Carbon availability affects already large species-specific differences in chemical composition of ectomycorrhizal fungal mycelia in pure culture

Abstract

Although ectomycorrhizal (ECM) contribution to soil organic matter processes receives increased attention, little is known about fundamental differences in chemical composition among species, and how that may be affected by carbon (C) availability. Here, we study how 16 species (incl. 19 isolates) grown in pure culture at three different C:N ratios (10:1, 20:1, and 40:1) vary in chemical structure, using Fourier transform infrared (FTIR) spectroscopy. We hypothesized that C availability impacts directly on chemical composition, expecting increased C availability to lead to more carbohydrates and less proteins in the mycelia. There were strong and significant effects of ECM species (R² = 0.873 and P = 0.001) and large species-specific differences in chemical composition. Chemical composition also changed significantly with C availability, and increased C led to more polysaccharides and less proteins for many species, but not all. Understanding how chemical composition change with altered C availability is a first step towards understanding their role in organic matter accumulation and decomposition.

Supplementary Information
The online version contains supplementary material available at 10.1007/s00572-023-01128-2.

Full text

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Mycorrhiza (2023) 33:303–319
https://doi.org/10.1007/s00572-023-01128-2
RESEARCH
Carbon availability affects already large species‑specific differences
inchemical composition ofectomycorrhizal fungal mycelia inpure culture
PetraFransson1 · A.H.JeanRobertson2· ColinD.Campbell2
Received: 24 February 2023 / Accepted: 27 September 2023 / Published online: 12 October 2023
© The Author(s) 2023
Abstract
Although ectomycorrhizal (ECM) contribution to soil organic matter processes receives increased attention, little is known
about fundamental differences in chemical composition among species, and how that may be affected by carbon (C) avail-
ability. Here, we study how 16 species (incl. 19 isolates) grown in pure culture at three different C:N ratios (10:1, 20:1, and
40:1) vary in chemical structure, using Fourier transform infrared (FTIR) spectroscopy. We hypothesized that C availability
impacts directly on chemical composition, expecting increased C availability to lead to more carbohydrates and less proteins
in the mycelia. There were strong and significant effects of ECM species (R2 = 0.873 and P = 0.001) and large species-specific
differences in chemical composition. Chemical composition also changed significantly with C availability, and increased C
led to more polysaccharides and less proteins for many species, but not all. Understanding how chemical composition change
with altered C availability is a first step towards understanding their role in organic matter accumulation and decomposition.
Keywords Carbohydrates· Cell wall composition· Fouriertransform infrared spectroscopy· Mycelia· Soil organic
carbon· C:N ratio
Introduction
The majority of plants form symbiosis with root-associated
fungi, and these fungi play critical roles in carbon (C) and
nutrient cycling. In northern forest biomes, ectomycorrhizal
(ECM) fungi belonging to Ascomycota and Basidiomycota
are most abundant (Brundrett 2009), and the influence of
ECM fungi on nutrient uptake is well documented. The
mycorrhizal contribution to soil organic matter processes
is receiving increased attention, as we are beginning to
recognize the importance of the ECM mycelium for soil
C accumulation and as potential drivers of organic matter
decomposition and related losses of C (Adamczyk etal.
2019; Averill and Hawkes 2016; Godbold etal. 2006;
Lindahl etal. 2021; Lindahl and Tunlid 2015; Zak etal.
2019). Belowground litter inputs may be more important
than aboveground litter input in many ecosystems (Kätterer
etal. 2011); Clemmensen and colleagues (2013) suggested
that organic layers grow from below through the continuous
addition of recently fixed C to the organic matter profile in
the form of remains from roots and associated mycelium.
Plants allocate up to 20% of their photosynthetically fixed
C belowground to mycorrhiza (Hobbie 2006), resulting in a
large standing biomass of extraradical mycelia in soils. Up
to 1.2kg mycelia may be produced per hectare every day in
young Scots pine forest (Hagenbo etal. 2017), and the ECM
mycelial production in coniferous forests has been estimated
to be several hundred kilogram per hectare per year in the
upper part of the soil (Ekblad etal. 2013), which is in the
same range as the biomass of fine roots. Mycelial turnover
will depend on chemistry, C:N stoichiometry, and morphol-
ogy as well as decomposer community activity and physical
protection by soil (Fernandez etal. 2016); therefore, organic
matter accumulation will be affected by which fungal spe-
cies are present in the community, and what their chemical
compositions are. For example, the presence of recalcitrant
chemical constituents such as melanin, oxidized polymers
of phenolic materials that require oxidative enzymes for
degradation, will potentially decrease decomposition rates
and at the same time increase organic matter accumulation
(Fernandez etal. 2016), while mycelia with high nitrogen
* Petra Fransson
petra.fransson@slu.se
1 Department ofForest Mycology andPlant Pathology,
Uppsala BioCenter, Swedish University ofAgricultural
Sciences, PO Box7026, SE-75007Uppsala, Sweden
2 The James Hutton Institute, Craigiebuckler,
AberdeenAB158QH, Scotland
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304 Mycorrhiza (2023) 33:303–319
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(N) concentration are subject to higher decomposition rates
(Koide and Malcolm 2009). The chemical composition of
ECM mycelia is thus key to a better understanding of which
factors may drive soil organic matter accumulation.
The fungal cell wall is central for the chemical composi-
tion of mycelia since up to 50% of the fungal biomass is
found in the cell wall (Ruiz-Herrera 1991). This represents
a considerable metabolic investment, and cell wall compo-
sition of different fungi is diverse and varies depending on
species, genotype, age and environment (Bowman and Free
2006; Feofilova 2010; Wessels 1994). Although the com-
position of non-cell wall components also can alter due to
environmental conditions (e.g., heat; Tereshina etal. 2011),
the cytoplasmic fraction of fungal tissue does not likely play
a significant role in the decomposition of ECM mycelia
since cytoplasm is moved to active parts of the mycelium
at senescence resulting in vacuolated hyphae (Cooke and
Rayner 1984). Furthermore, cytoplasm is highly labile and
readily taken up by decomposers (Nakas and Klein 1979).
The composition of the cell wall was suggested to exert the
most control on long-term decomposition of ECM fungal
necromass (Fernandez etal. 2016), and melanin and N
concentrations appear to be key biochemical controllers of
decomposition in fungal litter (Fernandez and Koide 2014).
The major cell wall component is polysaccharides (up to
80–90% of dry mass; Bartnicki-Garcia 1968), but there are
also lipids (up to 3%; Feofilova 2010) and glycoproteins
(15–30% of dry mass) with varying content, function, and
chemistry (Fernandez etal. 2016). The main structural ele-
ments are chitin microfibrils (the fungal-specific crystalline
homopolymer of linked N-acetyl-D-glucosamine residues)
and β-glucans (homopolymers of glucose making up the
central core of the cell wall), embedded in an amorphous
gel-like matrix (Feofilova 2010). Chitin, an important source
of N (Leake and Read 1990), is the structural equivalent
to plant lignin and concentrations range from ca. 1 to 10%
in ECM fungi (Ekblad etal. 1998; Fernandez and Koide
2012), while β-glucans constitute approximately 50–60%
of dry mass (Kapteyn etal. 1999). Cell walls also consist
of monosaccharides (glucose, mannose, and xylose), amino
acids, and hydrophobins, which are secretory surface pro-
teins important for ECM formation (Fernandez etal. 2016),
hydrophobicity, and adherence to substrata (Feofilova 2010).
