'Candidatus Phytoplasma lycopersici', a phytoplasma associated with 'hoja de perejil' disease in Bolivia

Hoja de perejil (parsley leaf) of tomato (THP) and brotes grandes (big bud) of potato (PBG) are two new phytoplasma diseases reportedly affecting these crop species (Jones et al., 2005a, b). PBG was associated with phytoplasmas of 16SrI group, Candidatus Phytoplasma asteris and related strains (Jones et al., 2005a), while phytoplasmas in association with THP and nearby Morrenia variegata with little leaf (MVLL) disease were tentatively identified as representatives of a novel phytoplasma group (Jones et al., 2005b).

Tomato plants affected with hoja de perejil disease are characterized by adventitious sprouting of axillary buds and rapid elongation of side shoots showing small and fern-like leaves, becoming large bushy plants as the season progresses.

Big bud diseases of tomato have been attributed to at least four distinct phytoplasma strains worldwide (Lee et al., 1993; Marcone et al., 1997; Davis et al., 1997b; Shaw et al., 1993). These include aster yellows subgroup (16SrI-A), peanut witches' broom subgroup (16SrII-E), clover proliferation subgroup (16SrVI-A) and stolbur subgroup (16SrXII-A) strains. A tomato yellow is associated with a subgroup 16SrI-B strain (Okuda et al., 1997).

Surveys in Italy have also revealed the presence of 16SrV group, Candidatus Phytoplasma ulmi, in tomato plants showing stunting, yellowing, proliferation of lateral shoots, adventitious roots and a reduced number of fruits (Del Serrone et al., 2001).

The present work reports results on the molecular characterization of phytoplasmas associated with THP and PBG from surveys carried out during 2000–2003 in potato and tomato fields located in Sucre and Santa Cruz provinces in Bolivia. Results of a study involving 16S rRNA gene PCR amplification, RFLP, sequence and phylogenetic analysis of THP and PBG compared with 39 other reference phytoplasmas representing the current classification scheme are reported, including the proposal of THP as a novel Candidatus Phytoplasma species.

Disease survey locations.
Tomato and potato production fields in east-central Bolivia were the focus of disease surveys conducted during 2000–2003. Survey sites included both tomato and potato fields located in the Chilon, Saipina, Pulquina and Comarapa valleys in Santa Cruz province and tomato fields at Limon Pampa, Río Chico, in Sucre province.

Plant samples and reference phytoplasma strains.
During the course of surveys, leaf samples for analysis were collected from a total of 44 diseased plants. These included 11 potato plants with typical PBG symptoms (Jones et al., 2005b), 13 tomato plants with THP symptoms (Jones et al., 2005b), eight Morrenia variegata (Griseb.) T. Mey. vines (Jones et al., 2005b) and six mora-mora (Serjania perulacea Radlk.) vines, both with little leaf symptoms, as well as six alfalfa (Medicago sativa L.) plants with witches' broom and little leaf. Morrenia and mora-mora plants were found growing in hedgerows near tomato crops near San Rafael, Santa Cruz, and potato fields in La Tranca, Sucre, respectively. Leaf samples from nine symptomless (presumably healthy) plants, including at least one plant of each species, were also collected for comparative purposes.

Phytoplasma strains belonging to five 16S rRNA RFLP groups (Lee et al., 1998) were included in the study for reference purposes. The strains and their respective group affiliations are as follows. Stolbur (STOL), grapevine yellows (VK) and Bois Noir (BN) (16SrXII, stolbur group) were kindly provided by Dr Giuseppe Firrao. American aster yellows (AAY) and apple chlorotic leaf roll (ACLR) (16SrI, aster yellows group), apple proliferation (AP) (16SrX, apple proliferation group), faba bean phyllody (FBP) (16SrII, peanut witches' broom group) and vaccinia witches' broom (VWB) (16SrIII, X-disease group) were all obtained from the phytoplasma collection at Rothamsted Research, UK.

DNA amplification and RFLP analysis.
DNA was extracted from 1.5 g samples of leaf tissue by the method of Doyle & Doyle (1990) and used as the template for a nested PCR assay primed by phytoplasma universal primer pairs P1 (Deng & Hiruki, 1991) and P7 (Schneider et al., 1995) for the first reaction and R16F2n/R16R2 (Gundersen & Lee, 1996) for the nested reaction, as described previously (Arocha et al., 2005). Nested PCR products were analysed by single-restriction endonuclease digestion with HaeIII, HinfI (both from Boehringer Mannheim), AluI, RsaI (both from Sigma), Sau3AI (Amersham Biosciences) or KpnI (Promega) according to the manufacturers' instructions. Digestion products were electrophoresed through 2.5–3 % agarose gels and visualized after staining with ethidium bromide by UV transillumination. The resulting RFLP patterns were compared with those described previously (Schneider et al., 1995; Gibb et al., 1996; Davis et al., 1997a; Lee et al., 1998, 2004; Arocha et al., 2005).

