Detection and phylogenetic analysis of phytoplasmas
Using qPCR on 227 leafhoppers, a positive signal was detected in 111 specimens. Only 14 samples with Cq value ≤ 30.38 were selected for further analysis (Table S1). The nested PCR primed by F2n/R2 amplified fragments of the 16S rRNA gene of 1,200 bp from 6 phytoplasma-infected samples (Fig. S1). A lower or different sensitivity of the direct/nested primers compared to the ones used in qPCR may have caused the negative results for the remaining 8 samples. Overall, 6 species tested positive:Leofa (Tortotettix ) dispar (Theron) (molecular sample codes: LH078), Pravistylus exquadratus (Naudé) (LH082),Macrosteles (Macrosteles ) sordidipennis (Stål) (LH102), Mayawa capitata (Kirkaldy) (LH133), Mayawa affinifacialis Dietrich (LH139), and Acharis ussuriensis(Melichar) (LH143) (Table 1, Fig. 2 A-F).
The phylogenetic trees included 27 phytoplasma strains, and the alignment of 16S rRNA consisted of 952 positions (including gaps). The ML phylogenetic tree recovered our new sequences in three main clusters (A, B and C in Fig. 3). The first well-supported cluster (A) includes a monophyletic group of 4 samples from this study (LH078, LH082, LH139, LH143) and 4 strains belonging to 16SrXI phytoplasma group (2 ‘Ca. Phytoplasma sacchari’ and 2 Goosegrass white leaf phytoplasma strains) and the Candidatus species ‘Ca.Phytoplasma oryzae’ (16SrXI) + ‘Ca. Phytoplasma cynodontis’ (16SrXIV). Although the internal branches of this clade are very short with low bootstrap support, both samples from South Africa (LH078, LH082) were recovered in the same subcluster. Samples from China (LH143) and Australia (LH139) are polyphyletic, with LH139 branching more deeply than LH143. A recent comprehensive ML tree for phytoplasmas recovered members of 16SrXI as paraphyletic with respect to 16SrXIV (Cao et al., 2020).
In the second cluster (B), LH133 is sister to ‘Ca. Phytoplasma brasiliense’ (16SrXV) and together these two strains are sister to the closely related phytoplasma strains in the 16SrII group. The last cluster (C) includes LH103 and members of the 16SrI phytoplasma group. The NJ analysis yielded the same topology (not shown).
Although the pairwise similarity of the sequences with closely related reference strains is >97.5%, further characterization revealed that all the phytoplasmas detected in this study represent new strains belonging to five new subgroups (Wei et al., in review).