-related strains in potato and sugar beet samples were carried out based on sequencing of informative genes. The DNA of phytoplasma-positive samples were used as the templates for PCR-amplification of stamp, vmp1, tuf, and secY genes using Stamp-F/R, Stamp‐F1/R1, fTuf1/rTuf1 and PosecF1/R1 primer pairs (Table S1) followed by nested-PCR with Stamp‐F1/R1, TYPH10F/R, fTufAY/rTufAY, and PosecF3/R3 primer pairs, respectively (Fabre et al., 2011, Cimerman et al., 2009, Fialová et al., 2009, Schneider & Gibb, 1997).

PCR products were sequenced in both directions using commercial service (Microsynth, Goettingen, Germany), using primer sets previously applied for amplification (Stamp-F1/R1, TYPH10F/R, fTufAY/rTufAY, and PosecF3/R3 primer pairs). To ensure ≥ 3x sequencing coverage per nucleotide position, for stamp, tuf, vmp1 and secY, three PCR products were separately amplified and sequenced for two randomly selected samples of each host crop. The obtained sequences were then assembled using BioEdit software version 7.2 (https://bioedit.software.informer.com/7.2/) and the consensus sequence for each gene was obtained.

A 1,244- nucleotide (nt) sequence of the almost complete 16S ribosomal RNA gene, a 992-nt partial sequence of the tuf gene, a 1,260-nt partial sequence of the secY gene and a 1,407-nt partial sequence of the stamp genes of the 'Ca. P. solani'-related strains for potato and sugar beet were deposited in GenBank.

In Silico RFLP Analyses

Since all amplified sequences were sequenced, a wet restriction fragment length polymorphism (RFLP) test was not carried out. Instead, in silico RFLP analysis was performed with the 16S rRNA gene sequences and 17 different restriction enzymes using iPhyClassifier (Zhao et al., 2009) to determine the structural diversity (Lee et al., 1998). Based on this assay, three restriction enzymes (AluI, MseI and TaqI) that showed differentiation patterns were further tested on 16S rRNA gene sequences along with a representative for each 16srXII subgroup.

Phylogenetic Analyses

To identify nucleotide sequence similarity of the ‘Ca. P. solani’ strains in potato and sugar beet with the publicly available strains in databases, NCBI GenBank database was searched using each consensus gene sequence in BLAST search (http://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on October 21 2023) and then aligned with reference strains of previously described 'Ca. Phytoplasma' species, using ClustalW under BioEdit software.

Evolutionary tree based on the 16S rRNA and also informative gene sequences of potato and sugar beet strains obtained in this study, representative strains of each subgroup (Wei & Zhao, 2022, Quaglino et al., 2009) (Bertaccini & Lee, 2018) and also the closest 'Candidatus Phytoplasma' species were tested using the Maximum-Likelihood (ML) method (MEGA X) and selected Kimura 2parameter model (Kumar et al., 2018). As an outgroup, 'Ca. Phytoplasma mali' or 'Ca. Phytoplasma vitis' was used to root the tree. To estimate the statistical significance of the clades, 1,000 bootstraps were? performed.

Pathogen detection and symptom observation

‘Ca. P. solani’ was detected in 18.7% (6/32) of potato tubers from three field locations (Meimsheim, Lauffen and Massenbachhausen) in Southern Germany (Fig. 1A). 15.6% (5/32) of tested samples also contained both phytoplasma and Arsenophonus bacteria (Table 1). The Cq value for phytoplasma samples was in range of 26 to 38 and samples with Cq < 35.1 were considered as positive. Most of the infected tuber with phytoplasma showed wilting and rubbery symptoms and some tubers displayed vascular brown discoloration (Fig. 1B). In the sugar beet samples, 60% (6/10) and 10% (1/10) of root with necrotic vascular tissues (Fig. 1C) were tested positive for only phytoplasma and Arsenophonus, respectively. Mix infection was observed in 30% (3/10) of root samples (Table 1). The Cq value of TaqMan qPCR was between 26 and 38. These roots were collected from plants with asymmetric, lancet-shaped leaf shapes, and yellowing of older leaves (Fig. 1C).

