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.