Introduction {#Sec1}
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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. [@CR4]). 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 (Chaturvedi et al. [@CR6]; Bertaccini [@CR3]). 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 [@CR10]).

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](#Tab1){ref-type="table"}). Attempts were made to confirm the phytoplasma etiology with the 17 symptomatic plants in the present study by PCR assays, phylogeny, and RFLP analysis.Table 1Survey, symptoms, locations, and PCR results for phytoplasma detection on ornamental plantsS. no.Plants and familySurvey periodLocationSymptomsPCR Results with 3Far/3Rev primer pair and Genbank Acc. No.Group and Subgroup1*Allamanda cathartica* L. (Apocynaceae)June 2016IARI, New DelhiLeaf yellowing+ve KX641019\
+ve KX64102016Sr VI-D2*Bougainvillea glabra* Choisy (Nyctaginaceae)April 2016IARI, New DelhiLeaf yellowing----3*Yucca aloifolia* L. (Asparagaceae)March 2016DFR, PuneLeaf chlorosis----4*Crossandra infundibuliformis* L. (Acanthaceae)June 2016BengaluruLeaf yellowing----5*Helichrysum italicum* Roth (Asteraceae)June 2016IARI, New DelhiYellowing and little leaf----6*Hibiscus rosa*-*sinensis* L. (Malvaceae)March 2016PuneLeaf yellowing and phyllody+ve KX64102316Sr VI-D7July 2016HaryanaLeaf yellowing----8July 2016Andhra PradeshLeaf chlorosis----9*Tagetes erecta* L. (Asteraceae)March 2016Baramati, PuneLittle leaf and witches'broom----10April 2016Andhra PradeshLittle leaf----11May 2016IARI, New DelhiStunting and phyllody----12July 2016HaryanaYellowing----13*Wrightia tinctoria* Roxb. (Apocynaceae)July 2016Andhra PradeshLeaf chlorosis----14*Saponaria officinalis* L. (Caryophyllaceae)March 2016DFR Field, PuneWitches'-broom+ve KX641021\
+ve KX64102216Sr VI-D15*Xanthostemon chrysanthus* (F.Muell.) Benth. (Myrtaceae)March 2016Andhra PradeshLeaf chlorosis and witches'broom----*IARI* Indian Agricultural research Institute, New Delhi, *DFR* Directorate of Floriculture Research, Pune

Materials and methods {#Sec2}
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Survey and symptomatology {#Sec3}
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Surveys of garden, nurseries, and experimental field/plots at New Delhi, Andhra Pradesh, Haryana, Bengaluru, and Pune was made during March to July 2016, and phytoplasma suspected symptoms were collected from 17 plants of ten different ornamental species, viz. *A. cathartica*, *B. glabra*, *Y. aloifolia*, *C. infundibuliformis*, *H. italicum*, *H. rosa*-*sinensis*, *T. erecta*, *W. tinctoria*, *S. officinalis,* and *Xanthostemon chrysanthus* (Table [1](#Tab1){ref-type="table"}).

DNA extraction {#Sec4}
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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 [@CR1]). Amplification of phytoplasma ribosomal DNA (rDNA) was performed with the universal phytoplasma primer pairs P1/P7 (Deng and Hiruki [@CR7]; Schneider et al. [@CR17]). Further nested PCR assays were performed with primer pairs 3Far/3Rev (Manimekalai et al. [@CR11]). The DNA isolated from toria phyllody phytoplasma infected *Catharanthus roseus* leaf tissue (Azadvar and Baranwal [@CR2]) 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 μl) contained 100 ng/μl 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 μl of product of the first round of PCR was used in nested PCR using internal primer pairs 3Far/3Rev (Manimekalai et al. [@CR11]). 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 {#Sec5}
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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 {#Sec6}
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The 16S rDNA sequence generated from the present study and reference phytoplasma strains sequence retrieved from GenBank were used to construct phylogeny by neighbor-joining method with 1000 replications for each bootstrap value using the MEGA 6.0 software version for '*Candidatus* Phytoplasma species' assignment (Tamura et al. [@CR20]). *Acholeplasma laidlawii* was used as out group to root the phylogenetic tree.

In silico RFLP analysis {#Sec7}
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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.

Results and discussion {#Sec8}
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Survey and symptomatology {#Sec9}
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During survey of garden nurseries and experimental field/plots in New Delhi, Andhra Pradesh, Haryana, Bengaluru, and Pune, phytoplasma suspected symptoms of little leaf, yellowing, chlorosis, phyllody, witches' broom, and stunting of plants were recorded on ten different ornamental plants species, viz. *A. cathartica*, *B. glabra*, *Y. aloifolia*, *C. infundibuliformis*, *H. italicum*, *H. rosa*-*sinensis*, *T. erecta*, *W. tinctoria*, *S. officinalis,* and *Xanthostemon chrysanthus* (Fig. [1](#Fig1){ref-type="fig"}; Table [1](#Tab1){ref-type="table"}).Fig. 1Phytoplasma suspected symptoms on different ornamental plant species **a** Witches' broom in *Saponaria officinalis* at Pune; **b** Leaf yellowing and phyllody in *Hisbiscus rosa*-*sinensis* at Pune. **c** Leaf chlorosis in *H. rosa*-*sinensis* at Andhra Pradesh. **d** Little leaf in *Tagetes erecta* at Andhra Pradesh; **e** stunting and phyllody in *Tagetes erecta* at Delhi. **f** Little and witches'broom in *Tagetes erecta* at Pune; **g** leaf chlorosis in *Wrightia tinctoria* at Rajahmundary, Andhra Pradesh. **h** Leaf yellowing in *Bougainvillea glabra* at Delhi; **i** leaf chlorosis and witches'broom in *Xanthostemon chrysanthus* at Rajahmundary, Andhra Pradesh, **j** Leaf chlorosis in *Yucca aloifolia* at Pune. **k** Leaf yellowing in *Allamanda cathartica* at Delhi. **l** Leaf yellowing in *Helichrysum italicum* at Delhi. **m** Leaf yellowing in *Crossandra infundibuliformis* at Bengaluru

