An abundant ' Candidatus Phytoplasma solani' tuf b strain is associated with grapevine, stinging nettle and Hyalesthes obsoletus

Abstract

Bois noir (BN) associated with 'Candidatus Phytoplasma solani' (Stolbur) is regularly found in Austrian vine growing regions. Investigations between 2003 and 2008 indicated sporadic presence of the confirmed disease vector Hyalesthes obsoletus and frequent infections of bindweed and grapevine. Infections of nettles were rare. In contrast present investigations revealed a mass occurrence of H. obsoletus almost exclusively on stinging nettle. The high population densities of H. obsoletus on Urtica dioica were accompanied by frequent occurrence of 'Ca. P. solani' in nettles and planthoppers. Sequence analysis of the molecular markers secY, stamp, tuf and vmp1 of stolbur revealed a single genotype named CPsM4_At1 in stinging nettles and more than 64 and 90 % abundance in grapevine and H. obsoletus, respectively. Interestingly, this genotype showed tuf b type restriction pattern previously attributed to bindweed associated 'Ca. P. solani' strains, but a different sequence assigned as tuf b2 compared to reference tuf b strains. All other marker genes of CPsM4_At1 clustered with tuf a and nettle derived genotypes verifying distinct nettle phytoplasma genotypes. Transmission experiments with H. obsoletus and Anaceratagallia ribauti resulted in successful transmission of five different strains including the major genotype to Catharanthus roseus and in transmission of the major genotype to U. dioica. Altogether, five nettle and nine bindweed associated genotypes were described. Bindweed types were verified in 34 % of grapevine samples, in few positive Reptalus panzeri, rarely in bindweeds and occasionally in Catharanthus roseus infected by H. obsoletus or A. ribauti. 'Candidatus Phytoplasma convolvuli' (bindweed yellows) was ascertained in nettle and bindweed samples.

Keywords: Bois noir; Stamp; Stolbur; secY; vmp1; ‘Candidatus phytoplasma convolvuli’.

Fig. 1

Infection rates of H. obsoletus at selected investigation sites listed in Table 1 in the Austrian vine growing Federal Provinces of Burgenland (Großhöflein, Rust1-4), Styria (Einöd) and Lower Austria (all other locations). Individuals from Großhöflein 1–3 and Niedersulz 1 and 2 were pooled

Fig. 2

Infection rates of weed samples at several investigation sites in the Austrian vine growing provinces of Burgenland (Rust1-3), Styria (Einöd) and Lower Austria (all other locations)

Fig. 3

Results of transmission experiments. Transmissions were performed with 10–50 field collected insects per trial in cylindrical cages as described in material and methods. U.d. – H.o. Experimental transmission to U. dioica by H. obsoletus. C.a. – H. o. Experimental transmission to Convolvulus arvensis by H. obsoletus; C.r. – H. o. Experimental transmission to Catharanthus roseus by H. obsoletus; C.r. – R.p. Experimental transmission to Catharanthus roseus by R. panzeri; C.r. – A. r. Experimental transmission to Catharanthus roseus by A. ribauti

Fig. 4

Analysis of the TUF-AY fragments of the elongation factor Tu (tuf) gene. a ‘Ca. P. solani’ types found in this study. Genotypically, 3 different sequences have been detected (tuf a, tuf b1 and tuf b2) corresponding to two HpaII types (tuf a and tuf b). Sequence differences are indicated for nucleotide (N) position after the Tuf start codon. Tuf a and tuf b reference strains from Germany and strains identified in this study (one example from each detected host species) are indicated. Vv: grapevine. Ca: bindweed. Ud: stinging nettle. Cr Catharanthus roseus; Ho H. obsoletus; Ar A. ribauti; Rp R. panzeri, aa: amino acid. Virtual (b) and sample (c) gel showing patterns of TUFAY fragments of a bindweed yellows (bwy) strain and of ‘Ca. P. solani’ tuf a and tuf b digested with HpaII

Fig. 5

SecY analysis showed a clear separation in two distinct groups containing a Hinf1 site allowing RFLP analysis. a Phylogenetic secY consensus tree of PosecF3/PosecR3 fragments and corresponding database entries (reference sequences from Cimerman et al. ; Cvrković et al. ; Fabre et al. ; Johannesen et al. ; Mitrovic et al. ; Semetey et al. unpublished; Seruga-Music et al. 2011) analysed by Maximum Likelihood. The numbers above the branches indicate the bootstrap values >60 (2,000 replicates). Differential Hinf1 site resulting in a corresponding amino acid substitution and 2 secY restriction types is indicated. b RFLP pattern obtained after digestion of PosecF3/PosecR3 fragments with Hinf1; SecY 1 corresponds to the “classical” tuf b1 type, SecY 2 to the tuf b2 type, NTC no template control. Tuf a strains were identical to tuf b2 strains in this analysis

Fig. 6

a Phylogenetic stamp tree of Stamp fw-0 and rv-0 fragments and b vmp1 tree of TYPH10F and TYPH10R fragments as well as corresponding database entries (reference sequences from Cimerman et al, ; Cvrković et al. ; Fabre et al., ; Johannesen et al., ; Mitrovic et al., ; Murolo et al., 2010; 2013) were analysed by Maximum Likelihood. The numbers above the branches indicate the bootstrap values >60 (2,000 replicates). Insertions (ins) and deletions (del) are indicated. Vm_At9, PO and 19–25 contain large deletions in the region of the repeated domain B (Cimerman et al, 2009). Corresponding tuf a and tuf b2 clusters are indicated

Fig. 7

Occurrence of ‘Ca. P. solani’ strains in different hosts V.v: grapevine. U.d. U. dioica; C.a. Convolvulus arvensis; H.o. H. obsoletus, all infected H. obsoletus collected from nettle. ‘Ca. P.solani’ genotypes either directly determined or in Catharanthus roseus after H. obsoletus transmission; R.p. R. panzeri; A.r. A. ribauti, determined or in Catharanthus roseus after A ribauti transmission. Genotypes were defined on basis of four sequenced marker genes. Genotype definition and database entries are shown in Figs. 5 and 6 and Table 2.