Ecological Indicators 141 (2022) 109136

IRMA PAYUK/K012231019
Contents lists available at ScienceDirect

Ecological Indicators
journal homepage: www.elsevier.com/locate/ecolind

Original Articles

Fungal genetic biodiversity and metabolic activity as an indicator of

potential biological weathering and soil formation – Case study of towards
a better understanding of Earth system dynamics
Anna Gałązka a, *, Anna Marzec-Grządziel a, Jarosław Grządziel a, Milan Varsadiya b,
Łukasz Pawlik c
a
Department of Agricultural Microbiology, Institute of Soil Science and Plant Cultivation-State Research Institute (IUNG-PIB), Czartoryskich 8, 24-100 Puławy, Poland
b
Department of Ecosystems Biology, University of South Bohemia in České Budějovice, Branišovská 31, 370 05 České Budějovice, Czech Republic
c
Institute of Earth Sciences, Faculty of Natural Sciences, University of Silesia in Katowice, Będzińska 60, 41-200 Sosnowiec, Poland

A R T I C L E I N F O A B S T R A C T

Keywords: Terrestrial plants act as ecosystem engineers modifying the flow of energy and matter and creating new habitats
Metataxonomics analysis of ITS rRNA next- for other organisms. This vital concept also encompasses plants’ effects on landforms and soils, crucial com­
generation sequencing ponents of forested landscapes worldwide. In the present study, we investigate how trees through their roots and
Fungal diversity
symbiotic organisms influence soil-weathering processes. The aim of the study was to answer one of the big
Tree root zone
Metabolic profiles of fungi
questions in Earth sciences: how do biological agents, including fungi, acting at the critical interface between the
Biological weathering biosphere and the abiotic environment, shape soil and landscape evolution? Within the present study we ask
Rock crack what is the level of fungal activity within root systems of trees and how can it influence biological weathering.
The area of interest is in the Poprad River gorge in the southern part of the Sącz Beskidy Mountains, the Outer
Western Carpathians. We applied the following analyses: 1) determination of the structural diversity of fungi
(ITS1) and 2) assessment of the metabolic profile of soils (Biolog FFPlates). The highest average number of
classified genera were fungi which simultaneously carried out pathotrophic, saprotrophic and symbiotrophic
functions. Boletales, Agaricales, Cantharellales and Archaeorhizomycetales were the most abundant orders, but
in one sample we also found a particularly high proportion of the order Mortierellales. The order Boletales and its
family Boletaceae were significantly enriched in rock crack samples, whereas the highest number of differentially
abundant taxa was observed in reference samples. The most frequently utilised substrates by fungi were: glycyl-L-
glutamic acids, L-ornithine, L-phenylalanine, L-proline, D-galacturonic acid, fumaric acids, D-saccharic acids,
succinic acids and N-acetyl-D-glucosamine. Our study demonstrates that the fungal community in the root zone is
geochemically active and the organic acids secreted by plant roots in oligotrophic conditions and nutrient
limitations significantly affect soil weathering.

1. Introduction formation (Nascimento et al., 2021), fungal communities (Uroz et al.,

Plants shape the Earth surface and impact the activity of geomorphic
and soil processes. Interactions between plants, soils, regolith and
bedrock are normally considered in a pedon scale but can be up-scaled to
the landscape scale when analyzed in a much longer time intervals
(Pawlik et al., 2020). Bedrock weathering by tree roots and associated
microorganisms (bacteria and fungi) has been considered an important
soil production agent since at least (Pawlik et al., 2016a). In recent years
the studies on microbial geomorphology (Viles, 2012), regolith
2009) and mycorrhizae (Bornyasz et al., 2005) has been significantly
improved through on aspect of biological weathering. Very important in
this context is the structural and functional diversity of fungi.
Within a plant’s rhizosphere, complex interactions between Earth’s
biotic and abiotic components occur (Amundson et al., 2007; Holbrook
et al., 2019). Many such interactions are poorly understood or even
await their full discovery (Brantley et al., 2011, 2017). One of the key
processes driving the evolution of the terrestrial system is biological
weathering (Zaharescu et al., 2020). However, owing to the complexity

* Corresponding author.
E-mail address: agalazka@iung.pulawy.pl (A. Gałązka).

