Tian Xiaoyan, Shi Yu, Geng Lili, Chu Haiyan, Zhang Jie, Song Fuping, Duan Jiangyan, Shu Changlong
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China.
School of Life Sciences, Shaanxi Normal University, Linfen, China.
Front Plant Sci. 2017 Sep 26;8:1623. doi: 10.3389/fpls.2017.01623. eCollection 2017.
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.
叶际微生物群落具有高度多样性并具有重要的生态意义；在此背景下，基于16S rRNA基因的细菌鉴定是一个重要的研究课题。在叶际微生物群落研究中，微孔过滤和离心用于收集微生物样本，但尚不清楚哪种方法具有更好的收集效率。在本研究中，我们比较了这两种微生物收集方法，并研究了DNA提取过程对微生物群落组成和结构估计的影响。考察了以下四种处理：（A）过滤、重悬和直接PCR；（B）过滤、DNA分离和PCR；（C）离心、重悬和直接PCR；（D）离心、DNA分离和PCR。我们的结果表明，叶绿体序列污染物的百分比受DNA提取过程的影响。处理A和C之间的细菌组成明显不同，表明收集方法对群落结构的测定有影响。与处理B和D相比，处理A和C产生的香农指数值更高，表明DNA提取过程可能会降低观察到的叶际微生物α多样性。然而，就群落结构而言，处理B和D产生了非常相似的结果，表明DNA提取过程消除了收集方法的影响。我们的研究结果提供了关键信息，以确保在叶际微生物研究中准确估计多样性和群落组成。