Table 1

Field locations and detection of Ca. P solani and Ca. A. phytopathogenicus in potato and sugar beet based on TaqMan real-time assay.
Host	Field location	Phytoplasma (%)	γ-proteobacterium (%)	Mixed infection (%)	Healthy (%)
Potato	Meimsheim	2/10 (20%)	4/10 (40%)	2/10 (20%)	2/10 (20%)
	Lauffen	2/16 (12.5%)	8/16 (50%)	3/16 (18.7%)	3/16 (18.7%)
	Massenbachhausen	2/6 (33.3%)	1/6 (16.6%)	1/6 (16.6%)	2/6 (33.3%)
	all potato fields	6/32 (18.75%)	13/32 (40.6%)	5/32 (15.6%)	7/32 (21.8%)
Sugar beet	Untereisesheim (Heilbronn)	6/10 (60%)	1/10 (10%)	3/10 (30%)	0/10 (0%)

Sequence typing by RFLP analyses and phylogenetic relationships

Identification of phytoplasma strains based on the 16S rRNA sequences and in silico RFLP patterns using iPhyClassifier showed that potato strains are identical (similarity coefficient 1.00) to the reference pattern of 16Sr group XII, subgroup A (GenBank accession: AF248959). BLAST search for the amplified 16S rRNA of potato samples showed highest identity (99.91%) to the grapevine strains in Turkey (MN398471) and Iran (MK392488) and a potato strain in Romania (HQ108386). The closest sequence reported in Germany is a sugar beet strain (OQ703963; isolate Z187) with 98.7% identity.

The sugar beet strain is different from all previously established 16Sr groups/subgroups. The most similar RFLP pattern was a reference pattern of the 16Sr group XII, subgroup I (GenBank accession: JX273494), with a similarity coefficient of 0.96, which is less than or equal to the threshold, 0.97. This strain may represent a new subgroup within the 16Sr group XII. BLAST search for the amplified 16S rRNA of sugar beet samples revealed that this is strain closest (99.8% identity) to a sugar beet strain (OQ717667) that was recently reported from Eastern Germany. Other strains from Salvia miltiorrhiza (China), Capsicum annuum (Japan), maize (Serbia), and insect (Italy) showed 98.8% identity to the sugar beet strain.

In silico RFLP analysis on the 16S rRNA gene sequences with 17 different restriction enzymes using iPhyClassifier showed that AluI, MseI and TaqI enzymes produce differential patterns for potato and the sugar beet strains (Fig. S1). The RFLP patterns for potato strains were similar but different from sugar beet strains (Fig. 2). Phylogenetic analysis of 16S rRNA sequences of potato and sugar beet associated strains along with representative 16SrXII subgroups confirmed that the sugar beet strain belongs to 16SrXII-P subgroup (Fig. 3), while potato strains from Southern Germany (This study) are also close to 16SrXII-N and 16SrXII-G subgroups (Fig. 3).

A part of the tuf gene was successfully amplified in sugar beet samples and potato samples using Tuf1f/r, and TufAYf/r primers which produced the expected ~ 0.95 kbp PCR product. BLAST search for the amplified and sequenced tuf gene in potato strain (P8 isolate) showed the highest identity (99.09%) with a grapevine strain (MW175421), while sugar beet strains in this study were closest (99.9%) to a recently reported sugar beet strain (OQ717008, strain Z187). Phylogenetic analysis shows that the sequenced tuf gene in sugar beet clustered together with the published tuf sequence from sugar beet from Eastern Germany (strain Z187). They are separated from the know tuf groups, Tuf -a, Tuf -b, Tuf -c and Tuf-d (Fig. 4a). However, tuf gene sequences from potato samples along with a published sequence from RTD sugar beet (MZ927455) belong to the Tuf-b1 group.