Older parts of the mycelia have increased levels of lipids
and pigments such as melanins, contributing to surface
properties and protection against stressors. Since we do not
know how ECM chemical composition varies between spe-
cies or in relation to different growth conditions, a better
understanding of their mycelial properties would improve
our understanding of how different ECM communities may
potentially feedback on soil organic matter processes.
Fourier transform infrared (FTIR) spectroscopy is a
widely used technique which gives the overall chemical
composition of a sample, including both organic and
inorganic components. The FTIR spectrum of a sample
arises from an interaction between IR radiation and the
vibrations of molecules present within the sample. IR
radiation is absorbed at frequencies which directly relate to
the frequencies of vibrations of functional groups present
in the sample, and the IR absorption bands observed
relate to all the constituents which make up its chemical
composition. The technique is thus capable of distinguishing
the principal chemical classes in, e.g., cell walls, and can
therefore provide a unique insight into differences in
chemical composition between samples of different species
or for samples of a single species grown under different
conditions. The technique has been extensively used for
bacterial cell walls, and to a growing extent to examine
fungi. Studies are published on changes in chemical
composition during hyphal growth and morphogenesis
(Adt etal. 2006; Jilkine etal. 2007; Szeghalmi etal.
2007), detection of fungi in different plant substrates
(Kos etal. 2002; Naumann etal. 2005), differentiation of
plant pathogen isolates, molds, and fungi in food industry
(Lecellier etal. 2015; Pomerantza etal. 2014; Salman etal.
2012; Shapaval etal. 2010), and processes involving fungi
such as substance transformations, decomposition, and
carbon sequestration (Jeewani etal. 2021; Mrnka etal.
2020). The method has also been suggested to distinguish
between fungal species (Adt etal. 2006; Essendoubi etal.
2005; Fischer etal. 2006; Lecellier etal. 2015; Naumann
etal. 2005; Rellini etal. 2009; Santos etal. 2010). Most
studies of species’ differences have used fungal isolates
grown under controlled growth conditions (e.g., Lecellier
etal. 2014; Naumann etal. 2005) to avoid alterations of
spectra due to fungal responses to environmental changes
(Pena etal. 2014). However, for mycorrhizal fungi, this
technique had not been commonly used. In a study by Pena
etal. (2014), the suitability and limitations of FTIR for the
distinction of ECM roots in field samples were tested. The
use of insitu collected material showed that environmental
variability did not limit discrimination between species. It
is worth noting though that ECM roots of the four species
tested are morphologically very distinct from each other. The
knowledge about the chemical composition in ECM fungal
mycelia based on FTIR analysis is thus still very limited.
Since ECM fungi are supported by belowground C alloca-
tion of plant photoassimilates (Hobbie 2006), and C avail-
ability is one key factor determining mycelial production
(Ekblad etal. 2013), mycelial production will be affected
by changes in C availability. We hypothesized that C avail-
ability impacts not only the production of mycelia but also
directly on the chemical composition of mycelia and expect
an increased C availability to lead to more carbohydrates
and less proteins. This was tested by growing 16 different
ECM fungal species in liquid media at three different C:N
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305Mycorrhiza (2023) 33:303–319
1 3
ratios, after which the chemical composition was determined
using FTIR analysis. By using a pure culture setup, we could
control for variation due to growth environment. Our sec-
ondary aim was to determine if FTIR spectroscopy could
be used to distinguish between different ECM species and
what this tells us about genetically determined differences.
We addressed the following questions: (1) Are ECM fungal
species different in chemical composition under the same
growth conditions? (2) Can ECM fungal species be distin-
guished from one another under the same growth conditions
by their chemical composition? (3) What are the potential
differences in chemical composition among ECM species
related to structurally? (4) How does increased C availability
affect chemical composition? (5) Are potential differences in
chemical composition among ECM species related to puta-
tive ecological role and/or functional traits? (6) How use-
ful is FTIR as a method to distinguish ECM species and to
explain differences in chemical composition? Understanding
differences in chemical composition of ECM fungi, and how
they change with altered C availability, is a key step towards
understanding their role in organic matter accumulation and
decomposition.
Materials andmethods
Fungal strains andexperimental conditions
A total of 19 fungal isolates, covering 16 different ECM
species, were used in the study (Table1). Identity of these
strains was confirmed by ITS sequence analysis using stand-
ard techniques (see Fransson and Johansson 2010). DNA
was extracted according to Gardes etal. (1991), and poly-
merase chain reactions (PCR) were carried out using the
primers ITS1 and ITS4 as described by White etal. (1990).
Fungal cultures were kept in darkness at 25°C on 25mL
agar plates containing cellophane covered Modified Melin
Norkrans (MMN) medium at pH 5.5, with the following
modifications: 2.5g L−1 glucose, 10g L−1 malt extract,
15g L−1 agar (Marx 1969). Fungal inoculum (three pieces à
5 × 5mm) were cut from the actively growing mycelial front
of each plate with a scalpel and placed in 90-mm Petri dishes
containing 30mL liquid Basal Norkrans (BN) medium
(Norkrans 1950) at pH 4.5. Care was taken to move the fun-
gal inoculum without any agar and cellophane; however, the
isolate of Laccaria laccata dissolved the cellophane which
made it difficult to avoid the agar completely for this par-
ticular species. The spectra for replicates of this species did
match closely; suggesting contamination was not an issue.
Petri dishes were sealed using Parafilm®.
To test for potential effects of C availability all fungi were
grown in duplicates at 25°C for 3weeks at three different
C:N ratios: 10:1, 20:1, and 40:1, containing a constant N
content of 0.236g L−1 (NH4)2SO4 and a C content of 1.25,
2.5, and 5g L−1 glucose, respectively. The C:N ratios were
chosen based on the direct measurements of field collected
mycelia reporting values of ca. 20:1 (Wallander etal. 2003),
and in effect moving from conditions of C limitation (C:N
10:1) to conditions of N limitation (C:N 40:1). At harvest,
fungal biomass was collected on muslin fabric, washed with
50mL double distilled H2O and freeze-dried using a Scan-
Vac™ CoolSafe 55–9 freeze drier (ScanLaf A/S, Denmark).
Biomass dry weight was recorded, and samples were ground
to a fine powder using a ball mill.
