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The Inuence of Mycorrhizal Hyphal Connections
and Neighbouring Plants on Plantago lanceolata
Physiology and Nutrient Uptake
Henry W. G. Birt
The University of Manchester
Lewis P. Allen
Natural History Museum
Sam Madge
University of Warwick
Clare H. Robinson
The University of Manchester
Richard D. Bardgett
Lancaster University
David Johnson
Lancaster University
Research Article
Keywords: photosynthesis, net ecosystem exchange, common mycorrhizal network, grassland, carbon,
phosphorus, magnesium, zinc, copper, sulphur
Posted Date: April 21st, 2025
DOI: https://doi.org/10.21203/rs.3.rs-6465715/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License.
Read Full License
Additional Declarations: No competing interests reported.
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Abstract
Most plants extend their zone of interaction with surrounding soils and plants via mycorrhizal hyphae,
which in some cases can form common mycorrhizal networks with hyphal continuity to other radial
plants. These interactions can impact plant health and ecosystem function, yet the role of these radial
plants in mycorrhizal interactions and subsequent plant performance remains underexplored. Here we
investigated the inuence of hyphal exploration and interaction with neighbouring mycorrhizal and non-
mycorrhizal plants on the performance of
Plantago lanceolata
, a mycotrophic perennial herb common to
many European grasslands, using mesh cores and the manipulation of neighbouring plant communities.
Allowing growth of hyphae beyond the mesh core increased carbon capture above-ground and release
below-ground as root exudates and resulted in the greater accumulation of elements relevant to plant
health in
P. lanceolata
. However, contrary to expectations, the presence of mycorrhizal or non-
mycorrhizal neighbours did not signicantly alter the benets of hyphal networks to
P. lanceolata
. Our
ndings demonstrate that enabling the development of a fungal network beyond the immediate host
rhizosphere signicantly inuences plant leaf elemental stoichiometry, enhances plant carbon capture,
and increases the amount of carbon they release via their roots as exudates.
Introduction
Arbuscular mycorrhizal (AM) fungi form symbiotic relationships with the vast majority of plant species
where they play a crucial role in various plant processes including nutrient uptake (Read and Perez-
Moreno 2003; Wipf et al. 2019) and stress tolerance (Bunn et al. 2009; Hohmann and Messmer 2017;
Bitterlich et al. 2018). More broadly, these AM fungal-plant interactions can mediate nutrient cycling,
thereby affecting ecosystem function (Yu et al. 2023). For example, mycorrhiza can affect both
photosynthetic processes and the subsequent release of carbon into the soil as root exudates (Gavito et
al. 2019; Muneer et al. 2020; Xu et al. 2023). This dual effect is likely to result in increased carbon
capture by the plant and a corresponding increase in carbon release below-ground due to the carbon
demands of the mycorrhizal symbiosis. However, how mycorrhizal fungi modify the interconnectedness
of carbon uptake above-ground and its release below-ground as root exudates remains unclear.
Elucidating how mycorrhiza may alter carbon cycling is important because the release of plant-derived
carbon below-ground is a primary driver of microbial metabolism in soil (Fan et al. 2022). Soil microbes
other than mycorrhiza are also responsible for essential functions, including plant growth promotion,
nutrient cycling, and pathogen protection (Wen et al. 2021; Fan et al. 2022). Furthermore, soil carbon
contributes to several important abiotic functions, such as maintaining soil structure and moisture, as
well as preventing soil erosion (Zhang et al. 2024). Understanding the role of mycorrhiza in carbon
cycling is also essential for developing accurate climate models, as below-ground carbon storage
mediated by these fungi represents a signicant, yet not fully quantied, carbon sink (Hawkins et al.
2023).
Carbon cycling is a crucial aspect of mycorrhizal function, yet the inuence of these fungi on the
accumulation of other nutrients is equally signicant. While the role of AM fungi in phosphorus and
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nitrogen uptake is well-documented (Smith and Read 2008), research suggests that they may also play a
role in the cycling of other essential elements, such as sulphur and calcium (Allen and Shachar-Hill 2009;
Fu et al. 2023). Furthermore, mycorrhiza can mediate the uptake and accumulation of heavy metals,
which can have profound impacts on plant health and ecosystem functions (Rosas-Moreno et al. 2023).