https://doi.org/10.1016/j.ecolind.2022.109136
Received 11 April 2022; Accepted 30 June 2022
Available online 9 July 2022
1470-160X/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
A. Gałązka et al.
Fig. 1. An orthophotomap and digital elevation model with the geographical position of the sampling site.
of processes and interactions standing behind different forms of bio­
logical weathering, additional thorough analyses are urgently needed
(Dontsova et al., 2020; Samuels et al., 2020). Our consideration of
conditions affecting fungal efficiency on biological weathering requires
a deep look into the biosphere, but also into the lithosphere and the
atmosphere.
In terms of biological weathering, trees may play a major role in
some circumstances. This can be partly explained by their size and
longevity (Miles, 1986; Binkley and Giardina, 1998; Pawlik, 2013;
Pawlik et al. 2016b). Plants are associated with their own microbiome
(White et al., 2016; Woźniak et al., 2022). Endophytic fungi, which live
inter- and intracellularly in plants and do not induce pathogenic
symptoms, interact with the host biochemically and genetically
(McNear, 2013; Woźniak and Gałązka, 2019). Endophytic fungi exhibit
mostly functions as plant growth and development. This is mainly due to
functions such as: defence promoters by synthesising phytohormones;
producing enzymes or precursors for secondary plant metabolites bio­
surfactants, controlling plant diseases, fixing atmospheric nitrogen and
CO2, as well as by providing a source for new bioactive natural products
(e.g. Wani et al., 2016). Fungi of soils are very sensitive indicators of
changes in the soil environment. Disturbances caused by anthropogenic
factors lead to changes in the fungal composition, which often manifests
itself in reduced biodiversity and redundancy (Nair and Padmavathy,
2014; White et al., 2016).
The rhizosphere is a part of the Critical Zone where microorganisms
(bacteria, fungi and mycorrhizal fungi) live in symbiosis and interact
with tree roots. Fungi play a important role in soil quality and func­
tioning and plant health. They determining nutrient cycling and soil
structure, and ultimately impacting plant performance through nutrient
mobilisation (Grządziel and Gałązka, 2019). Mycorrhizal fungi are also
producers of glomalins (fungal glicoproteins), which stabilize soil par­
ticles and prevent soil degradation (Gałązka and Gawryjołek, 2015). Our
previous studies have highlighted the importance of diverse below­
ground systems to maintain the stability and productivity of terrestrial
ecosystems and their multifunctionality (Woźniak and Gałązka, 2019).
These microorganisms benefit from carbon-rich root exudates; in
response, their living functions can accelerate weathering of minerals
through excretion of: siderophores, organic acids, protons and phenolic
compounds (Landeweert et al., 2001). Some authors argue that chemical
weathering mediated by microorganisms is more important than phys­
ical weathering because microbes can survive under the most severe
conditions for long periods of time (Burford et al., 2003).
The development of fungi in soil is conditioned by many factors, the
most important of which are: soil moisture and air access, the presence
of nutrients, temperature, pH and soil structure (Frąc et al., 2018). Fungi
live mainly in the upper soil horizons but have also been found several
kilometres below soil surface (Akob and Küsel, 2011; Gałązka and
Gawryjołek, 2015). Arbuscular mycorrhizal fungi (AMF) are one
important group of soil microorganisms; they form associations with
80%–90% of all terrestrial plants and have many beneficial functions for
their host plants as well as for their environments (Gałązka et al., 2017).
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Ecological Indicators 141 (2022) 109136
This aspect of root zone (rhizosphere) activity must be addressed to
understand and provide a more complete interpretation of the complex
way bedrock is transformed under the impact of tree roots and fungal
soil biota. At the end of the 20th century, researchers suggested that
these microorganisms are responsible for tunnelling of mineral grains
through the hyphae of ectomycorrhizal fungi. This phenomenon has
been documented mainly in Podzol soils, and the fungal type was named
rock-eating fungi. Mycorrhizal fungi have the ability to penetrate sites
that cannot be reached even by the thinnest fine roots (non-vascular
roots), such as intergranular mineral spaces (Pawlik et al., 2020).
Today, a growing amount of information on soil microbial diversity
and microbial functions is provided by metataxonomics analysis of next-
generation sequencing (NGS) based on the internal transcribed spacer
(ITS) of ribosomal RNA (rRNA). However, biodiversity characterisation
that takes into account only taxonomic aspects based on molecular data
(e.g. 16S rRNA for bacteria and ITS for fungi) as a measure of soil biota
structure and stability is under discussion (Ros et al., 2008; Grządziel
and Gałązka, 2019). On the basis of the ecological hypothesis of
redundancy (redundant hypothesis), which has existed for many years, a
new one is being built, investigating functional groups (a group of or­
ganisms that performs the same function in the ecosystem), rather than
species, which play an important role in the functioning of the ecosys­
tems (Morelli and Tryjanowski, 2016). Therefore, we aim to use meta­
genomics (NGS) and metabolic analysis (e.g. with the use of Biolog
FFPlates) simultaneously to examine plant performance parameters and
ecosystem functions, which should allow verification of hypotheses
based on the identification of fungi and evaluation of their functional
activity.
Within the present study we ask: what is the level of fungal biodi­
versity and activity within root systems of trees and how can it influence
biological weathering and subsequent soil production? The aim of the
study was to evaluate the fungal genetic and functional biodiversity in
the tree rhizosphere and rock cracks. We hypothesised that soil fungal
mycobiome and their metabolic activity is higher in places occupied by
living trees compared with places covered only by understory
vegetation.
2. Materials and methods
2.1. Study area and soil sampling
The area of interest was in the Poprad River gorge in the southern
part of the Sącz Beskidy Mountains, Poland. The mountain range belongs
to the Outer Western Carpathians and is predominantly built of flysch
rocks consisting of sandstones, mudstones and conglomerates. The area
is still neotectonically active. The study site is on a steep north-facing
valley side, approximately 250 m from the Poprad River (Fig. 1). The
area is known as prone to rock falling and for this reason metal nets were
installed along the road passing below the study site. The climate of the
area is temperate with a mean daily temperature of + 8.3 ◦ C (-2.65 ◦ C in
January, +18.2 ◦ C in July; data for 2010–2020), and a mean daily
A. Gałązka et al. Ecological Indicators 141 (2022) 109136

Fig. 2. The soil sampling scheme.

