Genetic Diversity of ‘Candidatus Phytoplasma solani’ in Plant Hosts and Insect Vectors in Winegrowing Regions in Germany

Abstract

The bois noir (BN) disease of grapevines is widespread in German winegrowing regions. It is associated with ‘Candidatus Phytoplasma solani’, which affects not only grapevines but also other wild and cultivated plants. This pathogen has a complex epidemiology including different insect vectors and various host plants. A study was carried out to investigate the genetic variability of ‘Ca. P. solani’ in different winegrowing regions in Germany. Between 2017 and 2023, samples of grapevine, stinging nettle, bindweed, and other herbaceous plants as well as specimens of different planthopper species colonizing viticultural habitats were analyzed for infection with ‘Ca. P. solani’. All positive tested samples were further characterized by multilocus sequence typing (MLST) based on the genes tuf, stamp, secY, and vmp1. The genetic variability was assessed by RFLP analyses of the tuf and vmp1 PCR products, coupled with sequencing of the stamp and secY amplification products. A total of 1274 grapevines, 35 bindweed, and 18 stinging nettle samples were infected with ‘Ca. P. solani’ but also five samples of other weed species. Among the known and putative insect vectors, specimens of Hyalesthes obsoletus, Reptalus spp., and Dictyophara europaea harbored the phytoplasma. In both plants and insects, two genotype combinations were predominantly associated with the classical bindweed and stinging nettle cycle, respectively. The MLST analysis revealed considerable differences between German isolates and data reported from other European regions and new genotype combinations were identified, indicating new host plant–vector associations.

1. Introduction

Bois noir (BN) is an important yellows disease of grapevine (Vitis vinifera), associated with the phytopathogenic bacterium ‘Candidatus Phytoplasma solani’ (CaPsol), taxonomic subgroup 16SrXII-A, commonly known as stolbur phytoplasma [1]. The phytoplasma is present in natural vegetation and it is transmitted from and to herbaceous plants mainly by planthoppers of the family Cixiidae [2,3,4,5]. Both the phytoplasma and vectors are endemic to Europe, probably originating from the Mediterranean basin [1,6]. CaPsol has also been reported in Chile, South Africa, the Middle East, and China [7]. While different polyphagous cixiid planthoppers (Hemiptera: Cixiidae) are reported to transmit CaPsol to many herbaceous and woody plants, Hyalesthes obsoletus Signoret is considered to be the principal vector to grapevine [6,8,9]. Hyalesthes obsoletus is a polyphagous cixiid living in Central Europe, preferentially on Urtica dioica L. (nettle) and Convolvulus arvensis L. (bindweed) [10], but it is able to develop on other herbaceous and woody hosts such as Crepis foetida, Lavandula angustifolia, Salvia sclarea, and Vitex agnus-castus [6,11,12,13,14]. The root feeding nymphs acquire the pathogen from their infected plant hosts [15]. Host-plant adaptation of H. obsoletus populations has led to the development of genetic host races at its northern range border restricted either to nettle or to bindweed [16]. These host races differ in morphology, phenology, and survival on their respective hosts [17,18,19,20,21]. In addition, the CaPsol pathogen has also adapted to the different host plants, resulting in nettle-associated and bindweed-associated strains, which can be distinguished by the molecular marker of the tuf (translation elongation factor Tu) gene [10]. Genotypes tuf-a and tuf-b2 are strictly limited to U. dioica while tuf-b1 is associated with C. arvensis and other herbaceous hosts [10,22]. In Germany, a genetic variant of tuf-b1 designated as tuf-c has been found in V. vinifera, Calystegia sepium, and H. obsoletus [10]. BN is the result of an occasional branching of the natural nettle and bindweed transmission cycles to grapevine, which is considered a dead-end host for the pathogen [15].

The distinct epidemiological cycles based on different reservoir plant species imply the risk that new plant/vector or plant/CaPsol strain combinations could result in altered disease cycles and changing infection pressure to grapevines. Possible reasons for such changes include altering environmental conditions or cultural practices, host plant shifts of phytoplasmas or vectors, or extensions of their range or dissemination [15,23]. It has been demonstrated that such scenarios have occurred in the past in northern wine growing regions such as Germany [24]. Bois noir in Germany was traditionally restricted to xerothermic viticultural sites on steep slopes of the river valleys, characterized by the presence of H. obsoletus on its sole host plant, C. arvensis. Locally, very high infestation rates in grapevine have been recorded [21]. Within a few years in the early 2000s, a rapid emergence of BN throughout the German wine growing regions was related to a sudden occurrence of H. obsoletus on U. dioica [24]. It is considered that this tuf-a related outbreak was likely the result of the host shift of local populations of H. obsoletus from bindweed to nettle in combination with the range extension of Italian H. obsoletus populations and an associated nettle-adapted tuf-a type strain to the north via Switzerland and France [24]. Within a few years, CaPsol tuf-a emerged to the predominant and most widespread strain of BN in Germany [21]. A similar situation has occurred in eastern Austria. There, immigration and a rapid demographic expansion of H. obsoletus on nettles, in combination with the uptake of the nettle-specific tuf-b2 genotype from another population, led to a sudden mass occurrence of the vector and the associated tuf-b2 genotype [22,25].

Beside H. obsoletus, other Auchenorrhyncha species may act as putative vectors. Among the family Cixiidae, the transmission of CaPsol to grapevines has been proven in restricted cases for Reptalus panzeri Löw [26]. Reptalus salicinus Tishechkin & Emeljanov (formerly known as R. quinquecostatus Dufour and confused with R. artemisiae Becker [27]) has been described as a vector of CaPsol to different crops [28,29,30], but transmission to grapevines has not yet been proven [28,31]. In contrast, Cvrkovic et al. [32] reported the transmission of CaPsol to grapevines by the Dictyopharidae Dictyophara europaea L. [32]. The Cicadellidae Anaceratagallia ribauti Ossiannilsson was able to inoculate Vicia faba test plants with CaPsol [33], and Mitrovic et al. [34] proved the transmission of CaPsol by Neoaliturus fenestratus Herrich-Schäffer to lettuce and carrots. Some more Auchenorrhyncha species were found to be infected with CaPsol [3,5,35] but overall, the role of these species in BN transmission has not yet been fully elucidated [23].

Today, BN is present in all Central European and Mediterranean regions [7]. In some regions, the abundance of H. obsoletus is low, and consequently there might be a lack of correspondence between disease spread and vector occurrence [25]. Aside from BN on grapevines, CaPsol is also the causal agent of various diseases of horticultural crops like potato stolbur [36,37], rubbery tap root disease (RTD) in sugar beets in Southeast Europe [38,39], and together with the γ-3-proteobacterium ‘Candidatus Arsenophonus phytopathogenicus’ (CaAp) it induces the syndrome ‘basses richesses’ (SBR) in sugar beets in Central Europe [40,41]. While CaPsol associated with RTD is transmitted by the cixiid R. salicinus [29], CaPsol in sugar beets together with CaAp is spread by the cixiid Pentastiridius leporinus L. [42]. Recently, P. leporinus has expanded its host range to potatoes [43] and carrots [44] in Germany, and both crops were found infected with both pathogens, CaPsol and CaAp. Furthermore, the vector capacity of P. leporinus for both pathogens was proven during transmission trials [45,46], indicating an important host-shift capacity of this planthopper and an increased vector potential.

