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1 National Centre for Animal and Plant Health (CENSA), Apdo 10, San José de Las Lajas, Havana, Cuba
2 CAI "Osvaldo Sánchez", Güines, Havana, Cuba
3 National Institute of Sugarcane Research (INICA), Havana, Cuba
4 Department of Biodiversity and Taxonomy, National Museums and Galleries of Wales, Cardiff, UK
5 Plant-Pathogen Interactions Division, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK
Correspondence
Yaima Arocha
yaimaarocha{at}yahoo.es
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences reported in this paper are given in Table 1
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and is associated with phytoplasmas (Arocha et al., 1999). The delphacid planthopper Saccharosydne saccharivora was recently identified as a vector of YLS, and phytoplasmas were shown to be the causal agent of the disease in Cuba (Arocha et al., 2005b). However, since phytoplasmas are naturally transmitted by many Auchenorryncha leafhoppers and planthoppers (Carraro et al., 2001; Gatineau et al., 2001; Lee et al., 2003), other species could play a role in the epidemiology of the disease.
Papaya bunchy top (PBT) disease has been associated with a bacterium-like organism from the 
-1 subgroup of the Proteobacteria in the genus Rickettsia, and is naturally spread by the leafhopper Empoasca papayae Oman (Davis et al., 1998). Mosaic, yellow crinkle and dieback diseases of papaya in Australia are known to be associated with phytoplasmas (Gibb et al., 1996, 1998; White et al., 1998; De La Rue et al., 1999), and are the main phytosanitary problems of the Australian papaya industry, causing losses of 100 % in some plantations (Guthrie et al., 1998). Epidemiological studies in Australia have identified Orosius leafhoppers species as target candidates for transmission studies (Padovan & Gibb, 2001). In Cuba, recent reports from papaya-growing areas have confirmed that PBT is spreading (Arocha et al., 2003), but no putative insect vectors have been identified as yet.
Little is known about the epidemiological role that weeds play in diseases of either sugarcane or papaya caused by phytoplasmas. In this paper, we report on the identification of phytoplasmas associated with YLS, PBT-like disease, weeds and putative vectors discovered during epidemiological studies of sugarcane and papaya plantations in Cuba.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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DNA extraction.
DNA was extracted from 1·5 g leaf tissue and batches of three insects by using the method of Doyle & Doyle (1990). Ethanol-precipitated nucleic acids were dried, resuspended in 100 µl TE buffer (50 mM Tris/HCl, pH 8·0 and 10 mM EDTA) and incubated with RNase for 1 h at 37 °C. Aliquots of final DNA preparations were used as templates for PCR.
DNA amplification and RFLP analysis.
A nested PCR (nPCR) assay was performed using puReTaq Ready-To-Go PCR beads (Amersham Biosciences) with phytoplasma 16S rDNA primers P1 (Deng & Hiruki, 1991) and P7 (Schneider et al., 1995).
PCR was done using a programmable thermocycler (MJ Research) with 30 cycles of denaturation at 95 °C for 30 s (2 min for first cycle), annealing at 53 °C for 1 min 15 s and extension at 72 °C for 1 min 30 s (10 min for the final cycle). The PCR products obtained were reamplified with 35 cycles of denaturation at 94 °C for 30 s (95 °C, 2 min for one cycle), annealing at 56 °C for 1 min and extension at 72 °C for 2 min, and a final extension step of 72 °C for 10 min, using the nested 16S rDNA primer pair R16F2n/R16R2 (Gundersen & Lee, 1996).
DNA from papaya and E. papayae samples were also analysed by using a PCR assay with the primer pair PBTF1/PBTR1, which amplify the common rickettsial flavoprotein subunit of the succinate dehydrogenase gene (sdhA). The PCR conditions used were according to Davis et al. (1998).
nPCR products were digested with HaeIII, AluI, Sau3AI, Tru9I, HpaII, HhaI or TaqI restriction enzymes, according to the manufacturer's instructions. Digestion products were electrophoresed in 1·5 % agarose gels, and visualized after staining with ethidium bromide by UV transillumination. RFLP patterns were compared with previously published patterns (Schneider et al., 1993; Lee et al., 1995, 1998; Davis et al., 1997; Marcone et al., 1997; Montano et al., 2001; 
eruga et al., 2003).
