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Cultivation Versus Molecular Analysis of Banana (Musa sp.) Shoot-Tip Tissue Reveals Enormous Diversity of Normally Uncultivable Endophytic Bacteria

  • Plant Microbe Interactions
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Abstract

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.

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Abbreviations

AP kit:

AxyPrep bacterial DNA isolation kit

FDW:

Filter sterilized distilled water post-autoclaving

PF kit:

MO BIO PowerFood microbial DNA isolation kit

NA:

Nutrient agar

TSA:

Trypticasein soy agar

UC kit:

MO BIO UltraClean microbial DNA isolation kit

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Acknowledgments

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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Authors and Affiliations

  1. Endophytic and Molecular Microbiology Laboratory, Division of Biotechnology, ICAR-Indian Institute of Horticultural Research (IIHR), Hessaraghatta Lake, Bengaluru, 560089, India

    Pious Thomas & Aparna Chandra Sekhar

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  1. Pious Thomas
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Correspondence to Pious Thomas.

Electronic Supplementary Material

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Table S1

Categorization of 100 clones derived from the two batches of 10 shoot-tips each of banana as per the ribotyping pattern and the suggested identity of the organisms (DOC 73 kb)

Table S2

16S rRNA metagene profiling data for MG07 and MG08 samples derived through Illumina MiSeq and phylogenetic distribution of OTUs as per direct QIIME analysis (QIIME-I) (DOC 61 kb)

Table S3

16S rRNA metagenome data for MG07 and MG08 samples in QIIME analysis-II excluding chloroplast, mitochondrial and unassigned reads showing the distribution of reads and OTUs (DOC 59 kb)

Table S4

Metagenome data for MG33-V3 and MG34-V3 samples derived through Illumina MiSeq and phylogenetic distribution of OTUs as per direct QIIME analysis (QIIME-I) of 16S rRNA V3 sequence data (DOC 55 kb)

Fig. S1

Phylogenetic distribution of cultivable endophytic bacterial strains isolated from banana ‘Grand Naine’ suckers in two batches (a and b) of 10 suckers; ×n indicates the number of strains (DOC 151 kb)

Fig. S2

OTU table heat map showing taxonomy assignment for each OTU. The OTU heat map displays raw OTU counts per sample where the counts are coloured based on the contribution of each OTU to the total OTU count present in that sample (blue: contributes low percentage of OTUs to sample; red: contributes high percentage of OTUs) (DOC 98 kb)

Fig. S3

Rarefaction plot of metagenome sequences of banana shoot-tip DNA derived through PowerFood (MG07) and AxyPrep (MG08) DNA isolation kits after QIIME-II analysis (DOC 73 kb)

Data set S1 (Excel file)

Taxonomic distribution of OTUs in MG07 sample (V3-V4 profiling) at phylum, class, order, family and genus levels as per QIIME-II analysis (XLS 71 kb)

Data set S2 (Excel file)

Taxonomic distribution of OTUs in MG08 sample (V3-V4 profiling) at phylum, class, order, family and genus levels as per QIIME-II analysis (XLS 39 kb)

Data set S3 (Excel file)

Taxonomic distribution as per OTUs and reads in MG33 and MG34 samples (V3 profiling) at genus level as per QIIME analysis-II (XLSX 12 kb)

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Thomas, P., Sekhar, A.C. Cultivation Versus Molecular Analysis of Banana (Musa sp.) Shoot-Tip Tissue Reveals Enormous Diversity of Normally Uncultivable Endophytic Bacteria. Microb Ecol 73, 885–899 (2017). https://doi.org/10.1007/s00248-016-0877-7

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