Box 3: Designing a successful experiment
Manipulating microbes can be challenging, but microbial treatments can
be created by isolating some fraction of the plant microbiome, for
example isolates of individual taxa, microbial washes, or whole soil
inoculum (for a discussion of efficacy see Howard et al. 2017,
Fig. 4A). Defining proper controls is also critical. For example,
researchers should isolate soil microbial effects without confounding
them with soil abiotic effects by using a sterilized soil mixture
(either a sterilized field soil mixture; e.g. Petipas et al. 2020
or sterilized potting mix; Howard et al. 2020, Fig. 4B) with a
small amount (1-5% soil volume) of live or sterilized inoculum added
back. The choice of soil sterilization methods can be taxon specific
(e.g. fungicide) or general (autoclaving or gamma irradiation). Each
method has advantages and disadvantages, and each method often has
effects beyond direct removal of microbes (Trevors 1996). In addition,
given that even sterilized treatments are rapidly colonized by weedy
microbes, they should be more likely considered low diversity microbial
treatments rather than no or low microbial abundance communities (Lau &
Lennon 2012).
Transplantation can occur directly in the soil using a mesh (0.45µm)
barrier to keep in microbial inoculum and exclude microbial taxa (fungi
and some bacterial taxa) from the surrounding soil environment (Mcguire
2007). Alternatively, plants can be transplanted into pots with
microbial treatments and placed in aboveground arrays in the two
habitats, although aboveground conditions in pots are often warmer and
subject to more rapid drying than in ground transplants (Fig. 4C). When
transplanting into the field (and even the greenhouse), especially if
plants are surrounded by the soil environment, contamination by
non-target microbes will always be an issue as mentioned above. For this
reason, the duration of the experiment will need to be planned
carefully. Since microbe-mediated adaptation experiments are
simultaneously manipulating multiple factors, proper planning of sample
sizes is important to have the power to detect significant interactive
effects.
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Table 1: Hypothetical linear model terms used to understand
microbe-mediated adaptation reciprocal transplant experiments. We also
include potential interpretations when main effects or interactions are
found to be significant and how that interaction type might be
interpreted in a quantitative genetics framework.