DNA sequencing.
P1/P7 amplicons were purified on spin columns (QIAquick gel extraction kit; Qiagen) and sequenced in the forward and reverse directions with primer pair P1/P7 by the Sequencing Service of the School of Life Sciences, University of Dundee, UK (), using Applied Biosystems Big-Dye version 3.1 chemistry on a 3730 automated capillary DNA sequencer.

16S rRNA gene sequence similarity, putative restriction sites and phylogenetic analysis.
Comparison of 16S rRNA gene sequences of Bolivian phytoplasmas with those archived in the nucleotide sequence database of the National Center for Biotechnology Information (NCBI) (Supplementary Table S1) was performed using the BLASTN program (Altschul et al., 1990). Sequence similarities were evaluated and putative restriction site maps of 16S rRNA genes were constructed using the RESearch program (Rothamsted Research). RFLP patterns of 16S rRNA genes were compared and similarity coefficients (F) for each pairwise comparison were calculated from virtual (in silico) restriction site analysis of rRNA gene sequences with enzymes AluI, RsaI, Sau3AI, KpnI and HinfI, according to the previously described formula F=2N_xy/(N_x+N_y) (Lee et al., 1998; Nei & Li, 1979), in which x and y are two given phytoplasmas under investigation, N_x and N_y are the total of number of fragments resulting from enzymic digestion in strains x and y, respectively, and N_xy is the number of fragments shared by the two strains.

Phytoplasma 16S rRNA gene sequences were aligned and edited using the multiple alignment program CLUSTAL W (Thompson et al., 1994) included in the MEGA program version 3.1 (Kumar et al., 2004). A phylogenetic analysis of the aligned sequences was performed using MEGA version 3.1 and a tree was constructed by the neighbour-joining method. Acholeplasma palmae ATCC 49389^T was used as an outgroup to root the tree. Bootstrap analyses (1000 replicates) were performed to estimate the stability and support for the inferred clades.

DNA amplification, analysis of RFLP profiles and putative restriction sites in phytoplasma rRNA operon sequences
Nested PCR products of about 1250 bp in size were amplified from all samples of each diseased plant species, whereas no products were amplified from any of the symptomless, presumably healthy plants included during this study. Sequence analysis of amplification products by comparison with the NCBI nucleotide sequence database confirmed their phytoplasma origin. As such, S. perulacea and M. variegata represent newly identified phytoplasma hosts.

Based on the combined RFLP data, no differences were evident between THP and MVLL; thus they appear to be very similar or identical strains. HaeIII profiles of 120 and 1000 bp (data not shown) were yielded by all positive PCR samples, confirming the presence of phytoplasma DNA. HinfI, AluI and RsaI profiles (Fig. 1) shown by THP and MVLL were similar to those of AAY, STOL, VK and the remaining Bolivian phytoplasmas, classifying them all as members of group 16SrI. However, the KpnI and Sau3AI profiles collectively differentiated THP and MVLL from the rest of the phytoplasmas analysed as a novel phytoplasma related to the 16SrI group.

(68K): Fig. 1. RFLP analysis of 16S rRNA genes amplified by nested PCRs with enzymes HinfI, KpnI, AluI, RsaI and Sau3AI. (a) Lanes: 1, 100 bp ladder (MBI Fermentas); 2–4 and 14–16, PBG, AlfWB and MMLL; 5–6 and 17–18, THP and MVLL; 7 and 19, AAY; 8 and 20, ACLR; 9 and 21, STOL; 10 and 22, VK; 11 and 23, AP; 12 and 24, VWB; 13 and 25, FBP. (b) Lanes: 1, 100 bp ladder (MBI Fermentas); 2–4, PBG, AlfWB and MMLL; 5–6, THP and MVLL; 7, AAY; 8, ACLR; 9, STOL; 10, VK; 11, AP; 12, VWB; 13, FBP.