Detection of phytoplasma by PCR assays {#Sec10}
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First round PCR amplification did not yield the expected 1.8 kb product of the 16S rRNA region from any of the 17 symptomatic ornamental test samples with primer pair P1/P7 (data not shown). However, 1.3 Kb amplicons were observed in nested PCR assays with 3Far/3Rev primer pair in five symptomatic plants samples of the three test ornamental species, *H. rosa*-*sinensis* (one isolate, Pune), *S. officinalis* (two isolates, Pune) and *A. cathartica* (two isolates, Haryana) and the positive control of toria phyllody phytoplasma infected *Catharanthus roseus* leaf tissue (Azadvar and Baranwal [@CR2]). No amplifications were observed in any of the rest twelve symptomatic ornamental samples and the non-symptomatic samples (Table [1](#Tab1){ref-type="table"}).

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](#Tab1){ref-type="table"}).

Sequence analysis and phylogenetic relationships {#Sec11}
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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](#Fig2){ref-type="fig"}).Fig. 2Phylogenetic tree constructed by neighbor-joining method showing the relationships among *H. rosa*-*sinensis*, *S. officinalis* and *A. cathartica*, and the reference phytoplasma strains. Accession numbers are specified in the tree. *A. laidlawii* was used as an out group

In silico RFLP analysis and phytoplasma classification {#Sec12}
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The phytoplasma sequence corresponding to the 3Far/3Rev region was subjected to in silico restriction enzyme digests and virtual gel plotting using the pDRAW32 program developed by AcaClone Software (<http://www.acaclone.com>). Comparison of restriction site maps with 17 restriction enzymes (*Alu*I, *Bam*HI, *Bfa*I, *Bst*UI (*Tha*I), *Dra*I, *Eco*RI, *Hae*III, *Hha*I, *Hin*fI, *Hpa*I, *Hpa*II, *Kpn*I, *Sau3*AI (*Mbo*I), *Mse*I, *Rsa*I, *Ssp*I, and *Taq*I) through pDraw analysis revealed that the all the three positive ornamental phytoplasma strains (*H. rosa*-*sinensis* KX641023, *A. cathartica* KX641019, and *Saponaria officinalis*, KX641021) produced a virtual RFLP profile identical to phytoplasma reference strain mollicutes sp. phytoplasma (X83431, 16Sr VI-D subgroup) (Fig. [3](#Fig3){ref-type="fig"}). Therefore, phytoplasma strains from all the three ornamental plant species in the present study were classified under 16Sr VI-D subgroup.Fig. 3Comparison of virtual RFLP patterns derived from in silico digestions of 1.3 kb 16S rDNA sequences of **a** Brinjal little leaf phytoplasma 16SrVI-D (X83431), **b** *S. officinalis* L. (KX641021), **c** *H. rosa*-*sinensis* L. (KX641023), and **d** *A. cathartica* L. (KX641019) digested using 17 different restriction endonucleases indicating that the *A. cathartica* L., *H. rosa*-*sinensis* L., and *S. officinalis* L. phytoplasma belonged to 16Sr VI-D phytoplasma subgroup

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 (Chaturvedi et al. [@CR6]; Madhupriya [@CR10]). Out of these eleven groups, the 16Sr I group is the most dominant group infecting major ornamental species in India (Chaturvedi et al. [@CR5]; Madhupriya [@CR10]). 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. [@CR8]), brinjal little leaf (Azadvar and Baranwal [@CR2]), *Withania* little leaf (Zaim and Samad [@CR21]; Samad et al. [@CR15]), *Portulaca* little leaf (Samad et al. [@CR16]), *Datura* little leaf (Raj et al. [@CR14]; Singh et al. [@CR19]), *Croton* leaf yellows (Madhupriya et al. [@CR13]), and *Calotropis gigantea* leaf yellows (Madhupriya et al. [@CR12]). However, 16SrVI-D subgroup of phytoplasma was only reported with brinjal little leaf (Azadvar and Baranwal [@CR2]) and *Catharanthus roseus* little leaf (Bertaccini [@CR3]). 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 [@CR9]).

An erratum to this article is available at <http://dx.doi.org/10.1007/s13205-016-0571-y>.

The authors are thankful to the ICAR-Extramural Research Project for providing financial assistance during the course of the study. The authors also wish to express sincere thanks to the Head, Division of Plant Pathology and the Director, Indian Agricultural Research Institute for providing laboratory facilities.

Conflict of interest {#FPar1}
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The authors declare that they have no conflict of interest.
