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Pore-Scale Monitoring of the Effect of Microarchitecture on Fungal Growth in a Two-Dimensional Soil-Like Micromodel

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In spite of the very significant role that fungi are called to play in agricultural production and climate change over the next two decades, very little is known at this point about the parameters that control the spread of fungal hyphae in the pore space of soils. Monitoring of this process in 3 dimensions is not technically feasible at the moment. The use of transparent micromodels simulating the internal geometry of real soils affords an opportunity to approach the problem in 2 dimensions, provided it is confirmed that fungi would actually want to propagate in such artificial systems. In this context, the key objectives of the research described in this article are to ascertain, first, that the fungus Rhizoctonia solani can indeed grow in a micromodel of a sandy loam soil, and, second, to identify and analyze in detail the pattern by which it spreads in the tortuous pores of the micromodel. Experimental observations show that hyphae penetrate easily inside the micromodel, where they bend frequently to adapt to the confinement to which they are subjected, and branch at irregular intervals, unlike in current computer models of the growth of hyphae, which tend to describe them as series of straight tubular segments. A portion of the time, hyphae in the micromodels also exhibit thigmotropism, i.e., tend to follow solid surfaces closely. Sub-apical branching, which in unconfined situations seems to be controlled by the fungus, appears to be closely connected with the bending of the hyphae, resulting from their interactions with surfaces. These different observations not only indicate different directions to follow to modify current mesoscopic models of fungal growth, so they can apply to soils, but they also suggest a wealth of further experiments using the same set-up, involving for example competing fungal hyphae, or the coexistence of fungi and bacteria in the same pore space.
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ORIGINAL RESEARCH
published: 03 July 2018
doi: 10.3389/fenvs.2018.00068
Frontiers in Environmental Science | www.frontiersin.org 1July 2018 | Volume 6 | Article 68
Edited by:
Luiz Fernando Wurdig Roesch,
Federal University of Pampa, Brazil
Reviewed by:
Ademir Araujo,
Federal University of Piauí, Brazil
Fernando San José Martínez,
Universidad Politécnica de Madrid
(UPM), Spain
*Correspondence:
Philippe C. Baveye
philippe.baveye@agroparistech.fr
Specialty section:
This article was submitted to
Soil Processes,
a section of the journal
Frontiers in Environmental Science
Received: 29 March 2018
Accepted: 12 June 2018
Published: 03 July 2018
Citation:
Soufan R, Delaunay Y, Gonod LV,
Shor LM, Garnier P, Otten W and
Baveye PC (2018) Pore-Scale
Monitoring of the Effect of
Microarchitecture on Fungal Growth in
a Two-Dimensional Soil-Like
Micromodel. Front. Environ. Sci. 6:68.
doi: 10.3389/fenvs.2018.00068
Pore-Scale Monitoring of the Effect
of Microarchitecture on Fungal
Growth in a Two-Dimensional
Soil-Like Micromodel
Raghad Soufan 1, Yolaine Delaunay 2, Laure Vieublé Gonod 1, Leslie M. Shor 3,
Patricia Garnier 2, Wilfred Otten 4and Philippe C. Baveye 1
*
1UMR EcoSys, AgroParisTech, Université Paris-Saclay, Thiverval-Grignon, France, 2UMR EcoSys, Institut National de la
Recherche Agronomique, Université Paris-Saclay, Thiverval-Grignon, France, 3Department of Chemical and Biomolecular
Engineering, University of Connecticut, Mansfield, CT, United States, 4School of Water, Energy and Environment, Cranfield
University, Cranfield, United Kingdom
In spite of the very significant role that fungi are called to play in agricultural production
and climate change over the next two decades, very little is known at this point about
the parameters that control the spread of fungal hyphae in the pore space of soils.
Monitoring of this process in 3 dimensions is not technically feasible at the moment.
The use of transparent micromodels simulating the internal geometry of real soils affords
an opportunity to approach the problem in 2 dimensions, provided it is confirmed that
fungi would actually want to propagate in such artificial systems. In this context, the key
objectives of the research described in this article are to ascertain, first, that the fungus
Rhizoctonia solani can indeed grow in a micromodel of a sandy loam soil, and, second,
to identify and analyze in detail the pattern by which it spreads in the tortuous pores
of the micromodel. Experimental observations show that hyphae penetrate easily inside
the micromodel, where they bend frequently to adapt to the confinement to which they
are subjected, and branch at irregular intervals, unlike in current computer models of the
growth of hyphae, which tend to describe them as series of straight tubular segments.
A portion of the time, hyphae in the micromodels also exhibit thigmotropism, i.e., tend
to follow solid surfaces closely. Sub-apical branching, which in unconfined situations
seems to be controlled by the fungus, appears to be closely connected with the bending
of the hyphae, resulting from their interactions with surfaces. These different observations
not only indicate different directions to follow to modify current mesoscopic models of
fungal growth, so they can apply to soils, but they also suggest a wealth of further
experiments using the same set-up, involving for example competing fungal hyphae,
or the coexistence of fungi and bacteria in the same pore space.
