Microbial community structure analysis inAcer palmatumbark and isolation of novel bacteria IAD-21 of the candidate division FBP

Kazuki Kobayashi,Hideki Aoyagi

doi:10.7717/peerj.7876

Abstract

BackgroundThe potential of unidentified microorganisms for academic and other applications is limitless. Plants have diverse microbial communities associated with their biomes. However, few studies have focused on the microbial community structure relevant to tree bark.MethodsIn this report, the microbial community structure of bark from the broad-leaved tree Acer palmatum was analyzed. Both a culture-independent approach using polymerase chain reaction (PCR) amplification and next generation sequencing, and bacterial isolation and sequence-based identification methods were used to explore the bark sample as a source of previously uncultured microorganisms. Molecular phylogenetic analyses based on PCR-amplified 16S rDNA sequences were performed.ResultsAt the phylum level, Proteobacteria and Bacteroidetes were relatively abundant in the A. palmatum bark. In addition, microorganisms from the phyla Acidobacteria, Gemmatimonadetes, Verrucomicrobia, Armatimonadetes, and candidate division FBP, which contain many uncultured microbial species, existed in the A. palmatum bark. Of the 30 genera present at relatively high abundance in the bark, some genera belonging to the phyla mentioned were detected. A total of 70 isolates could be isolated and cultured using the low-nutrient agar media DR2A and PE03. Strains belonging to the phylum Actinobacteria were isolated most frequently. In addition, the newly identified bacterial strain IAP-33, presumed to belong to Acidobacteria, was isolated on PE03 medium. Of the isolated bacteria, 44 strains demonstrated less than 97% 16S rDNA sequence-similarity with type strains. Molecular phylogenetic analysis of IAD-21 showed the lowest similarity (79%), and analyses suggested it belongs to candidate division FBP. Culture of the strain IAD-21 was deposited in Japan Collection of Microorganisms (JCM) and Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) as JCM 32665 and DSM 108248, respectively.DiscussionOur results suggest that a variety of uncultured microorganisms exist in A. palmatum bark. Microorganisms acquirable from the bark may prove valuable for academic pursuits, such as studying microbial ecology, and the bark might be a promising source of uncultured bacterial isolates.

Highlights

The total number of microorganisms existing on the earth is speculated to range from 1029 to 1030 organisms (Whitman, Coleman & Wiebe, 1998; Kallmeyer et al, 2012)
In order to confirm whether the above phyla could be universally detected from Acer palmatum bark, three samples were collected from two individual trees, and re-analysis of the 16S amplicon sequencing was performed (Fig. 2; Table S3)
We focused on one particular isolate, strain IAD-21, a potentially novel microorganism, cultured on DR2A medium (Table 2)

Summary

Introduction

The total number of microorganisms existing on the earth is speculated to range from 1029 to 1030 organisms (Whitman, Coleman & Wiebe, 1998; Kallmeyer et al, 2012). The number of bacterial species that currently have been isolated, investigated with regards to physiological properties, and assigned scientific names is about 15,000 (Parte, 2018) This is only 1% of the total number of bacterial species presumed to exist on earth, and the remaining 99% of uncultured microorganisms is called the “microbial dark matter” (Ledford, 2015). Methods: In this report, the microbial community structure of bark from the broad-leaved tree Acer palmatum was analyzed. Both a culture-independent approach using polymerase chain reaction (PCR) amplification and generation sequencing, and bacterial isolation and sequence-based identification methods were used to explore the bark sample as a source of previously uncultured microorganisms. Microorganisms acquirable from the bark may prove valuable for academic pursuits, such as studying microbial ecology, and the bark might be a promising source of uncultured bacterial isolates

Methods

Results

Discussion

Conclusion