A new phytoplasma associated with little leaf disease in azalea: multilocus sequence characterization reveals a distinct lineage within the aster yellows phytoplasma group

Plant samples

Leaf samples were collected from azalea plants showing little leaf, yellowing and witches'-broom growth symptoms in three locations of suburban Kunming, Yunnan Province, southwestern China in 2009 and 2010. Samples from asymptomatic plants in the same region were collected as negative controls.

Transmission electron microscopic observation

Leaf midrib samples of symptomatic and asymptomatic azalea plants were collected, cut into pieces of approximately 2 mm and fixed immediately with 2.5% glutaraldehyde in 0.1 M potassium phosphate buffer (pH 7.2) for 48 h at 4°C. The samples were subsequently postfixed with 2% osmium tetroxide, dehydrated through an ethanol series, infiltrated with propylene oxide and embedded in Epon 812 resin. After an overnight polymerization at 60°C, ultrathin sections were prepared using a microtome (LKB-2188, Bromma, Sweden). The sections were stained with 3% uranyl acetate and lead citrate before being examined under a transmission electron microscope (TEM; JEM-1200EXII, Tokyo, Japan).

DNA extraction and PCR

Total DNA was extracted from 0.5 g leaf tissues of each sample using a modified CTAB-DNeasy procedure as described previously (Cai et al., 2008). Nested PCR assays were performed for amplifications of three phytoplasma genomic segments that contain genes encoding 16S rRNA; ribosomal proteins (rp) S19, L22 and S3; and preprotein translocase SecY subunit, respectively. The primer sets used for the nested PCRs were P1/P7 followed by P1A/16S-SR for 16S rRNA gene amplification (Deng & Hiruki, 1991; Lee et al., 2004), rpF1/rpR1 followed by rp(I)F1/rp(I)R1A for rp gene amplification (Lee et al., 1998; Martini et al., 2007) and L15F1A(I)/MapR1A followed by SecYF1a/SecYR1(I) for secY gene amplification (Lee et al., 2010a). Each reaction mixture, in a total volume of 50 µL, contained 1 µL of the above prepared DNA extract, 25 pmol each of a forward and a reverse primer, 200 µM each of dATP, dCTP, dGTP and dTTP, 5 µL of GeneAmp High Fidelity 10× PCR buffer (with 25 mM MgCl₂) and 2.5 units of GeneAmp High Fidelity enzyme mix (Applied Biosystems, Foster City, CA). PCRs were carried out for 35 cycles under the following conditions: denaturation at 95°C for 60 s, annealing at 56°C for 60 s and extension at 72°C for 75 s. Amplicons were analysed by electrophoresis through agarose gel.

Amplicon cloning and sequencing

The phytoplasmal genomic segments amplified above were cloned into plasmid vector pCRII-TOPO (Invitrogen, Carlsbad, CA), and propagated in Escherichia coli. Both strands of cloned phytoplasmal DNA segments were sequenced to achieve at least 4× coverage per base position. DNA sequencing was performed with an ABI PRISM 377 automated DNA sequencer (Applied Biosystems).

Virtual and actual gel restriction fragment length polymorphism analyses and phytoplasma classification

For virtual RFLP analysis, 16S rDNA nucleotide sequences from reference strains of all previously delineated subgroups in the AY phytoplasma group (16SrI) were retrieved from the National Center for Biotechnology Information's nucleotide database. Together with the AzLL phytoplasma 16S rDNA sequences determined in the present study, the retrieved sequences were trimmed to the full F2nR2 region that was bounded by the two conserved nucleotide blocks corresponding to the annealing sites for phytoplasma-universal 16S rDNA primer pair R16F2n/R16R2 (Gundersen & Lee, 1996), and the trimmed sequences were subjected to computer-simulated restriction digestion and virtual gel plotting using iPhyClassifier (Zhao et al., 2009b). The virtual RFLP patterns were automatically compared and a similarity coefficient (F) was calculated for each pair of phytoplasma strains using a Perl program described previously (Wei et al., 2008b). For actual gel-based PCR-RFLP analysis, 16S rDNA F2nR2 fragments generated from nested PCR were subjected to digestions by key restriction enzymes that produced new patterns in virtual RFLP analysis; restriction fragments were separated by electrophoresis on a 3% agarose gel and visualised by ethidium bromide staining. The 16S rDNA RFLP patterns and pattern similarity coefficients were used as a basis for group and subgroup classification of the AzLL phytoplasma according to the criteria established previously (Lee et al., 1998; Wei et al., 2008b). Virtual and actual gel RFLP analyses of rp and secY genes were conducted essentially in the same way as described above for 16S rRNA genes.