Keywords: hyphae, spread, microfluidics, fungal highway, microscale
Soufan et al. Fungal Growth in Soil-Like Micromodel
INTRODUCTION
An estimated 1.5 million species of fungi are present in
terrestrial ecosystems (Hawksworth, 2001) where they fulfill a
wide array of essential ecological functions, in particular in the
global carbon cycle (Cromack and Caldwell, 1992). Their role
in soil-plant feedback processes in the rhizosphere is widely
regarded as key to achieving the estimated 100% increase in
overall food production that will be needed in the next 25
years, amidst decreasing availability of suitable land and already
overexploited surface- or groundwater resources (e.g., Sposito,
2013; Baveye, 2015; Baveye et al., 2018).
To maximize the benefits that can be derived from the
involvement of fungi in these different contexts, we can rely
on a wealth of qualitative information about these organisms.
For centuries, it has been known that fungal colonies grow
as an interconnected network of hyphae, collectively referred
to as mycelium (Fricker et al., 2017). In soils, fungal hyphae
absorb and mineralize stable biomolecules like cellulose or lignin.
Since they can access organic matter and nutrients located in
much tinier pores than those typically accessible to plant roots,
fungi are able to provide sustenance that otherwise would be
difficult for over 90% of vascular plants to take up on their own
(Boddy, 1993). Many soil-borne fungi are pathogenic to plants,
severely reducing crop production worldwide (Fisher et al., 2012),
whereas others have antagonistic properties, or hyperaccumulate
metal contaminants, makingthemparticularlysuitedtoremediate
polluted soils (Stamets, 2005). Last in this quick overview, but
certainly not least, fungi play a crucial role in stabilizing the
architecture of soils (e.g., Miller and Jastrow, 2000).
Underpinning these ecologically important processes is the
ability of fungi to invade the very convoluted pore space
in heterogeneous soil environments, with its tortuous paths,
multiple constrictions, and in some cases dead end spaces, all of
which may be variably filled with water (Otten et al., 2001; Pajor
et al., 2010). Tremendous technological advances over the last
two decades, in particular the development of advanced X-ray
computed tomography (CT) scanners, now allow the geometrical
features of the pore space in which fungal hyphae grow to be
determined at resolution of a few microns, which are adequate
given typical widths of hyphae of the order of 3–17 µm. Various
computer models have been developed in the last decade, which
use this information derived from CT images to predict the
spread of fungal biomass in soils (e.g., Falconer et al., 2012, 2015;
Cazelles et al., 2013). These models predict the amount of fungal
biomass that is likely to be present locally in the pore space, and
their outputs appear reasonable in light of the few macroscopic
observations available. These models have proven very useful to
understand the possible effects of various soil parameters, e.g., the
connectivity and tortuosity of the pore space, on the proliferation
of fungi or the interaction of competing fungal species in soils.
In a number of situations, for example during the bioclogging
of soils (e.g., Baveye et al., 1998) or when trying to understand
how the presence of fungal hyphae could affect the retention
and transport of water in soils, not just the amount of fungal
biomass likely to be present locally, but also the precise location
and configuration of fungal hyphae in soil pores, may have
a significant influence on processes of interest. Unfortunately,
the only experimental information available to us at this point,
at the microscopic scale, about the growth pattern of fungal
hyphae in soil pores has not evolved much in the last 30
years. Some progress has been made in the 3D visualization
of the configuration of fungal hyphae in systems constituted
of polystyrene beads (Lilje et al., 2013) or in wood. Recent
advances in the visualization of root hairs of similar diameter
as fungi in small samples using synchrotron X-ray CT does
demonstrate that at least in very small samples visualizing fungi
might be possible (Koebernick et al., 2017). It is however noted
that relative to the scale of fungal colonies and over which
nutrient can be translocated such sample sizes would not be
representative to capture colony development. Therefore, in
actual soils, the only way to visualize fungal hyphae is through
snapshots that one can get after preparing soil thin sections
(e.g., Harris et al., 2002, 2003), or stabilizing soil samples for
electron microscopy (e.g., Foster, 1988). The resulting images
provide us with very useful information about hyphae and
what surrounds them at discrete locations in soils at specific
instants of time. However, it has been so far impossible to derive
from these snapshots a reliable picture of the environmental
and morphological parameters that control the 3-dimensional
path followed by individual fungal hyphae in soil pores. Some
fungi, like Rhizoctonia solani, exhibit a remarkably constant,
undoubtedly genetically-determined behavior when grown in
Petri dishes, with virtually constant branching angles and average
internodal distances (Boswell and Hopkins, 2008; Boswell and
Davidson, 2012; Hopkins and Boswell, 2012; Choudhury et al.,
2018). It is tempting to assume that the same characteristics
are exhibited when this organism grows in the pore space of
a soil, but there is no reason at this point to believe that this
assumption is warranted. In fact, it seems safe to take as a working
hypothesis that the frequent presence of obstacles in the path
of the spreading hyphae in soils is likely to modify significantly
the behavior of R. solani compared to what it is in Petri dishes.
