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Biologische Bundesanstalt für Land- und Forstwirtschaft, Institut für Pflanzenschutz im Obstbau, D-69221 Dossenheim, Germany
Correspondence
Erich Seemüller
E.Seemueller{at}bba.de
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the P1/P7 amplimers (16S rRNA gene and 16S–23S rDNA spacer region) of phytoplasma strains AP15R, AP1/93, PD1R, ESFY-G1R and ESFY-G2 are AJ542541, AJ542542, AJ542543, AJ542544 and AJ542545, respectively.
Restriction maps of rDNA obtained from P1/P7 PCR products from the apple proliferation, pear decline and European stone fruit yellows phytoplasmas are available as supplementary material in IJSEM Online.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSTAXONOMIC EVIDENCEDISCUSSIONREFERENCES |
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). These diseases include three economically important disorders of temperate fruit trees: apple proliferation (AP), pear decline (PD) and European stone fruit yellows (ESFY). AP is only known in Europe and was first described in Italy (Rui et al., 1950). ESFY is mainly known in Europe, but has also been reported in Turkey (Jarausch et al., 2000a) and was first described as a decline of Japanese plum (Prunus salicina) in Italy (Goidanich, 1933). Until recently, the disease was known by the names plum leptonecrosis, apricot chlorotic leaf roll and others, according to the crop affected and the symptoms induced (Lorenz et al., 1994). PD was first reported in North America (McLarty, 1948; Woodbridge et al., 1957) and seems to have been introduced from Europe (Shalla et al., 1961; Seemüller, 1992). These data indicate that AP, PD and ESFY are of European origin and were present in Europe long before the first reports were published. In contrast, peach yellow leaf roll (PYLR), which is caused by a phytoplasma that is related closely to the AP, PD and ESFY agents, has only been reported in western North America (Seemüller, 1999).
Recent investigations, particularly sequence analysis of 16S rDNA, have revealed that phytoplasmas constitute a coherent, genus-level taxon. In the monophyletic phytoplasma clade, groups and subgroups have been delineated, many of which are being considered as putative species under the provisional status ‘Candidatus’ (Seemüller et al., 2002) for incompletely described prokaryotes, according to Murray & Stackebrandt (1995). Several provisional species have been described to date and rules for future putative species delineation have been defined (IRPCM Phytoplasma/Spiroplasma Working Team – Phytoplasma taxonomy group, 2004). According to these recommendations, ‘a phytoplasma can be described as a novel ‘Candidatus Phytoplasma’ species if its 16S rDNA sequence has <97·5 % similarity to that of any previously described ‘Candidatus Phytoplasma’ species’. In cases where phytoplasmas share 
97·5 % 16S rDNA sequence similarity, description as different ‘Candidatus’ species is only recommended if there is an indication that these phytoplasmas clearly represent separate populations, as evidenced by significant differences based on molecular markers other than 16S rDNA, antibody specificity, host range and vector transmission specificity.
The AP, PD and ESFY agents are among the phytoplasmas that have been studied most intensively, using both molecular and biological methods. 16S rDNA sequence data indicated that each of them represents a relatively uniform organism (Lorenz et al., 1995; Seemüller et al., 1998b; Kison & Seemüller, 2001). The results also revealed that the three pathogens are closely related phylogenetically and form, together with the PYLR phytoplasma, a cluster designated the ‘AP phytoplasma group’ (Seemüller et al., 1998b) or 16SrX group (Lee et al., 2000) within the AP subclade, which is one of the major branches of the phytoplasma clade. The four phytoplasmas differ from each other in <2·5 % of 16S rDNA nucleotide sequence positions. Thus, the objective of this work was to examine whether the above-mentioned requirements for differentiation of closely related phytoplasmas are fulfilled. The data examined include sequence and/or RFLP analyses of the 16S–23S rDNA spacer region, protein-encoding genes and randomly cloned DNA fragments, serological comparisons and differences in insect vector and host-plant specificity. Further data were generated in this work. In particular, rDNA sequences of more strains were examined and previously sequenced strains were re-examined in order to obtain unambiguous sequences. Also, RFLP analyses of macrorestriction fragments and Western blot studies were performed to demonstrate inter- and intraspecific differences of the phytoplasmas examined.
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METHODS |
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; Schneider et al., 1995) and extended from the 5' end of the 16S rRNA gene to the 5' region of the 23S rRNA gene, thus including the 16S–23S rDNA spacer region. RFLP analysis was carried out as described previously (Lorenz et al., 1995; Kison & Seemüller, 2001). For sequencing of strains AP1/93, PD1, ESFY-G1R and ESFY-G2 and resequencing of strains AT and AP15R, P1/P7 amplicons were cloned by using the pGEM-T vector system (Promega). Sequencing of both strands was done by a commercial service (SEQLAB, Göttingen, Germany) by using M13 forward and reverse primers and six primers that were complementary to conserved 16S rDNA regions (Seemüller et al., 1994). Sequencing data obtained with these primers usually provided threefold coverage of the sequences. To obtain unambiguous sequences, three or four clones were sequenced from each of strains AP15R, AT, PD1R and ESFY-G1R, which represent the three taxa treated in this description. One clone was sequenced from each of strains AP1/93 and ESFY-G2. Sequence evaluation, including manual alignment, was done by using the software package HUSAR (Biocomputing Service Group, German Cancer Research Center, Heidelberg, Germany). Gaps and ambiguities were removed from the final dataset. Phylogenetic and molecular evolutionary analyses were conducted by using the neighbour-joining program of the genetic analysis software MEGA, version 2.1 (Kumar et al., 2001). The data were resampled 500 times and bootstrap percentage values are given at the nodes of the tree. Phylogenetic distances were calculated by pairwise comparison. Numbering of nucleotide positions corresponds to that of the 16S rRNA gene of aster yellows phytoplasma strain OAY (Lim & Sears, 1989).
