New Disease Reports
New Disease Reports (2012) 26, 14. [http://dx.doi.org/10.5197/j.2044-0588.2012.026.014]
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First report of a 16SrII-D phytoplasma 'Candidatus Phytoplasma australasia' associated with a tomato disease in India

Jitender Singh 1*, Anchal Rani 1, Pankaj Kumar 1, V.K. Baranwal 2, P.L. Saroj 3 and Anil Sirohi 1

*jeets_80@rediffmail.com

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Received: 05 Jun 2012; Published: 19 Sep 2012

Keywords: ‘Candidatus Phytoplasma aurantifolia’, iPhyClassifier

 

Tomato (Solanum lycopersicum) is an important cash crop in India. Recently, symptoms of leaf yellowing and curling, little leaf, severe stunting and phyllody (Fig. 1) were observed in tomato fields in the Meerut district of western Uttar Pradesh with incidence of 5-8%. To investigate whether or not a phytoplasma is associated with the disease, eight tomato plants with indicative symptoms and eight without were collected from two fields belonging to Samaspur-Surani village, Meerut.

Total DNA was extracted from midribs (DNeasy Plant Mini Kit, Qiagen, Germany) and used as template in a nested PCR assay to amplify the phytoplasma 16S rRNA gene using universal phytoplasma primers P1/P7 (Deng & Hiruki, 1991) and R16F2/R16R2 (Gundersen & Lee, 1996). Bands of expected size (1.8 and 1.26 kb, respectively) were produced in all samples from symptom-bearing plants, but not for the symptomless ones (Fig. 2). Three PCR products amplified with P1/P7 samples were selected and purified (QIAquick Gel Extraction kit, Qiagen, Germany) and directly sequenced. The resultant 16S rDNA sequences showed 100% of sequence identity with each other, and the consensus sequence was deposited in GenBank (Accession No. JX104335). BLAST analysis showed the highest sequence identity (100%) with that of members of group 16SrII ‘Candidatus Phytoplasma aurantifolia’ including the Papaya yellow crinkle phytoplasma strain Y10097 designated as ‘Ca. Phytoplasma australasia’ that belongs to subgroup 16SrII-D. Phylogenetic analysis supported BLAST comparisons since the Meerut tomato phytoplasma (Tomato-TMT) clustered in a single distinct branch that embraces ‘Ca. Phytoplasma australasia’ (Fig. 3). iPhyClassifier (Zhao et al., 2009) was used to perform sequence similarity and generate virtual restriction fragment length polymorphism (RFLP) profiles. The 16S rDNA sequence of Tomato-TMT isolates exhibited RFLP profiles identical to those of the reference strain Y10097, ‘Ca. P. australasia’, subgroup 16SrII-D (Fig. 4). To our knowledge, this is the first report of a 16SrII-D phytoplasma in India affecting the tomato crop. The results have significant phytosanitary impact for the epidemiology of 16SrII phytoplasma diseases in the tomato industry of Uttar Pradesh, India.

 

Figure1+
Figure 1: Symptoms of phytoplasma infection on tomato in Samaspur-Surani village, Meerut, Uttar Pradesh, India. Symptoms include abnormal proliferation of shoots, reduced size of leaves, shortened internodes, phylllody and purpling of leaves and flowers.
Figure 1: Symptoms of phytoplasma infection on tomato in Samaspur-Surani village, Meerut, Uttar Pradesh, India. Symptoms include abnormal proliferation of shoots, reduced size of leaves, shortened internodes, phylllody and purpling of leaves and flowers.
Figure2+
Figure 2: PCR amplification of phytoplasma 16S rDNA fragment with PCR using DNA template from Tomato-TMT with P1/P7 and R16F2/R2 primers. Lane M: 1kb ladder; lanes 1 & 2: 1.8 kb amplified fragment by P1/P7; lane 3: negative control; lanes 4 & 5: 1.2 kb amplified fragment by R16F2/R2.
Figure 2: PCR amplification of phytoplasma 16S rDNA fragment with PCR using DNA template from Tomato-TMT with P1/P7 and R16F2/R2 primers. Lane M: 1kb ladder; lanes 1 & 2: 1.8 kb amplified fragment by P1/P7; lane 3: negative control; lanes 4 & 5: 1.2 kb amplified fragment by R16F2/R2.
Figure3+
Figure 3: Phylogenetic tree based on the 16S rDNA sequences of Tomato-TMT (Accession No. JX104335) with other selected phytoplasmas from GenBank, including strain Y10097, subgroup 16SrII-D. The 0.01 bar indicates one nucleotide change per 100 nucleotides. ‘Ca. P’ stands for 'Candidatus Phytoplasma sp.’; P stands for ‘Phytoplasma’; A laidlawii stands for Acholeplasma laidlawii as the outgroup.
Figure 3: Phylogenetic tree based on the 16S rDNA sequences of Tomato-TMT (Accession No. JX104335) with other selected phytoplasmas from GenBank, including strain Y10097, subgroup 16SrII-D. The 0.01 bar indicates one nucleotide change per 100 nucleotides. ‘Ca. P’ stands for 'Candidatus Phytoplasma sp.’; P stands for ‘Phytoplasma’; A laidlawii stands for Acholeplasma laidlawii as the outgroup.
Figure4+
Figure 4: Virtual RFLP patterns derived from in silico digestions of 1.2 kb 16S rDNA fragments (tomato-TMT, JX104335) with 17 restriction endonucleases. RFLP fragments were resolved by in silico electrophoresis through 3% agarose gel. MW, ΦX174 DNA-HaeIII digestion.
Figure 4: Virtual RFLP patterns derived from in silico digestions of 1.2 kb 16S rDNA fragments (tomato-TMT, JX104335) with 17 restriction endonucleases. RFLP fragments were resolved by in silico electrophoresis through 3% agarose gel. MW, ΦX174 DNA-HaeIII digestion.

Acknowledgements

This work was supported by Centre of Excellence in Agriculture Biotechnology, Council of Science & Technology, Uttar Pradesh and Bioinformatics Infrastructure Facility funded by DBT, Government of India.

References

  1. Deng S, Hiruki C, 1991. Amplification of 16 S rRNA genes from culturable and non-culturable mollicutes. Journal of Microbiological Methods 14, 53–61. [http://dx.doi.org/10.1016/0167-7012(91)90007-D]
  2. Gundersen DE, Lee IM, 1996. Ultrasensitive detection of phytoplasmas by nested-PCR assays using two universal primer sets. Phytopathologia Mediterranea 35, 144-151.
  3. Zhao Y, Wei W, Lee IM, Shao J, Suo X, Davis RE, 2009. Construction of an interactive online phytoplasma classification tool, iPhyClassifier, and its application in analysis of the peach X-disease phytoplasma group (16SrIII). International Journal of Systematic and Evolutionary Microbiology 59, 2582-2593. [http://dx.doi.org/10.1099/ijs.0.010249-0]
To cite this report: Singh J, Rani A, Kumar P, Baranwal VK, Saroj PL, Sirohi A, 2012. First report of a 16SrII-D phytoplasma 'Candidatus Phytoplasma australasia' associated with a tomato disease in India. New Disease Reports 26, 14. [http://dx.doi.org/10.5197/j.2044-0588.2012.026.014]

©2012 The Authors