A part of secY gene (~ 0.9 kbp bp) was amplified by PCR using PosecF1/R1 and PosecF3/R3 primer pairs and sequenced for two representative samples of each potato and sugar beet strains. The sugar beet strains were identical and according to the BLAST search, only one close sequence from sugar beet (OQ717010) showed a high identity (99.86%). Another published secY sequence (JQ977712) from stinging nettle (U. dioica) showed a much lower identity rate (77.06%). SecY gene sequence from the potato strain was 100% identical to secY genes reported from maize (KU374896, strain M7; Bosnia), R. XXXanzer (KP635228, isolate 202, Switzerland), periwinkle (JX645766, strin 204/10, Serbia), tobacco (JQ730747, strain 142, Serbia), and sugar beet (MT157233, strain 429/19, Serbia).

Phylogenetic analysis of ‘Ca. P. solani’-related strains infecting potato and sugar beet based on the secY gene indicates a separate cluster for sugar beet strains in this study together with the recently reported sequences from sugar beet in Eastern Germany. These strains are close to secY sequence from Ca. P. japonicum (AB738739) but separated from secY sequences of Ca. P. solani. However, the secY sequences from potato (P5 and P8) are close to the reported sequences of ‘Ca. P. solani’. They are close to secY4 and secY3 groups (Fig. 4b).

Vmp1 and stamp genes were amplified (~ 1.4 and 0.5 kbp size, respectively) and sequenced for two selected potato samples. However, amplification of these genes in sugar beet samples was not possible. BLAST search for the potato vmp1 (P8) showed the highest identity (99.91%) to vmp1 genes reported from cucurbit (ON645991, Strain 51, Turkey), sugar beet (OK041398, strain 216/20, Serbia) with RTD symptoms and other host plants including grapevine and periwinkle. Clustering vmp1 sequences with sequences from reported Vmp1 groups shows that potato strains are close to V4 and V17 groups (Fig. 5).

Stamp gene sequences of potato strains from different fields showed variation. BLAST search for P8 showed the closest identity (99%) to stamp sequences of ‘Ca. P. solani’ reported in grapevine (MF182868) and rose periwinkle (Catharanthus roseus; FN813270). This strain was clustered together with ‘Ca. P. solani’ in tomato (FN813257, strain Lot et Garonne, France), sugar beet (MZ604952, MZ604949, strain 1198/20 and 1204/20, Serbia) with RTD symptoms and H. obsoletus (FN813263, strain E) in Germany (Fig. 5).

We investigated for the first time the genetic diversity of potato stolbur in Southern Germany based on MLSA for 16SrRNA, tuf, vmp1, secY, stamp and tuf genes. Further, MLSA analysis was performed for sugar beet samples in the same geographical region to understand the possible similarity of stolbur in both fields.

Most of the potato plants displaying disease symptoms such as side shoots, wilting, and rubbery tubers were reported in association with Arsenophonus, in the absence of stolbur, therefore stolburlike symptoms in potato were anticipated to be triggered by Arsenophonus (Behrmann et al., 2023). However, in SBR disease in sugar beet and chlorosis disease in strawberry, disease symptoms Arsenophonus bacteria are convergent with those associated with phytoplasmas infections (Bressan, 2014). In this study, potato tubers showing stolbur-like symptoms including the formation of side shoots, wilting, and rubbery tubers and some with vascular discoloration contained both Arsenophonus and stolbur pathogen. Therefore, correlation of particular symptoms to each pathogen due to lack of Koch's postulates remains undefined. For the same reasons, we could not assign a particular symptom for ‘Ca. P. solani’ and/or ‘Ca. A. phytopathogenicus’ in sugar beet. However, we observed that sugar beet plants infected with ‘Ca. A. phytopathogenicus’ produce asymmetric young leaves and discoloration of vascular tissue in roots and ‘Ca. P. solani’ is associated more with rubbery taproot and yellowing leaves. Such symptoms have been also described by Bressan (Bressan et al.) and Ćurčić, (Ćurčić et al.) in SBR infected plants.

