Molecular Characterization and Phylogeny of a Phytoplasma Associated with Phyllody Disease of toria (Brassica rapa L. subsp. dichotoma (Roxb.)) in India

Abstract

Samples from toria plants (Brassica rapa L. subsp. dichotoma (Roxb.)) exhibiting phyllody, virescence, witches broom, extensive malformation of floral parts, formation of bladder like siliquae and flower sterility were collected from four different locations in India. Sequencing and phylogenetic analysis of the 16S rRNA, a part of 23S rRNA, partial sec A genes, rp gene and 16S–23S intergenic spacer region indicated that the phytoplasmas associated with toria phyllody (TP) symptoms were identical and belonged to 16SrIX phytoplasma Pigeon pea witches’-broom (PPWB) group. The iPhyClassifier generated virtual RFLP pattern of 1.25 kb 16S rDNA sequences indicated that TP phytoplasma belongs to 16SrIX-C phytoplasma subgroup. Complete 23S rRNA gene of TP phytoplasma had 2,787 nucleotides and is the first sequence of 16SrIX phytoplasma group. Restriction digestion of 16S rDNA and 23S rDNA PCR products has also shown that TP phytoplasmas from all the four locations in India were identical. Toria is a previously unreported host for a phytoplasma in16SrIX-C subgroup.

Introduction

Toria (Brassica rapa L. subsp. dichotoma (Roxb.)), an oilseed crop in India, has been affected by phyllody disease since 1958 [5, 6]. The associated phytoplasma was determined to be a member of 16SrIX Pigeon pea witches’-broom group phytoplasma on the basis of 16S rDNA sequencing [3].

Phytoplasmas have been classified according to 16S-rDNA phylogeny and RFLP profiles into 30 phylogenetic groups and 28 ‘Candidatus Phytoplasma’ species [31]. The phytoplasma strains within a species should share at least 97.5% sequence identity within the 16S rRNA gene (Firrao et al. 2005). Subsequently in silico virtual RFLP analysis has been used as a powerful tool for groups and subgroups phytoplasma identification [15, 29–31]. However, because of its highly conserved nature, the 16S rRNA gene may not be adequate for finer differentiation of closely related but distinct phytoplasmas strains. The additional markers such as secY, tuf and rp genes as well as 16S–23S intergenic spacer region genes sequencing have been used for subgroup differentiation of 16SrI phytoplasmas which were divided into 11 subgroups [7, 14, 17]. Similarly tuf gene and rp operon have been used to subdivide the members of 16SrXII phytoplasma group [24]. SecY, map and uvrB–degV genes were used to analyse strains in the 16SrV cluster [1]. As five subgroups in 16SrIX phytoplasma group have been identified [8, 19, 31], it was important to identify the phytoplasma subgroup associated with toria phyllody (TP) disease. Therefore, in silico virtual RFLP and phylogenetic analysis of 16S rRNA gene, actual RFLP of 16S rRNA and 23S rRNA genes, sequencing and phylogenetic analysis of ribosomal protein gene, 16S–23S rRNA intergenic spacer region, 23S rRNA and secA genes were attempted for characterization and phylogeny of a phytoplasma associated with TP disease using samples from four different locations in India.

Materials and Methods

Collection of Phyllody Samples

During 2009, plant samples of toria affected with phyllody were collected at the time of flowering from four major toria growing states of India viz., Delhi (Pusa campus, IARI, New Delhi), Uttar Pradesh (NRC for cropping system, Meerut), Punjab (Punjab Agricultural University, Ludhiana) and Rajasthan (Directorate of Rapeseed and Mustard, Bharatpur). Leaf midribs of symptomatic toria plants were used freshly or stored at −80°C for further study. Toria plants grown from the seeds in the glasshouse were used as healthy control.