FTIR spectroscopy
Spectral characterization of fungal samples was performed
by FTIR spectroscopy. IR spectra were recorded on a Bruker
Vertex 70 FTIR spectrometer (Bruker, Ettlingen, Germany)
fitted with a potassium bromide beam splitter and a deutro-
glycine sulfate detector for two replicates of each species. A
Diamond Attenuated Total Reflectance (DATR) sampling
accessory, with a single reflectance system, was used to
produce “transmission-like” spectra, then the IR transmit-
tance spectra were converted to absorbance spectra using
the spectral software which applies the formula A = log10T
where A is absorbance and T is transmittance. Samples
were placed directly on a DATR/KRS-5 crystal, and a flat
tip powder press was used to achieve even distribution and
contact. Spectra were acquired by averaging 200 scans at
4 cm−1 resolution over the range 4000–370 cm−1. A correc-
tion was made to the ATR spectra to allow for differences
in depth of beam penetration at different wavelengths, using
OPUS software (Bruker, Ettlingen, Germany, version 6.0).
The spectra were also baseline corrected. No correction was
required for water vapor and CO2 as the spectrometer is con-
tinuously flushed with dry air. Technical replication formed
part of the performance checks for the FTIR instrument and
method used. FTIR data was normalized by subtraction of
the minimum value and subsequent division by the average
of all data points per sample prior to statistical analyses.
FTIR raw data has been deposited in Dryad (Fransson etal.
2023).
Statistics
FTIR data were analyzed by principal components analysis
(PCA) using Genstat for Windows (8th edition, VSN Interna-
tional, UK), followed by canonical variate analysis (CVA) of
the PC scores using CAP (Community Analysis Package: a
multipurpose package for community data, including classifi-
cation and ordination) software (see Anderson 2004; Anderson
and Willis 2003) for visualization of data. Data was stand-
ardized using David Hirst’s standardization (subtract mean
and divide by standard deviation on a per sample basis). The
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306 Mycorrhiza (2023) 33:303–319
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Table 1 Ectomycorrhizal fungal isolates used to study chemical composition after growth in liquid medium under three different C:N ratios
(10:1, 20:1, and 40:1), and classification of fungal species according to their putative ecological role and traits
a Isolated from ectomycorrhizal root tip
b Mycorrhizal root tip name (Brand etal. 1992) corresponding to the characteristic black mycorrhizas formed by ascomycetous fungi in the
Rhizoscyphus ericae species complex, now referred to as Meliniomyces bicolor Hambleton and Siegler, sp. nov. (Hambleton and Sigler 2005)
c Isolated from fruitbody by P. Fransson
d Sequenced by Anna Rosling (Rosling etal. 2004)
e Sequenced by Trude Vrålstad (Vrålstad etal. 2000)
f Pattern of mycelial differentiation according toAgerer (2001)
g Taxonomic level set to class
h Accordning toHobbie and Agerer (2010)
i The genus Hebeloma classified as uncertain hydrophobicity according to Hobbie and Agerer (2010); the isolate displays hydrophobic properties
in liquid culture
ECM fungal
isolate Isolate code GenBank
accession No Exploration
type fOrder Hydrophobicity hN tolerance lSuccession p
Amanita muscaria
(L.:Fr.) Hook UP3 DQ179118 Medium-distance/
smooth Agaricales Hydrophobic High Early
Amphinema
byssoides
(Pers.:Fr.) J.
Erikss
A705 EF493272 Medium-distance/
fringe Atheliales Hydrophobic Low(-high) mEarly
Cenococcum
geophilum Fr. aVe-95–12 DQ179119 Short-distance Dothideomycetes
gHydrophilic Low(-high) mMulti
Cortinarius
glaucopus
(Sch.:Fr) Fr
UP21 DQ179120 Medium-distance/
fringe Agaricales Hydrophobic Low Late
C. scaurus (Fr.:
Fr.) Fr UP22 - Medium-distance/
fringe Agaricales Hydrophobic Low Late
Hebeloma velutipes
Bruchet UP184 AF432845 dShort-distance Agaricales Hydrophobic iHigh nEarly-mid
Hebeloma sp. Sp 1 - Medium-distance/
fringe Agaricales Hydrophilic High Early-mid
Laccaria bicolor
(Maire) Orton CRBF581 DQ179121 Medium-distance/
fringe Agaricales Hydrophilic High Early
L. laccata
(Scop.:Fr.) Berk.
and Br
Sk33 - Medium-distance/
fringe Agaricales Hydrophilic High Early-mid
Paxillus involutus
(Batsch:Fr.) Fr G05 DQ179126 Long-distance Boletales Hydrophobic High Late?
“Piceirhiza
bicolorata” a, b ARON2938.S AJ430152 eContact Helotiales Hydrophilic/
hydrophobi jLow oLate
Piloderma
byssinum (P.
Karst.) Jülich a
UP185 DQ179124 Medium-distance/
fringe Atheliales Hydrophobic Low Late
P. fallax (Liberta)
Stalpers aUP113 DQ179125 Medium-distance/
fringe Atheliales Hydrophobic Low Multi
Rhizopogon
roseolus (Corda)
Th. M. Fr
UP175 DQ179127 Long-distance Boletales Hydrophobic kLow Late
Suillus bovinus
(L.:Fr.) Roussel BL DQ179128 Long-distance Boletales Hydrophobic Low Late
UP63c-
UP592 EF493250
S. variegatus
(Sw.:Fr.) O.
Kuntze
UP60 DQ179130 Long-distance Boletales Hydrophobic Low
UP597 EF493256
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307Mycorrhiza (2023) 33:303–319
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PCA step was used to reduce the number of variables for CVA
so that the sample number exceeded the number of variates
for CVA, and the minimum numbers of PC scores needed to
explain more than 95% of the total data variability were used
for CVA. The adjusted loadings (or coefficients of linear dis-
criminants for each absorbance value) for the first two original
variates were plotted to indicate informative regions of the
FTIR spectra. To test for overall treatment effects and parti-
tion the variation in FTIR spectra depending on ECM species
and C:N ratio, permutational multivariate analysis of variance
(perMANOVA) was run based on the first two variates, with
Euclidean distance and 999 permutations using the adonis2
function from R (R core team 2023) Vegan package (Oksanen
etal 2022). Since the treatments Piloderma fallax and Suillus
variegatus UP60 were not fully replicated, they were excluded
from the perMANOVA. Term position was changed to ensure
that order does not matter, and in a second analysis, biomass
was added as a covariate term (additive). Additionally, fungal
species were classified according to their putative ecological
role and/or according to functional traits (Table1), including
exploration type (pattern of mycelial differentiation; Agerer
2001), hydrophobicity (Hobbie and Agerer 2010), N toler-
ance (based on where ECM root tips were found along a N
deposition gradient; Lilleskov etal. 2002), and successional
stage (based on when during forest succession the species is
preferentially found, adapted from Deacon and Fleming 1992).
