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SecY gene sequence analysis for finer differentiation of diversestrains in the aster yellows phytoplasma group
Molecular and Cellular Probes 20 (2006) 87–91 www.elsevier.com/locate/ymcpr
SecY gene sequence analysis for finer differentiation of diverse strains in the aster yellows phytoplasma group I.-M. Lee *, Y. Zhao, K.D. Bottner USDA-ARS Molecular Plant Pathology Laboratory, Beltsville, MD 20705, USA Received 30 June 2005; accepted for publication 4 October 2005 Available online 5 December 2005
Abstract Aster yellows (AY) group (16SrI) phytoplasmas are associated with more than 100 economically important diseases worldwide and represent the most diverse and widespread phytoplasma group. Phylogenetic analysis of secY gene sequences resolved 10 genetically distinct lineages. The 10 lineages coincide with those delineated by phylogenetic analysis based on ribosomal protein (rp) gene sequences. However, greater genetic variability among the 10 lineages was revealed based on secY gene sequences. The distinct phylogenetic lineages can be readily identified through restriction fragment length polymorphism (RFLP) analysis of secY gene sequences. Ten subgroups were differentiated among the AY group phytoplasmas based on RFLP analysis of secY gene sequences. Phylogenetic analysis based on secY gene sequences in this study, and previous studies on the 16S rRNA gene, tuf gene, and rp gene sequences reinforced the notion that most subgroups identified by RFLP analysis of secY and rp gene sequences represent distinct phylogenetic lineages. Published by Elsevier Ltd. Keywords: Aster yellows; Phytoplasma; Ribosomal protein gene; SecY gene
1. Introduction The aster yellows (AY) phytoplasma group (16SrI) comprises AY and numerous related phytoplasmas that are associated with more than 100 economically important diseases worldwide, representing the most diverse phytoplasma group [1–3]. In nature, the AY group phytoplasmas occupy diverse ecological niches [2]. They are transmitted by many species of leafhoppers and cause diseases in more than 100 plant species [3–5,6]. While many AY group phytoplasma strains share common vectors or plant hosts, some strains are carried by specific vectors and have specific plant hosts, and are often restricted to certain geographical regions [7]. Phylogenetic analyses of 16S rDNA sequences indicate the AY group phytoplasmas form a discrete subclade within the phytoplasma clade [8–11]. Within the AY subclade, six distinct phylogenetic lineages were revealed [2]. Fifteen 16SrI subgroups have been differentiated by RFLP analysis of 16S rDNA sequences. Because members of the AY phytoplasma group share at least 97% homology in their 16S * Corresponding author. Tel.: C1 301 504 6024; fax: C1 301 504 5449. E-mail address: [email protected] (I.-M. Lee).
0890-8508/$ - see front matter Published by Elsevier Ltd. doi:10.1016/j.mcp.2005.10.001
rDNA sequences, thus far there is no consensus whether the AY group phytoplasmas should represent more than one phytoplasma species. Currently, based on 16S rDNA sequences a single species, ‘Candidatus phytoplasma asteris’ was proposed to represent the entire group [2]. However, phylogenetic analysis based on less conserved ribosomal protein (rp) gene sequences revealed substantial genetic variations among members of the AY phytoplasma group, implying that the AY group phytoplasmas are more genetically diverse than indicated by phylogenetic analysis based on highly conserved 16S rRNA gene sequences [2]. Rp-based phylogeny delineates 10 distinct phylogenetic lineages or subgroups within the AY group. The rp-subgroups are consistent with their unique ecological niches and biological properties. The new phylogenetic criteria together with differential biological properties (such as plant host range and insect vectors) are consistent with the concept that the AY phytoplasma group may comprise more than one species. The deficiency of conserved 16S rDNA sequences as the sole phylogenetic parameter for use in phytoplasma speciation prompts us to seek less conserved genes as phylogenetic parameters that are more useful for accurate determination of true phylogenetic relationships among closely related but distinct phytoplasma strains. Thus, phytoplasma species can
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Table 1 Classification of aster yellows group phytoplasmas based on restriction length polymorphism analyses (RFLP) of 16S rRNA, ribosomal protein and secY gene sequences Disease caused, strain Chrysanthemum yellows, CHRYM Gray dogwood stunt, GD1 Hydrangea phyllody, HYDP Plantago virescence, PVM Tomato big bud, BB Hydrangea phyllody, HyPH1 Maize bushy stunt, MBS Maryland aster yellows, AY1 Primrose virescence, PRIVC Clover phyllody, CPh Clover phyllody, KVE and KVGZKV Paulownia witches’-broom, PaWB Blueberry stunt, BBS3 Apricot chlorotic leaf roll, ACLR-AY Leafhopper borne, CVB Strawberry multiplier, STRAWB2 Aster yellows, AV2192 Aster yellows, AVUT Ipomoea obscura witches’-broom, IOWB a
Natural host
Country/state
Chrysanthemum frutescens (marguerite) Cornus racemosa (gray dogwood) Hydrangea macrophylla (hydrangea) Plantago coronopus (plantago) Lycopersicon esculentum (tomato) Hydrangea macrophylla (hydrangea) Zea mays (corn) Catharantus rosea (periwinkle) Primula sp. (primrose) Trifolium sativum (red clover) Trifolium repens (white clover) Paulownia spp. (paulownia) Blueberry Prunus aremeniaca (apricot) Leafhopper Fragaria x ananassa (strawberry) Callistephus chinensis (China aster) Callistephus chinensis (China aster) Ipomoea obscura (dwarf morning glory)
Germany New York Belgium Germany Arkansas Italy Mexico Maryland Germany Canada Germany Taiwan Michigan Spain Germany Florida Germany Germany Taiwan
RFLP subgroup classification 16SrIa
rpI
SecY-I
A A A A A B B B B C C D E F F K L M N
A M A A A K L B B C C D E N N J B B F
A M A A A B L B B C C D E N N J B B F
16SrI, 16S rRNA gene RFLP group I; rpI, ribosomal protein gene RFLP group I; secY-I, secY gene RFLP group I.
be better identified and defined by using multiple phylogenetic parameters. In this work, we present an additional phylogenetic parameter, the secY gene sequence, which proves to be useful for finer differentiation among diverse strains in the aster yellows phytoplasma group. 2. Materials and methods
(Bio-Rad, Hercules, CA) and cloned in Escherichia coli using the TOPO TA cloning kit (Invitrogen, Carlsbad, CA) according to the manufacturers’ instructions. Sequencing was performed with an automated DNA sequencer (ABI Prism Model 3100) at the Center for Agricultural Biotechnology, University of Maryland, College Park, MD. The cloned nucleotide sequences were deposited in the GenBank database and accession numbers are shown in Fig. 1.
