Soil Biology and Biochemistry 159 (2021) 108305

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Soil Biology and Biochemistry

journal homepage: www.elsevier.com/locate/soilbio

Disentangling the abiotic and biotic components of AMF suppressive soils

Carla Cruz-Paredes a, *, Tomas Diera b, Marie Davey c, Maria Monrad Rieckmann a,
Peter Christensen b, Majbrit Dela Cruz b, Kristian Holst Laursen a, Erik J. Joner c,
Jan H. Christensen b, Ole Nybroe d, Iver Jakobsen a
a
Section for Plant and Soil Science, Department of Plant and Environmental Sciences, Thorvaldsensvej 40, 1871, Frederiksberg C, Denmark
b
Section for Environmental Chemistry and Physics, Department of Plant and Environmental Sciences, Thorvaldsensvej 40, 1871, Frederiksberg C, Denmark
c
Division of Environment and Natural Resources, Norwegian Institute of Bioeconomy Research, Høgskoleveien 7, 1433, Ås, Norway
d
Section for Microbial Ecology and Biotechnology, Department of Plant and Environmental Sciences, Thorvaldsensvej 40, 1871, Frederiksberg C, Denmark

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

Keywords: Arbuscular mycorrhizal fungi (AMF) are important in plant nutrient uptake, but their function is prone to
Aluminum environmental constraints including soil factors that may suppress AMF transfer of phosphorus (P) from the soil
Arbuscular mycorrhizal fungi to the plant. The objective of this study was to disentangle the biotic and abiotic components of AMF-suppressive
Fusarium
soils. Suppression was measured in terms of AMF-mediated plant uptake of 33P mixed into a patch of soil and
pH
Soil suppression
treatments included soil sterilization, soil mixing, pH manipulation and inoculation with isolated soil fungi. The
Volatile organic compounds degree of suppression was compared to volatile organic compound (VOC) production by isolated fungi and to
multi-element analysis of soils. For a selected suppressive soil, sterilization and soil mixing experiments
confirmed a biotic component of suppression. A Fusarium isolate from that soil suppressed the AMF activity and
produced greater amounts than other fungal isolates of the antimicrobial VOC trichodiene (a trichothecene toxin
precursor), beta-chamigrene, alpha-cuprenene and p-xylene. These metabolites deserve further attention when
unravelling the chemical background behind the suppression of AMF activity by soil microorganisms. For the
abiotic component of suppression, soil liming and acidification experiments confirmed that suppression was
strongest at low pH. The pH effect might be associated with changed availability of specific suppressive elements.
Indeed 33P uptake from the soil patches correlated negatively to Al levels and Al toxicity seems to play a major
role in the AMF suppressiveness at pH below 5.0–5.2. However, the documentation of a biotic component of
suppression for both low and high pH soils leads to the conclusion that biotic and abiotic components of sup­
pression may act in parallel in some soils. The current insight into the components of soil suppressiveness of the
AMF activity aids to develop management practices that allow for optimization of AMF functionality.

1. Introduction not to other members of the soil biota. However, in natural (non-sterile)
soil a multitude of organisms interacts with the AMF and modulates
Arbuscular mycorrhizal fungi (AMF) form symbiotic associations their function. For example, the growth of ERM was suppressed in
with the majority of known plants (Smith and Read, 2008). They are response to the addition of a bacterial soil filtrate (Leigh et al., 2011) and
important to ecosystem functions through effects on carbon and mineral P uptake by AMF-colonized roots was much smaller from natural
nutrient cycles, plant stress tolerance, plant diversity and soil aggrega­ (non-sterile) soil than from semi-sterile soil (Hetrick et al., 1988).
tion (van der Heijden et al., 2015). The extra-radical mycelium (ERM) is Such suppression of AMF activity in non-sterile soils was investigated
a key structural component of the AMF symbiosis as it links colonized in more detail in two recent papers using one subset of soils from 16
roots with the soil matrix where nutrients such as phosphorus (P) are Scandinavian cultivated field sites (Svenningsen et al., 2018) and
taken up and transferred to the plant (Leake et al., 2004). another subset from 19 Danish non-cultivated sites (Cruz-Paredes et al.,
Most experiments addressing the function of the ERM in nutrient 2019). Both subsets covered a range of soil characteristics for texture,
uptake and transfer have used (semi)-sterile soils to ensure that observed pH and nutrient availability. For the experiments, a mesh-enclosed
effects on plant nutrition could be attributed to the introduced AMF, and patch of each soil was labelled with 33P and embedded in semi-sterile

* Corresponding author.
E-mail address: [email protected] (C. Cruz-Paredes).

https://doi.org/10.1016/j.soilbio.2021.108305
Received 31 March 2021; Accepted 14 May 2021
Available online 21 May 2021
0038-0717/© 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
C. Cruz-Paredes et al. Soil Biology and Biochemistry 159 (2021) 108305