area of interest).
Table 1
Soil sampling description.
2.2. Sampling strategy
Symbol Explanation of Soil sampling description
abbreviations
Soil samples were collected in October 2020 from three types of sites:
Tree root zone
TR1S_20 T – tree, R – root, S – soil Rhizosphere soil; depth 20 cm
1) tree root zone, 2) rock cracks and 3) a reference site (Fig. 2, Table 1).
TR1S_40 T – tree, R – root, S – soil Rhizosphere soil; depth 40 cm Following our initial assumption that highlights the root zone as a
TR1S_60 T – tree, R – root, S – soil Rhizosphere soil; depth 60 cm hotspot of higher fungal activity and potential weathering processes, we
TR1S_80 T – tree, R – root, S – soil Rhizosphere soil; depth 80 cm took five soil samples from the rhizosphere zone of the main Norway
TR1S_100 T – tree, R – root, S – soil Rhizosphere soil; depth 100 cm
spruce root. The sampling interval was 20 cm starting from the zone
TS_Ref T – tree, Ref – reference, S A soil sample taken 30 cm from the tree
– soil below the tree stump and reaching a depth of 100 cm. In the first group,
Tref H1 T – tree, R – root Horizon O/A (needle, leaves) we also included three samples taken from a distance close to the tree
Tref H2 T – tree, R – root Horizon O/A (moss) stump, assuming that the microbiological profile is different from the
reference samples (Table 1).
Rock crack The second group consisting of various localities with rock cracks are
TMS T – tree, M – mycorrhiza, S Soil with mycorrhiza from rock crack, considered zones of initial soil development. They are places of my­
– soil without root
corrhiza and root activity, but also of weathering of potentially different
TR2C2 T – tree, R – root, C – rock Sample from rock crack with root R2
crack magnitudes. All these factors contribute to the soil formation at the site.
C1R2 C – rock crack, R – root Sample from rock crack with root R2; Rock cracks are a result of physical weathering, but in the current study
depth 100 cm we assumed that the presence and activity of roots and mycorrhiza could
C2R2M C – rock crack, R – root, M Sample from rock crack with root and amplify weathering processes (thus contributing to biological weath­
– mycorrhiza mycorrhiza
ering) and soil development in those specific conditions. We took seven
C3M C – rock crack, M – Sample from rock crack with mycorrhiza
mycorrhiza without root; depth 130 cm soil samples consisting of roots and mycorrhiza. One case (TMS) was just
TR3S T – tree, R – root, S – soil Soil from rock crack with root R3; depth soil with mycorrhiza and another case (C2R2M) was a sample with root
60/40 cm and mycorrhiza (Table 1).
TR4 T – tree, R – root Sample from rock crack with root; depth
To have a background for a direct comparison of microbial activity
88/44 cm
related to tree roots in different localities, the third group consists of
samples taken from the reference site, characterised by the lack of living
Reference site Distance from the top edge of the soil pit/distance from
or dead trees. The site was on the same slope surface but within a
the left edge of the soil pit/depth
Pref_1 P – soil pit 10 cm/20 cm/10 cm minimum distance of 2 m from any tree. We did not notice any remnants
Pref_2 P – soil pit 10 cm/50 cm/10 cm of decomposed wood that would suggest occupation of that site by a tree
Pref_3 P – soil pit 10 cm/90 cm/10 cm in the past. We took 10 reference samples from this site. The maximum
Pref_4 P – soil pit 36 cm/0 cm/25 cm
sampling depth was 40 cm.
Pref_5 P – soil pit 105 cm/20 cm/40 cm
Pref_6 P – soil pit 80 cm/50 cm/40 cm
Pref_7 P – soil pit 65 cm/50 cm/40 cm 2.3. DNA extraction and region ITS1 sequencing
Pref_8 P – soil pit 50 cm/0 cm/20 cm
Pref_9 P – soil pit 60 cm/80 cm/20 cm
Pref_10 P – soil pit 105 cm/10 cm/25 cm 300–350 mg of each sample was collected and DNA extraction was
performed using a commercially available kit (FastDNA™ SPIN Kit for
Soil, MP Biomedical). The DNA concentration and purity were measured
precipitation total of 2.3 mm (mean annual precipitation total for by using a NanoDrop 1000 Spectrophotometer (Thermo Scientific). Each
2010–2020 was 902.6 mm; https://danepubliczne.imgw.pl/) measured DNA sample was diluted to (10 ng µl− 1) with sterile MilliQ water.
at the Piwniczna-Zdrój meteorological station (10 km south-west of our Sequencing was performed by an external company (Genomed S.A,

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A. Gałązka et al. Ecological Indicators 141 (2022) 109136

Fig. 3. Fungal structural diversity: A – phylum, B – order and C – class.

4
A. Gałązka et al.
Fig. 4. Phylogenetic tree based on metataxonomics analysis of the internal transcribed spacer (ITS) of the ribosomal RNA (rRNA) gene. The heatmap represents the
number of amplicon sequence variants (ASVs) identified in each sample. The bar charts represent the frequency of the core microbiome genera in each sample (%).
Warsaw, Poland) with 2 × 300 base pair (bp) paired-end technology
using the Illumina MiSeq system. PCR amplification of the selected ITS1
region was performed by using Q5 Hot Start High-Fidelity 2x Master
Mix, with primers ITS1Fl2 (5′ -GAACCWGCGGARGGATCA-3′ ) and 5.8S
(5′ -CGCTGCGTTCTTCATCG-3′ ) (Schmidt et al., 2013). The ITS ampli­
con sequencing raw data (fastq) have been deposited in the NCBI
Sequence Read Archive (SRA) under the BioProject PRJNA780801.
Different alpha diversity indices (species richness – Chao1; diversity
– Shannon; evenness – Simpson) were calculated using the Microbiome
package in R 3.5.3 (Lahti and Shetty, 2017). Functional annotation of
each identified fungus was done by assigning each amplicon sequence
variant (ASV) to a trophic mode and lifestyle level based on the Fun­
galTraits database (Põlme et al., 2020).
2.4. Functional diversity analysis using Biolog FFPlates
The metabolic potential of the fungal communities present in the
selected soil samples was evaluated using the Biolog FFPlate method
(Biolog Inc., Hayward, CA, USA). The metabolic capacities of soil fungi
present in each sample were determined by ability of decomposition of
96 different carbon sources (Garland and Millis, 1999; Preston-Mafham
et al., 2002; Cartwright, 2015). Each soil sample was prepared as fol­
lows: 1 g of soil sample was transferred to conical flasks with 99 cm3
sterile 0.9% NaCl, vortexed for 30 min at 150 rpm (25 ◦ C), and cooled
for 30 min (4 ◦ C). Next, 100 mm3 of each sample was transferred to each
well in a FFPlate. The plates were incubated in the dark for 144 h
(25 ◦ C). Each samples was analysed in three technical replications. The
results were read every 24 h using MicroStation ID systems by Biolog®.
The reduction of colourless tetrazolium chloride to red formazan (λ =
590 nm; Gałązka et al., 2017) determined the decomposition of analysed
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Ecological Indicators 141 (2022) 109136
carbon sourcesthrough. The most intense reaction was observed after
120 h of incubation. All analysed carbon sourceshave been grouped into
carbohydrates, amines and amides, polymers, amino acids, carboxylic
acids (Preston-Mafham et al., 2002).
2.5. Statistical and bioinformatics analysis
All bioinformatics was performed in R version 4.0.3 (R Core Team,
2020). Exact amplicon sequence variants (ASVs) were resolved from
forward and reverse reads by using the DADA2 v. 1.18 package (Call­
ahan et al., 2016). All obtained reads were trimmed to 250 nucleotides
(nt); the leftmost 20 nt containing low-quality bases were removed. The
primer sequences were removed by using cutadapt (Martin, 2011). The
sequences were filtered as follows: maxN = 0, maxEE = 2 and truncQ =
2 (other parameters were set to default). The standard error rates were
estimated by learnErrors (n-reads set to 1 × 106). The replicated reads
were removed by using derepFastq (default parameters), and exact ASVs
were obtained. The chimeric sequences were removed with remov­
eBimeraDenovo. The taxonomy was assigned by using a Naïve Bayesian
Classifier against the UNITE database (v. 8.3, built 2020.12.11) (Wang
et al., 2007). All the taxa other than fungi kingdom were removed and
resulting taxonomy was analysed by using phyloseq package (McMurdie
and Holmes, 2013).
The statistical analysis was performed by using STATISTICA 10
(StatSoft. Inc., Tulsa, OK, USA). The variance of the obtained data were
analysed for comparison of means (ANOVA). The Tukey’s post hoc test
was performed for significant differences calculations (P < 0.05). The
statistical analyses were performed on standardised data from Biolog
FFPlate (the average absorbance values at 120 h). The obtained results
were also analysed by the principal component analysis (PCA).
A. Gałązka et al. Ecological Indicators 141 (2022) 109136