Full-length chromosomes of the AP, PD and ESFY phytoplasmas were isolated from diseased tobacco plants and RFLP analysis was performed as described previously (Lauer & Seemüller, 2000). ELISA and Western blot experiments were carried out by using standard procedures (Ausubel et al., 1987). The strains examined in this work are listed in Table 1.
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TAXONOMIC EVIDENCE |
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). Of these phytoplasmas, which were collected in several European countries and California and, in the case of the ESFY agent, from several stone fruit species (Table 1
), strains AP15R, PD1R and ESFY-G1R were selected as reference strains. AP15R is known to occur in several European countries and is probably the most common type of AP agent (Kison et al., 1994; Jarausch et al., 2000b).
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).
The AP, PD and ESFY phytoplasmas are closely related phylogenetically. In interspecific comparisons of the AP/PD, AP/ESFY and PD/ESFY agents, differences in 16S rDNA sequences were 1·0–1·1, 1·3–1·5 and 1·2–1·3 %, respectively. Strain PYLR1 is related most closely to the PD strains, with which it shares 99·6 % sequence similarity, whereas similarity values between the AP and ESFY strains were 98·4–98·6 %. Sequence similarity values within taxa and divergence between taxa largely confirm the results of previous work (Seemüller et al., 1994, 1998b).
Phylogenetic relatedness of the phytoplasmas examined is depicted in Fig. 1. Strains of each of the three pathogens cluster tightly together and form three distinct branches. The PYLR agent clusters with the PD strains. The closest relatives of the AP-group phytoplasmas are the spartium witches’-broom (SpaWB; Marcone et al., 1996b) and buckthorn witches’-broom (BWB; Mäurer & Seemüller, 1996) agents, which compose, together with the fruit tree agents, the AP subclade. Their 16S rDNA sequence similarity with the fruit tree phytoplasmas is slightly less than 97·5 % and is thus below the defined 2·5 % threshold. Other selected phytoplasmas that represent other major subclades differ from the AP-group agents in >7 % of 16S rDNA nucleotide positions.
16S rDNA signature sequences.
The 16S rRNA gene sequences from the AP, PD and ESFY agents were aligned with those of phytoplasmas that represent most phylogenetic groups. Several oligonucleotides that are unique for the AP-group phytoplasmas were selected and used as query sequences in a BLAST 2.0 search (Altschul et al., 1997). This analysis resulted in the determination of signature sequences that are characteristic for the AP-group phytoplasmas and are absent in other organisms or prokaryotes. Sequences identified as unique for the AP group were at positions 1393–1409, where 5'-AATACTCGAAACCAGTA-3' and 5'-AATACCCGAAACCAGTA-3' were specific for the AP and ESFY phytoplasmas, respectively, and 5'-AATACTCAAAACCAGTA-3' was specific for the PD and PYLR agents. Another sequence that was only shared by PD and PYLR strains was 5'-TTAATAAGTCTATGGTCT-3' (positions 570–587). PD and PYLR agents could be distinguished from each other by the sequence 5'-ATACGGCCCAAACTCATACGGA-3' at positions 329–351, which was specific for PYLR. A second ESFY-specific sequence was 5'-TGAAGTTTTGAGGCATCTCGAA-3' at positions 159–180.
Sequence analysis of the 16S–23S rDNA spacer region.
The 16S–23S rDNA spacer of AP-group phytoplasmas consists of about 210 bp. The gene that encodes tRNAIle was present and identical in all strains examined, which comprised AP15R, AT, AP1/93, APS (GenBank accession no. X76426), PD1R, PD3, PD5 (accession no. U54989), ESFY-G1R, ESFY-G2, ESFY3, ESFY4, ESFY6 (accession no. U54988), PYLR1 and PYLR2 (accession no. U54990) (see Table 1 for strain identities and Fig. 1 for other accession numbers). Flanking sequences within these phytoplasmas were also highly conserved; those of strains of each the AP and ESFY phytoplasmas were identical, whereas one polymorphism in the PD spacer (in the form of a deletion of an A residue in PD1R and PD5, just downstream of the tRNAIle gene) was identified. Divergence between taxa was slightly higher in the 16S–23S rDNA spacer region than in the 16S rDNA sequence, being 1·9, 3·0 and 1·5 % for the comparisons AP/PD, AP/ESFY and PD/ESFY, respectively. The sequence of PYLR strains PYLR1 and PYLR2 was identical to that of strain PD3. Relationships between the phytoplasmas examined here agree with results of previous comparisons that were made with fewer strains (Kirkpatrick et al., 1994; Schneider et al., 1995).
Sequence analyses of non-ribosomal DNA.