The differential uptake of these nutrients and elements, mediated by mycorrhizal associations, can alter
plant tissue chemistry, affecting herbivory, decomposition rates, and ultimately, biogeochemical cycling
(Hartley and Gange 2009). It is therefore likely that mycorrhiza will inuence the accumulation of a broad
range of minerals, yet quantitative assessment of the accumulation of a range of minerals
simultaneously in plants subjected to mycorrhizal treatments is lacking in the current literature (Sardans
et al. 2023). Therefore, to gain a holistic view of their ecological signicance, a more comprehensive
understanding and reporting of how mycorrhizal fungal networks inuence the acquisition of a broader
range of elements—beyond the common focus on phosphorus and nitrogen—is crucial.
An emergent property of AM fungi-plant relationships is the development of hyphal networks. The
structure and function of these networks is important in the transport of nutrients from areas that plant
roots cannot access (Wipf et al. 2019). They can also provide a physical connection between individual
plants of the same or different species, forming common mycorrhizal networks (CMNs) (Alaux et al.
2021). These networks can mediate competition between adult plants (Lin et al. 2015; Tedersoo et al.
2020; Bahadur et al. 2023), and have varying effects (positive, negative, or neutral) on establishing
seedlings (Van Der Heijden and Horton 2009). Fungal hyphae can also interact with neighbouring plants
without forming a direct physical connection. Therefore, common mycorrhizal networks that exhibit
direct hyphal connections (CMN-HC) between roots represent a specic subset within the broader
category of fungal networks (Rillig et al. 2024). Thus, it follows that the functioning of a host plant may
be affected when its extra-radical hyphae can interact with neighbouring plants regardless of whether
hyphal continuity occurs.
Various studies have demonstrated that involvement in a CMN can alter plant uptake and allocation of
nutrients. For instance, when connected via a CMN, ax (
Linum usitatissimum
) obtained substantial
nutrients for minimal carbon, while sorghum (
Sorghum bicolor
) acquired few nutrients despite a large
carbon investment (Walder et al. 2012). When these species were combined in a CMN ax gained
biomass without the loss of biomass from sorghum thus demonstrating that a CMN can maximise
resource eciency within a plant community. Interspecies dynamics has shown to inuence CMN
nutrient dynamics in other species as well (Graves et al. 1997; Bougoure et al. 2014). While CMN-HCs
have the potential to facilitate resource transfer between plants, the presence of other plants may also
alter CMN dynamics through the creation of nutrient gradients, production of inhibitory and stimulatory
chemicals (Barto et al. 2011), and competition for fungal resources (Wang et al. 2022). These effects
could be substantial, and may also be caused by non-mycorrhizal plants, which can still compete for
resources and alter the soil environment (Lambers and Teste 2013). Yet, it remains unclear how non-
mycorrhizal plants relative to mycorrhizal plants inuence neighbouring plants via interaction with
CMNs. Nevertheless, it is likely that these effects could be substantial and mediated by whether
neighbouring plants form mycorrhizas or not. Furthermore, when investigating the role of CMNs, it is
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important to consider the total volume of soil that mycorrhizal fungi can explore within a system (Karst
et al., 2023). When AM fungi are limited to resources within a smaller soil volume, they have far less
nutrients available to forage than in a larger soil volume. Therefore, differences in plant performance
may be attributable to a ‘soil foraging volume effect’. This potential effect has been highlighted for eld-
based experiments on ectomycorrhizal fungi, but also applies to other mycorrhizal types including AM
fungi (Karst et al. 2023).
To address these knowledge gaps, we explored how the extension of extra-radical mycorrhizal hyphae,
and their interaction with different neighbouring plants, inuence plant physiology and nutrient uptake.
To achieve this, we conducted a greenhouse experiment using
Plantago lanceolata
as a model plant. It is
a mycotrophic perennial herb common to European grasslands that has been extensively used as a
model for mycorrhizal interactions.
P. lanceolata
was grown inside mesh containers that allowed fungal
hyphae, but not roots, to grow outward. The cores were placed inside larger pots (mesocosms) that
contained one of three treatments: 1)
P. lanceolata
plants (mycorrhizal); 2)
Rumex acetosa
plants (a non-
mycorrhizal species); or 3) fallow (no plants). This design allowed us to manipulate the connections via
extra-radical mycorrhizal mycelium to the outside of the core by rotating half of the cores once per week.