Indeed is has been shown that colony geometry is to a large extent
determined by connected tortuous pathways on soil (Otten and
Gilligan, 1998; Otten et al., 1999). Following Watts et al. (1998),
one might for example assume that fungal hyphae in soils are
likely to manifest some type of thigmotropism, by which they
would tend to remain in contact with solid surfaces after they
encountered them during their foraging in the soil pore space.
Direct dynamic observations of the spread of fungal hyphae
in soils are clearly direly needed, to find out to what extent
the spreading and branching patterns of fungal hyphae in soils
differ from those on Petri dishes. At the moment, the best
opportunity we have to get a glimpse of the dynamics of hyphae
in soil pores appears to be in two dimensions, by using so-
called micromodels or microfuidic devices (e.g., Karadimitriou
and Hassanizadeh, 2012; Stanley et al., 2016). Various authors
(Hanson et al., 2006; Held et al., 2010, 2011; Hopkins and
Boswell, 2012), a few years back, have used micromodels to
visualize the spread of fungi. Their micromodels had rectilinear
pores intersecting at right angle and of a width just a little
bigger than that of hyphae. Since these early investigations,
the design and manufacture of micromodels have evolved
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Soufan et al. Fungal Growth in Soil-Like Micromodel
noticeably. It is now possible to replicate faithfully the pore
geometry of soils, using an inexpensive and biocompatible
polymer, polydimethylsiloxane (PDMS), that offers excellent
optical clarity. Deng et al. (2015) and Rubinstein et al. (2015) have
used such a soil-like micromodel to observe the effect of bacterial
activity on water or particle retention and movement in larger
pores. Similar work with fungal hyphae has yet to be carried out.
In this general context, the key objective of the research
described in the present article was to find out, apparently for
the first time, if micromodels can indeed be used to monitor
the growth of fungi in confined pore spaces similar to those
found in soils, and to elucidate the mechanisms that control
this growth. R. solani was selected as the target organism in
part for the fact that it does not produce spores, which would
complicate the dynamics, and for its remarkably predictable
behavior in unconfined situations, but also because its growth in
Petri dishes is described with particularly striking realism by a
computer model developed by Hopkins and Boswell (2012) and
extended recently to three dimensions (Vidal-Diez de Ulzurrun
et al., 2017). The key ingredients of this model are briefly outlined
in the section that follows this introduction, and serve as a
guide later on to determine to what extent the growth pattern
of hyphae observed in the microcosms differs from the “normal”
2-dimensional behavior out in the open. The article concludes
with a quick overview of the many perspectives the preliminary
results obtained so far open up for future experimental research
and modeling.
THEORETICAL ASPECTS
In the model of Hopkins and Boswell (2012), the mycelium is
thought of as a network of inter-connected tubes (representing
hyphae) through which various substances (including carbon,
nitrogen, trace metals, and tip vesicles) are translocated as part
of an internal cytoplasm. Hyphae are modeled as a discrete series
of straight line segments. After every time interval of length 1t,
the local substrate concentration changes due to translocation,
uptake and diffusion. New line segments are included in the
fungal network, corresponding to the processes of lengthening
of existing tubes (apical extension), and creation of new tubes
(subapical branching), according to a set of stochastic rules that
depend in part on the local concentrations of internal substrate.
A further transformation of the hyphal network may result from
the fusion of hyphae that come into contact with each other,
a process known as anastomosis. Hopkins and Boswell’s (2012)
model involves many aspects related to the translocation of
chemicals or materials inside the hyphae, as well as a description
of the response of hyphal tips to external gradients of an
inhibitor produced by the fungus itself, and which diffuses in the
surrounding medium. The components of the model that interest
us most here, however, are related to parameters that control the
elongation and branching of the hyphae.
Apical extension is represented schematically by the creation
of a new (virtual) line segment of nominal length 1x that extends
from the unconnected end of an existing line segment and
represents the movement of the hyphal tip over a discrete time
interval 1t. In addition to different tropisms associated with
gradients in nutrient- or inhibitor concentrations, hyphal tips
also display small stochastic variations in their growth axis. To
simulate the process of tip movement, a “velocity-jump” model
is used, which basically assumes that the velocity of hyphal tip
undergoes a biased circular random walk with its orientation
remaining the same or changing by an angle ±1θ(termed a
velocity jump) between successive time intervals and where the
localized concentration of the inhibitor induces bias so that
model tips have a tendency to move in the direction of lower
inhibitor concentrations, according to detailed mathematical
formulas for the probability of tip re-orientation by an angle 1θ,
clockwise or anti-clockwise.
