Assessing Bacterial and Fungal Diversity in the Plant Endosphere

Plants are colonized various microorganisms including endophytes. These microbes can play an important role in agricultural production as they promote plant growth and/or enhance the resistance of their host plant against diseases and environmental stress conditions. Although culture-independent molecular approaches such as DNA barcoding have greatly enhanced our understanding of bacterial and fungal endophyte communities, there are some methodical problems when investigating endophyte diversity. One main issue are sequence contaminations such as plastid-derived rRNA gene sequences which are co-amplified due to their high homology to bacterial 16S rRNA genes. The same is true for plant and fungal ITS sequences. The application of highly specific-primers suppressing co-amplification of these sequence contaminations is a good solution for this issue. Here, we describe a detailed protocol for assessing bacterial and fungal endophyte diversity in plants using these primers in combination with next-generation sequencing.