These classifications into groups (exploration types: contact,
short-distance, medium-distance smooth, medium-distance/
fringe and long-distance; hydrophobicity: hydrophobic,
hydrophilic and hydrophobic/hydrophilic; N tolerance: low,
low(-high), and high; and successional stage: early, early-mid,
late, and multi) were used to potentially explain the variation
in chemical composition. The Multi Response Permutation
Procedure (MRPP) was applied to test whether there was a
significant difference between the groups, using Euclidean dis-
tance and 999 permutations in the R-package Vegan (Oksanen
etal 2022). MRPP provides a P value and an A value that is a
measure of effect size, representing homogeneity within the
group compared to that expected randomly. For example, per-
fect homogeneity in the group gives A = 1 whereas A values
between 0 and 1 indicate that heterogeneity between the groups
is more than that expected by chance. As a proxy for mycelial
C:N ratio, we semi-quantitatively compared the relative height
of the main C-O stretching vibration (~ 1030 cm−1, associated
with polysaccharides) to the relative height of the amide bands
(amide I ~ 1650, amide II ~ 1550 cm−1, associated with N) for
Amanita muscaria and Laccaria bicolor. Two-way ANOVA
was used to test for effect of fungal species and C availabil-
ity (C:N ratios 10:1, 20:1, and 40:1) on biomass dry weights,
using Minitab 16.0 (Minitab Inc., State College, PA, USA).
When interpreting the IR spectrum of a sample as a “chemi-
cal fingerprint,” the important thing to note, when comparing
spectra (e.g., biological replicates), is whether the same bands
are present (no additional or missing bands) and if the bands
are present with comparable relative intensities. This would
indicate a very similar chemical composition. Furthermore,
the reproducibility of ECM fungal spectra was evaluated based
on the average coefficient of variation for the two replicate
IR spectra for each treatment, after first calculating the coef-
ficients of variation (standard deviation/sample average) for all
individual wavenumbers (n = 1894). The lower the coefficient
of variation the higher is the reproducibility. Three treatments
without replicates were excluded (P. fallax C:N 20:1, and S.
variegatus 20:1 and 40:1). Outliers were excluded from the
calculation of averages when the coefficients of variation for
individual wavenumbers exceeded 240. This was applicable
to seven treatments in total, where either one (six treatments)
or two individual wavenumbers (one treatment) were affected
(see TableS1 for details).
Results
Characteristics andreproducibility ofthefungal
FTIR spectrum
In the case of ECM samples, the main constituents of the
cell walls typically include polysaccharides, lipids, pro-
teins, chitin, monosaccharides, amino acids, and β-glucans.
The FTIR spectrum of the fungal mycelium (Fig.1) was
characterized by a strong, broad OH stretching absorb-
ance band at ~ 3300 cm−1 and a strong C-O stretching
j Predominantly hydrophilic according to Agerer (2006); the isolate displays both hydrophilic and hydrophobic properties in liquid culture
k The genus Rhizopogon has either hydrophobic or hydrophilic rhizomorphs according to Hobbie and Agerer (2010); the isolate displays hydro-
phobic properties in liquid culture
l Based on where species from ECM root tips were found predominantly over a N deposition gradient according toLilleskov etal. (2002)
m Low N level dominating species according to Lilleskov etal. (2002), but frequently occurring in nurseries (Menkis etal. 2005; Rudawska etal.
2006) and after fertilization (Fransson etal. 2000)
n Intermediate N level dominating species according to Lilleskov etal. (2002), but the genera is frequently occurring in nurseries (Rudawska
etal. 2006)
o Meliniomyces sp. decrease in abundance with increasing N availability (Kjøller etal. 2012; Dean etal. 2014)
p Successional stage, adapted from Deacon and Fleming (1992) and Twieg etal. (2007), and references therein
Table 1 (continued)
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308 Mycorrhiza (2023) 33:303–319
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absorbance band at 1030 cm−1 which both relate to the
polysaccharides, present in relatively high proportions.
Sharp CH2 stretching absorbance bands at 2920 and
2850 cm−1 are indicative of long chain C compounds, or
waxy compounds, such as alkanes, acids, or esters. The
ester functional group can be detected by an absorption
band at around 1740 cm−1, with acids at a lower frequency,
closer to 1710 cm−1. Amide functional groups (amide
I ~ 1650 cm−1 and amide II ~ 1545 cm−1), which can arise
from amino acids, chitin, or proteins, also form a distinc-
tive part of the fungal spectrum. An NH stretching peak at
3260 cm−1 may be detectable but is sometimes concealed
by the broad OH stretch. Water contributes to the broad OH
stretching absorbance band and a related OH bending band
at ~ 1630 cm−1. This band often overlaps with the amide I
band, as does a band due to a carboxylate functional group.
Other features, such as the presence of aromatic stretching
absorbance bands (~ 1510 cm−1), can indicate the pres-
ence of phenolic compounds, e.g., melanin. In addition,
CH stretches for unsaturated CH or for the CH3 functional
group can sometimes be detected (3010 and 2870 cm−1,
respectively) in the fungal spectrum. Reproducibility of
fungal spectra was high, and the average coefficient of
variation when comparing replicates within treatments
(ECM species × C:N ratio) ranged between 0.05 and 15.4%
(TableS1), with 48 out of 54 treatments below 10%. The
replicate spectra for each of the specific C:N ratios for
Rhizopogon roseolus could be almost exactly overlaid
(Fig.S1), indicating how close their chemical compositions
were. This was also the case for each of the other species
studied (not shown).
Ectomycorrhizal fungal species differences inchemical
composition underthesame growth conditions
The IR spectra of different ECM fungal species, grown
under the same conditions, showed noticeable differences
from each other, as illustrated in Fig.2 (see Fig.S3 for spec-
tra plotted overlaid). All consisted of similar components,
e.g., predominantly polysaccharide and amide/protein, but
with different relative proportions and some other differ-
ences such as the extent to which waxy compounds were
present, or how distinct bands relating to ring linkages were.
For example, Amphinema byssoides, Cenococcum geophi-
lum, and Cortinarius scaurus and Piloderma byssinum all
had relatively high amide/protein content in relation to the
polysaccharide, whereas Piceirhiza bicolorata and Suillus
bovinus had a far greater proportion of polysaccharide rela-
tive to amide/protein (Fig.2). Piceirhiza bicolorata also had
very intense absorption bands consistent with an unsaturated
waxy ester compound (Fig.2b), which the spectra of the
other species did not show.