Examination of putative translation products of immunodominant membrane protein (IMP) genes of the AP, PD, ESFY and PYLR phytoplasmas revealed a similar size for all. The IMPs consisted of a transmembrane region towards the N-terminus, with only a short N-terminal intracellular sequence (together, about 35 aa), and a large hydrophilic C-terminal domain (about 130 aa), probably held on the outside of the cell membrane. The extracellular hydrophilic domain, which may play a role in pathogen–host interactions, is not conserved. Similarities of nucleotide and deduced amino acid sequences were 47–76 and 31–57 %, respectively. Sequence similarity values for the combined N-terminal and transmembrane domains were higher, ranging from 82 to 98 % and 65 to 100 %, respectively. Whilst the PD and PYLR phytoplasmas differed significantly in the nucleotide and amino acid sequences of their large hydrophilic domain (54 and 31 % similarity, respectively), sequences of their combined N-terminal and transmembrane domains were nearly identical or identical. However, sequence similarity of the entire gene was highest between the PD and AP phytoplasmas (Berg et al., 1999; Barbara et al., 2001; Morton et al., 2003). There is no sequence similarity in antigenic membrane proteins between AP-group and aster yellows-group phytoplasmas (Barbara et al., 2002).
The DNA sequence of randomly cloned DNA fragment AT67 from strain AT of the AP phytoplasma (Schneider & Seemüller, 1994b), which contains three ORFs that encode a putative ATP-binding protein and two putative permease proteins, was compared with analogous, PCR-amplified sequences of Californian, German and Italian PD phytoplasma isolates and a Californian PYLR phytoplasma isolate. This analysis revealed that the Californian and German PD isolates and the Californian PYLR isolate had identical sequences. DNA sequence similarity of these isolates to the Italian PD isolate was 98 % and to AP strain AT was 92 % (Guerra, 1997).
RFLP analysis of ribosomal and non-ribosomal DNA.
rDNA, PCR-amplified with universal phytoplasma primer pairs fU5/rU3 or P1/P7 or with primers fO1/rO1, fPD/rO1 or P1/PYLRint, which specifically amplify some or all phytoplasmas of the AP group, was predominantly used in RFLP analysis (Seemüller et al., 1998a). Following AluI digestion, the AP, PD and ESFY phytoplasmas showed the same restriction pattern, which was also shared by the PYLR agent (Kison et al., 1997). The AP, PD and ESFY phytoplasmas can be distinguished from each other by separate restriction of 16S rDNA with RsaI and SspI. Following RsaI digestion, the ESFY agent differs from the AP and PD phytoplasmas by one additional restriction site at position 420, whereas SspI digestion cleaves the AP phytoplasma at position 413, a site that is absent in the PD and ESFY agents. Differentiation of AP and PD from ESFY phytoplasmas is also possible with BsaAI and SfcI. The ESFY agent shows a BsaAI site at position 422 that is absent in the two other phytoplasmas, whilst both the PD and ESFY phytoplasmas have an SfcI site at position 624 that is absent in AP. In addition, the PD and ESFY agents share a unique SspI site in the 16S–23S rDNA spacer region that does not occur in the AP phytoplasma (Fig. 2). A supplementary figure showing restriction maps of rDNA from the AP, PD and ESFY agents is available in IJSEM Online. In all comparisons, the PYLR agent resembled the PD phytoplasma (Lorenz et al., 1995; Marcone et al., 1996a; Guerra, 1997; Kison et al., 1997; Del Serrone et al., 1998; Seemüller et al., 1998a; Topchiiska et al., 2000; Kison & Seemüller, 2001). Hundreds of AP-group phytoplasma isolates from various locations in European countries and California, including the ESFY agent taken from several Prunus species, have been examined to date by using these differentiation approaches. With the above-mentioned restriction enzymes and several others, no variation among the different AP-group phytoplasmas was observed.
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), also proved to be useful for AP-group phytoplasma characterization and differentiation. Work by Jarausch et al. (1994, 2000a, 2000b) showed that this fragment contains three ORFs, including a putative nitroreductase gene. Restriction enzyme digestion with RsaI and HincII of a product obtained with primers AP9/AP10 enabled the differentiation of AP from PD and ESFY agents. All three phytoplasmas could be distinguished from each other by digestion of an AP3/AP10 amplicon with these enzymes. One hundred and seventy-five ESFY phytoplasma isolates, which were collected from many Prunus species and hybrids in four Mediterranean countries, responded identically in this work. However, by examining a 1·5 kbp fragment that was obtained from AP phytoplasma isolates with primers AP8/AP10, three RFLP types were identified by restriction digestion with RcaI and HincII. Nucleotide sequence analysis revealed that the three RFLP types are nearly identical and differ only by a point mutation in each of the two restriction sites that are responsible for the polymorphisms.
Primers that were derived from the non-ribosomal DNA fragment AT67 (see above) of strain AT were used to amplify the analogous sequence of the PD phytoplasma. The primers designed from the sequence obtained were PD-specific and were used to examine 11 Californian PD isolates and a German reference strain. All showed the same RFLP profile following digestion with AluI and RsaI (Guerra, 1997). In another experiment, AP and PD phytoplasmas were distinguished by digestion of an 835 bp tuf gene fragment with both HinfI and HpaII (Berg, 1998). The PYLR agent also showed the same profiles as the PD phytoplasma in the present study.
Southern blot hybridizations.
