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1 Dipartimento di Biologia, Difesa e Biotecnologie Agro-Forestali, Università degli Studi della Basilicata, Campus Macchia Romana, I-85100 Potenza, Italy
2 Northern Territory University, Darwin, Northern Territory 0909, Australia
3 Biologische Bundesanstalt für Land- und Forstwirtschaft, Institut für Pflanzenschutz im Obstbau, D-69221, Dossenheim, Germany
Correspondence
C. Marcone
marcone{at}unibas.it
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Published online ahead of print on 28 November 2003 as DOI 10.1099/ijs.0.02838-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rDNA and 16S–23S rDNA spacer region sequences of the AlloY phytoplasma and for the 16S–23S rDNA spacer region of the BWB phytoplasma are AY135523 and AJ583009, respectively.
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). They are associated with diseases in several hundred plant species, including many woody shrubs or small trees (McCoy et al., 1989; Seemüller et al., 1998a). Sequence analysis of 16S rDNA and other conserved genes reveals that phytoplasmas comprise a discrete, monophyletic clade that is related more closely to acholeplasmas than to other mollicutes (Gundersen et al., 1994; Seemüller et al., 1994). According to phylogenetic data based on 16S rDNA sequences, approximately 20 major phylogenetic groups or subclades have been identified within the phytoplasma clade. This number is generally in accordance with the phytoplasma strain clusters that have been established by RFLP analysis of PCR-amplified rDNA (Lee et al., 1998, 2000; Seemüller et al., 1998a). Each phytoplasma subclade is considered to represent at least one distinct species under the provisional taxonomic prefix Candidatus (International Committee on Systematic Bacteriology Subcommittee on the Taxonomy of Mollicutes, 1997, 2000; Seemüller et al., 2002).
Spartium witches'-broom (SpaWB), buckthorn witches'-broom (BWB) and allocasuarina yellows (AlloY) are lethal witches'-broom and yellows diseases of Spartium junceum (Spanish broom), Rhamnus catharticus (buckthorn) and Allocasuarina muelleriana (Slaty she-oak), respectively. SpaWB is known to occur in Italy and Spain (Marcone et al., 1996; Torres et al., 2002) and BWB disease is present in south-western Germany (Seemüller et al., 1994; Mäurer & Seemüller, 1996), whereas AlloY disease occurs in Australia (Gibb et al., 2003). The causal agents, the SpaWB, BWB and AlloY phytoplasmas, are distinct phytoplasmas that are phylogenetically related more closely to phytoplasmas of the apple proliferation (AP) group than to other phytoplasma subclades (Seemüller et al., 1994, 1998a, 2002; Mäurer & Seemüller, 1996; Marcone et al., 1996; Lee et al., 1998; Gibb et al., 2003). In this paper, it is proposed to consider the SpaWB, BWB and AlloY phytoplasmas as novel ‘Candidatus Phytoplasma’ species. Most of the data on which this proposal is based were published previously. Additional data were generated in this work. In particular, the rDNA of the AlloY phytoplasma, for which a partial 16S rDNA sequence (GenBank accession no. AY135523) had been determined previously by Gibb et al. (2003), was resequenced in order to obtain the full-length 16S rDNA and 16S–23S rDNA spacer region sequences. For this purpose, rDNA of the AlloY pathogen was amplified by PCR using the universal phytoplasma primer pairs P1/rU3 (Deng & Hiruki, 1991; Lorenz et al., 1995) and fU5/m23SR (Lorenz et al., 1995; Padovan et al., 1995). PCR products were purified by using a QIAquick gel extraction kit (Qiagen), according to the manufacturer's instructions. Sequencing of both strands was performed by using an ABI Prism Big Dye Cycle Sequencing Ready Reaction kit (Applied Biosystems) with the following primers: P1, fP3, fU5, 16R723f and m23SR (Deng & Hiruki, 1991; Lorenz et al., 1995; Padovan et al., 