Absorbance
Wavenumbers (cm
)
3500 3000 25002000150010004000
-1
500
OH, polysaccharide,
water unsaturated
CH
CH2 long chains
CH3amide I, carboxylate,
water
C-O polysaccharide
amide II
aromatic
C=C
ester
acid
0.1
0.2
0.3
0.4
0.5
0.6
Fig. 1 Characteristic bands of the major biochemical descriptors in ECM fungal FTIR spectra; based on fungal mycelia (P. bicolorata) grown in
liquid culture at C:N ratio 20:1, with an inorganic N source ((NH4)2SO4)
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309Mycorrhiza (2023) 33:303–319
1 3
Differences in chemical composition among the 19
isolates were visualized in the CVA (Fig.3a), where the
first three CVA axes together accounted for 98% of the
variation. The adjusted loadings for the first two variates
showing which regions of the FTIR spectra are important
for separating samples in multivariate space are shown in
Fig.S2. There were strong and significant effects of ECM
species (perMANOVA F = 266.2, R2 = 0.873, and P = 0.001;
Table2) and large species-specific differences in chemical
composition (Fig.3a). Species were generally separated from
each other in multivariate space within a single C:N ratio
treatment (Fig.S4), with few exceptions. In C:N ratio 10:1
Hebeloma sp. 1 and S. bovinus UP592, Hebeloma velutipes
and R. roseolus overlapped with each other, and in C:N ratio
40:1 H. velutipes and S. bovinus UP63 overlapped. The two
isolates of S. variegatus (UP597 and UP60) were similar in
chemical composition in C:N ratio 40:1. The spread among
species within the individual treatments was similar irre-
spective of C availability (Fig.S4). The separation of species
in multivariate space was therefore primarily explained by
species’ differences. However, when taking all three C:N
ratios into account at the same time species start overlapping
(Fig.3a), and only some species (e.g., C. geophilum, A. bys-
soides, and Paxillus involutus) remained clearly separated
from all the other species. Closely related species pairs such
as the two Piloderma spp., the two Cortinarius spp., and the
two Hebeloma isolates grouped relatively closely together
within genus in the ordination (Fig.3a), and the Piloderma
spp. were also clearly separated as a group from all other
species. The similarity within these genera was also obvi-
ous from the IR spectra (not shown). Laccaria bicolor and
L. laccata, on the other hand, showed a large spread from
each other in the multivariate analysis, also within sepa-
rate C:N treatments (Fig.S4). This separation was possibly
Absorbance
3500 30002500 2000 1500 10004000
0.1
0.2
0.3
0.4
0.5
0.6
Wavenumbers (cm )
-1
0.7
500
0.8
0.9
1.0
1.1
1.2
1.3
1.4
1.5
1.6
C. scaurus
C. geophilum
A. byssoides
A
3500 30002500 200015001000 500
S. bovinus
P. bicolorata
P. byssinum
B
Fig. 2 Species variation in FTIR spectra for different ECM fun-
gal species, showing (A) A. byssoides, C. geophilum and C. scarus,
and (B) P. bicolorata, P. byssinum and S. bovinus. Mycelia were
grown in pure culture at C:N ratio 20:1 with an inorganic N source
((NH4)2SO4). Absorbance (spectral signals) was normalized to give
relative absorbance/abundance, and spectra are shown off set. Black
arrows in a indicate high amide/protein content in relation to the
polysaccharide. Black arrows in b indicate the intense amide peaks
seen in P. byssinum, and dashed arrows indicate a greater proportion
of polysaccharide relative to amide/protein
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310 Mycorrhiza (2023) 33:303–319
1 3
Am. byssoides
H. velutipes
P. involutus
Pi. bicolorata
Cort. scaurus
S. bovinus BL
L. bicolor
Heb. sp.
S. bov UP592
S. bov UP63
S. var UP597
S. var UP60
R. roseolus
Pilo. fallax
Pilo. byssinum
L. laccata
C. geophilum
Cort. glaucopus
A. muscaria
A
B
CVA axis 1 (80.6%)
CVA axis 2 (13.8%)
C:N 10:1
C:N 20:1
C:N 40:1
10 mg
25 mg
50 mg
Fig. 3 Large species-specific differences in chemical composition,
and varied responses to increased C availability. Canonical variate
analysis of FTIR spectra for ECM fungal isolates grown for 3weeks
in liquid media at three different C:N ratios (10:1, 20:1, and 40:1)
with an inorganic N source ((NH4)2SO4), showing the difference
between (a) fungal species, and (b) C:N ratios. For each treatment,
there were two replicates. Biomass for each individual sample is indi-
cated by the size of the circle in b, and arrows exemplify the direc-
tion of change in chemical composition when a species experienced
increasing C availability
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311Mycorrhiza (2023) 33:303–319
1 3
explained by differences in relative proportions of chitin.
The L. laccata spectra show absorption bands in the polysac-
charide region (1200–900 cm−1) which have a pattern very
similar to that of chitin and also show a relatively strong
band at 1311 cm−1, which is also present in the spectrum
of chitin. Conversely the L. bicolor spectra have a different
pattern of bands in this region (more like reference spectra
of isomaltose, possibly more glucan like). The suilloid iso-
lates (6 isolates corresponding to three species: S. bovinus,
S. variegatus, and R. roseolus) grouped together (however
fully overlapping with other species) with the exception of
S. bovinus BL that differed in chemical composition (Fig.3).
This was mainly due to the presence of an aromatic peak and
less distinct polysaccharide backbones in S. bovinus BL but
not in the other suilloid isolates (Fig.S5).
Effects ofincreasing C‑availability
onchemical composition
FTIR data showed a significant effect of C:N ratio on chemi-
cal composition, but C:N ratio explained a much smaller part
of the variation compared to ECM species (perMANOVA
F = 23.9, R2 = 0.0098, and P = 0.001; Table 2). However,
there was also a significant interaction between ECM
species and C:N ratio explaining 11% of the variation in
chemical composition (perMANOVA F = 16.3, R2 = 0. 107,
and P = 0.001; Table2). The multivariate analysis showed
varied responses in terms of chemical composition among
different species, and also among isolates of the same
species, to increasing C:N ratio (Fig.3b). The responses
ranged from no shift in chemical composition irrespective
of C availability (A. byssoides and P. fallax; Fig.3a), to
large differences in composition so that the C:N ratio
treatments were completely separated (e.g., P. bicolorata,
L. bicolor, and A. muscaria; Fig.3a). Separation in mul-
tivariate space was significantly explained by exploration
type (MRPP A = 0.2034, P = 0.001; Fig.4), hydrophobicity
(MRPP A = 0.112, P = 0.001; Fig.S6a), N tolerance (MRPP
A = 0.08632, P = 0.001; Fig.S6b), and succession (MRPP
A = 0.05828, P = 0.001; Fig.S6c) when ECM fungal species
were classified according to these traits (see classifications
in Table1). When classifying ECM species according to
their pattern of mycelial differentiation and putative ecologi-
cal roles, the chemical composition of short-distance explo-
ration types was mostly separated from medium-distance
fringe subtypes, while long-distance exploration types partly
overlapped with both former groups (Fig.4).
The IR spectra for different species varied mark-
edly in response to different C:N ratios, and four groups
of responses could be distinguished. Firstly, many of the
most obvious changes were related to the relative propor-
tions of the amide (or protein) bands and the polysaccha-
ride bands, with a clear reduction in amide with increasing
C availability (e.g., A. muscaria; Fig.5a; see Fig.S7 for
the same spectra plotted overlaid). Amanita muscaria also
had very distinct ring linkages (1074, 572, and 528 cm−1).
The pattern with increasing polysaccharide bands was also
similar but less pronounced for P. bicolorata (Fig.5c), and
C. geophilum, Cortinarius glaucopus, L. laccata, P. bys-
sinum, S. bovinus UP592, and S. variegatus UP60 (not
shown). Piloderma byssinum had intense amide peaks, and
possible contribution from a carboxylate band in the amide
region (Fig.2a). Secondly, one group moves in the oppo-
site direction with increasing relative proportions of amide
with increasing C availability (e.g., L. bicolor; Fig.5b; S.
bovinus UP63; Fig.S5). Laccaria bicolor showed subtle
changes in waxy character with most CH2 and ester at C:N
ratio 10:1 (Fig.5b), and P. bicolorata had a strong waxy
character with very sharp CH2 bands and evidence for an
acid, in addition to ester, in C:N 20:1 (Fig.5c). As a proxy
for how mycelial C:N ratio was affected by C availability for
A. muscaria and L. bicolor, representing the two first groups
of responses mentioned above, the relative height of the
main polysaccharide peak was compared to that of the amide
peaks (Table3). This showed a decreased amount of pro-
tein for A. muscaria (ratio increasing from 1.7 to 3.4), and
increased amount of protein for L. bicolor (ratio decreasing
from 2.1 to 1.2), respectively, with increased C availability.