Table 2 was found in sample TR2C2. In the same sample, the highest relative
The amplicon sequence variants (ASVs). order proportion was unidentified_120. Boletales, Agaricales, Canthar­
Sample ID ASVs Classified ASVs (%) NCBI ID ellales and Archaeorhizomycetales were the most abundant orders, but
Sequence Read Archive (SRA) sample TR2C2 also had a particularly high relative proportion of Mor­
Tree root zone tierellales (Fig. 3b). Boletales was dominant in rock crack samples. The
TR1S_20 69 69.565 SRR16955238 highest relative proportion of classes in all samples was Agaricomycetes,
TR1S_40 87 68.966 SRR16955237 Eurotiomycetes, Leotiomycetes and Mortierellomycetes (Fig. 3c). Class
TR1S_60 68 70.588 SRR16955226 unidentified_143 was confirmed in samples TR2C2, TR1S_20, TR1S_40,
TR1S_80 87 66.667 SRR16955220
TR1S_100 38 65.789 SRR16955219
TR1S_60 (Fig. 3c). The highest number of ASVs was detected in sample
TS_Ref 42 64.286 SRR16955235 Tref_H2 (1 7 1), and the lowest was in sample TR1S_100 (38) (Fig. 4).
T_Ref_H1 171 64.327 SRR16955233 The percentage of classified ASVs was the highest in sample TR2C2
T_Ref_H1 112 64.286 SRR16955232 (73.239%) (Table 2). The fungal core microbiome represents seven ASVs
(24.587% of each sample, on average) (Fig. 4). Bioinformatics analysis
Rock crack of individual samples revealed the presence of 77 unique fungal ASVs,
TMS 44 63.636 SRR16955218 the highest number of which was found in sample T_Ref_H1 (47 ASVs,
TR2C2 71 73.239 SRR16955217
27.485% of the sample).
C1R2 43 69.767 SRR16955216
C2R2M 51 64.706 SRR16955215 The most abundant genera present in samples taken from tree root
C3M 44 72.727 SRR16955214 zone were Boletus, Discosia, Exophiala, Mortierella, Penicillium, Pseudo­
TR3S 65 66.154 SRR16955236 tomentella, Tricholoma and Wilcoxina. For rock crack samples, the most
TR4 58 63.793 SRR16955234
abundant genera were Boletus, Exophiala, Oidiodendron, Penicillium,
Sagenomella and Tricholoma (Fig. 5a and 5b).
Reference samples To find differentially abundant families among the groups, we used
Pref_1 87 71.264 SRR16955231
LefSe. Reference samples were significantly enriched with orders Tre­
Pref_2 95 69.474 SRR16955230
Pref_3 108 70.370 SRR16955229 chisporales and Polyporales and family Tricholomataceae from order
Pref_4 115 70.435 SRR16955228 Agaricales compared with rock crack and references site samples. We
Pref_5 80 70.000 SRR16955227 found only order Boletales and its family Boletaceae were significantly
Pref_6 100 67.000 SRR16955225 enriched from rock crack samples, whereas the highest number of
Pref_7 83 66.265 SRR16955224
Pref_8 111 70.270 SRR16955223
differentially abundant taxa was observed for reference site samples.
Pref_9 104 67.308 SRR16955222 Phylum Mortierellomycota and its members were significantly enriched
Pref_10 83 68.675 SRR16955221 in reference site samples compared with tree root zone samples. Inter­
estingly, we found classes Leotiomycetes and Archaeorhizomycetes and
their members were significantly enriched; however, this difference was
Two nonparametric test, Kruskal–Wallis sum-rank and the Wilcoxon,
not observed at the phylum level. LefSe also revealed that families
were used to test the results for consistent differences in family taxa
Myxotrichaceae, Russulaceae, Sebacinaceae, Cortinariaceae, Athelia­
abundance among different groups (P < 0.05). Both tests were prepared
ceae and Hydnaceae had significantly higher abundance in comparison
with linear discriminant analysis (LDA) to estimate the effect size of
to reference site samples (Fig. 6).
taxonomical covariates driving the group difference (LefSe) (Segata
Analysis of pooled samples (tree root zone, rock crack and reference
et al., 2011; Varsadiya et al., 2021). The threshold of the logarithmic
site samples) revealed the presence of 137 unique ASVs, mostly in tree
LDA score was set to 4. For a more general comparison of our results, we
root zone samples (86). The core microbiome of the pooled samples
have searched for rhizosphere soil fungi in the Global Fungi Atlas (https
comprises 92 ASVs (Fig. 7). The five uniquely classified ASVs which
://globalfungi.com/). We have filtered the database and selected the
were found in rock crack samples belong to genera Aureobasidium,
most abundant fungi genus taxa found in our study. The results were
Curvibasidium, Paraphoma, Rhodosporidiobolus and Simplicillium. One
then mapped using R.
ASV was not classified at either the genus or family level (order: Sor­
dariales) (Fig. 7). This uncultured fungus was previously found in forest
3. Results
soil habitat and was associated with Fagus sp. Root (GENBANK:
JF519087.1).
3.1. iTS NGS
3.1.2. Fungal life style and fungal biodiversity indices
3.1.1. Fungal structural diversity based on ITS NGS
Fungal biodiversity indices were evaluated based on the data ob­
Analysis of fungal sequencing data revealed the presence of 304
tained from ITS sequencing. The Fungal alpha diversity indices for each
ASVs belonging to 200 different genera, 128 families, 65 orders, 23
of the soil samples were presented in Fig. 8. The soil collected from the
classes and 8 phyla. There were 96 ASVs unclassified genera (63 un­
tree root zone, rock crack and references site were characterised by
classified families, 28 unclassified orders, 13 unclassified classes and 1
significant differences in the indicators (Chao1 and Shannon diversity).
unclassified phylum).
The highest Chao1 diversity was identified in tree root zone samples.
The most abundant phylum was Basidiomycota, followed by Asco­
The Simpson evenness index was similar at all sites (Fig. 8).
mycota and Mortierellomycota (Fig. 3a). In general, we found a higher
LDA was used to estimate the effect size of taxonomical covariates
relative proportion of the phyla Basidiomycota, Ascomycota and Mor­
driving the group difference procedure implemented in LefSe; it showed
tierellomycota in all samples belonging to the three study groups: tree
that mycorrhizal fungi dominated in the all three groups (Fig. 9). This
root zone, rock crack and the reference site. We found a particularly high
confirms our earlier hypotheses about the significant participation of
relative proportion of Mortierellomycota in the TR1S_20, TR1S_40,
ectomycorrhizal fungi in the process of biological soil weathering.
TR1S_60 and TR2C2 samples, which may indicate intensive biological
Moreover, all sample groups had a high share of unspecified saprotrophs
weathering processes. Mortierella species are soil saprotrophs in soil.
and unclassified fungal families. Soil saprotrophs also dominated in the
Trichoderma, Mucor, Penicillium, and Mortierella species belong to an
reference site group.
ecological group representing the first organisms that grow on roots and
The highest average number of classified genera were fungi that
initiate the processes formation of humic substances and soil. In addi­
simultaneously functioned as a pathotroph, saprotroph and symbiotroph
tion, a particularly high relative proportion of the phylum ‘unidentified’
(25.229% of all samples). Pathotrophs accounted for an average of