soil with plants colonized by a model AMF. The AMF activity was The present work aims to disentangle the abiotic and biotic com­
measured as the uptake and transfer of 33P from each patch and into the ponents of AMF-suppressive soils and to identify some underlying
plants. Suppression of the AMF activity varied greatly for both soil mechanisms; the following hypotheses were tested: 1) biotic suppression
subsets and both biotic and abiotic soil factors appeared to be involved may be alleviated by soil sterilization, shift of soil pH or mixing with
(Cruz-Paredes et al., 2019; Svenningsen et al., 2018). non-suppressive soil, 2) suppressive soil fungi may be identified after
Suppression of the AMF activity is mitigated by soil pasteurization inoculation of sterile soil with fungi isolated from a suppressive soil, 3)
and has a dominating biotic component (Cruz-Paredes et al., 2019; suppression may be related to microbial production of AMF-toxic VOCs
Svenningsen et al., 2018). Indeed, those authors found that bacterial and and 4) abiotic suppression is caused by specific toxic elements in addi­
fungal communities differ between suppressive and non-suppressive tion to direct pH effects. Patches of soils to be tested were labelled with
33
soils, but also observed so-called general suppression, which is often P, enclosed by meshes and placed in irradiated soil with AMF-
expressed as a continuum of suppression levels and involves the com­ colonized plants. This model system served to assess the degree of
mon action of several taxa. The bacterial taxa that contributed most to suppression of AMF by quantifying the hyphal transfer of 33P from soil
the variation between suppressive and non-suppressive soils were patches to the plant.
Acidobacteria subgroup 1 and Xanthomonadacea, and both groups have
been associated with soils suppressive towards fungal pathogens as 2. Materials and methods
Rhizoctonia, Fusarium and Gaeumannomyces (Campos et al., 2016;
Mendes et al., 2011; Sanguin et al., 2009; Shen et al., 2015). Moreover, 2.1. Model system, soils, plants and AMF
Cruz-Paredes et al. (2019) found that fungi such as Mycena, Mortierella
and Archaeorhizomycetes species were more abundant in The model system was modified from Smith et al. (2003) (Supple­
AMF-suppressive soils. Mycena spp. produces organic acids (Rosling mentary, methods and Fig. S1). In brief, Medicago truncatula colonized
et al., 2004), which might contribute to AMF-suppression, while by Rhizophagus irregularis and growing in pots with semi-sterile soil
Archaeorhizomycetes co-occurs with AMF (Choma et al., 2016), but served as donor association for the production of ERM, with the aim to
there is limited knowledge about their lifestyle and ecological role study the ERM uptake up of 33P from an unsterile mesh-enclosed soil
(Rosling et al., 2011). patch. Six experiments were carried out using soils selected from a
Examples of antagonism by microorganisms towards AMF include subset of Scandinavian cultivated soils and a subset of non-cultivated
fungal parasitism of AMF spores and hyphae (Paulitz and Menge, 1986; soils from the Danish BIOWIDE project (http://www.biowide.dk/)
Rousseau et al., 1996) as well as disruption of the ERM in the presence of that were already tested for suppressiveness against AMF in previous
Trichoderma harzianum (de Jaeger et al., 2011). The latter study even experiments (Svenningsen et al., 2018; Cruz-Parades et al., 2019). See
reported that the AMF activity was reduced (general metabolic activity Table S1 for geographical and physiochemical details for soils used in
as well as specific 33P translocation within the AMF mycelium and this study. Each experiment had three replicate pots per soil or soil
consequently the transfer of 33P to the host plant). Soil microorganisms treatment. The plants were harvested after 35 days and the shoot 33P
may adversely affect AMF activities in soil e.g., by competing for re­ content was quantified and used as a proxy for the P uptake activity of
sources, or by producing unfavorable metabolites, but the mechanism the ERM (Supplementary, methods).
behind the above antagonism was not identified. For other soils that are
suppressive towards plant pathogenic fungi (Hol et al., 2015; Raaij­ 2.2. Modifications of soils in mesh-enclosed soil patches
makers and Mazzola, 2016) suppression is sometimes caused by sec­
ondary metabolites produced by soil microorganisms including volatile 2.2.1. Experiment 1: pasteurization and irradiation
organic compounds (VOC’s; Cha et al., 2016; Cordovez et al., 2015). The aim of this experiment was to use pasteurization (water bath at
These metabolites are involved in signaling and antagonism (Kan­ 80 ◦ C for 1h) and/or irradiation (2 × 18 kGy electron beam) to remove
chiswamy, 2015) and will help the producer organism to survive abiotic the biotic components from the soils and determine whether biotic soil
and biotic stress (Khan et al., 2018). While some VOCs produced by factors were responsible for suppression of the AMF activity. Four sup­
microorganisms can indeed suppress growth of fungi (De Boer et al., pressive soils were selected and were exposed to both sterilization
2019; Effmert et al., 2012; Garbeva et al., 2011; Garbeva and Weisskopf, treatments (Moystad E2 and Rodekro, cultivated) or to irradiation alone
2020), their effect on growth and activity of AMF remains unknown. (Toftlund, cultivated and SV93, heathland). Two non-suppressive soils
The suppression of the AMF activity is generally higher in low pH served as controls and were also irradiated (VO40, grassland and VO41,
soils (Svenningsen et al., 2018; Cruz-Paredes et al., 2019) and can be dune; Table S1 provides soil characteristics).
mitigated by liming (Svenningsen et al., 2018). Soil pH plays a major
role in shaping microbial communities (Lauber et al., 2009; Rousk et al, 2.2.2. Experiment 2: mixing of soils
2009, 2010). Consequently, soil pH may affect soil suppressiveness to­ Different soil mixing treatments were used to investigate trans­
wards AMF through indirect effects of the microbial community ferability of suppression and hence further address whether AMF sup­
composition. Soil pH also directly affects the root colonization intensity pression has a biological basis. Pasteurized E2 soil was mixed with the
and ERM production by AMF (van Aarle et al., 2002; Wang et al., 1993). suppressive E2 or with the non-suppressive Trelleborg soil (Table S1) in
In this context, Cruz-Paredes et al. (2020) found that AMF isolates the following ratios: 0:1, 1:9, 1:1, 9:1, and 1:0. These ratios were also
differed in their susceptibility to suppression, but for most isolates, some used for mixing the suppressive E2 soil and the non-suppressive Trel­
soils could be identified as suppressive and others as non-suppressive. leborg soil.
Moreover, soil pH directly affects the amount of nutrients and
chemicals in the soil solution and hence their biological availability to 2.2.3. Experiment 3: soil liming or acidification
the soil microbiota. For example, the availability of B, Cu, Fe, Mn, and To determine whether a decrease in soil pH induces soil suppressive
Zn usually increases, and Mo availability decreases as soil pH decreases activity, the non-suppressive soils, Risø stored (Table S1) and Trelleborg,
(Fageria and Nascente, 2014). Some of these elements are potentially which had baseline pH of 7.3 and 5.9 respectively, were supplemented
toxic to AMF. Hence, growth of AMF is severely inhibited by bioavail­ with HCl to target new soil pH levels of 4.8 and 4.4 respectively.
able Cu at concentrations that do not affect growth of other main mi­ Furthermore, it was determined if the AMF-suppressive effect of the E2
crobial groups in soil (Hagerberg et al., 2011). Even Al toxicity is usually soil could be mitigated by increasing soil pH from 4.4 to 7.1 by adding
restricted to acidic conditions (Illmer and Buttinger, 2006) and 4.0 g of CaCO3 per kg soil as in Svenningsen et al. (2018).
Göransson et al. (2008) found that high levels of easily reacting Al
showed a strong negative correlation with root colonization by AMF.

