Molecular characterization of phytoplasmas of ‘Clover proliferation’ group associated with three ornamental plant species in India

Suspected phytoplasma symptoms of little leaf, yellowing, chlorosis, phyllody, witches’ broom, and stunting were observed on ten different ornamental plant species at New Delhi, Andhra Pradesh, Haryana, Bengaluru, and Pune, India, during March to July 2016. To investigate the possibility of phytoplasma etiology, PCR assays were performed using universal primer pairs (P1/P7 followed by 3Far/3Rev) specific to the phytoplasma 16Sr RNA gene. First round PCR amplification with primer pair P1/P7 did not yield expected 1.8 kb product of 16S rRNA region from any of the 17 symptomatic samples. However, 1.3 Kb amplicons were observed in nested PCR assays with 3Far/3Rev primer pair in symptomatic leaf samples of Hibiscus rosa-sinensis L. (Pune isolate), Saponaria officinalis L. (Pune isolate), and Allamanda cathartica L. (Delhi isolate). No amplifications were observed in any of the other tested symptomatic and non-symptomatic plant samples either in first round or second round of nested PCR assays with phytoplasma specific primer pairs. Pairwise sequence comparison of 16S rDNA sequences of the five positive phytoplasma strains of A. catharica, H. rosa-sinensis, and S. officinalis in the present study revealed 99–100% sequence identities with strains of ‘clover proliferation’ (16SrVI) group. Phylogenetic and virtual RFLP analysis of 16S rDNA sequences of the five identified phytoplasma strains belonging to three ornamental species further confirmed their clustering and grouping with member strains of ‘clover proliferation’ subgroup D. This is the first record of the phytoplasma association of ‘clover proliferation’ subgroup D with H. rosa-sinensis, S. officinalis, and A. cathartica in the world.


Introduction
Phytoplasmas, formerly known as mycoplasma-like organisms (MLOs), are cell wall less Mollicutes that colonize plant phloem sieve tube elements and insects gut wall. They are known to cause devastating losses in crops and natural ecosystems worldwide. They are transmitted from one plant to another by phloem-feeding insects, primarily leafhoppers, plant hoppers, and psyllids (Bertaccini et al. 2014). Phytoplasmas cause diseases in several commercial ornamental plants causing serious economic losses all over the world. Phytoplasma diseases are the major constraint in commercial ornamental plant production by lowering the quantum and quality gaining international importance. Phytoplasma causes different symptoms of general yellowing and stunting of plants, proliferation of shoots, phyllody, virescence, reduced size of flowers in many ornamental plants which affects their economic value Bertaccini 2015). The 'Ca. P. asteris' 16SrI group is the major group infecting ornamental species worldwide. So far, more than 60 ornamental plant species have been reported to be infected with phytoplasma worldwide (Madhupriya 2016).
During a recent survey in five states (New Delhi, Andhra Pradesh, Haryana, Bengaluru, and Pune) of India, phytoplasma suspected symptoms were observed on 17 plants belonging to ten different ornamental plant species (Table 1). Attempts were made to confirm the phytoplasma etiology with the 17 symptomatic plants in the present study by PCR assays, phylogeny, and RFLP analysis.

DNA extraction
DNA from three healthy and three symptomatic plant tissues (midrib and leaf veins) of 17 ornamental samples were extracted following a described procedure (Ahrens and Seemuller 1992). Amplification of phytoplasma ribosomal DNA (rDNA) was performed with the universal phytoplasma primer pairs P1/P7 (Deng and Hiruki 1991;Schneider et al. 1995). Further nested PCR assays were performed with primer pairs 3Far/3Rev (Manimekalai et al. 2010). The DNA isolated from toria phyllody phytoplasma infected Catharanthus roseus leaf tissue (Azadvar and Baranwal 2012) was used as positive control.
PCR reactions were carried out in a thermal cycler (Eppendorf, Germany) and the cycling protocol used for the first round PCR using P1/P7 primer pair with initial denaturation at 94°C for 5 min, followed by 35 cycles consisting of denaturation at 94°C for 45 s, annealing at 55°C for 1 min. and extension at 72°C for 2 min, with the final extension for 10 min at 72°C. Total PCR mixture (50 ll) contained 100 ng/ll of total nucleic acid, 20 pmol of 3Far/3Rev primers, 1.0 unit of Taq DNA polymerase (G-biosciences), 0.2 mM of dNTP, 2.0 mM MgCl 2, and 1X PCR buffer. Two ll of product of the first round of PCR was used in nested PCR using internal primer pairs 3Far/3Rev (Manimekalai et al. 2010). Reaction mixture and condition of nested PCR used were similar as first round PCR except for annealing at 63°C for 1 min. The PCR product was subjected to electrophoresis in a 1.0% (w/v) agarose gel, stained with ethidium bromide, and observed under UV transilluminator.