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Fig. 5. Chord diagrams based on metataxonomics analysis of internal transcribed spacer (ITS) of the ribosomal RNA (rRNA) gene. A – tree root zone and B – rock
crack. The upper parts of the diagrams represent samples taken from the tree root zone (A) and rock crack (B). The lower parts of the diagrams show the genera
present with > 2% abundance on average in the analysed samples.

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A. Gałązka et al. Ecological Indicators 141 (2022) 109136

Fig. 6. Linear discriminant analysis effect size (LefSe) representing differentially abundant families between the tree root zone, rock crack and references
site samples.

0.272% of each sample, saprotrophs accounted for 9.487% and sym­

biotrophs accounted for 13.006%. According to the place of sampling,
samples taken from the tree root zone and rock crack showed the highest
composition of fungi with pathotrophic, saprotrophic and symbio­
trophic functions (24.263% and 52.037%, respectively). For reference
samples taken from places without biological weathering symptoms,
fungi with symbiotrophic function (31.601%) were the most abundant
(Fig. 10). Within the microorganisms present only in rock crack samples,
one showed pathotrophic, saprotrophic and symbiotrophic functions;
two showed pathotrophic and saprotrophic functions; and one showed
pathotrophic function (in addition, 1 ASV was unclassified and 1 was not
assigned to any function).

3.2. Evaluation of fungal physiological profile and metabolism based on

Biolog FF plates

3.2.1. Fungal physiological and metabolic profiles

Fig. 7. Venn diagram prepared based on internal transcribed spacer (ITS) of the
ribosomal RNA (rRNA) gene sequencing. Each circle represents pooled samples
from three different sampling locations. The data presented in the graph refer to
the site-specific unique amplicon sequences variants (ASVs) and the
core microbiome.
Based on analysis of the metabolic profile results, the most abundant
group of compounds used by fungi were carbohydrates, followed by
carboxylic acids, amino acids, and polymers. The percentage relation­
ship changed slightly among the groups of compounds in individual
samples (Table 3). The following samples showed the highest decom­
position of amines and amides: TR2C2, TR1S20, T_Ref_H1 and Pref_10.
The strong use of amino acids by fungi was confirmed in samples
TR1S_20, TS_Ref, C3M, TR3S and Pref_10. Carbohydrates were most
degraded by fungi in samples TR1S_20, TR1S_80, TR1S_100 and TMS.
The greatest use of polymers by fungi was found in samples C2R2M,
C1R2 and Pref_6 (Table 3).
The most frequently utilised amino acids were: glycyl-L-glutamic
acids, L-ornithine, L-phenylalanine and L-proline. L-Phenylalanine was
used in particular by fungi in sample C3M, which showed hyphae of

8
A. Gałązka et al.
Fig. 8. Fungal biodiversity indices: Chao1, Shannon and evenness Simpson.
Fig. 9. Lifestyle analysis in a group of individual soil samples.
Fig. 10. Heatmap presenting the functions of classified genera prepared based
on internal transcribed spacer (ITS) of the ribosomal RNA (ITS) rRNA gene
sequencing. The results represent samples pooled based on the sampling site.
The data presented on the graph are scaled.
9
Ecological Indicators 141 (2022) 109136
mycorrhizal fungi. L-Threonine was the least metabolised amino acid in
all samples (Fig. 11A). D-Galacturonic acid was the most metabolised
carboxylic acid in all samples. Fumaric acids, D-saccharic acids and
succinic acids were also actively used by fungi. The highest metabolic
potential in the use of carboxylic acids was found in sample Pref_10
(Fig. 11B). N-Acetyl-D-glucosamine was the most actively utilised car­
bohydrate, especially in sample C3M. The highest metabolic potential of
carbohydrate utilisation was found in rock crack samples. Moreover, the
high functional potential of fungi was confirmed by the utilisation of the
following compounds: D-sorbitol, N-acetyl-D-glucosamine and D-mele­
zitose (Fig. 11C).
3.2.2. Fungal functional biodiversity indices
The fungal biodiversity indices were calculated on the data (the
absorbance values) of Biolog FFPlates after 120 h of incubation are
presented in Table 4. The Shannon’s general diversity index (H’), sub­
strate evenness (E), substrate richness R and average well colour
development at 590 nm (AWCD590) were statistically significant dif­
ferences within the groups. The highest H’ index was observed in sam­
ples TR1S_20, TR1S_40, TR1S_60, TR1S_80, TS_Ref, TR2C2, Pref_1,
Pref_3, Pref_5 and Pref_10 (Table 4). The highest values of the R index
and the AWCD590 index were also found in the same samples, which may
prove both the high metabolic activity of the fungi in these samples and
their high biodiversity. In turn, the substrate evenness index (E)
remained at the same level in all samples.
A. Gałązka et al. Ecological Indicators 141 (2022) 109136

Table 3
The percentage (%) utilisation of substrate groups in soil samples. Treatment means with different letters are significantly different (Tukey’s mean separation test, p <
0.05; n = 3).
Sample ID Amines and amides Amino acids Carbohydrates Carboxylic acids Miscellaneous Polymers

