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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 Biologische Bundesanstalt für Land- und Forstwirtschaft, Institut für Pflanzenschutz im Obstbau, D-69221, Dossenheim, Germany
Correspondence
Carmine Marcone
marcone{at}unibas.it
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Published online ahead of print on 19 December 2003 as DOI 10.1099/ijs.0.02837-0.
The GenBank/EMBL/DDBJ accession numbers for the P1/P7 amplimer (16S rRNA gene and 16S–23S rDNA spacer region) of phytoplasma strains BGWL-C1, BGWL-C2 and BGWL-CA are AJ550984, AJ550985 and AJ550986, respectively.
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). The disease is known to occur in several Asian countries (Chen et al., 1972; Zahoor et al., 1995; Viswanathan, 1997; Sdoodee et al., 1999), Sudan (Dafalla & Cousin, 1988) and Italy (Marcone et al., 1997). The most characteristic symptoms are extensive chlorosis, proliferation of axillary shoots, bushy growing habit, small leaves, shortened stolons and rhizomes, stunting and death of the plants. A similar disease, termed cynodon white leaf (CWL), has been reported in Australia. The causal agent appeared to be related closely or identical to the BGWL phytoplasma (Padovan et al., 1999; Tran-Nguyen et al., 2000; Blanche et al., 2003).
Phytoplasmas associated with Bermuda grass diseases form, together with the agents of white leaf diseases of other gramineous plants [including Brachiaria distachya (brachiaria grass), Poa annua (annual blue grass) and probably Dactyloctenium aegyptum (growfoot grass)], the BGWL phytoplasma group or 16SrXIV-A group (Lee et al., 1998, 2000; Seemüller et al., 1998; Sdoodee et al., 1999). There is evidence that phytoplasmas associated with diseases of date palms (Phoenix dactylifera) (Cronjé et al., 2000a, b) and carpet grass (Axonopus compressus) white leaf (CGWL) (Padovan et al., 1999) are related closely to this group. More distantly related to the Bermuda grass pathogens are the phytoplasmas that are associated with sugarcane white leaf (SCWL), sugarcane grassy shoot (SCGS), rice yellow dwarf (RYD) and sorghum (Sorghum stipoideum) grassy shoot (SGS). Aside from these monocot diseases, BGWL-related phytoplasmas have been identified in two dicotyledons, Cirsium arvensis (Canada thistle) and Galactia tenuifolia, and in the leafhopper Psammotettix cephalothes (Seemüller et al., 1994; Schneider et al., 1997).
The above-mentioned phytoplasmas form a major branch, thereafter referred to as the SCWL branch, in the monophyletic phytoplasma clade. Previous taxonomic studies on these phytoplasmas, based on sequence analysis of PCR-amplified 16S rDNA, showed that most of them share >97·5 % sequence similarity, the standard threshold for defining putative phytoplasma species under the provisional status ‘Candidatus’ for incompletely described prokaryotes according to Murray & Stackebrandt (1995). However, the phytoplasmas that cluster in this branch appeared to be too diverse to be included in a single species. Thus, the objectives of this work were to examine (i) whether the phytoplasmas identified in Bermuda grass were sufficiently homogeneous to be included in a single species, and (ii) whether the Bermuda grass agents are distinctly different from other phytoplasmas of the SCWL branch to justify separation at the putative species level. It is recommended that phytoplasmas differing in <2·5 % of 16S rDNA nucleotide positions should only be regarded as putative species when the separation is supported by data based on molecular markers other than 16S rDNA, such as plant host range, insect vector transmission and serological comparisons (IRPCM, 2000). Based on data obtained in previous work and additional sequence analysis carried out in this study, our comparisons indicate that the phytoplasmas associated with BGWL and CWL are largely identical and represent a distinct taxonomic entity that we propose should be distinguished at the putative species level.
Phylogenetic analysis of rDNA
Phytoplasmas examined and GenBank accession numbers of retrieved and newly submitted rDNA sequences are listed in Table 1. Strain BGWL-C1, examined previously by RFLP and sequence analysis under the designations Cy.da. and BGWL (accession no. Y16388; Marcone et al., 1997; Seemüller et al., 1998), was resequenced in order to obtain unambiguous data. Sequencing of both strands of P1/P7 PCR products (Schneider et al., 1995) was performed by a commercial service (Bio Molecular Research and Biotechnology Centre, University of Padua, Padua, Italy) using internal primers, as described previously (Seemüller et al., 1994; Marcone et al., 1997). rDNA sequences were analysed by using the software package HUSAR (Biocomputing Service Group, German Cancer Research Center, Heidelberg). 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; bootstrap percentage values are given at nodes of the trees. Maximum-likelihood and parsimony methods yielded similar results in tree construction. Phylogenetic distances were calculated by pairwise comparison. Numbering of nucleotide positions corresponds to that of the 16S rDNA molecule of aster yellows phytoplasma strain OAY (Lim & Sears, 1989).