Randomly cloned chromosomal DNA fragments from strain AT were employed in Southern blot analysis to characterize and differentiate AP-group phytoplasmas. HindIII fragments IH184 and IH196 (Bonnet et al., 1990) and EcoRI fragments AT17, AT27, AT67 and AT72 (Schneider & Seemüller, 1994b) were used as probes (see above for inserts IH196 and AT67). With both sets of fragments, which were employed mostly as cocktail probes, distinctly different RFLP patterns were obtained with HindIII- and EcoRI-digested DNA from plants that were infected with the AP or ESFY agents (Ahrens et al., 1993; Lorenz et al., 1994; Kison et al., 1997). The EcoRI probes also enabled clear differentiation of the PYLR agent from the AP and ESFY phytoplasmas. In contrast, following hybridization with probes IH184 and IH196, HindIII-digested PYLR phytoplasma DNA showed the same RFLP profile as strain AP15T, which was different from that of strain AT (Kison et al., 1997). Hybridization experiments using HindIII- or EcoRI-digested DNA from PD-diseased pear trees with both sets of probes gave inconclusive results; this is probably due to too low phytoplasma DNA concentrations in extracts from PD-diseased trees. The probes used were specific for AP-group phytoplasmas and did not hybridize or hybridized only weakly to DNA of phytoplasmas from other phylogenetic groups. When Southern blots of EcoRI-digested DNA were probed with IMP gene 318B from strain AT, hybridization signals were also obtained from the PD and ESFY phytoplasmas. However, the fragment size was different for each phytoplasma (Berg et al., 1999).
Southern blot analysis also revealed sequence heterogeneity between isolates of the AP phytoplasma. After hybridization of HindIII-digested DNA from 34 isolates of the AP agent with HindIII fragments IH184 and IH196, five different RFLP patterns were observed. Two of them were represented by strains AT and AP15R (Bonnet et al., 1990; Kison et al., 1994). These two genotypes correspond to PCR-RFLP subtypes AT-1 and AP of Jarausch et al. (2000b). Sequence heterogeneity within the AP agent was also observed when HindIII-digested DNA from three AP phytoplasma isolates was hybridized with a 5 kbp probe from an aster yellows phytoplasma, which comprised part of an rDNA operon and a 2·8 kbp upstream region. In this case, each strain showed a different RFLP pattern (Schneider & Seemüller, 1994a). However, no polymorphisms were observed following hybridization of EcoRI-digested AP DNA with EcoRI fragments AT17, AT27 and AT67 (Kison et al., 1997). By hybridization of HindIII- or EcoRI-digested DNA from over 50 ESFY phytoplasma isolates with cocktail probes IH184 and IH196 or AT17, AT27 and AT67, polymorphisms were only observed in two samples following hybridization with the HindIII fragments as probes. No heterogeneity was observed in the PYLR phytoplasma following hybridization with both sets of probes (Ahrens et al., 1993; Lorenz et al., 1994; Kison et al., 1997).
Genome sizes, chromosome restriction maps and G+C content.
PFGE was used to estimate genome sizes of the AP, PD and ESFY phytoplasmas by resolving full-length chromosomes and/or chromosomal macrorestriction fragments that were generated by single or double digestions with rare-cutting enzymes (Marcone et al., 1999b; Lauer & Seemüller, 2000). PFGE analysis showed that the chromosome sizes of the three fruit-tree pathogens are in the lower range for phytoplasmas. Estimated sizes of the chromosomes of strains AT and AP15R of the AP agent were 645 and 690 kbp, respectively, whereas the chromosome length of ESFY phytoplasma strains ESFY1, ESFY-G1R and ESFY-G2 was uniformly 630 kbp and that of strain PD1R was 660 kbp. Physical maps were constructed from the chromosomes of strains AT and ESFY-G1R (Lauer & Seemüller, 2000; Marcone & Seemüller, 2001). Although there were differences in the restriction enzymes that were suitable for map construction and in the frequency and location of restriction sites, the overall arrangement of the chromosomes was similar, as evidenced by gene positions on the maps. Interspecific variations were detected by digestion of full-length chromosomes from strains AT, AP15R, PD1R and ESFY-G2 with BssHII. Also, polymorphisms were observed between strains AT and AP15R (Fig. 3). The G+C content of the chromosome of strain AT was estimated to be 23·7 mol% (Kollar & Seemüller, 1989).
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). In our Western blot experiments, in which five different strains of the AP agent were included, these mAbs reacted with only three strains (AP15R, AP1/93 and AP12/93) and poorly with IMP 318B from strain AT (see above). A polyclonal antiserum that was raised to IMP 318B of strain AT (Berg et al., 1999) gave different results when the same samples were analysed in Western blot experiments. This antiserum reacted with the recombinant protein and antigens of strains AT and AP5/93, but not with antigens of strains AP15R, AP1/93 or AP12/93 (Fig. 4). In another experiment, polyclonal antibodies purified from antisera that were raised to antigens from the AP, PD and ESFY phytoplasmas recognized, in Western blot assays, only the homologous antigens and did not cross-react (Davies & Adams, 2001).
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; Seemüller, 1992; Jarausch et al., 2001; Kison & Seemüller, 2001; Carraro et al., 2002). Using universal phytoplasma primers or AP group-specific primers, phytoplasmas showing rDNA RFLP profiles that were specific for the three fruit-tree agents were occasionally identified in plants other than the typical host. On the basis of these patterns, additional AP phytoplasma hosts include stone fruits and both European and Asian pear (Pyrus communis and Pyrus pyrifolia) (Lee et al., 1995; Del Serrone et al., 1998; E. Seemüller, unpublished results). AP-group phytoplasmas were also detected in hazel (Corylus avellana) (Marcone et al., 1996c), ash (Fraxinus excelsior), dog rose (Rosa canina), hackberry (Celtis australis) (Jarausch et al., 2001), hawthorn (Crataegus monogyna), oak (Quercus robur and Quercus rubra), hornbeam (Carpinus betulus) and bindweed (Convolvulus arvensis) (Schneider et al., 1997; Berges, 1999; Seemüller, 2002). However, the identity of the phytoplasmas detected in these plants remains to be confirmed by pathological and vector transmission studies and by examining additional molecular markers.