This demonstrates the complexity of the epidemiological scenario, including the broad range of tentative CaPsol host plants and vectors and the considerable genetic diversity among CaPsol strains [47]. Multigene genotyping techniques have been developed to distinguish different phytoplasma strains. A common multilocus sequence typing approach (MLST) is based on two housekeeping genes, tuf and secY, and two variable membrane proteins, vmp1 and stamp [10,48,49,50]. Similar to the tuf gene, the genotypes of the secY gene, encoding for a major membrane protein in the secretory pathway, correspond to the separation between nettle and bindweed systems [22,24,51]. However, the two genes that encode for membrane proteins, vmp1 [48] and stamp [49], are more variable and enable a finer differentiation of CaPsol strains [23]. Both genes are subjected to positive selection pressure and might be involved in the interaction of the phytoplasma with its plant and insect hosts [48,49,50].

The history of BN in Germany dates back to the 1930s, when a new disease with typical symptoms of BN was reported from the steep-slope vineyards of the winegrowing regions of Mosel and Middle-Rhine [52]. With rare exceptions, the disease remained confined to these regions. The typical temporal dynamics of epidemic outbreaks and endemic periods [15] were already being observed in the 1950s. Infestation levels between 23% and 95% in individual plots were recorded in the 1960s in the Middle-Rhine valley [53], where BN still reaches the highest infestation levels in Germany. The historic emergence of BN was based on C. arvensis as the sole natural host plant with the tuf-b1 genotype of CaPsol. It was likely linked to the immigration of the BN vector H. obsoletus to Germany [24]. The BN situation changed in the early 2000s, with a sudden spread of BN to so far unaffected regions.

The present survey aimed to collect current data on the BN incidence and distribution in different winegrowing regions in Germany and to characterize the predominant CaPsol genotypes in plant and insect hosts, thereby to identify potential alternative host plants and vectors of CaPsol in order to gain insights into the occurrence of known or new epidemiological BN pathways in German vineyards.

2. Materials and Methods

2.1. Plant and Insect Samples

Grapevine shoots with typical Grapevine Yellows (GY) symptoms were sampled between July and September during surveys conducted in different winegrowing regions in Germany in the years 2017–2023. In addition, between 2022 and 2023, 24 plots were selected for vine-by-vine monitoring to assess the current BN incidence in reference vineyards in Pfalz (17 plots), Mittelrhein (4 plots), and Mosel (3 plots) regions.

From every symptomatic shoot, five leaves were taken for a molecular analysis of the phytoplasma infection. Samples from weeds known to be confirmed or putative host plants of CaPsol were taken within or on the borders of the selected vineyards with high incidence of GY symptoms throughout the years 2021–2023 at different localities in the Pfalz region.

Adult Auchenorrhyncha species were collected with sweep nets and mouth aspirators from herbaceous hosts from the inter-rows or borders of reference plots with high incidence of BN in the regions of Franken, Mittelrhein, Mosel, Pfalz, and Württemberg. They were transported to the lab alive in 50 mL Falcon tubes and frozen at −20 °C. Dead insects were morphologically identified using the taxonomic key by Biedermann and Niedringhaus [54]. Determinations of the different Reptalus spp. was based on specific structures of the terminalia for males, while females from the same location were randomly subjected to molecular identification by COI barcoding [55] according to the EPPO standard protocol PM 7/129 [56]. Known and presumed vectors of CaPsol of the suborder Auchenorrhyncha were sorted out for further molecular analysis. All samples were tested for CaPsol infection. Genotyping by tuf and MLST was then applied to positive samples.

Table 1. Plant and insect samples used for genotyping by tuf (first value) and MLST (second value).

		Plant Samples			Insect Samples
Region	N° Sites	Vitis vinifera	Urtica dioica	Convolvulus arvensis	Hyalesthes obsoletus	Reptalus spp.
Baden	3	7/3
Franken	9	62/0
Mittelrhein	1	20/9
Mosel	4	52/24			59/33	25/11
Nahe	2	6/0
Pfalz	22	945/116	18/9	35/34	36/32	5/5
Rheinhessen	2	12/1
Württemberg	12	170/15				2/1
total		1274/168	18/9	35/34	95/65	32/17

gives an overview of the origin of plant and insect samples that were used for genotyping.

2.2. TNA Extraction

Total nucleic acids (TNA) for both plants and insects, were extracted using a modified CTAB method according to Maixner et al. [57]. For plant material, 150 mg of the leaves’ mid-ribs and petioles were ground with 3 mL of buffer (Doyle buffer mixed with 0.2% mercaptoethanol). As the detection of phytoplasmas in nettle leaves was difficult, in this case TNA was also extracted from roots, which usually harbor a higher pathogen concentration. Insect TNA extraction was conducted as described by Witczak et al. [44].

Pellets from plants and from adult leafhoppers were resuspended in 150 μL of TE buffer, and the TNA content was measured using a UV/Vis spectrophotometer NP80 (Implen GmbH, Munich, Germany).

2.3. Phytoplasma Detection

The presence of CaPsol in 1:10 prediluted extracts from grapevine leaves, leaves from herbaceous hosts, and root extracts from nettle was assessed by nested PCR amplification using primers U5 and P7 for the first round of PCR [58,59] followed by amplification of 1:100 diluted U5/P7 PCR products via a second round of PCR using 16SrXIIA-group-specific primers fStol/rStol [57]. Infection with CaPsol in extracts from insects was confirmed by direct generic PCR amplification using 16SrXIIA-group-specific primers fStol/rStol [57]. Additionally, in grapevine samples, the phytoplasma was identified by two different TaqMan triplex real-time PCR assays as simultaneous detection of 16SrV- and 16SrXII-A group phytoplasmas [60] according to the protocols in the EPPO standard PM7/079 [61]. Extracts from a phytoplasma collection at the Julius Kühn Institute (JKI), Siebeldingen, Germany, in Madagascar periwinkle (Catharanthus roseus (L.) that maintained different CaPsol reference strains were used as positive controls.

2.4. Molecular Characterization of 16SrXII Isolates

Samples that tested positive for infection with CaPsol were genetically characterized by restriction fragment length polymorphism (RFLP) and/or multilocus sequence typing (MLST) analyses based on amplified fragments of tuf, stamp, secY, and vmp1 genes. The tuf gene was amplified in a nested PCR analysis using the primer pairs Tuf1f/r and TufAYf/r as described by Langer and Maixner [10]. PCR products were subjected to restriction analysis using HpaII endonuclease (R0171S, New England Biolabs, Ipswich, USA). Reference strains for tuf-a, tuf-b1, and tuf-c maintained in periwinkle served as positive controls. The restriction fragments were separated on a 2% agarose gel stained with SERVA DNA Stain Clear G (SERVA Electrophoresis GmbH, Heidelberg, Germany). The RFLP tuf-typing results were confirmed by sequence analysis of the tufAY amplicon obtained from random samples of plants and insects after nested PCR. The sequences of a 903 bp fragment of the tuf gene were compared to reference sequences for tuf-a (Accession No. GU220558), tuf-b1 (Accession No. FJ394552), tuf-b2 (Accession No. LT899727), tuf-b3 (Accession No. LT899726), tuf-b5 (Accession No. MT505885), tuf-b6 (Accession No. MT505896), tuf-d (Accession No. MT157234) and tuf-e (Accession No. PP270188). In addition, the sequences were compared to the tuf-c reference strain DE30003 transmitted to periwinkle by H. obsoletus captured on infected C. sepium (M. Maixner pers. comm.). The sequence of tuf-c was deposited in the NCBI Genbank under Accession No. PZ246496.