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. Italian strains BGWL-C1 (proposed as the reference strain), BGWL-C2 and BGWL-CA were identical in 16S rDNA sequence, whereas Thai strain BGWL-T differed from them in four nucleotide positions. Australian strain CWL showed one polymorphism in comparison to strain BGWL-T and three polymorphisms in comparison to the Italian strains. From this data, it can be concluded that the Bermuda grass phytoplasma strains examined are highly uniform and that strain CWL can be included with the BGWL phytoplasma. The BraWL agent is related very closely to the BGWL phytoplasma. It differs in five positions from strain BGWL-T and in nine positions from the Italian strain, corresponding to 99·7 and 99·4 % 16S rDNA sequence similarity, respectively. Of the other phytoplasmas that cluster in the SCWL branch, those related most closely to the BGWL phytoplasma are the SGS and SCWL agents, which share 98·3–98·5 and 98·2–98·4 % 16S rDNA sequence similarity with them. By showing divergences of 3·3–3·4 and 2·6 %, respectively, strains CirP and GaLL from dicotyledonous hosts are related most distantly to the BGWL agent in the SCWL branch. Phytoplasmas from other phylogenetic groups differ from the BGWL agent in >5 % of 16S rDNA positions (Fig. 1). Comparisons of 16S rRNA gene sequences from the BGWL strains with those of phytoplasmas representing the major phylogenetic groups revealed that the BGWL phytoplasma has the following unique signature sequence: 5'-AATTAGAAGGCATCTTTTAAT-3' (positions 169–189), which is not present in any other organism.
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. The tRNAIle gene was present in all strains and the sequence of this gene was identical in all BGWL strains and most other SCWL-branch phytoplasmas. Also, the regions flanking the tRNAIle gene were highly conserved. Strains BGWL-CA, BGWL-C1, BGWL-C2, BGWL-S, BGWL-T, BGWL-I and CWL and the two date palm strains, DP-WTD and DP-SD, were identical. Strains BGWL-C, BraWL and CGWL differed from them in two positions, upstream and downstream of the tRNAIle gene, where G residues were replaced by A residues. These data confirm the results of the 16S rDNA analysis, i.e. that strains from Bermuda grass are identical or nearly identical and that the BraWL agent is related to them very closely. It also appears that the phytoplasmas associated with CGWL and date palm diseases are very similar to the BGWL agent. These findings largely confirm the results of previous work (Wongkaew et al., 1997; Padovan et al., 1999; Sdoodee et al., 1999; Cronjé et al., 2000a, b; Tran-Nguyen et al., 2000). 16S–23S rDNA spacer sequence similarity of the other SCWL-branch phytoplasmas to the BGWL agent is considerably lower than on the 16S rDNA level, ranging from 93·3 % for the RYD phytoplasma to 86·3 % for the GaLL agent.
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; Wongkaew et al., 1997; Lee et al., 1998; Tran-Nguyen et al., 2000). Also, no differences were found between a white leaf agent from annual blue grass and a BGWL phytoplasma strain from Thailand (Lee et al., 1997). Although there is no firm evidence from previous work that phytoplasmas associated with BraWL, CGWL and date palm diseases can be differentiated from the closely related BGWL agent by RFLP analysis of rDNA, putative restriction site mapping carried out in this work revealed a TaqI site at position 1424 in the BraWL agent that was not present in the BGWL phytoplasma strains. In contrast, a MaeIII site at position 1067 was identified in the BGWL phytoplasma that does not occur in the BraWL agent. In all previous comparisons, the BGWL phytoplasma could be distinguished clearly by RFLP analysis from several more distantly related SCWL-branch phytoplasmas, including the SCWL, SCGS and SGS agents (Marcone et al., 1997, 2001; Padovan et al., 1999; Sdoodee et al., 1999; Tran-Nguyen et al., 2000; Blanche et al., 2003).
