In boreal forests an important part of the photo assimilates are allocated
belowground to support symbiosis of ectomycorrhizal fungi (EMF). The
production of EMF extramatrical mycelium can contribute to carbon (C)
sequestration in soils, but the extent of this contribution depends on the
composition of the EMF community. Some species can decrease soil C stocks by
degrading soil organic matter (SOM), and certain species may enhance soil C
stocks by producing hydrophobic mycelia which can reduce the rate of SOM
decomposition. To test how EMF communities contribute to the development of
hydrophobicity in SOM, we incubated sand-filled fungal-ingrowth mesh bags
amended with maize compost for one, two or three growing seasons in
non-fertilized and fertilized plots in a young Norway spruce (Picea abies) forest. We
measured hydrophobicity as determined by the contact angle and the C/N ratios
in the mesh bags contents along with the amount of new C entering the
mesh bags from outside (determined by C3 input to C4 substrate), and related
that to the fungal community composition. The proportion of EMF species
increased over time to become the dominant fungal guild after three growing
seasons. Fertilization significantly reduced fungal growth and altered EMF
communities. In the control plots the most abundant EMF species was
Piloderma olivaceum, which was absent in the fertilized plots. The hydrophobicity of the
mesh bag contents reached the highest values after three growing seasons only
in the unfertilized controls plots and was positively related to the
abundance of P. olivaceum, the C/N ratios of the mesh bag contents and the amount of new C
in the mesh bags. These results suggest that some EMF species are associated
with higher hydrophobicity of SOM and that EMF community shifts induced by
fertilization may result in reduced hydrophobicity of soil organic matter,
which in turn may reduce C sequestration rates.
Introduction
Fertilization of forests has been suggested as a way to increase C
sequestration to mitigate climate change (Jörgensen et al., 2021). In
support of this, Bergh et al. (2008) found more than doubling of
aboveground growth of young Norway spruce forests in response to yearly
additions of a complete fertilizer in experimental sites in Sweden. A major
part of gross primary production, between 25 % and 63 % according to
Litton et al. (2007), is however allocated belowground to roots and
associated ectomycorrhizal fungi, and this portion usually declines in
response to fertilization (Högberg et al., 2010). In support of this, reduced
growth of mycelium of ectomycorrhizal fungi (EMF) was found in the young fertilized Norway spruce
stands studied by Bergh et al. (2008) (Wallander et al., 2011).
EMF species form extensive mycelial networks, which efficiently distribute C in the
soil (Smith and Read, 2008), and this mycelium turns into necromass when the
mycelium dies. Necromass from different EMF species decomposes at different
rates (Koide and Malcolm, 2009). Melanin content appears to have a negative
influence on necromass decomposition, but physical protection is also an
important factor to reduce decomposition according to Fernadez and Kennedy (2016). Soil organic matter (SOM) can be protected from decomposition in aggregates where
hydrophobic coatings of mineral particles change the physical properties of
the particles, reduce water films around them and limit water penetration
inside the aggregates. This affects the mobility of microbial decomposers
and extracellular enzymes from the soil solution and reduces organic matter
decomposition (Leelamanie and Liyanage, 2016; Goebel et al., 2011; von
Lützow et al., 2008), and hydrophobic SOM generally decomposes more slowly than hydrophilic SOM (Nguyen and Harvey, 2003, 2001). Since some EMF
species form hydrophobic, while others form hydrophilic, mycelia (Unestam and
Sun, 1995), the composition of the EMF community may thus have fundamental
importance for the SOM properties and subsequently for carbon sequestration
rates in the soil.
In contrast to carbon-accumulating activities by EMF species, certain species may
also reduce soil C stocks by oxidizing organic matter to release nitrogen
and phosphorus. Some EMF species use “brown-rot” Fenton chemistry and some
use “white-rot” peroxidases to decompose SOM (Shah et al., 2015; Lindahl
and Tunlid, 2015; Bödeker et al., 2014). This can result in a 30 %
decrease in SOM according to Lindahl et al. (2021). Ectomycorrhizal fungi may
thus have opposing effects on the amount of SOM, and differences in
community composition were proposed as one explanation for different C
accumulation rates in boreal forests in northern Sweden (Clemmensen et al.,
2015, 2013): later successional stages that accumulated more C were
dominated by ericoid mycorrhizal fungi with recalcitrant necromass, while
younger successional stages that accumulated less C were dominated by EMF species of
long-distance-exploration types with a high capacity to degrade soil organic
matter. Certain EMF species may have exceptional importance for organic
matter degradation as the presence of Cortinarius acutus (which has retained the enzymatic
capability to break down SOM to access nutrients) was linked to 33 % lower
C storage in the organic topsoils in 359 investigated stands in boreal
forests in Sweden (Lindahl et al., 2021).
It is well known that fertilization with N has a strong impact on growth and
composition of EMF species (Lilleskov et al., 2011; Wallenda and Kottke, 1988).