Thirdly, one group (A. byssoides, P. fallax, and S. variegatus
UP597) showed little change in response to C availability
(not shown). Amphinema byssoides had some of the highest
amide peaks of all species (Fig.2a), and P. fallax had an oxa-
late peak (Fig.S8; 1317 cm−1). Fourthly, the remaining spe-
cies showed a mixture of responses not including the amide/
Table 2 Variation in chemical composition based on FTIR spectra
(perMANOVA analysis) in relation to ECM species and C-availability
for 17 ECM isolates grown in pure culture at C:N ratios 10:1, 20:1,
and 40:1, testing overall effects of (a) ECM species and C:N ratio,
and (b) ECM species, C:N ratio, and biomass. Values of degrees of
freedom (DF) and sums of squares (SS) are shown
DF SS R2FPr (> F)
(a)
Species 16 21,278.0 0.87283 266.224 0.001
C:N ratio 2 239.0 0.00980 23.922 0.001
Species * C:N ratio 32 2606.4 0.10691 16.305 0.001
Residual 51 254.8 0.01045
Total 101 24,378.2 1.00000
(b)
Species 16 21,278.0 0.87283 265.2366 0.001
C:N ratio 2 239.0 0.00980 23.8333 0.001
Biomass 1 50.0 0.00205 9.9744 0.001
Species * C:N ratio 32 2560.4 0.10503 15.9582 0.001
Residual 50 250.7
Total 101 24,378.2
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312 Mycorrhiza (2023) 33:303–319
1 3
polysaccharide response. Cortinarius scaurus had intense
amide bands in all treatments (Fig.2a, C:N ratio 20:1) and
greater CH2 intensity at C:N ratio 10:1. Hebeloma velutipes
had slightly greater CH2 intensity but overall little change
in pattern, and Hebeloma sp. 1 showed some more aromatic
character at C:N ratio 40:1 (not shown). Furthermore, P.
involutus had an obvious aromatic peak at 1515 cm−1 (see
Fig.S5 for the aromatic peak in S. bovinus), and ring link-
ages (1074, 572, and 528 cm−1). Finally, R. roseolus showed
no changes in amide bands but a large change in waxy ester
bands especially at C:N ratio 40:1 (Fig.S1), and S. bovinus
BL had a distinct aromatic component which decreased at
low C availability (Fig.S5). In addition to these four groups
of responses to increased C availability, there were also more
subtle changes in the nature of the polysaccharide noted in
some cases, in particular how distinct the bands relating to
ring linkages changed with C:N ratio.
Mycelial biomass
Although chemical composition was significant influenced
by biomass, the variance explained was very small (per-
MANOVA F = 9.97, R2 = 0.002, and P = 0.001). Biomass
dry weight for each sample is depicted in the sample plot
from the multivariate analysis (Fig.3b), ranged from ca. 3
to 27mg, 5 to 49mg, and 11 to 60mg at C:N ratios 10:1,
20:1, and 40:1, respectively (TableS2), and was signifi-
cantly affected by both fungal species (two-way ANOVA
P < 0.001) and C availability (two-way ANOVA P < 0.001).
In addition, there was a significant interaction effect (two-
way ANOVA fungal species × C availability P < 0.001).
Most of the tested isolates (13) increased biomass with
increasing C availability (producing the smallest biomass
at C:N ratio 10:1 and the largest at C:N ratio 40:1), while
two isolates (P. fallax and S. bovinus UP592) produced very
Medium-distance fringe subtype
Short-distance
Contact
Long-distance
Medium-distance smooth subtype
CVA axis 1
CVA axis 2
Fig. 4 Separation in multivariate space was significantly explained by
exploration types (MRPP A = 0.2034, P = 0.001), although the various
exploration types partly overlap in terms of chemical composition.
Canonical variate analysis of FTIR spectra for ECM fungal isolates
grown at three different C:N ratios (10:1, 20:1, and 40:1; same CVA
as Fig.3). ECM species were classified according to their pattern of
mycelial differentiation and putative ecological role (Agerer 2001)
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313Mycorrhiza (2023) 33:303–319
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similar amount of biomass at C:N ratios 10:1 and 20:1 and
one isolate (S. variegatus UP60) produced similar biomass
at C:N ratios 20:1 and 40:1. Three species (A. muscaria,
C. geophilum, and P. bicolorata) produced maximum bio-
mass at intermediate C availability (C:N ratio 20:1). Iso-
lates with the largest increase in biomass at C:N ratio 40:1
compared to 10:1 also exhibit large differences in chemi-
cal composition, e.g., S. bovinus BL, UP63 and UP592, L.
bicolor, and H. velutipes (Fig.3), although not exclusively
so (e.g., A. muscaria decrease in biomass at C:N ratio 40:1
compared to 10:1 but still show large differences). The two
ascomycetous isolates (C. geophilum and P. bicolor) showed
large differences in chemical composition when comparing
C:N ratio 40:1 and 10:1 despite small increases in biomass
(Fig.3). Finally, there were some isolates with smaller bio-
mass increases at C:N ratio 40:1 compared to 10:1 that also
showed small differences in chemical composition, e.g., A.
byssoides and P. fallax (Fig.3).