2.1. Phytoplasma strains and nucleic acid preparation Twenty representative AY group (16SrI) phytoplasma strains were used in the present study (Table 1). Total nucleic acid was extracted, according to the method described by Ahrens et al. [12] or Lee et al. [13], from leaf midribs or other tissues of the original natural host plants or of periwinkle plants that had been experimentally inoculated with phytoplasma strains belonging to the AY phytoplasma group. These phytoplasma strains were previously characterized and identified on the basis of RFLP analysis of rDNA or tuf gene sequences [14–16]. 2.2. PCR and sequencing of secY gene A polymerase chain reaction (PCR) using a generic primer pair AYsecYF1/AYsecYR1(5 0 -CAGCCATTTTAGCAGTTGGTGG-3 0 /5 0 -CAGAAGCTTGAGTGCCTTTACC3 0 ) was employed to amplify a DNA fragment (about 1.4 kb) that encodes near full-length secY gene sequence. The primer pair was designed on the basis of phytoplasma translocation protein gene (secY) sequences available in GenBank. PCR was performed as described previously [2]. PCR amplicons were purified using Quantum Prep PCR Kleen Spin Columns
2.3. Phylogenetic analysis Phylogenetic interrelationships among strains of the AY group were assessed based on secY gene sequences. Sequences of the secY gene from members of the AY phytoplasma group were aligned by using CLUSTAL, version V [17], and DNASTAR’s Laser Gene software (DNASTAR, Madison, WI, USA). Cladistic analyses were performed with PAUP version 4.0 written by D.L. Swofford (University of Illinois), on a Power Mac G4. Uninformative characters were excluded from analyses. A phylogenetic tree was constructed via random stepwise addition by 100 replicates of a heuristic search employing the tree bisection and reconnection algorithm to find optimal trees. Strain EY1 (16SrV) was selected as the outgroup to root the tree. Bootstrap analyses (100 replicates) were performed to estimate the stability and support for the inferred clades. Sequence variations among members of the AY phytoplasma group were compared based on both 16S rRNA and secY gene sequences. 16Sr RNA gene sequences of the AY group, except strain GD1 (DQ112021), were obtained from GenBank.
I.-M. Lee et al. / Molecular and Cellular Probes 20 (2006) 87–91
89
100/100 99.6/99.8 99.2/95.2 100/100 99.0/96.8 99.0/96.9 99.5/96.3 100/100 99.2/95.9 99.0/94.6 99.1/94.7 99.6/98.0 100/100 99.2/95.4 99.0/97.7 98.8/96.5 99.0/96.6 99.3/95.8
PaWB
100/100 99.7/100 99.2/95.4 99.1/97.7 98.9/96.5 99.0/96.6 99.4/95.8
KVG
100/100 99.5/95.6 99.4/95.6 99.7/99.5 99.4/96.0 99.2/94.6 99.3/94.7 99.8/98.3
CPh MBS
16SrRNA gene sequence homology/secY gene sequence homology.
100/100 97.9/96.2 98.8/95.2 98.9/95.0 99.0/94.9 98.8/96.4 98.8/96.4 98.8/94.8 98.8/97.1 98.6/96.4 98.6/96.5 99.0/95.1
GDI
100/100 98.7/95.0 98.8/94.8 98.9/94.8 98.5/96.7 98.5/96.7 98.0/94.7 98.0/97.1 97.7/96.2 98.5/96.0 98.5/95.4
AYI
100/100 99.7/99.8 99.7/99.5 99.2/95.8 99.2/95.8 99.5/99.3 99.2/96.3 99.0/95.0 99.1/95.1 99.6/98.6
AVUT
100/100 99.8/99.2 99.4/95.6 93.3/95.6 99.6/99.1 99.3/96.1 99.1/94.8 99.2/94.9 99.7/98.4 a
Fig. 1. Phylogenetic tree constructed by parsimony analysis (PAUP version 4.0b, D. Swofford) of the secY gene sequences from representative phytoplasma strains in the AY phytoplasma group (16SrI). Sequences were aligned with CLUSTAL version V (DNAStar Lasergene software). EY was employed as the outgroup to root the tree. Branch lengths are proportional to the number of inferred character state transformations. Bootstrap values (measures of support for the inferred subclades) are shown on main branches. Strain abbreviations are listed in Table 1. Bar, 10 inferred character state changes.