Lilleskov et al. (2011) demonstrated that EMF species sensitive to N (e.g., Cortinarius, Tricholoma, Suillus and Piloderma) usually produce hydrophobic mycelia, while N-tolerant species often produce
hydrophilic mycelia (e.g., Laccaria). Loss of hydrophobic EMF species at high N input
could thus have consequences for SOM formation and C sequestration rates,
but it is not well known to what extent EMF abundance has a significant
effect on the overall hydrophobicity of SOM.
In our study with young Norway spruce forests reported above (Wallander et
al., 2011), we used mesh bags amended with maize compost (C4 plant material
enriched in 13C) to estimate EMF growth in control and
fertilized plots. In the present study we analyzed the fungal communities as
well as the hydrophobicity of the same mesh bag contents. The mesh bags were
harvested after one, two or three growing seasons in order to follow fungal
succession and development of hydrophobicity over time. All samples were
subjected to 454 sequencing in order to characterize the fungal communities.
We expected community composition to be influenced by fertilization and
hydrophobicity to increase over time when EMF biomass and necromass
accumulates. We also expected more N to be removed by EMF species from the mesh bags
in the control than in the fertilized treatment. In addition, we expected
higher hydrophobicity in control versus fertilized plots due to a higher
proportion of hydrophobic species.
Material and methodStudy site
The experimental forest was located close to Ebbegärde in southeastern
Sweden (56∘53′ N, 16∘15′ E) in a 10-year-old Norway
spruce forest at time of sampling. The soil is a podzol on coarse sandy
glacial till (site index G29), and the depth of the humus layer varied
between 3 and 8 cm.
The treatments were designed in randomized block design with three fertilization
treatments and three blocks per treatment (n=3). The plot size was 40×40 m.
The fertilization treatments were the unfertilized control plots and two
fertilization regimes. In the fertilization treatments specific amounts of
nitrogen (N) (ammonium and nitrate) were applied to optimize plant growth
without inducing leaching. The amount of N addition was based on needle N
determinations and monitoring of N in soil water (Bergh et al., 2008). Thus,
the fertilization was applied by hand as 50–100 kg N ha-1 every year
for the first fertilization regime and as 100–150 kg N ha-1 every
second year in the second fertilization regime (fertilization began in
2002). To avoid nutrient imbalance caused by fertilization, the quantity of
micronutrients was adjusted to optimum nutrient proportions for Picea abies (as
calculated by Ingestad, 1979). For a more detailed description of the
fertilization regime see Linder (1995) and Bergh et al. (2008). For this
study both fertilization regimes were treated as one fertilization
treatment.
Experimental design
We used triangular-shaped ingrowth bags made of nylon mesh (50 µm
mesh size, 10 cm side, ∼ 1 cm thick) to capture fungi growing
in the soil. This mesh size allows the ingrowth of fungal hyphae but not of
roots (Wallander et al., 2001). The mesh bags were filled with 30 g of acid-washed quartz sand (0.36–2.0 mm, 99.6 % SiO2, Ahlsell AB, Sweden)
heated to a temperature of 600 ∘C overnight to remove all organic
carbon. The sand was then mixed with 0.8 % (w/w) maize compost. Maize
compost was used since it has a unique C isotopic signature, which makes it
possible to estimate C influx into the mesh bags. Results from these
measurements are presented in Wallander et al. (2011). Maize compost was
produced by cutting maize leaves into small pieces and composting them in an
isolated plastic compost bin for 12 months. After that the compost was kept
at +4 ∘C. Fresh compost was forced through a 2 mm mesh and then
mixed with dry sand to make a uniform mixture. The sand maize mixture had a
carbon content of 0.4 %. The bags were buried at approximately 5 cm depth
in the interface between the organic horizon and the mineral soil where ectomycorrhizal
fungi are abundant (Lindahl et al., 2007). The first harvest was done in
November 2007, after 8 months of incubation. The second harvest was done in
November 2008, and the third harvest was done in November 2009. Four mesh bags
were pooled to make one composite sample for each block, year and treatment.
In the laboratory the mesh bags were opened and the contents from the four
replicate mesh bags from each experimental plot were carefully pooled and
mixed. Subsamples were taken for subsequent analyses (ergosterol,
hydrophobicity, C and N content, fungal community) and immediately frozen.
The abundance of δ13C as well as total C and N content was
analyzed using an elemental analyzer (model EuroEA3024; Eurovector, Milan,
Italy) connected to an Isoprime isotope-ratio mass spectrometer (Isoprime,
Manchester, UK) as described by Wallander et al. (2011). The isotopic shift
that occurred when 13C-depleted C (mainly EMF mycelia) entered the bags
from outside was used to calculate the amount of new C in the mesh bags. To
estimate ectomycorrhizal growth, the fungal cell membrane compound
ergosterol was measured as a biomarker of fungal biomass. Ergosterol was
extracted from 5 g of the pooled sand–maize mixture from the mesh bag.
Briefly the sample was subjected to saponification using a solution of 10 % KOH in methanol, and the non-polar phase (where the ergosterol is
present) was separated using cyclohexane. The ergosterol was quantified by a
high-performance liquid chromatograph (Hitachi model L2130) and a UV detector
(Hitachi model L2400). For more detail regarding the protocol see
Wallander et al. (2011).