Sub-apical branching is modeled by the creation of new line
segments emerging from the ends of existing line segments. Since
turgor pressure is thought to be implicated in the branching
process (Gow and Gadd, 1995; Riquelme and Bartnicki-Garcia,
2004), the model assumes that in the time interval 1t, the
probability of an existing line segment k to generate a new line
segment from its end position is zero unless the internal substrate
concentration exceeds a critical concentration β. The new line
segment is oriented at an angle ±φrelative to the existing line
segment with equal probability (Paulitz and Schroeder, 2005),
and the internal substrate is uniformly divided between the
existing and the new line segment.
Hopkins and Boswell (2012) parameterize their model with
data from the literature, relative to R. solani. The hyphal line
segment length 1x and the angular step size are taken to be
50 µm and π/12 radians (9), respectively, following Riquelme
et al. (1998). The branching angle φis considered to be normally
distributed, with mean of 79.2and a standard deviation of
3.16. This value of the branching angle may seem a little low,
since many authors have pointed out that R. solani branches
at right angle (90). Nevertheless, the lower value adopted by
Hopkins and Boswell (2012) has been borne out by recent
experimental data. The very detailed monitoring of the growth
of several fungi in Petri dishes over a 75 h timeframe, carried
out by Vidal-Diez et al. (2015) using image analysis techniques,
indicates that hyphae of R. solani branch at an angle that is
in fact slightly lower than 90, at 81.93 ±1.15. Nevertheless,
the small standard deviation shows that it is still reasonable
to view this value as virtually constant over time. The same
feature seems to be also manifested by a parameter, the internodal
length, which is not involved in Hopkins and Boswell’s (2012)
model, but is straightforward to measure in images of fungal
hyphae. It corresponds to the average distance between septa
(internal cross-walls separating cells in the hyphae). Vidal-Diez
et al. (2015) report that the internodal length of R. solani first
decreases from 175 to 171 µm over the first 17 h of growth, then
increases stepwise to reach 180 µm at the end of 75 h. Overall, the
average internodal distance they report is 179.29 ±11.27 µm.
MATERIALS AND METHODS
Micromodel Concept and Fabrication
The microfluidic device, or micromodel, concept adopted in
this research, as well as its manufacturing, have been described
in detail in the recent article by Deng et al. (2015), which
contains full references to earlier work as well as equipment
information. To make the present article as self-contained as
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Soufan et al. Fungal Growth in Soil-Like Micromodel
possible, we shall reproduce here some information on the design
and manufacturing of the micromodels. The original, much more
thorough description of Deng et al. (2015) should however be
consulted to obtain complete specifications.
In a nutshell, each micromodel is comprised of three parallel
channels each one mm wide and 34 µm high connected to a
single inlet well and a single outlet well (Figures 1A,B). The
central, 10-mm long portion of each of the channels consists
of a microstructured region, with pillars of varying sizes and
shapes representing a two-dimensional slice of the solid phase
of a simulated sandy loam soil (Figure 1C). The geometry of
the microstructured region is based on a realistic computer-
generated three-dimensional packing of ellipsoidal particles. The
size distribution of the particles is based on an experimentally-
determined sandy loam particle size distribution comprised of
56% fine sand and 44% very fine sand (USDA size ranges: 125–
250 mm and 50–125 mm, respectively).
To create the soil geometry, ellipsoidal particles were
randomly placed in a three-dimensional computational domain,
and the packing algorithm DigiPac (Jia and Williams, 2001) was
employed to create realistic particle-particle contacts. Then, a
two-dimensional slice of the packed three-dimensional domain
with a suitable level of pore connectivity was selected. The
selected slice was then manually traced using the Raster Design
toolset in AutoCAD 2010 and partitioned to completely fill
the 1 mm x 10 mm microstructured region in a high-resolution
chrome on-glass photomask. The geometrical features of the
pseudo-2D soil pattern are as follows: particle diameters averaged
110 µm and ranged from 10 to 300 µm, and the hydraulic
pore radius averaged 44 µm and ranged from 16 to 130 µm. In
contrast with typical porosities of sandy loam soils, which are in
the range of 25–35%, the porosity of Deng’s et al. (2015) pseudo-
2D emulated soil micromodel is 57%. This increase in porosity is
a result of selecting in the simulated porous medium a slice that
maintains pore connectivity in 2-D.
The photomask described above was then used to fabricate the
reusable casting mold, called the “master,” via photolithography.