The CaPsol-specific stamp gene was amplified by nested PCR with StampF/R0 primers followed by StampF1/R1, with PCR conditions according to Fabre et al. [49]. The secY gene was amplified using the primer pair PosecF1/PosecR1 followed by PosecF3/PosecR3 as described by Fialová et al. [51].

A fragment of vmp1 was amplified with the primer pairs StolH10F1/StolH10R1 followed by TYPH10F/TYPH10R according to the PCR protocol by Fialová et al. [51]. Amplicons were digested with RsaΙ restriction enzyme (ER1121, Thermo Fisher Scientific Inc., Darmstadt, Germany). In order to discriminate between V2 and V2-TA profiles, amplificates were digested with AluI restriction enzyme (E2010-01, EURx, Roboklon, Berlin, Germany) according to Cvrkovic et al. [26]. The restriction fragments were separated on a 2% agarose gel stained with SERVA DNA Stain Clear G and a SERVA FastLoad 50 bp DNA ladder (SERVA Electrophoresis GmbH, Heidelberg, Germany) was used for correct evaluation of vmp1 profiles in the RFLP analysis.

Reference genotypes, description, and sequence accession numbers for the gene markers tuf, secY, stamp, and vmp1 are shown in Appendix A.

2.5. Sequencing and Phylogenetic Analyses

The gene amplicons of tuf, secY, and stamp were directly sequenced in both directions by Microsynth Seqlab GmbH (Göttingen, Germany) using the Sanger method. Assembly of raw chromatograms and alignment of the German isolates was performed with the CLC main workbench software 25.0.3 (Qiagen, Hilden, Germany). The partially amplified sequences of tuf, secY, and stamp were compared with sequences from the GenBank database using the BLAST algorithms (version 12, http://www.ncbi.nlm.nih.gov/blast; accessed on 31 March and 7 April 2026). Phylogenetic analyses were conducted with MEGA12 [62] using the maximum-likelihood algorithm with 1000 bootstrap replicates [63].

3. Results

3.1. Diversity of Tuf Genotypes in German Viticultural Areas

Symptomatic grapevines were sampled in most of the major winegrowing regions in Germany and analyzed for infection with ‘Ca. P. solani’. A majority of the CaPsol-positive V. vinifera samples was first analyzed with the diagnostic tuf gene primers and RFLP analysis of the restriction fragments. A part of these samples was further subjected to MLST using various CaPsol specific gene markers (Table 1). Details on the markers used and on the specific location for each sample are listed in Table S1. The tuf typing revealed that all grapevine samples were infected either by the tuf-a type or tuf-b type of CaPsol. Sequence analysis of the tuf gene of random samples from Mosel, Palatinate, and Württemberg showed that all isolates corresponded to the tuf-b1 type. The repartition of tuf-a and tuf-b types in BN-affected grapevines in the different regions is illustrated in Figure 1.

The graph shows a heterogeneity in the distribution of the types tuf-a and tuf-b among the different viticultural areas with a more frequent occurrence of the tuf-b type in the geographically more northern parts of Germany and the traditional BN sites with steep-slope vineyards, like Mittelrhein and Mosel.

In the Mittelrhein region, tuf-b is the only tuf type detected at the investigated site. However, tuf-a type strains have been found in previous work at specific sites of the Mittelrhein region [21]. Tuf-a slightly dominated in the Mosel region, but there is an equal repartition in the region Franken. In all of the other more southern winegrowing regions, the tuf-a type was the most frequent or only tuf type detected in Rheinhessen and Baden. This type was also the dominant one in Pfalz and Württemberg. As the tuf marker is linked to different epidemiological cycles, the composition of the tuf types can give indications for the most prominent epidemiological cycles in the different regions.

In this regard, the evolution of the tuf types in vineyards in the Pfalz region during the last decade was remarkable. In the first period of our study (2017–2020), the tuf-a type was highly prevalent at 93.5% of all samples (143/153), compared with only 6.5% tuf-b (10/153 samples). In the period between 2021 and 2023, a significant increase of tuf-b was observed, reaching 33.4% of all samples (268/802), while the portion of tuf-a types dropped to 65.2% (524/802 samples). It must be noted that the sampling in both periods was dependent on the occurrence of symptomatic grapevines in different vineyards, and the plots for both periods are not completely identical.

3.2. Current BN Incidence and Tuf Genotypes in Reference Plots

3.2.1. Grapevine

Recent data recording the current CaPsol incidence were collected in selected reference vineyards in the Pfalz, Mittelrhein, and Mosel regions between 2022 and 2023. The determination of the overall incidence in these plots comprised PCR results or visual inspection for BN symptoms. As shown in

Table 2. Incidence of Bois noir (BN) in reference vineyards in Pfalz, Mittelrhein, and Mosel regions in 2022/2023 and distribution of tuf types in the various plots.

Region	Municipality	Plot ID	Cultivar	BN Incidence *	Tuf-Typing Results Total N° of Samples Tested Tuf-a/Tuf-b
Pfalz	Albersweiler	AW01	Cabernet Dorsa	38/235 (16.2%)	30 (15/15)
	Albersweiler	AW02	Dornfelder	19/523 (3.6%)	14 (9/5)
	Albersweiler	AW03	Scheurebe	24/604 (4.0%)	16 (10/6)
	Birkweiler	BW01	Riesling	10/1162 (0.9%)	7 (7 tuf-a)
	Birkweiler	BW03	Chardonnay	36/1075 (3.3%)	34 (31/3)
	Burrweiler	BU02	Riesling	63/2065 (3.1%)	47 (31/16)
	Burrweiler	BU04	Pinot Meunier	12/2698 (0.44%)	10 (4/6)
	Burrweiler	BU05	Riesling	51/2213 (2.3%)	19 (14/5)
	Burrweiler	BU06	Kerner	38/1883 (2.0%)	14 (13/1)
	Hainfeld	BE	Scheurebe	1/499 (0.2%)	1 (1 tuf-a)
	Leinsweiler	LEI03	Kerner	13/973 (1.3%)	nt
	Leinsweiler	LEI07	Dornfelder	13/1384 (0.94%)	7 (7 tuf-a)
	Leinsweiler	LEI08	Scheurebe	17/501 (3.4%)	17 (14/3)
	Edenkoben	ED01	Cabernet Dorsa	22/180 (12.2%)	13 (13 tuf-b)
	Edenkoben	ED02	St. Laurent	5/222 (2.3%)	3 (3 tuf-b)
	Edenkoben	ED03	Dornfelder	19/398 (4.8%)	16 (2/14)
	Mussbach	MUP	St. Laurent	29/1210 (2.4%)	26 (20/6)
Mittelrhein	Boppard	DidBe	Riesling	195/1168 (16.6%)	8 (8 tuf-b)
	Boppard	DidJu	Riesling	55/423 (13.0%)	1 (1 tuf-b)
	Boppard	LzRiJ	Riesling	135/609 (22.2%)	7 (7 tuf-b)
	Boppard	LzSBJ	Pinot Noir	10/815 (8.2%)	4 (4 tuf-b)
Mosel	Kesten	KeKar	Riesling	64/679 (9.4%)	nt
	Klotten	Klot	Riesling	114/5588 (2.0%)	21 (14/7)
	Pommern	Pom15	Riesling	120/3340 (3.6%)	24 (11/13)

* Number of symptomatic vines per total number of monitored plants; nt = not tested.

