Clover proliferation phytoplasma: ‘Candidatus Phytoplasma trifolii’

Biological properties of the CP^R phytoplasma

At least 10 naturally field-infected alsike clover (Trifolium hybridum) plants were maintained in the greenhouse for observation and as stock cultures. Symptoms such as virescence and proliferation of shoots were observed as described by Chiykowsky (1965). Identical symptoms were produced on young alsike clover after being fed on by the six-spotted leafhopper (Macrosteles fascifrons), according to the method of Chiykowsky (1965). Periwinkle (Catharanthus roseus) showed virescence and mildly dwarfed, compact growth after infection (Hiruki & Chen, 1984).

Dodder transmission from diseased alsike clover to Catharanthus roseus by means of Cuscuta subinclusa was achieved by using a method reported previously (Deng & Hiruki, 1991), resulting in the induction of typical virescence and dwarfed growth (Chiykowsky, 1965), and also to tomato (Lycopersicon esculentum cv. Earliana) plants, in which typical big-bud symptoms, as well as yellows, were produced. In similar experiments, witches'-broom symptoms were produced when young potato (Solanum tuberosum cv. Russet Burbank) plants were inoculated.

Vector specificity of leafhopper transmission in certain phytoplasma strains of the CP group has been shown, but is not completely delineated. The CP^R phytoplasma was transmitted by M. fascifrons from T. hybridum to Callistephus chinensis, Catharanthus roseus, Daucus carota and Nicotiana rustica (Chiykowski, 1965). No transmission of CP^R was obtained with Scaphytopius acutus (Chiykowski, 1965), or of alfalfa witches'-broom (AWB) by Aceratagallia sp., Neokolla hieroglyphica or Cuerna septentrionalis, although accumulation of the CP^R 16S rRNA gene was detected in N. hieroglyphica and Cuerna septentrionalis (Khadhair et al., 1997a). Circulifer tenellus transmitted beet leafhopper-transmitted virescence (BLTV) and tomato big bud in California (TBBc), but not aster yellows (AY), whereas M. fascifrons transmitted AY, but not BLTV or TBBc (Shaw et al., 1993).

Detection by nucleic acid hybridization and PCR

The CP^R phytoplasma, which was originally obtained from alsike clover that showed typical proliferation symptoms in the field in 1970, was transmitted to Catharanthus roseus seedlings by Cuscuta subinclusa and maintained in a greenhouse (Hiruki & Chen, 1984). Restriction fragments of total DNA that was isolated from CP^R-infected periwinkle plants 40 days after graft-inoculation were cloned into plasmid pUC19. The cloned, 2·99 kbp DNA fragment was labelled radioactively or with biotin and used as a probe in dot hybridization for detection of the CP^R phytoplasma and for analysis of its genetic relatedness to other phytoplasmas. The results indicated that up to 2 ng CP^R phytoplasma DNA could be detected with biotinylated DNA probes (Deng & Hiruki, 1990). The probes also hybridized with DNA that was isolated from potato witches'-broom (PWB)-infected periwinkle plants, but not with DNA extracts from those phytoplasmas associated with clover phyllody (CPh), hydrangea virescence (HV), eastern aster yellows (EAY) and AY (AY27) (Deng & Hiruki, 1991). CP^R phytoplasma-specific primers, designed on the basis of the cloned CP^R chromosomal DNA sequence, were used successfully for PCR amplification from CP^R and PWB DNA, but not from DNA from CPh, HV, EAY or AY27 (Deng & Hiruki, 1991). A similar study using CP^R phytoplasma-specific DNA probes in dot hybridization revealed that phytoplasmas associated with BLTV and TBBc were related to the CP^R phytoplasma (Lee et al., 1991). Southern blot analysis of extrachromosomal DNA indicated that the TBBc and BLTV phytoplasmas were related closely to each other, which was confirmed by cross-infection of tomato with the BLTV phytoplasma (Shaw et al., 1993).

RFLP analysis of PCR-amplified 16S rRNA genes

Partial 16S rRNA gene fragments were amplified from DNA samples of CP^R and other phytoplasmas by using universal primers R16F2n and R16R2 (Lee et al., 1993) and were digested with various restriction endonucleases. The results showed repeatedly that the AWB, BLTV, CP^R, PWB and TBBc phytoplasmas produced unique RFLP profiles with restriction endonucleases AluI and MseI and shared very similar or identical RFLP patterns with other endonucleases, such as EcoRII, HaeIII, HhaI, HinfI, HpaII, KpnI RsaI, Sau3AI and ThaI (Lee et al., 1993, 1998; Khadhair et al., 1997a; Wang et al., 1998; Wang & Hiruki, 2001). Collective RFLP patterns of 16S rRNA genes amplified from the phytoplasmas associated with strawberry (Fragaria multicipita) multiplier disease (FM) and Illinois elm yellows (ILEY) indicated that these phytoplasmas were only differentiated from the CP^R phytoplasma by a single restriction site (AluI for FM and HhaI for ILEY) among 12 endonucleases that were used (Jomantiene et al., 1998; Jacobs et al., 2003). These results suggested that these phytoplasmas were similar or identical to one another, but distinctly different from other phytoplasmas; they were thus were assigned to the CP group, but to different subgroups (Table 1⇓).