DNA extraction, PCR and 454 sequencing
A total of 10 g of the sand–maize mixture from the composite samples was
homogenized using a ball mill without a ball (Retsch, Haan, Germany). DNA
was extracted from the homogenized samples by adding CTAB buffer (2 % cetyltrimetylammoniumbromid, 2 mM EDTA, 150 mM tris HCl, pH 8), vortexing
and then incubating at 65 ∘C for 1.5 h, followed by chloroform
addition, vortexing, supernatant removal, and isopropanol and ethanol
precipitation. The pellet was resuspended in 50 µL of Milli-Q water
(Millipore) and further cleaned using a Wizard DNA cleanup kit (Promega,
Madison, WI, USA).
PCR was carried out for each sample in three triplicate 25 µL reactions,
using the fungal-specific primers ITS1-F (Gardes and Bruns, 1993) and ITS4
(White, 1990). Each primer was elongated with adaptors required for
454 pyrosequencing (ITS1-F/A adaptor and ITS4/B adaptor). The ITS4 also
contained a sample-specific tag consisting of eight bases; ITS1-F/A
5′-CCTATCCCCTGTGTGCCTTGGCAGTCTCAGCTTGGTCATTTAGAGGAAGTAA-3′ and ITS4/B 5′-CCATCTCATCCCTGCGTGTCTCCGACTCAGXXXXXXXXTCCTCCGCTTATTGATATGC-3′. PCR
products were purified with Agencourt AMPure kit (Agencourt Bioscence
Corporation, Beverly, MA, USA) in order to remove residual salts, primers
and primer dimers. The concentration of the purified PCR products was
measured with the PicoGreen dsDNA quantification kit (Molecular Probes,
Eugene, OR, USA) on a FLUOstar OPTIMA (BMG Labtech Gmbh, Ortenberg,
Germany). Equal amounts of DNA from each sample were pooled into one single
pool and submitted for 454 pyrosequencing. Sequencing was performed on an FLX 454 (Roche Applied Biosystems) using the Lib-L chemistry at
the pyrosequencing facility at Lund University, Lund, Sweden.
Bioinformatic analysis
After sequencing, sequences were trimmed and filtered using Mothur v1.34
(Schloss et al., 2009). The trim sequence
operation was run with the following exclusion parameters: all sequences
that mismatched the sample ID barcode at more than one position, those that mismatched
the primers at more than two positions, those that had homopolymers longer than 10 bp, those that
were shorter than 150 bp or those that had an average base call quality score below 20
over a moveable window of 40 bases.
Sequences outside the ITS2 region and chimeric sequences were removed using ITSx
extractor v1.5.0 (Bengtsson-Palme et
al., 2013). After filtering, a Bayesian clustering was applied to the
sequences using the Gaussian mixture model CROP
(Hao et al., 2011) at 97 % sequence
similarity, and a set of operational taxonomic units (OTUs) was thus
obtained. Clusters that were only found in one mesh bag sample (one PCR
reaction) were excluded, further reducing the possibility that any chimeric
sequences were used in our analysis. Search for sequence identities were
performed by iteratively BLASTing (BLAST denotes “basic local alignment search tool”)
against two different sequence databases: the first was the UNITE (Koljalg et
al., 2005, http://unite.ut.ee/index.php, last access: 10 August 2022) reference/representative sequence
database (21 000 sequences, dynamic taxa threshold, release date 9 February 2014), and
the second was the full UNITE+INSD sequence database (377 000 sequences,
dynamic threshold, release date 15 February 2014) (Karsch-Mizrachi et al., 2012).
The UNITE and INSD databases were purged of all sequences, nearly 25 % of
the total, that did not have any taxonomic information, primarily
environmental samples from soils and roots using Boolean terms (excluding
Environmental, uncultured, root endophyte, unidentified). Sequences were
assigned to species when there was at least 97 % similarity between the query
sequence and top hit. Sequences that failed to match at this threshold were
excluded. Separate clusters that matched the same database sequence were
subsequently lumped into one OTU.
Using names and taxonomy associated with the OTUs, the total fungal
community was divided by both phylum (Basidiomycota, Ascomycota,
Mucoromycota, Zoopagomycota and Chytridiomycota) and function (known
ectomycorrhizal fungi, unknown ectomycorrhizal status and saprotrophic fungi);
OTUs were considered known ectomycorrhizal fungi based on the knowledge of
the ecology of known close relatives (genera or below) according to Tedersoo
et al. (2010).
After filtering, each sample was rarified to the median number of reads
using the “rrarefy” function in the vegan package (Oksanen et al., 2017) in
R (R Core Team, 2013). For community comparison (total or for
ectomycorrhizal fungi), all read abundances were converted to relative
abundance such that the read abundances for all OTUs for each sample
totaled 1.
Hydrophobicity
The hydrophobicity was evaluated in terms of contact angle (CA) with the
sessile drop method (Bachmann et al., 2003), using a CCD-equipped CA
microscope (OCA 15, DataPhysics, Filderstadt, Germany). Here the angle a
drop of water forms at the solid–liquid–vapor interphase is
measured. This contact angle is used to describe the wettability of the
surface; CA ≥ 90∘ indicates a hydrophobic and a zero CA a hydrophilic
surface. A CA > 0 and < 90∘ indicates
subcritical water repellency.