We then assessed how these treatments (outer compartment species and connection by extra-radical
mycelium) inuenced a range of plant attributes including biomass, chlorophyll uorescence, gross
ecosystem exchange of carbon and primary production, and leaf elemental composition. We
hypothesised that plants connected to an outer soil compartment via extra-radical mycorrhizal mycelium
would exhibit increased carbon exchange compared to unconnected plants. In addition, we expected
connection to an outer soil compartment via extra-radical mycorrhizal mycelium to alter the pattern of
accumulation of a broad range of elements in the connected plant compared to unconnected plants.
Together with increased carbon assimilation, we expected this alleviation of nutrient limitation to result
in increased plant biomass. Finally, we hypothesised that focal plants neighbouring other mycorrhizal
plants (potentially forming CMN-HC) would demonstrate enhanced nutrient uptake compared to focal
plants neighbouring non-mycorrhizal plants or surrounded by bare soil because of the increased
resource use eciency afforded by CMNs.
Methods
Mesocosms and greenhouse conditions
Soil for the greenhouse experiment was collected from the top 20 cm from land adjacent to Formby
Beach, Liverpool, UK (Latitude: 53.56113; Longitude: -3.08637). The area sampled is a semi-natural dune
grassland dominated by
Ammophila arenaria
but which also included
Plantago lanceolata
. The sandy
soil facilitated the sampling of root exudates with minimal damage, yet contained natural mycorrhizal
inoculum, which was conrmed through a preliminary pilot trial. The soil was passed through a sieve (8
mm) and homogenised.
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Sieved soil was placed into 26 litre pots (43 cm x 34 cm x 18 cm). An empty cylindrical core, measuring
12 cm in height with 5 cm diameter with 4 cm x 8 cm windows cut in the side, was covered in nylon
mesh with a 40 µm aperture and placed into the centre of each pot. The 40 µm mesh allows penetration
by AM fungal hyphae but prevents penetration by roots and rotation of the mesh disrupts fungal hyphae
(Johnson et al. 2001).
Plantago lanceolata
seeds (Emorsgate seeds, King’s Lynn, UK) were placed 2.5 cm apart in between two
sheets of heavy weight seed germination paper then rolled up and placed vertically in deionised water
and incubated in the dark at room temperature for 5 days. Following germination, seedlings were placed
into the outer compartment of the pots approximately 5 cm apart for an initial soil conditioning stage to
provide the sole source of mycorrhizal inoculum for plants growing in the central mesh core; this
resulted in 24 plants outside of the core. Plants were grown for six weeks and then removed.
Three planting treatments were then applied to the outer (‘radial’) compartment of the conditioned soils
(
Plantago lanceolata
[mycorrhizal],
Rumex acetosa
[non-mycorrhizal], and fallow, i.e., no plants) (Fig.1).
Radial plants were placed into pots at the same rate as the conditioning stage. The central core was then
lled with 230 g of autoclaved soil and three
Plantago lanceolata
seedlings were planted into the core
(referred to hereafter as the core plant). Autoclaved soil was added to the centre to ensure mycorrhizal
colonisation of the central plants occurred by growth of extra-radical mycelium from outside of the core,
likely from a CMN, but potentially from independent spores. Throughout the experiment cores were
rotated approximately 45° once per week in half of all pots to sever mycorrhizal hyphal connections
(Johnson et al. 2001). Pots were arranged in a randomised block design to account for any differences
attributable to environmental variation in the greenhouse.
Plants grew for two weeks and were then thinned to the single strongest plant. Growing conditions were
maintained at 14–25°C and sixteen hours of light, with supplementary lighting to replicate summer
conditions in the UK for all growing periods. To prevent soil moisture loss and to protect the seedlings
from soil pests, a 2 cm depth of perlite was spread across the soil surface. Soil moisture was
maintained every three days at 60% water holding capacity (WHC). WHC was measured using a soil
moisture probe (Theta probe ML3 attached to a HH2 Moisture Meter, Delta-T Devices, Cambridge, UK).
Each pot was an independent replicate, and eight pots were allocated per treatment for a total of 48
replicates across all treatments (Fig.1).