, the overall BN incidence was highest in the Mittelrhein region, where up to 22.2% of the plants were infected. It was lower in the Mosel and Pfalz regions, with a high variability between different plots ranging from 2 to 9.4% in the Mosel region and 0.2 to 16.2% in the Pfalz region. Double-digit infestation rates in the Pfalz were only measured in two different plots with the cultivar ‘Cabernet Dorsa’.

A part of the samples was further examined by molecular means, first of all by analyzing the diagnostic tuf gene marker. As displayed in Table 2, the tuf-a and tuf-b types were unevenly distributed in the reference vineyards. In the Mittelrhein plots, only tuf-b was present, while in the Mosel plots either tuf-a or tuf-b dominated in different plots. Interestingly, although the tuf-a type was more widely distributed in the Pfalz vineyards, the tuf-b type was equal or dominant in some plots; the location Edenkoben was characterized by a clear tuf-b prevalence.

As the tuf types are indicators for different epidemiological cycles related to the herbaceous host plants nettle or bindweed, the local differences of the tuf types in the reference vineyards could be explained by the different repartition of nettle and bindweed inside and outside of the vineyards.

3.2.2. Weeds

To identify putative herbaceous reservoirs of CaPsol, spontaneous weeds were sampled in reference plots in the Pfalz region, which had increased BN incidence and presence of vector species. The sampling was focused on the most abundant interrow vegetation but also on confirmed host plants of CaPsol, such as U. dioica and C. arvensis. Urtica dioica was not widespread in our plots and did not show any symptoms of a phytoplasma infection, therefore root samples were taken randomly. Convolvulus arvensis was widespread, and the sampling was focused on plants with typical symptoms of CaPsol infection, expressed as stunted internodes and curled and sometimes reddish leaves. In some plots, Tanacetum vulgare or Potentilla reptans were rather abundant. As T. vulgare showed no symptoms, leaf samples were taken randomly. For P. reptans, plants with suspicious symptoms were tested. The 35 C. arvensis sampled exhibited typical symptoms and were all positive for CaPsol. Molecular typing by RFLP showed that all samples were of the tuf-b type, and sequence analysis of selected samples confirmed an association with tuf-b1. Among 39 symptomless U. dioica root samples, all 18 positives were infected with tuf-type a. In contrast, one sample out of 25 T. vulgare and four out of five P. reptans tested positive for CaPsol. All of these weed samples harbored tuf-type b. Regarding further alternative host plants [10], the only sample of Artemisia vulgaris investigated was negative. C. sepium did not occur in the reference plots. A summary of these data and details for MLST are presented in Table S2.

3.2.3. Insects

During the survey of the Auchenorrhyncha fauna in BN-affected vineyards in the Mosel and Pfalz regions, more than 1400 specimens belonging to the families Cixiidae, Cicadellidae, and Dictyopharidae were collected and analyzed for infection with CaPsol and RFLP patterns of the tuf gene were identified (

Table 3. Results of CaPsol detection and tuf typing in different plant- and leafhopper species (nd = not determined).

Insect Species	N° Positive/Total	Tuf-a	Tuf-b	Tuf-c	nd
H. obsoletus (total N°)	144/664 (22%)	41	53	1	49
Pfalz	65/501 (13%)	31	5	0	29
Mosel	79/163 (48%)	10	48	1	20
Reptalus spp. (total N°)	1/75 (1%)	0	0	0	1
Mittelrhein	0/6
Pfalz	0/62
Württemberg	1/7 (14%)	0	0	0	1
R. salicinus (total N°)	4/28 (14%)	0	4	0	0
Franken	0/4
Württemberg	1/12 (8%)	0	1	0	0
Pfalz	3/12 (25%)	0	3	0	0
R. panzeri (total N°)	29/183 (15%)	2	25	0	2
Mittelrhein	0/7
Pfalz	4/12 (33%)	0	3	0	1
Mosel	25/164 (15%)	2	22	0	1
Dictyophara europea	2/203 (1%)	0	1	1	0
Agallia spp.	0/187 (0%)
Anaceratagallia ribauti	0/10 (0%)
Cixius spp.	0/7 (0%)
Neoaliturus fenestratus	0/5 (0%)
Psammotettix spp.	0/48 (0%)
total	1410

). A part of the CaPsol-positive specimens was further characterized by MLST of the genes sec-Y, stamp, and vmp1 (Table S3).

The investigation revealed that H. obsoletus was the most abundant cixiid planthopper in both regions, but the natural infection rate in the Mosel region was four times higher than in the Pfalz region. While in the Pfalz, H. obsoletus was mainly associated with the tuf-a type, characteristic of the nettle cycle, the tuf-b type was predominant in the Mosel region, indicating a relation with the bindweed cycle. One specimen from the Mosel region harbored tuf-c [10].

Among the Reptalus species, R. panzeri was predominant in the investigated vineyards, and it was almost always associated with the tuf-b type. Interestingly, the infection rate of the less abundant R. salicinus was similar to that of R. panzeri and tuf-b type was found in all specimens of R. salicinus. Another described vector species, D. europaea, was also frequently found in some BN-affected plots. Among the 203 individuals tested, two specimens were infected with CaPsol. In one individual, tuf-type c was identified, while the second positive D. europaea was infected with tuf-b type. All other plant and leafhopper species were negative for infection with CaPsol (Table 3).

3.3. Multilocus Sequence Typing (MLST)

After the identification of the tuf genotype, a selection of 289 samples comprising 164 grapevine samples, 42 weed samples, and 83 insect samples was further subjected to a molecular characterization of the secY, vmp1, and stamp genes.

3.3.1. SecY

The sequence analysis of the secY amplicons led to the identification of the sequence variants secY-1, secY-4, and secY-6 that were identical with the previously reported se-quences, according to the SEE-ERA.NET nomenclature [64]. The diverse correlations of the secY gene with the other gene markers are summarized in Figure 2. The tuf-a genotype was almost entirely assigned to secY-6 genotype (98% of samples), while the tuf-b genotype was more variable, being 63% associated by with the secY-1 and 32% with the secY-4 genotype. The tuf-c genotype in H. obsoletus from the Mosel region was associated with the secY-4 genotype. In 99% of the samples, secY-6 was correlated with stamp St11, followed by secY-1 in combination with stamp St10 (74%). The association of the genotype secY-4 with stamp genotypes was highly variable. The correlation of secY genotypes with the RFLP profiles of the vmp1 gene was dominated by the combination secY-6 with vmp1-V1, while the secY-1 genotype was mainly linked to the vmp1-V15 profile, but also with a new profile found in this study (V-VvKL). Again, the secY-4 genotype showed the highest variability in combinations with different vmp1 patterns, including the second new vmp1 profile detected in this study (V-CaIL). Overall, the genotypes secY-1 and secY-6 were prevalent in grapevine and insects, while secY-4 was the most frequent genotype in weeds (Figure 2,

Table 4. CaPsol haplotypes identified in different plant (Vitis vinifera, Urtica dioica, and Convolvulus arvensis) and insect (H. obsoletus, R. salicinus, and R. panzeri) hosts.