1995). Sequences were then assembled and edited by using MacVector and Assembly Lign (Eastman Kodak) and a consensus sequence was generated. The full-length AlloY sequence has been deposited in GenBank (accession no. AY135523). Phylogenetic distances were calculated by pairwise comparisons using the GCG 8.1.0 (Genetics Computer Group, Madison, WI, USA) program GAP (gap weight, 5·0; length weight, 0·3) and multiple alignments were performed by using PILEUP, obtained through the Australian National Genomic Information Centre, Sydney, NSW, Australia. The rDNA sequences of the following phytoplasmas were retrieved from GenBank and used in this study: SpaWB, accession no. X92869; BWB, X76431; AP, AJ542541; pear decline (PD), AJ542543; European stone fruit yellows (ESFY), AJ542544; peach yellow leaf roll (PYLR), Y16394 and U54990; aster yellows, M30790; stolbur, X76427; X-disease, L04682; ‘Candidatus Phytoplasma aurantifolia’, U15442; Bermuda grass white leaf, AJ550984; elm yellows, X68376; ‘Candidatus Phytoplasma fraxini’, AF092209; clover proliferation, L33761; ‘Candidatus Phytoplasma phoenicium’, AF515636; coconut lethal yellowing, U18747; loofah witches'-broom, L33764; and sugarcane white leaf, X76432. Also, the 16S–23S rDNA spacer region sequence of the BWB phytoplasma, which had been determined earlier by Mäurer & Seemüller (1996), was deposited in GenBank (accession no. AJ583009) and included in the study. Numbering of nucleotide positions corresponds to that of the 16S rDNA molecule of aster yellows phytoplasma strain OAY (Lim & Sears, 1989).
Nucleotide sequence analysis of the PCR-amplified rDNA revealed that the SpaWB phytoplasma is related most closely to the AP, PD, ESFY and PYLR phytoplasmas of the AP group. The SpaWB phytoplasma shows 97·1–97·2 % 16S rDNA sequence similarity to the mentioned fruit tree phytoplasmas. Greater differences exist in the sequences of the 16S–23S rDNA spacer region. In this fragment of about 210 bp, the SpaWB phytoplasma differs from the AP and PD phytoplasmas in 12 % of positions and from the ESFY and PYLR phytoplasmas in 13 and 18 % of nucleotide positions, respectively. These findings largely confirm the results of previous work (Marcone et al., 1996; Seemüller et al., 1998a). Like all members of the AP group, the SpaWB agent has a unique PvuII site following position 518 of the 16S rDNA, which is lacking in phytoplasmas of other groups (Fig. 1). It differs from the four fruit tree phytoplasmas by the presence of two additional AluI sites following positions 234 and 627. The SpaWB phytoplasma can be further distinguished from the AP–fruit tree phytoplasmas by differences in other putative restriction sites that are suitable for characterizing members of the AP group (Lorenz et al., 1995; Kison et al., 1997). The SpaWB agent lacks the SspI site following position 419, which is present in the AP phytoplasma, but not in the PD, ESFY or PYLR phytoplasmas. It also shows an SfcI site following position 630, whereas the ESFY, PD and PYLR phytoplasmas have an additional SfcI site following position 1000 and the AP phytoplasma shows an SfcI site only at the latter position (Fig. 1).
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, 1998a; Mäurer & Seemüller, 1996) and 95 % to the SpaWB agent. There are greater differences in the sequences of the 16S–23S rDNA spacer region, where the BWB agent differs from the AP, PD, ESFY and PYLR phytoplasmas in 14–17 % of nucleotide positions and from the SpaWB phytoplasma in 16 % of positions. The BWB phytoplasma lacks the SspI and SfcI restriction sites in the 16S rDNA. Like the ESFY phytoplasma, the BWB agent shows an additional BsaAI site following position 428. The 16S–23S rDNA spacer region of the BWB phytoplasma shows a PvuII site that is not present in the AP–fruit tree agents or related phytoplasmas, as well as an additional AluI site (Fig. 1
).