Tree root zone

TR1S_20 5.73a ± 0.60 21.68a ± 0.94 38.91 ± 1.54 20.31a ± 0.30 8.76b ± 1.28 4.61b ± 0.80
TR1S_40 5.34a ± 0.27 17.48b ± 0.17 44.27b ± 1.57 18.26a ± 1.15 9.75a ± 0.46 4.89b ± 0.51
TR1S_60 5.63a ± 0.08 16.34b ± 0.38 48.75a ± 0.51 17.07b ± 0.29 7.80b ± 0.55 4.42b ± 0.25
TR1S_80 5.60a ± 0.38 16.39b ± 0.31 47.54a ± 0.92 18.06a ± 0.33 7.89b ± 0.46 4.51b ± 0.48
TR1S_100 5.27a ± 0.72 16.36b ± 2.58 47.70a ± 4.35 15.89b ± 0.92 9.50a ± 1.91 5.26b ± 2.48
TS_Ref 3.97c ± 1.07 25.58a ± 3.52 37.42c ± 3.17 25.44a ± 0.61 5.32c ± 0.34 2.27c ± 0.58
T_Ref_H1 5.76a ± 0.63 17.16b ± 0.36 42.75b ± 1.12 19.62a ± 0.13 9.61a ± 1.18 5.30d ± 0.36
T_Ref_H2 5.51a ± 0.21 16.57b ± 0.42 41.84b ± 3.75 19.36a ± 1.63 1.26d ± 0.53 6.46a ± 1.48

Rock crack
C2R2M 3.63c ± 0.26 16.83b ± 1.63 42.55b ± 2.19 16.71b ± 0.85 11.00a ± 0.67 9.28a ± 1.51
C3M 5.16b ± 0.74 21.30a ± 0.12 41.39b ± 0.92 21.67a ± 0.54 7.55b ± 0.82 2.93c ± 0.39
TR3S 4.99b ± 0.28 22.15a ± 0.77 41.76b ± 1.87 21.76a ± 1.83 6.10c ± 0.49 3.23c ± 0.55
TR4 4.51b ± 0.39 17.73b ± 0.42 43.69b ± 1.51 16.65b ± 1.01 10.71a ± 1.10 6.71a ± 0.68
TMS 3.24c ± 0.12 17.09b ± 1.50 48.09a ± 0.92 16.82b ± 1.45 9.36a ± 0.50 5.40b ± 0.86
TR2C2 6.22a ± 0.42 15.48b ± 2.14 43.86b ± 1.61 16.69b ± 0.58 12.83a ± 1.14 4.92b ± 0.80
C1R2 3.63c ± 0.22 16.83b ± 1.41 42.55b ± 0.84 16.71b ± 1.20 11.00a ± 0.78 9.28a ± 0.28

Reference site
Pref_1 4.76b ± 0.37 17.15b ± 0.59 46.29a ± 1.21 21.59a ± 0.30 6.97b ± 0.88 3.22c ± 0.12
Pref_2 5.56a ± 0.35 17.39b ± 0.77 41.29b ± 2.91 22.75a ± 0.70 8.42b ± 1.29 4.60b ± 1.43
Pref_3 4.73b ± 0.24 19.44a ± 0.47 44.63b ± 2.48 18.45a ± 1.10 8.32b ± 0.75 4.42b ± 0.59
Pref_4 5.09b ± 0.12 16.47b ± 0.98 44.20b ± 2.19 18.21a ± 0.92 10.21a ± 1.27 5.81b ± 0.31
Pref_5 4.67b ± 0.41 18.13a ± 1.42 45.05a ± 1.50 19.46a ± 0.17 8.05b ± 0.40 4.64b ± 0.82
Pref_6 4.46b ± 0.29 15.98b ± 1.50 43.04a ± 3.92 17.19b ± 0.78 11.28a ± 0.91 8.05a ± 1.71
Pref_7 5.12b ± 0.32 15.51b ± 1.51 46.24a ± 3.01 16.61b ± 0.57 10.30a ± 0.79 6.22a ± 1.33
Pref_8 4.56b ± 0.10 17.82b ± 0.97 42.43b ± 1.08 18.29a ± 0.33 10.27a ± 0.11 6.62a ± 0.21
Pref_9 5.22b ± 0.17 16.55b ± 0.22 44.71a ± 0.57 18.69a ± 0.73 9.29a ± 0.52 5.53b ± 0.47
Pref_10 5.86a ± 0.55 21.87a ± 0.32 41.03b ± 0.99 21.07a ± 0.50 6.36c ± 1.54 3.80c ± 0.20