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2.2.4. Experiment 4: inoculation with fungal isolates from E2 soil to screen for potentially AMF-toxic VOCs. Particular attention was paid
Fungi were isolated from the suppressive E2 soil with the aim to to VOCs produced by fungi found to be AMF suppressive. Fungi were
screen for the ability of specific isolates to suppress AMF activity. Pure grown from 2 mm (diameter) agar plugs placed on PDA in pre-
cultures of fungi growing on potato dextrose agar (PDA) were obtained autoclaved 20 ml headspace vials. The vials were closed with 18 mm,
(Supplementary, methods) and their preliminary identity was deter­ magnetic HDSP caps (PTFE/Sil, Supelco, Bellefonte, PA, United States).
mined by morphology. This identification was further detailed by The fungal cultures were grown at room temperature for six weeks and
sequencing of the ITS region of rDNA using the primers ITS1-F and ITS4 the headspace vials were then transferred to the GC-MS autosampler for
(Gardes and Bruns, 1993; White et al., 1990) (Supplementary, methods). analysis of VOCs, Sampling was performed by solid-phase micro­
Fungal isolates of Archaeorhizomycetes and Mycena were also obtained extraction (SPME) and VOCs were analyzed by GC-MS using a 7890A GC
from private culture collections as these groups were apparently interfaced to a 5973N MS (Agilent technologies, CA, USA) (Supple­
enriched in suppressive non-cultivated soils (Cruz-Paredes et al., 2019). mentary, methods). For three out of the 23 fungal cultures little or no
Mycena galopus and M. epipterygia were provided by Ella Thoen, Uni­ growth was observed in the four replicates, which was later confirmed
versity of Oslo and Archaeorhizomyces finlayi and A. borealis by Anna by the VOC analysis. Results for these cultures were therefore left out of
Rosling, Uppsala University. The Mycena isolates were cultured on PDA the statistical analysis.
while Archaeorhizomyces isolates were cultured in liquid modified Melin
Norkrans media (Supplementary, methods). 2.4. Experiment 6: multi-elemental analysis of cultivated and non-
Inoculum of the E2 and the Mycena isolates was prepared as follows. cultivated soils
Mycelium from a two week old culture on PDA was scraped from the
surface of the agar, combined with 5 ml sterile distilled water and The possible role of specific elements as abiotic components behind
fragmented using a ball bearing homogenizer. Sterile distilled water was AMF suppression was analyzed by correlating the concentration of ele­
then added to the mycelium slurry to give a final volume of 40 ml. Serial ments extracted from cultivated and non-cultivated soils (Table S3)
dilutions of the inocula were plated on PDA and colony forming units against the shoot 33P content as previously determined in Svenningsen
were counted after 5–10 days growth. For the Archaeorhizomyces spe­ et al. (2018) and Cruz-Paredes et al. (2019). Four suppressive and six
cies, inoculum was prepared by filtering mycelium from liquid cultures non-suppressive, cultivated soils (Tables S1 and S3) were extracted by
on sterile filter paper and homogenizing as described above. shaking 8 g of soil in 16 ml of 0.01 M CaCl2 for 16 h. Samples were then
The inoculum of each fungus (Table 1) was introduced into electron centrifuged for 15 min at 1800 g. After centrifugation, the supernatant
beam-irradiated (2 × 18 kGy) E2 soil after the soil had been labelled was passed through 0.45 μm Q-Max syringe filters (Frisenette, Knebel,
with 33P and filled into the plastic cylinders. Each soil patch was inoc­ Denmark) and extracts were kept frozen until analysis. The elemental
ulated with 5 ml of the 40 ml suspension of fungal fragments or spores in composition of all extracts was measured by ICP-OES (Optima 5300DV,
sterile distilled water and the influence of the inocula on AMF activity PerkinElmer, MA, USA).
was determined. A suppressive Fusarium isolate was identified and used Six suppressive and 12 non-suppressive, non-cultivated soils
in a second experiment including two- and three-fold dilutions. That (Tables S1 and S3) were extracted by shaking 100 mg of soil in 0.5 ml
experiment also included a mixture of the Fusarium isolate and a 0.5 M NH4Ac. Samples were centrifuged for 15–20 min at 10,000 g and
tentatively suppressive Penicillium isolate to test for synergetic effects. the supernatant collected. The residual soil pellet was re-extracted and
the two supernatants were pooled, filtered through 0.2 μm nylon spin
2.3. Experiment 5: VOCs produced by fungal isolates from E2 soil filters (Spin-X, Costar, Merck, Darmstadt, Germany), centrifuged for 8
min as above and acidified with HNO3 to an acid percentage of 3.5%
The fungi isolated from the suppressive E2 soil (Experiment 4) were followed by ICP-MS analysis (Agilent 7900, CA, USA).
analyzed for production of volatile organic compounds (VOCs) in order
2.5. Data analysis
Table 1
Fungal isolates from the E2 soil and existing fungal isolates assessed for their
Student’s t-tests were used to compare shoot 33P content in plants
AMF-suppressiveness. Isolates were submitted in GenBank with submission ID: grown in soils with the original and the modified pH and to compare
2445755. between soils inoculated with candidate fungi and uninoculated soils
(control). One-way analysis of variance (ANOVA) was conducted to
Fungal isolate used Source Genbank Accession No.
compare the shoot 33P content in plants grown in the soil pasteurization,
Archaeorhizomyces borealis Culture collection irradiation, and transfer experiments, with Tukey’s post hoc tests
Archaeorhizomyces finlayi Culture collection
Fusarium cf. solani Isolated from E2 soil MW837838
implemented when appropriate. Non-normally distributed data (as per
Fusarium sp. 1 Isolated from E2 soil MW837831 Shapiro–Wilk test), were log-transformed prior to statistical analyses.
Fusarium sp. 2 Isolated from E2 soil No Sequence Data was also checked for homogeneity of variances (as per Bartlett’s
Fusarium sp. 3 Isolated from E2 soil MW837836 test). Pearson correlation was used to find relationships between 33P and
Mortierella sp. 1 Isolated from E2 soil MW837833
the different element concentration from the elemental analysis. Sta­
Mortierella sp. 2 Isolated from E2 soil MW837834
Mortierella sp. 3 Isolated from E2 soil MW837840 tistical analyses were performed using R (R Core Team, 2020).
Mycena epipterygia Culture collection For VOC analyses, peak detection and deconvolution in the GC-MS
Mycena galopus Culture collection SCAN data was done in AMDIS 2.73 (Mallard, 2014) providing a peak
Penicillium sp. 1 Isolated from E2 soil MW837835 list of retention times, quantifier, qualifier ions etc. VOCs were identified
Penicillium sp. 2 Isolated from E2 soil MW837837
Penicillium sp. 3 Isolated from E2 soil MW837839
by comparison of the deconvoluted mass spectra with a reference
Sterile culture 1 Isolated from E2 soil No Sequence spectral library (NIST14) and their linear retention index. Compounds
Sterile culture 2 Isolated from E2 soil No Sequence were considered tentatively identified if they had a NIST mass spectral
Sterile culture 3 Isolated from E2 soil No Sequence match factor >80% and a deviation in the retention index between 1 and
Sterile culture 4 Isolated from E2 soil No Sequence
2% from reported values in literature (e.g., NIST14). The AMDIS peak
Sterile culture 5 Isolated from E2 soil No Sequence
Sterile culture 6 Isolated from E2 soil No Sequence list and raw GC-MS data were then imported into MATLAB 9.3.0
Talaromyces sp. 1 Isolated from E2 soil MW837832 (R2017b, MathWork Inc., USA) using the graphical user interface
Trichocladium sp. 1 Isolated from E2 soil MW837830 Gavin3 0.96 (Behrends et al., 2011) for visual confirmation and
Trichoderma sp. 1 Isolated from E2 soil No Sequence adjustment of the retention time, and peak areas for the quantified ion.
Trichoderma sp. 2 Isolated from E2 soil No Sequence
VOCs detected in the fungal samples were compared to VOCs in the

