Template Preparation Affects 16S rRNA High-Throughput Sequencing Analysis of Phyllosphere Microbial Communities.

Xiaoyan Tian,Yu Shi,Haiyan Chu,Fuping Song,Changlong Shu,Lili Geng,Jie Zhang,Jiangyan Duan

doi:10.3389/fpls.2017.01623

Abstract

Phyllosphere microbial communities are highly diverse and have important ecological implications; in that context, bacterial identification based on 16S rRNA genes is an important research issue. In studies of phyllosphere microbial communities, microporous filtration and centrifugation are used to collect microorganism samples, but it is unclear which one has a better collection efficiency. In this study, we compared these two microorganism collection methods and investigated the effects of the DNA extraction process on the estimation of microbial community composition and organization. The following four treatments were examined: (A) filtration, resuspension, and direct PCR; (B) filtration, DNA isolation, and PCR; (C) centrifugation, resuspension, and direct PCR; (D) centrifugation, DNA isolation, and PCR. Our results showed that the percentage of chloroplast sequence contaminants was affected by the DNA extraction process. The bacterial compositions clearly differed between treatments A and C, suggesting that the collection method has an influence on the determination of community structure. Compared with treatments B and D, treatments A and C resulted in higher Shannon index values, indicating that the DNA extraction process might reduce the observed phyllosphere microbial alpha diversity. However, with respect to community structure, treatments B and D yielded very similar results, suggesting that the DNA extraction process erases the effect of the collection method. Our findings provide key information to ensure accurate estimates of diversity and community composition in studies of phyllosphere microorganisms.

Highlights

The phyllosphere is an important habitat for microbes and is colonized by a large number of microbial taxa
The microbial community from B. napus L. leaves obtained using the four treatment methods were dominated by Proteobacteria
Previous work has demonstrated the importance of characterizing the phyllosphere microbial community, which contains many functionally important microbes

Summary

Introduction

The phyllosphere is an important habitat for microbes and is colonized by a large number of microbial taxa. Culture-based isolation studies have shown that phyllosphere microbial communities include many important taxa, including plant pathogens, ice nucleationactive bacteria (Hirano and Upper, 2000), decomposers (Hicks and Silvester, 1985), phytohormone. Phototrophic microbes colonize some plant leaves, including rhodopsin-based ones, absorbing different fractions of the light spectrum (Atamna-Ismaeel et al, 2012b). These findings indicate that the phyllosphere microbial community is closely related to phyllosphere functions and further studies are likely to discover new ecological interactions. Both culturedependent and culture-independent methods are used to analyze phyllosphere microorganisms. The culture-independent methods can detect a broader microbial diversity and are widely used for microbial community analyses, primarily using 16S rRNA gene amplicon sequencing to determine the composition, organization, and spatiotemporal patterns of microbial communities

Methods

Results

Conclusion