Experimental design of nutrient manipulations
Nutrient availability was manipulated in growth media differing in
complexity and C sources, namely defined glucose medium, defined medium
with cellulose as C source and soil-extract agar (SEA) manipulated by N,
P, glucose and cellulose additions. Eight fungal isolates covering all
three phyla were used (RLCS10, RLCS01, RLCS16, RLCS12, RLCS27, RLCS17,
RLCS28, RLCS09; see Table S1 for information on these isolates); in SEA
medium RLCS10 was replaced by RLCS22, since its growth in SEA was too
limited for further analyses.
Glucose medium . N and P availability were manipulated in defined
medium with known growth conditions and nutrient limitation patterns.
This medium was designed in accordance with the law of the minimum,
providing all elements and conditions in non-limiting supply to ensure N
or P limitation of fungal growth only (Camenzind et al. 2020).
The resulting medium contained 5 g L-1 glucose, 0.5 g
L-1 MgSO4, 0.5 g KCl, 0.1 g NaFeEDTA,
5 mg L-1 ZnSO4, 0.05 mg
L-1 Na2Mo4, 0.05 mg
L-1 MnSO4, 0.05 mg
L-1 H3BO4, 0.01 mg
L-1 CuSO4, 1 mg L-1Thiamine HCl, 0.05 mg L-1 biotin and 20 g
L-1 agar. N supply was manipulated by adding
NH4NO3 in a defined quantity as
universal fungal N source (Jennings 1995), five levels of N supply were
tested (molar C:N = 5, 20, 40, 80 and 200 (C:P = 100)). P availability
was manipulated similarly by adding
NaH2PO4 (molar C:P = 20, 100, 5000,
1000, 3000 (C:N = 20)), since Na was previously shown to not affect
fungal growth (Camenzind et al. 2018b). Levels of high N and P
supply were based on expected fungal demands derived from published
C:N:P contents (Mouginot et al. 2014; Zhang & Elser 2017),
whereas values for low nutrient supply were based on lower limits
reported in litter and soil (McGroddy et al. 2004; Cleveland &
Liptzin 2007). NH4NO3 or
NaH2PO4 additions did not change the pH
of the base medium (~ 4.5). In case of manipulations of
P supply, 16 g L-1 special purified agar was used
(A7921, Sigma-Aldrich, Darmstadt, Germany; 0.04 mg P
g-1 agar (ICP-OES analysis)).
Cellulose medium . For this experimental approach glucose was
replaced by cellulose (SigmaCell - highly purified fibers,
Sigma-Aldrich, St. Louis, US) maintaining molar C contents. In a
separate pre-test we confirmed that no other elements or conditions were
limiting and fungi were able to use cellulose as a C source, though to
varying degrees (Table S2). N was manipulated as described above.
Nutrient manipulation in SEA . In order to obtain a more natural
fungal growth substrate, we prepared an additional growth medium based
on soil extract. Soil sampled at the original grassland site was used
(average soil characteristics: pH 5.77, C content 11.9 mg
g-1, N content 0.9 mg g-1, P content
7.32 mg kg-1 (Horn et al. 2014)). Samples were
autoclaved, mixed thoroughly with demineralized H2O 1:3
(v:v) and sieved through 20 µm. The resulting liquid was supplemented
with 20 g L-1 agar. For nutrient manipulation, only
the addition of N and P is possible in uncontrolled organic substrate.
However, by concomitantly increased C availability by adding glucose
(Glu) or cellulose (Cel), respectively, nutrient limitation was induced
experimentally. Consequently, treatments comprised a control (Ctr;
non-manipulated SEA), +N, +P, +Glu, +Glu+N, +Glu+P, +Cel, +Cel+N and
+Cel+P. Glucose and Cellulose were added in equal molar C quantities,
mimicking conditions in controlled media – 5 g L-1glucose and 4.5 g L-1 cellulose. For N addition, 1.28
g L-1 NH4NO3 was
added, lowering soil C:N ratios of 15 experimentally to 5. P was added
as 0.27 g L-1 NaH2PO4to shift soil C:P ratios of 1500 to 100 (based on original soil sample
analyses). The parallel addition of C and N or C and P resulted in C:N
ratios of ~5.2 and C:P ratios of ~75,
respectively. pH of SEA was determined as 7.35, and slightly decreased
by N additions (7.14) and P additions (6.6), independently of C supply.
For media preparation, glucose/cellulose and phosphate were autoclaved
separately, since glucose may caramelize in the presence of salts, and
phosphate forms insoluble precipitates or provokes toxic conditions
(Moore et al. 2011; Tanaka et al. 2014).
In glucose and cellulose media, fungi were grown in petri dishes in the
dark for 12 days at 20°C (Ø 9mm), only the fast-growing isolate RLCS01
was kept for 7 days in glucose media, while RLCS28 was grown for 27 days
to obtain sufficient biomass. In SEA, all fungal strains were cultured
for 26 days to ensure sufficient biomass formation for analyses.