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growth media (‘blank sample’). Among VOCs showing significantly (p < soil with pasteurized E2 soil or non-suppressive Trelleborg soil. In both
0.05, t-test) larger peak area of the compounds in the fungal samples cases, the AMF activity, i.e. shoot 33P content, was gradually suppressed
compared to the blank sample, a total of 330 compounds with at least 10 with increasing proportions of E2 soil. This suppression had reached
times higher peak areas were included in the intensity peak table. The 80–90% (p < 0.001) when unsterile E2 soil constituted 50% of the
intensity peak table was cube root transformed, and Pareto scaled (van mixture with pasteurized E2 soil (Fig. 2A) or non-suppressive Trelleborg
den Berg et al., 2006). The data distribution was assessed according to its soil (Fig. 2B), respectively. The mixing of pasteurized E2 soil into the
skewness, kurtosis, and visual inspection of histograms (Vinaixa et al., non-suppressive Trelleborg soil also led to decreases in shoot 33P con­
2012) for each compound. Box-whisker plots (Filipiak et al., 2012) were tent; however, these decreases followed a more linear pattern and were
used to identify compounds with significantly larger intensities in significant only when pasteurized E2 soil constituted as much as 90% of
Fusarium cf. solani cultures than in cultures of the other fungi. the mixture (Fig. 2C). We noted that although soil pH was consistently
Mann-Whitney U test calculated in R, with the function “Wilcox.test” lowered by increasing the proportion of the E2 soil (Fig. 2) the AMF
was used to determine the significance of individual compounds. Prin­ activity was significantly more reduced (p = 0.01) by the E2 soil than by
cipal component analysis (PCA) was performed on the cube root trans­ the pasteurized E2 soil at similar pH (compare 1:1 mixing ratios in
formed, and Pareto scaled intensity peak table (Bro and Smilde, 2014). Fig. 2B and C).
The data filtration was set to exclude compounds with more than 70%
missing data, resulting in 73 compounds in the final peak table. The 3.1.2. Suppression of AMF activity by fungi isolated from E2 soil
reliability of the contribution from each of the compounds was calcu­ Both the soil pasteurization and the mixing experiments indicated
lated by jackknife confidence intervals, based on a 7-fold full that a biotic component of the E2 soil was involved in the suppression of
cross-validation. Partial least squares regression (PLS) was used to AMF activity and a collection of soil fungi was therefore established
investigate the relationship between VOC profiles and suppression using from this soil (Table 1). Briefly, we obtained several isolates from Tri­
the filtered peak intensity table as X and range scaled values for the chocladium, Talaromyces, Fusarium, Penicillium, Trichoderma, Mortierella
suppression; i.e. differences from the control shoot 33P content. and several sterile cultures. The obtained sequences were submitted to
GenBank with the submission ID: 2445755.
3. Results Inoculation of the fungal isolates into the soil patches of the plant
model system had in most cases no significant effect on the shoot 33P
3.1. Biotic component of suppression of AMF activity in the E2 soil content when compared to uptake from uninoculated irradiated soil
(Control) (Fig. 3A). However, the shoot 33P content was consistently
3.1.1. Pasteurization, irradiation and mixing of soils reduced by the isolate Fusarium cf. solani (p = 0.008) when tested at
Initially, the highly suppressive E2 soil was exposed to pasteurization variable inoculum strength (p < 0.001; p = 0.01), and in mixture with
as well as irradiation. The 33P shoot content was significantly (p < Penicillium sp.1 (p < 0.001), as compared to uninoculated irradiated soil
0.001) higher for irradiated and pasteurized soil than for not treated soil (Fig. 3B).
(Fig. 1) supporting that a biotic component suppressed AMF activity in
this soil. However, pH of the E2 soil increased by up to 0.4 pH units due 3.1.3. VOC production by fungal isolates
to pasteurization or irradiation (Fig. 1). In four of the soils, the 33P shoot The total number of detected fungal VOCs was 330, of which 189
content was not enhanced by pasteurization or irradiation; this will be compounds were tentatively identified (see Table S2). The number of
considered in section 3.2. detected VOCs for the 20 individual fungal strains is shown in Fig. S2.
Subsequently, transfer of suppressiveness was studied by mixing E2 Many of the 141 unidentified compounds exhibited mass spectral