			Plant Hosts			Insect Hosts
Haplotype	Haplotype Acronym	N°	V. vinifera	U. dioica	C. arvensis	H. obsoletus	R. salicinus	R. panzeri
tuf-a_secY-1_St11_V1		1 (0.3%)	1
tuf-a_secY-4_St11_V1		1 (0.3%)				1
tuf-a_secY-6_St11_V1	H1	100 (34.6%)	54	9		35		2
tuf-a_secY-6_St11_V5	H6	4 (1.4%)	2			2
tuf-b_secY-1_St1_V4	H7	23 (8.0%)	14			4		5
tuf-b_secY-1_St1_V15		2 (0.7%)	2
tuf-b_secY-1_St1_V-VvKL		3 (1.0%)	3
tuf-b_secY-1_St5_V1		1 (0.3%)	1
tuf-b_secY-1_St10_V1		1 (0.3%)	1
tuf-b_secY-1_St10_V4	H10	6 (2.1%)	5		1
tuf-b_secY-1_St10_V15	H2	78 (27.0%)	52		9	9	2	6
tuf-b_secY-4_St5_V1	H11	8 (2.8%)	2		6
tuf-b_secY-4_St5_V2	H3	7 (2.4%)	1		2	3	1
tuf-b_secY-4_St5_V2-TA	H4	8 (2.8%)	4		3	1
tuf-b_secY-4_St5_V4		1 (0.3%)			1
tuf-b_secY-4_St5_V15		1 (0.3%)					1
tuf-b_secY-4_St10_V1	H8	7 (2.4%)	3			3		1
tuf-b_secY-4_St10_V4		4 (1.4%)			4
tuf-b_secY-4_St46_V2		1 (0.3%)	1
tuf-b_secY-4_St46_V4		1 (0.3%)	1
tuf-b_secY-4_St-CaIL_V-CaIL		1 (0.3%)			1
tuf-b_secY-4_St-HoBKS_V1		1 (0.3%)				1
tuf-b_secY-4_FR622/23_V1		1 (0.3%)				1
tuf-b_secY-4_FR662/23_V4	H5	6 (2.1%)	3		1	2
tuf-b_secY-4_Z187_V1		1 (0.3%)	1
tuf-b_secY-4_Z187_V4	H12	10 (3.5%)	5		5
tuf-b_secY-6_St10_V1		1 (0.3%)				1
tuf-b_secY-6_St11_V1	H9	8 (2.8%)	7			1
tuf-b_secY-6_St11_V2		1 (0.3%)	1
tuf-c_secY-4_St10_V1		1 (0.3%)				1
total		289

). The concrete number of samples per combination is shown in Figure 2.

3.3.2. Vmp1

The vmp1 typing through digestion of nested PCR amplicons of TYPH10 fragments using the RsaI enzyme revealed the presence of 6 RFLP profiles attributed to identified vmp1 types V1, V2, V2-TA, V4, V5, and V15 according to the SEE-ERA.NET nomenclature [23,64] (Figure 2). Two profiles did not correspond to any of the formerly described patterns, designated here as V-CaIL and V-VvKL. The RsaI digestion of V-CaIL (1 sample from C. arvensis in the Pfalz region, details in Table S2) yielded fragments of 889, 252, 174, and 117 bp. The new profile, designated as V-VvKL, was found in three grapevine samples from a single location in the Mosel region (details in Table S1) and yielded fragments of approximately 1044, 252, and 117 bp. New RFLP profiles were confirmed by sequencing (V-CaIL, Accession No. PZ246497; V-VvKL, Accession No. PZ246498) and in silico digestion.

Ninety-six percent of the tuf-a type samples were associated with the V1 profile, while only 16% of the tuf-b type samples and the tuf-c type identified in this study were linked to V1. While all samples expressing a vmp1 V5 pattern were linked to the tuf-a type, all samples with V2, V2-TA, V4, and V15 profiles were associated with the tuf-b type, as were the newly found profiles designated as V-CaIL and V-VvKL. With regard to the correlation with the secY gene marker, the samples showing a V1 restriction profile were mainly assigned to the secY-6 type, and smaller portions were associated with the secY-1 and secY-4 types (Figure 2). Almost all samples with a V15 restriction pattern were linked to the secY-1 type, while V4-related samples were distributed between secY-1 and secY-4 genotypes. Samples showing V2 and V2-TA patterns were associated with the secY- 4 genotype as well as the new profile V-CaIL, whereas the second new vmp1 pattern V-VvKL could be attributed to the secY-1 genotype. The vmp1 pattern V1 was mainly associated with stamp genotype St11 (83%) and the V15 profile was prevalently linked to the stamp St10 genotype (96%). All three samples that showed the new RFLP profile V-VvKL in this study was combined with stamp genotype St1. Interestingly, the isolate V-CaIL could not be attributed to any known vmp1 pattern, but it was associated with a new stamp type, St-CaIL.

3.3.3. Stamp

The sequence identity analysis performed on stamp amplicons enabled the identification of five variants that corresponded with previously reported genotypes [65]: St1, St5, St10, St11, and St46. In addition, the recently reported stamp genotypes Z187 ([38]; Acc.no. MZ604974) and FR622/23 ([66]; Acc.no. PP731988) were detected. Two new genetic variants were identified for the first time in this study and designated as St-CaIL and St-HoBKS. The nucleotide sequences of these variants were deposited in the NCBI GenBank repository under the Accession Numbers PZ232207 and PZ232208. As recorded in Figure 2, all tuf-a type samples had the genotype St11, and this combination accounted for 92% of all St11 samples. All other stamp genotypes identified in this study were linked to tuf-b genotypes, with a prevalence of the association of tuf-b with St10 in 53% of tuf-b samples. Also, the tuf-c variant detected in this study was linked to the genotype St10. Regarding the correlation with secY genotypes, secY-1 was predominantly associated with stamp genotype St10 (74%), secY-4 genotype with St5 (42%), and secY-6 was almost always linked to genotype St11 (99%). Remarkably, the less abundant genotypes FR622/23, Z187, and St46, as well as the novel variants described in this study (St-CaIL and St-HoBKS) were always associated with genotype secY-4. Finally, the different combinations between stamp sequence variants and vmp1 RFLP profiles exhibited a strong correlation between St1 and V4 (82%), St11 and V1 (96%), FR622/23 and V4 (86%), Z187 and V4 (91%), and St10 with V15 (80%) of all samples. Notably, genotype St46 was equally associated with the V2 and V4 profiles, and the new vmp1 profile V-VvKL was linked to genotype St1. The V2-TA profile was exclusively correlated to St5. Interestingly, one sample of C. arvensis exhibited a new stamp variant (St-CaIL) as well as a new vmp1 profile (V-CaIL). The actual numbers of samples for all genotype combinations are shown in Figure 2. In summary, St10 and St11 were the most prevalent stamp genotypes in grapevines, while St11 was dominant in insects and St10 in weeds, accompanied by a high host variability of other stamp genotypes.

