High taxonomic diversity of cultivation-recalcitrant endophytic bacteria in grapevine field shoots, their in vitro introduction, and unsuspected persistence
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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.
KeywordsEndophytic bacterial community Metagenomics Video microscopy Non-cultivable bacteria Plant microbe interactions Plant tissue culture Vitis vinifera L.
Filter-sterilized distilled water post autoclaving
Spotting- and tilt-spreading
Single plate-serial dilution spotting
Trypticase soy agar
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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