Net Ecosystem Carbon exchange
After seven weeks of growth, measurements of gross CO2 exchange were made from the core plant over
135 second intervals with a PP systems EGM5 portable IRGA coupled to a cylindrical chamber, 7 cm
diameter and 19 cm height (Ward et al. 2007, 2013). One chamber was transparent to permit light
inltration and tted with lights to ensure even light between all replicates, and the other chamber was
opaque. These contrasting chamber congurations enabled the measurement of gross CO2 ux through
subtracting ecosystem respiration (dark conditions) from net CO2 ux (light conditions).
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Sampling procedure
Following CO2 exchange measurements, the core plants were checked for signs of stress using a
FluorPen FP100. The FluorPen quanties chlorophyll A by measuring the intensity of red uorescence
(685 nm) emitted after blue light excitation (470 nm), with lower uorescence indicating reduced
chlorophyll concentration, plant stress, and potentially decreased photosynthetic rates (Padhi et al.
2021). Three FluorPen measurements were taken on the second emerged leaf of the plant that had
previously been darkened by being wrapped in aluminium foil for at least one hour. Plants were then
carefully removed from the core and roots were washed under tap water. Biomass was separated into
above and belowground parts. The roots were dabbed dry with paper towel and weighed wet; a
proportion were then weighed and separated for an assessment of AM fungal root colonisation and
stored in 70% ethanol at 4°C. The remaining biomass was then dried at 65°C for 24 hours and weighed.
The amount of dried biomass removed for AM fungal root colonisation was calculated through the loss
of water weight from the remaining roots. The roots of ten
Rumex
plants were randomly sampled and
checked for AM fungal colonisation.
Root exudate sampling and analysis
Six replicates from every treatment were subjected to root exudate sampling. Root exudates were
collected according to the method of Williams et al. (2021). Briey, plants were removed from pots and
loosely adhered soil was removed. Roots were separated from the remaining soil by submerging the
plants 3–5 times in a beaker of deionised water until the water appeared clear. Plants were then allowed
to recover for three days in 150 mL of soil hydroponics solution aerated using an aeration stone and air
pump. Plants were maintained under the same greenhouse conditions as the previous stage of the
experiment. The soil hydroponic solution was created by combining 200 g of soil from the same source
that had been used for the pot experiment with 1 L of water. This mixture was placed on an orbital
shaker for 1 h at 90 rpm and then centrifuged at 3,200 RCF for 5 mins and the supernatant kept. After
three days of recovery, plants were washed again in deionised water and placed into 150 mL of aerated
fresh Milli-Q water on ice under the same greenhouse conditions. After two hours of collection, plants
were removed, and the remaining solution was passed through a 0.22 µm lter. After root exudates had
been collected, the plants were sampled in the same manner as other plants. A preliminary trial was
conducted to check the impact of root exudate collection on histological observations of root structures
and no signicant difference was found. Root exudates were analysed for total C on a Shimadzu TOC-L
CPN E200 TOC analyser (CITY Japan).
Quantication of AM fungal root colonisation and hyphal length
The prevalence of AM fungal structures within root segments was evaluated using a magnied gridline
intersections method (McGonigle et al. 1990; Vierheilig et al. 1998). Following ethanol removal, root
segments were immersed in 10 mL of 10% KOH at room temperature for 1 hour 10 minutes to facilitate
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clearing. Subsequently, the cleared samples were rinsed with deionised water, subjected to acidication
by submersion in 1% HCl for 30 seconds, and then transferred to test tubes containing 10 mL of 1%
Parker's Quink Blue ink solution in 1% HCl at room temperature for 1 hour. Excess stain was removed by
thorough washing with water. Finally, the stained root segments were transferred to 15 mL falcon tubes
containing a de-staining solution composed of glycerol, water, and lactic acid in a 1:1:1 ratio. Following
incubation in the de-staining solution for at least 24 hours, 5 root segments of 4 cm in length were
mounted onto microscope slides using lactoglycerol (1:1:1 lactic acid, glycerol, and water) as a
mounting medium. For each slide, 50 root-graticule intersections were examined to determine the
proportion containing visible fungal hyphae, arbuscules, and vesicles.
