Harvest, fluorescent analysis and molecular analysis
We harvested all plates three months after inoculation. We discarded
contaminated plates and plates in which the fungal network did not cross
into the central compartment. We removed roots from the plates and dried
them in paper bags and extracted extraradical hyphae from the MSR medium
as described in Whitesideet al. (2019). We weighed the dried root and fungal material and
subsampled the roots for fluorescent analysis (~7 mg)
and DNA extraction (~20 mg). We measured phosphorus
transfer to the roots by measuring the quantum-dot apatite fluorescence
in the focal root systems with a Bio-Tek Synergy MX plate reader as
described in Whitesideet al. (2019). To measure intraradical fungal colonization in the
whole plant greenhouse and the in-vitro root organ culture
experiment, and extraradical fungal abundance in the in-vitroroot organ culture experiment, we first isolated fungal DNA using the
DNeasy Plant Mini kit (Qiagen, Hombrechtikon, Switzerland), and then
analyzed the fungal abundance with real time (qPCR) on DNA as described
in Whiteside et al.2019. qPCR allowed us to obtain total copy numbers of intra- and
extraradical colonization in all replicates. It also allowed us to
distinguish between R. irregularis and R.aggregatum when grown in combination. In contrast, R.irregularis strain A5 and R. irregularis B12 are
too genetically similar to use qPCR to differentiate their abundances.
In those cases, only total abundance was measured.
Statistical
analyses
We performed all statistical analysis in R version 3.3.1. We tested all
data for normality with a Shapiro test and transformed data if
necessary. We analyzed the data using linear models, with the
independent variable as the partner strain (A5, B12 or Agg). We tested
the homogeneity of the variances with a Leneve’s test and checked the
distribution of the residuals by eye with a normal QQ plot. We produced
ANOVA type II tables with the R package car
(Fox et al. 2016).
To assess the statistical differences between the groups, we used a
Tukey HSD test as post-hoc test. We calculated the ratio of intraradical
colonization of focal/partner plant in the whole plant experiment by
dividing the intraradical colonization of the focal root over the
intraradical colonization of the partner root, (Engelmoer et al.2014) we then analyzed the logarithm of the ratio. For thein-vitro root organ culture experiment, we analyzed the logarithm
of the intraradical and the extraradical colonization because the
residuals were not normally distributed. We then calculated the
investment ratio as a metric to quantify fungal investment into
intraradical growth versus extraradical growth
(Engelmoer et al.2014), by dividing the total extraradical copy number per plate over the
total amount of intraradical copy number per plate. To calculate the
network efficiency, we calculated the amount of quantum-dot apatite per
total focal root over the extra-radical hyphal abundance in the focal
compartment.