100/100 99.2/99.9 98.5/96.4 99.0/95.4 99.2/95.1 99.2/95.1 99.1/96.5 99.0/96.5 99.0/94.9 99.0/97.2 98.7/96.6 98.8/96.7 99.2/95.3
10 changes
BB CHRYM GDI AYI AVUT MBS CPh KVG PaWB BBS3 ACLR-AY CVB IOWB
EY1 (AY197691)
CHRYM
100
HYDP (AY803181) PVM (AY803185) BB (AY803178) CVB (AY803172) ACLR-AY (AY803166)
a
83
100
GD1 (AY803171) CPh (AY803179) 100 KVE (AY803174) KVG (AY803183) BBS3 (AY803169) STRAWB2 (AY803180) CHRYM (AY803170)
BB
85
Table 2 Comparative sequence homologies of 16S rDNA and secY genes among aster yellows phytoplasma group (16SrI) strains
99
BBS3
3. Results and discussion
AV2192 (AY803167) AVUT (AY803168) AY1 (AY803177) HyPH (AY803173) PRIVC (AY803176) 59 MBS (AY803175) PaWB (AY803184) IOWB (AY803182)
100/100 99.2/95.3
IOWB ACLR-AY
CVB
PCR products of secY sequences were digested with MseI, Tsp509I, AluI, HinfI, DdeI, BfaI, RsaI, HhaI, and HpaII. The restriction products were then separated by electrophoresis through a 5% (12% for MseI and Tsp509I digests of secY products) polyacrylamide gel, stained in ethidium bromide, and visualized with a UV transilluminator.
The secY gene exhibited greater sequence variation than 16Sr RNA gene among members of the AY phytoplasma group. Sequence homologies ranged from 94.7 to 98.8% based on secY gene sequences compared to 98.5 to 99.5% based on 16S rDNA sequences between two given 16SrI subgroups (Table 2). A phylogenetic tree, derived by analysis of secY gene sequences, delineated 10 distinct phylogenetic lineages among 20 strains analyzed (Fig. 1). Six corresponded to 16SrI subgroups 16SrI-C (strains KVG, KVE, and CPh), 16SrI-D (strain PaWB), 16SrI-E (strain BBS3), 16SrI-F (strains ACLR-AY and CVB), 16SrI-K (strain STRAWB2), and 16SrI-N (strain IOWB), respectively. One lineage contained subgroups 16SrI-B (strains AY1, PRIVC, HyPH), 16SrI-L (strains AV2192), and 16SrI-M (strain AVUT), while strain MBS (a member of subgroup 16SrI-B) formed a lineage
100/100
2.4. RFLP analysis
MseI (d)
HinfI (g)
RsaI
(b)
Tsp509I (e)
DdeI (h)
HhaI
S BB CHRYM GD1 HYDP PVM AY1 HyPH PRIVC MBS CPh KVG PaWB BBS3 ACLR-AY STRAWB2 AV2192 AVUT IOWB S
(a)
S BB CHRYM GD1 HYDP PVM AY1 HyPH PRIVC MBS CPh KVG PaWB BBS3 ACLR-AY STRAWB2 AV2192 AVUT IOWB S
I.-M. Lee et al. / Molecular and Cellular Probes 20 (2006) 87–91 S BB CHRYM GD1 HYDP PVM AY1 HyPH PRIVC MBS CPh KVG PaWB BBS3 ACLR-AY STRAWB2 AV2192 AVUT IOWB S
90
(c)
AluI (f)
BfaI (i)
HpaII
Fig. 2. RFLP profiles of secY sequences (1.4 kb) amplified by PCR with primer pair AYsecYF1/AYsecYR1 from representative phytoplasma strains in the AY phytoplasma group (16SrI). PCR products were digested with (a) MseI, (b) Tsp509I, (c) AluI, (d) HinfI, (e) DdeI, (f) BfaI, (g) RsaI, (h) HhaI, or (i) HpaII. AluI, HinfI, DdeI, BfaI, RsaI, HhaI, and HpaII digests were separated by electrophoresis through 5% acrylamide gels and MseI and Tsp509I digests were separated by electrophoresis through 12% acrylamide gels. Lane S, FX174 RFI DNA HaeIII digest fragment sizes (bp) from top to bottom for 5% gel: 1353, 1078, 872, 603, 310, 281, 271, 234, 194, 118, 72; fragment sizes (bp) from top to bottom for 12% gel: 1078, 872, 603, 310, 281, 271, 234, 194, 118, 72. Other abbreviations are defined in Table 1.
divergent from the other members of 16SrI-B. Members of 16SrI-A (strains PVM, HYDP, CHRYM, BB and GD1) were delineated into two distinct lineages, strain GD1 alone representing one. Phylogenetic analysis based on amino acid sequences of the secY gene resolved the same lineages as those delineated by analysis of nucleotide sequences (data not shown). Composite profiles obtained from digests with restriction enzymes MseI, Tsp509I, and AluI differentiated the AY group into 10 distinct secY RFLP subgroups (Fig. 2 and Table 1). The 10 distinct lineages delineated by phylogenetic analysis of secY gene sequences were readily differentiated by RFLP analyses of PCR products of secY gene sequences. SecY gene proved to be another efficient molecular tool for differentiation of distinct phytoplasma strains that cannot be readily resolved by highly conserved 16S rRNA gene. For example, strains PaWB (subgroup 16SrI-D) and MBS
(subgroup 16SrI-B), which share O98% 16S rRNA sequence homology with members of subgroup 16SrI-B, were clustered with 16SrI-B strains by 16S rDNA-based phylogeny, but secY-based phylogeny clearly indicated that they represented two distinct lineages (bootstrap values 99–100%). In nature, strain PaWB is associated with paulownia witches’-broom disease in Southeastern Asia, and strain MBS is associated with maize bushy stunt in the Americas. These two strains share neither insect hosts nor plant hosts with other members of subgroup 16SrI-B. Other strains, BBS3 (subgroup 16SrIE), ACLR-AY (16SrI-F), STRAWB2 (16SrI-K), and IOWB (16SrI-N) also occupy uniquely different ecological niches; they are associated with specific plant and insect hosts and/or are restricted to isolated geographical regions [2,4,6]. Phylogeny inferred by secY gene sequence analysis was nearly congruent with that inferred by rp gene analysis [2]. Phylogenetic analyses based on secY gene in the present study and on rp and tuf
I.-M. Lee et al. / Molecular and Cellular Probes 20 (2006) 87–91