Whilst other phytoplasmas are transmitted by either leafhoppers (Cicadellidae) or planthoppers (Xixiidae), the three fruit-tree phytoplasmas are vectored by psyllids (Psylloidea). AP is transmitted by both Cacopsylla picta (synonym, Psylla costalis) (Frisinghelli et al., 2000) and Cacopsylla melanoneura (Tedeschi et al., 2002), ESFY by Cacopsylla pruni (Carraro et al., 1998b) and PD by both Cacopsylla pyricola (Jensen et al., 1964) and Cacopsylla pyri (Carraro et al., 1998a). C. pyricola is also a vector of PYLR (Blomquist & Kirkpatrick, 2002).
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DISCUSSION |
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; Lee et al., 1992; Schneider & Seemüller, 1994b; Griffiths et al., 1999).
Phylogenetic analyses revealed that the AP, PD and ESFY phytoplasmas are closely related, differing by only 16–19 nucleotide positions in their 16S rDNA, which corresponds to 98·6–99·1 % sequence similarity. An even closer relationship occurs in other mollicute species. For example, Mycoplasma cottewii and Mycoplasma yeatsii share 99·7 % sequence similarity (Heldtander et al., 1998) and Mycoplasma gallisepticum and Mycoplasma imitans differ by only 2 nt in their 16S rDNA sequences. However, the latter two taxa have other characteristics that show that they can be regarded as different species (Bradbury et al., 1993). Similarly, the AP, PD and ESFY phytoplasmas differed clearly in all comparisons made, in addition to 16S rDNA analyses. From this, it can be concluded that the requirements for defining phylogenetically closely related phytoplasmas as putative species are fulfilled and that the AP, PD and ESFY phytoplasmas represent discrete, coherent taxa.
In contrast to the AP, PD and ESFY agents, there is not sufficient evidence to justify a formal taxonomic classification of the PYLR phytoplasma at present. This pathogen is phylogenetically closely related to, but not identical to, the PD agent. In most molecular analyses in which ribosomal and non-ribosomal DNA sequences were included, this pathogen was indistinguishable from the PD agent. Only recent comparisons of structurally similar IMP genes showed distinct differences between the two phytoplasmas (Morton et al., 2003). This supports geographical and pathological evidence that indicates that PD and PYLR are caused by different organisms. Based on these results, it appears to be most appropriate to regard the PYLR agent as a subtype of the PD phytoplasma.
Taxonomic descriptions
To facilitate reference to unique phytoplasma lineages and to have names by which distinct phytoplasmas can be known, we propose to designate the AP, PD and ESFY phytoplasmas as novel ‘Candidatus’ species, according to the convention proposed by Murray & Schleifer (1994) for prokaryotes that can be only incompletely described.
We propose that the AP, PD and ESFY phytoplasmas be designated as novel, distinct ‘Candidatus’ species: ‘Candidatus Phytoplasma mali’, ‘Candidatus Phytoplasma pyri’ and ‘Candidatus Phytoplasma prunorum’, respectively, with the following descriptions.
‘Candidatus Phytoplasma mali’ (mali, epithet referring to the plant host) [(Mollicutes) NC; NA; O, wall-less; NAS (GenBank accession no. AJ542541); oligonucleotide sequence complementary to unique region of the 16S rRNA gene is 5'-AATACTCGAAACCAGTA-3'; P (Malus, phloem); M].
‘Candidatus Phytoplasma pyri’ (pyri, epithet referring to the plant host) [(Mollicutes) NC; NA;O, wall-less; NAS (GenBank accession no. AJ542543); oligonucleotide sequences complementary to regions of the 16S rRNA gene, which are only shared by the PYLR agent, are 5'-AATACTCAAAACCAGTA-3' and 5'-ATACGGCCCAAACTCATACGGA-3'; P (Pyrus, phloem); M]. We propose to regard the PYLR agent as a subtype of ‘Candidatus Phytoplasma pyri’.
‘Candidatus Phytoplasma prunorum’ (prunorum, epithet referring to the plant host) [(Mollicutes) NC; NA; O, wall-less; NAS (GenBank accession no. AJ542544); oligonucleotide sequences complementary to unique regions of the 16S rRNA gene 5'- AATACCCGAAACCAGTA -3' and 5'-TGAAGTTTTGAGGCATCTCGAA -3'; P (Prunus, phloem); M].
Reference strains AP15R, PD1R and ESFY-G1R and other strains mentioned herein that are maintained in Catharanthus roseus are available from the authors.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25, 3389–3402.
Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. & Struhl, K. (1987). Current Protocols in Molecular Biology. New York: Wiley.
Barbara, D. J., Morton, A., Clark, M. F. & Davies, D. L. (2001). Molecular variation in immunodominant membrane proteins from phytoplasmas. Acta Hortic 550, 405–408.
Barbara, D. J., Morton, A., Clark, M. F. & Davies, D. L. (2002). Immunodominant membrane proteins from two phytoplasmas in the aster yellows clade (chlorante aster yellows and clover phyllody) are highly divergent in the major hydrophilic region. Microbiology 148, 157–167.
Berg, M. (1998). Elongationsfaktoren und immunogene Membranproteine des Apfeltriebsucht-Phytoplasmas. PhD thesis, Technische Universität Darmstadt, Germany (in German).
