High taxonomic diversity of cultivation-recalcitrant endophytic bacteria in grapevine field shoots, their in vitro introduction, and unsuspected persistence

Abstract

Main conclusion

Molecular and microscopic analyses reveal enormous non-cultivable endophytic bacteria in grapevine field shoots with functional significance. Diverse bacteria enter tissue cultures through surface-sterilized tissues and survive surreptitiously with varying taxonomic realignments.

The study was envisaged to assess the extent of endophytic bacterial association with field shoot tissues of grapevine and the likelihood of introduction of such internally colonizing bacteria in vitro adopting molecular techniques targeting the non-cultivable bacterial community. PowerFood®-kit derived DNA from surface-sterilized field shoot tips of grapevine Flame Seedless was employed in a preliminary bacterial class-specific PCR screening proving positive for major prokaryotic taxa including Archaea. Taxonomic and functional diversity were analyzed through whole metagenome profiling (WMG) which revealed predominantly phylum Actinobacteria, Proteobacteria, and minor shares of Firmicutes, Bacteroidetes, and Deinococcus-Thermus with varying functional roles ascribable to the whole bacterial community. Field shoot tip tissues and callus derived from stem segments were further employed in 16S rRNA V3–V4 amplicon taxonomic profiling. This revealed elevated taxonomic diversity in field shoots over WMG, predominantly Proteobacteria succeeded by Actinobacteria, Firmicutes, Bacteroidetes, and 15 other phyla including several candidate phyla (135 families, 179 genera). Callus stocks also displayed broad bacterial diversity (16 phyla; 96 families; 141 genera) bearing resemblance to field tissues with Proteobacterial dominance but a reduction in its share, enrichment of Actinobacteria and Firmicutes, disappearance of some field-associated phyla and detection of a few additional taxonomic groups over field community. Similar results were documented during 16S V3–V4 amplicon taxonomic profiling on Thompson Seedless field shoot tip and callus tissues. Video microscopy on tissue homogenates corroborated enormous endophytic bacteria. This study elucidates a vast diversity of cultivation-recalcitrant endophytic bacteria prevailing in grapevine field shoots, their in vitro introduction, and unsuspecting sustenance with possible silent participation in tissue culture processes.

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Abbreviations

FDW:

Filter-sterilized distilled water post autoclaving

NA:

Nutrient agar

SATS:

Spotting- and tilt-spreading

SP-SDS:

Single plate-serial dilution spotting

TSA:

Trypticase soy agar

WMG:

Whole metagenome

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Acknowledgements

The study was funded partly under the ICAR-AMAAS Net-work project ‘Exploration of the endophytic microbial diversity in horticultural crops through metagenomics and cultivation’ funded through the ICAR-National Bureau of Agriculturally Important Microorganisms, Mau Nath Bhanjan, Uttar Pradesh, India and partly by ICAR-Indian Institute of Horticultural Research, Bengaluru, India under the sub-project: ‘Tissue culture systems in horticultural crops with reference to management and exploitation of endophytes’. The author thanks Dr. T. P. Rajendran (Former Assistant Director General-Plant Protection, ICAR and Acting Director, National Institute of Biotic Stress Management, Raipur, India) for the critical reading of the manuscript and the suggestions. This publication bears ICAR-IIHR Contribution No. 21/2017.

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Correspondence to Pious Thomas.

Electronic supplementary material

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425_2017_2733_MOESM2_ESM.mp4

Movie 01. Tissue homogenate from surface-sterilized field-shoot-tip tissues of grape ‘Flame Seedless’ showing abundant motile bacterial cocci and bacilli besides the tissue debris under bright field microscopy (1000×) (MP4 10590 kb)

425_2017_2733_MOESM3_ESM.mp4

Movie 02. Tissue homogenate from healthy 1 year plus old callus culture of grape ‘Flame Seedless’ showing abundant motile bacterial cocci and bacilli corresponding to bacteria under bright field microscopy (1000×) (MP4 4597 kb)

425_2017_2733_MOESM4_ESM.mp4

Movie 03. Pure culture of Pantoea ananatis (Proteobacterium) isolated from index-positive grape callus ‘Flame Seedless’ under bright field microscopy (1000×) showing condensed rods with motility (MP4 10209 kb)

425_2017_2733_MOESM5_ESM.mp4

Movie 04. Pure culture of Staphylococcus haemolyticus (Firmicute) isolated from index-positive grape callus ‘Flame Seedless’ under bright field microscopy (1000×) with small cocci displaying motility (MP4 8711 kb)

Supplementary material 1 (DOC 567 kb)

Movie 01. Tissue homogenate from surface-sterilized field-shoot-tip tissues of grape ‘Flame Seedless’ showing abundant motile bacterial cocci and bacilli besides the tissue debris under bright field microscopy (1000×) (MP4 10590 kb)

Movie 02. Tissue homogenate from healthy 1 year plus old callus culture of grape ‘Flame Seedless’ showing abundant motile bacterial cocci and bacilli corresponding to bacteria under bright field microscopy (1000×) (MP4 4597 kb)

Movie 03. Pure culture of Pantoea ananatis (Proteobacterium) isolated from index-positive grape callus ‘Flame Seedless’ under bright field microscopy (1000×) showing condensed rods with motility (MP4 10209 kb)

Movie 04. Pure culture of Staphylococcus haemolyticus (Firmicute) isolated from index-positive grape callus ‘Flame Seedless’ under bright field microscopy (1000×) with small cocci displaying motility (MP4 8711 kb)

Data set 1: Detailed path analysis WMG profiling on Flame Seedless field-shoot-tip functional analysis after filtering the contigs corresponding to Viridiplantae through MEGAN SEED classification (XLS 36 kb)

Data set 2: Detailed path analysis WMG profiling on Flame Seedless field-shoot-tip functional analysis after filtering the contigs corresponding to Viridiplantae through KEGG pathway analysis (XLS 38 kb)

Data set 3. Comparison of Flame Seedless and Thompson Seedless field and callus tissues for taxonomic diversity at family level (XLS 184 kb)

Data set 4. Comparison of Flame Seedless and Thompson Seedless field and callus tissues for taxonomic diversity at genus level (XLS 183 kb)

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Thomas, P., Sekhar, A.C. & Shaik, S.P. High taxonomic diversity of cultivation-recalcitrant endophytic bacteria in grapevine field shoots, their in vitro introduction, and unsuspected persistence. Planta 246, 879–898 (2017). https://doi.org/10.1007/s00425-017-2733-5

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Keywords

  • Endophytic bacterial community
  • Metagenomics
  • Video microscopy
  • Non-cultivable bacteria
  • Plant microbe interactions
  • Plant tissue culture
  • Vitis vinifera L.