For measurement, material from the mesh bags contents was fixed on a glass
slide with double-sided adhesive tape in an ideally one-grain layer.
Placement of a water drop is recorded and the initial CA evaluated after
ending of mechanical disturbances by drop shape analysis (ellipsoidal fit)
and fitting tangents on the left and right side of the drop, using the
software SCA 20 (DataPhysics, Filderstadt, Germany; Goebel et al., 2013). CA
is given as the mean CA of the left and right side of the drop. For an
estimate about CA stability, CA was evaluated again after 1 s (denoted as
CA1s) and after 5 s (denoted as CA5s; Bachmann et al., 2021).
Three replicates from each treatment (control or fertilized) and each
incubation period (2007, 2008, 2009) were used in the measurements. One
slide per replicate was prepared, and for each slide six drops were placed
and averaged to obtain one CA per replicate (n=6). Two slides containing
the non-incubated sand–maize compost mix were also analyzed as a non-treated
reference material. Due to the coarse texture of the mesh bag material, the
drop volume was 6 µL.
Statistical analysis
The statistical analyses for the fungal communities were performed using the
vegan package (Oksanen et al., 2017) in R (R Core Team, 2013). Fungal
communities were visualized with ordination using non-parametric
multidimensional scaling (NMDS) using the metaMDS function. Differences in
community structure were visually compared with centroids and the associated
95 % confidence interval associated with a t distribution around the
standard error of the centroid. To detect if the fungal communities were
significantly influenced by the treatments (fertilization and incubation
periods), permutational multivariate analysis of variance (PERMANOVA;
Anderson, 2017) was performed. Pairwise comparisons between treatments were
tested using the pairwise Adonis test.
To test for differences in hydrophobicity (contact angle), C/N ratios, new C
inside the mesh bags and ergosterol, ANOVA and two-way ANOVA were performed
using the car package (Fox and Weisberg, 2019) in R (R Core Team, 2013). To
test for differences in the relative abundance of EMF species between the
treatments, Dunn's test for non-parametrical samples was performed (Dinno,
2015).
Principal component analysis (PCA) was used to analyze the relationships
between the most abundant fungal species and the properties of the mesh bag
contents (hydrophobicity (contact angle), C/N ratios, new carbon inside the
mesh bags, ergosterol) using the package FactoMineR (Lê et al., 2008) in
R (R Core Team, 2013).
ResultsFungal biomass
The concentration of ergosterol, as an estimate of fungal biomass, in the
mesh bags has been reported earlier (Wallander et al., 2011) and is
summarized in Table 1. In brief, ergosterol content increased from a
starting value of 0.7 (original maize compost) to 2.2 mg g-1 in the
mesh bags after incubation for one growing season in control plots. After
this the concentration did not change significantly over the following 2 years. In fertilized plots the concentration was significantly lower than in
the control plots (ANOVA, F=13.4, p<0.01).
Average and standard error of the ergosterol concentrations, total C %,
C/N ratio, amount of new carbon (C3 mainly from EMF species), percentage of EMF DNA reads
and contact angle determined 5 s after placement of water droplets
placed on mesh bags material amended with maize compost (CA5s;
estimation of contact angle stability). Low score letters refer to
statistical differences according to the post hoc Tukey test and pairwise Dunn
test. Asterisks correspond to statistical differences for CA after 5 s between the mesh bag contents and the non-incubated reference material.
Incubation in the field significantly increased hydrophobicity of the
mesh bag contents in the unfertilized control plots as indicated by CA1s
and CA5s (ANOVA, F=6.2 and p<0.05 and ANOVA, F=10.2 and
p<0.01, respectively). CA of the control plots increased with
incubation time, but only CA1s and CA5s were significantly
different from the reference material (non-incubated sand–maize compost
mix); i.e., stability of CA was increased (Fig. 1a).
Contact angle (CA) comparisons between (a) control treatment and
reference material, (b) fertilization treatment and reference material, and (c) control and fertilized treatments. Shown is the initial CA (ini), determined
directly after placement of the water drop and CA determined 1
and 5 s after placement of the water drop. Bars represent
standard deviation (n=3).
Incubation in the field also affected hydrophobicity of the mesh bag contents
in the fertilized plots as indicated by the initial CA and CA1s and
CA5s (ANOVA, F=5.2 and p<0.05; ANOVA, F=4.1 and p=0.06; and
ANOVA, F=3.9 and p=0.05, respectively). The CA stability (CA1s and
CA5s) was increased compared to the reference material only in the
1-year-incubation mesh bags. As the time of incubation in the soil increased,
however, CA decreased. After 3 years of incubation the initial CA became
significantly smaller in comparison with the reference material (Fig. 1b).