The phylogenetic analysis of the stamp genotypes identified in the study (Figure 3) showed that the sequences were members of the subcluster stamp a1, subcluster stamp b1, and subcluster stamp b2. The new genotype St-CaIL clustered with stamp b1 sequences while the new genotype St-HoBKS clustered with stamp b2 sequences.

3.3.4. Combining the Gene Markers to Haplotypes

By combining the gene markers tuf, sec-Y, stamp, and vmp1, 30 different haplotypes were identified in the different German viticultural areas in different plant and insect hosts (Table 4). For details of the geographic locations, see Tables S1–S3. Two haplotypes (H1 and H2) were dominant and accounted for more than 60% of the samples.

The haplotype tuf-a_secY-6_St11_V1 (H1) is the typical nettle type and was found in Vitis, U. dioica, and H. obsoletus. Two samples of R. panzeri also harbored this haplotype. H1 occurred in all winegrowing regions. It was predominant in Pfalz (36% of all samples) and in Baden-Württemberg (63%) (Figure 4). The haplotype tuf-b_secY-1_St10_V15 (H2) was typically correlated to the bindweed epidemiological cycle and was found in Vitis and C. arvensis as well as in all insect vector species. It was putatively also found in P. reptans, although the secY marker is missing (Table S2). While H2 was not detected in Baden-Württemberg, it was dominant (33% of samples) in the Pfalz region together with H1. Further haplotypes that were found in grapevine, bindweed, and one of the insect vector species were considered as potentially epidemic CaPsol haplotypes. These were tuf-b_secY-4_St5_V2 (H3), which was detected in all regions, and tuf-b_secY-4_St5_V2-TA (H4), which was only found in the Pfalz region. The new combination tuf-b_secY-4_FR622/23_V4 (H5) was identified for the first time in grapevine, bindweed, and H. obsoletus. It was restricted to the Pfalz region.

Other haplotypes were found only in grapevine and in at least one planthopper species, but the tuf-type strongly indicated a weedy plant host. For example, tuf-a_secY-6_St11_V5 (H6) is a nettle genotype restricted to the Pfalz. In contrast, the haplotype tuf-b_secY-4_St1_V4 (H7) was exclusively found in the Mosel–Mittelrhein region and had the highest frequency of all haplotypes (32% of all samples) in both H. obsoletus and R. panzeri. Haplotype tuf-b_secY4_St10_V1 (H8) was also restricted to the Mosel–Mittelrhein, except for one sample in the Pfalz region. In contrast, haplotype tuf-b_secY-6_St11_V1 (H9) was restricted to Pfalz and Württemberg.

A third group of haplotypes was only found in grapevine and bindweed. Thus, the respective vector species remains unclear. Except for H10, which was present in Baden-Württemberg, these haplotypes were restricted to the Pfalz region: tuf-b_secY-1_St10_V4 (H10), tuf-b_secY-4_St5_V1 (H11), and tuf-b_secY-4_Z187_V4 (H12). In addition, the combination tuf-b_St10_V1 was identified in the new host T. vulgare, indicating an association with the haplotype H10, although the secY marker was not included (Table S2).

All other haplotypes were found only once or in low numbers in either grapevines or an insect sample. E.g., the new vmp1-genotype VvKL was identified in three samples of a single location in the combination tuf-b_secY-1_St1_VvKL. The tuf-c_secY4_St10_V1 haplotype was detected in H. obsoletus from the Mosel but likely also in one D. europaea from the Pfalz, although in the latter case the secY marker was not determined (Table S3).

The major haplotypes identified in this study suggested different propagation pathways, as shown in Figure 5. The epidemic haplotype H1 was exclusive for nettle, while H2–H5 were associated with bindweed and are transmitted by H. obsoletus and/or Reptalus spp. The haplotype H5 has recently been identified in carrots [44], which however might be a dead-end host, like grapevine.

4. Discussion

Bois noir disease in Germany has been characterized in the past by short epidemic outbreaks with high infection rates and longer endemic periods with low level infection rates [15]. The actual monitoring of 24 vineyards in the Mosel, Mittelrhein, and Pfalz regions showed that the incidence of BN is rather low compared to previous reports, wherein the rate of symptomatic vines reached up to 50% in the Mittelrhein region. A recorded infection rate of 22% in a Riesling vineyard in this region, monitored every year since 1996 [21], indicates that BN is currently in a stable endemic phase. The incidence in the other regions (Mosel and Pfalz) varied considerably and were also related to the cultivar. Particularly high rates were observed in cv. ‘Cabernet Dorsa’, which is a descendant of the cv. ‘Lemberger’, the most susceptible cultivar known in Germany [67]. Interestingly, the highly FD-susceptible cultivar ‘Chardonnay’ had a relatively low BN incidence in the Pfalz region, despite a severe expression of GY symptoms. The analysis of the repartition of the tuf-a and tuf-b types in the reference vineyards indicated different local epidemiological cycles related to the availability of the respective host plants, nettle and bindweed. An increase of tuf-b types in the Pfalz region was observed, which is new for this region [21,68]. It could be linked to alternative epidemiological pathways as well as to global warming, since H. obsoletus requires higher temperatures for its development but also completes its life cycle faster on bindweed [69]. Environmental conditions such as summer drought stress could reduce stinging nettle populations and changed weed control practices may favor bindweed within vineyards.

The German-wide analysis of tuf types revealed striking differences between northern and southern winegrowing regions. The differences can be attributed to different cultural practices, e.g., steep-slope vineyards with xerothermic conditions in the north, or to different epidemiological cycles based on the availability of the host plant. Regional differences in the tuf-a and tuf-b strains have also been reported in Italy, where tuf-a was prevalent in northern Italy [5,70,71] while in central and Southern Italy tuf-b was dominant [2,65,71,72]. In Slovenia, the repartition of tuf types is split into two regions: tuf-b2 is increasing in the northeastern part toward the Austrian border while tuf-a and tuf-b1 dominate in the southwestern part on the border to Italy [23], which mirrors the situation in central Italy, where only tuf-a and tuf-b1 are present [73].

Since the analysis of tuf types in our study was based on RFLP analysis including HpaII-digestion, no distinction between different b-types could be made. However, sequence analysis of random samples of tuf-b isolates from different regions, plants, and insects revealed that all isolates corresponded to the tuf-b1 type. Hence, the tuf-b2 type has not been detected in Germany so far. Other tuf types have only been found in Asian countries (tuf-b3, tuf-b5, tuf-b6) [22] or in sugar beets (tuf-d, tuf-e) [39,40] and are thus unlikely to be detected in grapevines in Germany. In contrast, RFLP with HpaII enables identification of the tuf-c type, which had previously been reported only in the lower Mosel region in Germany by Langer and Maixner [10]. This tuf-b1 variant has been continuously found (Maixner, personal communication) in H. obsoletus since then. In this study, however, we detected the tuf-c type for the first time in the Pfalz region and in another confirmed CaPsol vector, D. europaea, suggesting that this tuf-type is more widespread than previously thought.

