Summary
Although it used to be common practice, multiple studies now suggest that duplicate and triplicate PCR reactions are unnecessary for fungi and bacteria (Egen et al ., 2018; Marotz et al ., 2019). However, based on the results of this study, we recommend the inclusion of a different kind of technical replicate (i.e., multiple extraction reps from a single plant), in addition to biological replicates (multiple plants in a single population), especially when studying factors that may generate subtler differences in plant associated microbial communities. We show that extracting DNA from the standard 25-30 mg (dry weight) per plant can result in microbial communities that vary by as much as 100% and extractions of 250 mg from a single plant can vary by as much as 79%. The need for increased replication is particularly important if site, treatment, or seasonal differences may be obscured by other environmental drivers. Striving for a more comprehensive understanding of the depth and structure of plant microbiomes and their response to their surrounding environments will help us to better understand the exact functions of plant-microbe associations and how we might manipulate plant microbiomes in order to reduce disease or increase plant productivity in the future.
Sample size and sampling effort when studying plant-associated microbes have surpassed sequencing depth and cultivation as the bottleneck when characterizing microbial communities. Clearly, we need to be able to justify both effort and size when developing a sufficient sampling design. A good sampling design is essential to approximate underlying patterns in microbial community composition in a reproducible manner. Schloss (2018) elaborates on the concern of replicability and reproducibility with the growing use of Illumina-based studies of microbial communities, and describes PCR bias, sequencing errors, and cryptic or poorly described bioinformatics as preventing data from being generalizable to other environments. Undersampling and poor to absent descriptions of sampling effort and strategy also contribute to this problem, and the current frequency of undersampling should be concerning. The differences we see here between sampling strategies and the extreme variation among replicates suggest that many studies of plant-associated microbial communities may not be sufficiently replicable or reproducible.
Due to variation in community structure among AMF, bacteria, and non-AM fungi, standardizing a sampling protocol for all organisms is difficult, and best practices will, to some degree, depend on the question being asked. Since neither sampling approach appeared to outperform the other, in many studies the overall sampling effort may be of greater importance. For example, when investigating landscape-scale differences in abundant or species poor microorganisms, a smaller sampling effort is often sufficient. However, we suggest that more diverse plant-associated microbial communities, such as foliar fungal endophytes and root-associated bacteria, necessitate a more robust sampling effort than what is currently practiced in the literature. Per sample richness, relative to the estimated total community richness should always be considered when determining the optimum sampling strategy for any system. For example, sampling strategies and volumes sufficient for sampling AMF communities in extreme environments are likely not adequate for sampling fungal endophytes in the tropics where richness is high (Arnold et al ., 2000). The need for increased sampling effort is especially pertinent if noise associated with sites, treatments or sample processing may potentially obscure the differences among them. In studies that fail to see differences in microbial communities among sites or treatments, sampling effort should always be examined as a potential impediment.