First, a thin layer of SU-8 2025 photoresist was spin-coated onto
a 4-inch diameter Si wafer. The thickness of the photoresist
coating was 34 ±3µm as determined by profilometry. Then,
the photoresist was patterned by selectively exposing transparent
regions in the photolithography mask to 26.4 mWcm2
ultraviolet light for 6.1 s then finished by cross-linking and
developing steps. Finally, the master was “silanized,” or coated
with (tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane.
Individual emulated soil micromodels were cast 1 cm thick in
PDMS. First, Sylgard 184 base and curing agent were mixed in
a 10:1 ratio, degassed at 75 kPa gage for 30 min, then poured
over the master and cured at 60C for 4 h. Cured castings were
carefully peeled from the master (silanization facilitates release
of the cured PDMS from the master), trimmed, and access ports
were manually punched from the patterned side using a 4 mm
biopsy punch. Finally, each casting was treated with O2plasma
for 45 s in an evacuated air atmosphere and irreversibly bonded
featured-side down to a clean glass microscope slide. The plasma
treatment is desirable in order for the micromodels to better
FIGURE 1 | (A) Picture of the experimental system showing the rows of inlet and outlet wells. (B) Each channel has a micro-structured region 10 mm long, 1 mm
wide, and 34 ±3µm-deep, sandwiched between 5 mm-long open channels. Access ports are 1 cm high and 4 mm in diameter. (C) Micrograph of the 2-D pore
structure, with pores (darker color) located between simulated soil particles (lighter color).
Frontiers in Environmental Science | www.frontiersin.org 4July 2018 | Volume 6 | Article 68
Soufan et al. Fungal Growth in Soil-Like Micromodel
emulate soil since it results in PDMS having a surface charge
similar to quartz sand (Roman and Culbertson, 2006), at least
as long as the surface of the PDMS remains covered by water.
Observations made by Cruz et al. (2017) suggests that the plasma
treatment and the emulation of quartz-like surface chemistry are
not permanent under unsaturated conditions. In such cases, the
macromolecular mobility of the polymer at room temperature
allows re-configuration at the surface, and the latter is relatively
likely to have properties typical of untreated PDMS.
Cultivation of R. solani and Inoculation of
Poppy Seeds
Potato dextrose agar (PDA) plates were inoculated with an
anastomosis group (5) isolate of R. solani and incubated for 3 days
at 23C. Small plugs were cut from the edge of the plates and used
as a source of inoculum. Following the inoculation technique
adopted by Otten et al. (2012), poppy seeds (Papaver rhoeas) were
autoclaved twice at 120C at 1.1 Atm for 1 h over a 48 h period.
Sterilized seeds were subsequently sprinkled over the PDA plates
previously colonized by R. solani, and incubated at 23C for 3
days.
Operation of Micromodels
The microporous portions of the channels were partially filled
with sterile distilled water by injecting a small amount of water
inside the access well on one side of the micromodel, and letting
the water diffuse in the microporous region over time. Inspection
under the microscope was used to determine, for each amount of
distilled water applied, the portion of the channel porosity that
was saturated.
Once the microporous region had reached equilibrium in
terms of the water phase, colonized seeds were removed from the
Petri dishes with the PDA, and were placed inside the access wells
on the other side of the micromodel, relative to the access wells
used to inject water. At this stage, the micromodel was introduced
in a sterile Petri dish to maintain a suitable moisture level, but at
the same time allow the exchange of oxygen and carbon dioxide
with the atmosphere. The Petri dish was incubated for an initial
period of 24 h before the microscopic observation of the fungal
hyphae began.
Microscopy and Image Processing
Fungal spread in the channels of the micromodels was observed
with a Brunel inverted light microscope (Brunel Microscope
Limited, Wiltshire, U.K.). Pictures of the hyphae were typically
collected as time series at regular intervals, usually one frame
every 4 min. at selected locations, before the lens was repositioned
on a different spot in the micromodel.
To enhance the quality and contrast of the images obtained
with the light microscope, and allow easier visualization of
the hyphae, the micrographs showing features of interest were
processed with the imaging software Photoshop (Adobe Systems,
San José, California), by selecting the green channel in the RGB
format and changing its contrast setting. In some cases, false
colors were added with an image analysis software (GIMP) to
the liquid phase and to the simulated solid particles of the
micromodels, in order to make it clearer where the fungal hyphae
spread.
RESULTS AND DISCUSSION
Spread of Hyphae in the Inlet Portion of the
Micromodels
Prior to the experiments, it was not clear at all that R. solani
would manifest any inclination to enter the 34 µm-high inlet
section in the micromodels, leading to the microporous region
(see Figure 1). Our expectation, encouraged by the opinion of
several experts we consulted, was that R. solani would prefer
to stay in the much roomier access well where the poppy
seed was deposited, and would have to be enticed to go inside
the channel inlet section. This enticement could in principle
be carried out in a number of ways, for example via a piece
of fresh wood placed in the opposite access well. Based on
Fries (1973) observations, the release of volatile compounds by
the wood, which would diffuse through the partially saturated
microporous section, might be enough of an incentive for the
hyphae to penetrate the micromodel. Another option would
be to add a source of dissolved carbon to the distilled water
injected inside the micromodel, which would have attracted the
fungus.