Isolation of Total DNA and PCR Amplification

Total genomic DNA was isolated from 100 mg of leaf midrib tissues using DNeasy plant mini kit (Qiagen Gmbh, Hilden, Germany) according to the manufacturer’s instruction. Five microliters of DNA was evaluated for phytoplasma DNA by a PCR assay employing universal phytoplasma primer pair P1/P7 [23]. For further confirmation, resulting amplification products were diluted to 1:30 with sterile distilled water. Two microliters were used for nested PCR with primer pair R16F2n/R16R2 [9]. The thermal conditions were as follows: denaturation at 94°C for 40 s (5 min for the first cycle), annealing at 55°C for 1 min and extension at 72°C for 2 min. The last cycle was extended for an additional 10 min at 72°C. Annealing temperature for nested PCR was 56°C. Subsequently a modified R16F2n forward primer (m(IX)R16F2n) was designed on the basis of sequence result of 16S rDNA product for better amplification of TP phytoplasma.

Amplification of 23S rRNA gene was performed using P23S5F3/A23S3R3 (5′-GTGGATGCCTTGGCACTAAGAGCC-3′/5′-ACTTACACACCTGGCCTATCAACC-3′) primer pair as described by Guo et al. [10]. For amplification of secA gene, SecAfor1/SecArev3 (5′-GARATGAAAACTGGRGAAGG-3′/5′-GTTTTRGCAGTTCCTGTCATNCC-3′) phytoplasma universal primers were used [12]. PCR was also performed using 16SrIX group specific primer pair rp(IX)F2/rp(IX)R2 (5′-GCACAAGCTATTTTAATGTTTACACC-3′/5′-CAAAGGGACTAAACCTAAAG-3′) for amplification of a part of ribosomal protein (rp) gene. The thermal conditions were as follows: denaturation at 94°C for 1 min (5 min for the first cycle), annealing at 60°C for 2 min and extension at 72°C for 3.5 min. The last cycle was extended for an additional 10 min at 72°C [19].

All the PCR amplifications were carried out in a thermal cycler (Mycycler, Bio-Rad, USA) and the resulting PCR products were viewed in gel documentation unit (XR documentation system, Bio-Rad, USA).

Restriction Digestion of PCR Products

The PCR products of 16S rDNA (using R16F2n/R16R2 primer pair) and 23S rDNA (using P23S5F3/A23S3R3 primer pair) were purified by QIAquick PCR purification kit (Qiagen Gmbh, Hilden, Germany) according to the manufacturer’s instruction. A total volume of 7 μl of PCR products were digested with restriction endonucleases AluI, HaeIII, HhaI, HinfI, MseI and RsaI (New England BioLabs, Waverley, MA, USA) at 37°C for 3 h. Resultant restriction fragments were visualized by electrophoresis through 2.8% agarose gel with Tris–EDTA as running buffer. DNA fragment profiles in gels were visualized and recorded in gel documentation unit (XR documentation system, Bio-Rad, USA).

Cloning of PCR Products and DNA Sequencing

PCR products obtained by use of different sets of primers were purified by QIAquick PCR purification kit (Qiagen Gmbh, Hilden, Germany), as per manufacturer’s instructions and cloned in pGEM-T Easy vector (Promega, USA) and transformed into Escherichia coli DH5-α cells (Invitrogen Life Technologies, Carlsbad, CA, USA). Recombinants were screened by blue and white screening method as per standard protocol [22]. Sequencing of 2–3 representative clones was performed with automated sequencing facility at Chromos Biotech (Bangalore, India). In addition purified PCR products were directly used for sequencing.