A membrane lter technique was used to measure hyphal length (Hanssen et al. 1974). Briey, a 2 g
sample of soil from the central core was mixed with 500 mL dH2O for 2 minutes at high speed with a
magnetic stirrer. A 200 mL sub-sample was then decanted from the top, span again, and then counter-
stirred to allow large particles to sink. A 30 mL sub-sample was then ltered through 25 mm diameter,
1.2 µm pore size, white polycarbonate lters (Cytiva, MA, USA) placed on top of a Buchner funnel held
under vacuum. The lter was then incubated with a 5% Parker's Quink Blue ink and 5% acetic acid
solution for 5 mins and then rinsed three times with 5 mL dH2O. Filters were mounted on slides with a
50% glycerol solution. Using a LEICA DM2500 microscope at 100x magnication, gridded sections were
counted on a lter until the variation in the mean number of hyphal intersections varied by < 1% three
times in a row. Hyphal length (H) was calculated as H = (IπA)/(2L), where I = average intersections per
grid, A = grid area, and L = total grid line length. Finally, total fungal hyphae length (F) in mm mg− 1 of soil
was determined using F = H × (A/B)*(1/S), where A = lter area, B = grid area, and S = soil amount ltered.
Leaf elemental composition
To assess leaf elemental composition, 50 mg of dried leaf biomass was weighed into glass acid-washed
test tubes. For each digestion a set of 38 tubes, 2 tubes of blanks and 2 tubes of hay powder as a
certied reference material (Commission of the European Communities, Community Bureau of
Reference, reference material 129), to assess recovery rates, were also prepared. To each tube, 0.42 mL
of concentrated nitric acid (70%) was added and left overnight to release nitrogen dioxide. Subsequently,
0.42 mL of hydrogen peroxide (30%) was added, and the tubes were placed in a heat block at 100°C for 1
hour, then 120°C for a further hour, and nally 140°C for 2 hours. Samples were then diluted to 2% of
HNO3 using MiliQ water and syringe ltered using a 0.22 µm lter. These solutions were run on an ICP-
OES (Agilent 5800) using a 1.2 kW Argon RF plasma at 12 L min− 1 and auxiliary gas running at 1 L min−
1. Sample concentrations were analysed and reported as the average of three by ve-second
integrations. Sample uptake into the ICP-OES was done at 0.7 L min− 1 using a Mira-Mist nebuliser.
Statistical analyses
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Univariate variables were assessed using mixed-effects linear models with experimental block as a xed
effect; this analysis was implemented through the R package
lme4
(Bates et al. 2015). Post-hoc analysis
was conducted using the R package
emmeans
through analysis of estimated marginal means with
Benjamin-Hochberg corrections (Lenth 2023). The
vegan
R package was used to assess multivariate
signicant differences between leaf elemental composition by testing Euclidean distance between
samples using PERMANOVA (Oksanen et al. 2022).
Results
Chlorophyll uorescence and biomass
Plants across all treatments exhibited chlorophyll uorescence values indicative of healthy plants (~ 0.8,
Fig S1, Zhori et al. 2015).
The rotation of the core inuenced above-ground biomass, but did not inuence root biomass of the core
plants. Rotated plants had 15.4% less above-ground biomass than those with static cores. Plants
without any radial plants (fallow) also had more above-ground biomass than plants surrounded by either
R. acetosa
or
P. lanceolata
(on average, 25.1% and 22.0%, respectively; Fig. 2). However, radial plants had
no inuence on root biomass. There was no observed interaction between core rotation and radial plant
community.
Table 2The effect of radial plant community (radial) and core rotation (rotation) on the biomass of
Plantago lanceolata
.
Leaf elemental composition
The elemental composition of leaves was inuenced by the rotation of the in-growth core; however,
radial plants had no inuence on plant leaf elemental composition and no interaction was observed
between the treatments (Table 3, Fig S2). Leaf concentrations of phosphorus, a key element foraged by
mycorrhiza, were 51% higher on average in plants grown in static cores (Fig 3). Radial plants did not
inuence leaf phosphorus concentration (Fig 3). In addition, magnesium, sulphur, copper, and zinc were
all elevated in plants grown in static cores (Table 4).
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Table 3PERMANOVA results showing the effect of soil volume (size), radial plant community (radial),
and core rotation (rotation) on the multivariate elemental composition of the leaves of
Plantago
lanceolata
.
Table 4The effect of radial plant community (radial) and core rotation (rotation) on the mean elemental
composition of the leaves of
Plantago lanceolata
. Signicantly highest mean values are coloured green,
and the lowest values red. ± indicates the standard error of the mean.