Heteroduplex mobility assay (HMA) analysis of the 16S rRNA gene and the 16S–23S spacer region

DNA fragments that consisted of the entire 16S rRNA gene and 16S–23S spacer region sequences were amplified from various phytoplasma isolates of CP, PWB and AWB by using the universal primers P1 (Deng & Hiruki, 1991) and P7 (Smart et al., 1996), and were used as templates to amplify the 16S rRNA gene by using nested primers R16F2n/R2, as well as the 16S–23S spacer region by using primers P3/P7 (Smart et al., 1996). HMA analysis of the nested PCR-amplified DNA fragments was carried out by using an equal quantity of each PCR product with the sequence driver (AY27). DNA heteroduplexes were formed between two different DNA sequences after denaturation and reannealing and detected by electrophoresis in a 5 % polyacrylamide gel under non-denaturing conditions. Two different HMA profiles were observed for both the 16S rRNA gene and the 16S–23S spacer region, among 23 isolates of the CP, PWB and AWB phytoplasmas (Wang & Hiruki, 2001). The results indicate that although they are related closely to each other, diversity exists within each of the CP, PWB and AWB phytoplasmas. This conclusion was confirmed by nucleotide sequence analysis of the 16S–23S spacer region, with differences occurring at positions 78 (G/A), 237 (T/C) and deletion or insertion of guanine at position 99. This result also showed that HMA analysis is a rapid and highly sensitive means to investigate genetic diversity in closely related phytoplasmas.

Putative restriction sites in the 16S rRNA gene

The DNA fragment comprising the 16S rRNA gene and 16S–23S spacer region, which was PCR-amplified from the CP^R phytoplasma by using primers P1/P7 and Pfu DNA polymerase (Stratagene), was cloned into a pGEM-T vector (Promega) and sequenced by using standard dideoxy termination methods and automated DNA sequencing. The nucleotide sequence was deposited in GenBank under accession no. AY390261. The 16S rRNA gene sequences of the brinjal little leaf (BLL) (X83431), ILEY (AF268895), ILEY1 (AF409069), ILEY2 (AF409070) and FM (AF036354) phytoplasmas were obtained from GenBank. Putative restriction site maps of the 16S rRNA gene sequences of these phytoplasmas were generated by using the MapDraw option of the dnastar program (dnastar Inc.), as illustrated in Fig. 1⇓. The expected sizes, based on analysis of the putative restriction sites, were in agreement with the fragment sizes obtained by enzymic RFLP analysis of the amplified 16S rRNA gene (Lee et al., 1993, 1998; Khadhair et al., 1997b; Jomantiene et al., 1998; Wang et al., 1998; Wang & Hiruki, 2001; Jacobs et al., 2003). Analysis of putative restriction sites of the partial 16S rRNA gene revealed that these phytoplasmas share very similar or identical restriction profiles, whereas the BLL, FM, ILEY1 and ILEY2 phytoplasmas were distinguished from the CP^R phytoplasma by the absence of the HhaI site at position 1089, AluI site at position 993, HhaI site at position 234 and MseI site at position 944 in the 16S rRNA gene sequence of the CP^R phytoplasma.

Phylogenetic relationships

The 16S rRNA gene sequence of the CP^R phytoplasma was aligned with those of most reported phytoplasmas, including all Candidatus phytoplasma species and at least one phytoplasma in each previously delineated group (subclade) (Lee et al., 1998; Seemüller et al., 1998) by using the program clustal w (Thompson et al., 1994). The degree of 16S rRNA gene sequence divergence between the CP^R phytoplasma and other phytoplasmas ranged from 0·1 % (between CP^R and ILEY) to 12·1 % [between CP^R and the American aster yellows (AAY) phytoplasma]. Phylogenetic analysis indicated clearly that the CP^R phytoplasma and its close relatives, the BLL, FM and ILEY phytoplasmas, formed a subcluster and were different from all other phytoplasmas, with sequence divergences of ⩾2·5 %. The working team on phytoplasmas of the International Research Programme of Comparative Mycoplasmology (IRPCM) set a threshold of 2·5 % for defining phytoplasma groups (International Committee on Systematic Bacteriology Subcommittee on the Taxonomy of Mollicutes, 1995, 1997). On the basis of such criteria, the BLL, FM, ILEY and CP phytoplasmas were defined as the CP group, with CP^R as the reference strain. These phytoplamas were related most closely to those in the ash yellows (AshY) group, followed by the elm yellows (EY) and jujube witches'-broom (JWB) groups (Fig. 2⇓).

Phytoplasma signature sequences

The 16S rRNA gene sequence of the CP^R phytoplasma was compared with those of 64 other phytoplasmas. The results revealed that the CP^R phytoplasma contains all the sequences that were previously reported as being unique to phytoplasmas (Namba et al., 1993; Gundersen et al., 1994). Two signature sequences were only present in the 16S rDNA of phytoplasmas in the CP group. The sequence 5′-TTCTTACGA-3′ at positions 201–209 of the CP^R phytoplasma 16S rRNA gene sequence (AY390261) differed at three to seven positions from the corresponding sequences of phytoplasmas in other subclades, and the sequence 5′-TAGAGTAAAAGCC-3′ at positions 252–264 differed at three to five positions from the sequences in other phytoplasmas.

Conclusions

According to the scheme for assigning incompletely described prokaryotes to the provisional status Candidatus, implemented by the International Committee on Systematic Bacteriology (Murray & Stackebrandt, 1995), we propose that the clover proliferation phytoplasma be designated as a Candidatus species, ‘Candidatus Phytoplasma trifolii’, with the following description: ‘Candidatus Phytoplasma trifolii’ [(Mollicutes): NC; NA; O; NAS (GenBank accession no. AY390261); oligonucleotide sequences of unique regions of the 16S rRNA gene 5′-TTCTTACGA-3′ (201–209) and 5′-TAGAGTAAAAGCC-3′ (252–264) P (Trifolium, phloem); M]. Other strains that are related to CP^R include AWB, BLL, BLTV, FM, ILEY, PWB, TBBc and potato yellows (PY).