Phylogenetic Analysis

Sequences of PCR products and cloned DNA of PCR products were assembled using BioEdit software [11]. A database search of homologous sequences was performed by BLAST analysis at NCBI (http://ncbi.nlm.nih.gov/BLAST). 16S rRNA, 23S rRNA, rp and secA gene sequences were aligned with phytoplasma group/subgroup representatives available in GenBank using Clustal W [26]. Phylogenetic relationship of four isolates of TP phytoplasma with other group representative phytoplasma sequences available in GenBank were assessed based on sequences of 16S rRNA gene between primers R16F2n and R16R2, secA gene between primers SecAfor2 and SecArev3 [12], 23S rRNA gene between primers P7 and 23Srev [12], rp gene between primers rp(IX)F2 and rp(IX)R2 [19] and 16S–23S rRNA intergenic spacer region between primers P3 and P7 [12]. Sequence alignments were performed using CLUSTAL W [26]. The phylogenetic distance tree and molecular evolutionary analyses were performed with MEGA 4.0 software [25] using the maximum parsimony method with default values and 1,000 replications for bootstrap analysis. Acholeplasma palmae was used as out groups to root the trees.

In Silico Restriction Enzyme Digestion of 16S rDNA and Virtual Gel Plotting

1.25 kb of 16S rDNA sequences (R16F2n/R16R2 primer primed) derived from all the four TP phytoplasma strains from different locations of India were submitted to iPhyClassifier online tool (http://www.plantpathology.ba.ars.usda.gov/cgibin/resource/iphyclassifier.cgi) as described by Zhao et al. [30]. In silico restriction analysis and virtual gel plotting with restriction endonucleases AluI, BamHI, BfaI, BstUI (ThaI), DraI, EcoRI, HaeIII, HhaI, HinfI, HpaI, HpaII, KpnI, Sau3AI (MboI), MseI, RsaI, SspI and TaqI were obtained and compared with the virtual RFLP gel from 16SrIX phytoplasma subgroups by the same restriction enzymes [30].

Results

The major symptoms of phytoplasma diseases of toria consisted of flower sterility, virescence, phyllody, formation of hollow bladders and pod malformation (Fig. 1). An approximately 2.7 kb PCR product was obtained from four TP phytoplasma isolates using P23S5F3/A23S3R3 primer pair. No amplification obtained in healthy toria samples (Fig. 2).

Fig. 1.

Symptoms of phyllody disease on toria (Brassica rapa L. subsp. dichotoma (Roxb.)); a healthy plant, b flower sterility, phyllody, virescence, bladder-like pod and pod malformation on infected plant

Fig. 2.

Agarose gel electrophoresis of PCR products amplified from phytoplasma infected toria with Primer pair P23S5F3/A23S3R3; H, healthy toria; TPN, toria phyllody isolate New Delhi; TPM, toria phyllody isolate Meerut; TPB, toria phyllody isolate Bharatpur; TPL, toria phyllody isolate Ludhiana; M, 1 kb DNA ladder

Actual RFLP Analysis of 16S rDNA and 23S rDNA PCR Products

Nested PCR products of 16S rDNA using P1/P7 followed by R16F2n/R16R2 primers of TP phytoplasma isolates from New Delhi, Meerut, Bharatpur and Ludhiana digested with AluI, HaeIII, HhaI, HinfI, MseI and RsaI endonuclease were completely identical (Fig. 3). Similarly PCR products of 23S rDNA of these isolates by P23SF3/A23SR3 primers produced the identical RFLP patterns. No differences in restriction fragment profiles were evident among all the four TP isolates indicating that they all contained the same phytoplasma (Fig. 4).

Fig. 3.

Actual RFLP analyses of 1.25 kb of 16S rDNA nested-PCR products of TP phytoplasma (amplified using primer pair R16F2n/R16R2) digested with HinfI, HaeIII, RsaI, AluI, HhaI and MseI restriction enzymes; TPN, toria phyllody strain New Delhi; TPM, toria phyllody strain Meerut; TPB, toria phyllody strain Bharatpur; TPL, toria phyllody strain Ludhiana; M, phi X174/HaeIII marker (1353, 1078, 872, 603, 310, 281, 271, 234, 194, 118, 72)

Fig. 4.