As the dispersal of BN could not always be explained by H. obsoletus-associated nettle- or bindweed pathosystems [26,31,33], one focus of the study was on putative vectors and alternative epidemiological cycles. While Reptalus spp. are important CaPsol vectors in Eastern Europe, they are generally rare in Germany [74]. In the past, R. panzeri was found in high populations only in xerothermic steep-slope vineyards in Mosel and Mittelrhein [74]. Our recent data show, that the CaPsol infection rates of both Reptalus species were in the same range of 15%, which corresponded to the infection rate of H. obsoletus in the Pfalz. This is a considerable increase compared to the infection rate of 1.1% reported by Lang et al. [74] for the period of 2009–2014 in the Mosel region. The infection rates measured in Reptalus spp. corresponded to those found in Eastern Europe, with ca. 18% for R. salicinus and about 22% for R. panzeri [26]. This indicates that Reptalus spp. might contribute an increasing part to the spread of tuf-b, since almost all Reptalus spp. were infected with this tuf type. However, two individuals of R. panzeri captured in the Pfalz were infected with tuf-a, confirming that R. panzeri is able to acquire tuf-a from nettles and transmit it to Catharanthus test plants [74]. Among other collected Auchenorrhyncha species that had been reported as putative vectors from other European countries [3,5,22,32], only two out of 203 specimens of D. europaea were found to be infected. One specimen harbored the comprehensive genotype tuf-c_St10_V1, which was found only once in H. obsoletus from the Mosel region, which suggests that a D. europaea has a different epidemiological cycle in Germany than in Eastern European countries [32].

Besides alternative insect vectors, multiple reservoir plants can play a role in the CaPsol ecology and thus contribute to the spread of BN, at least at a local level. Two groundcover weed species in vineyards were found to be infected with CaPsol in this study: P. reptans and, for the first time, T. vulgare. This confirms a previous report by Credi et al. [75] from borthern northern Italy that P. reptans represents a host for CaPsol. Interestingly, all four positive samples were infected by the same marker gene combination, tuf-b_St10_V15, and this comprehensive genotype was also identified in two R. salicinus specimens from the same location, designated as complete haplotype H2. This could imply the existence of a local epidemiological CaPsol cycle. Although multiple spontaneous weeds were examined in diverse studies [47,70,75] the potential of T. vulgare as a CaPsol host plant was not analyzed. Lessio et al. [76] only mentioned that adults of H. obsoletus were found on T. vulgare, but they did not further elucidate its role as a putative CaPsol reservoir. In some plots in this study, T. vulgare was quite common, and we collected both H. obsoletus and Reptalus spp. from these plants. The genetic characterization of the CaPsol isolate in the infected plant revealed the combination tuf-b_St10_V4. This combination has also been detected in one H. obsoletus from another plot but also as comprehensive genotype in C. arvensis, indicating a possible role of T. vulgare in the BN epidemiology, at least on a local level.

A molecular typing of the CaPsol strains was conducted to decipher potential new epidemiological cycles in Germany. While many recent studies have investigated the diversity of CaPsol in Eastern and Southern Europe [6,23,32,65,71,72,77,78], data for Germany were missing. Therefore, the genetic variability of CaPsol isolates in Germany was assessed by combining the data sets of four epidemiologically relevant genes (tuf, secY, stamp, and vmp1) from different plant and insect hosts to yield informative haplotypes of CaPsol. Two out of 30 different haplotypes were dominant: H1 (tuf-a_secY-6_St11_V1) and H2 (tuf-b_secY-1_St10_V15). H1 is specific to nettles, accounted for 99% of all tuf-a type strains in Germany, and is widespread in all German winegrowing regions. It corresponds to the type N1s1-V1 reported by Johannessen et al. [24] and was identified in 92% of samples from nettle and H. obsoletus collected between 2005 and 2010. It is considered as the type strain of the nettle epidemic cycle in Germany due to its very stable association with U. dioica and H. obsoletus. However, H1 is not reported in other Central and Eastern European countries. H1 was also detected in a few cases in R. panzer panzeri, confirming previous data that R. panzeri is also able to transmit this nettle type strain [74]. H2 can be regarded as a typical strain of the bindweed cycle in Germany, as it was only found in bindweed and accounted for 43% of all tuf-b haplotypes. It is widespread in Mosel and Pfalz but was not detected in Mittelrhein or Württemberg. This implies that H2 is predominantly transmitted by the bindweed-adapted host-race of H. obsoletus [16], albeit this haplotype is also associated with the alternative vector species R. panzeri and R. salicinus.

Tuf-a type strains showed a very low genetic variability, with high associations of the secY-6 genotype, the stamp type St11, and the vmp1 type V1. Interestingly, the tuf-a specific combination secY-6_St11_V1 was also found in combination with tuf-b (haplotype H9) in grapevine and H. obsoletus. This could indicate a potential recombination event in H. obsoletus that led to a host plant-shift from nettle to bindweed. The genetic monomorphism of tuf-a strains seems to be particular to Germany and adjacent regions. E.g., several different stamp types were found in Eastern and Southern Europe, clustering in the stamp subclusters stamp-a1 and stamp-a2 related to tuf-a [65]. None of the additional gene marker genotypes have been identified in Germany. These results thus corroborate the observation reported by Johannesen et al. [24].

Conversely, tuf-b type strains exhibited a much higher genetic variability due to the combination with all three secY genotypes, all nine stamp genotypes, and seven out of eight vmp1 genotypes identified in the present study. This is in agreement with the data of many other studies from Eastern and Southern Europe. The second most abundant tuf-b haplotype H7 (tuf-b_secY-1_St1_V4) was found in Mittelrhein and Mosel in grapevines but also in equal percentages in H. obsoletus and R. panzeri. This haplotype was also reported from various hosts in Serbia [26,79], Macedonia [77], Montenegro [6], Slovenia, Styria [23], and Croatia [4], and could represent a link between Eastern and Western European strains. The combination tuf-b_secY-1_St1 found in the Mosel region also combined with vmp1 types V15 and V-VvKL. While V15 is the major vmp1 type in combination with tuf-b, V-VvKL is so far unreported. Interestingly, stamp type St1 (Reptalus associated isolate Rqg50, Acc.no. KC703019 [26]) was only found in the Mosel–Mittelrhein area with locally high populations of R. panzeri. No herbaceous host plant could be correlated to this genotype. This raises the question of whether other host plants of St1 exist in this region. Nevertheless, this study reveals that in Germany the stamp genotype St1 appears to be restricted to xerothermic viticultural sites where R. panzeri is regularly found. Haplotype tuf-b_secY4_St10_V1 (H8) is also more abundant in the Mosel region than elsewhere in Germany. This is consistent with data from the central Italian vineyards of Tuscany [3,80], where St10 was dominant in grapevine samples as well as in R. salicinus, and suggests the existence of a new epidemiological pattern of CaPsol in Tuscany vineyards including grapevine, R. salicinus, and weeds and comprising an extremely specialized population of R. salicinus.

Analysis of the data on the level of the individual gene marker showed that the genetic variability of CaPsol in Germany is strikingly different from other European regions. Only three out of more than 30 known secY genotypes (SEE-ERA.Net database [64]) have been found in the present study. They represent the three major secY types dominant in Western Europe: secY-1, secY-4, and secY-6. However, secY-1 is widespread all over Europe [22,23,49,81]. In Germany, secY-1 was associated with only three stamp genotypes, St1, St5, and St10, from which St1 and St10 were found in most of the Reptalus spp.

