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. 2020 Feb 3;10(3):83. doi: 10.1007/s13205-020-2069-x

Characterization and genetic diversity of phytoplasmas associated with elephant foot yam (Amorphophallus paeoniifolius) in India

G P Rao 1,✉, Madem Gurivi Reddy 1, Madhu Priya 1, Amar Bahadur 2, Durgesh K Dubey 3
PMCID: PMC6997314  PMID: 32089978

Abstract

During the growing season of 2015 and 2016, leaf yellowing, stunting, and declining symptoms were observed on elephant foot yam in three states of India.The 1.3 kb 16S rDNA fragments were amplified from genomic DNA extracted from all the symptomatic elephant foot yam samples in nested PCR assays using primer pairs, P1/P7 followed by 3F/3R. Pair wise sequence comparison, virtual RFLP and phylogenetic analysis of 16S rRNA gene sequences confirmed association of ‘Candidatus Phytoplasma trifolii’ (16SrVI-D) and ‘Candidatus Phytoplasma oryzae’ (16SrXI-B) related strains in Uttar Pradesh, Uttarakhand and Tripura states, respectively. To our knowledge, this is the first report of association of ‘Ca. P. trifolii’ and ‘Ca. P. oryzae’ related strains in elephant foot yam in world. In the present study, we also reported Datura stramonium showing witches' broom as a natural weed host for ‘Ca. P. trifolii’ phytoplasma in Gorakhpur and Kushinagar districts of Uttar Pradesh state, India.

Keywords: Suran, ‘Ca. p. Trifolii’, ‘Ca. p. Oryzae’, Datura stramonium

Introduction

The elephant foot yam (EFY) [Amorphophallus paeoniifolius (Dennst.) Nicolson, fam Araceae], is the “king of tuber crops” popularly known as "oal" or “suran” and is one of the important aroid tuber vegetable crop in Africa, Asia, and the tropical Pacific islands (Ravi et al. 2009). The large underground tuber is edible and used as a vegetable which are the most important carbohydrate source after rice and maize. This crop is propagated mainly through vegetative propagules (corms and tubers). In India, EFY is extensively cultivated in the states of Andhra Pradesh, West Bengal, Gujarat, Kerala, Tamil Nadu, Maharashtra, Uttar Pradesh, Jharkhand, and Tripura in ~ 30,000 ha area with production of 774,000 metric tons (Anonymous 2018).

The elephant foot yam (EFY) is affected by several biotic stress like Konjac mosaic virus and Dasheen mosaic virus (Pandit et al. 2001; Padmavathi et al. 2013) and collar rot caused by Sclerotium rolfsii (Lal et al. 2015). Phytoplasma disease has been recorded on some of the tuber crops like cassava in South America and South East Asia (Rao et al. 2018a), but no record is available on EFY worldwide. In the present study, an attempt was made to identify and characterize phytoplasma association with leaf yellowing, stunting and declining of EFY in three states of India.

Materials and methods

Phytoplasma suspected symptoms were observed on EFY crops in Gorakhpur, Kushinagar, Pantnagar, and Tripura states during field surveys in 2015 and 2016. The incidence of disease was recorded on EFY at different locations based on the visual observation of symptoms in the field by counting number of symptomatic plants showing symptoms. Phytoplasma suspected symptoms on a weed, Datura stramonium (DS) plants grown nearby EFY fields were also recorded at Gorakhpur and Kushinagar states. Leaf samples were collected from five symptomatic and two non-symptomatic plants both from EFY and DS at different surveyed locations (Table 1).

Table 1.

Distribution, incidence and identification of phytoplasma in elephant foot yam and Datura stramonium