gene sequences in the previous studies reinforced the notion that most subgroups identified by RFLP analysis of 16S rDNA sequences represent distinct phylogenetic lineages. Such distinctions are consistent with phylogenetic delineations based on analysis of other genomic DNA fragments [18,19]. Currently, ‘Candidatus phytoplasma asteris’ has been proposed to represent the entire AY group phytoplasma. In light of the substantial genetic variability and differences in biological properties among the diverse AY group phytoplasmas, it seems justified to assign more than one ‘Candidatus phytoplasma’ species in the AY group according to the guidelines proposed by the International Committee of Systematic Bacteriology Subcommittee on the Taxonomy of Mollicutes [20]. For practical diagnostic and quarantine purposes, it would be advantageous if such biologically divergent strains could be designated as distinct taxonomic entities. Both secY and rp genes are useful phylogenetic parameters that can be employed for accurate identification of closely related but distinct AY phytoplasma strains. Thus, each distinct lineage can be better defined by employing multiple phylogenetic parameters for analysis. Acknowledgements We thank E. Seemu¨ller, C. Marcone, and others for providing phytoplasma strains used in this study. References [1] Lee I-M, Davis RE. Aster yellows. In: Maloy O, Murray T, editors. Encyclopedia of plant pathology. New York: Wiley; 2000. p. 60–3. [2] Lee I-M, Gundersen-Rindal DE, Davis RE, Bottner KD, Marcone C, Seemu¨ller E. ‘Candidatus Phytoplasma asteris’, a novel phytoplasma taxxon associated with aster yellows and related disease. Int J Syst Evol Microbiol 2004;54:1037–48. [3] McCoy RE, Cauldwell A, Chang CJ, Chen TA, Chiykowski LN, Cousin MT, et al. Plant diseases associated with mycoplasma-like organisms. In: Whitcomb RF, Tully JG, editors. The mycoplasmas, vol. 5. New York: Academic Press; 1989. [4] Brca´k J. Leafhopper and planthopper vectors of plant disease agents in central and southern Europe. In: Maramorosch K, Harris KF, editors. Leafhopper vectors and plant disease agents. New York: Academic Press; 1979. p. 97–154. [5] Chiykowski LN. Vector–pathogen–host plant relationships of clover phyllody mycoplasmalike orgainsm and the vector leafhopper Paraphlepsius irroratus. Can J Plant Pathol 1991;13:11–18.
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[6] Tsai JH. Vector transmission of mycoplasmal agents of plant diseases. In: Whitcomb RE, Tully JG, editors. The mycoplasmas, vol. 3. New York: Academic Press; 1979. p. 265–307. [7] Lee I-M, Gundersen-Rindal DE, Bertaccini A. Phytoplasma: ecology and genomic diversity. Phytopathology 1998;88:1359–66. [8] Gundersen DE, Lee I-M, Rehner SA, Davis RE, Kingsbury DT. Phylogeny of mycoplasmalike organisms (phytoplasmas): a basis for their classification. J Bacteriol 1994;176:5244–54. [9] Lee I-M, Davis RE, Gundersen-Rindal DE. Phytoplasma: phytopathogenic mollicutes. Annu Rev Microbiol 2000;54:221–55. [10] Seemu¨ller E, Schneider B, Ma¨urer R, Ahrens U, Daire X, Kison H, et al. Phylogenetic classification of phytopathogenic mollicutes by sequence analysis of 16S ribosomal DNA. Int J Syst Bacteriol 1994;44:440–6. [11] Seemu¨ller E, Marcone C, Lauer U, Ragozzino A, Go¨schl M. Current status of molecular classification of the phytoplasmas. J Plant Pathol 1998;80:3–26. [12] Ahrens U, Lorenz K-H, Seemu¨ller E. Genetic diversity among mycoplasmalike organisms associated with stone fruit diseases. Mol Plant Microbe Interact 1993;6:686–91. [13] Lee I-M, Davis RE, Sinclair WA, DeWitt ND, Conti M. Genetic relatedness of mycoplasmalike organisms detected in Ulmus spp. in the United States and Italy by means of DNA probes and polymerase chain reactions. Phytopathology 1993;83:829–33. [14] Lee I-M, Hammond RW, Davis RE, Gundersen DE. Universal amplification and analysis of pathogen 16S rDNA for classification and identification of mycoplasmalike organisms. Phytopathology 1993;83: 834–42. [15] Lee I-M, Gundersen-Rindal DE, Davis RE, Bartoszyk IM. Revised classification scheme of phytoplasmas based on RFLP analyses of 16SrRNA and ribosomal protein gene sequences. Int J Syst Bacteriol 1998;48:1153–69. [16] Marcone C, Lee I-M, Davis RE, Ragozzino A, Seemu¨ller E. Classification of aster yellows-group phytoplasmas based on combined analyses of rRNA ans tuf gene sequences. Int J Syst Evol Microbiol 2000;50: 1703–13. [17] Higgins DG, Sharp PM. CLUSTAL: a package for performing multiple sequence alignments on a microcomputer. Genetic 1988;73:237–44. [18] Gundersen DE, Lee I-M, Schaff DA, Harrison NA, Chang CJ, Davis RE, et al. Genomic diversity and differentiation among phytoplasma strains in 16S rRNA groups I (aster yellows and related phytoplasmas) and III (X-disease and related phytoplasmas). Int J Syst Bacteriol 1996;46:64–75. [19] Lee I-M, Davis RE, Chen T-A, Chiykowski LN, Fletcher J, Hiruki C, et al. A genotype-based system for identification and classification of mycoplasmalike organisms (MLOs) in the aster yellows MLO strain cluster. Phytopathology 1992;82:977–86. [20] IRPCM. Report of the working team for phytoplasmas, spiroplasmas, mesoplasmas and entomoplasmas. In report of consultations of the internatioanl research program on comparative mycoplasmology (IRPCM) of the international organization for mycoplasmology (IOM). Fukuoka, Japan: IOM; 2000.
