Cultivation Versus Molecular Analysis of Banana (Musa sp.) Shoot-Tip Tissue Reveals Enormous Diversity of Normally Uncultivable Endophytic Bacteria
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The interior of plants constitutes a unique environment for microorganisms with various organisms inhabiting as endophytes. Unlike subterranean plant parts, aboveground parts are relatively less explored for endophytic microbial diversity. We employed a combination of cultivation and molecular approaches to study the endophytic bacterial diversity in banana shoot-tips. Cultivable bacteria from 20 sucker shoot-tips of cv. Grand Naine included 37 strains under 16 genera and three phyla (Proteobacteria, Actinobacteria, Firmicutes). 16S rRNA gene-ribotyping approach on 799f and 1492r PCR-amplicons to avoid plant organelle sequences was ineffective showing limited bacterial diversity. 16S rRNA metagene profiling targeting the V3-V4 hypervariable region after filtering out the chloroplast (74.2 %), mitochondrial (22.9 %), and unknown sequences (1.1 %) revealed enormous bacterial diversity. Proteobacteria formed the predominant phylum (64 %) succeeded by Firmicutes (12.1 %), Actinobacteria (9.5 %), Bacteroidetes (6.4 %), Planctomycetes, Cyanobacteria, and minor shares (<1 %) of 14 phyla including several candidate phyla besides the domain Euryarchaeota (0.2 %). Microbiome analysis of single shoot-tips through 16S rRNA V3 region profiling showed similar taxonomic richness and diversity and was less affected by plant sequence interferences. DNA extraction kit ominously influenced the phylogenetic diversity. The study has revealed vast diversity of normally uncultivable endophytic bacteria prevailing in banana shoot-tips (20 phyla, 46 classes) with about 2.6 % of the deciphered 269 genera and 1.5 % of the 656 observed species from the same source of shoot-tips attained through cultivation. The predominant genera included several agriculturally important bacteria. The study reveals an immense ecosystem of endophytic bacteria in banana shoot tissues endorsing the earlier documentation of intracellular “Cytobacts” and “Peribacts” with possible roles in plant holobiome and hologenome.
Keywords16S rRNA metagenomics Bacterial diversity Endophytic microorganisms Musa sp Plant-microbe interactions
- AP kit
AxyPrep bacterial DNA isolation kit
Filter sterilized distilled water post-autoclaving
- PF kit
MO BIO PowerFood microbial DNA isolation kit
Trypticasein soy agar
- UC kit
MO BIO UltraClean microbial DNA isolation kit
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 National Bureau of Agriculturally Important Microorganisms, Mau Nath Bhanjan, Uttar Pradesh, India, and partly by ICAR-Indian Institute of Horticultural Research, Bengaluru, India. The authors thank Dr. T. P. Rajendran, Former Acting Director, National Institute of Biotic Stress Management, Raipur, India, for the critical reading of the manuscript and the suggestions. The supports provided by Bipul Banerjee, Srinivas Vudathala, Chandan Badapanda (Xcelris Labs), Beena, P. S., Rajadurai C. P. and Jubina Benny (SciGenom Labs) during the metagene profiling studies are gratefully acknowledged. Thanks to Sadiq Pasha and N. Shivarudriah for the technical help during the conduct of some of the experiments. The publication bears ICAR-IIHR contribution No. 97/2015.
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