Impacts on above- and below-ground carbon ux
Plants grown in static cores had a signicantly lower (18.2% less on average) gross ecosystem
exchange (GEE) compared to those in rotated cores (Table 5, Fig. 4), indicating greater xation of carbon
by plants in static cores. Radial plant community did not inuence GEE. In addition, rotation of the mesh
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core surrounding the plant signicantly reduced the total amount of carbon exudated by plant roots by
24.2% on average (Table 5, Fig. 4). No signicant impact of radial plants on the total amount of carbon
exudated by plant roots was detected. There was a signicant correlation between GEE and the total
amount of carbon in root exudates with biomass (Fig. 4). When both effects were scaled by above-
ground biomass, they were no longer signicant (Table S1, Fig. S3). There was no signicant impact on
soil respiration (Fig S4).
Table 5The effect of radial plant community (radial) and core rotation (rotation) on the total amount of
carbon exudated and gross ecosystem exchange (GEE) of
Plantago lanceolata
.
AM fungal colonisation
No evidence of mycorrhizal colonisation was observed in any radial
Rumex
roots sampled, conrming
their status as non-mycorrhizal plants. Although all
P. lanceolata
roots from the mesh cores were
colonised by mycorrhiza, fungal hyphae and vesicles were signicantly more abundant in the roots that
were in static as opposed to rotated cores. Radial plants did not inuence the presence of hyphae or
vesicles in the roots (Table 6 and Fig. 5). No treatment inuenced the presence of arbuscules (Table 6).
Hyphal length in soil from inside the core was signicantly greater in static compared to rotated cores
(Table 6 and Fig. 5). Radial plants did not inuence hyphal length.
Table 6The effect of soil volume (size), radial plant community (radial), and core rotation (rotation) on
root associated AM fungal structures in
Plantago lanceolata
. Asterisks indicate signicant differences.
Discussion
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This study investigated how the extension of extra-radical mycorrhizal hyphae, and their interaction with
different neighbouring plants, inuence the physiology and nutrient uptake of
Plantago lanceolata
.
Enabling growth of hyphae into the radial compartment increased plant carbon capture above-ground
and release below-ground as root exudates, and resulted in the greater accumulation of elements key to
plant health, especially phosphorus, copper, sulphur, and zinc; although 13 other elements remained
unaffected by the treatments. However, contrary to expectations, the presence of mycorrhizal or non-
mycorrhizal neighbours did not signicantly alter the benets
Plantago lanceolata
derived from having
greater exploratory extra-radical mycorrhizal hyphae. This nding highlights the importance of hyphal
exploration of soil regardless of the presence of a simple radial plant community.
Biomass and Carbon Dynamics
Plants that had hyphae that could penetrate the radial compartment had greater above-ground biomass
than those that could not (Fig.2). This was in line with our expectation that mycorrhiza would alleviate
nutrient limitation resulting in greater plant biomass. Other studies have found that association with AM
fungi can promote (Zhang et al. 2015), have no impact (Karasawa et al. 2012), or decrease biomass of
P.
lanceolata
(Qu et al. 2021). These differing results may arise from differences among studies in soil
conditions, especially nutrient availability, and AM fungal genotypes. Here, we prioritised sourcing AM
fungi from an environment where
P. lanceolata
grew wild, as the AM fungal community would likely be
adapted to promoting
P. lanceolata
. Although AM fungal association has been reported to promote
below-ground biomass (Zhang et al. 2015), no signicant impacts were detected in our study, although
there was a promotion of above-ground biomass. This may have been due to the mesh cores restricting
growth, although no evidence of roots being pot bound was apparent at sampling. The promotion of
above-ground biomass may be due to the additional carbon sink from the connected mycorrhizal
hyphae, that in turns stimulates leaf growth to supply additional carbon (Kaschuk et al. 2009). This
theory is in-line with our hypothesis that mycorrhiza would stimulate carbon absorption and release
below-ground as root exudates and was conrmed by our experimental results (Fig.4). Other literature
also supports that disconnection from mycorrhizal hyphae reduces plant photosynthetic rate (Gavito et
al. 2019). These ndings has implications for the role that AM fungi play in terrestrial carbon cycling
(Hawkins et al. 2023).