Phylogenetic analysis

Phylogenetic trees were constructed with the maximum parsimony method using the PAUP* program. The parsimony analyses were performed with the heuristic search option via random stepwise sequence addition. Uninformative characters were excluded from the analyses. The reliability of the phylogenetic analyses was subjected to a bootstrap test with 1000 replicates. Acholeplasma laidlawii was designated as the out group to root the tree.

Protein structure predictions

AzLL and OYM phytoplasma SecY protein sequences were deduced from corresponding secY gene nucleotide sequences. Three-dimensional structures were predicted using the online SWISS-MODEL program (http://swissmodel.expasy.org/). Predicted three-dimensional structures were visualised and analysed with Swiss PDB-Viewer (Guex & Peitsch, 1997).

AzLL disease symptoms and evidence of phytoplasma infection

Azalea plants growing in suburban areas of the capital city Kunming of Yunnan Province, China, exhibited abnormal growth patterns that included reduced leaf size (little leaf), shortened internodal length (stunting), excessive branching (witches'-broom-growth) and leaf chlorosis (yellowing) (Fig. 1). Although the symptom combinations and the severity of the symptoms varied from plant to plant, the little leaf symptom was present in all affected plants and was most prominent compared with other symptoms; therefore, the disease was designated as AzLL disease. The disease first occurred in 2009 and then again in 2010. Three locations where the diseased plants were observed belonged to the same municipal district. Because the AzLL disease occurred in a region where multiple phytoplasmal diseases had been reported (Cai, 2007; Cai et al., 2008), and because all symptoms of the AzLL disease were suggestive of phytoplasma infection, tools for diagnosis of phytoplasmal diseases were utilised.

A TEM study revealed the presence of single-membrane-bounded, ovoid to spherical bodies in phloem sieve elements of leaf vein and stem samples from diseased azalea plants (Fig. 2), but not in the samples from asymptomatic control plants. These membrane-bound bodies were concentrated near sieve plates, and were within the size range of phytoplasma cells (200 to 1000 nm in diameter), a further indication of phytoplasmal infection.

DNA amplicons of approximately 1.5 kb were obtained in nested PCR that were primed by phytoplasma-universal rDNA primer pairs P1/P7 and P1A/16S-SR using template DNA extracted from diseased azalea plants. DNAs amplified from five individual samples in separate independent nested PCRs were cloned and sequenced, and found to be identical 16S rRNA gene sequences; a representative sequence was deposited (GenBank no HQ285917). The sequence contained a signature motif, 5′-caagaybatkatgtktagcyggdct-3′ (IRPCM, 2004), characteristic of phytoplasma 16S rRNA genes. Amplification and nucleotide sequencing of near-full-length 16S rRNA gene provided convincing evidence connecting AzLL disease and phytoplasmal infection.

AzLL phytoplasma represents a new subgroup in the aster yellows phytoplasma group

The AzLL phytoplasma's near-full-length 16S rRNA gene shares 99.5% sequence identity with that of ‘Candidatus Phytoplasma asteris' reference strain Oenothera phytoplasma 86-7 (OAY, GenBank accession M30790); therefore, AzLL phytoplasma is a ‘Ca. Phytoplasma asteris'-related strain. Virtual RFLP analysis of 16S rDNA confirmed this relationship and showed that AzLL belongs to the AY phytoplasma group (group 16SrI), the largest and most diverse phytoplasma group known (Marcone et al., 2000, Lee et al., 2004).

Prior to this study, phytoplasmas in the AY group had been differentiated into no less than 17 subgroups based on RFLP analysis of 16S rDNA sequences (Lee et al., 2004; Jomantiene et al., 2011). In the present study, a virtual RFLP analysis was performed on the AzLL phytoplasma 16SrDNA F2nR2 fragment with a defined set of 17 enzymes routinely used for identification and classification of phytoplasmas [AluI, BamHI, BfaI, BstUI (ThaI), DraI, EcoRI, HaeIII, HhaI, HinfI, HpaI, HpaII, KpnI, Sau3AI (MboI), MseI, RsaI, SspI and TaqI; Lee et al., 1998]. The analysis resulted in a unique restriction profile (Fig. 3A), which differed from those of representative strains of all previously delineated AY subgroups. The similarity coefficients between the collective 16S rDNA F2nR2 RFLP profile of AzLL phytoplasma and those of the representative strains of the previously delineated subgroups were ≤92.5%. This value is significantly lower than the threshold for recognition of a new subgroup (97%; Lee et al., 1998; Wei et al., 2008b), supporting recognition of a new 16S rDNA RFLP subgroup in the AY group. RFLP patterns from in silico digestions of two restriction enzymes, BstUI and RsaI, effectively distinguished AzLL phytoplasma from strains belonging to all previously delineated subgroups (Fig. 3B and Fig. 3C). An actual gel-based RFLP analysis, using the two distinguishing enzymes, generated restriction profiles (Fig. 3D) identical to those produced by virtual RFLP analysis (Fig. 3B and Fig. 3C), validating the results of the virtual RFLP analysis and further justifying recognition of the AzLL phytoplasma as the representative of a new 16SrI subgroup, hereby designated as 16SrI-T.

