Insufficient sampling constrains our characterization of plant microbiomes

Area of Science:

• Plant microbiology
• Microbial ecology
• Molecular sampling techniques

Background:

• Plants host diverse microbial communities, crucial for host health and ecosystem function.
• Standardized methods for sampling plant-associated microbes are lacking, leading to inconsistent findings.
• The consequences of different sampling strategies on microbial community characterization are not well understood.

Purpose of the Study:

• To compare the effectiveness of two common DNA extraction strategies for characterizing plant microbial communities.
• To assess the extent of undersampling for bacteria, arbuscular mycorrhizal (AM) fungi, non-AM fungi, and foliar fungal endophytes (FFE).
• To evaluate the impact of sampling intensity on microbial community composition and the detection of seasonal dynamics.

Main Methods:

• Comparison of two sampling strategies: homogenizing after subsampling vs. homogenizing bulk tissue before subsampling.
• Targeted analysis of bacteria, AM fungi, non-AM fungi in roots, and FFE in leaves of showy milkweed (Asclepias speciosa).
• DNA extraction from varying tissue masses to quantify FFE undersampling and assess seasonal variations.

Main Results:

• Bacterial and FFE community compositions differed significantly between the two sampling strategies.
• Increased DNA extraction mass (10x) led to a ~10x increase in FFE richness, confirming severe undersampling.
• Despite undersampling, seasonal patterns in FFE communities were detectable, indicating the influence of strong ecological drivers.

Conclusions:

• Current plant microbial sampling practices often lead to poor characterization of microbial communities, particularly FFE.
• Increased sampling intensity and extraction mass are crucial for enhancing reproducibility and detecting subtle microbial patterns.
• Future research should focus on optimizing sampling protocols to accurately capture the full diversity of plant-associated microbes.