Genotype secY-4 is dominant in France and seems to be related to weeds. In Austria, it has been found in grapevine [22] and in Croatia in grapevine and bindweed [78]. In Germany, the highest proportion of secY-4 strains was detected in bindweed, followed by grapevine and H. obsoletus. Furthermore, secY-4 was detected in association with the widest variety of different stamp and vmp1 types, suggesting the possibility of new epidemiological cycles emerging. The combination tuf-b_secY-4_St5 was detected in several epidemic haplotypes: tuf-b_secY-4_St5_V2 (H3), tuf-b_secY-4_St5_V2-TA (H4), tuf-b_secY-4_St5_V1 (H11), and tuf-b_secY-4_St5_V4. Compared to other combinations, tuf-b_secY-4_St5 was primarily detected in bindweed and rarely in H. obsoletus and Reptalus spp., raising the question of whether there is another vector that does not transmit to grapevine. In other European countries like Austria [22], Croatia [4], Italy [82,83] and Hungary [82], St5 was detected in a wide range of different plants and insects.

The secY-6 type is known from Western Europe as a tuf-a_secY-6 combination in the typical nettle haplotype (H1). The combination tuf-a_secY-6_St11 has also been reported locally from grapevines in Slovenia [23]. In Austria, secY-6 and St11 are associated with tuf-b2 [22,23]. In addition, the combination secY-6_St11 has also been found in association with tuf-b1 in Slovenia [23] as well as in Germany, where it accounted for about 3% of the secY-6 type isolates. The corresponding epidemic haplotype H9 is potentially transmitted by H. obsoletus. In Southeastern Europe, secY-6 has much more variable associations with other markers including tuf, stamp, and vmp1 [4,23].

Despite the high genetic variability of the stamp gene, with more than 60 different known variants ([3,65]; present data), only few genotypes were prevalent in Germany compared to other countries. The two major genotypes St10 and St11 were related to the dominant bindweed (haplotype H2) or nettle (haplotype H1) epidemiological cycles, respectively. However, no clear correlation of one of the genotypes with either H. obsoletus or Reptalus spp. was observed. Almost all stamp genotypes were also found in H. obsoletus. The stamp genotype St11 (Stamp-6 according to the SEE-ERA-Net nomenclature) has been mainly recorded for isolates from different plant hosts and insects from Serbia [8,26,79,82] and is a major stamp genotype for grapevines and H. obsoletus captured on grapevines, and for U. dioica in Austria and Croatia [22,78]. The finding of genotype St46 was unexpected, as this genotype was originally described from a R. salicinus (former R. quinquecostatus) isolate in Southern France (RQ161, Acc.no. LN823951 [28]). In Germany, this genotype has now been described from grapevine samples obtained from two different regions, Mosel and Pfalz, where diverse Reptalus spp. are present.

Although the gene marker vmp1 is considered to play a role in the phytoplasma-insect interaction, no clear correlation of one of the genotypes with either H. obsoletus or Reptalus spp. was observed. Six vmp1 profiles were found in both grapevine and H. obsoletus. Only the profiles V4 and V15 were identified in R. panzeri in substantial numbers compared to H. obsoletus. This is not surprising, since these bindweed-related vmp1 profiles were the major profiles found in Germany. However, this is in contrast to the situation in Eastern Europe, where V4 is rarely identified and V15 is absent [6,26,79]. Both profiles are also rare in Southern Italy [84]. Another vmp1 genotype confirming the distinct molecular CaPsol pattern between Western and Eastern Europe is V2-TA, which is widespread in Eastern Europe [4,6,26,32,79] but was sparse in Germany. Similarly, common Eastern European profiles like V14 [4,6,26,32,79] were not identified in our survey. However, one case of V14 was recently reported from carrots in Germany [44]. On the other hand, the dominant vmp1 profile in Germany, V1, is absent in Eastern Europe [6,26,32,79]. This profile is part of the nettle haplotype H1 but was also found in bindweed and R. panzeri, indicating a less conserved host specialization than the H1 haplotype itself.

Another interesting result is the detection of stamp genotypes in grapevine that were recently described only in sugar beet samples. Genotype Z187 was reported by Cvrcic et al. [38] from a single plant in South of Hessen in Germany, a location not very far from Rheinhessen and Pfalz. Genotype FR622/23 was detected by Duduk et al. [66] in a sugar beet field in Northwestern France. The present data show that these genotypes are widespread in Germany, as they were detected in Mosel, Pfalz, and Württemberg. Both genotypes were related to bindweed and the genotypes were assigned to tuf-b and secY-4 markers. In addition, the two haplotypes tuf-b_secY-4_FR622/23_V1 and tuf-b_secY-4_FR662/23_V4 (H5) were found in H. obsoletus, suggesting a propagative cycle via this vector. Since stamp Z187 was not detected in any insect vector but the haplotypes tuf-b_secY-4_Z187_V1 and tuf-b_secY-4_Z187_V4 were comparable to the FR622/23-related genotypes, there is strong evidence that these haplotypes are also transmitted by H. obsoletus. However, the haplotype H5 has recently been described in severely infected carrots [44] in two different fields in Rheinhessen and Pfalz. These carrot fields were not only infected with CaPsol (tuf-b1) but also with CaAp and the new 16SrXII-P strain. As the latter pathogens are transmitted by P. leporinus, the question arises whether the 16SrXII-A haplotype H5 is also transmitted by this new 16SrXII-vector.

Thus, the detection of sugar beet-related CaPsol isolates in grapevine and bindweed extends the epidemiological significance of the present study to other cultural crops and vectors in Germany. In the last years, severe stolbur epidemics occurred not only in sugar beets and potatoes [43] but now spread further to other vegetable crops [85,86].

5. Conclusions

This study aimed to investigate the current status of the spread of Bois noir in the most important winegrowing regions in Germany and to elucidate the actual epidemiological cycles. The genetic diversity of CaPsol isolates in known and potential plant and insect hosts was analyzed. Monitoring data from different winegrowing regions showed that the disease is currently in a stable endemic phase. The analysis of the tuf gene marker indicated different epidemiological cycles in northern and southern German viticultural regions. The data suggest an increasing prevalence of the tuf-b1 type that may be linked to changing climatic conditions. While H. obsoletus remained the most important vector both of the nettle and of the bindweed cycle, the proportion of CaPsol infected Reptalus spp. increased considerably. This is accompanied by the growing importance of bindweed as a CaPsol reservoir plant. In addition, CaPsol infections were also detected for the first time in Germany in P. reptans and T. vulgare as well as in the known vector D. europaea.

The CaPsol isolates of infected samples were characterized by MLST and RFLP analyses of a set of genetic markers, including the genes tuf, secY, stamp, and vmp. The data revealed a remarkable difference between Germany and other European regions in terms of the occurrence and distribution of the individual markers. New stamp and vmp1 genotypes were detected. While the genetic diversity among tuf-a type isolates was very low, it was high for tuf-b1 isolates. Based on the marker combinations, comprehensive genotypes could be generated and defined as specific haplotypes. Two major haplotypes related to the nettle (haplotype H1) and bindweed (haplotype H2) epidemiological cycles, respectively, were identified. No specific haplotype could be associated with the BN spread by H. obsoletus or Reptalus spp. However, the new haplotype H5 indicated a new link to the epidemic spread of CaPsol in sugar beets and carrots in Germany, which is related to P. leporinus. Further studies are needed to clarify if this vector is also able to transmit CaPsol to grapevine.