There were significant differences in the CA (initial, CA1s and
CA5s) between mesh bags from the control and fertilized plots in the
3-year-incubation bags with smaller CA (initial, CA1s and CA5s)
for the fertilized plots compared to the control (2009; Fig. 1c; ANOVA, F=3.2 and p<0.05; F=3.1 and p=0.05, and F=2.8 and p=0.06,
respectively) but not for the first and second incubation year (2007, 2008)
The concentration of C in the mesh bags was not influenced by time or
fertilization, but the amount of new C (C3 C presumably from EMF species) in the mesh
bags was significantly affected by fertilization and was higher in the
control plots than in the fertilized plots according to the two-way ANOVA
(F=5.3, p<0.05). The amount of new C tended to increase with
incubation time in the control plots (Table 1). The interaction between
fertilization and incubation time was not significant.
There was a positive correlation between the amount of new C and the
hydrophobicity for both CA1s and CA5s (Pearson, T=2,
p=0.06, Cor = 0.45) (Fig. 2a and b, respectively). When breaking down the
data by fertilization regime, there was a positive correlation between
amount of new C and the hydrophobicity for the CA5s in the control
plots (Pearson, T=2.2, p=0.06, Cor = 0.63) (Fig. 2c), but the
correlation tended to be negative in the fertilized plots (Fig. 2d).
Correlation between the amount of new C and the hydrophobicity of
the mesh bag contents measured as the contact angle (CA). The CA was
determined directly after placement of the water drop at (a) 1 s and
(b) 5 s. (c) Correlation between the amount of new C and the
hydrophobicity of the mesh bag at 5 s in the control plots. (d) Correlation
between the amount of new C and the hydrophobicity of the mesh bag at 5 s
in the fertilized plots.
The C/N ratio of the mesh bag content was 11.9 in the initial material,
which increased to an average of 14.6 and 12.2 after 3 years of incubation
in the control and fertilized plots, respectively (Table 1). According to the
two-way ANOVA, fertilization had a significant effect on the C/N ratios of
the mesh bags (ANOVA, F=6.1, p<0.05). The impact of incubation
time or the interaction between fertilization and incubation was not
significant. During the first 2 incubation years (2007, 2008) there were
no differences between the C/N ratios in the control and fertilized samples.
During the third incubation year (2009) the C/N ratios in the control
samples were significantly higher than the C/N ratios in the fertilized
samples.
Effects of fertilization and incubation time on fungal community
composition
After all bioinformatic processing and quality filtering, followed by
rarefaction to a maximum of 1200 sequence reads per sample (minimum 612)
and elimination of all operational taxonomic units (OTUs) that were only found
in one sample, 26 943 sequence reads were recovered that were apportioned to
146 OTUs.
The total fungal communities were significantly influenced by incubation
time and by fertilization according to the PERMANOVA analysis (p<0.001 and F=5.4 and p<0.001 and F=8.4, respectively) (Fig. 3a).
Response of the fungal communities in the mesh bags to the
fertilization treatment and incubation time. (a) NMDS ordination analysis of
the fungal communities. (b) Relative abundance of the different fungal species
and their corresponding mycelium exploration type and hydrophobic (Ho) or
hydrophilic (Hi) properties. (c) Relative abundance of the genus Piloderma. (d) Relative
abundance of the genus Amphinema. (e) Relative abundance of the genus Tylospora. (f) Correlation
between the proportion hydrophobic EMF species and the averaged contact
angle (initial CA, CA at 1 s and CA at 5 s) in the control plots.
Fertilization had no significant effect on the total fungal community during
the first year, but during the second year and third year the fertilization
effect was found to be significant (pairwise Adonis, p=0.06 and F=2 and
p=0.02 and F=5.3, respectively).
The proportion of EMF sequences increased significantly over time in the
mesh bags (Dunn test, χ2=18, p<0.0001) (Fig. 3b). Of the sequences, 11 % and 7 % were EMF species during the first growing period in
the control and fertilized plots, respectively. These values increased to
24 % and 31 % after 2 years of incubation in the control and
fertilized plots, respectively, and to 78 % and 72 % after three growing
seasons in the control and fertilized plots, respectively (Table 1). The
number of EMF reads was significantly correlated with the new C in the
mesh bags (Pearson, T=2.4, p<0.05, Cor = 0.46).
The more abundant hydrophobic EMF genera were Piloderma and Amphinema (Fig. 3c and d,
respectively), while the more abundant hydrophilic genus was Tylospora (Fig. 3e).
The proportion of hydrophobic EMF species (the sum of the relative abundance of
fungal reads belonging to hydrophobic EMF species) tended to be higher in
the control plots (up to 57 % of the total fungal reads) in comparison
with the fertilized plots (up to 44 % of the total fungal reads) in the
3-year-incubation bags, but this increase was not significant.
Additionally, the proportion of hydrophobic EMF species in relation to
hydrophilic EMF species in the control plots tended to be higher than in the
fertilized plots in the 3-year bags, but this was not significant. When
both treatments (control and fertilization) where analyzed together, there
was no correlation between the proportion of hydrophobic species and the
contact angle. The proportion of hydrophobic EMF species was positively
correlated with the averaged contact angle (initial CA, CA at 1 s and CA
at 5 s) in the control plots (Pearson, T=2.9, p<0.04,
Cor = 0.68) (Fig. 3f) but not in the fertilized plots.
