Metaprofiling of wheat phylloplane microbial endophyte communities

The potential of crop associated microbiota research is realized and being harnessed essentially for the benefit of plant/crop. In this scenario the need for optimized amplicon sequencing protocols is there for complex samples like phyllosphere endophytes which could face the problem of host originated sequences. Endophyte research usually consists of processes like surface sterilization to remove epiphytes and freezing of samples for storage. Also, in this regard, no systematic research has been done to evaluate how the sequence of freezing and sterilization effects the host associated microbiome identification. In this study, I have developed optimized protocols to profile the bacterial and fungal endophytic communities associated with matured wheat leaves in the field. Also I have studied the effect of timing/sequence of surface sterilization and freezing on the structure of endophytic microbial community data of wheat phylloplane. As a third objective I have analyzed the impact of field sites on alpha and beta diversities of bacterial and fungal communities. It was found that first frozen (FF) samples have higher fungal diversity while first sterilized (FS) samples have more bacterial diversity but not significant. Within the field, bacterial communities are more diverse than between the fields. While fungal communities are more different between the fields compared to within field diversity. This difference is also found to be statistically not significant. Zymoseptoria and Blumeria are significantly abundant in the samples from fungicide free plot of Karkendamm compared to the samples of Hohenschulen field.Table of Contents 1. Introduction ........................................................................................................................ 1 1.1 Methods for exploring microbial diversity.................................................................. 3 1.1.1 Culture Methods................................................................................................... 3 1.1.2 Culture-independent methods .............................................................................. 3 1.1.3 High throughput Metabarcoding .......................................................................... 5 1.2 Challenges in Phyllosphere endophytic microbial diversity research ......................... 7 1.2.1 Highly similar host cellular DNA sequences ....................................................... 7 1.3 Previous research in phyllosphere microbial diversity ............................................... 9 1.4 Thesis aim ................................................................................................................. 10 2. Materials and Methods ..................................................................................................... 12 1.5 Study design .............................................................................................................. 12 1.6 Sampling of wheat leaves .......................................................................................... 12 1.7 Leaf surface sterilization ........................................................................................... 12 1.7.1 Microbial growth assay ...................................................................................... 14 1.7.2 PCR assay .......................................................................................................... 14 1.8 Methods for DNA extraction .................................................................................... 14 1.8.1 Qiagen kit method .............................................................................................. 14 1.8.2 Modified CTAB (Cetyltrimethylammonium bromide) method ........................ 15 1.8.3 Phenol-Chloroform based DNA extraction protocol ......................................... 15 1.9 Design of blocking primers ....................................................................................... 16 1.10 Clone library method ................................................................................................. 17 1.11 Next-generation sequencing ...................................................................................... 17 1.11.1 Amplicon library preparation ............................................................................. 18 1.11.2 Amplicon Sequencing ........................................................................................ 18 1.12 Data Analyzes ........................................................................................................... 19 3. Results .............................................................................................................................. 21 1.13 Optimization of surface sterilization ......................................................................... 21 1.13.1 Results from Microbial growth assay ................................................................ 21 1.13.2 Results from PCR assay ..................................................................................... 21 1.14 Comparison of DNA extraction methodologies ........................................................ 22 1.14.1 Results from Qiagen kit method ........................................................................ 23 1.14.2 Results from modified CTAB method ............................................................... 23 1.14.3 Results from Phenol-Chloroform based DNA extraction method ..................... 23 1.15 Design of blocking primers ....................................................................................... 24 1.16 Preliminary screening of endophytes with Clone library method ............................. 27 1.17 Library preparation and Amplicon sequencing ......................................................... 28 1.18 Data pre-processing and OTU picking ...................................................................... 29 1.19 Structure of bacterial and fungal endophyte communities ........................................ 30 1.19.1 Taxonomy, relative abundances and taxa summary .......................................... 30 1.20 Diversity analyzes ..................................................................................................... 32 1.20.1 Alpha Diversity .................................................................................................. 32 1.20.2 Beta Diversity .................................................................................................... 34 1.20.3 PCoA and NMDS .............................................................................................. 35 1.21 Metabolic pathways prediction in bacterial communities ......................................... 36 1.22 Identification of differentially abundant taxa ............................................................ 37 4. Discussion ........................................................................................................................ 38 1.23 Highly efficient protocol for metaprofiling of wheat phyllosphere endophyte communities ......................................................................................................................... 38 1.24 The timing of sterilization and freezing process doesn’t have significant effect on the microbial diversity ......................................................................................................... 42 1.25 Structure of bacterial and fungal communities and the impact of field site on the microbial diversity ............................................................................................................... 43 1.26 Conclusions and future perspectives ......................................................................... 46 5. Acknowledgement ........................................................................................................... 48 6. References ........................................................................................................................ 4