DNA sequencing.
Phytoplasma rDNA amplified by PCR using the primer pair P1/P7 was purified on spin columns (QIAquick gel extraction kit; QIAGEN). The PCR products were forward- and reverse-sequenced using primer pair P1/P7 by the Sequencing Service, School of Life Sciences, University of Dundee, UK (http://www.dnaseq.co.uk), with Applied Biosystems Big-Dye version 3.1 chemistry on an Applied Biosystems model 3730 automated capillary DNA sequencer.
Sequence similarity, similarity coefficient calculations and putative restriction-site analysis.
The 16S rRNA gene sequences obtained were compared with others in GenBank (Table 1). Sequence editing and alignment were performed using the programs SEQED, LINEUP and PILEUP in the Wisconsin GCG version 10 package (Devereux et al., 1984). Alignments of sequences were generated, sequence similarities were evaluated and putative restriction-site maps were produced with the enzymes HpaII, TaqI, DraI, BfaI, AluI, KpnI, HaeIII, Tru9I, Sau3AI, HinfI, RsaI and HhaI, using the RESearch program (Invitro; Rothamsted Research). Similarity coefficients (F) between the 16S rRNA gene sequences of phytoplasmas identified from sugarcane, weeds, papaya and putative vectors, and of other reference phytoplasmas, were calculated as described by Montano et al. (2001). F was calculated as F=2Nxy/(Nx+Ny), where x and y are the strains of two given phytoplasmas, Nx and Ny are the number of fragments resulting from enzymic digestion of strains x and y, respectively, and Nxy is the number of fragments shared by the two strains.
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). Phylogenetic trees were constructed from the aligned sequences by using a parsimony method (DNAPARS) and 1000 bootstrap datasets generated by the program SEQBOOT from the original dataset. The consensus tree was generated by using the program CONSENSE, with Acholeplasma laidlawii as the outgroup sequence to root the phylogenetic tree. The consensus tree was displayed with TreeView (Page, 1996). |
RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. PCR amplifications were not obtained with the DNA of any papaya or E. papayae using the primer pair PBTF1/PBTR1.
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) indicated that, although some phytoplasmas associated with sugarcane, papaya, weeds, Saccharosydne saccharivora, Cedusa spp. and E. papayae showed slight differences among their 16S rRNA gene RFLP patterns, when compared with the reference phytoplasmas, they could be distinguished from each other and from the rest of the strains analysed. On the other hand, their putative rRNA operon sequence restriction-site maps showed contrasting results (Fig. 4), when compared with those of the other phytoplasma groups analysed.
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), which might explain why putative restriction-site analysis of their 16S rRNA gene sequences showed an additional AluI site (Fig. 4) that was absent from the other known phytoplasma groups analysed.
The Tru9I RFLP profiles of the 16S rRNA gene of phytoplasmas identified in sugarcane, weeds and Saccharosydne saccharivora were similar to those of Stolbur and AAY reference strains (Fig. 1). However, restriction profiles of Cedusa spp., papaya and E. papayae phytoplasmas had an additional band at 600 bp, which was present in AAY and ACLR reference phytoplasmas, but not in the 16S rRNA genes of phytoplasmas of sugarcane, Saccharosydne saccharivora and weeds. Putative restriction analysis (Fig. 4) revealed a Tru9I site in the 16S rRNA genes of sugarcane, Saccharosydne saccharivora and weed phytoplasmas, which was also present in the 16S rRNA gene sequence of the Stolbur reference phytoplasma, but not in those of papaya and E. papayae. Moreover, there were other additional Tru9I sites that distinguished phytoplasmas in sugarcane, Saccharosydne saccharivora, weeds, Cedusa spp., papaya and E. papayae from the reference strains they were compared with.