Fig. 1. Shoot uptake of 33P by arbuscular mycorrhizal fungi from soil patches exposed to irradiation (irradiated) and/or pasteurization (past) compared to shoot 33P
uptake from untreated soils (none) are shown as bars. Soil pH values from soil patches at the end of the experiment are shown as (●). Data are presented as mean
values ± SEM (n = 3). Different letters indicate significant differences in shoot uptake of 33P (p < 0.05).

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Fig. 2. Shoot uptake of 33P by arbuscular mycorrhizal fungi (bars) from soil patches containing A) suppressive E2 and pasteurized suppressive E2 soils mixed at
different ratios (1:0, 9:1, 1:1, 1:9, 0:1); B) suppressive E2 soil and non-suppressive Trelleborg soil mixed at different ratios; and C) pasteurized suppressive E2 soil and
non-suppressive Trelleborg soil mixed at different ratios. For all panels, soil pH values from soil patches at the end of the experiment are shown as (●). Data are
presented as means ± SEM (n = 3). Different letters indicate significant differences in shoot uptake of 33P (p < 0.05).*.

similarity (>80%) to mono- and sesquiterpenes, but with a deviation in 3.2. Abiotic components of suppression of AMF activity in soil
the retention index >1–2%; which indicate other isomers than the ones
in the NIST14 library. 3.2.1. Irradiation did not alleviate suppression in all soils
PCA was used to investigate differences and similarities in the VOC Among the soils subjected to pasteurization and/or irradiation, four
profiles of the 20 fungi (Fig. 4). PC1 and PC3 seemed to describe relevant soils showed no significant increase in 33P shoot content, which was in
chemical information on the VOCs while for PC2 the confidence in­ contrast to the increase seen for the suppressive E2 soil (Fig. 1). Two of
tervals indicated that this component is unreliable. PC1 explained these, VO40 and VO41, could be assigned as non-suppressive as 33P
31.6% of the variation; the separation can be explained by negative uptake was high also from unsterile soil patches. Besides, the strongly
loading coefficients for acetic acid, while positive contribution was suppressive SV93 had a very low pH. The general observation that pH
found for several alcohols and ketones such as 1-pentanol, 2-nonanone, tended to be higher in pasteurized or irradiated soils than in untreated
phenyl ethyl alcohol, ethyl acetate, 3-methyl-1-butanol, 2-methyl-1- soils (Fig. 1), prompted us to investigate the effect of soil pH manipu­
butanol, and their esters. PC1 is related to fungal VOC production, so lation on AMF activity (Fig. S4). Liming of the E2 suppressive soil from
that fungi with high scores generally have high intensities (high pro­ pH 4.4 to 7.0 increased the shoot 33P content significantly (p < 0.001).
duction) and produce a variety of VOCs. PC3 explained 10.3% of the In contrast, HCl-mediated acidification of two non-suppressive soils,
variation, and negative loadings were dominated by 1-octen-3-ol, 2,3 selected for their high pH, resulted in shoot 33P contents being signifi­
butanol, acetoin, octane, while the positive loadings were dominated by cantly decreased for the Trelleborg soil (p = 0.01), but not for the Risø
2-heptanol, ethyl acetate, butanoic acid ethyl ester, 2-nonanol, and beta- stored soil with higher pH values. Similar decreases in shoot 33P contents
phellandrene. The PC1 and PC3 loading plots are shown in Fig. S3. were obtained when the two soils had been pasteurized before acidifi­
However, the PCA did not enable identification of specific compounds cation (data not shown). In this experiment, the acidification-generated
related to the observed suppression. pH values (4.8 for Risø and 4.4 for Trelleborg) had increased to 5.9 and
To identify specific compounds related to the suppression mediated 5.0 at the end of the experiment. The observed abiotic effects of pH on
by Fusarium cf. solani, we compared the peak area of individual com­ AMF suppression in some soils led us to investigate whether the pH ef­
pounds produced by Fusarium cf. solani with median peak area measured fect was associated with changed availability of specific suppressive
for the rest of the isolates (Fig. 5). We found significantly higher in­ elements.
tensity of p-xylene and several terpenes including the sesquiterpene
trichodiene and the compounds beta-chamigrene and alpha-cuprenene. 3.2.2. Multi-elemental analysis of soils
Furthermore, an unidentified sesquiterpene (C17H28O2), with retention The correlation analyses of the concentration of elements extracted
index = 1721 and high spectral similarity to cedryl acetate, was only from cultivated and non-cultivated soils (Table S3) and the shoot 33P
found for Fusarium cf. solani. uptake showed for the cultivated soils that correlations were significant
and negative between 33P shoot content and Al (r = − 0.50, p = 0.009), K
(r = − 0.47, p = 0.01), Mn (r = − 0.53, p = 0.005), S (r = − 0.41, p =

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Fig. 3. Differences in shoot 33P uptake by arbuscular mycorrhizal fungi from soil pathces containing control soil (irradiated E2 soil) and soil patches inoculated with
fungal isolates obtained from E2 soil. A) All fungal isolates, non-mycorrhizal (NM) and E2 soil with no irradiation treatment, B) Dilutions and mixtures of selected
fungal isolates. Data are presented as means ± SEM (n = 3). Significant differences are indicated by * (p < 0.05).