We observed a high infection rate for Arsenophonus (40.6%) and low infection rate for the stolbur pathogen (18.7%) in potato tubers. A similar trend was also reported for the Arsenophonus (80%) and stolbur (5%) infection in potato tubers (Behrmann et al., 2023). The high rate of Arsenophonus infection can be explained by the endosymbionts relationship of Arsenophonus bacteria and the vertical transmission of this pathogen to the insect progeny (Bressan et al., 2009b). This may also to some extend reflect the unequal distribution of Arsenophonus and phytoplasmas in the infected plant tissues.

The MLSA results for 16S rRNA, stamp, secY, tuf, and vmp1 genes of ‘Ca. P. solani’ indicate clear genetic variations between potato and sugar beet strains in fields with close distance in Southern Germany. This indicates separate sources and/or insect vectors of ‘Ca. P, solani’ in these two crops with no cross infection between two crops in the same region yet. Although, potato related “Ca. P. solani’ strains are close to a single strain, called Z187, reported from SBR sugar beet in a far distance field (> 100 Km) in Bickenbach (Ćurčić et al., 2021a). Therefore, a continuous monitoring of the pathogen in both crops and also surrounding weeds is essential to better understand the epidemiology of sugar beet SBR and potato stolbur in Germany and Central Europe.

Based on the 16S rRNA sequences and in silico RFLP patterns, sugar beet related strains in this study are different from all previously established 16Sr groups/subgroups. These strains are close (99.8% identity) to a novel subgroup, 16SrXII-P, that has been recently described in sugar beet in Eastern Germany (Duduk et al., 2023), but different from a single strain, Z187, of 'Ca. P. solani' reported in sugar beet in Bickenbach, Germany (Ćurčić et al., 2021a). This may indicate the prevalence of 16SrXIIP in Eastern and Southern Germany but with possible variation in other sugar beet growing regions such as Bickenbach. Further studies are required to cover all SBR infected sugar beet growing regions in Germany and neighboring countries that SBR and the insect vector have been spread. This is to better understand the prevalent 'Ca. P. solani' strains and their epidemiology.

Based on the in silico RFLP patterns of 16SrRNA sequences, potato related strains are different from sugar beet related strains and belong to 16Sr group XII, subgroup A, although, the phylogenetic tree shows that potato related strains are also close to 16SrXII-N and 16SrXII-G subgroups. Subgroup A is common in potato and has been also reported from potato fields in Greece (Holeva et al., 2014) and Serbia (Jović et al., 2011). In addition to subgroup A, subgroup B and C have been also reported in association with potato stolbur in Russia (Girsova et al., 2016). This suggests, a wide diversity of Ca. P. solani strains in a single crop in this region.

It needs to note that although the conserved 16SrRNA gene has been the main basis for the classification of phytoplasmas into ribosomal subgroups, because of the high level of rRNA nucleotide sequence conservation across several phytoplasma lineages, identification based on only 16SrRNA sequence gene has some limitations (Lee et al., 2010). The epidemiological informative and housekeeping gene data or additional molecular markers that exhibit moderate genetic variability need to be analysed. Therefore, we included analysis of informative genes to better characterize the ‘Ca. P. solani’ associated with potato and sugar beet.