The AlloY phytoplasma is related most closely to the BWB agent, sharing 96 % 16S rDNA sequence similarity, whereas 16S rDNA sequence similarity with each of the SpaWB, AP, PD, ESFY and PYLR phytoplasmas is 94 %. These sequence similarity values are lower than those reported previously by Gibb et al. (2003), who examined only a 1152 bp 16S rDNA fragment. At the 16S–23S rDNA spacer region level, the AlloY agent differs from AP–fruit tree phytoplasmas in 11–15 % of nucleotide positions and from the SpaWB and BWB phytoplasmas in 17 and 18 % of nucleotide positions, respectively. The AlloY phytoplasma lacks the SspI site in the 16S rDNA, as well as the AluI site following position 1000, which is present in the AP–fruit tree agents SpaWB and BWB (Fig. 1). However, it has the unique PvuII site following position 518. Also, it shows the same SfcI restriction sites as the ESFY, PD and PYLR phytoplasmas and has an additional HpaII restriction site in the 16S–23S rDNA spacer region. Phytoplasmas from other phylogenetic groups usually differ from the SpaWB, BWB and AlloY agents in >8 % of 16S rDNA positions.
In previous work, RFLP analysis of a PCR-amplified 1800 bp rDNA fragment that included the 16S rDNA and the 16S–23S rDNA spacer region revealed that the SpaWB phytoplasma had the same RsaI, Sau3AI, HhaI and BsaAI restriction profiles as the AP phytoplasma. However, the SpaWB phytoplasma differed from the AP agent when the same rDNA fragments were digested with the restriction endonucleases AluI and SspI (Marcone et al., 1996, 1997). The fragment sizes obtained by enzymic RFLP analysis of PCR-amplified rDNA are in agreement with the deduced fragment sizes based on the putative restriction site analysis that was carried out in this work (Fig. 1). Also, RFLP analysis of the PCR-amplified 16S rDNA and the 16S–23S rDNA spacer region sequences by using the endonucleases AluI, RsaI, Sau3AI, MseI, TaqI, SspI and BsaAI revealed that the SpaWB agent is indistinguishable from the phytoplasma associated with the sarothamnus witches'-broom (SSWB) disease of Sarothamnus scoparius, which is known to occur in Italy (Marcone et al., 1996, 1997; Seemüller et al., 1998a). Distinction of the SpaWB and SSWB phytoplasmas is possible by digestion of the amplified sequences with HhaI. In this case, the profile of the SpaWB phytoplasma is different, as it lacks a cleavage site (Marcone et al., 1997). Following separate digestion of the P1/P7 amplimer (Schneider et al., 1995) with AluI and RsaI, the BWB phytoplasma showed restriction patterns that were identical to those of the AP phytoplasma with each of these enzymes (Mäurer & Seemüller, 1996). These findings are consistent with data obtained by putative restriction site analysis. RFLP analysis of a PCR-amplified 880 bp 16S rDNA fragment by using MseI revealed that the AlloY phytoplasma is indistinguishable from the AP and PD phytoplasmas (Gibb et al., 2003). However, when the same rDNA fragments were digested with HpaII, the AlloY phytoplasma proved to be different from both the AP and PD phytoplasmas. Following digestion with SfcI, the AlloY phytoplasma showed the same restriction patterns as the PD phytoplasma, but a different pattern from those of the AP agent.
