DNA amplification and sequencing of phytoplasmas
TaqMan real time PCR (qPCR) analysis of the 16S ribosomal gene was carried out on DNA extracted from the 227 specimens to identify the presence of phytoplasmas, with the primers and probe described by Christensen et al. (2004). The assays were performed in 96-well plates on a CFX96 thermal cycler (Biorad), according to the protocol of Angelini et al. (2007). The reaction in 10 µl contained 4 µl of DNA template diluted 1:2, 5 µl Platinum Quantitative PCR Supermix-UDG (ThermoFisher scientific), 160 nM for each primer and probe. Because this protocol may yield false positives for other bacteria (e.g.,Bacillus spp.), samples with Cq value ≤ 30.38 (according to Christensen et al., 2004) were tested using nested PCR of the 16S ribosomal RNA gene to confirm the phytoplasma identity. In the 16S rRNA region, nested PCR was performed using universal primer pair P1/P7 (Deng & Hiruki 1991; Smart et al., 1996) followed by F2n/R2 (Gundersen & Lee, 1996). Amplicons were visualized on 1% agarose gel stained with GelRed (Biotium Inc.) under a GelDoc XR UV transilluminator (Biorad). The DNA of ALY (Italian alder yellows) phytoplasma, obtained from experimentally-infected periwinkle (Catharanthus roseus ), was used as a positive reference strain in all the amplification reactions. Sequencing of the F2n/R2 amplicons was carried out in both directions using automated equipment (BMR Service, Padua, Italy). Forward and reverse reads were assembled using Gap4 and Pregap (Bonfield et al., 1995), followed by manual editing. Nucleotide sequences were deposited in the GenBank database under the accession numbers listed in Table 1. An initial BLAST query (Altschul et al., 1990) was performed in order to evaluate the similarity of newly obtained sequences to phytoplasma reference sequences, the top two similar sequences were included in the dataset for further phylogenetic analyses. The final reference sequence dataset consisted of 21 sequences obtained from the National Center for Biotechnology Information (NCBI) database (Federhen, 2012). The ingroup included 20 phytoplasma strains (11 described as ‘Ca.Phytoplasma’ species, including an incidental citation) representing different countries and isolated from distantly related hosts (Table S2) and the outgroup included Acholeplasma palmae(Acholeplasmataceae ). Electropherograms were corrected and aligned using the Muscle algorithm as implemented in MEGA 7.0 (Edgar, 2004; Kumar et al., 2016) with default settings. Phylogenetic trees were constructed with the Maximum Likelihood (ML) and Neighbor Joining (NJ) methods. Branch support was measured using a bootstrap test with 1,000 replicates.