Berg, M., Davies, D. L., Clark, M. F., Vetten, H. J., Maier, G., Marcone, C. & Seemüller, E. (1999). Isolation of the gene encoding an immunodominant membrane protein of the apple proliferation phytoplasma, and expression and characterization of the gene product. Microbiology 145, 1937–1943.[Abstract]
Berges, R. (1999). Untersuchungen zur molekularbiologischen Identifizierung und ätiologischen Bedeutung von Phytoplasmen in Wald- und Feldgehölzen und zur Quantifizierung der Erreger in Wirtspflanzen mit unterschiedlicher Besiedlungsdichte. PhD thesis, Universität Heidelberg, Germany (in German).
Blomquist, C. L. & Kirkpatrick, B. C. (2002). Identification of phytoplasma taxa and insect vectors of peach yellow leaf roll disease in California. Plant Dis 86, 759–763.[Medline]
Bonnet, F., Saillard, C., Kollar, A., Seemüller, E. & Bové, J. M. (1990). Detection and differentiation of the mycoplasmalike organism associated with apple proliferation disease using cloned DNA probes. Mol Plant–Microbe Interact 3, 438–443.
Bradbury, J. M., Abdul-Wahab, O. M. S., Yavari, C. A., Dupiellet, J.-P. & Bové, J. M. (1993). Mycoplasma imitans sp. nov. is related to Mycoplasma gallisepticum and found in birds. Int J Syst Bacteriol 43, 721–728.[CrossRef][Medline]
Carraro, L., Osler, R., Refatti, E. & Poggi Pollini, C. (1988). Transmission of the possible agent of apple proliferation to Vinca rosea by dodder. Riv Patol Veg 24, 43–52.
Carraro, L., Loi, N., Ermacora, P., Gregoris, A. & Osler, R. (1998a). Transmission of pear decline by using naturally infected Cacopsylla pyri L. Acta Hortic 472, 665–668.
Carraro, L., Osler, R., Loi, N., Ermacora, P. & Refatti, E. (1998b). Transmission of European stone fruit yellows phytoplasma by Cacopsylla pruni. J Plant Pathol 80, 233–239.[Medline]
Carraro, L., Ferrini, F., Ermacora, P. & Loi, N. (2002). Role of wild Prunus species in the epidemiology of European stone fruit yellows. Plant Pathol 51, 513–517.[CrossRef][Medline]
Christiansen, G. (1992). Genetic variation in natural populations. In Mycoplasmas: Molecular Biology and Pathogenesis, pp. 561–573. Edited by J. Maniloff, R. N. McElhaney, L. R. Finch & J. B. Baseman. Washington, DC: American Society for Microbiology.
Davies, D. L. & Adams, A. N. (2001). European stone fruit yellows phytoplasmas in southern England. Acta Hortic 550, 389–393.
Del Serrone, P., La Starza, S., Krystai, L., Kölber, M. & Barba, M. (1998). Occurrence of apple proliferation and pear decline phytoplasmas in diseased pear trees in Hungary. J Plant Pathol 80, 53–58.[Medline]
Deng, S. & Hiruki, C. (1991). Amplification of 16S rRNA genes from culturable and nonculturable mollicutes. J Microbiol Methods 14, 53–61.
Frisinghelli, C., Delaiti, L., Grando, M. S., Forti, D. & Vindimian, M. E. (2000). Cacopsylla costalis (Flor 1861), as a vector of apple proliferation in Trentino. J Phytopathol 148, 425–431.[CrossRef][Medline]
Goidanich, G. (1933). Un deperimento dei susini. Boll Stn Patol Veg Roma 13, 160–173 (in Italian).[Medline]
Griffiths, H. M., Sinclair, W. A., Smart, C. D. & Davis, R. E. (1999). The phytoplasma associated with ash yellows and lilac witches’-broom: ‘Candidatus Phytoplasma fraxini’. Int J Syst Bacteriol 49, 1605–1614.[CrossRef][Medline]
Guerra, L. J. (1997). Biological and molecular characterization of phytoplasmas infecting fruit and nut trees in California. PhD thesis, University of California, CA, USA.
Heldtander, M., Pettersson, B., Tully, J. G. & Johansson, K.-E. (1998). Sequences of the 16S rRNA genes and phylogeny of the goat mycoplasmas Mycoplasma adleri, Mycoplasma auris, Mycoplasma cottewii and Mycoplasma yeatsii. Int J Syst Bacteriol 48, 263–268.[CrossRef][Medline]
IRPCM Phytoplasma/Spiroplasma Working Team – Phytoplasma taxonomy group (2004). ‘Candidatus Phytoplasma’, a taxon for the wall-less, non-helical prokaryotes that colonize plant phloem and insects. Int J Syst Evol Microbiol 54, 1257–1269.
Jarausch, W., Saillard, C., Dosba, F. & Bové, J.-M. (1994). Differentiation of mycoplasmalike organisms (MLOs) in European fruit trees by PCR using specific primers derived from the sequence of a chromosomal fragment of the apple proliferation MLO. Appl Environ Microbiol 60, 2916–2923.
Jarausch, W., Saillard, C., Broquaire, J. M., Garnier, M. & Dosba, F. (2000a). PCR-RFLP and sequence analysis of a non-ribosomal fragment for genetic characterization of European stone fruit yellows phytoplasmas infecting various Prunus species. Mol Cell Probes 14, 171–179.[CrossRef][Medline]
Jarausch, W., Saillard, C., Helliot, B., Garnier, M. & Dosba, F. (2000b). Genetic variability of apple proliferation phytoplasmas as determined by PCR-RFLP and sequencing of a non-ribosomal fragment. Mol Cell Probes 14, 17–24.[CrossRef][Medline]
Jarausch, W., Jarausch-Wehrheim, B., Danet, J. L., Broquaire, J. M., Dosba, F., Saillard, C. & Garnier, M. (2001). Detection and identification of European stone fruit yellows and other phytoplasmas in wild plants in the surroundings of apricot chlororic leaf roll-affected orchards in southern France. Eur J Plant Pathol 107, 209–217.[CrossRef][Medline]
Jensen, D. D., Griggs, W. H., Gonzales, C. Q. & Schneider, H. (1964). Pear decline virus transmission by pear psylla. Phytopathology 54, 1346–1351.