Our ndings conrmed our hypothesis that AM fungi induce an increase in photosynthetic rate and this
is linked to increased carbon in root exudation. This nding therefore connects above-ground and below-
ground effects of mycorrhizal symbiosis that have been reported separately (Xu et al. 2023). We
observed a strong positive correlation between leaf biomass and both above-ground carbon capture and
below-ground carbon release as root exudation (Fig.4). This suggests that the effect is mediated by an
increase in photosynthetic tissue as opposed to some metabolic shift. Nevertheless, the observed
enhancements in photosynthesis and below-ground carbon allocation may be constrained in
environments where mycorrhizal networks cannot provide sucient resources to support increased
biomass. This limitation of growth could occur in nutrient-poor soils, such as used here, or when other
environmental factors, such as temperature, restrict plant growth.
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Nutrient uptake
Plants grown in static cores had, on average, a 51% higher concentration of phosphorus in their leaves,
likely due to the connection to mycorrhizal hyphae outside of the core (Fig.3), and supports the well-
established role that mycorrhiza play in plants obtaining this nutrient (Smith and Read 2008; Jiang et al.
2021; Tibbett et al. 2022). In addition, magnesium, sulphur, copper, and zinc were all elevated (Table4),
which are key to plant health. For example, magnesium is the central atom in the chlorophyll molecule
(Shaul 2002); sulphur is a key constituent in some amino acids (Hawkesford and De Kok 2006); copper
serves as a cofactor for electron-transfer proteins (Burkhead et al. 2009); and zinc is a co-factor in
thousands of proteins, including those involved in CO2 xation (Broadley et al. 2007). Therefore, this
study adds to evidence that mycorrhiza help plants to accumulate a range of elements that are essential
to their survival (Allen and Shachar-Hill 2009; Lehmann and Rillig 2015; Zare-Maivan et al. 2017; Rosas-
Moreno et al. 2021). The accumulation of these elements in plants due to mycorrhizal interactions could
have important implications for plant-herbivory interactions (Ballu-Fry et al. 2022), and the nutritional
status of crops (Lehmann and Rillig 2015).
Mycorrhizal colonisation
The presence of vesicles and hyphae in roots, as well as the average hyphal length inside the in-growth
core was reduced in rotated cores (Fig.5). This indicates that the rotation treatment disrupted
connections between the focal plant and mycorrhiza outside of the core. Vesicles and hyphal density are
often-used as proxies of performance of mycorrhizal fungi but do not always correlate with performance
benets for the plant (Gange and Ayres 1999), although in our experiment plant performance was better
in the static treatments. Arbuscule formation was not different between treatments, although these
structures are transient (2–8 days) and the sampling represents a single time point sampling
(Luginbuehl and Oldroyd 2017). Therefore, differences in arbuscules present in the root may have been
more apparent at different stages of the symbiosis.
Lack of Radial Plant Effect
We found no signicant effect of the presence or identity of radial plants on the biomass of focal
P.
lanceolata
. This effect included mycorrhizal plants of the same species (i.e.
P. lanceolata
) and non-
mycorrhizal plants of a different species (
Rumex aceotosa
). This nding counters our initial hypothesis
that the presence of a radial community of mycorrhizal plants would enhance ecient resource use via a
CMN and therefore improve plant performance. Instead, our ndings suggest that soil foraging effects
are important whereby plant biomass increases when its mycorrhizal network is given access to a
greater volume of soil (e.g., the soil outside the in-growth core (Karst et al. 2023). Like many
manipulations of CMNs, our study is limited in that we could not establish the extent that a continuous
function CMN had formed, which typically requires use of isotopes (Lekberg et al. 2024). However, our
study demonstrates the importance of additional controls (areas with no plants and non-mycorrhizal
plants, which likely modify edaphic conditions in a similar way to mycorrhizal plants, but which cannot
form CMN-HCs) that help prevent attributing observed effects to a CMN. This methodological approach
Page 13/22
is important as isotopes or other similar methods can be restrictive due to cost, access to material, and
restrictions on radio-isotope use (e.g., in eld experiments).
It is possible that the duration of plant growth in this study (seven weeks) was insucient to establish a
CMN-HC. Although an initial condition phase was included, the removal and replacement of plants may
have disrupted an established CMN. Furthermore, other studies have suggested that CMN effects may
be most evident when there are imbalances within the network, such as differences in carbon costs to a
plant for mycorrhiza-obtained nutrients (Walder et al. 2012), or plant access to water (Egerton-Warburton
et al. 2007).