Piloderma increased in abundance over time in the control plots to become the
dominating genus (up to 47 % of the relative abundance) after 3 years
of incubation (Fig. 3c). The most dominant species in the control plots was
Piloderma olivaceum, which was reduced to 0 % in the fertilized plots independent of incubation
time (Fig. 3b). Tylospora fibrillosa was also reduced in response to fertilization (Dunn test,
χ2=13.4, p<0.0001), while T. asterophora showed an opposite trend
(Dunn test, χ2=4.4, p<0.05) (Fig. 3b). Amphinema sp. 5 was the
most abundant species in the fertilized plots and was enhanced by
fertilization (Dunn test, χ2=3.8, p<0.05) (Fig. 3b).
Principal component analysis
The principal component analysis (Fig. 4) separated the samples by incubation
time along principal component 1. This component explained 34 % of the
variance. Samples belonging to the 3-year-incubation bags were
ordinated to the right of principal component 2. Along principal
component 2 (PC2) the samples were separated by the fertilization treatment. This
component explained 25.7 % of the variance. Samples belonging to the
unfertilized controls were ordinated above principal component 1 (PC1). The
linear model showed that the fertilization/incubation treatments were
significantly associated PC1 (F=8.3, p<0.01) and
PC2 (F=18, p<0.0001). The proportion of EMF species and Basidiomycota
increased over time while the proportion of saprotrophic fungi and
Ascomycota decreased with increasing incubation time. The EMF species
Tylospora fibrillosa and Piloderma olivaceum were positively related to the CA (initial CA, CA1s, CA5s),
with the C/N ratios and with the amount of new carbon inside the mesh bags;
and their vectors were directed towards longer incubation time and the opposite
to the fertilization treatments.
Principal component analysis of the most abundant fungal species
and the properties of the organic material inside the mesh bags.
DiscussionEffect of incubation and fertilization on the total fungal communities
As expected, the fungal communities were influenced by the fertilization and
by incubation time and there was a significant increase in the percentage of
EMF reads over time. It should, however, be noted that the ingrowth of EMF species
in relation to other fungal groups was surprisingly low during the first
growing season (< 12 % of the fungal sequences), which is much
lower than what has been found in earlier studies (Parrent and Vilgalys,
2007; Wallander et al., 2010). Some of this variation may be due to
different weather conditions – the first year was wetter than normal, while
the third was close to normal in precipitation (Wallander et al., 2011) – or
due to larger belowground carbon allocation when the trees approach canopy
closure during the third year, as discussed in Wallander et al. (2010).
Whether shifts in EMF species were due to selection of later succession fungal taxa
or variation in climatic conditions remains unclear but is ultimately not
particularly important in terms of understanding how shifts in EMF species relate to
soil organic matter cycling. Thus, the EMF abundance was highest during the
third year, and this increase was associated with higher C/N ratios and
hydrophobicity in the control plots and higher input of new C in the control
and fertilized plots. This suggests a strong relation between EMF species and the
changes in the properties of the organic material in the mesh bags.
The most dominant EMF genera in our study were Amphinema, Piloderma and Tylospora, which are also common in
other studies of EMF communities in coniferous forests (Almeida et al.,
2019; Walker et al., 2014; Tedersoo et al., 2008). In the control plots, the
most dominant species was P. olivaceum, which did not colonize the mesh bags collected
from fertilized plots. Piloderma is a common genus in boreal forests and is reported
to be more abundant in soils rich in organic N (Heinonsalo et al., 2015;
Lilleskov et al., 2002b) and to decline in response to inorganic N
fertilization (Teste et al., 2012) and elevated N deposition (Kjöller
et al., 2012; Lilleskov et al., 2011, 2002a; Taylor et
al., 2000). The decline in Piloderma in the fertilized plots in the present study is
not surprising since this genus produces abundant hydrophobic rhizomorphs
that might constitute a large C cost for the host (Defrenne et al., 2019),
which is not economical for symbiosis at high mineral N concentrations.
Other more direct effects of the fertilizer on the growth of Piloderma mycelium are
also possible. The increase in the C/N ratios of the mesh bag substrates from
the control treatment might thus be an effect of biomass accumulation of
Piloderma species, since EMF species in general have a higher C/N ratio than maize
compost (Wallander et al., 2003). Additionally, it has been shown that P. olivaceum produces
proteases that improve the ability of the host trees to utilize N from
organic compounds (Heinonsalo et al., 2015). Therefore, N released from the
maize compost by this fungus could have been transferred to the host plants,
which would contribute to the increase in C/N ratios in the control plots in
comparison with the fertilized plots. This explanation is consistent with
results described by Nicolás et al. (2017), who used FTIR and NEXAFS to
analyze chemical changes of similar maize compost incubated in mesh bags
over one growing season in a Norway spruce forest in southwestern Sweden.
They found that heterocyclic-N compounds declined in mesh bags in comparison
with non-incubated reference material, which was interpreted as an effect of
removal by EMF species and transfer to the host trees. This decline was higher in
the unfertilized control plots compared with fertilized plots. In the
fertilized plots of the present study, the amount of new C tended to
increase in the 3-year-incubation bags where the C/N ratios reached the
lowest values, indicating limited N removal by the EMF species colonizing these
bags.