SecY gene sequence analysis for finer differentiation of diverse strains in the aster yellows phytoplasma group I.-M. Lee *, Y. Zhao, K.D. Bottner USDA-ARS Molecular Plant Pathology Laboratory, Beltsville, MD 20705, USA Received 30 June 2005; accepted for publication 4 October 2005 Available online 5 December 2005
Abstract Aster yellows (AY) group (16SrI) phytoplasmas are associated with more than 100 economically important diseases worldwide and represent the most diverse and widespread phytoplasma group. Phylogenetic analysis of secY gene sequences resolved 10 genetically distinct lineages. The 10 lineages coincide with those delineated by phylogenetic analysis based on ribosomal protein (rp) gene sequences. However, greater genetic variability among the 10 lineages was revealed based on secY gene sequences. The distinct phylogenetic lineages can be readily identified through restriction fragment length polymorphism (RFLP) analysis of secY gene sequences. Ten subgroups were differentiated among the AY group phytoplasmas based on RFLP analysis of secY gene sequences. Phylogenetic analysis based on secY gene sequences in this study, and previous studies on the 16S rRNA gene, tuf gene, and rp gene sequences reinforced the notion that most subgroups identified by RFLP analysis of secY and rp gene sequences represent distinct phylogenetic lineages. Published by Elsevier Ltd. Keywords: Aster yellows; Phytoplasma; Ribosomal protein gene; SecY gene
1. Introduction The aster yellows (AY) phytoplasma group (16SrI) comprises AY and numerous related phytoplasmas that are associated with more than 100 economically important diseases worldwide, representing the most diverse phytoplasma group [1–3]. In nature, the AY group phytoplasmas occupy diverse ecological niches [2]. They are transmitted by many species of leafhoppers and cause diseases in more than 100 plant species [3–5,6]. While many AY group phytoplasma strains share common vectors or plant hosts, some strains are carried by specific vectors and have specific plant hosts, and are often restricted to certain geographical regions [7]. Phylogenetic analyses of 16S rDNA sequences indicate the AY group phytoplasmas form a discrete subclade within the phytoplasma clade [8–11]. Within the AY subclade, six distinct phylogenetic lineages were revealed [2]. Fifteen 16SrI subgroups have been differentiated by RFLP analysis of 16S rDNA sequences. Because members of the AY phytoplasma group share at least 97% homology in their 16S * Corresponding author. Tel.: C1 301 504 6024; fax: C1 301 504 5449. E-mail address: [email protected] (I.-M. Lee).
0890-8508/$ - see front matter Published by Elsevier Ltd. doi:10.1016/j.mcp.2005.10.001
rDNA sequences, thus far there is no consensus whether the AY group phytoplasmas should represent more than one phytoplasma species. Currently, based on 16S rDNA sequences a single species, ‘Candidatus phytoplasma asteris’ was proposed to represent the entire group [2]. However, phylogenetic analysis based on less conserved ribosomal protein (rp) gene sequences revealed substantial genetic variations among members of the AY phytoplasma group, implying that the AY group phytoplasmas are more genetically diverse than indicated by phylogenetic analysis based on highly conserved 16S rRNA gene sequences [2]. Rp-based phylogeny delineates 10 distinct phylogenetic lineages or subgroups within the AY group. The rp-subgroups are consistent with their unique ecological niches and biological properties. The new phylogenetic criteria together with differential biological properties (such as plant host range and insect vectors) are consistent with the concept that the AY phytoplasma group may comprise more than one species. The deficiency of conserved 16S rDNA sequences as the sole phylogenetic parameter for use in phytoplasma speciation prompts us to seek less conserved genes as phylogenetic parameters that are more useful for accurate determination of true phylogenetic relationships among closely related but distinct phytoplasma strains. Thus, phytoplasma species can
88
I.-M. Lee et al. / Molecular and Cellular Probes 20 (2006) 87–91
Table 1 Classification of aster yellows group phytoplasmas based on restriction length polymorphism analyses (RFLP) of 16S rRNA, ribosomal protein and secY gene sequences Disease caused, strain Chrysanthemum yellows, CHRYM Gray dogwood stunt, GD1 Hydrangea phyllody, HYDP Plantago virescence, PVM Tomato big bud, BB Hydrangea phyllody, HyPH1 Maize bushy stunt, MBS Maryland aster yellows, AY1 Primrose virescence, PRIVC Clover phyllody, CPh Clover phyllody, KVE and KVGZKV Paulownia witches’-broom, PaWB Blueberry stunt, BBS3 Apricot chlorotic leaf roll, ACLR-AY Leafhopper borne, CVB Strawberry multiplier, STRAWB2 Aster yellows, AV2192 Aster yellows, AVUT Ipomoea obscura witches’-broom, IOWB a
Natural host
Country/state
Chrysanthemum frutescens (marguerite) Cornus racemosa (gray dogwood) Hydrangea macrophylla (hydrangea) Plantago coronopus (plantago) Lycopersicon esculentum (tomato) Hydrangea macrophylla (hydrangea) Zea mays (corn) Catharantus rosea (periwinkle) Primula sp. (primrose) Trifolium sativum (red clover) Trifolium repens (white clover) Paulownia spp. (paulownia) Blueberry Prunus aremeniaca (apricot) Leafhopper Fragaria x ananassa (strawberry) Callistephus chinensis (China aster) Callistephus chinensis (China aster) Ipomoea obscura (dwarf morning glory)
Germany New York Belgium Germany Arkansas Italy Mexico Maryland Germany Canada Germany Taiwan Michigan Spain Germany Florida Germany Germany Taiwan
RFLP subgroup classification 16SrIa
rpI
SecY-I
A A A A A B B B B C C D E F F K L M N
A M A A A K L B B C C D E N N J B B F
A M A A A B L B B C C D E N N J B B F
16SrI, 16S rRNA gene RFLP group I; rpI, ribosomal protein gene RFLP group I; secY-I, secY gene RFLP group I.
be better identified and defined by using multiple phylogenetic parameters. In this work, we present an additional phylogenetic parameter, the secY gene sequence, which proves to be useful for finer differentiation among diverse strains in the aster yellows phytoplasma group. 2. Materials and methods
(Bio-Rad, Hercules, CA) and cloned in Escherichia coli using the TOPO TA cloning kit (Invitrogen, Carlsbad, CA) according to the manufacturers’ instructions. Sequencing was performed with an automated DNA sequencer (ABI Prism Model 3100) at the Center for Agricultural Biotechnology, University of Maryland, College Park, MD. The cloned nucleotide sequences were deposited in the GenBank database and accession numbers are shown in Fig. 1.