The Sau3AI restriction profiles (Fig. 3) of phytoplasmas in sugarcane, weeds and Saccharosydne saccharivora were similar to that of the VK reference phytoplasma strain; however, we noted that all the bands of the VK strain profile were slightly larger in size than those of the unknown phytoplasmas. In addition, the Sau3AI RFLP patterns of phytoplasmas identified in papaya, Cedusa spp. and E. papayae were more similar to that of the ACLR reference phytoplasma, but the latter also showed a band that was slightly larger in size. From putative restriction-map comparisons (Fig. 4), all of the 16S rRNA gene sequences of the phytoplasmas analysed shared a Sau3AI site at the position specified by the arrows, except for the papaya and E. papayae phytoplasmas; this site is characteristic of the latter phytoplasmas, and could explain their RFLP profiles. However, differentiation could also be justified by the presence of an additional Sau3AI site in the 16S rRNA gene sequences of sugarcane, Saccharosydne saccharivora and weed phytoplasmas, which distinguished them from the other phytoplasmas analysed.
The HpaII 16S rRNA gene RFLP patterns (Fig. 3) of phytoplasmas in papaya and E. papayae were similar to those of the AAY and ACLR reference strains. However, in the case of the sugarcane, Saccharosydne saccharivora, weeds and Cedusa spp. 16S rRNA gene profiles, a band of approximately 270 bp distinguished them from the other phytoplasmas analysed. This might be supported by the putative restriction analysis (Fig. 4), as their 16S rRNA gene sequences showed additional HpaII sites that were not present in those of the other phytoplasmas mapped.
The TaqI 16S rRNA gene RFLP patterns of phytoplasmas (Fig. 3) in sugarcane, weeds and Saccharosydne saccharivora, which were similar to that of the VK reference strain, showed that the bottom three bands of the profiles were slightly smaller in size, which distinguished them from VK and the other phytoplasmas analysed. However, the TaqI 16S rRNA gene RFLP profiles of phytoplasmas in Cedusa spp., papaya and E. papayae were similar to that of the ACLR reference strain. Putative restriction analysis (Fig. 4) revealed the lack of a TaqI site in the 16S rRNA gene sequences of papaya and E. papayae phytoplasmas, and the displacement of this site in the 16S rRNA gene sequences of phytoplasmas in sugarcane, weeds and Saccharosydne saccharivora, when compared with those of VK and Stolbur reference strains, which might explain the slight differences in their RFLP patterns.
The HhaI 16S rRNA gene RFLP patterns of phytoplasmas in sugarcane and Saccharosydne saccharivora were similar to that of the VK reference strain (Fig. 2), whereas those of the other phytoplasmas were similar to the AAY HhaI profile. From putative restriction-map comparisons (Fig. 4), the 16S rRNA gene sequences of all the phytoplasmas analysed shared one HhaI site at the positions specified by the arrows; however, an additional HhaI site distinguished phytoplasmas identified in papaya and E. papayae from the other strains analysed.
No differences were found among the HaeIII 16S rRNA gene RFLP patterns of phytoplasmas analysed (Fig. 2). According to the putative restriction-map analysis (Fig. 4), the 16S rRNA gene sequences of phytoplasmas in papaya and E. papayae only showed two additional HaeIII sites, which distinguished them from the other strains mapped.
Putative restriction analysis (Fig. 4) also revealed that phytoplasmas in sugarcane, weeds, Saccharosydne saccharivora and Cedusa spp. could be distinguished from the other strains analysed in that their 16S rRNA gene sequences showed an additional KpnI site, which was not present in the 16S rRNA gene of all other 16SrXII phytoplasmas mapped.
The 16S rRNA gene sequence of the phytoplasma identified in Cedusa spp. had an additional HpaII site and lacked a HinfI site, which are the distinguishing characteristics of this phytoplasma.
