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1 Laboratory of Plant Pathology, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
2 Laboratory of Bioresource Technology, University of Tokyo, 202 Frontier Bioscience Building, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan
Correspondence
Shigetou Namba
snamba{at}mail.ims.u-tokyo.ac.jp
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rDNA sequences of jujube witches'-broom phytoplasma reported in this paper are AB052875–AB052879.
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); it has also been reported in at least three prefectures in Japan (Ohashi et al., 1996). This disease is a common problem, being found in jujube orchards in different locations, which grow different cultivars and have different management practices.
JWB disease was first described, tentatively, as a graft-transmissible viral disease of jujube trees in Korea (Kim, 1965). However, transmission electron microscopy showed that a phytoplasma was associated with the disease (Yi & La, 1973). Sequence analysis of amplified 16S rDNA from the Korean isolate (Namba et al., 1993a) revealed that the pathogen is distinct from mulberry dwarf (MD) and paulownia witches'-broom (PaWB) phytoplasmas, both of which are classified in the aster yellows subgroup of 16S-group I (Jung et al., 2002). More recently, additional studies (Zhu et al., 1996; Tian et al., 2000) that used 16S rDNA RFLP and sequence analysis have revealed that the JWB phytoplasma from China is related to elm yellows (EY) phytoplasma, which currently belongs to 16S-group VIII (the EY group) (Jung et al., 2002), and that the pathogen may constitute a distinct subgroup as it is divergent from other 16S-group VIII members, such as EY and flavescence dorée (FD). To determine whether the phytoplasmas associated with JWB form a discrete, coherent taxon, we analysed JWB phytoplasma isolates from four different areas of Korea and Japan and compared them with other JWB isolates and most other phytoplasmas analysed so far.
More than 20 samples from naturally diseased jujube trees that displayed symptoms of witches'-broom were collected from fields in Kyoto (JWB-Ky), Gifu (JWB-G) and Fukui (JWB-F) prefectures in Japan from 1993 to 1995, and from fields in Gyeongbuk Province, Korea (JWB-Kor), in 1993. As these jujube phytoplasma lines produced indistinguishable symptoms, the JWB phytoplasma isolates were named after their places of origin. Non-symptomatic plants were also collected from the same locations. Healthy tissues were collected from greenhouse-grown jujube seedlings. Total nucleic acids were extracted from tissues as described elsewhere (Namba et al., 1993b), for use as PCR templates. A pair of primers that consisted of a previously designed primer (SN910610; Namba et al., 1993b) plus SN011119 (5'-TCGCCGTTAATTGCGTCCTT-3') was used to amplify 16S rDNA from each sample tested. The following thermal cycling programs were used: 30 cycles of 30 s at 94 °C, 30 s at 55 °C and 1 min 30 s at 72 °C, with a final elongation step of 7 min at 72 °C. Direct PCR with the primer-pair SN910610/SN011119 amplified an approximately 1·8 kbp fragment of the phytoplasma 16S rDNA gene from all diseased jujube trees examined. Under the same conditions, no products were amplified from asymptomatic plants collected from the same areas or from healthy plants grown in a greenhouse (data not shown). The PCR results indicated that there was an association between phytoplasma and JWB disease.
The PCR products of JWB phytoplasmas were sequenced using six primers that have been used previously to sequence phytoplasma 16S rDNA (Namba et al., 1993b, c). Primers 1505F (5'-GGTATCCCTACCGGAAG-3') and 1840R (5'-TCGCCGTTAATTGCGTCCTT-3') were used to sequence the 16S–23S rRNA spacer region. The PCR-amplified products of at least three independent JWB-infected plant samples were sequenced by using Dye Terminator Cycle Sequencing kits (Applied Biosystems); similarity levels were calculated among these JWB phytoplasmas and other phytoplasma sequences available in GenBank (see Table 1 for accession numbers). All 16S rDNA sequences of JWB phytoplasma, isolated from four different regions in Japan and Korea, were virtually identical to each other and to the sequences of two isolates, JWB-Kor2 and JWB-Ch, which were deposited in GenBank. The sequence similarity among these strains exceeded 99·5 %. The representative JWB phytoplasma line (JWB-G1T) with the most conserved sequence was compared with other phytoplasma lines that represented the major phylogenetic groups. Sequence comparisons also revealed that the JWB phytoplasma 16S rDNA sequences were most similar to those of the EY strains, with similarities that ranged from 97·9 (JWB-Kor1 vs FD) to 99·0 (JWB-G1T vs EY2) %.