Crop State Symptoms Disease incidence (%) GenBank Acc. Nos 16S rDNA groups
Elephant foot yam Gorakhpur Leaf yellowing 6 MH198668 16Sr VI-D
Kushinagar Leaf yellowing 8 MH198669 16Sr VI-D
Pantnagar Stunting and leaf chlorosis 3 MH198671 16Sr XI-B
Tripura Leaf yellowing and wilting 30 MH198672 16Sr XI-B
Datura stramonium Gorakhpur Witches’ broom – KY078230 16Sr VI-D
Kushinagar Little leaf and witches’ broom – KY078231 16Sr VI-D
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Total genomic DNA was extracted from the leaf veins of the collected EFY and DS samples using CTAB method (Ahrens and Seemüller 1992). The extracted DNA was amplified with the universal phytoplasma primers P1/P7 in a direct PCR assay (Deng and Hiruki 1991; Schneider et al. 1995) followed by 3F/3R primer pair in a nested PCR assay (Manimekalai et al. 2010). DNA extracted from asymptomatic EFY and DS plants served as negative controls, while the DNA from brinjal little leaf phytoplasma (‘Ca. P. trifolii’ related strain, Acc. No. KX421261) was used as a positive control. PCR reactions were carried out in a thermal cycler (Mastercycler, Eppendorf, Hamsburg, Germany) and the cycling protocol used for PCR assay was followed as earlier described (Manimekalai et al. 2010; Rao et al. 2014). The PCR product was subjected to electrophoresis in a 1.0% (w/v) agarose gel, stained with ethidium bromide and observed under UV transilluminator. Amplified DNA products from all the symptomatic EFY and DS samples under study were purified using the WizardRSV Gel and PCR Clean-up System (Promega). The nested amplified PCR products were then ligated into pGEM®T vector (Promega) and cloned in Escherichia coli (DH5-α) following the manufacturer’s instructions. Cloned products were out sourced for sequencing in both directions at Agrigenome, Kerala, India.

The sequences obtained from PCR products were assembled using DNA Base V.4 (https://www.dnabaser.com) online tool and submitted to GenBank. A database search of homologous sequences was performed by BLASTn analysis at NCBI (www.ncbi.org). The16S rRNA gene sequences were aligned with phytoplasma group/subgroup representatives available in GenBank using Clustal W (Thompson et al. 1994). The sequences generated in the present study and the reference phytoplasma strains sequences retrieved from the GenBank were used to construct phylogeny tree through MEGA7.0 version software (Kumar et al. 2016) using 1000 bootstrap replications. The 16S rRNA sequence of Acholeplasma laidlawii (Acc. no. AB680603) was used to root the phylogenetic tree. 16Sr group/subgroup classification of EFY phytoplasma strains were done based on virtual RFLP profile of 16S rDNA sequences. The phytoplasma sequence corresponding to the 16Sr region was subjected to in silico restriction digestion with enzymes universally accepted for phytoplasma classification (Lee et al. 1998) and virtual gel plotting using the pDRAW32 program was developed by AcaClone Software (http://www.acaclone.com).

Results

Bright leaf yellows symptoms were recorded on EFY plants during field survey at Gorakhpur and Kushinagar districts of Uttar Pradesh (Fig. 1c, f) and the incidence of the disease was recorded as 6 to 8%, respectively (Table 1). However, severe stunting with leaf chlorosis symptoms on EFY was noticed at Pantnagar with disease incidence of 3% (Fig. 1b, Table 1). At Nagicherra, Tripura, a typical leaf yellowing, wilting and declining symptoms were observed on EFY plants (Fig. 1d, e) and the disease incidence was recorded up to 30% in different fields (Table 1). Severe witches’ broom symptoms were also observed on Datura stramonium plants grown as weeds nearby EFY fields at Gorakhpur and Kushinagar in Uttar Pradesh (Fig. 1h, i).

Fig. 1.

Fig. 1

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Symptoms on elephant foot yam: (a) healthy elephant foot yam; (b) stunting and leaf chlorosis at Pantnagar; (c) leaf yellowing at Gorakhpur; (d) leaf yellowing and declining symptoms at Tripura; (e) leaf yellowing and wilting at Tripura; (f) leaf yellowing at Kushinagar; (g) healthy Datura stramonium; (h) little leaf of D. stramonium at Gorakhpur; (i) little leaf and witches’ broom of D. stramonium at Kushinagar

The DNA fragments of 1.8 and 1.3 kb were amplified from all the collected five symptomatic EFY samples from Gorakhpur, Kushinagar, Pantnagar and Tripura regions and DS samples from Gorakhpur and Kushinagar regions by utilizing P1/P7 and 3F/3R primer pairs, respectively (data not shown). However, the DNA extracted from asymptomatic EFY and DS (two samples each) from all the locations under study did not produce any amplicons with phytoplasma specific primer pairs. The sequences of the representative EFY and DS samples were analyzed and deposited in GenBank (Table 1).