2.1. Phytoplasma strains and nucleic acid preparation Twenty representative AY group (16SrI) phytoplasma strains were used in the present study (Table 1). Total nucleic acid was extracted, according to the method described by Ahrens et al. [12] or Lee et al. [13], from leaf midribs or other tissues of the original natural host plants or of periwinkle plants that had been experimentally inoculated with phytoplasma strains belonging to the AY phytoplasma group. These phytoplasma strains were previously characterized and identified on the basis of RFLP analysis of rDNA or tuf gene sequences [14–16]. 2.2. PCR and sequencing of secY gene A polymerase chain reaction (PCR) using a generic primer pair AYsecYF1/AYsecYR1(5 0 -CAGCCATTTTAGCAGTTGGTGG-3 0 /5 0 -CAGAAGCTTGAGTGCCTTTACC3 0 ) was employed to amplify a DNA fragment (about 1.4 kb) that encodes near full-length secY gene sequence. The primer pair was designed on the basis of phytoplasma translocation protein gene (secY) sequences available in GenBank. PCR was performed as described previously [2]. PCR amplicons were purified using Quantum Prep PCR Kleen Spin Columns
2.3. Phylogenetic analysis Phylogenetic interrelationships among strains of the AY group were assessed based on secY gene sequences. Sequences of the secY gene from members of the AY phytoplasma group were aligned by using CLUSTAL, version V [17], and DNASTAR’s Laser Gene software (DNASTAR, Madison, WI, USA). Cladistic analyses were performed with PAUP version 4.0 written by D.L. Swofford (University of Illinois), on a Power Mac G4. Uninformative characters were excluded from analyses. A phylogenetic tree was constructed via random stepwise addition by 100 replicates of a heuristic search employing the tree bisection and reconnection algorithm to find optimal trees. Strain EY1 (16SrV) was selected as the outgroup to root the tree. Bootstrap analyses (100 replicates) were performed to estimate the stability and support for the inferred clades. Sequence variations among members of the AY phytoplasma group were compared based on both 16S rRNA and secY gene sequences. 16Sr RNA gene sequences of the AY group, except strain GD1 (DQ112021), were obtained from GenBank.
I.-M. Lee et al. / Molecular and Cellular Probes 20 (2006) 87–91
89
100/100 99.6/99.8 99.2/95.2 100/100 99.0/96.8 99.0/96.9 99.5/96.3 100/100 99.2/95.9 99.0/94.6 99.1/94.7 99.6/98.0 100/100 99.2/95.4 99.0/97.7 98.8/96.5 99.0/96.6 99.3/95.8
PaWB
100/100 99.7/100 99.2/95.4 99.1/97.7 98.9/96.5 99.0/96.6 99.4/95.8
KVG
100/100 99.5/95.6 99.4/95.6 99.7/99.5 99.4/96.0 99.2/94.6 99.3/94.7 99.8/98.3
CPh MBS
16SrRNA gene sequence homology/secY gene sequence homology.
100/100 97.9/96.2 98.8/95.2 98.9/95.0 99.0/94.9 98.8/96.4 98.8/96.4 98.8/94.8 98.8/97.1 98.6/96.4 98.6/96.5 99.0/95.1
GDI
100/100 98.7/95.0 98.8/94.8 98.9/94.8 98.5/96.7 98.5/96.7 98.0/94.7 98.0/97.1 97.7/96.2 98.5/96.0 98.5/95.4
AYI
100/100 99.7/99.8 99.7/99.5 99.2/95.8 99.2/95.8 99.5/99.3 99.2/96.3 99.0/95.0 99.1/95.1 99.6/98.6
AVUT
100/100 99.8/99.2 99.4/95.6 93.3/95.6 99.6/99.1 99.3/96.1 99.1/94.8 99.2/94.9 99.7/98.4 a
Fig. 1. Phylogenetic tree constructed by parsimony analysis (PAUP version 4.0b, D. Swofford) of the secY gene sequences from representative phytoplasma strains in the AY phytoplasma group (16SrI). Sequences were aligned with CLUSTAL version V (DNAStar Lasergene software). EY was employed as the outgroup to root the tree. Branch lengths are proportional to the number of inferred character state transformations. Bootstrap values (measures of support for the inferred subclades) are shown on main branches. Strain abbreviations are listed in Table 1. Bar, 10 inferred character state changes.
