Advertisement

3 Biotech

, 9:420 | Cite as

Multilocus gene-specific characterization of ‘Candidatus Phytoplasma australasia’ associated with shoot proliferation disease of small cardamom in India

  • Swarup Mishra
  • Surabhi Mitra
  • N. S. Radhika
  • Amit Yadav
  • G. P. RaoEmail author
Original Article
  • 23 Downloads

Abstract

Symptoms of excessive shoot proliferation were observed in the Njallani cultivar of small cardamom accompanied by stunting of stalks with fewer degenerated capsules at Nedumkandam Panchayat of Idukki district of Kerala in 2017. Five symptomatic Elettaria cardamomum shoot proliferation (ECSP) plant samples were collected and processed for DNA extraction and PCR assays utilizing universal phytoplasma 16S ribosomal-specific primers pair, P1/P7 followed by R16F2n/R16R2. Sequence comparison analysis of the R16F2n/R16R2 region of 16SrRNA gene showed 100% sequence identity with the ‘Candidatus Phytoplasma australasia’- related strain. Phylogeny and virtual RFLP analyses of 16S rRNA gene sequences confirmed the association of ‘Ca. P. australasia’ strain subgroup D with ECSP disease. The association of 16SrII group was further established and validated by amplifying phytoplasma-specific multilocus candidate genes by utilizing specific primers of secA, secY, SAP11, and tuf genes. The multilocus gene sequence comparison analysis again confirmed the association of ‘Ca. P. australasia’ with the ECSP phytoplasma isolate. This is the first report of phytoplasma association with small cardamom.

Keywords

Candidatus Phytoplasma australasia’ Elettaria cardamomum shoot proliferation (ECSP) 16SrII-D subgroup 

Notes

Acknowledgements

The authors extend their gratitude to the Indian Council of Agricultural Research, New Delhi, India, for providing financial assistance during the course of the study. The authors wish to express their sincere thanks to the Head, Division of Plant Pathology and the Directors of Indian Agricultural Research Institute and National Centre for Microbial Resource, Pune for providing laboratory facilities.

Author contributions

SM did the analytical work of processing samples for DNA extraction, PCR assays and sequence submission. SM did the analysis of sequences for the identification of phytoplasma strain on small cardamom. NSR helped in survey and collection of samples and recordi9nf incidence of the disease. AY helped in sequencing of phytoplasma isolates. GPR helped in preparing and editingthe ms and analyzing sequence data.

Compliance with ethical standards

Conflict of interest

No conflict of interest.

GenBank submission

All the 16Sr, secA, secY, tuf and SAP11 gene sequences have been submitted in GenBank and appeared in public database.

Supplementary material

13205_2019_1944_MOESM1_ESM.jpg (201 kb)
Supplementary Fig. 1 Agarose gel electrophoresis of PCR assay results from small cardamom plants with primer pairs (a) R16F2n/R16R2; M: ladder; N: negative; P: Chickpea phyllody phytoplasma; Lane 1-5: symptomatic ECSP isolates; Lane 6: negative; (b) SecAfor5/SecARev2; M: ladder; N: negative; P: Chickpea phyllody phytoplasma; Lane 1-2: symptomatic ECSP isolates; (c) SecYF2 (II)/SecYR1 (II); M: ladder; P: Chickpea phyllody phytoplasma; Lane 1- 2: symptomatic ECSP isolates; N: negative; (h) TUF-II-F2/ TUF-II-R1; M: ladder; Lanes 1-2: symptomatic ECSP isolates; N: negative control; P: Chickpea phyllody phytoplasma; (e) SAP11(II)DF2/ SAP11(II)DR2; M: ladder; P: Chickpea phyllody phytoplasma; Lanes 1-2: symptomatic ECSP isolates; N: negative (JPEG 202 kb)
13205_2019_1944_MOESM2_ESM.jpg (375 kb)
Supplementary Fig. 2 Phylogenetic tree constructed by neighbor-joining method of (a) secA, (b) secY, (c) tuf and (d) SAP11 gene sequences from 16SrII-D subgroup reference phytoplasma strains and isolates of Elettaria cardamomum shoot proliferation (ECSP) phytoplasma isolates (black triangles). Accession numbers are specified in the tree. Numbers on branches are bootstrap values obtained for 1000 bootstrap replicates. The bar represents a phylogenetic distance of 0.050, 0.20, 0.50 and 0.50 respectively (JPEG 375 kb)