A BLASTn identity search of GenBank database revealed that the EFY phytoplasma isolates from Gorakhpur and Kushinagar shared 99.8–100% 16S rRNA gene sequence homology with ‘Ca. P. trifolii’ related strains like periwinkle little leaf (Acc. No. AF228053) and brinjal little leaf (Acc. Nos. AF228052). The EFY phytoplasma strain from Pantnagar and Tripura had 100% 16S rRNA sequence identity with ‘Ca. P. oryzae’ strains like sugarcane grassy shoot (Acc. Nos. MG745892, MG745893) and sugarcane white leaf (Acc. Nos. KR020689, JX862179). A phylogenic tree was deduced by neighbor-joining algorithm, showed clustering of Gorakhpur and Kushinagar EFY and DS phytoplasma strains together with ‘Ca. P. trifolii’ related strains while Pantnagar and Tripura EFY isolates were clustered together with ‘Ca.P. oryzae’ related strains (Fig. 2).

Fig. 2.

Fig. 2

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Phylogenetic tree constructed by neighbour-joining method showing the relationships among elephant foot yam phytoplasma strains and reference phytoplasma strains based on 16S rDNA sequences. Accession numbers are specified in the tree and ‘Acholeplasma laidlawii was used as an outgroup. Numbers on branches are bootstrap values obtained for 1000 replicates

Virtual RFLP of the 16S rDNA fragments of phytoplasma strains from symptomatic EFY and DS weed was done for the 16Sr subgroup assignment. Comparison of the restriction profiles of the representative EFY phytoplasma strains from Gorakhpur (Acc. No. MH198668) produced identical RFLP patterns with periwinkle little leaf phytoplasma reference strain (16SrVI-D, Acc. No. AF228053) (Fig. 3a, b). However, EFY phytoplasma strains from Pantnagar (Acc. Nos. MH198671) produced similar identical RFLP profiles to sugarcane white leaf phytoplasma reference strain (16SrXI-B, Acc. No. AB052874) (Fig. 3c, d). The DS witches’ broom phytoplasma strains from Gorakhpur (Acc. No. KY078230) produced a virtual RFLP profiles identical to periwinkle little leaf phytoplasma strain belonging to 16SrVI-D subgroup (Acc. No. AF228053) (Fig. 3e, f).

Fig. 3.

Fig. 3

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Comparison of restriction gel plots of reference phytoplasma subgroup with 17 restriction enzymes (AluI, BamHI, BfaI, BstUI, DraI, EcoRI, HaeIII, HhaI, HinfI, HpaI, HpaII, KpnI, MboI (Sau3AI), MseI, RsaI, SspI, and TaqI) generated using pDRAW software with NEB 100 bp ladder as a marker. a Reference phytoplasma strain of 16SrVI-D (Periwinkle little leaf phytoplasma, Acc. No. AF228053); b elephant foot yam Gorakhpur isolate (Acc. No: MH198668); c reference phytoplasma strain of 16SrXI-B (sugarcane white leaf phytoplasma, Acc. No. AB052874); d elephant foot yam Pantnagar isolate (Acc. No. MH198672); e reference phytoplasma strain of 16SrVI-D (Periwinkle little leaf phytoplasma, Acc. No. AF228053); f Datura stramonium Gorakhpur isolate (Acc. No. KY078230)

Discussion

In the present study, we have reported the occurrence of 16SrVI-D and 16SrXI-B subgroups of phytoplasmas in EFY from Uttar Pradesh, Uttarakhand and Tripura states of India. Out of different places surveyed in the study, maximum incidence of phytoplasma infection was reported at Nagicherra orchard in Tripura (Table 1), where EFY is being grown as commercial intercrop in mango and lychee fruit orchards. EFY is also a major stable food crop for the tribal people of Tripura state and identification of phytoplasma disease in EFY poses a serious threat to EFY cultivation in the state. However, EFY is grown in small areas in Uttar Pradesh and Uttarakhand states of India for self-consumption purpose and sale in local markets. Since the EFY is a vegetative propagated crop, there are maximum chances of transmission of phytoplasma using phytoplasma infected EFY corm as a source of propagation materials (Rao et al. 2018a). Hence, it would be always better to index EFY tubers/corms for phytoplasma infection before use as seed material and it would be important to grow healthy EFY nurseries in Tripura state.