4. Discussion
Biomechanical weathering encompasses the ability of living organ­
isms to physically change soils, regolith and bedrock. The type of bio­
logical weathering has rarely been studied; far greater attention has
been directed to biomechanical effects, which include soil mixing, bio­
turbation and biotransport, and root growth (Pawlik et al. 2016b).
Contributions of tree roots to mineral weathering of forest soils include
some critical processes (Landeweert et al., 2001; McNear, 2013; Pawlik
et al., 2016a). One of them is the participation of microorganisms (rhi­
zobacteria and fungi) in the stabilisation of mineral particles, leading to
the release of inorganic nutrients (Uroz et al., 2009). Another important
factor is the plant root secretion of active low-molecular-weight com­
pounds (LMW) such as acids, anions, organic acids and carbon-rich ex­
udates (Landeweert et al., 2001). In the process of biological
weathering, an increased concentration of high-molecular-weight
organic acids in the soil solution is also observed. It is manifested as a
pH decrease caused by increased partial CO2 of soil solutions due to
plant respiration and evapotranspiration, and elevated soil moisture
content, allowing for enrichment of water in base cations and other
plants nutrients (Sokolova, 2015; Mommer et al., 2016a).
The main assumptions have motivated us to look for definitive an­
swers regarding the potential ability of tree roots to weather fractured
bedrock and, in association with microorganisms, chemically alter it and
make it more susceptible to further penetration by tree roots. This
strictly biological function of roots in the context of bedrock weathering
and soil formation is one of the most important scientific problems of the
recently defined hillslope biomorphodynamics concept and bio­
geomorphology discipline (Goudie and Viles, 2012; Pawlik et al., 2016a;
Šamonil et al., 2018).
4.1. The role of fungal structural diversity in biological weathering
Fungi are soil-forming factors involved in the process of shaping soil
fertility and providing plants with nutrients and detoxifying harmful
chemical compounds (Frąc et al., 2018). Fungi have various character­
istics that allow them to master all habitats suitable for life. In soil, their
structural diversity is determined by biotic and abiotic factors (Soko­
lova, 2011). Greater structural diversity of fungi has been observed in
places richer in chemical compounds and organic matter, including
forest soil (Mommer et al., 2016a, b). The development of fungi in soils
is conditioned by many factors. Among them, the most important are
soil moisture, air access, the presence of nutrients, temperature, pH and
soil structure. It is believed that the basic process carried out by fungi
during biological weathering is the use of oxygen for the decomposition
of organic matter and nitrates as oxidants (Sokolova, 2011). An
important regulator of the growth of microorganisms is soil pH, because
it affects the solubility of various mineral substances, and thus their
bioavailability. Certain types or species of fungi live in an environment
with a specific range of the hydronium ion concentration (Frąc et al.,
2018). In addition, the quantitative and qualitative composition of fungi
may be influenced indirectly by many natural and anthropogenic
factors.
We found a particularly high structural biodiversity of fungi in the
rhizosphere of trees and in the sampling sites where plant roots were
present. In our study, Boletus, Discosia, Exophiala, Mortierella, Penicillium,
Pseudotomentella, Tricholoma and Wilcoxina were the most abundant
genera in the tree root zone. On the other hand, Oidiodendron, Penicil­
lium, Sagenomella and Tricholoma were the most abundant genera in rock
crack samples. These results are consistent with other authors (Calvar­
uso et al., 2009; Mommer et al., 2016a, 2016b; Ortega-Morales et al.,
2016). Globally, there were only few studies that reported similar set of
fungi genus taxa. Most of them were found in the northern hemisphere
(Fig. 12). Global Fungi Atlas contains 482 records of Boletus, Discosia,
Exophiala, Mortierella, Penicillium, Pseudotomentella, Tricholoma, Wil­
coxina, Oidiodendron, and Sagenomella found in the rhizosphere soil.
Cecia et al. (2015) documented fungal biotransformation of mimetite
and vanadinite by Aspergillus niger. This is crucial in the context of the

10
A. Gałązka et al. Ecological Indicators 141 (2022) 109136

Fig. 11. Heatmaps presenting the metabolic potential of fungi in soil: A – amino acids, B – carboxylic acids and C – carbohydrates.

11
A. Gałązka et al. Ecological Indicators 141 (2022) 109136

Table 4 as Xylaria, Annulohypoxylon and Rosellinia forming biofilms on the stone

Evaluation of changes of fungal metabolic diversity obtained in the Biolog surface. The authors confirmed that Rosellinia sp., Annulohypoxylon,
FFPlates incubated for 120 h. Treatment means with different letters are Penicillium oxalicum and were very active in biological weathering ex­
significantly different (Tukey’s mean separation test, p < 0.05; n = 3). periments with limestone-containing mesocosms. These geochemically
Sample ID H’ R E AWCD590 active fungi have the ability to secrete organic acids under oligotrophic
Tree root zone conditions. For example, oxalic acid released by fungi may deteriorate or
TR1S_20 3.12a ± 0.07 57.33b ± 0.09 0.77a ± 0.01 0.58b ± 0.08 stabilise limestone surfaces (Ortega-Morales et al., 2016). In our
TR1S_40 3.15a ± 0.01 71.67a ± 0.02 0.74a ± 0.02 0.81a ± 0.02 research, we noticed the high utilisation of carboxylic acids (for
TR1S_60 3.22a ± 0.03 78.00a ± 0.03 0.74a ± 0.01 0.93a ± 0.04 example: D-galacturonic acid, fumaric acids, D-saccharic acids and
TR1S_80 3.23a ± 0.04 78.67a ± 0.03 0.74a ± 0.01 0.93a ± 0.01
TR1S_100 2.58b ± 0.18 49.67c ± 0.44 0.67a ± 0.06 0.61b ± 0.07
succinic acids) by fungi present in the tree root zone. In addition, they
TS_Ref 3.15a ± 0.09 62.33b ± 0.15 0.76a ± 0.01 0.65b ± 0.06 are of particular importance in the process of biological weathering
T_Ref_H1 3.02a ± 0.05 64.33b ± 0.10 0.76a ± 0.01 0.72b ± 0.05 under the conditions of the mutualistic symbioses of lichens and my­
T_Ref_H2 2.87b ± 0.09 56.00b ± 0.06 0.71a ± 0.01 0.65b ± 0.09 corrhizas (Gadd, 2007). These aerobic processes can take place in soils,
rock and mineral surfaces as well as in the plant root–soil interface. Our
Rock crack research also confirmed a high percentage of mycorrhizal fungi in the
C2R2M 2.60b ± 0.16 51.33c ± 0.33 0.66a ± 0.06 0.64b ± 0.16 tested samples. Undoubtedly, the presence of mycorrhizae in tree rhi­
C3M 2.88b ± 0.11 62.00b ± 0.16 0.70a ± 0.03 0.72b ± 0.06
zospheres as well as in rock crevices could initiate biological weathering
TR3S 2.91a ± 0.04 59.33b ± 0.05 0.71a ± 0.01 0.76b ± 0.04
TR4 2.31b ± 0.04 40.33c ± 0.24 0.63a ± 0.03 0.48c ± 0.07 (Hoffland et al., 2004; Finlay et al., 2009).
TMS 2.28b ± 0.03 40.67c ± 0.11 0.62a ± 0.04 0.41c ± 0.03 In forest soils, microbes are most active within the rhizosphere and
TR2C2 3.21a ± 0.05 70.33a ± 0.07 0.76a ± 0.01 0.86a ± 0.06 mycorrhizosphere, where mineral weathering is more rapid than in a
C1R2 3.12a ± 0.03 55.67b ± 0.07 0.78a ± 0.01 0.43c ± 0.04
bulk soil without roots (Calvaruso et al., 2009; Uroz et al., 2009; Malik
et al., 2019). AMF are in symbiosis with almost all agricultural plants,
Reference site including legumes and some trees, shrubs and grasses (Quirk et al.,
Pref_1 3.20a ± 0.01 74.67a ± 0.02 0.74a ± 0.01 0.88a ± 0.28
2012; Gałązka and Gawryjołek, 2015). AMF have the ability to penetrate
Pref_2 3.04a ± 0.11 62.33b ± 0.13 0.74a ± 0.02 0.70a ± 0.04
Pref_3 3.18a ± 0.07 71.67a ± 0.10 0.75a ± 0.01 0.82a ± 0.06 an increased volume of soil. The outer structures of AMF are soil-
Pref_4 3.09a ± 0.02 67.33a ± 0.08 0.73a ± 0.01 0.69b ± 0.08 penetrating hyphae and single resting spores (van Schöll et al., 2007).
Pref_5 3.23a ± 0.06 68.00a ± 0.06 0.77a ± 0.01 0.57b ± 0.05 The uptake of nutrients from the soil, mainly phosphorus, and their
Pref_6 2.46b ± 0.09 45.67c ± 0.26 0.65a ± 0.05 0.53b ± 0.02 translocation is one of the basic functions of AMF that is beneficial for
Pref_7 2.94a ± 0.09 58.67b ± 0.03 0.72a ± 0.01 0.57b ± 0.07
Pref_8 2.49b ± 0.04 43.33c ± 0.11 0.66a ± 0.03 0.52b ± 0.02
plants, apart from the production of glomalins (Gałązka and Gawryjołek,
Pref_9 2.96a ± 0.03 59.00b ± 0.04 0.73a ± 0.01 0.58b ± 0.04 2015).
Pref_10 3.21a ± 0.04 72.67a ± 0.01 0.75a ± 0.03 0.84a ± 0.05
4.1.1. The role of fungal metabolic diversity in biological weathering of soil
A significant part of the biomass of trees and undergrowth plants
biological transformation of apatites. On the other hand, Daghino et al.
(leaves, roots) goes to the forest soil directly or after being partially
(2008) confirmed the participation of Aspergillus fumigatus, Paecilomyces
processed by consumers. It is broken down by microorganisms with the
lilacinus and Verticillium leptobactrum in the biotransformation of
release of nutrients (Burghelea et al., 2015). These are mainly enzymatic
asbestos found in serpentine rocks.
processes. In addition, ecological microniches are formed in the soil,
In one study, common fungi such as Penicillium, Trichoderma, Fusa­
which comprise plant remains, live and dead microflora and microfauna
rium and Pestalotiopsis were obtained from limestone surfaces (Ortega-
or bodies of dead animals.
Morales et al., 2016). In contrast, the authors found unusual fungi such
The participation of plants and, above all, increased biological