References

  1. Adkar-Purushothama CR, Casati P, Quaglino F, Durante G, Bianco PA (2009) First report of a ‘Candidatus Phytoplasma asteris’-related strain associated with a yellows disease of black pepper (Piper nigrum) in India. Plant Pathol 58:789CrossRefGoogle Scholar
  2. Ahrens U, Seemüller E (1992) Detection of DNA of plant pathogenic mycoplasma like organisms by a polymerase chain reaction that amplifies a sequence of the 16S rRNA gene. Phytopathology 82:828–832CrossRefGoogle Scholar
  3. Al-Subhi AM, Hogenhout SA, Al-Yahyai RA, Al-Sadi AM (2018) Detection, identification, and molecular characterization of the 16SrII-D phytoplasmas infecting vegetable and field crops in Oman. Plant Dis 102:576–588CrossRefGoogle Scholar
  4. Anonymous (2018) Spices Board, Ministry of Commerce and Industry, Government of IndiaGoogle Scholar
  5. Arocha Y, Piñol B, Picornell B, Almeida R, Jones P, Boa E (2006) Basil little leaf: a new disease associated with a phytoplasma of the 16SrI (Aster Yellows) group in Cuba. Plant Pathol 55:822CrossRefGoogle Scholar
  6. Bhai RS, Thomas J (2003) Diseases of cardamom (fungal, bacterial and nematode diseases). In: Ravindran PN, Madhusoodanan KJ (eds) cardamom. CRC Press, Florida, pp 176–195Google Scholar
  7. Bhat AI, Madhubala R, Hareesh PS, Anandaraj M (2006) Detection and characterization of the phytoplasma associated with a phyllody disease of black pepper (Piper nigrum L.) in India. Sci Hortic 107:200–204CrossRefGoogle Scholar
  8. Bhat AI, Jiby MV, Anandaraj M, Bhadramurthy V, Patel KD, Patel NR, Agalodia AV (2008) Occurrence and partial characterization of a phytoplasma associated with phyllody disease of fennel (Foeniculum vulgare Mill.) in India. J Phytopathol 156:758–761CrossRefGoogle Scholar
  9. Choueiri E, Salar P, Jreijiri F, Zammar El, Massaad S, Abdul-Nour H, Foissac X (2007) Occurrence and distribution of ‘Candidatus Phytoplasmatrifolii’ associated with diseases of solanaceous crops in Lebanon. Eur J Plant Pathol 118:411–416CrossRefGoogle Scholar
  10. Deng S, Hiruki C (1991) Amplification of 16S rRNA genes from culturable and nonculturable mollicutes. J Microbiol Methods 14:53–61CrossRefGoogle Scholar
  11. Galdeano E, Guzmán FA, Fernández F, Conci LR (2013) Genetic diversity of 16SrIII group phytoplasmas in Argentina. Predominance of subgroups 16SrIII-J and B and two new subgroups 16SrIII-W and X. Eur J Plant Pathol 137:753–764CrossRefGoogle Scholar
  12. Gundersen DE, Lee IM (1996) Ultrasensitive detection of phytoplasmas by nested-PCR assays using two universal primer pairs. Phytopathol Mediterrenea 35:144–151Google Scholar
  13. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98Google Scholar
  14. Hodgetts J, Boonham N, Mumford R, Harrison N, Dickinson M (2008) Phytoplasma phylogenetics based on analysis of secA and 23S rRNA gene sequences for improved resolution of candidate species of ‘Candidatus Phytoplasma’. Int J Syst Evol Microbiol 58:1826–1837CrossRefGoogle Scholar
  15. Khan MS, Raj SK (2006) First report of molecular detection of an Aster yellows phytoplasma isolate infecting chilli (Capsicum annuum) in India. Plant Pathol 55:822CrossRefGoogle Scholar
  16. Kumar S, Stecher G, Tamura K (2016) MEGA 7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874CrossRefGoogle Scholar
  17. Lebsky V, Hernandez-Gonzalez J, Arguello-Astorga G, Cardenas-Conejo Y, Poghosyan A, Bertaccini A, Maini S (2011) Detection of phytoplasmas in mixed infection with begomoviruses: a case study of tomato and pepper in Mexico. Bull Insectol 64:55–56Google Scholar
  18. Lee IM, Bottner-Parker Zhao K D, Davis RE, Harrison NA (2010) Phylogenetic analysis and delineation of phytoplasmas based on secY gene sequences. Int J Syst Evol Microbiol 60:2887–2897CrossRefGoogle Scholar
  19. Randall JJ, Bosland PW, Hanson SF (2009) Brotegrande, a new phytoplasma-associated disease of Chile peppers. Plant Dis 93:968CrossRefGoogle Scholar
  20. Rao GP, Reddy MG, Mishra S, Panda P (2018) First report of ‘Candidatus Phytopalsma asteris’ subgroup 16SrI-B association with a witches’ broom disease of fennel. Phytopathogenic Mollicutes 8:102–105CrossRefGoogle Scholar
  21. Siddiqui MA, Akram M, Mohiddin FA, Haque Z (2019) Bio-intensive approaches for management of pests and diseases in small cardamom and black pepper. In: Khan MR, Mukhopadhyay AN, Pandey RN, Thakur MP, Singh D, Siddiqui MA, Akram M, Mohiddin FA, Haque Z (eds) Bio-intensive approaches: application and effectiveness in the management of plant nematodes, insects and weeds. Today and Tomorrow’s Printers and Publishers, New Delhi, pp 549–585Google Scholar
  22. Yang Y, Jiang L, Che HY, Cao XR, Yang JY, Sang LW, Luo DQ (2016) Molecular identification of a 16SrII-A group related phytoplasma associated with cinnamon yellow leaf disease in China. J Phytopathol 164:52–55CrossRefGoogle Scholar
  23. Zhao Y, Wei W, Lee M, 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). Int J Syst Evol Microbiol 59:25Google Scholar

Copyright information

© King Abdulaziz City for Science and Technology 2019

Authors and Affiliations

  1. 1.Division of Plant PathologyIndian Agricultural Research InstituteNew DelhiIndia
  2. 2.College of Agriculture, VellayaniThiruvananthapuramIndia
  3. 3.National Centre for Microbial ResourcePuneIndia

Personalised recommendations