Amphinema sp. 5 responded positively to fertilization in our study, which is supported
by a study by Kranabetter (2009), who found a strong increase in the abundance
of Amphinema-colonized root tips along productivity gradients in Canada. While a
reduced abundance of T. fibrillosa was observed in the fertilized plots, T. asterophora responded
positively. Similarly contrasting effects between these two species were
found in other studies as well (Teste et al., 2012; Kjöller et al.,
2012; Toljander et al., 2006). In an N deposition gradient Kjøller et
al. (2012) found increased abundance of Tylospora asterophora in areas with high N throughfall,
while T. fibrillosa abundance decreased with higher N deposition. Reduction in T. fibrillosa in
response to fertilization may be a result of C starvation since it has been
shown that this species is more dependent on C transferred from a living
host in order to colonize new seedlings on a clear-cut compared to
Amphinema sp., which readily colonized saplings on clear-cuts (Walker and Jones,
2013).
Effect of incubation and fertilization on hydrophobicity
As expected, hydrophobicity increased over time in respect to the reference
material (non-incubated maize–sand mixture), and this increase occurred only
in the unfertilized controls at the last sampling when the fungal
communities in the mesh bags were dominated by EMF species. This increase in
hydrophobicity was expected to be an effect of the accumulation of fungal
biomass and necromass over time as it has been shown that organic C (Woche
et al., 2017; Mataix-Solera and Doerr, 2004; Chenu et al., 2000) and microbial
biomass and necromass contribute to the hydrophobicity of soils (Schurig et
al., 2013; Šimon et al., 2009; Capriel, 1997). However, the total amount of C
was similar for all the incubation times and was not affected by
fertilization, indicating that C content alone could not explain the
variations in hydrophobicity. Instead, the amount of new C entering the
mesh bags from outside was found to be significantly correlated with
hydrophobicity (CA1s and CA5s). This new C is expected to be of
EMF origin as discussed by Wallander et al. (2011). Since saprotrophic fungi
utilize the maize compost material as their C source, it is expected that
new C inputs come from plant photoassimilates and are brought by EMF species
(Wallander et al., 2011). Therefore, these results suggest that the
accumulation of biomass and necromass of EMF origin over time might
contribute to the buildup of hydrophobicity in SOM in the control plots.
Our results show that fertilization reduced ergosterol concentration in the
mesh bags in comparison with the control samples (Wallander et al., 2011) and
this coincided with a decrease in the hydrophobicity over time in comparison
with the unfertilized controls and the non-incubated reference material. It
has been shown that fungi may enhance soil water repellency of soil
particles since some filamentous fungi produce insoluble substances like
ergosterol and hydrophobins (Mao et al., 2019; Rillig et al., 2010). For
instance, Hallet et al. (2001) found that soil hydrophobicity decreased when
fungi were killed after fungicide additions. Therefore, it is possible that
the lower fungal biomass in the fertilized plots in our study led to a
decrease in hydrophobicity as incubation time in the soil increased. However
the concentration of ergosterol in the mesh bags from the control plots did
not increase with incubation time and even tended to decline in the last
incubation sampling when hydrophobicity increased, indicating that
ergosterol alone is not a good predictor of hydrophobicity. It is possible
that high ergosterol values after one growing season was an effect of high
abundance of yeast like Guehomyces, Cryptococcus, Rhodotorula and Candida, which are unlikely to contribute to
hydrophobicity but dominated the fungal communities of the mesh bags during
the first growing seasons. These fungi decreased drastically in abundance in
the 3-year-incubation bags. The ergosterol content per dry mass of
yeasts is much higher than in filamentous fungi (Pasanen et al., 1999),
which might explain the high ergosterol values in the first incubation
periods. From these results we conclude that hydrophobicity is more
associated with EMF colonization (measured as the amount of new C)
than with total fungal biomass (measured by ergosterol). It should also be
noted that we cannot rule out the possibility that other compounds from the
soil entered the mesh bags during the underground incubation. In soils,
polymeric substances coming from SOM, root or microbial exudates can have
hydrophobic properties (Vogelmann et al., 2013). Hence, the hydrophobic
changes in the material could be partly explained by sources other than EMF
mycelium. However, the significant correlation between the new carbon in the
bags and the EMF reads and the negative effect of fertilization on the CA
might suggest that hydrophobicity changes in the mesh bag content are caused
mainly by EMF species.
Given the apparent association of EMF colonization with higher
hydrophobicity over time, some EMF species may be expected to be more
important than others for this process. We expected higher hydrophobicity in
the control plots in response to a higher proportion of hydrophobic
long-distance-exploration-type species. Indeed, the proportion of
hydrophobic EMF species in the control plots tended to be higher in
comparison with the fertilized plots in the mesh bags incubated for 3 years. From the hydrophobic species in the control plots, Piloderma spp. constituted
the majority of fungal species with up to 47 % of the total fungal reads.