100/100 99.2/99.9 98.5/96.4 99.0/95.4 99.2/95.1 99.2/95.1 99.1/96.5 99.0/96.5 99.0/94.9 99.0/97.2 98.7/96.6 98.8/96.7 99.2/95.3
10 changes
BB CHRYM GDI AYI AVUT MBS CPh KVG PaWB BBS3 ACLR-AY CVB IOWB
EY1 (AY197691)
CHRYM
100
HYDP (AY803181) PVM (AY803185) BB (AY803178) CVB (AY803172) ACLR-AY (AY803166)
a
83
100
GD1 (AY803171) CPh (AY803179) 100 KVE (AY803174) KVG (AY803183) BBS3 (AY803169) STRAWB2 (AY803180) CHRYM (AY803170)
BB
85
Table 2 Comparative sequence homologies of 16S rDNA and secY genes among aster yellows phytoplasma group (16SrI) strains
99
BBS3
3. Results and discussion
AV2192 (AY803167) AVUT (AY803168) AY1 (AY803177) HyPH (AY803173) PRIVC (AY803176) 59 MBS (AY803175) PaWB (AY803184) IOWB (AY803182)
100/100 99.2/95.3
IOWB ACLR-AY
CVB
PCR products of secY sequences were digested with MseI, Tsp509I, AluI, HinfI, DdeI, BfaI, RsaI, HhaI, and HpaII. The restriction products were then separated by electrophoresis through a 5% (12% for MseI and Tsp509I digests of secY products) polyacrylamide gel, stained in ethidium bromide, and visualized with a UV transilluminator.
The secY gene exhibited greater sequence variation than 16Sr RNA gene among members of the AY phytoplasma group. Sequence homologies ranged from 94.7 to 98.8% based on secY gene sequences compared to 98.5 to 99.5% based on 16S rDNA sequences between two given 16SrI subgroups (Table 2). A phylogenetic tree, derived by analysis of secY gene sequences, delineated 10 distinct phylogenetic lineages among 20 strains analyzed (Fig. 1). Six corresponded to 16SrI subgroups 16SrI-C (strains KVG, KVE, and CPh), 16SrI-D (strain PaWB), 16SrI-E (strain BBS3), 16SrI-F (strains ACLR-AY and CVB), 16SrI-K (strain STRAWB2), and 16SrI-N (strain IOWB), respectively. One lineage contained subgroups 16SrI-B (strains AY1, PRIVC, HyPH), 16SrI-L (strains AV2192), and 16SrI-M (strain AVUT), while strain MBS (a member of subgroup 16SrI-B) formed a lineage
100/100
2.4. RFLP analysis
MseI (d)
HinfI (g)
RsaI
(b)
Tsp509I (e)
DdeI (h)
HhaI
S BB CHRYM GD1 HYDP PVM AY1 HyPH PRIVC MBS CPh KVG PaWB BBS3 ACLR-AY STRAWB2 AV2192 AVUT IOWB S
(a)
S BB CHRYM GD1 HYDP PVM AY1 HyPH PRIVC MBS CPh KVG PaWB BBS3 ACLR-AY STRAWB2 AV2192 AVUT IOWB S
I.-M. Lee et al. / Molecular and Cellular Probes 20 (2006) 87–91 S BB CHRYM GD1 HYDP PVM AY1 HyPH PRIVC MBS CPh KVG PaWB BBS3 ACLR-AY STRAWB2 AV2192 AVUT IOWB S
90
(c)
AluI (f)
BfaI (i)
HpaII
Fig. 2. RFLP profiles of secY sequences (1.4 kb) amplified by PCR with primer pair AYsecYF1/AYsecYR1 from representative phytoplasma strains in the AY phytoplasma group (16SrI). PCR products were digested with (a) MseI, (b) Tsp509I, (c) AluI, (d) HinfI, (e) DdeI, (f) BfaI, (g) RsaI, (h) HhaI, or (i) HpaII. AluI, HinfI, DdeI, BfaI, RsaI, HhaI, and HpaII digests were separated by electrophoresis through 5% acrylamide gels and MseI and Tsp509I digests were separated by electrophoresis through 12% acrylamide gels. Lane S, FX174 RFI DNA HaeIII digest fragment sizes (bp) from top to bottom for 5% gel: 1353, 1078, 872, 603, 310, 281, 271, 234, 194, 118, 72; fragment sizes (bp) from top to bottom for 12% gel: 1078, 872, 603, 310, 281, 271, 234, 194, 118, 72. Other abbreviations are defined in Table 1.
divergent from the other members of 16SrI-B. Members of 16SrI-A (strains PVM, HYDP, CHRYM, BB and GD1) were delineated into two distinct lineages, strain GD1 alone representing one. Phylogenetic analysis based on amino acid sequences of the secY gene resolved the same lineages as those delineated by analysis of nucleotide sequences (data not shown). Composite profiles obtained from digests with restriction enzymes MseI, Tsp509I, and AluI differentiated the AY group into 10 distinct secY RFLP subgroups (Fig. 2 and Table 1). The 10 distinct lineages delineated by phylogenetic analysis of secY gene sequences were readily differentiated by RFLP analyses of PCR products of secY gene sequences. SecY gene proved to be another efficient molecular tool for differentiation of distinct phytoplasma strains that cannot be readily resolved by highly conserved 16S rRNA gene. For example, strains PaWB (subgroup 16SrI-D) and MBS
(subgroup 16SrI-B), which share O98% 16S rRNA sequence homology with members of subgroup 16SrI-B, were clustered with 16SrI-B strains by 16S rDNA-based phylogeny, but secY-based phylogeny clearly indicated that they represented two distinct lineages (bootstrap values 99–100%). In nature, strain PaWB is associated with paulownia witches’-broom disease in Southeastern Asia, and strain MBS is associated with maize bushy stunt in the Americas. These two strains share neither insect hosts nor plant hosts with other members of subgroup 16SrI-B. Other strains, BBS3 (subgroup 16SrIE), ACLR-AY (16SrI-F), STRAWB2 (16SrI-K), and IOWB (16SrI-N) also occupy uniquely different ecological niches; they are associated with specific plant and insect hosts and/or are restricted to isolated geographical regions [2,4,6]. Phylogeny inferred by secY gene sequence analysis was nearly congruent with that inferred by rp gene analysis [2]. Phylogenetic analyses based on secY gene in the present study and on rp and tuf
I.-M. Lee et al. / Molecular and Cellular Probes 20 (2006) 87–91