Sequence similarity
The 16S rRNA gene sequences of phytoplasmas from sugarcane and Saccharosydne saccharivora planthoppers were identical (100 % similarity), and shared 99 % similarity with the 16S rRNA genes amplified from Cynodon dactylon, Sorghum halepense, Conyza canadensis and Macroptilium lathyroides, and 98 % similarity with the 16S rRNA gene sequence from Cedusa spp. planthopper (DP). The phytoplasma 16S rRNA gene sequences from papaya (PAY) and an E. papayae sample (Emp3) also showed 100 % similarity, whereas the sequence similarities were 95 % when compared with the 16S rRNA genes from sugarcane, Saccharosydne saccharivora and weeds and 95·5 % when compared with the 16S rRNA gene from Cedusa spp.
The sequence similarities of the phytoplasma 16S rRNA genes from sugarcane, Saccharosydne saccharivora, Cynodon dactylon, Sorghum halepense, Conyza canadensis and Macroptilium lathyroides were 97·5 % to those of previously characterized phytoplasma strains STOL, VK and Phormium yellow leaf (PYL1); 97 % to Capsicum anuum to Catharanthus roseus phytoplasma (STOLS); 95 % to PYL2, ‘Candidatus Phytoplasma australiense’, Papaya dieback (PDB), Strawberry green petal (SGP) and Strawberry lethal yellows (SLY); 94·5 % to ‘Candidatus Phytoplasma japonicum’; and 93·5 % to AAY and ‘Candidatus Phytoplasma asteris’. The similarity of the amplified 16S rRNA gene from Cedusa spp. was 96 % to those of STOL, VK and STOLS; 94·5 % to PYL1, PYL2, ‘Ca. P. australiense’, PDB and SGP; 93·5 % to SLY; and 93 % to Periwinkle virescence (MPV), ‘Ca. P. japonicum’, AAY and ‘Ca. P. asteris’. Similarities to other known phytoplasma group representatives ranged from 86 to 89·7 %.
The sequence similarities of 16S rRNA genes amplified from PAY and Emp3 phytoplasmas were 97 % to those of STOL, VK and STOLS; 95·5 % to ‘Ca. P. japonicum’; 95 % to PYL1, PYL2, ‘Ca. P. australiense’, PDB, SGP and SLY; and 93·5 % to MPV, AAY and ‘Ca. P. asteris’. Similarities to other phytoplasma groups ranged from 86 to 89·7 %.
The 16S rRNA gene sequence of the phytoplasma identified in Empoasca sample 5 (Emp5) was 98 % similar to that of ‘Candidatus Phytoplasma allocasuarinae’; 93 % to Pear decline (PD), ‘Candidatus Phytoplasma pyri’, ‘Candidatus Phytoplasma prunorum’ and ‘Candidatus Phytoplasma rhamni’; 92 % to Apple proliferation (AP) and ‘Candidatus Phytoplasma mali’; 90 % to representatives from groups 16SrI, 16SrII, 16SrVI, 16SrXI, 16SrXIV and 16SrXV; 89 % to representatives from groups 16SrIII, 16SrIV, 16SrV, 16SrVII, 16SrVIII, 16SrIX, 16SrXII, 16SrXIII, papaya and Emp3; and 88·5 % to phytoplasmas in sugarcane, Saccharosydne saccharivora, weeds and Cedusa spp.
Similarity coefficients
Similarity coefficients derived from RFLP analysis were calculated on the basis of putative restriction-site analysis of nucleotide sequences of 16S rRNA genes (Table 3). The unknown phytoplasma sequences were compared with those of 16S rRNA gene phytoplasma groups included in the analysis of sequence similarity. This analysis revealed that members of the same 16S rRNA gene RFLP group shared values that were >0·82. Stolbur (16SrXII) phytoplasmas shared values ranging from 0·83 (PYL2 with STOLS) to 0·98 (SLY with SGP), whereas RFLP similarity coefficients between phytoplasmas from the Aster yellows group (16SrI) reached 0·99 (‘Ca. P. asteris' with AAY). The 16S rRNA gene similarity coefficients between phytoplasmas in sugarcane, Saccharosydne saccharivora, Macroptilium lathyroides, Conyza canadensis, Sorghum halepense, Cynodon dactylon and Cedusa spp. ranged from 0·82 (PAY and Emp3 with Cedusa spp.) to 0·99 [YLS phytoplasma (SCYLP) and Saccharosydne saccharivora with Sorghum halepense and Conyza canadensis, as well as Sorghum halepense and Macroptilium lathyroides with Cynodon dactylon].