Putative restriction maps of rRNA operon sequences support results obtained from actual digestion of phytoplasma rRNA gene products amplified by PCR. The differences in the KpnI profile between THP and MVLL on the one hand and the remaining Bolivian phytoplasmas are reflected by the addition and displacement of KpnI sites in the 16S rRNA gene sequence of THP and MVLL when compared with those of PBG, AlfWB and MMLL, besides AshWB, BD, AAY, Ca. Phytoplasma asteris and Ca. Phytoplasma fragariae phytoplasmas, except for ACLR (Fig. 2). Similarly, the differences in the Sau3AI RFLP profile exhibited by THP and MVLL when compared with the Bolivian and 16SrI group phytoplasmas are reflected by the displacement of two Sau3AI sites in the 16S rRNA gene sequence of THP and MVLL, which are specified by the downward arrows in the 16S rRNA gene sequences of the Bolivian and 16SrI group phytoplasmas in Fig. 2.

(43K): Fig. 2. Putative restriction sites of phytoplasma 16S rRNA gene sequences. Maps were generated using the RESearch program (Rothamsted Research) for comparison of recognition sites for the restriction endonucleases AluI, HinfI, KpnI, RsaI and Sau3AI. Downward arrows indicate sites that are common between the Bolivian phytoplasmas and the reference strains analysed. Frames enclose different putative restriction sites.

In addition, comparisons of putative restriction sites between the THP and MVLL 16S rRNA gene sequences with that of Ca. Phytoplasma asteris exhibited additional RsaI and AluI sites for the latter and one additional HinfI site for THP and MVLL. Comparison with the closest relative, Ca. Phytoplasma fragariae, showed one and two additional AluI sites for the latter when compared with Ca. Phytoplasma asteris and with THP and MVLL, respectively. The 16S rRNA gene sequence of Ca. Phytoplasma fragariae also lacked the RsaI and AluI sites present in Ca. Phytoplasma asteris, as well as the additional HinfI site of the 16S rRNA gene of THP and MVLL.

Similarity coefficients
The similarity coefficients of the RFLP patterns of the novel phytoplasma (Supplementary Table S2) derived from putative restriction map analysis of 16S rRNA gene sequences using five restriction enzymes were 91–96 % with 16SrI phytoplasmas, including Ca. Phytoplasma asteris, 96 % with Ca. Phytoplasma caricae, 95 % with Ca. Phytoplasma japonicum, 93 % with Ca. Phytoplasma australiense, 92 % with Ca. Phytoplasma graminis, 88 % with Ca. Phytoplasma fragariae, 84 % with Ca. Phytoplasma americanum, 77 % with Ca. Phytoplasma allocasuarinae and 82 % with Ca. Phytoplasma brasiliense. These results support recognition of THP and MVLL as a novel phytoplasma of the 16SrI group.

Sequence similarity
Comparisons of 16S rRNA gene sequences revealed that those of novel phytoplasmas THP and MVLL were co-identical and shared only 96.5 % similarity with those of Bolivian strains PBG, AlfWB and MMLL, 97.26 % similarity with that of BD and AshWB, 96 % with that of AAY and ACLR, 97.5 % with that of Ca. Phytoplasma asteris and less than 95 % with the remaining Candidatus Phytoplasma species. Sequence similarity values are tabulated in Supplementary Table S3.

Phylogenetic analysis
The phylogenetic relatedness of THP and MVLL and the rest of the phytoplasmas analysed, including A. palmae, is depicted in Fig. 3. The tree is in good agreement with previous findings according to the bootstrapping values that support most branches, indicating the robustness of the branching order (Arocha et al., 2005; Lee et al., 2006). THP and MVLL were included in the same branch corresponding to PBG, AlfWB and MMLL and the rest of the phytoplasmas of group 16SrI. In addition, the phylogenetic analysis indicated that THP and MVLL and the remaining Bolivian phytoplasmas have a common ancestor with Ca. Phytoplasma japonicum, Ca. Phytoplasma australiense, Ca. Phytoplasma graminis, Ca. Phytoplasma caricae, Ca. Phytoplasma fragariae and Ca. Phytoplasma americanum (Valiunas et al., 2006).

(32K): Fig. 3. Phylogenetic tree of 16S rRNA gene sequences constructed by the neighbour-joining method showing relationships between the phytoplasmas identified in THP, MVLL, PBG, AlfWB and MMLL and reference phytoplasmas from GenBank. Numbers above or below branches are bootstrap values obtained for 1000 replicates. Branch lengths are proportional to the number of inferred character-state transformations. A. palmae ATCC 49389T was used as an outgroup. Abbreviations of phytoplasma names are defined in Supplementary Table S1.

Sequences unique to phytoplasmas in the 16S rRNA gene of the new phytoplasmas identified
The 16S rRNA gene sequence from THP and MVLL was aligned with sequences from 39 phytoplasmas representing the current phytoplasma grouping (IRPCM, 2004), including those of the remaining Bolivian phytoplasmas.