Our study had similar genotypes growing in a relatively homogeneous environment. As such, biological
market theory would suggest that with no variation in supply or demand (even soil resources), and no
partner choice driving competition (similar genotype), a single, uniform "exchange rate" (the price of
nutrients for photosynthate) would emerge across the entire population, thus with little trading
advantage to be gained (Bunn et al. 2024). Future studies may consider designs with likely imbalances in
the CMN partners. Our ndings also counters the hypothesis that non-mycorrhizal plants may induce
CMN-like effects (Wang et al. 2022) as well as our hypothesis that radial mycorrhizal plants would
modulate mycorrhizal effects. This is evident as we did not observe an impact of radial
R. aceotosa
on
plant performance in the static cores relative to those with no radial plants or those with
P. lanceolata
as
radial plants. However, the inclusion of non-mycorrhizal plants in a greater range of environmental
conditions and plant communities is required to fully establish their role in CMN function. Beyond
nutrient transfer, other CMN effects, such as herbivory signalling, may have been present, but were not
tested in this study (Babikova et al. 2013).
Conclusion
This study demonstrates that enabling the development of a fungal network beyond the immediate host
rhizosphere signicantly enhances carbon capture by plants and increases the amount of carbon they
release at their roots as exudates, and that this effect is strongly correlated with increases in leaf
biomass. Furthermore, our results indicate that mycorrhiza facilitate plant accumulation of a range of
elements in addition to phosphorus, especially micronutrients zinc, copper, and sulphur. Critically, this
study also underscores the importance of including non-mycorrhizal and no-plant controls to accurately
attribute plant performance to CMN effects, rather than mycorrhizal soil foraging effects.
Declarations
Author Contribution
H. B.: Conceptualisation (support); Methodology (co-lead); Visualisation (lead); Investigation (equal);
Writing – original draft (lead); Investigation (lead); Writing – review and editing (support). L.A.:
Investigation (equal); Methodology (support); Writing – review and editing (support).S.M.: Investigation
Page 14/22
(equal); Methodology (support); Writing – review and editing (support).C.R.: Funding Acquisition
(support); Project administration (support); Supervision (support); Writing – review and editing
(support).R.B.:Funding Acquisition (support); Methodology (support); Project administration (support);
Supervision (support); Writing – review and editing (support).D.J.: Conceptualisation (lead); Funding
Acquisition (lead); Methodology (co-lead); Investigation (support); Project administration (lead);
Supervision (lead); Writing – original draft (support); Writing – review and editing (lead).
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Figures
Page 19/22
Figure 1
A schematic of the experimental design in this study
Page 20/22
Figure 2
The impact of the experimental treatments on plant biomass. A) The radial plant community and B) the
rotation of an in-growth core on dry leaf biomass as well as C) their interaction on the dry root biomass
of
Plantago lanceolata
. Boxplots show the range, median, and interquartile range of each treatment.
Dots overlaid show individual data points. Signicance between treatments is shown: *** < 0.001, ** <
0.01, and * < 0.05.
Page 21/22
Figure 3
The impact of the experimental treatments on phosphorus accumulation. The effect of A) the rotation of
an in-growth core and B) the radial plant community on phosphorus leaf content in
Plantago lanceolata
.
Boxplots show the range, median, and interquartile range of each treatment. Dots overlaid show
individual data points. Signicance between main effect of rotation treatment is shown: *** < 0.001, ** <
0.01, and * < 0.05.
Figure 4
Page 22/22
The impact of the experimental treatments on carbon exchange. The effect of the rotation of an in-
growth core on A) gross ecosystem exchange and B) total carbon in root exudates of
Plantago
lanceolata.
C) The relationship between gross ecosystem exchange and D) total dissolved carbon in root
exudates with leaf biomass. Boxplots show the range, median, and interquartile range of each treatment.
Dots overlaid show individual data points. Signicance between treatments is shown: *** < 0.001, ** <
0.01, and * < 0.05.
Figure 5
The impact of the experimental treatment on fungal colonisation. The effect of the rotation of the in-
growth core on the presence of A) vesicles and B) hyphae in the roots of
Plantago lanceolata
as well as
C) the impact of core rotation on the average hyphal length found in the soil within the core. Boxplots
show the range, median, and interquartile range of each treatment. Dots overlaid show individual data
points. Signicance between treatments is shown: *** < 0.001, ** < 0.01, and * < 0.05.
Supplementary Files
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Birtetal2025MycorrhizaandPlantagoPhysiologysupplementary.docx