Specific amplification of BGWL phytoplasma rDNA is possible by using reverse primer rCWL, derived from the 16S–23S rDNA spacer sequence of strain CWL in combination with primer fSCWL, deduced from the 16S rDNA sequence of the SCWL phytoplasma (Tran-Nguyen et al., 2000). No amplification products were obtained from the SCWL, SCGS and SGS phytoplasmas. On the other hand, the SGS-specific primer rSGS in combination with primer fSCWL did not amplify the target DNA from the BGWL phytoplasma (Tran-Nguyen et al., 2000).
PFGE analysis revealed a chromosome size of 530 kbp for two BGWL isolates from southern Italy (Marcone et al., 1999). This value was confirmed by examining five other BGWL isolates that were collected at different locations in the same area (C. Marcone, unpublished results). The estimated genome size represents not only the smallest mollicute chromosome reported to date, but also the smallest genome known for any self-replicating organism.
Serological differentiation and plant host and insect vector specificity
Polyclonal antisera raised to the BGWL and SCWL phytoplasmas revealed no cross-reactivity in serological comparisons (Sarindu & Clark, 1993; Viswanathan, 1997, 2001). Also, an antiserum raised to BGWL phytoplasma antigens did not react with BraWL and DacWL phytoplasma antigens (Sarindu & Clark, 1993). This indicates that the BraWL and DacWL agents are not identical to the BGWL phytoplasma.
By serological methods, PCR amplification using various primers and RFLP analysis of amplified DNA, the BGWL phytoplasma sensu stricto has only been identified in white leaf-diseased Bermuda grass and not in other plants. In reciprocal transmission experiments with the leafhopper Matsumuratettix hiroglyphicus, the natural vector of the SCWL phytoplasma, there was no evidence for transmission of the BGWL phytoplasma from Bermuda grass to sugarcane or vice versa (Anonymous, 1979).
Conclusion and taxonomic description
This taxonomic study showed that, based on 16S rDNA and 16S–23S rDNA spacer sequence analysis, phytoplasma isolates from Bermuda grass are identical or nearly identical. At the 16S rDNA level, they differ from most other phytoplasmas in the SCWL branch, including the SCWL, SGS and RYD agents, in 1·5–2·3 % of nucleotide positions. Thus, divergence of the BGWL agent from these phytoplasmas is below the recommended threshold of 97·5 % sequence similarity for defining a novel phytoplasma species under the provisional status ‘Candidatus’, according to the International Research Program on Comparative Mycoplasmology (IRPCM, 2000). However, from sequence analysis of the 16S–23S rDNA spacer region, serological comparisons, vector transmission specificity and plant host preferences, there is supporting evidence that the BGWL agent is sufficiently different from these phytoplasmas to be described as a novel putative species.
At the 16S rDNA and/or 16S–23S rDNA spacer sequence level, several phytoplasmas, including the BraWL and CGWL agents and isolates associated with diseases of date palms, proved to be identical or nearly identical to the BGWL phytoplasma. However, their classification does not presently seem advisable, as only 16S–23S rDNA sequence data are available from the most closely related phytoplasmas (from date palms); this is a less significant taxonomic tool than 16S rDNA sequence. Also, there is little information on other taxonomic markers of these phytoplasmas. It is only known that antibodies raised to the BGWL agent did not react with BraWL phytoplasma antigens. Thus, taxonomic assignment of these phytoplasmas must await results of additional investigations, particularly on biological properties, such as vector and plant host specificity. It is conceivable that they are pathovars or other subtypes of the BGWL phytoplasma.
Due to its unique properties, we propose to designate the BGWL phytoplasma as a novel ‘Candidatus’ species according to guidelines proposed by Murray & Schleifer (1994) for uncultivated prokaryotes whose uniqueness is defined only by very limited characteristics. The BGWL phytoplasma is designated as a novel distinct species with the following description: ‘Candidatus Phytoplasma cynodontis' (cynodontis epithet referring to the plant host) [(Mollicutes) NC; NA; O, wall-less; NAS (GenBank accession no. AJ550984; oligonucleotide sequence complementary to unique region of the 16S RNA gene is 5'-AATTAGAAGGCATCTTTTAAT-3'; P (Cynodon dactylon); M]. The reference strain is BGWL-C1. DNA samples from this strain are available from C. M.
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ACKNOWLEDGEMENTS |
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