The 16SrVI-D subgroup has already been reported as a major widespread group of phytoplasma infecting several crops viz. brinjal (Rao and Kumar 2017), ornamental plants (Madhupriya and Rao 2017), medicinal plants (Rao et al. 2018b) and sesame (Madhupriya et al. 2015a) in India. However, the occurrence of ‘Ca. P. oryzae’ (16SrXI) group was reported earlier on sugarcane (Rao et al. 2014), potato (Rao et al. 2018c), jasmine (Madhupriya et al. 2015b), areca nut and coconut (Manimekalai et al. 2010, 2011), onion (Goel et al. 2017) and wheat (Rao et al. 2017) from India. The earlier report of wider natural spread of 16SrVI-D and 16SrXI-B subgroups of phytoplasmas infecting several crop species and their natural transmission through leafhoppers vectors in India (Rao et al. 2017), poses a serious threat of these phytoplasma infection and spread to other cultivated species. So far, only cassava tuber crop has been reported to be identified with association of different phytoplasma strains in South America and South East Asia (Rao et al. 2018a). No report is available on association of phytoplasma with EFY. Thus, the confirmation of association of 16SrVI-D and 16SrXI-B subgroups of phytoplasmas with EFY in the present study is the first report worldwide. Our study also suggested role of D. stramonium weed growing in and around EFY fields as a putative natural host reservoir of 16SrVI-D subgroup of phytoplasma.

Acknowledgements

Authors wish to thank Director, IARI, New Delhi for providing lab facilities and Principal, College of Agriculture, Lembucherra, Tripura for field survey work.

Author contributions

GPR carried out survey, collected the samples and helped in editing of the manuscript. MGR prepared the manuscript and did the analysis of sequences for the identification of phytoplasma strains. MP processed the samples for PCR assay and sequence submission. AB helped in survey, collection of samples and recording the disease incidence. DKD helped in survey, collection of samples and recording the disease incidence.

Compliance with ethical standards

Conflict of interest

All authors declare that they have no conflict of interest.

GenBank submission

All the 16Sr RNA gene sequences has been submitted in GenBank and appeared in public database.

