New Disease Reports (2009) 19, 17.

First report of Candidatus Phytoplasma asteris associated with witches broom of Crotalaria tetragona in India

P. Baiswar 1*, Y. Arocha 2, S. Chandra 1 and S.V. Ngachan 1

*pbaiswar@yahoo.com

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Accepted: 20 Mar 2009

Crotalaria tetragona is mainly used as a green manure in Northeast India, since farmers in this region are averse to using chemicals. It is also used in contour hedgerow farming systems in the jhum fields (Ya and Tulachan, 2003). During field visits to Barapani, Meghalaya (Northeast India) in June-July, 2008, typical witches’ broomsymptoms were observed in almost 46% of C. tetragona plants (Fig. 1).

Total DNA was extracted from leaf samples of three randomly collected plants with symptoms and two symptomless plants. Nested PCR with the 16S rDNA phytoplasma universal primers R16F2m/R1-fU5/rU3 was carried out that yielded PCR amplicons in all tested plants with symptoms. No such products were obtained from symptomless plants. PCR products were purified (QIAquick gel extraction kit, USA), cloned (pGEM-T Easy vector, Promega) and sequenced in both directions using M13 forward and reverse sequencing primers (www.dnaseq.co.uk). The 16S rDNA sequences from the Crotalaria phytoplasma isolates showed 100% similarity with each other, and a representative was submitted to GenBank (Accession No. FJ185141). BLAST search analyses revealed that the Crotalaria phytoplasma sequence scored 99% similarity with members of group 16SrI, ‘Candidatus Phytoplasma asteris’. Phylogeny analyses (MEGA version 3.1) using reference phytoplasmas from GenBank confirmed sequencing results and placed the Indian Crotalaria phytoplasma in this group (Fig 2).

Crotalaria spp. have been recognized as a plant host for phytoplasmas from group 16SrII, ‘Ca. Phytoplasma aurantifolia’ in Asian countries, including India (IRPCM, 2004). The 16SrI ‘Ca. Phytoplasma asteris’ group has been reported from a number of plant hosts in India, including sesame (Khan et al., 2007), Cannabis sativa (Raj et al., 2008) and sandal (Khan et al., 2008). To our knowledge, this is the first record of a 16SrI ‘Ca. Phytoplasma asteris’ isolate in Crotalaria spp.

Figure1+
Figure 1: (A) Healthy plants of C. tetragona. (B) Symptoms of (witches’ broom) in twigs of C. tetragona.
Figure 1: (A) Healthy plants of C. tetragona. (B) Symptoms of (witches’ broom) in twigs of C. tetragona.
Figure2+
Figure 2: Phylogenetic tree showing relationships between the Indian Crotalaria phytoplasma (Accession No. FJ185141) and reference phytoplasmas from GenBank.
Figure 2: Phylogenetic tree showing relationships between the Indian Crotalaria phytoplasma (Accession No. FJ185141) and reference phytoplasmas from GenBank.

Acknowledgements

Work in the UK was done under Plant Health Licence No. PHL 199B/5745(11/2007).


References

  1. The IRPCM Phytoplasma/Spiroplasma Working Team – Phytoplasma taxonomy group, 2004.Candidatus Phytoplasma, a taxon for the wall-less, non-helical prokaryotes that colonize plant phloem and insects. International Journal of Systematic and Evolutionary Microbiology54, 1243-1255.
  2. Khan MS, Raj SK, Snehi SK, 2007. First report of 'Candidatus Phytoplasma asteris' affecting sesame cultivation in India. Journal of Plant Pathology 89, 301-5.
  3. Khan MS, Singh SK, Ahmad J, 2008. Characterization and phylogeny of a phytoplasma inducing sandal spike disease in sandal (Santhalum album). Annals of Applied Biology 153, 365-372.
  4. Raj SK, Snehi SK, Khan MS, Kumar S, 2008. 'Candidatus Phytoplasma asteris' (group 16SrI) associated with a witches' broom disease of Cannabis sativa in India. Plant Pathology 57, 1173.
  5. Ya T, Tulachan PM, 2003. Contour hedgerow farming in Northeast India, Mountain agriculture in the Hindu Kush Himalayan region. In: Ya T, Tulachan PM eds. Proceedings of an International Symposium on Integrated Mountain Development, 2001. Kathmandu, Nepal: International Centre for Integrated Mountain Development, 85-90.

This report was formally published in Plant Pathology

©2009 The Authors