Absorbance
3500 3000 2500 2000 1500 10004000
0.1
0.2
0.3
0.4
0.5
0.6
0.7
500
0.8
0.9
1.0
1.1
1.2
1.3
1.4
1.5
1.6 AC:N 40:1
C:N 20:1
C:N 10:1
Wavenumbers (cm )
-1
3500 3000 2500 2000 1500 1000 500
B
35003000 25002000 15001000 500
C
Fig. 5 Changed C availability alters chemical composition of ECM
fungi differently depending on species. FTIR spectra for (a) A. mus-
caria, (b) L. bicolor, and (c) P. bicolorata mycelia grown at three dif-
ferent C:N ratios with an inorganic N source ((NH4)2SO4). Arrows
in a indicate reductions of amide peaks with increasing C:N ratio
for A. muscaria, representing the most obvious change in chemical
composition in response to increased C availability. In b, the arrows
indicate the amide peak which was highest with C:N ratio 40:1, and
L. bicolor represents a group of species together with P. bicolorata
that showed increasing relative proportions of amide with increas-
ing C availability. In C, black arrows indicate large CH2 peaks and
the dashed arrows indicate the ester peak for P. bicolorata, showing
subtle changes in waxy character in the C:N ratio 10:1. Absorbance
(spectral signals) was normalized to give relative absorbance/abun-
dance, and spectra are shown off set
Table 3 Comparison between the relative height of the main C-O
stretching vibration (~ 1030 cm − 1, associated with polysaccharides)
and the relative height of the amide bands (amide I ~ 1650, amide
II ~ 1550 cm − 1, associated with N) for Amanita muscaria and Lac-
caria bicolor, used as a proxy for mycelial C:N ratio. Relative height
is given corrected for baseline. Uncorrected peak height in ()
Species C:N ratio C-O peak height
(a) Amide 1 peak height
(b) Amide 2 peak height
(c) Ratio
a/(b + c)
A. muscaria 10:1 0.405 (0.449) 0.183 (0.288) 0.057 (0.177) 1.688
20:1 0.526 (0.591) 0.182 (0.298) 0.055 (0.185) 2.219
40:1 0.619 (0.663) 0.143 (0.217) 0.037 (0.121) 3.439
L. bicolor 10:1 0.511 (0.591) 0.18 (0.296) 0.059 (0.186) 2.138
20:1 0.468 (0.514) 0.203(0.290) 0.076 (0.178) 1.677
40:1 0.266 (0.326) 0.158 (0.248) 0.065 (0.166) 1.193
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314 Mycorrhiza (2023) 33:303–319
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Discussion
Carbon availability has been suggested to be the key fac-
tor determining mycelial production and possibly also its
standing biomass in boreal and temperate forests, and any
factors regulating C availability from the host plant such
as global change, forest age (Hagenbo etal. 2017), for-
estry management, and plant properties as well as fungal
C use (Hagenbo etal. 2019) can potentially cause large
variations in mycelial production of ECM fungi (Ekblad
etal. 2013). In this study, we tested the hypothesis that C
availability impacts not only on the production but also
directly on the chemical composition of mycelia, and we
expected an increased C availability to lead to more car-
bohydrates and less proteins. We showed that C availabil-
ity significantly impacted chemical composition, and for
many species, the levels of carbohydrates increased while
proteins decreased. However, this was only part of the pat-
tern since different species varied markedly in response to
C:N ratios, and ECM species explained by far the largest
part of the variation in IR spectra.
Large species‑specific differences
inchemical composition
When C availability increased, more than 70% of the tested
isolates responded with increased biomass production,
although the magnitude differed depending on species and/
or isolate. This biomass increase was in most cases associ-
ated with differences in chemical composition, confirm-
ing our overall hypothesis that C availability impacts both
production (Ekblad etal. 2013) and chemical composition
of mycelia. The 19 ECM isolates showed large species-
specific variation in chemical composition (and mycelial
production) and in their response to C availability as was
evident from the significant interaction between ECM
species and C:N ratio (perMANOVA), and species could
mostly be distinguished from each other under the same
growth conditions (C:N ratio), by using FTIR fingerprint-
ing. Ectomycorrhizal fungi typically show large species-
specific variation in growth and function (e.g., Abuzinidah
and Read 1986; Agerer 2001; Cairney 1999; Koide and
Malcolm 2009), but also intraspecific variation may be
important (Hazard etal. 2017). Changes in the utilization
of C and N among ECM fungi have been reported from
many studies, with increased mycelial biomass at higher C
availability (but depending on total C and N levels) in pure
culture (e.g., Alexander 1983; Baar etal. 1997; Fransson
etal. 2007; Itoo and Reshi2014) and pots and field experi-
ments (CO2 meta-analysis; Alberton etal. 2005). Variation
in chemical composition (beyond C and N content) among
ECM species is less well studied. Species-specific patterns
have been reported for ECM fungi in pure culture, sym-
biosis, and fruitbodies, for example, whole cell fatty acid
composition (Karliński etal. 2007), fungal soluble carbo-
hydrate concentrations (Koide etal. 2000), and N, starch,
and soluble sugar concentrations (Trocha etal. 2010). For
ECM, mycelia chemical composition has mostly received
attention in the context of decomposition and soil organic
matter sequestration. Species-specific differences in chem-
ical composition in this context were reported for melani-
zation (Fernandez and Koide 2014; Meliniomyces bicolor
intra-specific variation: Fernandez and Kennedy 2018),
N concentration (Koide and Malcolm 2009), and chitin
(Fernandez and Koide 2012). Fernandez and colleagues
(2016) concluded that the C:N ratio of ECM necromass
is an important factor governing decomposition, but that
its role is modulated by the nature of the C and N in the
mycelia. Finally, Yang and colleagues (2019) investigated
how leaf litter quality influenced the biochemical profiles
of mycorrhizal root tips by planting young beech trees in
an oak forest and replacing the natural leaf litter with other
trees species’ litter. They found that chemical composi-
tion changed with leaf litter species in a species-specific
manner, with apparent changes in the infrared absorption
bands assigned to functional groups of lipids, amides,
and carbohydrates, similarly to the present study. Yang
etal. (2019) suggested that the biochemical composition
of ECM mycelium is a fungal response trait, sensitive to
environmental changes. These findings together point to
the importance of understanding mycelial chemical com-
position, and how it varies among species and under dif-
ferent conditions.
Species variation inchemical composition related
tostructural components
The predominant compounds identified by FTIR were
polysaccharides and amide (proteins), which is in line with
what is previously known about fungal cell wall composi-
tion (Bartnicki-Garcia 1968; Fernandez etal. 2016). What
some of the other constituents responsible for differences in
chemical composition among ECM species may be related to
structurally was sometimes not clear. The waxy compounds
correspond to long chain aliphatic hydrocarbons and could
either be CH-based only, or attached to ester or acid func-
tional groups. The unsaturated waxy esters in the ascomy-
cete P. bicolorata were unique to this species, but we do
not know the function. Of the six closely related suilloid
isolates, all but one shared similar cell wall composition;
the presence of an aromatic peak in S. bovinus BL may cor-
respond to melanin or other similar compounds, separating
this isolate from the others in multivariate space.
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315Mycorrhiza (2023) 33:303–319
1 3
Melanins, a group of complex polymers of phenolic or
indolic monomers forming negatively charged, hydrophobic
pigments of high molecular weight (White 1958), result in
the black or brown color of, e.g., C. geophilum and P. bicol-
orata. Ascomycetes produce different types of melanin com-
pared to basidiomycetes, and in cell walls, melanin is likely
cross-linked to polysaccharides (Butler and Day 1998).
Melanins are important for protection and stress tolerance,
and the recalcitrance and decomposition of fungal myce-
lia (Fernandez and Koide 2014). An observed difference in
aromatic compounds in C. geophilum mycorrhizal root tips
grown with beech litter compared to other leaf litter species
in a field experiment may have been explained by a higher
melanin content (Yang etal. 2019). Furthermore, mela-
nin correlated negatively with fungal biomass production
across 62 fungal species (Siletti etal. 2017), suggesting a
physiological trade-off. The isolates we tested, however, did
not follow this pattern clearly since P. bicolorata produced
relatively large biomass. Chitin is usually complexed with
other compounds such as glucans, proteins, and melanins
(Bartnicki-Garcia 1968), but could be detected in, e.g., L.
laccata. Chitin is used as an indicator of all fungal biomass
integrated over the lifespan (Ekblad etal. 1998). Similar
to melanin, chitin has been suggested to affect decomposi-
tion of mycelia, but Fernandez and Koide (2012) showed
that chitin was not recalcitrant relative to other compounds
in fungal tissues, and that its concentration was positively
related to the decomposition. Unlike other structural poly-
saccharides, chitin contains substantial concentrations of N,
likely contributing to the decomposability.