References

  1. Ahrens U, Seemüller E. Detection of plant pathogenic mycoplasma like organisms by a polymerase chain reaction that amplifies a sequence of 16S rRNA gene. Phytopathology. 1992;82:828–832. doi: 10.1094/Phyto-82-828. [DOI] [Google Scholar]
  2. Deng S, Hiruki C. Amplification of 16S rRNA genes from culturable and nonculturable mollicutes. J of Microbiol Methods. 1991;14:53–61. doi: 10.1016/0167-7012(91)90007-D. [DOI] [Google Scholar]
  3. Goel S, Madhupriya, Rao GP. Identification and characterization of 16SrIX and 16SrXI groups of phytoplasmas associated with leaf yellows and declining disease of garlic and onion in India. Indian Phytopathol. 2017;70:368–372. [Google Scholar]
  4. Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016;33:1870–1874. doi: 10.1093/molbev/msw054. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Lal HC, Kumar P, Sengupta S, Ekka S, Kumar N. Integrated disease management of collar rot in elephant foot yam (EFY) caused by Sclerotium rolfsii Sacc. J Mycol Plant Pathol. 2015;45:309–313. [Google Scholar]
  6. Lee IM, Gundersen DE, Davis RE, Bartoszyk IM. Revised classification scheme of phytoplasmas based on RFLP analysis of 16S rRNA and ribosomal protein gene sequences. Int J Syst Evol Microbiol. 1998;48:1153–1169. [Google Scholar]
  7. Madhupriya, Rao GP. Phytoplasma diseases of ornamental plants: an Indian overview. J Ornam Hortic. 2017;20:87–102. doi: 10.5958/2249-880X.2017.00001.9. [DOI] [Google Scholar]
  8. Madhupriya, Rao GP, Kumar A, Baranwal VK. Classification of the sesame phytoplasma strains in India at the 16Sr subgroup level. J Plant Pathol. 2015;97:3. [Google Scholar]
  9. Madhupriya, Rao GP, Khurana SMP. Rice yellow dwarf phytoplasma (16SrXI-B subgroup) infecting Jasminum sambac in India. Phytoparasitica. 2015;43:77–80. doi: 10.1007/s12600-014-0425-2. [DOI] [Google Scholar]
  10. Manimekalai R, Soumya VP, Sathish KR, Selvarajan R, Reddy K, Thomas GV, Sasikala M, Rajeev G, Baranwal VK. Molecular detection of 16SrXI group phytoplasma associated with root (wilt) disease of coconut (Cocosnucifera) in India. Plant Dis. 2010;94:636. doi: 10.1094/PDIS-94-5-0636B. [DOI] [PubMed] [Google Scholar]
  11. Manimekalai R, Sathish KR, Soumya VP, Thomas GV. Molecular detection of phytoplasma associated with yellow leaf disease in areca palms (Areca catechu) in India. Acta Phytopathol Entomol Hung. 2011;46:59–99. doi: 10.1556/APhyt.46.2011.1.7. [DOI] [PubMed] [Google Scholar]
  12. Padmavathi M, Srinivas KP, Hema M, Sreenivasulu P. First report of Konjacmosaicvirus in elephant foot yam (Amorphophalluspaeoniifolius) from India. Aus Plant Dis Notes. 2013;8:27–29. doi: 10.1007/s13314-012-0088-0. [DOI] [Google Scholar]
  13. Pandit MK, Nath PS, Mukhopadhyay S, Devonshire BJ, Jones P. First report of Dasheen mosaic virus in elephant foot yam in India. Plant Pathol. 2001;50:802. doi: 10.1046/j.1365-3059.2001.00625.x. [DOI] [Google Scholar]
  14. Rao GP, Kumar M. Status of phytoplasma diseases associated with eggplant. Crop Prot. 2017;96:22–29. doi: 10.1016/j.cropro.2017.01.005. [DOI] [Google Scholar]
  15. Rao GP, Madhupriya, Tiwari AK, Kumar S, Baranwal VK. Identification of sugarcane grassy shoot-associated phytoplasma and one of its putative vectors in India. Phytoparasitica. 2014;42:349–354. doi: 10.1007/s12600-013-0366-1. [DOI] [Google Scholar]
  16. Rao GP, Madhupriya, Manimekalai R, Tiwari AK, Yadav A. A century progress of research on phytoplasma diseases in India. Phytopathogenic Mollicutes. 2017;7:1–38. doi: 10.5958/2249-4677.2017.00001.9. [DOI] [Google Scholar]
  17. Rao GP, Alvarez E, Yadav A. Phytoplasma diseases of Industrial Crops. In: Rao GP, Bertaccini A, Fiore N, Liefting LW, editors. Phytoplasmas: plant pathogenic bacteria—I, Characterisation and epidemiology of phytoplasma-associated diseases. Berlin: Springer; 2018. pp. 91–122. [Google Scholar]
  18. Rao GP, Marcone C, Bellardi MG, Madhupriya (2018b) Phytoplasma disease of medicinal crops. In: GP Rao A Bertaccini N Fiore LW Liefting (eds) Phytoplasmas: plant pathogenic bacteria—I, characterisation and epidemiology of phytoplasma-associated diseases, Springer, Germany pp 235–266
  19. Rao GP, Mishra A, Mishra MK, Rao A, Goel S. Identification and characterization of Candidatus Phytoplasma trifolii (16SrVI-D) inducing shoot proliferation disease of potato in India. Indian Phytopathol. 2018;71:75–81. doi: 10.1007/s42360-018-0011-5. [DOI] [Google Scholar]
  20. Ravi V, Ravindran CS, Suja G. Growth and productivity of elephant foot yam (Amorphophalluspaeoniifolius) (Dennst. Nicolson): an overview. J Root Crops. 2009;35:131–142. [Google Scholar]
  21. Schneider B, Seemüller E, Smart CD, Kirkpatrick BC. Phylogenetic classification of plant pathogenic mycoplasma-like organisms or phytoplasmas. In: Razin S, Tully JG, editors. Molecular and Diagnostic Procedures in Mycoplasmology. CA: San Diego; 1995. pp. 369–380. [Google Scholar]
  22. Thompson JD, Higgins DG, Gibson TJ. ClustalW-improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994;22:4673–4680. doi: 10.1093/nar/22.22.4673. [DOI] [PMC free article] [PubMed] [Google Scholar]

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