Germination and growing conditions
We first performed whole plant greenhouse experiments. We usedMedicago truncatula as a host (genotype Jemalong A17, courtesy of
dr. Bettina Hause, Leibniz Institute of Plant Biochemistry, Halle,
Germany), as previously
(Kiers et al. 2011;
Whiteside et al. 2019). We scarified M. truncatula seeds
by submerging them in 95% sulfuric acid for 5-10 minutes, after which
we rinsed the seeds with excess ddH2O. We stored the
seeds in petri dishes on moist filters, first two days in the dark at 5
˚C, then one day at 20 ˚C in the dark, followed by two days in the light
at 20 ˚C. We planted the germinated seeds in autoclaved germination
soil. After 11 days, we selected seedlings of 3-4 cm with at least three
leaves to transplant to three-compartment boxes with a 6 L capacity
(Garcia et al.2006). The boxes were
divided into three equal compartments with a 50 µm pore size nylon mesh
(Cell Micro Sieves, Gentaur) glued onto a PVC window. This limited the
plant roots to the outermost compartments, but allowed the fungal hyphae
to grow in the central fungus-only compartment. We filled each
compartment with autoclaved quartz sand and supplemented the central
fungus-only compartment with 1 g hydroxyapatite per kilogram quartz sand
as a phosphorus source (Pel et al.2018). We planted oneM. truncatula seedling in each of the two outer compartments of
the three-compartment box.
As fungal inoculum, we homogenized in-vitro Ri T-DNA Daucus
carota L. transformed root organ cultures containing each fungus and
added 16 mL of the mixture to the roots (~700 spores).
We randomly assigned one plant as the “focal plant”. This focal plant
was consistently inoculated with the strain ‘A5’. The other root was
designated as the partner plant, and inoculated with either A5
(n =8), B12 (n =9) or Agg (n =9) (Fig. 1a). After
inoculation, we added 10 mL water to the roots and we fertilized the
plants once with an adjusted Hoagland solution with 25% phosphorus (5.5
mM KNO3; 4.0 mM
CaCl2.2H2O; 7.25 mM
NH4NO3; 0.5 mM
KH2PO4; 1.0 mM 20 mM
MgSO4.6H2O;
Fe(Na)EDTA; 1.0 mL/L micronutrients). We placed all experimental units
in a randomized grid, and we rotated them every two weeks to avoid local
effects. After a week, we covered the sand with a one cm layer of
sterile low-density polyethylene beads (Fardem Packaging, Edam, the
Netherlands) to limit evaporation. Temperature in the greenhouse
fluctuated between 20 ˚C and 30 ˚C. We watered the plants twice a week
with dH2O keeping the water content between 10 and 12.5
% and fertilized the plants every fourth day with 35 mL of an adjusted
Hoagland solution, containing no phosphorus but extra nitrogen (5.5 mM
KNO3; 4.0 mM
CaCl2.2H2O; 7.25 mM
NH4NO3; 0.5 mM KCl; 1.0 mM
MgSO4.6H2O; 20 mM
Fe(Na)EDTA; 1.0 mL/L micronutrients). To confirm that our three fungal
strains did not differ significantly in their mutualistic quality, we
also grew single M. truncatula plants in standard pots of 880 mL,
filled with sterile quartz sand. We inoculated host plants with either
A5, B12, or Agg, and grew and fertilized plants as above. We found no
statistically significant difference in either root or shoot biomass of
plants grown with our three strains, confirming that they did not
significantly differ in their nutrient provisioning strategy (Fig. S3).