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), enabling the comparison of 1370 nucleotide positions. Comparative analysis of the putative restriction sites in the sequenced DNA is summarized in Fig. 1. RFLP patterns that used 17 restriction enzymes were identical in all JWB isolates; therefore, we assume that the JWB phytoplasmas are relatively homogeneous. The JWB phytoplasmas can also be distinguished from other phytoplasmas by RFLP analysis of 16S rDNA. Although JWB and other phytoplasmas that belong to 16S-group VIII had most of the restriction sites in common, either the HpaII or RsaI restriction sites clearly distinguished JWB phytoplasmas from all other members of the EY subgroup, supporting the hypothesis that the JWB phytoplasmas represent a distinct novel subgroup.
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, except that the annealing temperature was 54 °C and 30 cycles were run. A primer set was designed from the JWB phytoplasma 16S rDNA sequence to detect JWB phytoplasma specifically. The forward primer was JWBF1 (5'-TAAAAAGGCATCTTTTTGTT-3', corresponding to nucleotides 168–177 in the JWB 16S rDNA sequence) and the reverse primer was JWBR1 (5'-AATCCGGACTAAGACTGT-3', complementary to nucleotides 1268–1285). PCR using primers JWBF1 and JWBR1 amplified a 1119 bp DNA fragment when the template DNA was derived from plants that were naturally infected with JWB phytoplasma. No amplification was observed when the template was derived from healthy plants (data not shown). Conversely, EY-specific primer-pair R16(V)F1/R1, designed to amplify only the 16S rDNA of EY phytoplasma strains (Lee et al., 1994), did not amplify DNA from any of the jujube samples with witches'-broom disease (data not shown). This result supports the hypothesis that this phytoplasma is unique within the EY group.
Nearly complete 16S rDNA sequences from most reported phytoplasmas and Acholeplasma palmae (Table 1
) were aligned with the JWB phytoplasma sequences by using the program CLUSTAL W (Thompson et al., 1994). The base positions were numbered by using a previously described system (Namba et al., 1993c). Nucleotide substitution rates (Knuc values) were calculated (Kimura, 1980) and a phylogenetic tree was constructed by using the neighbour-joining method (Saitou & Nei, 1987), with A. palmae as the outgroup. The neighbour-joining tree constructed by phylogenetic analysis of 16S rDNA sequences from 42 diverse phytoplasmas, three JWB phytoplasmas and A. palmae is shown in Fig. 2. Bootstrap analysis revealed that the phylogenetic tree was reliable and in good agreement with a tree constructed previously by using 110 phytoplasma sequences (Jung et al., 2002). In the phylogenetic analysis, the JWB phytoplasma isolates were included in 16S-group VIII (the EY group) and were most closely related to the EY subgroup (Jung et al., 2002). However, the three representative JWB phytoplasma isolates clustered tightly together and constituted a branch that was distinct from all other 16S-group VIII phytoplasmas, with 100 % bootstrap support. This is in good agreement with previous reports (Zhu et al., 1996; Tian et al., 2000), implying that JWB in China is a member of the EY group (16S-group VIII). These data support the notion that the JWB phytoplasmas represent an independent subgroup that is distinct from the other subgroups that belong to 16S-group VIII.