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The 16S rRNA gene similarity coefficients of unknown phytoplasmas identified in Cuba ranged from 0·78 (Cedusa spp. with PYL1) to 0·94 (SCYLP and Saccharosydne saccharivora with VK and STOLS), when compared with phytoplasmas from the 16SrXII group; from 0·5 (Cynodon dactylon) to 0·82 (SCYLP and Saccharosydne saccharivora), when compared with MPV from the 16SrXIII group; from 0·6 (Sorghum halepense with ‘Ca. P. asteris’) to 0·88 (PAY and Emp3 with AAY), when compared with the 16SrI group; and from 0·42 (Sorghum halepense with ‘Ca. P. allocasuarinae’) to 0·75 (SCYLP and Saccharosydne saccharivora with AP), when compared with the 16SrX group.
These results clearly distinguished phytoplasmas in sugarcane, weeds, Saccharosydne saccharivora and Cedusa spp. from those present in papaya and Emp3 and from the reference strains analysed. The findings support recognition of the phytoplasmas present in sugarcane, Saccharosydne saccharivora, Macroptilium lathyroides, Conyza canadensis, Sorghum halepense, Cynodon dactylon and Cedusa spp., and those from papaya and Emp3, as representing two new 16S rRNA gene RFLP groups.
Phylogenetic analysis
Phylogenetic analysis of 62 phytoplasmas, Acholeplasma palmae and Acholeplasma laidlawii produced the consensus tree illustrated in Fig. 5. Bootstrapping values strongly supported most branches, indicating a robust tree whose branching order is in good agreement with previous findings (Lee et al., 1998; White et al., 1998; Jung et al., 2003, 2004). However, phytoplasmas in Cuban sugarcane, weeds, Saccharosydne saccharivora and the Cedusa spp. gave rise to a new branch, whereas phytoplasmas in papaya and Emp3 formed a second new branch, compared with previously published phylogenetic trees in which 15 phytoplasma subclades were identified (IRPCM Phytoplasma/Spiroplasma Working Team–Phytoplasma Taxonomy Group, 2004). Although these two new branches are closely related to the 16SrXII Stolbur group, according to the species definition of Stackebrandt & Goebel (1994) and the classification system of Lee et al. (1998), differences in 16S rRNA gene RFLP patterns and sequence similarities show that the phytoplasmas from sugarcane, weeds, Saccharosydne saccharivora and Cedusa spp., and those from papaya and Emp3, represent two new lineages (16SrXVI and 16SrXVII, respectively) that are distinct from 16SrXII (Stolbur) and all other phytoplasma groups.
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). This analysis revealed that these phytoplasmas contain sequences unique to phytoplasmas and sequences that also distinguish them from other known phytoplasmas.
Sequences previously reported to be unique to phytoplasmas (Gundersen et al., 1994; Davis et al., 1997), 5'-TTTTAAAAG-3' at positions 167–175, 5'-GTGT-3' at positions 256–259, 5'-TGGAGG-3' at positions 347–352, 5'-GGCAAG-3' at positions 632–637, 5'-ATCAG-3' at positions 992–996, 5'-TAGC-3' at positions 1212–1215 and 5'-AGTT-3' at positions 1290–1293, were present in the 16S rRNA gene of the novel phytoplasmas identified. The other signature sequence 5'-ACTGGA-3' also occurred at positions 135–140 but, in the case of the Cedusa spp. phytoplasma, was replaced by TCTGGA.
Unique sequences from the 16S rRNA genes of the phytoplasmas from sugarcane, weeds, Saccharosydne saccharivora and Cedusa derbid were 5'-TTTG-3' at positions 424–427, 5'-TTG-3' at positions 436–439, 5'-GGG-3' at positions 1490–1492, 5'-TAA-3' at positions 1326–1328 and 5'-ATTTACGTTTCTG-3' at positions 1330–1342. These differ at all positions from corresponding sequences from phytoplasmas in other subclades. The Cedusa sp. phytoplasma was the only one to contain the unique sequence 5'-CCC-3' at positions 508–510 with respect to the 16S rRNA gene of the other phytoplasmas analysed, including those identified in Cuba.