Sequencing and BLAST analysis
The *1.3 kb nested PCR products were sequenced directly in both directions using 3Far/3Rev primers. The sequences were assembled using DNA baser V.4 program and were further aligned using CLUSTAL W method of Bio-Edit software. Aligned sequences were deposited in NCBI GenBank and used as query sequence in BLASTn search analysis with related submitted sequences in GenBank.

Phylogenetic analysis
The 16S rDNA sequence generated from the present study and reference phytoplasma strains sequence retrieved from GenBank were used to construct phylogeny by neighborjoining method with 1000 replications for each bootstrap value using the MEGA 6.0 software version for 'Candidatus Phytoplasma species' assignment (Tamura et al. 2013). Acholeplasma laidlawii was used as out group to root the phylogenetic tree.

In silico RFLP analysis
The phytoplasma sequences corresponding to the 3Far/ 3Rev region was subjected to in silico RFLP analysis using pDRAW32 program developed by AcaClone Software (http://www.acaclone.com) and compared with representative sequences of the mollicutes sp. phytoplasma 16Sr VI-D (Ac. No. X83431) subgroup for assigning 16Sr subgroups to ornamental phytoplasma strains analysed by the same restriction mapping utilizing AcaClone software generated RFLP sequences.

Detection of phytoplasma by PCR assays
First round PCR amplification did not yield the expected 1.8 kb product of the 16S rRNA region from any of the 17   isolates, Haryana) and the positive control of toria phyllody phytoplasma infected Catharanthus roseus leaf tissue (Azadvar and Baranwal 2012). No amplifications were observed in any of the rest twelve symptomatic ornamental samples and the non-symptomatic samples (Table 1). Nested PCR products of five positive amplified products were directly sequenced, and the partial 16Sr RNA sequences of 1326, 1258, 1333, 1339, and 1252 bp, respectively, were deposited in the GenBank database under the Accession numbers as A. cathartica (KX641019, KX641020), H. rosa-sinensis (KX641023), and S. officinalis (KX641021, KX641022) (Table 1).

Sequence analysis and phylogenetic relationships
Pairwise sequence comparison of the partial 16S rRNA gene sequences of the five positive ornamental phytoplasma strains of H. rosa-sinensis, S. officinalis, and A. cathartica revealed 99% (H. rosa-sinensis L. Acc No. KX641023) and 100% (A. cathartica L. Acc No. KX641019, KX641020; Saponaria officinalis L., Acc No. KX641021, KX641022) sequence identity with strains of clover proliferation. Phylogenetic analysis based on 16S rDNA sequences of all the five ornamental phytoplasma strains in the present study revealed their close relationship with strains of clover proliferation group (16SrVI) (Fig. 2).
So far, eleven different groups (16SrI,16SrII,16SrIII,16SrV,16SrVI,16SrVII,16SrIX,16SrX,16SrXII,16SrXIII, and 16SrXIV) of phytoplasmas were identified in ornamental plants in the world Madhupriya 2016). Out of these eleven groups, the 16Sr I group is the most dominant group infecting major ornamental species in India (Chaturvedi et al. 2009;Madhupriya 2016). In the present study, we have reported occurrence of 16SrVI group on three ornamental plants species. In India, 16SrVI group of phytoplasmas was earlier reported to be associated with several diseases of plants, viz. Araucaria little leaf (Gupta et al. 2009), brinjal little leaf (Azadvar and Baranwal 2012), Withania little leaf (Zaim and Samad 1995;Samad et al. 2006), Portulaca little leaf (Samad et al. 2008), Datura little leaf Singh et al. 2012), Croton leaf yellows (Madhupriya et al. 2016), and Calotropis gigantea leaf yellows (Madhupriya et al. 2010). However, 16SrVI-D subgroup of phytoplasma was only reported with brinjal little leaf (Azadvar and Baranwal 2012) and Catharanthus roseus little leaf (Bertaccini 2015). Hence, in our study, all the three ornamental species are new host records of 'clover proliferation' subgroup D in the world. There is a possibility of natural transmission of the 16Sr VI-D subgroup phytoplasma from ornamental species to brinjal crops which is already reported as natural host of 16Sr VI-D subgroup phytoplasma in India (Kumar 2015).