As it turns out, the hyphae do not need any kind of incentive
to penetrate the micromodels. Evidence indicates that they do
so easily and spread readily away from the poppy seeds, into
the inlet portion of the micromodels, and eventually in the
microporous sections as well. Near the poppy seeds (Figure 2a),
branching tends to be abundant, and anastomosis is frequent,
making it difficult to determine the range of values exhibited for
the branching angle, the hyphal line segment, or the intermodal
distance. Close to the entrance of the microporous portion of the
micromodel (Figure 2b), whenever Rhizoctonia does not grow
along the wall of the cavity, the branching pattern is very similar
to what was observed earlier in the PDA agar plates, which itself
was in line with accounts published in the literature. In Figure 2b,
hyphae, with a constant width of 7 µm, branch at angles of 62,
63, 78, and 63, respectively, from bottom to top. Branching
systematically occurs immediately before the septa on the main
hypha, and the segment length is equal to the internodal distance,
respectively 227, 236, and 256 µm for the three segments shown
in Figure 2b. These values for the internodal distances are slightly
larger than those of 179.29 ±11.27 µm measured by Vidal-Diez
et al. (2015).
One has to be careful in assigning values to the branching
angles in the case of these experiments. Indeed, when growing
on agar plates, fungal hyphae have a major incentive to branch
out strictly at the surface of the agar, from which they derive
energy and carbon. In the experiments described here, however,
hyphae derive their sustenance strictly from the poppy seeds,
and are therefore not bound metabolically to spread along the
bottom surface of the micromodels, as they would be expected
to do when growing in agar (even though, even in these cases,
it is not infrequent to see them shoot upward as well). Inside
the micromodels, branching hyphae can shoot upward at least
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Soufan et al. Fungal Growth in Soil-Like Micromodel
FIGURE 2 | Illustrative example of the growth of R. solani in the inlet section of the micromodel, (a) near the inoculation point, in the inlet well, and (b) hypha with 3
very regular segments, further toward the porous section of the micromodel.
initially, until they reach the 34 µm-high ceiling of the cavities
inside the micromodels and are then forced to move horizontally
or come back down. Analysis of the resulting images at too
coarse a magnification gives the misleading impression of a
branching angle that is very different than one would expect,
when in fact, close analysis of the images shows sharp bends of
the hyphae right after branching. This same process occurs within
the microporous regions in the micromodels. In this article,
whenever branching angles are mentioned, it is after careful
evaluation of the branching at different focal distances of the
microscope, to avoid gross misrepresentations.
Influence of Liquid Phase on Fungal Spread
After an initial period during which the hyphae propagate in
the inlet regions of the channels, some hyphae tips reach the
microporous region, which is variably saturated with distilled
water. Based on previous observations by several authors (e.g.,
Otten et al., 1999), one anticipates at that point that the hyphae
would tend to spread preferentially inside larger pores, which
are not water filled. Indeed, this behavior is clearly evinced in
our experiments (see Figure 3). Bundles of hyphae are seen in
several images to converge to single air-filled pores and to grow
there in preference to other portions of the pore space that are
saturated with liquid. One needs to be careful in interpreting
these observations because of the fact that the surface of the
micromodels in the portions of the pores that are unsaturated
are likely not to have properties similar to those of sand particles,
because the plasma treatment of the PDMS is not permanent
under these conditions. This point will need to be taken into
account in future research. Be that as it may, the apparent
preference for the unsaturated part of the pore space is not
FIGURE 3 | Preferential spread of R. solani in air-filled pores. Flas colors have
been added to highlight the different phases. The water is represented in blue,
and the solid particles in brown. Hyphae are clearly seen to prefer growing in
pores without water, even though some hyphae manage to grow inside the
liquid phase. The width of the image corresponds to 1 mm.
exclusive. As many authors have pointed out, hyphae are capable
of growing through water-filled space if need be, as seen in the
water-filled parts of Figure 3. Indeed in a series of papers it was
shown that R. solani used in this study spreads preferentially
through water filled pores, larger pores and readily crosses cracks,
but when given little choice does spread through smaller and
water filled pores (Otten et al., 1999, 2004a,b).