Fig. 12. Global redistribution of fungi genus taxa similar to these found in our study.

12
A. Gałązka et al.
activity in their root zone can lead to effective rock weathering (Sullivan
et al., 2016). In our research, we found increased metabolic activity of
fungi in the tree root zone. Moreover, the highest values of biodiversity
indices were in tree root zone samples. Tree root fungi actively metab­
olised carboxylic acids, carbohydrates as well as amines. Metabolic ac­
tivity in the rhizosphere of plants has also been observed by other
authors (Puente et al., 2004; Akob and Kusel, 2011; Audet, 2012;
Mitchell et al., 2016).
Nascimento et al. (2021) evaluated the dependence between plants
and quartzite rocky slopes of the campos rupestres. They paid particular
attention to the functioning of family Velloziaceae. Velosioid roots were
particularly involved in the weathering process. Plants in rock crevices
caused the release of carboxylates, which led to the dissolution of the
rocks. In addition, the active substances secreted by plant roots were
involved in this process. Our results obtained from the Biolog FFPlate
analysis confirm a very high share of fungi in the utilisation of a number
of compounds. This active group of fungi can also take part in bio­
weathering. The most frequently utilised substrates by fungi were D-
galacturonic acid, L-phenylalanine, glycyl-L-glutamic acids, L-proline,
fumaric acids, D-saccharic acids, succinic acids and N-acetyl-D-glucos­
amine. Kirtzel et al. (2020) also confirmed that root secretions and
Schizophyllum commune activity led to the degradation of the rock rich in
organic carbon.
Trees are biogeomorphic ecosystem engineers; as such, they alter the
ecosystem they form and live in and modify the availability of resources
to other taxa (Phillips and Marion, 2006). For example, it has been
suggested that in karst regions trees can act as biogeomorphic ecosystem
engineers, because under favourable conditions they can cause car­
bonate rock dissolution (Schenk, 2008; Zseni, 2009). The formation of
subsoil corrosion channels is accelerated by the root acids of greater
plants and intense CO2 production of fungi in rhizosphere (Poot et al.,
2012; Brantley et al., 2017).
5. Conclusions
One of the fundamental issues we have tried to address is whether
tree roots in cooperation with mycorrhizal fungi are active participants
in biological weathering, initial soil formation and hillslope relief
development. Our study has clearly demonstrated the fungal community
in the rhizosphere of trees was much more geochemically active than in
in places without plants. The organic acids secreted by plant roots in
oligotrophic conditions and nutrient limitations significantly affect soil
weathering. Increased activity of fungi in the root zone may have a
direct impact on the weathering processes and, consequently, on the
development of soils. In our future research, we will also explore the role
of bacteria in this process. The root zone of plants is a wealth of diversity
not only of fungi but also bacteria.
CRediT authorship contribution statement
Anna Gałązka: Conceptualization, Methodology, Writing – original
draft, Data curation, Supervision, Formal analysis, Investigation, Fund­
ing acquisition, Resources. Anna Marzec-Grządziel: Data curation,
Writing – review & editing, Validation, Formal analysis. Jarosław
Grządziel: Data curation, Software, Validation, Writing – review &
editing. Milan Varsadiya: Writing – review & editing, Software, Vali­
dation. Łukasz Pawlik: Conceptualization, Methodology, Writing –
original draft, Supervision, Funding acquisition, Investigation.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
13
Ecological Indicators 141 (2022) 109136
Acknowledgments
This work was conducted under the project NCN2019/33/B/ST10/
01009 (to Ł.P. and A.G.) and TreesBEEs – Trees as biogeomorphic
ecosystem engineers – biological weathering, initial soil production and
hillslope relief formation caused by tree roots, rhizospheric bacteria, and
mycorrhizal fungi (2020–2023). We acknowledge financial support
from the Polish National Science Centre through the above-mentioned
grant. This publication was partly supported by a research grant ob­
tained under the Research Excellence Initiative (Inicjatywa Doskona­
łości Badawczej) of the Silesia University in Katowice (to Ł.P.). We
acknowledge Paweł Kroh’s support during the fieldwork.
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