The presence of Piloderma species like P. olivaceum, known to form hydrophobic mycelia (Lilleskov
et al., 2011; Agerer, 2001) and which was totally absent in the fertilized
plots, is likely to contribute significantly to hydrophobicity of SOM. In the
fertilized plots there was also an increase over time in the proportion of
hydrophobic EMF species (Amphinema being the most abundant hydrophobic genus), which
was not accompanied by an increase in hydrophobicity. This may suggest that
necromass from Amphinema does not accumulate to the same extent as for Piloderma and is probably
not associated with the hydrophobicity in the mesh bags. These findings
suggest that hydrophobicity of living mycelium might not necessary influence
the water retention of the organic material to a large extent. This is
consistent with the findings of Zheng et al. (2014), who found that the
hydrophobicity of EMF mycelium does not necessary enhance soil water
repellency. They tested how different EMF strains were inoculated on Pinus sylvestris-affected
water repellency of sandy loamy soil. The mycelium hydrophobicity of the
fungal strains used in their experiment was previously tested by drop
immersion on fungal mycelium growing on pure cultures. The authors found
that the mycelium from hydrophobic species generally enhanced water
repellency but not all hydrophobic isolates had a positive effect on soil
hydrophobicity. It was suggested that beside mycelium hydrophobicity, other
species-dependent factors like growth patterns, the degree of soil particles
coverage or the quantity of hydrophobic substances produced by the fungus
might influence soil water repellency. In the present study the difference
in hydrophobicity between treatments might be related to not only the
exploration types of the abundant species but also species-dependent
features. For example, the characteristic yellow color of Piloderma comes from an
insoluble pigment called corticrocin (Gray and Kernaghan, 2020; Schreiner et
al., 1998). Moreover, the hyphae of Piloderma are reported to be coated with calcium
oxalate crystals (Arocena et al., 2001), probably as a strategy against
grazers or to repel water to avoid microbial predation (Gray and Kernaghan,
2020; Whitney and Arnott, 1987). Thus, these particular features of
Piloderma make it a good candidate to explain the enhanced hydrophobicity of the
material in the control mesh bags, which is supported by the association
between the abundance of this fungus, the new C in the mesh bags and the CA.
Ecological significance
The effect of fertilization on fungal communities and its significance for C
sequestration has been largely discussed (see Jörgensen et al., 2021;
Almeida et al., 2019; Högberg et al., 2010; Janssens et al., 2010;
Treseder, 2004). Additions of inorganic N may have a strong positive effect
on plant net primary production (Binkley and Högberg, 2016) but have
also been shown to decrease belowground C allocation (Högberg et al., 2010)
and consequently decrease EMF biomass (Almeida et al., 2019; Bahr et al.,
2015; Högberg et al., 2007, 2010; Nilsson and Wallander, 2003), which
will reduce the input of C to the soils and may reduce C sequestration.
However, Bödeker et al. (2014), for example, showed that addition of
inorganic N significantly decreased the abundance of Cortinariusacutus, a species that can
enhance SOM decomposition in order to take up N (Lindahl et al., 2021). The
decrease in Cortinarius sp. was accompanied by a decrease in the enzymatic oxidation in
the humus layer of the soil. Therefore, it has been suggested that
fertilization might improve C sequestration by suppressing SOM decomposition
by some key EMF genera like Cortinarius (Lindahl and Tunlid, 2015; Bödeker et
al., 2014). In the current study we show that Piloderma, another common species from
northern-forested ecosystems, is negatively affected by fertilization and
that its decrease might be associated with a decrease in the organic-material hydrophobicity. These findings suggest that even if fertilization
could reduce the abundance of EMF species with decomposer capabilities, it may also
reduce the accumulation of hydrophobic fungal mycelium that could enhance
SOM formation and C sequestration rates. Therefore, the role of different
abundant EMF genera like Piloderma and Cortinarius in boreal forests for establishment and
destruction of hydrophobicity and the effect of fertilization on them
warrant further research.
Code availability
The pipelines and software used to analyze the data in this study are described in the references provided in Sect. 2.4 and 2.6 of this article.
Data availability
For further information on the data, the readers should contact the corresponding author of the article.
Author contributions
JPA contributed in terms of conceptualization of the research goals and aims, data curation and
analysis, and manuscript writing. NPR contributed in terms of data acquisition, curation and analysis. SKW contributed in terms of data acquisition, curation and analysis. GG contributed in terms of conceptualization and development of the methodology. HW contributed in terms of conceptualization and development of the methodology, research goals and
aims and manuscript writing.
Competing interests
The contact author has declared that none of the authors has any competing interests.
Disclaimer
Publisher’s note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Special issue statement
This article is part of the special issue “Global change effects on terrestrial biogeochemistry at the plant–soil interface”. It is not associated with a conference.
Acknowledgements
We would like to thank the Swedish Research Council for Sustainable Development (Formas) for financial support.
Financial support
This research was supported by a grant from Formas (grant no. 2018-00634) for Håkan Wallander.
Review statement
This paper was edited by Serita Frey and reviewed by Mark Anthony, Christopher Fernandez, and one anonymous referee.
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