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Front. Plant Sci. | doi: 10.3389/fpls.2017.01623

Template Preparation Affects 16S rRNA High-throughput Sequencing Analysis of Phyllosphere Microbial

Xiaoyan Tian1, 2, Yu Shi3, Lili Geng1, Haiyan Chu3, Jie Zhang1, Fuping Song1, Jangyan Duan2* and Changlong Shu1*
  • 1State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, China
  • 2School of Life Sciences, Shanxi Normal University, China
  • 3State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, China

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.

Keywords: phyllosphere, template, Method, Amplicon sequencing, 16S rRNA gene

Received: 06 Apr 2017; Accepted: 05 Sep 2017.

Edited by:

Abd El-Latif Hesham, Assiut University, Egypt

Reviewed by:

Andrea Campisano, Fondazione Edmund Mach, Italy
Birgit Mitter, Austrian Institute of Technology, Austria  

Copyright: © 2017 Tian, Shi, Geng, Chu, Zhang, Song, Duan and Shu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence:
Prof. Jangyan Duan, Shanxi Normal University, School of Life Sciences, Linfen, China,
Dr. Changlong Shu, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, State Key Laboratory for Biology of Plant Diseases and Insect Pests, Beijing, China,