gene sequences in the previous studies reinforced the notion that most subgroups identified by RFLP analysis of 16S rDNA sequences represent distinct phylogenetic lineages. Such distinctions are consistent with phylogenetic delineations based on analysis of other genomic DNA fragments [18,19]. Currently, ‘Candidatus phytoplasma asteris’ has been proposed to represent the entire AY group phytoplasma. In light of the substantial genetic variability and differences in biological properties among the diverse AY group phytoplasmas, it seems justified to assign more than one ‘Candidatus phytoplasma’ species in the AY group according to the guidelines proposed by the International Committee of Systematic Bacteriology Subcommittee on the Taxonomy of Mollicutes [20]. For practical diagnostic and quarantine purposes, it would be advantageous if such biologically divergent strains could be designated as distinct taxonomic entities. Both secY and rp genes are useful phylogenetic parameters that can be employed for accurate identification of closely related but distinct AY phytoplasma strains. Thus, each distinct lineage can be better defined by employing multiple phylogenetic parameters for analysis. Acknowledgements We thank E. Seemu¨ller, C. Marcone, and others for providing phytoplasma strains used in this study. References [1] Lee I-M, Davis RE. Aster yellows. In: Maloy O, Murray T, editors. Encyclopedia of plant pathology. New York: Wiley; 2000. p. 60–3. [2] Lee I-M, Gundersen-Rindal DE, Davis RE, Bottner KD, Marcone C, Seemu¨ller E. ‘Candidatus Phytoplasma asteris’, a novel phytoplasma taxxon associated with aster yellows and related disease. Int J Syst Evol Microbiol 2004;54:1037–48. [3] McCoy RE, Cauldwell A, Chang CJ, Chen TA, Chiykowski LN, Cousin MT, et al. Plant diseases associated with mycoplasma-like organisms. In: Whitcomb RF, Tully JG, editors. The mycoplasmas, vol. 5. New York: Academic Press; 1989. [4] Brca´k J. Leafhopper and planthopper vectors of plant disease agents in central and southern Europe. In: Maramorosch K, Harris KF, editors. Leafhopper vectors and plant disease agents. New York: Academic Press; 1979. p. 97–154. [5] Chiykowski LN. Vector–pathogen–host plant relationships of clover phyllody mycoplasmalike orgainsm and the vector leafhopper Paraphlepsius irroratus. Can J Plant Pathol 1991;13:11–18.
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[6] Tsai JH. Vector transmission of mycoplasmal agents of plant diseases. In: Whitcomb RE, Tully JG, editors. The mycoplasmas, vol. 3. New York: Academic Press; 1979. p. 265–307. [7] Lee I-M, Gundersen-Rindal DE, Bertaccini A. Phytoplasma: ecology and genomic diversity. Phytopathology 1998;88:1359–66. [8] Gundersen DE, Lee I-M, Rehner SA, Davis RE, Kingsbury DT. Phylogeny of mycoplasmalike organisms (phytoplasmas): a basis for their classification. J Bacteriol 1994;176:5244–54. [9] Lee I-M, Davis RE, Gundersen-Rindal DE. Phytoplasma: phytopathogenic mollicutes. Annu Rev Microbiol 2000;54:221–55. [10] Seemu¨ller E, Schneider B, Ma¨urer R, Ahrens U, Daire X, Kison H, et al. Phylogenetic classification of phytopathogenic mollicutes by sequence analysis of 16S ribosomal DNA. Int J Syst Bacteriol 1994;44:440–6. [11] Seemu¨ller E, Marcone C, Lauer U, Ragozzino A, Go¨schl M. Current status of molecular classification of the phytoplasmas. J Plant Pathol 1998;80:3–26. [12] Ahrens U, Lorenz K-H, Seemu¨ller E. Genetic diversity among mycoplasmalike organisms associated with stone fruit diseases. Mol Plant Microbe Interact 1993;6:686–91. [13] Lee I-M, Davis RE, Sinclair WA, DeWitt ND, Conti M. Genetic relatedness of mycoplasmalike organisms detected in Ulmus spp. in the United States and Italy by means of DNA probes and polymerase chain reactions. Phytopathology 1993;83:829–33. [14] Lee I-M, Hammond RW, Davis RE, Gundersen DE. Universal amplification and analysis of pathogen 16S rDNA for classification and identification of mycoplasmalike organisms. Phytopathology 1993;83: 834–42. [15] Lee I-M, Gundersen-Rindal DE, Davis RE, Bartoszyk IM. Revised classification scheme of phytoplasmas based on RFLP analyses of 16SrRNA and ribosomal protein gene sequences. Int J Syst Bacteriol 1998;48:1153–69. [16] Marcone C, Lee I-M, Davis RE, Ragozzino A, Seemu¨ller E. Classification of aster yellows-group phytoplasmas based on combined analyses of rRNA ans tuf gene sequences. Int J Syst Evol Microbiol 2000;50: 1703–13. [17] Higgins DG, Sharp PM. CLUSTAL: a package for performing multiple sequence alignments on a microcomputer. Genetic 1988;73:237–44. [18] Gundersen DE, Lee I-M, Schaff DA, Harrison NA, Chang CJ, Davis RE, et al. Genomic diversity and differentiation among phytoplasma strains in 16S rRNA groups I (aster yellows and related phytoplasmas) and III (X-disease and related phytoplasmas). Int J Syst Bacteriol 1996;46:64–75. [19] Lee I-M, Davis RE, Chen T-A, Chiykowski LN, Fletcher J, Hiruki C, et al. A genotype-based system for identification and classification of mycoplasmalike organisms (MLOs) in the aster yellows MLO strain cluster. Phytopathology 1992;82:977–86. [20] IRPCM. Report of the working team for phytoplasmas, spiroplasmas, mesoplasmas and entomoplasmas. In report of consultations of the internatioanl research program on comparative mycoplasmology (IRPCM) of the international organization for mycoplasmology (IOM). Fukuoka, Japan: IOM; 2000.