Kartte, S. & Seemüller, E. (1991). Susceptibility of grafted Malus taxa and hybrids to apple proliferation disease. J Phytopathol 131, 137–148.[Medline]
Kirkpatrick, B. C., Smart, C. D., Gardner, S. & 9 other authors (1994). Phylogenetic relationship of plant pathogenic MLOs established by 16/23S rDNA spacer sequences. IOM Lett 3, 228–229.[Medline]
Kison, H. & Seemüller, E. (2001). Differences in strain virulence of the European stone fruit yellows phytoplasma and susceptibility of stone fruit trees on various rootstocks to this pathogen. J Phytopathol 149, 533–541.[CrossRef][Medline]
Kison, H., Schneider, B. & Seemüller, E. (1994). Restriction fragment length polymorphism within the apple proliferation mycoplasmalike organism. J Phytopathol 141, 395–401.[Medline]
Kison, H., Kirkpatrick, B. C. & Seemüller, E. (1997). Genetic comparison of the peach yellow leaf roll agent with European fruit tree phytoplasmas of the apple proliferation group. Plant Pathol 46, 538–544.[CrossRef][Medline]
Kollar, A. & Seemüller, E. (1989). Base composition of the DNA of mycoplasmalike organisms associated with various plant diseases. J Phytopathol 127, 177–186.[Medline]
Kumar, S., Tamura, K., Jakobsen, I. B. & Nei, M. (2001). MEGA2: molecular evolutionary genetic analysis software. Bioinformatics 17, 1244–1245.
Lauer, U. & Seemüller, E. (2000). Physical map of the chromosome of the apple proliferation phytoplasma. J Bacteriol 182, 1415–1418.
Lee, I.-M., Davis, R. E., Chen, T. A., Chiykowski, L. N., Fletcher, J., Hiruki, C. & Schaff, D. A. (1992). A genotype-based system for identification and classification of mycoplasmalike organisms (MLOs) in the aster yellows MLO strain cluster. Phytopathology 82, 977–986.
Lee, I.-M., Bertaccini, A., Vibio, M. & Gundersen, D. E. (1995). Detection of multiple phytoplasmas in perennial fruit trees with decline symptoms in Italy. Phytopathology 85, 728–735.[CrossRef]
Lee, I.-M., Davis, R. E. & Gundersen-Rindal, D. E. (2000). Phytoplasma: phytopathogenic mollicutes. Annu Rev Microbiol 54, 221–255.[CrossRef][Medline]
Lim, P.-O. & Sears, B. B. (1989). 16S rRNA sequence indicates that plant-pathogenic mycoplasmalike organisms are evolutionarily distinct from animal mycoplasmas. J Bacteriol 171, 5901–5906.
Loi, N., Carraro, L., Musetti, R., Pertot, I. & Osler, R. (1995). Dodder transmission of two different MLOs from plum trees affected by "leptonecrosis". Acta Hortic 386, 465–470.
Loi, N., Ermacora, P., Carraro, L., Osler, R. & Chen, T. A. (2002). Production of monoclonal antibodies against apple proliferation phytoplasma and their use in serological detection. Eur J Plant Pathol 108, 81–86.[CrossRef][Medline]
Lorenz, K.-H., Dosba, F., Poggi Pollini, C., Llacer, G. & Seemüller, E. (1994). Phytoplasma diseases of Prunus species in Europe are caused by genetically similar organisms. Z Pflanzenkr Pflanzenschutz 101, 567–575.[Medline]
Lorenz, K.-H., Schneider, B., Ahrens, U. & Seemüller, E. (1995). Detection of the apple proliferation and pear decline phytoplasmas by PCR amplification of ribosomal and nonribosomal DNA. Phytopathology 85, 771–776.[CrossRef]
Marcone, C. & Seemüller, E. (2001). A chromosome map of the European stone fruit yellows phytoplasma. Microbiology 147, 1213–1221.
Marcone, C., Ragozzino, A., Del Serrone, P., Aloj, B., Barba, M. & Seemüller, E. (1996a). Detection of apple proliferation and pear decline in southern Italy. Petria 6, 149–157.
Marcone, C., Ragozzino, A., Schneider, B., Lauer, U., Smart, C. D. & Seemüller, E. (1996b). Genetic characterization and classification of two phytoplasmas associated with spartium witches’-broom disease. Plant Dis 80, 365–371.