Another feature that is manifested in this same image is the
fact that, after a while, as the hyphae undoubtedly consume some
of the liquid phase around them, or as the water slowly evaporates
from the microcosms, the configuration of the liquid phase that
Frontiers in Environmental Science | www.frontiersin.org 6July 2018 | Volume 6 | Article 68
Soufan et al. Fungal Growth in Soil-Like Micromodel
remains in the pores tends in places to adjust to the presence of
the hyphae. Pockets of water exhibit external surfaces that appear
to be unphysical from the standpoint of the theory of capillarity,
e.g., with a concavity opposite to what one might expect based on
the geometry of nearby solid surfaces. However, in many cases,
these conflicting observations can be resolved once one realizes
upon scrutiny of the micrographs that these interfaces are held in
place by one or more fungal hyphae acting as a restraining net.
Linear Apical Extension and Growth Along
Pore Walls
In many of the images of the water-saturated microporous
regions of the micromodels, hyphae appear to be extending
linearly for hundreds of microns without branching (Figure 4).
Again, one needs to be very careful in that context, and make
sure by changing the focal plane of the microscope that one does
not miss branching that may occur vertically. But in the absence
of such branching, the very long internodal distances that are
apparent in these images are in sharp contrast with what has been
routinely observed on agar plates.
When a hypha encounters a pore wall, as in Figure 4 (at point
b) and in Figure 5 (at point a), there is a clear tendency for it
to stay in contact with it for a while, as expected according to
Watts et al. (1998), a phenomenon termed thigmotropism. This
behavior is not entirely surprising and may be due in this case
to some extent to electrostatic interactions. R. solani might react
positively to electrical surface charge, as small as it might be
(similar to that on sand particles) on the walls of the pillars in the
micromodels. Common wisdom is that if one drags one’s finger
FIGURE 4 | Example of particularly long extension of hyphae in the
water-saturated portion of the micromodel. (a) this very long hyphal segment
does not show any appearance of branching yet, at the time the picture was
taken. (b) At that point, the hypha touches the surface of the pore, and stays in
contact with it for a little while, but eventually separates from the surface to
return to the pore space.
on a flat surface, producing static electricity in the process, fungal
hyphae subsequently colonizing the surface will have a tendency
to follow closely the path of the finger. By the same process,
hyphae approaching a surface tangentially would have a tendency
to keep following it closely afterwards, even if the surfaces curves.
Nevertheless, it is clear from Figures 4,5, that this tendency does
not associate the hyphae and surfaces indefinitely. At different
stages in the progression of the hypha in Figure 4, and at point b
in Figure 5, the hyphae begin to separate from the surfaces and
eventually foray into the open pore space.
Hyphae Encountering Pore Walls “Head
on”
Less predictable initially was what happens to fungal hyphae that
run straight into a pore wall, as in Figure 6. As it touches the
wall, the hypha in this image does not branch, as one might have
expected. Instead, it seems to keep elongating. The apical region
does not move, but the part of the hypha behind it progressively
bends to accommodate the extra length that is generated over
time. As the bending intensifies, the angle the apex makes with
the surface reduces progressively, until the apex is eventually
not encumbered by the surface any more, and can grow again,
alongside it. This sequence of events, which is observed in many
of the pictures we took, clearly deviates from the sequence of
steps described by the model of Hopkins and Boswell (2012).
In the presence of confining surfaces, fungal hyphae cannot be
viewed as series of rigid, straight tubes connected with each other.
Provision needs to be made in models for connected tubes to
bend in response to constraints.
Branching Pattern
In a previous section, it was mentioned that hyphae can elongate
sometimes more than a mm without branching (as in Figure 4),
unlike what has been routinely observed on Petri dishes. This
FIGURE 5 | Illustration of the tendency of hyphae to stay in close contact with
pore walls once they encounter them (at point a). Nevertheless, this
thigmotrophic process does not extend indefinitely, as the hypha eventually
dissociates from the surface (at point b).
Frontiers in Environmental Science | www.frontiersin.org 7July 2018 | Volume 6 | Article 68
Soufan et al. Fungal Growth in Soil-Like Micromodel
FIGURE 6 | Time sequence of 6 successive snapshots (1 to 6) of the propagation and bending of a hypha and its encounter “head on” with a pore wall.
FIGURE 7 | Location and time sequence of the branching of a hypha. (a) at this point, immediately preceding a septum, branching seems to be very much like that
observed in Petri dishes or in the inlet portion of the micromodel, whereas at (b) the branching seems to be closely associated with the strong bending of the hypha.
Frontiers in Environmental Science | www.frontiersin.org 8July 2018 | Volume 6 | Article 68
Soufan et al. Fungal Growth in Soil-Like Micromodel
behavior may be due to the fact that hyphae in our experiments
are surrounded by distilled water. There is therefore very little
reason for the hyphae to branch out to scavenge more nutrients
and energy from their environment. Nevertheless, hyphae do
branch out at various times. Some of this branching, as in
Figure 7 at point “a,” just before a septum, seems to be typical
of what happens in Petri dish. But many cases of branching in
the hundreds of images that we have taken seem to be as at point
“b” in Figure 7, associated with bending of the hyphae, following
a “head-on collision” with pore walls. The common explanation
for the branching process, as mentioned earlier, is that it is related
to turgor pressure inside the cell that eventually branch. Turgor
pressure is a strictly osmotic process, related to the concentration
of electrolyte inside the cytoplasm. However, it could be that the
pressure felt inside the branching cell in soil pores is in fact more
mechanical than osmotic. As the hypha elongates and is forced to
bend, cells walls may be under sizeable stress, just as they would
under regular turgor pressure.