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; Gundersen et al., 1994), ACTGGA at positions 155–160, TTTTAAAAG at positions 187–195 and GCTT at positions 224–227, were conserved in the JWB phytoplasma 16S rDNA sequence. Whereas the 16S rDNA of EY subgroup phytoplasmas has the unique sequence CTTCAAAA at positions 63–70, the JWB phytoplasmas have CCTCAAAA at these positions, underscoring the difference between JWB and EY subgroup members. Several other unique sequences that also distinguish JWB phytoplasmas from other EY subgroup phytoplasmas were found. For example, the sequence ATAAAAAG at positions 167–174 differed at two positions from the corresponding sequences of other EY subgroup phytoplasmas. Three sequences (at positions 823–825, 982–987 and 1096–1102) were present only in the 16S rDNA of the JWB phytoplasmas. Additionally, the sequences at positions 1220–1223 and 1268–1279 in JWB differed from the sequences in the other EY subgroup phytoplasmas.
In nature, JWB phytoplasmas have been identified only from jujube trees and not from related tree species. This apparent host limitation of JWB phytoplasmas may be due to insect vector feeding preferences rather than to the resistance of particular plants, as these phytoplasmas can be transmitted experimentally into periwinkle plants with the help of a dodder (La & Woo, 1980). The disease is transmitted experimentally by only one species, the leafhopper Hishimonus sellatus (La & Woo, 1980). This ecological property supports the concept of JWB phytoplasmas as a relatively homogeneous taxon, distinct from other phytoplasmas.
According to our survey, JWB phytoplasma appears to be restricted to far-eastern Asia, including China, Japan and Korea, although jujube trees also grow in the Americas and Europe. By contrast, EY and closely related phytoplasmas appear to be restricted to the Americas and Europe. In fact, some results have revealed that many phytoplasmas tend to be distributed locally rather than uniformly, over all continents (Lee et al., 1994; Seemüller et al., 1998). For example, the apple proliferation, peanut witches'-broom, ash yellows and clover proliferation group phytoplasmas each appear to be localized to specific regions (Lee et al., 1998). The geographical isolation of these phytoplasmas may be correlated with the distribution of their host plants and the insect vectors that are native to a particular region. As mentioned before, JWB phytoplasma is transmitted only by one species of leafhopper, H. sellatus, and the EY phytoplasma in America is transmitted by the monophagous or oligophagous vector Scaphoideus luteolus, which feeds only on a few plant species, mostly in the genus Ulmus (Lee et al., 1992). These factors may contribute to the diversification of two related phytoplasma populations, such as the JWB and EY subgroups.
Our findings are consistent with other data and support recognition of the JWB phytoplasma as a unique novel taxon. The degree of divergence of JWB from EY and other phytoplasmas warrants its delineation as a separate lineage. The phylogenetic analysis, the results of base-by-base comparisons of the JWB and EY phytoplasma sequences and the results of an analysis of putative restriction sites in their 16S rDNA sequences, are consistent with the hypothesis that two distinct gene pools have evolved: the EY and JWB phytoplasma. The geographical distribution of JWB phytoplasma in far-eastern Asia may have provided the ecological isolation that favoured the evolution of the distinct JWB phytoplasma.
In a PCR approach that used a primer-pair specific to JWB (JWBF1/JWBR1), specific amplification provided evidence for a novel phytoplasma species, JWB phytoplasma. The presence of JWB-specific signature sequences and sequences unique to JWB phytoplasma in the 16S rDNA sequence also support this proposition and provide evidence for the genetic divergence of this pathogen from other phytoplasmas.
Therefore, we propose that the JWB phytoplasma should be designated as a novel, distinct ‘Candidatus’ species, namely ‘Candidatus Phytoplasma ziziphi’, with the following description: ‘Candidatus Phytoplasma ziziphi’ [(Mollicutes) NC; NA; O; NAS (GenBank no. AB052875–AB052879), oligonucleotide sequences of unique regions of the 16S rRNA gene 5'-TAAAAAGGCATCTTTTTGTT-3' and 5'-AATCCGGACTAAGACTGT-3', P (jujube, phloem); M].
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Jung, H.-Y., Sawayanagi, T., Kakizawa, S. & 7 other authors (2002). ‘Candidatus Phytoplasma castaneae’, a novel phytoplasma taxon associated with chestnut witches' broom disease. Int J Syst Evol Microbiol 52, 1543–1549.[Abstract]
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