Specific detection of SpaWB and AlloY phytoplasmas is possible by using the reverse primers rSP and rAllo1 (Marcone et al., 1996; Gibb et al., 2003). The rSP primer was designed from the 16S–23S rDNA spacer sequence of the SpaWB phytoplasma (Marcone et al., 1996). This oligonucleotide (5'-GCTAATTAGAAATATCAACTA-3'), which is complementary to a downstream region of the conserved tRNAIle gene, amplified the target DNA from SpaWB and SSWB phytoplasmas when paired with the universal forward primers fU5 or P1. No amplification products were obtained from either the AP phytoplasma or more distantly related phytoplasmas (Marcone et al., 1996, 1997). Primer rAllo1 (5'-GAGTCCCCAACTAAATGAT-3'), derived from the 16S rDNA sequence of the AlloY agent, amplified the target DNA from AlloY and AP phytoplasmas when used in combination with the AP group-specific primer fO1 (Lorenz et al., 1995). However, DNA from the PD agent or from more distantly related phytoplasmas was not amplified (Gibb et al., 2003). This confirmed other reports (Lorenz et al., 1995; Seemüller et al., 1998b) that ribosomal primers designed for specific detection of a given pathogen or group of pathogens cross-amplified target DNA from other related phytoplasmas. PCR primers for specific detection of the BWB phytoplasma have not been reported. Thus far, host range testing for the SpaWB, BWB and AlloY phytoplasmas by PCR amplification using universal and specific phytoplasma primers indicates that these phytoplasmas have only been detected in diseased Spanish broom, buckthorn and Slaty she-oak plants, respectively (Mäurer & Seemüller, 1996; Marcone et al., 1996; Gibb et al., 2003).
Conclusion and taxonomic description
The great importance given to 16S rDNA sequences in mycoplasma phylogeny, taxonomy and species identification (Razin et al., 1998) has led the International Committee on Systematic Bacteriology Subcommittee on the Taxonomy of Mollicutes (1997) to recommend the inclusion of 16S rDNA sequences in any description of a novel mollicute species. For uncultured phytoplasmas, a novel putative species may be described when its 16S rDNA sequence (>1200 bp) has 
97·5 % similarity to any previously described ‘Candidatus Phytoplasma’ species. The SpaWB, BWB and AlloY phytoplasmas share <97·5 % 16S rDNA sequence similarity with each other and with other known phytoplasmas, including the AP group phytoplasmas. Also, the SpaWB, BWB and AlloY phytoplasmas each have a different natural plant host that seems to be unique. The difference in geographical distribution of the AlloY phytoplasma from the closely related AP group phytoplasmas supports its placement in a distinct taxonomic unit. Based on their unique properties, we propose to designate the SpaWB, BWB and AlloY phytoplasmas as novel ‘Candidatus’ species, according to guidelines proposed by Murray & Schleifer (1994) for uncultivated prokaryotes whose uniqueness is defined only by very limited characteristics.
The SpaWB phytoplasma is proposed as a distinct, novel species with the following description: ‘Candidatus Phytoplasma spartii’ (spartii, epithet referring to the plant host) [(Mollicutes) NC; NA; O, wall-less; NAS (GenBank accession no. X92869; oligonucleotide sequence complementary to a unique region of the 16S rDNA is 5'-TTATCCGCGTTAC-3'; P (Spartium junceum, phloem); M]. Reference strain is SpaWB. DNA samples from this strain are available from C. M.
The BWB phytoplasma is proposed as a distinct, novel species with the following description: ‘Candidatus Phytoplasma rhamni’ (rhamni, epithet referring to the plant host) [(Mollicutes) NC; NA; O, wall-less; NAS (GenBank accession nos X76431 and AJ583009; oligonucleotide sequence complementary to a unique region of the 16S rDNA is 5'-CGAAGTATTTCGATAC-3'; P (Rhamnus catharticus, phloem); M]. Reference strain is BWB. DNA samples from this strain are available from B. S.
The AlloY phytoplasma is proposed as a distinct, novel species with the following description: ‘Candidatus Phytoplasma allocasuarinae’ (allocasuarinae, epithet referring to the plant host) [(Mollicutes) NC; NA; O, wall-less; NAS (GenBank accession no. AY135523; oligonucleotide sequence complementary to a unique region of the 16S rDNA are 5'-TTTATTCGAGAGGGCG-3'; P (Allocasuarina muelleriana, phloem); M]. Reference strain is AlloY. DNA samples from this strain are available from K. S. G.
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