Enriching plant microbiota for a metagenomic library construction

Abstract

Plant microbiota (the microorganisms that live in any associations with plant tissues) represents a rather unexplored area of metagenomic research compared with soils and oceans. Constructing a metagenomic library for plant microbiota is technically challenging. Using all the biomass without pre-enrichment could lead to vast proportions of the host plant DNA in the metagenomic library, doubtless obliterating the microbial contribution. Therefore, the first and essential step is to enrich for the constituent microorganisms from plant tissues. Here, a strong enrichment for plant microbiota was achieved by coupling SDS (sodium dodecyl sulfate) with NaCl, creating a predominantly microbial metagenomic library that contains 88% bacterial inserts. 16S rDNA sequence analysis revealed that the metagenomic DNA of enrichments originates from very diverse microorganisms. At least 74 distinct ribotypes (at a 97% threshold) from seven different bacterial phyla were identified and mainly distributed among Actinobacteria and Proteobacteria. Additionally, a simplified version of Amplified Ribosomal DNA Restriction Analysis (ARDRA) was developed for a quick and efficient assessment of the enriching procedures. This work opens further insight into the great biotechnical potential of plant microbiota, holding more potential for drug discovery through a metagenomic strategy, and paving the way for recovery and biochemical characterization of functional gene repertoire from plant microbiota.

Biosynthetic Potential of Phylogenetically Unique Endophytic Actinomycetes from Tropical Plants

“…Micromonosporaceae clade 1 is comprised of six endophytic strains isolated from plants on Mborokua Island and the uncultured bacterium clone 1-12D from the metagenomic library derived from Mallotus nudiflorus stem barks collected in Yunnan, China (42). Similar to clone 1-12D, all of our endophytic strains were isolated from either the stem bark of an unidentified tree or the prop root bark of a Pandanus sp., suggesting that this genus may specifically inhabit tissues outside of the vascular cambium.…”

“…Clade 1 also includes the uncultured bacterium clone 1-12D (accession number EU289478), which is the most closely related sequence in GenBank. Interestingly, the 16S rRNA gene of clone 1-12D is from the microbial-enriched DNA of the metagenomic library of the tree Mallotus nudiflorus (42 (2,4,34). Since the strains in clade 1 are equally related by sequence identity to the nearest type strains of three different genera and are all plant derived, they may represent a new genus in Micromonosporaceae.…”

Isolation, Diversity, and Antimicrobial Activity of Rare Actinobacteria from Medicinal Plants of Tropical Rain Forests in Xishuangbanna, China

“…It was first successfully applied to the isolation of diverse endophytic actinobacteria from culture in our study. Recently, Wang et al (84) reported on the diversity of uncultured plant-associated microbes obtained by an improved DNA extraction method and increased differential centrifugation at a force of 5,000 ϫ g. Actinobacteria were the dominant microbial group, accounting for 37.7% of the 16S rRNA gene library, which also demonstrated the feasibility of using pretreatment method 3 for the isolation of endophytic actinobacteria.…”

Isolation and Genetic Characterization of Aurantimonas and Methylobacterium Strains from Stems of Hypernodulated Soybeans

“…3B and Table S4). Some references were derived from plant materials such as alpine shoots (47) and bark of the tropic tree Mallotus nudiflorus (54). The phylogenetic analysis revealed that Cluster I mainly including phyllosphereassociated Aurantimonas strains was phylogenetically distantly related to Cluster II mainly consisting of oceanassociated Aurantimonas strains (Fig.…”