This analysis revealed that THP, MVLL and the remaining Bolivian phytoplasmas contained the six sequences previously reported to be unique to phytoplasmas (Gundersen et al., 1994); however, signature sequences unique to THP and MVLL distinguished them from the other phytoplasmas analysed.

Unique sequences not present in the 16S rRNA gene sequences of other phytoplasmas were found in those of THP and MVLL: 5'-CTTA-3' at positions 175–178, 5'-AATGGT-3' at positions 198–203, 5'-ATA-3' at positions 229–231, 5'-TTGGAGGAA-3' at positions 234–242, 5'-CACG-3' at positions 302–305, 5'-TCT-3' at positions 315–317, 5'-GCT-3' at positions 334–336, 5'-TAT-3' at positions 336–338, 5'-TAC-3' at positions 413–415 and 5'-AGC-3' at positions 434–436.

Based on RFLP results, sequence similarity coefficients and phylogenetic analysis of 16S rRNA gene sequences, THP, MVLL and the remaining Bolivian phytoplasmas were definitively placed as members of the 16SrI group, Ca. Phytoplasma asteris and related strains. However, both Sau3AI profiles and putative restriction site analysis distinguished THP and MVLL from PBG, AlfWB and MMLL and the other phytoplasmas analysed. The 16S rRNA gene sequence similarity of THP and MVLL in comparison with those from the remaining Bolivian phytoplasmas was 96.5 % and ranged from 96–97.26 % in comparison with the other members of the 16SrI group.

THP and MVLL showed clear differences in RsaI and Sau3AI putative restriction sites when compared with Ca. Phytoplasma asteris, which also belongs to the 16SrI group. Comparisons of 16S rRNA gene sequences of THP and MVLL reached 97.5 % when compared with Ca. Phytoplasma asteris.

The International Committee on Systematics of Prokaryotes Subcommittee on the taxonomy of Mollicutes has recommended the inclusion of 16S rRNA gene sequences for any description of a novel mollicute species (Marcone et al., 2004a, b). Novel putative species of uncultured phytoplasmas may be described when their 16S rRNA gene sequence (1200 bp) has ≤97.5 % similarity to any previously described Candidatus Phytoplasma species (IRPCM, 2004).

Previous BLAST analysis of the 16S/23S rRNA intergenic sequence of THP and MVLL showed the highest similarity of 91 % with those of other phytoplasmas in the 16SrI group, Ca. Phytoplasma asteris (Jones et al., 2005b). From our study, phylogenetic analysis showed that THP, MVLL and Ca. Phytoplasma asteris share the same branch of phytoplasmas belonging to the 16SrI group. However, THP and MVLL exhibited 97.5 % 16S rRNA gene sequence similarity to Ca. Phytoplasma asteris and 95 % or less when compared with the closest relative, Ca. Phytoplasma fragariae, and other previously described Candidatus Phytoplasma species and phytoplasma strains representing other unnamed phytoplasma groups or subgroups. Differences have been also supported by means of 16S rRNA gene putative restriction maps and sequence similarity coefficients.

In addition, signature sequences not found in the 16S rRNA gene sequences of any other Ca. Phytoplasma species described previously have been described from the 16S rRNA gene sequences of THP and MVLL.

We propose to designate to THP as the reference strain of a novel Candidatus, according to the scheme for assigning incompletely described prokaryotes to the provisional status Candidatus implemented by the International Committee on Systematics of Prokaryotes (Murray & Stackebrandt, 1995). We propose that THP is designated Candidatus Phytoplasma lycopersici, with the following description.

Candidatus Phytoplasma lycopersici (N.L. n. Lycopersicon a botanical genus; N.L. gen. n. lycopersici of Lycopersicon esculentum, referring to the plant host, tomato) [(Mollicutes) NC; NA; O, wall-less; NAS (GenBank accession no. AY787136); oligonucleotide sequences of unique regions of the 16S rRNA gene: 5'-CTTA-3' (positions 175–178), 5'-AATGGT-3' (198–203), 5'-ATA-3' (229–231), 5'-TTGGAGGAA-3' (234–242), 5'-CACG-3' (302–305), 5'-TCT-3' (315–317), 5'-GCT-3' (334–336), 5'-TAT-3' (336–338), 5'-TAC-3' (413–415) and 5'-AGC-3' (434–436); P (Lycopersicon esculentum, phloem; M]. DNA samples from these strains are available from the authors.

We are grateful to Dr G. Firrao (University of Udine, Italy) for kindly providing DNA of stolbur group phytoplasmas for comparison. Work in the UK was done under the DEFRA plant health licence no. PHL:174B/4612(09/2003) and was supported by the Biotechnology and Biological Sciences Research Council, UK.