Additional evidence of AzLL phytoplasma as representative of a distinct, new lineage

Previous studies indicated that rp and secY genes carry valuable genetic markers and can be used, in conjunction with 16S rRNA genes, to identify and distinguish closely related, but distinct phytoplasma strains and lineages (Lee et al., 1998, 2006, 2010a; Martini et al., 2007; Jomantiene et al., 2011). To further characterise the AzLL phytoplasma, genomic segments that contain genes encoding preprotein translocase SecY subunit (secY) and ribosomal proteins S19 (rpsS), L22 (rplV) and S3 (rpsC) were cloned and sequenced.

Results from virtual RFLP analysis of the 1.2 kb AzLL phytoplasma rpsS-rplV-rpsC locus (GenBank accession no. HQ285918) revealed new BfaI and Sau3AI RFLP patterns (Fig. 4A and Fig. 4B) having recognition sites that were absent from this locus in representative strains of all previously established rp(I) subgroups, indicating the AzLL phytoplasma represented a distinct rp subgroup. To validate the new BfaI and Sau3AI RFLP patterns, conventional RFLP analysis was performed; results from this actual gel electrophoresis confirmed the new patterns produced from the virtual RFLP analysis (Fig. 4C). In accordance with the criteria and naming convention established for recognition of new rp subgroups (Lee et al., 1998), we hereby designate AzLL phytoplasma as the representative of a new rp subgroup, rp(I)-P.

Distinction between AzLL phytoplasma and strains representative of other AY group phytoplasmas was further evidenced by the results from comparative analysis of the genomic locus bearing the secY gene. Nested PCR amplification primed by primer pairs L15F1A(I)/MapR1A and SecYF1a/SecYR1(I) resulted in an amplicon of 1776 bp, which was subsequently cloned and sequenced. The nucleotide sequence data confirmed that the genomic segment covered a partial rplO gene, a complete secY gene and a partial adk gene (GenBank accession no. HQ285919). For comparative analyses, the 1776 bp sequence was trimmed to a 1358 bp segment bounded by the annealing sites for primer pair AYsecYF1/AYsecYR1 in accordance with Lee et al. (2006, 2010a). On the basis of percentage sequence identity and RFLP pattern similarity coefficients derived from comparative analyses of the secY locus, the AzLL phytoplasma was most distantly related to AYWB (94.3% sequence identity and 0.63 RFLP pattern similarity coefficient) and most closely related to OnP1 (99.0% sequence identity and 0.95 RFLP pattern similarity coefficient) and OYM (98.9% sequence identity and 0.90 RFLP pattern similarity coefficient), among AY phytoplasmas studied thus far. RFLP analysis using only one restriction enzyme, TaqI, was sufficient to distinguish AzLL phytoplasma from representatives of all other secY(I) subgroups (Fig. 5). According to previously established criterion (Lee et al., 2006), AzLL represents a new secY lineage, secY(I)-O. It is pertinent to note that the deduced AzLL SecY amino acid sequence differed from the SecY sequence of OYM, a closely related AY phytoplasma strain, by nine amino acid residues, with most of the altered amino acid residues being located in the amphipathic regions of the proteins and four of them causing differences in charge or polarity of the affected positions (Fig. 6). As SecY is the main transmembrane subunit of the bacterial protein secretory pathway, such amino acid substitutions may have functional implications, signifying the lineage difference between AzLL and other AY phytoplasmas.