Fig. 4. VOCs produced by 20 fungal strains isolated from suppressive E2 soil. The PCA score plot shows PC1 and PC3 for the dataset consisting of VOCs detected in
cultures of 20 fungi strains with four biological replicates and of Fusarium cf. solani with12 biological replicates. The data were cube root transformed, and Pareto
scaled (van den Berg et al., 2006). The dataset was filtered to exclude compounds with more than 70% missing values. PC1 explaning 31.6% and PC3 10.3%
of variation.

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study by means of a plant-AMF model system with a mesh-enclosed

patch of 33P-labelled unsterile field soils. As the soil patches could be
accessed by AMF hyphae only and not by roots, suppression could be
investigated without confounding influence from the rhizosphere (e.g.
pH changes and root exudation) and without the impact of the AMF
suppressive soil on root colonization, which in all cases was higher than
90%, by the model AMF R. irregularis. The model system proved suitable
to show that the degree of suppression varies between soils and that the
suppression of the AMF activity involves both abiotic and biotic com­
ponents (Svenningsen et al., 2018; Cruz-Paredes et al., 2019). Disen­
tangling the abiotic and biotic components of AMF-suppressive soils and
understanding some underlying mechanisms were the focal points of
this study.

4.1. Biotic factors behind suppression

The experiments aiming at identifying a biotic component of sup­

pression involved the E2 soil previously shown to be suppressive
(Svenningsen et al., 2018). In disease-suppressive soils, suppression is
eliminated by pasteurization (Garbeva et al., 2011). Likewise, for the E2
soil, we here show that pasteurization as well as irradiation mitigates
suppression, hence confirming our first hypothesis and expanding pre­
vious observations made for this soil (Svenningsen et al., 2018). Transfer
of the suppressive effect of E2 soil to pasteurized E2 soil and to the
conducive Trelleborg soil confirmed a biotic component, but transfer of
suppression occurred only at a 1:1 ratio. For disease-suppressive soils, a
distinction is made between specific and general suppression. Specific
suppression is caused by a single microorganism and is transferable by
adding small amounts (1–10%) of suppressive soil to a non-suppressive
soil (Henry, 1931; Schlatter et al., 2017). Hence, the current results
indicate that the suppression of the AMF activity is a general suppres­
sion. General suppression does not depend on the presence of the target
organism, here AMF (Schlatter et al., 2017), and involves the collective
competitive and antagonistic activity of the soil microbiota (Weller
et al., 2002). Accordingly, Svenningsen et al. (2018) and Cruz-Paredes
et al. (2019) compared the composition of bacterial and fungal com­
munities for selected AMF-suppressive versus AMF-conducive soils and
found several taxa that had higher relative abundance in suppressive
soils and therefore might contribute to suppression.
The second hypothesis was confirmed by our identification of Fusa­
rium cf. solani as an isolate that significantly suppressed the AMF activity
Fig. 5. Box-Whisker plot showing Log 10 transformed measured intensities for when it was introduced into soil patches. This result differs from most
alpha-Cuprenene, beta-Chamigrene, para-Xylene and Trichodiene which had previous reports on negative interactions between saprotrophic fungi
significantly higher (p < 0.05) intensity in Fusarium cf. solani than in any other and AMF, which mostly reported effects of soil fungi on AMF spore
fungal culture. The box plots show the median, the two hinges that correspond germination and growth (Paulitz and Menge, 1984, 1986), abundance
to the 25th and the 75th percentiles, and the upper and lower whiskers, which and viability of AMF hyphae (De Jaeger et al., 2011; Rousseau et al.,
extend from the higher and lower hinges to the largest and smallest values no 1996) and root colonization (McAllister et al., 1994; Ravnskov et al.,
further than 1.5 times the inter-quartile range. The black points repre­ 2006). Taxa belonging to the genus Fusarium are widespread in soil but
sent outliers. their occurrence and their ability to cause plant disease vary between
soils (Orr and Nelson, 2018; Venkatesh and Keller, 2019). This vari­
0.03) and Zn (r = − 0.53, p = 0.005) (Fig. 6A). For the non-cultivated ability is often caused by biotic (antagonistic) interactions, but these
soils, significant negative correlations were observed for Al (r = biotic interactions even depend on the abiotic characteristics of the soil
− 0.70, p < 0.001) and Pb (r = − 0.51, p = 0.03) (Fig. 6B). Hence, high such as the pH, the availability of elements, and the soil texture (Orr and
soil Al levels clearly correlated with suppression of AMF activity across Nelson, 2018; Siegel-Hertz et al., 2018). Moreover, the impact of Fusa­
all cultivated and non-cultivated soils. Since Al toxicity prevails in low rium on the AMF-to-plant transfer of 33P seems inconsistent. One report
pH soils, the correlations between 33P uptake, pH and Al were further points to suppression of growth and P uptake in AMF colonized chickpea
explored (Fig. 7). In general, 33P uptake was low at pH < 5.0–5.2 by F. oxysporum (Shukla et al., 2015), while another found no suppres­
(Fig. 7A, D) and this was associated with markedly increased Al levels sion of AMF growth or P transport by F. culmorum in a model system
(Fig. 7B, E). In consequence, 33P uptake from the soil patches correlated resembling the one used in the current study (Larsen et al., 1998).
negatively to Al levels (Fig. 7C, F). It appears that Al becomes toxic to the Hence, more studies are needed to confirm a general role of Fusarium as
activity of R. irregularis at 1–2 μg Al g− 1 soil or higher, depending on the part of AMF-suppressive consortia in other soils that the E2 soil currently
extraction agent (Fig. 7). investigated. The variable results for antagonism of Fusarium against the
AMF activity might even depend on the metabolites produced by the
4. Discussion specific strains under study.
In the current study, we identified 189 fungal VOCs produced by 20
Soil-associated suppression of AMF activity was demonstrated in this different fungal cultures. The VOCs produced by these fungi include the