Comparative phylogenetic analyses based on 16S rRNA and secY gene sequences from 80 and 83 phytoplasma strains, respectively, showed that analysis of secY gene-based phylogeny was nearly consistent with that inferred by 16S rRNA gene-based phylogeny (Lee et al., 2010). Additionally, phylogenetic analysis based on the secY gene enabled finer differentiation of phytoplasma strains within a given 16Sr group The amplified secY sequences in sugar beet samples grouped together with the published sequences from ‘Ca. P. japonicum’(Takinami et al., 2013) and a sugar beet strain from Eastern Germany (Duduk et al., 2023), which is the only close secY sequence to the sugar beet strain in this study. This further indicates that secY gene sequence in SBR phytoplasma in Southern (This study) and Eastern (Duduk et al., 2023) Germany is relatively unique and can be used to separate these stains from other published phytoplasma diseases. Indeed, phylogenetic analysis of diverse strains in the aster yellows phytoplasma group based on secY has indicated that most subgroups identified by RFLP analysis of secY represent distinct phylogenetic lineages (Lee et al., 2006). In contrast to the sugar beet strain, secY sequences from potato samples (this study) are highly identical (100%) to the published secY sequences of ‘Ca P. solani’ in various host plants including maize, tobacco, sugar beet (the causal agent of RTD) and also an insect vector, R. panzeri, but they are clearly different from secY sequences (44% identity) of sugar beet with SBR symptoms (this study) and the reported secY sequence in sugar beet samples (with 67% identity) in Eastern Germany. Additionally, there is variation between two secY sequences from two potato samples (97% identity) in close-distance fields, 4 km distance. These secY sequences in potato samples are close to SecY-3 and SecY-V4 groups. Similarly, the SecY-4 type has been also reported in a potato strain in Turkey (Çağlar & Şimşek, 2022). This may support similarity of the potato strain in Germany and other counties and possible distribution of the Ca. P. solani via seed tubers.

Similar to secY sequence genes from sugar beet with SBR symptoms, phylogenetic analysis of tuf sequences from sugar beet showed a unique and independent clade of all known tuf groups. This data supports the proposition of a novel 'Ca. Phytoplasma' species for the sugar beet infecting phytoplasma in Germany (Duduk et al., 2023). Since, at least two thresholds of 97.5% for the tuf and rp genes and 95.0% for the secY housekeeping gene are met for the novel 16SrXII-P strain (Bertaccini et al., 2022), Tuf-b is known to be linked to field bindweed (C. arvensis) (Johannesen et al., 2012). Therefore, potato strains in this study may also be linked to C. arvensis which can be the primary source of ‘Ca. P. solani’-related strains in potato in Southern Germany. Supporting this hypothesis, an epidemic cycle for Tuf-b strains of ‘Ca. P. solani’ in C. arvensis has been reported in Germany (Langer and Maixner 2004, Johannesen et al. 2012). In addition, the presence of Tuf-b stains in potato fields in Romania, Southern Russia (Ember et al., 2011) and Turkey (Çağlar & Şimşek, 2022) and also in weeds (C. arvensis, Cuscuta sp. and Euphorbia falcata) in the vicinity of the potato fields suggested that Tuf-b strains are associated with C. arvensis. Finally, vmp1 sequence genes from potato in this study are also close (99.91% identity) to a ‘Ca. P. solani’ (JQ977738) reported from C. arvensis in Germany. This evidence additionally supports the possible association of ‘Ca. P. solani’ in potato fields with C. arvensis in Southern Germany. Further studies need/are needed? to monitor ‘Ca. P. solani’ in weeds including C. arvensis and other crops in the bordering potato fields for better management of ‘Ca. P. solani’ sources.

Similar to the reported 16SrXII-P subgroup of ‘Ca. P. solani’ (Duduk et al., 2023), we could not amplify vmp1 and stamp genes in sugar beet samples. This further reflects the high diversity between sugar beet strains in Germany (Eastern and Southern) and all established 16Sr groups/subgroups. The vmp1 gene is more variable than the other housekeeping genes and involves in protein translation, secretion and maturation and may therefore be involved in phytoplasma-host interactions. Because of an additional copy of a repeated domain, this gene is also variable in size (Cimerman et al., 2009). This may eexplain the difficulties for amplification of vmp1 and stamp genes in sugar beet samples which are infected with the novel 16SrXII-subgroup. Further investigations are required to identify and characterise vmp1 sequence variation in sugar beet samples which potentially can explain the interaction of 16SrXII-P subgroup with the main insect vector, P. leporinus. Vmp1 and stamp genes were successfully amplified and sequenced in potato samples and showed variation between sampled fields. In potato, vmp1 genes are close to V4 and V14 of Vmp groups. Supporting such high variability for vmp1 sequences, another Vmp group,V1, has been reported from the same crop in Turkey (Çağlar & Şimşek, 2022).

