Plant Microbiomes: Do Different Preservation Approaches and Primer Sets Alter Our Capacity to Assess Microbial Diversity and Community Composition?

Zhiguang Qiu, Juntao Wang, Manuel Delgado-Baquerizo, Pankaj Trivedi, Eleonora Egidi, Yi-Min Chen, Haiyang Zhang, Brajesh K. Singh

The microbial communities associated with plants (the plant microbiome) play critical roles in regulating plant health and productivity. Because of this, in recent years, there have been significant increase in studies targeting the plant microbiome. Amplicon sequencing is widely used to investigate the plant microbiome and to develop sustainable microbial agricultural tools. However, performing large microbiome surveys at the regional and global scales pose several logistic challenges. One of these challenges is related with the preservation of plant materials for sequencing aiming to maintain the integrity of the original diversity and community composition of the plant microbiome. Another significant challenge involves the existence of multiple primer sets used in amplicon sequencing that, especially for bacterial communities, hampers the comparability of datasets across studies. Here, we aimed to examine the effect of different preservation approaches (snap freezing, fresh and kept on ice, and air drying) on the bacterial and fungal diversity and community composition on plant leaves, stems and roots from seven plant species from contrasting functional groups (e.g. C3, C4, N-Fixers, etc.). Another major challenge comes when comparing plant to soil microbiomes, as different primers sets are often used for plant vs. soil microbiomes. Thus, we also investigated if widely used 16S rRNA primer set (779F/1193R) for plant microbiome studies provides comparable data to those often used for soil microbiomes (341F/805R) using 86 soil samples. We found that the community composition and diversity of bacteria or fungi were robust to contrasting preservation methods. The primer sets often used for plants provided similar results to those often used for soil studies suggesting that simultaneous studies on plant and soil microbiomes are possible. Our findings provide novel evidence that preservation approaches do not significantly impact plant microbiome data interpretation and primer differences do not impact the treatment effect, which has significant implication for future large-scale and global surveys of plant microbiomes.