Marcone, C., Ragozzino, A. & Seemüller, E. (1996c). Association of phytoplasmas with the decline of European hazel in southern Italy. Plant Pathol (Oxf) 45, 857–863.[CrossRef][Medline]
Marcone, C., Hergenhahn, F., Ragozzino, A. & Seemüller, E. (1999a). Dodder transmission of pear decline, European stone fruit yellows, rubus stunt, picris echioides yellows and cotton phyllody phytoplasmas to periwinkle. J Phytopathol 147, 187–192.[CrossRef][Medline]
Marcone, C., Neimark, H., Ragozzino, A., Lauer, U. & Seemüller, E. (1999b). Chromosome sizes of phytoplasmas composing major phylogenetic groups and subgroups. Phytopathology 89, 805–810.[CrossRef]
Marwitz, R., Petzold, H. & Özel, M. (1974). Untersuchungen zur Übertragbarkeit des möglichen Erregers der Triebsucht des Apfels auf einen krautigen Wirt. Phytopathol Z 81, 85–91 (in German).[Medline]
Mäurer, R. & Seemüller, E. (1996). Witches' broom of Rhamnus catharticus: a new phytoplasma disease. J Phytopathol 144, 221–223.[Medline]
McLarty, H. R. (1948). Killing of pear trees. Ann Rep Canad Plant Dis Surv 28, 77.[Medline]
Morton, A., Davies, D. L., Blomquist, C. L. & Barbara, D. J. (2003). Characterization of homologues of the apple proliferation immunodominant membrane protein gene from three related phytoplasmas. Mol Plant Pathol 4, 109–114.
Murray, R. G. E. & Schleifer, K. H. (1994). Taxonomic notes: a proposal for recording the properties of putative taxa of procaryotes. Int J Syst Bacteriol 44, 174–176.[CrossRef][Medline]
Murray, R. G. E. & Stackebrandt, E. (1995). Taxonomic note: implementation of the provisional status Candidatus for incompletely described procaryotes. Int J Syst Bacteriol 45, 186–187.[CrossRef][Medline]
Rui, D., Ciferri, R. & Refatti, E. (1950). La virosi degli "scopazzi del melo" nel Veronese. Not Mal Piante 13, 7–11 (in Italian).[Medline]
Schneider, B. & Seemüller, E. (1994a). Presence of two sets of ribosomal genes in phytopathogenic mollicutes. Appl Environ Microbiol 60, 3409–3412.
Schneider, B. & Seemüller, E. (1994b). Studies on the taxonomic relationships of mycoplasmalike organisms by Southern blot analysis. J Phytopathol 141, 173–185.[Medline]
Schneider, B., Seemüller, E., Smart, C. D. & Kirkpatrick, B. C. (1995). Phylogenetic classification of plant pathogenic mycoplasma-like organisms or phytoplasmas. In Molecular and Diagnostic Procedures in Mycoplasmology, vol. 1, pp. 369–380. Edited by S. Razin & J. G. Tully. San Diego, CA: Academic Press.
Schneider, B., Marcone, C., Kampmann, M., Ragozzino, A., Lederer, W., Cousin, M.-T. & Seemüller, E. (1997). Characterization and classification of phytoplasmas from wild and cultivated plants by RFLP and sequence analysis of ribosomal DNA. Eur J Plant Pathol 103, 675–686.[CrossRef][Medline]
Seemüller, E. (1992). Pear decline. In Plant Diseases of International Importance: Diseases of Fruit Crops, pp. 308–334. Edited by J. Kumar, U. S. Singh, A. N. Mukhopadhyay & H. S. Chaube. Englewood Cliffs, NJ: Prentice Hall.
Seemüller, E. (1999). On some problems in temperate fruit tree phytoplasma research. In First Internet Conference on Phytopathogenic Mollicutes (http://www.Uniud.It/phytoplasma/pap/seem2700.html).
Seemüller, E. (2002). Apple proliferation: etiology, epidemiology and detection. In Atti Giornata Fitopatologiche, pp. 3–6. Baselga di Piné (Trento), 7–11 April 2002.
Seemüller, E., Schneider, B., Mäurer, R. & 8 other authors (1994). Phylogenetic classification of phytopathogenic mollicutes by sequence analysis of 16S ribosomal DNA. Int J Syst Bacteriol 44, 440–446.[CrossRef][Medline]
Seemüller, E., Kison, H., Lorenz, K.-H., Schneider, B., Marcone, C., Smart, C. D. & Kirkpatrick, B. C. (1998a). Detection and identification of fruit tree phytoplasmas by PCR amplification of ribosomal and nonribosomal DNA. In COST 823: New Technologies to Improve Phytodiagnosis. Advances in the Detection of Plant Pathogens by Polymerase Chain Reaction, pp. 56–66. Edited by C. Manceau. Luxembourg: European Community.
Seemüller, E., Marcone, C., Lauer, U., Ragozzino, A. & Göschl, M. (1998b). Current status of molecular classification of the phytoplasmas. J Plant Pathol 80, 3–26.
Seemüller, E., Garnier, M. & Schneider, B. (2002). Mycoplasmas of plants and insects. In Molecular Biology and Pathology of Mycoplasmas, pp. 91–116. Edited by S. Razin & R. Herrmann. London: Kluwer Academic/Plenum Publishers.
Shalla, T. A., Chiarappa, L., Blodgett, E. C., Refatti, E. & Baldacci, E. (1961). The probable coidentity of the moria disease of pear in Italy and pear decline in North America. Plant Dis Rep 45, 912–915.[Medline]
Tedeschi, R., Bosco, D. & Alma, A. (2002). Population dynamics of Cacopsylla melanoneura (Homoptera: Psyllidae), a vector of apple proliferation phytoplasma in northwestern Italy. J Econ Entomol 95, 544–551.[Medline]
Topchiiska, M., Marcone, C. & Seemüller, E. (2000). Detection of pear decline and European stone fruit yellows in Bulgaria. Z Pflanzenkr Pflanzenschutz 107, 658–663.[Medline]
Woodbridge, C. G., Blodgett, E. C. & Diener, T. O. (1957). Pear decline in the Pacific Northwest. Plant Dis Rep 41, 569–572.[Medline]
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