Actual RFLP analyses of 2.7 kb of 23S rDNA PCR products of TP phytoplasma (amplified using primer pair P23S5F3/A23S3R3) digested with HinfI, HaeIII, RsaI, AluI, HhaI and MseI restriction enzymes; TPN, toria phyllody strain New Delhi; TPM, toria phyllody strain Meerut; TPB, toria phyllody strain Bharatpur; TPL, toria phyllody strain Ludhiana; M, phi X174/HaeIII marker (1353, 1078, 872, 603, 310, 281, 271, 234, 194, 118, 72)

Sequence Analysis

16S–23S rDNA sequences of all the four TP phytoplasma isolates of India collected from New Delhi (GenBank Acc. GU11154), Meerut (HM559247), Bharatpur (HM559246) and Ludhiana (HM559245) were completely identical and had 1,845 nt. Comparison of 16S–23S rDNA sequences of four TP phytoplasma Indian isolates to other phytoplasma sequences revealed that the phytoplasma associated with TP had maximum identity of 99% to 16S rRNA gene sequence of Khafr (Iran) almond witches’-broom (DQ195209) and Pigeon pea witches’-broom (AF248957) and 98% to Juniperus occidentalis witches’-broom (GQ925918) phytoplasmas all belonging to 16SrIX phytoplasma group.

Comparison of 1.25 kb of F2n/R2 primed sequence of 16S rDNA of four TP phytoplasma isolates to other phytoplasma sequences reported in GenBank using the BLAST tool revealed that the phytoplasma associated with toria phyllody had maximum identity of 99% to Knautia arvensis phytoplasma, KAP strain (EF186823), Khafr (Iran) almond witches’ broom (DQ195209) and some other phytoplasmas in 16SrIX group.

Comparison of 838 nucleotide sequences of TP phytoplasma obtained by SecAfor1/SecArev3 primers to other phytoplasma sequences revealed that the phytoplasma associated with TP had maximum identity of 80% to the secA gene sequence of Jujube witches’-broom phytoplasma (GU471770) of 16SrV group. However, when 483 bp sequences of secA gene of TP phytoplasma (primed SecAfor2/SecArev3 primer pair) of all the four TP phytoplasma isolates (HM559249, HM559250, HM559251 and HM559252) were compared, they showed maximum identity of 93% to secA gene sequence of Pigeon pea witches’-broom phytoplasma (EU168746) of 16SrIX group.

Sequences of complete 23S rRNA gene of all the four TP phytoplasma isolates (HM559253, HM559254, HM559255) and HM559256) were also identical and had 2,787 nucleotides. Blast analysis of 23S rRNA gene sequence of TP phytoplasma isolates to other phytoplasma sequences revealed that the TP phytoplasma had maximum identity of 91% with Jujube witches’-broom phytoplasma (GU723425) and Loofah witches’-broom phytoplasma (AF086621) of 16SrV group.

Comparison of 808 bp ribosomal protein (rp) gene sequences of all the four TP phytoplasma isolates (HM559257, HM559258, HM559269 and HM559260) to other phytoplasma sequences revealed that the phytoplasma associated with TP had maximum identity of 99% to the rp gene sequence of K. arvensis phytoplasma, KAP strain (EF186801) which belongs to 16SrIX-C subgroup.

In Silico Analysis of 16S rDNA Sequences

Analysis of R16F2n/R16R2 primed 16S rDNA sequences of TP phytoplasma isolates using iPhyClassifier online tool [30] showed that all TP phytoplasma isolates collected from four different locations in India shared 99.12% identity with 16S rDNA sequence of the ‘Ca. Phytoplasma phoenicium’ reference strain (AF515636) which indicates TP phytoplasma is a ‘Ca. Phytoplasma phoenicium’-related strain.

