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Characterization of Dickeya fangzhongdai causing bacterial soft rot disease on Dendrobium nobile in India

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Abstract

Soft-rot disease on Dendrobium nobile observed in a commercial farm in the Nilgiris district of Tamil Nadu, India was investigated. Disease etiology is reported along with phenotypic, molecular identification of the pathogen. Infected leaves showed rot symptoms characterized by irregular water-soaked lesions on the leaf margin that rapidly spread over the entire leaf. Milky exudation on the cut end of infected leaves indicated the bacterial etiology of the disease. The bacterial colonies isolated were irregular, whitish-yellow, spindle-shaped with a characteristic raised center. All isolates showed identical phenotypic traits and induced a hypersensitive reaction on Nicotiana tabacum leaves. Pathogenicity assay performed on D. nobile confirmed their pathogenic nature as all three bacterial isolates incited typical soft-rot within 12 h post-inoculation (hpi). In vitro host range studies revealed that all the isolates could cause rot on cut slices of other vegetable crops including potato, carrot, and onion. Additionally, the bacterium incited soft rot on Aloe vera, but not on chrysanthemum and banana transplants. The identity of the bacterial isolates was confirmed as Dickeya fangzhongdai using 16S rRNA gene sequences as well as by their pathogenic behavior. Complete understanding of the etiology of the disease and of the pathogen would be useful to devise suitable management practices for the containment of disease spread in the future.

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Acknowledgements

Authors are thankful to Director, ICAR-IARI for providing necessary facilities to complete this work successfully. Biolog facility provided by the NAHEP-CAAST project, ICAR-IARI, New Delhi is thankfully acknowledged.

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Authors and Affiliations

  1. Department of Plant Pathology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, 641003, India

    A. Balamurugan & M. Karthikeyan

  2. Division of Plant Pathology, ICAR - Indian Agricultural Research Institute, New Delhi, 110012, India

    A. Kumar & Kuleshwar Prasad Sahu

  3. Crop Protection Section, ICAR - Indian Institute of Oilseed Research, Hyderabad, Telangana, 500030, India

    K. Sakthivel

  4. Seed Pathology Lab, ICAR - Indian Institute of Seed Science, Mau, Uttar Pradesh, 275103, India

    M. Ashajyothi

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  1. A. Balamurugan
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Correspondence to A. Kumar.

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Electronic supplementary material

Supplementary Fig 1

Hypersensitive reaction on N. tabacum leaf. Tobacco leaf showing brown, necrotic hypersensitive response at 60 h post inoculation induced by the Dickeya fangzhongdai (108 cfu/ml) Supplementary Fig. 1 represents the results of all three D. fangzhongdai isolates (JPG 2765 kb)

Supplementary Fig 2

PCR confirmation of Dickeya fangzhongdai isolates through 16S rRNA sequence analysis. M 1 kb ladder (DNA size marker), 1 DDf_Kot1; 2 DDf_Kot2 and 3 DDf_Kot5, Note: Amplicon at 1500 bp specific to 16S rRNA gene (PNG 74 kb)

Supplementary Fig 3

Biolog based phenotypic finger printing assay matched with Dickeya dianthicola (isolate Kot1) with 0.56 similarity index and 0.897% probability recorded on 24 h post inoculation at 32 °C (PNG 195 kb)

Supplementary Fig 4a

Tissue maceration by Dickeya fangzhongdai on potato cut slice. (a) Potato cut slice with 0 h post inoculation (hpi) of the bacterium, (b) Rotting symptoms with definite spreading layer on 12 hpi, (c) Rotting sign continued to form second layer on 24 hpi, (d) Third layer of rotting on 36 hpi, (e) Fourth layer of rotting on 48 hpi, (f) Complete rotting of potato slice with disintegrated tissues due to maceration of the bacterium on 60 hpi, hpi: hours post inoculation, Supplementary Fig. 4a represents the results of all three D. fangzhongdai isolates (PNG 566 kb)

Supplementary Fig 4b

Tissue maceration by Dickeya fangzhongdai on carrot cut slice. (a) Un-inoculated cut slice of carrot, (b) Drop inoculation of the bacterium, 20 μl at 0 hpi (black arrow), (c) Rotting symptoms initiated within 12 hpi, (d) Rotting advanced upto periphery of the slice on 24 hpi, (e) Tissue maceration of the bacterium on 36 hpi, (f) Cut slice get disintegrated with spongy appearance on 60 hpi, hpi: hours post inoculation, Supplementary Fig. 4b represents the results of all three D. fangzhongdai isolates (PNG 444 kb)

Supplementary Fig 4c

Tissue maceration by Dickeya fangzhongdai on onion cut slice. (a) Drop inoculation of the bacterium, 20 μl on cut slice of onion at 0 hpi (red arrow), (b)Yellowish brown rotten symptom at 24 hpi, (c) Tissue maceration at 36 hpi, (d) Complete disintegration of cut slice with gummy appearance on 48 hpi, hpi: hours post inoculation, Supplementary Fig. 4c represents the results of all three D. fangzhongdai isolates (PNG 413 kb)

Supplementary Fig 5

Tissue maceration by Dickeya fangzhongdai on Aloe vera plants. (a) Syringe inoculation of the bacterium, 20 μl onto healthy Aloe vera leaf at 0 hpi (red arrow), (b) Water soaked, dark-green color lesion on 24 hpi, (c) Lesion advanced on 32 hpi, (d) Typical soft-rot symptoms on 48 hpi, (e) Intercellular spread of the bacterium led to tissue maceration and disintegration was recorded 60 hpi, (f) Systemic infection of Aloe vera leaves upon inoculation of D. fangzhongdai isolate recorded on 96 h post inoculation, hpi: hours post inoculation, Supplementary Fig. 5 represents the results of all three D. fangzhongdai isolates (PNG 641 kb)

Supplementary Fig. 6

Pathogenic assay of Dickeya fangzhongdai on chrysanthemum (Chrysanthemum indicum L. cv. locally grown yellow flower type) and banana (Musa x paradisiaca L. cv. grand nain) transplants. (a) Inoculation made through injection on chrysanthemum stem and (c) banana pseudostem using syringe - loaded with test bacterium (yellow arrows), The transplants did not infected by the bacterium indicated the non-pathogenic nature to three unique D. fangzhongdai isolates was recorded in chrysanthemum (b) and banana (d) and appeared as healthy, The R1, R2 and R3 represent replicated results for each inoculated plants, Control plants inoculated with sterile water remains healthy, Image was taken 30 days after inoculation of the bacterium (PNG 1496 kb)

Supplementary Fig. 7

The original phylogenetic tree constructed using Maximum Likelihood phylogeny by Kishino-Yano method (Hasegawa et al. 1985) based on the fragment of the 16S rRNA gene with a distance scale (PNG 25 kb)

Supplementary Table 1

16S rRNA gene sequence identity of Dickeya fangzhongdai causing soft rot of Dendrobium nobile with other type-strains representing diverse hosts or habitats (DOCX 16 kb)

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Balamurugan, A., Kumar, A., Sakthivel, K. et al. Characterization of Dickeya fangzhongdai causing bacterial soft rot disease on Dendrobium nobile in India. Eur J Plant Pathol 158, 773–780 (2020). https://doi.org/10.1007/s10658-020-02094-7

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