Phytoplasmas lack cell wall, therefore cell membrane proteins expected to play role in phytoplasmahost interactions (Cimerman et al., 2009, Arricau-Bouvery et al., 2018). The vmp1 gene provides information about the phytoplasma vector-pathogen-weed host relationships and the molecular epidemiology of the pathogen (Cimerman et al., 2009). This gene could be informative for understanding the molecular epidemiology of ‘Ca. P. solani’ (Cvrković et al., 2014, Johannesen et al., 2012). Thus, the new vmp1 genotypes in potato samples could raise the potential for new insect vector(s) such as P. leporinus or host plant(s) to be involved in the disease epidemiology of ‘Ca. P. solani’ in Germany and Central Europe.

Different insect vectors were found to either contain or transmit stolbur into potato plants. For example, phytoplasmas from 16SrXII-A subgroup were identified in P. leporinus and H. obsoletus in Russia (Kastal’eva et al., 2016). In Serbia, R. panzeri, H. obsoletus, and R. quinquecostatus tested positive for ‘Ca. P. solani’. R. panzeri and H. obsoletus were able to successfully transmit the phytoplasma to potato plants and induce stolbur disease symptoms (Mitrović et al., 2015). However, the authors could not find a clear species specific association of certain ‘Ca. P. solani’ genotypes with plausible insect vectors. P. leporinus is the main economic vector for SBR disease in sugar beet (Bressan et al., 2008bmétey et al., 2007a, Pfitzer et al., 2020) which has recently been spread into potato fields. Both pathogens, Ca. A. phytopathogenicus and stolbur were detected in potato, sugar beet and the insect vector (Behrmann et al., 2023). Detection of stolbur was based on TaqMan qPCR using general primers that target 16SrRNA (Behrmann et al., 2023) which is unable to discriminate specific subgroup or pathogen variation in potato and sugar beet fields. Further, it is impossible to detect the novel 16SrXII-P phytoplasma strain with specific PCR primers routinely used for the detection and/or characterization of 'Ca. P. solani' (Duduk et al., 2023). In addition, transmission of these pathogens from one crop to another one by P. leporinus was not confirmed yet and only nymphs were shown to transmit both pathogens into sugar beet and only Arsenophonus into potato plants under controlled conditions (Behrmann et al., 2023)(Behrmann et al., 2023)(Behrmann et al., 2023)(Behrmann et al. 2023). Our MLSA data clearly show that phytoplasma strains in sugar beet are different from potato strains in Southern Germany. The sugar beet strains are close to the newly reported 16SrXII-P subgroup (Duduk et al., 2023) based on sequence analysis of 16SrRNA, secY and tuf genes. It seems that this subgroup is dominant is sugar beet growing regions in Eastern and Southern Germany. Further studies are required to understand the interaction between the 16SrXII-P subgroup with P. leporinus and monitoring this pathogen in sugar beet and other crops including potato and weeds in the close-distance fields to understand the epidemiology and potential source of the pathogen.

Acknowledgements

We thank S. Hartje and K. Buhr from BNA (Böhm-Nordkartoffel Agrarproduktion) for providing potato samples and their scientific comments on this research.

Conflict of interest

The authors declare no conflict of interest

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Molecular detection and multilocus sequence analysis of Candidatus Phytoplasma solani-related strains infecting potato and sugar beet plants in Southern Germany

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