The 16S rRNA gene of the phytoplasma amplified from both papaya and Emp3 had the following signature sequences: 5'-AAA-3' at positions 161–163, 5'-ATT-3' at positions 558–560, 5'-AGGCGCC-3' at positions 1039–1045 and 5'-GCGGATTTAGTCACTTTTCAGGC-3' at positions 1324–1346, which differ from those of all other known phytoplasmas at all positions.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) was obtained from diseased sugarcane, weeds and papaya and from putative leafhopper vectors Saccharosydne saccharivora, Cedusa spp. and E. papayae. The presence of sequences unique to phytoplasmas from both papaya and E. papayae DNA and the absence of any rickettsial amplimers establish that the organisms associated with sugarcane YLS, papaya PBT-like disease and those present in weeds and putative vectors are phytoplasmas.
Phytoplasmas were detected in Saccharosydne saccharivora collected from YLS-infected sugarcane fields, supporting its role as the vector of the phytoplasma associated with YLS (Arocha et al., 2005b). Leafhoppers and planthoppers are the most prolific natural vectors of phytoplasma diseases worldwide (Fletcher et al., 1998; Carraro et al., 2001), so the detection and identification of the phytoplasma present in Cedusa spp. suggests that they might be a putative vector of YLS and should be a target for future transmission studies.
Although leafhopper vectors of phytoplasmas infecting papaya are unknown, a species of Orosius has been identified as a candidate for transmission studies of papaya diseases in Australia (Padovan & Gibb, 2001). The leafhopper E. papayae has been reported to be a natural vector of PBT disease (Davis et al., 1998); however, from our study, all E. papayae captured from the papaya plantations showing PBT-like symptoms were negative when indexed by PCR using the specific rickettsial primer pair PBTF1/PBTR1, whereas 104 of 106 plants tested clearly showed 16S rRNA gene signatures. This strongly indicates that phytoplasmas are consistently associated with the disease in Cuba, playing a fundamental role in its development, and that E. papayae is a putative vector.
Cynodon dactylon and Conyza canadensis, which tested positive for phytoplasma, displayed typical white leaf and witches'-broom symptoms, respectively. Symptoms of white leaf Cynodon dactylon plants have been previously associated with the Cynodon white leaf phytoplasma (Arocha et al., 2005a). However, although Macroptilium lathyroides and Sorghum halepense tested positive for phytoplasma, they were asymptomatic, suggesting that weeds present in and surrounding sugarcane and papaya plantations serve as phytoplasma reservoirs. Further studies will be required to determine the various factors involved in their roles in the epidemiology of YLS and PBT-like diseases.
Symptomless phytoplasma infections in sugarcane occur widely (Bailey et al., 1996; Cronjé et al., 1998; Arocha et al., 2000; Tran-Nguyen et al., 2000; Aljanabi et al., 2001), and the relatively long growth period of this crop allows infections to be carried through seasonal barriers and crop cycles. In this study, 14·2 % of sugarcane plants without symptoms were infected with phytoplasmas. Similarly, 10·6 % of symptomless papaya plants contained phytoplasmas, demonstrating that latent infections can also occur in papaya.
RFLP analysis has been found to be useful for general classification (Schneider et al., 1993; Lee et al., 1998; Seemüller et al., 1998) although, in some cases, phytoplasma groups classified on the basis of this method were not always consistent with phylogenetic grouping (Lee et al., 1998). However, RFLP analysis has proved to be a simple and rapid tool for the preliminary classification and identification of unknown phytoplasmas in a relatively short time (Lee et al., 1998; Seemüller et al., 1998). HaeIII and HhaI 16S rRNA gene RFLP patterns obtained from our study could not distinguish the unknown phytoplasmas from reference phytoplasmas; however, AluI, Sau3AI, HpaII and TaqI enzymes yielded restriction profiles that could differentiate phytoplasmas in sugarcane, weeds and Saccharosydne saccharivora as a distinct 16S rRNA gene RFLP group from those in papaya and E. papayae (Emp3) and the other phytoplasmas analysed. This was also supported by clear differences in putative 16S rRNA gene restriction maps, including similarity coefficient calculations and sequence similarity analysis.
