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.
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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.
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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.
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All the 16Sr, secA, secY, tuf and SAP11 gene sequences have been submitted in GenBank and appeared in public database.
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13205_2019_1944_MOESM1_ESM.jpg
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
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)
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Mishra, S., Mitra, S., Radhika, N.S. et al. Multilocus gene-specific characterization of ‘Candidatus Phytoplasma australasia’ associated with shoot proliferation disease of small cardamom in India. 3 Biotech 9, 420 (2019). https://doi.org/10.1007/s13205-019-1944-9
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DOI: https://doi.org/10.1007/s13205-019-1944-9