The oxalate peak in P. fallax showed an oxalate presence.
This species produces ornamented hyphae that are coated
with crystals of calcium oxalate or other crystalline deposits
(Brand 1991a, b; Taylor and Alexander 2005), hypothesized
to make them unattractive as food for fungal grazers such as
mites. Exudation of oxalate, the most commonly produced
organic acid, varies significantly between ECM species
and responds to, e.g., increased C availability (Johansson
etal. 2009), and plays important roles for biological min-
eral weathering and in formation of secondary minerals via
the oxalate–carbonate pathway (Finlay etal. 2020). Organic
acids and other exudates such as amino acids, sugars, and
pigments that are water soluble should however mostly not
impact the chemical composition of mycelia when they are
continuously released into the surrounding soil solution.
Chitin is usually complexed with other compounds such as
glucans, proteins, and melanins (Bartnicki-Garcia 1968),
but could be detected in e.g., L. laccata. Changes in ECM
community composition and species variation in chemi-
cal composition may ultimately lead to changes in soil C
sequestration if species with a higher proportion of recalci-
trant compounds such as melanin become more common in
response to changed environmental conditions.
Large differences inthetype ofresponse toincreased
C‑availability amongECM fungal species
Carbon availability directly and significantly impacted
the chemical composition of ECM mycelia, but not for
all tested species. The expected increase in carbohydrates
and reduction in amide (less proteins) in the cell walls was
observed for half of the isolates. The two other types of
strategies in response to increased C availability included
increasing relative proportions of amide, which is the exact
opposite to what we expected, and no change. This revealed
that responses to C availability among ECM species are
not general, instead changes in C utilization are complex.
Even when looking at closely related species, for example,
the suilloid fungi, all three main responses were present
among the six tested isolates in this group. Suilloid spe-
cies are known to be C demanding (Kuikka etal. 2003),
produce abundant extraradical mycelia and extensive rhizo-
morph systems (Agerer 2001), and were highly responsive to
increasing C availability in terms of biomass production and
respiration (Fransson etal. 2007). We expected this group
to behave in a similar way, increase in carbohydrates and
decrease in proteins with high C availability, but instead
they displayed increased polysaccharide bands together with
decreased amide, increased relative proportions of amides,
or little change in chemical composition. It should, however,
be noted that the nature of the amide/protein material does
vary from species to species—differences in the amide I
and II position and shape can be indicative of different pro-
tein structures or may relate to different amide containing
material. Furthermore, different responses to C availability
may reflect ecological differences between groups or spe-
cies, and we could relate both mycelial differentiation type
(exploration type), and putative ecological roles such as
N tolerance, hydrophobicity, and succession significantly
to chemical composition in the present study. Exploration
types, a functional trait used to explore, e.g., nutrient acqui-
sition, have also been proposed to be linked to fungal C
demand and host photosynthetic capacity (Defrenne etal.
2019; Köhler etal. 2018; Wasyliw and Karst 2020). How
exploration types or other functional traits relate to chemical
composition of mycorrhizal fungi would need to be explored
further in detail to elucidate possible patterns and to under-
stand the differences among ECM species.
Usefulness oftheFTIR method todistinguish ECM
species andexplain differences inchemical composition
Despite the fact that we analyzed only two replicates per
treatment, reproducibility of fungal FTIR spectra was gener-
ally high. The replicate spectra could be almost exactly over-
laid which indicates that the chemical compositions were
nearly identical, and the coefficients of variation were low
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

316 Mycorrhiza (2023) 33:303–319
1 3
for the majority of treatments. Differences between spec-
tra could therefore be reliably taken to show differences
in chemical composition for the fungal mycelium growing
under different conditions. Interpretation of the FTIR spec-
tra for all the species in this study has provided a wealth of
information on the variation in chemical composition. We
determined that FTIR spectroscopy can be used to distin-
guish between different ectomycorrhizal species under the
current set up, especially when they were grown under the
same growth conditions, and that species explained as much
as 87% of the variation in chemical composition. Since the
experiment was done under highly controlled conditions,
the ecological relevance of our finding may be questioned
since the fungal performance together with its host interact-
ing with the environment may be quite different. Due to the
very limited knowledge about the chemical composition of
ECM mycelia, investigating the potential differences that
ECM species can display is still valuable. The use of insitu
collected mycorrhizal root material by Pena etal. (2014)
showed that four distinctly different morphotypes could be
readily distinguished from each other; however, there are
issues with recording spectra of insitu spectra as mineral
material present will significantly alter the FTIR spectrum.
How well our results correspond to differences in field col-
lected extraradical mycelia grown under different conditions,
and whether the differences in chemical composition may
be as large and useful for distinguishing a variety of ECM
fungal species remains to be tested.
Conclusions
Fernandez and colleagues (2016) concluded that the C:N
ratio of ECM necromass is an important factor governing
decomposition, but that its role is modulated by the nature of
the C and N in the mycelia. Variation in chemical composi-
tion among ECM species is, however, not well understood.
To understand the decomposition process of mycelia, we
need more studies on how growth conditions in terms of C
or nutrient availability may impact on the chemical com-
position of mycorrhizal mycelia in the soil, and how this
couples to the ECM community composition. We showed
that C availability impacted significantly on the chemical
composition of mycelia, and we conclude that ECM species
explained the major part of the variation in chemical com-
position and can mostly be discriminated from each other by
FTIR, especially when compared within the same treatment
using a pure culture setup. Given that the responses of differ-
ent species (and isolates) varied markedly in response to C:N
ratios and that the species variation in chemical composition
related to different types of structural components, changes
in community composition may ultimately lead to changes
in soil C sequestration if species with a higher proportion
of recalcitrant compounds such as melanin become more
common in response to changed environmental conditions.
The approach of using FTIR looks promising and further
species could be tested under a wider set of environmental
and physiological controls to further elucidate linkages to
their ecological role.
Supplementary Information The online version contains supplemen-
tary material available at https:// doi. org/ 10. 1007/ s00572- 023- 01128-2.
Acknowledgements We thank David Rönnlund for assisting with
sample preparation, Claire Cameron for running Genstat analyses, and
Christopher Jones for running R analyses.
Author contribution CC conceived the idea, and PF and CC designed
the study. PF conducted all laboratory work, and JR performed all
FTIR analysis and interpreted the FTIR spectra. PF and CC performed
statistical analysis. PF wrote the first draft and all authors contributed
to data interpretations, writing, and revisions.
Funding Open access funding provided by Swedish University of Agri-
cultural Sciences. PF was supported by the Swedish Research Council
FORMAS (2016–01107).
Availability of data and materials FTIR raw data are available in Dryad
(Fransson etal. 2023).
Declarations
Competing interests The authors declare no competing interests.
Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article’s Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
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