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C. Cruz-Paredes et al. Soil Biology and Biochemistry 159 (2021) 108305

Fig. 6. Pearson correlations between 33P shoot content and soil elements measured by ICP-MS for experiments in A) 0.01M CaCl2 extracts of cultivated soils and B)
0.5M NH4Ac extracts of non-cultivated soils. Circles show significant correlations, red circles represent negative correlations and blue circles represent positive
correlations, the size of the circles reflects r-values. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of
this article.)

dominating compounds 1-pentanol, 2-nonanone, phenyl ethyl alcohol, upregulated during co-culture with the fungus Alternaria tennuissima
ethyl acetate, 3-methyl-1-butanol, 2-methyl-1-butanol, and their esters (Venkatesh and Keller, 2019). The interactions between Fusarium and
which are commonly produced by soil fungi and detected in fungal VOC AMF may be more complex as the AMF Glomus (Rhizophagus) irregulare
experiments (Dickschat, 2014; Kanchiswamy, 2015). can down-regulate production of the trichothecene toxin 4,15 diac­
Fusarium cf. solani produced significantly higher amounts of the etoxyscirpenol by a plant pathogenic F. sambucinum (Ismail et al., 2013).
volatile sesquiterpenes trichodiene, beta-chamigrene, and alpha- Future studies should consequently address how the chemical dialogue
cuprenene, which are related to trichodiene synthesis (Vedula et al., between AMF and Fusarium affect the fitness of the AMF, and even have
2008). Trichodiene has previously been isolated from a number of attention on the plant symbiont as trichothecenes may be highly
Fusarium species, and is one of the first intermediates in the synthesis of phytotoxic (Venkatesh and Keller, 2019).
trichothecene toxins (Perkowski et al., 2008), and trichodiene produc­ While the above experiments clearly documented a biotic component
tion has been correlated to the production of these toxins (Kramer and of suppression, at least for the E2 soil, several of our observations point
Abraham, 2012). Beta-chamigrene production has also been docu­ to an additional role for pH as previously observed by (Svenningsen
mented for Fusarium and some fungi produce mixtures of VOCs et al., 2018). In brief, both the current transfer experiments with
including beta-chamigrene for defense against enemies (Kramer and pasteurized versus field E2 soil, the pH adjustment experiments for
Abraham, 2012). Hence, these results point to a potential role of the suppressive and non-suppressive soils, and the lack of an effect of soil
above volatile sesquiterpenes as well as of trichothecene toxins in the irradiation/pasteurization for several soils support a role for pH or
antagonism of Fusarium cf. solani towards AMF. This potential mecha­ pH-dependent factors in AMF activity suppression or reduced P avail­
nism of antagonism is in line with the third hypothesis and points to the ability across a wider panel of soils.
need for future effect studies, which should even address broader
ecological interactions since other volatile sesquiterpenes from Fusarium
4.2. Abiotic factors behind suppression
play important roles in e.g. antagonism against nematodes and promo­
tion of growth and disease resistance in plants (Li et al., 2016; Werner
Low pH might be suppressive due to the direct toxicity of H+, due to
et al., 2016).
pH-related toxicity factors such as Al3+ or deficiency of nutrients such as
The trichothecenes is a group of potent mycotoxins with prominent
P. Plants growth is often inhibited by H+ toxicity in organic, acid soils,
antibiotic properties (Peres de Carvalho et al., 2016). In accordance,
low in Al and with pH below ~4; in contrast, Al3+ toxicity and/or P
antifungal activity of trichothecenes from Fusarium towards other fungi
deficiency are the limiting factors in mineral soils at pH 4–5 (Kidd and
has previously been documented (Campos et al., 2011), and it has been
Proctor, 2001; Rahman et al., 2018). This is the pH range where cation
proposed that these metabolites play a role in securing the environ­
exchange buffering shifts to Al buffering. In the present study, AMF
mental niche of the producer (Venkatesh and Keller, 2019). In support of
suppression was severe in soils with pH ~5 or less and the highly sig­
this notion, production of deoxynivalenol by F. culmorum was
nificant negative correlations between extractable Al and pH in

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C. Cruz-Paredes et al. Soil Biology and Biochemistry 159 (2021) 108305

Fig. 7. Pearson correlations between 33P shoot content and soil pH (A, D), soil pH and soil aluminium concentration (B,E) and 33P shoot content and soil aluminium
concentration (C,F) in cultivated (A-C) and non-cultivated soils (D-F). Different colors indicate different soils. Cultivated soils E2 ( ) and E7 ( ) were sampled from
the same long-term field experiment, but received NPK fertilizer and manure, respectively. Al was extracted with 0.01 M CaCl2 in cultivated soils and with 0.5 M
NH4Ac in non-cultivated soils. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