Relationship between the azalea little leaf phytoplasma and new 16SrI strains from East Asia

Over the past decade, new phytoplasmas associated with plant diseases worldwide have been discovered at an increasingly rapid pace, and recent studies have unveiled remarkable genetic diversity and lineage-specific evolution among phytoplasmas (Lee et al., 2000; Davis et al., 2003, 2005; Cai et al., 2008). Although many phytoplasmas can be differentiated based on 16S rRNA gene nucleotide sequences alone, some require analysis of other genetic loci (Marcone et al., 2000; Andersen et al., 2006; Arnaud et al., 2007; Pacifico et al., 2009; Lee et al., 2010b). Results from multilocus analysis have revealed that phytoplasma strains belonging to a given 16Sr subgroup may represent separate rp, secY, tuf, vmp1 or other gene lineages (Lee et al., 2006; Arnaud et al., 2007; Martini et al., 2007; Fialováet al., 2009; Pacifico et al., 2009; Murolo et al., 2010); on the other hand, strains belonging to one rp or secY gene lineage may be affiliated with different 16SrI subgroups (Lee et al., 2006; Martini et al., 2007; Jomantiene et al., 2011).

In the present study, we found that the AzLL phytoplasma rpsS-rplV-rpsC and secY loci share high levels of sequence identity with the corresponding loci of several new strains from East Asia. These East Asian strains include chinaberry witches'-broom phytoplasma strains CbWB-Hainan and CbWB-Wsh from China; mulberry dwarf phytoplasma strains MD-TS (or MD-TA), MD-NY and MD-XT from China; mulberry dwarf phytoplasma strain MD-Gyeongbuk from Korea; amaranth virescence phytoplasma AV from China; goldenrain phytoplasma strain GRP from Korea and mulberry yellow dwarf phytoplasma strain MYD-AnH from China (Table 1).

Interestingly, most of the new East Asian strains belong to subgroup 16SrI-B on the basis of RFLP analysis of 16S rDNA sequences. Yet, these strains shared higher levels of nucleotide sequence identity of rpsS-rplV-rpsC and secY loci with AzLL (16SrI-T) than with well-known Asian strain OYM (a member of subgroup 16SrI-B), and exhibited collective RFLP patterns of these loci that were more comparable to those of AzLL than to those of OYM. For example, all of the new East Asian strains share the unique BfaI restriction pattern that is characteristic of the AzLL rp lineage [rp(I)-P] (Fig. 7A) and the unique TaqI restriction pattern characteristic of the AzLL secY lineage [secY(I)-O] (Fig. 7B).

Relationships between AzLL and strains classified in subgroup 16SrI-B were further assessed by phylogenetic analysis of the 16S rRNA gene, the rpsS-rplV-rpsC locus and the secY gene. Phylogenetic relationships inferred from these analyses (Fig. 8) were in agreement with the results from RFLP analyses of these genetic loci and were consistent with the concept that AzLL represents a new 16SrI, rp(I) and secY(I) lineage. In the 16S rDNA-based tree, the high degree of nucleotide sequence conservation among 16 rRNA genes largely blurred distinctions among closely related subgroups in group 16SrI, but in spite of the low resolving power of 16S rDNA, strain AzLL was distinguished from subgroup 16SrI-B strains and the other strains studied (Fig. 8A). The rp-based tree indicated that the rpsS-rplV-rpsC locus in AzLL and other strains from China shared a recent ancestral nucleotide sequence, distinct from that of strains originating in other regions (Fig. 8B). Similarly, the secY genes of AzLL and other strains from China appeared in the same subclade, indicating that these genes also shared a common recent ancestral nucleotide sequence, distinct from that of strains originating in other regions (Fig. 8C).

Multilocus analysis of closely related phytoplasma strains in the AY group employing differentially conserved nucleotide sequences, such as those of 16S rDNA, rp and secY loci, presents significant advantages over single gene analysis. In particular, differences in the resolving power of such loci enable distinctions among phytoplasmas that share different levels of relatedness (Lee et al., 2010b). Thus, while 16S rRNA gene sequences are valuable for distinguishing clearly divergent strains at species and higher taxonomic levels, the less highly conserved rp and secY gene sequences offer greater potential for distinguishing closely related lineages that may or may not represent evolutionary divergence at the level of species. Multilocus analysis in this study revealed the association of a new disease with a previously unknown phytoplasma, characterised three genetic loci of the new phytoplasma lineage and identified genetic markers of geographic strain origins. Multilocus characterization, such as that in the present study, forms a step towards fuller description of newly found phytoplasmas and provides the kind of gene lineage information that should be useful for eventual delineation and description of new species in a yet-to-be-adopted formal system of phytoplasma taxonomy, phasing out the current provisional Candidatus status.