The International Committee on Systematics of Prokaryotes Subcommittee on the Taxonomy of Mollicutes has recommended the inclusion of 16S rRNA gene sequences in any description of a novel mollicute species (Marcone et al., 2004a, b). According to Stackebrandt & Goebel (1994), at sequence similarity values below about 97·5 % (in the 16S rRNA gene), it is unlikely that two organisms have more than 60 to 70 % DNA relatedness and hence that they are related at the species level. For uncultured phytoplasmas, a novel putative species may be described when its 16S rRNA gene sequence (1200 bp) has 
97·5 % similarity to any previously described ‘Candidatus Phytoplasma’ species (IRPCM Phytoplasma/Spiroplasma Working Team–Phytoplasma Taxonomy Group, 2004).
A mean sequence similarity of 95·58 % between phytoplasmas in the 16SrXII Stolbur group and those detected in sugarcane, Saccharosydne saccharivora, Cynodon dactylon, Sorghum halepense, Conyza canadensis, Macroptilium lathyroides and Cedusa spp. was demonstrated in this study, whereas similarities between the latter strains and PAY or Emp3 phytoplasmas are slightly greater, at 95·8 %. The unknown phytoplasmas also showed 85–93 % similarity in their 16S rRNA gene sequences compared with representatives of other established phytoplasma groups. Although they are most closely related to phytoplasmas of the Stolbur 16SrXII group, all data presented here and the presence of signature sequences in their respective 16S rRNA genes demonstrate that these phytoplasmas represent two novel provisional species. This conclusion is supported by differences in 16S rRNA gene RFLP patterns and putative restriction maps of unknown phytoplasmas when compared with the reference groups analysed, which, together with the phylogenetic analysis, have demonstrated these phytoplasmas to be representatives of two new 16S rRNA gene RFLP groups (16SrXVI and 16SrXVII, respectively), which are distinct from other phytoplasma groups.
We propose that these phytoplasmas should be given Candidatus status, 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 phytoplasma in sugarcane in Cuba should be designated ‘Candidatus Phytoplasma graminis' and that strains identified in Saccharosydne saccharivora, Cedusa spp., Cynodon dactylon, Conyza canadensis, Macroptilium lathyroides and Sorghum halepense should be considered to be related to this novel species. For the phytoplasma in papaya, including the related strain in E. papayae, we propose the name ‘Candidatus Phytoplasma caricae’, with the following descriptions.
‘Candidatus Phytoplasma graminis' (L. gen. n. graminis of grass, herb; epithet referring to the plant host) [(Mollicutes) NC; NA; O, wall-less; NAS (GenBank accession no. AY725228); oligonucleotide sequences of unique regions of 16S rRNA gene: 5'-TTTG-3', 5'-TTG-3', 5'-GGG-3', 5'-TAA-3' and 5'-ATTTACGTTTCTG-3'; P (Saccharum officinarum; phloem); M]. Reference strain is SCYLP from Cuban sugarcane. DNA samples from this strain are available from the authors.
‘Candidatus Phytoplasma caricae’ (N.L. gen. n. caricae of Carica, the scientific generic name of papaya; epithet referring to the plant host) [(Mollicutes) NC; NA; O, wall-less; NAS (GenBank accession no. AY725234); oligonucleotide sequences of unique regions of 16S rRNA gene: 5'-AAA-3', 5'-ATT-3', 5'-AGGCGCC-3' and 5'-GCGGATTTAGTCACTTTTCAGGC-3'; P (Carica papaya; phloem); M]. Reference strain is PAY from papaya. DNA samples from this strain are available from the authors.
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ACKNOWLEDGEMENTS |
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