cultivated as well as non-cultivated soils had a ‘break-point’ around pH 2015). Nevertheless, for R. irregularis we can generally distinguish be­
5.0–5.2. This suggests a role for Al in the suppression of AMF activity tween suppressive and non-suppressive soils and propose that the cur­
(33P uptake) which was indeed negatively correlated to extractable Al. rent results are relevant for a wide range of AMF inoculants.
The correlative evidence for Al toxicity-related suppression of AMF
function in P uptake in low pH soils supports our fourth hypothesis and 5. Conclusions and outlook
extends previous reports on low pH effects on AMF colonization and
abundance (Aliasgharzad et al., 2010; Göransson et al., 2008; Zhang This study extends our previous reports on soil-borne suppression of
et al., 2015). AMF P transfer from soil to plant (Svenningsen et al., 2018; Cruz-Par­
Sorption of P and 33P to Al and Fe oxides/hydroxides represents an edes, 2019) by unequivocally showing the involvement of both biotic
alternative potential explanation for the suppressed 33P uptake in low and abiotic factors. The demonstrated suppression of AMF activity by
pH soils. However, 33P uptake did not correlate with P availability Fusarium cf. solani isolated from an AMF suppressive soil is novel, as is
(Svenningsen et al., 2018) as confirmed by data from the suppressive E2 our identification of Fusarium-derived trichodiene, an intermediate for
(pH 4.4) and the non-suppressive E7 soils (pH 5.6) of the Moystad potentially AMF-toxic trichothecenes. Future work in this area will
long-term field experiment. Aqueous extracts of these soils contained hopefully improve our understanding of the chemical dialogue under­
twice as much P for E2 than for E7 (12.6 vs 6.4 μg P g− 1 soil) and also the pinning the interactions between AMF and other soil fungi. However,
most 33P in extracts from E2 (533 vs 427 Bq g− 1 soil). Since the other our confirmation of a general suppression even shows that the interplay
soils represented in Fig. 7 were also poorly weathered it can be assumed between several microorganisms in the soil microbial web needs to be
that the low uptake of 33P from low pH and high Al soils was probably resolved in more detail. This study focused on suppression of AMF in
not caused by fixation of the added 33P, and hence by a lower avail­ terms of P transfer only and it will be relevant to investigate the sup­
ability of 33P for AMF uptake and transport. pression of other AMF characteristics such as growth, sporulation and
Rhizophagus irregularis BEG87 was the AMF model strain throughout transport of other nutrients by the ERM.
this study and an involvement of Al in suppression of P uptake by this Our finding that suppression of AMF function is most severe in low
strain corroborates previous observations where Al3+ was highly toxic pH soils, and related to Al toxicity, has relevance for the management of
towards colonization of soybean roots by another isolate of the same AMF towards optimal function under field conditions. In case the low pH
AMF species (Zhang et al., 2015). Indeed, a previous study reported a dependent suppression occurs in the field, the soil pH should be kept
limited variation in soil suppression of AMF activity among five higher than 5.0–5.2 by liming and fertilizing. These standard manage­
R. irregularis isolates, while three other Rhizophagus species showed ment practices might have a hitherto overlooked potential for enhancing
different patterns (Cruz-Paredes et al., 2020). Such variation among the function of native and introduced AMF in the field.
AMF in their tolerance to low soil pH has been demonstrated for spore
germination, hyphal growth and root colonization (Bartolome-Esteban
and Schenck, 1994; Kelly et al., 2005; Rohyadi et al., 2004; Zhang et al.,

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C. Cruz-Paredes et al. Soil Biology and Biochemistry 159 (2021) 108305

Declaration of competing interest Filipiak, W., Sponring, A., Baur, M., Filipiak, A., Ager, C., Wiesenhofer, H., Nagl, M.,
Troppmair, J., Amann, A., 2012. Molecular analysis of volatile metabolites released
specifically by Staphylococcus aureus and Pseudomonas aeruginosa. BMC Microbiology
The authors declare that they have no known competing financial 12, 113. https://doi.org/10.1186/1471-2180-12-113.
interests or personal relationships that could have appeared to influence Garbeva, P., Hol, W.H.G., Termorshuizen, A.J., Kowalchuk, G.A., De Boer, W., 2011.
the work reported in this paper. Fungistasis and general soil biostasis - a new synthesis. Soil Biology and
Biochemistry 43, 469–477. https://doi.org/10.1016/j.soilbio.2010.11.020.
Garbeva, P., Weisskopf, L., 2020. Airborne medicine: bacterial volatiles and their
Acknowledgements influence on plant health. New Phytologist 226, 32–43. https://doi.org/10.1111/
nph.16282.
Gardes, M., Bruns, T.D., 1993. ITS primers with enhanced specificity for basidiomycetes–
We would like to thank Lena Asta Byrgesen, Mette Sylvan and application to the identification of mycorrhizae and rusts. Molecular Ecology 2,
Morten Læssøe Stephensen for their help in the laboratory and in the 113–118.
plant growth facilities. We would also like to thank Tobias Frøslev for Göransson, P., Olsson, P.A., Postma, J., Falkengren-Grerup, U., 2008. Colonisation by
arbuscular mycorrhizal and fine endophytic fungi in four woodland grasses -
providing access to the BIOWIDE soils. We are also pleased to variation in relation to pH and aluminium. Soil Biology and Biochemistry 40,
acknowledge Richard J. Simpson for helpful suggestions on components 2260–2265. https://doi.org/10.1016/j.soilbio.2008.05.002.
of abiotic suppression. Funding was provided by the Novo Nordisk Hagerberg, D., Manique, N., Brandt, K.K., Larsen, J., Nybroe, O., Olsson, S., 2011. Low
concentration of copper inhibits colonization of soil by the arbuscular mycorrhizal
Foundation, grant NNF16OC0021576 and from Innovation Fund fungus Glomus intraradices and changes the microbial community structure.
Denmark, grant 6151-00002A - SoilTracker. Microbial Ecology 61, 844–852. https://doi.org/10.1007/s00248-010-9795-2.
Henry, A.W., 1931. The natural microflora of the soil in relation to the foot-rot problem
of wheat. Canadian Journal of Research 4, 69–77. https://doi.org/10.1139/cjr31-
Appendix A. Supplementary data 006.
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Supplementary data to this article can be found online at https://doi. microorganisms on mycorrhizal contribution to growth of big bluestem grass in non-
sterile soil. Soil Biology and Biochemistry 20, 501–507. https://doi.org/10.1016/
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Hol, W.H.G., Garbeva, P., Hordijk, C., Hundscheid, M.P.J., Klein Gunnewiek, P.J.A., Van
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