The virtual RFLP pattern derived from in silico analysis of R16F2n/R16R2 primed sequence of 16S rRNA of all the four Indian TP phytoplasma isolates using iPhyClassifier tool with 17 selected restriction enzymes showed that all four TP phytoplasma isolates had identical RFLP pattern with a similarity coefficient of 0.98 to the reference pattern of 16Sr group IX, subgroup C (Y16389) indicating phytoplasma associated with TP disease in India is a member of 16SrIX-C subgroup in the phytoplasma group classification scheme. Among the 17 restriction enzymes, AluI, DraI, HaeIII, HhaI, HinfI, RsaI and TaqI were differential restriction enzymes for subgroup identification in pigeon pea witches’-broom phytoplasma group and all of the 16SrIX group produced the same RFLP patterns when rest of the restriction enzymes were used (data not shown).

Phylogenetic Analysis

Phylogenetic analysis of 16S rDNA sequences showed that all the four TP phytoplasma isolates from India grouped together with 16SrIX-C phytoplasma subgroup, K. arvensis phytoplasma KAP strain (EF186823) and they in turn grouped with 16SrIX subgroups E (GQ925919), A (AF248957), D (AF515636) and B (AF455041) of 16SrIX group phytoplasma. All subgroups in the group 16SrIX clustered with Cassia witches’-broom (EF666054) of 16SrXXIX group (Fig. 5).

Fig. 5.

Dendrogram constructed by the maximum parsimony method showing the phylogenetic relationships for 16S rDNA sequences (between primers R16F2n and R16R2) for TP phytoplasma isolates and representatives from other 16Sr groups. GenBank accession numbers are shown in parentheses. Bootstrap values greater than 70% (expressed as percentages of 1,000 replications) are shown

Discussion

Toria phyllody has been recorded in India since 1958 and causes reduced yield and oil content in TP infected plants by as much as 79 and 28% respectively [13]. The incidence of infection by phytoplasmas based on symptoms expressed in different accessions of toria was variable and reached 11% in toria field in New Delhi over a period of 2 years [2]. The major symptoms of phytoplasma diseases of toria consisted of virescence, phyllody and formation of hollow bladders but not stunting, leaf yellowing or purpling as reported for phytoplasma infected oilseed brassicas in other countries [4, 16, 20] indicating that the phytoplasmas associated with toria phyllody might be distinct from phytoplasmas infecting brassicas in other countries. Comparison of 16S rRNA gene sequence of toria phyllody showed that the toria phyllody phytoplasma shared maximum identity of 99% to that of K. arvensis phyllody (Y18052) from Italy and Khafr (Iran) almond witches’-broom phytoplasma (DQ195209) from Iran. It showed 98% identity with Pigeon pea witches’-broom phytoplasma (AF248957) from the USA and ‘Ca. Phytoplasma phoenicium’ (AF515636) from Lebanon and Iran [27]. All these phytoplasmas belong to 16SrIX phytoplasma group. Phytoplasma associated with oilseed brassicas in Canada, Czech Republic, Italy and more recently in Greece on the other hand belonged to 16SrI group [4, 16, 20, 28]. Further study involving in silico analysis and virtual RFLP of 16S rDNA sequence including the application of iPhyClassifier indicated that all the four Indian isolates of TP phytoplasma were identical and belonged to 16SrIX-C phytoplasma subgroup and this was further supported by phylogenetic analyses of 16S rRNA, rp genes and 16S–23S rRNA intergenic region spacer sequences. Association of 16SrIX-C subgroup of phytoplasma is the first record with plants of Brassicaceae family. There are only two other phytoplasma in this sub group viz. phytoplasma associated with K. arvensis phyllody in Italy [18] and almond witches’-broom in Iran [21].

The 23S rRNA gene has not been sequenced completely for most of the phytoplasmas [10, 12]. Our study of complete 23S rRNA gene sequence from TP phytoplasma showed that it has maximum identity with Jujube witches’-broom phytoplasmas of 16SrV group. Whether rRNA gene, encoding 23S can be used for finer differentiation of phytoplasmas in a group or not will be known when more sequences of phytoplasma in a group become available. There was only one sequence of secA gene in 16SrIX group i.e. pigeon pea witches’-broom phytoplasma and TP phytoplasma had clustered with it but more sequences of secA gene would be required for its application for further sub grouping.