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Analysis of the diversity and distribution of leaf endophytic bacterial communities

Ding, Tao
Scope and Method of Study:
This research project aimed to reveal the basic composition of leaf endophytic bacterial communities, to detect the dominant and significant bacterial groups and to study the environmental influences on the structure of leaf endophytic bacterial communities; specifically, to see the relationship between host plants and endophytic bacteria and to track the dynamics of the endophytic bacteria during the host plant growing season. This research employed cultivation-independent methods to analyze bacterial communities, including total DNA extraction, polymerase chain reaction (PCR), terminal restriction fragment length polymorphism (T-RFLP) and tagged 454 pyrosequencing. Research data was analyzed using statistical software including R, SAS and CANOCO. Sequencing data was analyzed using Mothur and quantitative insight into microbial ecology (QIIME).
Findings and Conclusions:
T-RFLP has helped to study the environmental influences on the leaf endophytic bacterial communities quantitatively. Three major environmental factors, including host plant species, sampling date and collecting locations, were all tested significant using the profiles of the proportion of terminal restriction fragments (T-RF) by partial Canonical Correlation Analysis (pCCA). Dominant T-RFs were detected and host-specific T-RFs were also defined.
Tagged 454 pyrosequencing allowed revealing the leaf endophytic bacterial communities at a deeper level. Sequences (64,591) of the 16S rDNA fragments were obtained, and after alignment and distance calculation were categorized into 3,291 Operational Taxonomic Units (OTUs) at 97% similarity level. Bacteria species from 16 phyla were detected with the dominant group, from Proteobacteria, represented by 1982 OTUs, followed by Bacteriodetes and Actinobacteria. Environmental influences were also evaluated. Host-specific OTUs were recognized. Three main types of trends of the OTU dynamics during the host plant growing season were observed. Alphaproteobacteria was significantly more abundant in Asclepias viridis among the five host plant species, and also expanded greatly in July in the whole leaf endophytic bacterial community. Pyrosequencing data were also used to identify the dominant T-RFs, showing that the result of T-RFLP is consistent with pyrosequencing.