CONCLUSION
The research described above corresponds to a first attempt
to use a soil-like micromodel to identify the parameters that
control the growth of fungal hyphae in the confining pore
space of soils. The results suggest that R. solani, introduced in
the micromodel on a poppy seed from which it subsequently
propagates, is indeed able to penetrate into the microporous
portion of the micromodel, without having to be enticed to do
so. Once the fungus has penetrated inside the micromodel, a
working hypothesis in the research was that the geometry of the
pores, as well as the presence of hard surfaces in the path of the
hyphae would influence the latter’s behavior significantly. The
experimental results support this hypothesis. Indeed, both the
branching pattern as well as the apical elongation of the hyphae
appear to be strongly affected by the presence of “obstacles” in
soil pores. In particular, far from being series of straight and
rigid tubes, hyphae of R. solani are able to bend after the forward
movement of the apex has stopped. The observations reported
in this article therefore suggest that the modeling of hyphal
growth in soils cannot simply be viewed as a special case of
growth in more open environments. A model tailored to soils
will have to encompass very different growth mechanisms and
characteristics.
This preliminary experiment shows that it is feasible to use
micromodels to study the behavior of fungi under conditions
that, although nearly 2-dimensional, are in many respects like
those found in real soils. It will be interesting, in future
experiments, to try to grasp better, quantitatively, the different
parameters that control the growth of R. solani, and other fungi
as well, in soil pores. This will require systematic replication
so that statistics can be computed and the behavior of hyphae
characterized in great detail. Further experiments could also
address other aspects of the spread of fungal hyphae about
which little is yet known, like what happens when different
fungal species propagate in the same pore space in a soil, or
when bacteria, hopping onto the external surfaces of hyphae,
are carried along as the hyphae grows (a process often referred
to as “hitchhiking on the fungal highway”). Clearly, there are
a lot of avenues that can be pursued in this general context,
all of which would result in a far better understanding than
is currently available of the ecology of fungi in terrestrial
environments.
AUTHOR CONTRIBUTIONS
PB, WO, and RS came up with the idea. LS provided the
micromodels and advice on how to use them. RS carried out the
laboratory work, under the supervision of YD, LG, and PB, and
wrote a preliminary draft of the paper. PB did the final editing of
the manuscript, to the revision of which RS, WO, LS, and LVG
participated.
ACKNOWLEDGMENTS
The research described in this article was made possible in part
through a grant from the Agence Nationale de la Recherche
(ANR, France) to project Soilµ3D, which provided an internship
to RS, and to NPRP grant #9-390-1-088 from the Qatar National
Research Fund (Project Simupor) during the final preparation
of the manuscript. LS contribution was made possible through
grant DE-SC0014522 from the U.S. Department of Energy.
WO acknowledges funding from the National Environment and
Research Council (NE/P014208/1). The assistance of Dr. Cécilia
Cammas, who gave us access to the microscopes of the
Soil Micromorphology Laboratory (INRAP-AgroParisTech), is
gratefully acknowledged.
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Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Copyright © 2018 Soufan, Delaunay, Gonod, Shor, Garnier, Otten and Baveye.
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... These results agree with the visual observations made by Harris et al. (2003) in soil thin sections (Otten et al., 2006). One explanation would be that the fungus has less opportunity to branch when the pore space is reduced as observed by Otten et al. (1999) and Soufan et al. (2018). In another set of modelling scenarios where detailed soil architecture is considered through the use of CT images of sandy soil samples repacked at different densities, Pajor et al. (2010) also found that the colonization rate of the fungus is highest for the repacked sandy soils with the lowest density. ...
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... This has been demonstrated in microfluidic devices to facilitate microbial persistence by providing physical compartmentalisation and protection from external stressors (Nadell et al., 2017, Donlan, 2002. Soil micromodels (i.e., 2D or 3D models of soil structure within a microfluidic system) provide excellent examples of integration with real-world structures (Deng et al., 2015, Soufan et al., 2018. ...
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Ubiquitously, the environments of microorganisms have three-dimensional structure that create heterogeneous distributions of the space in which microorganisms reside. In particular, the soil environment is a complex porous medium inhabited by a vast array of microorganisms, essential for Earth processes like biogeochemical cycling. However, these microorganisms are subject to environmental perturbation. The physiological impacts of environmental stress on microorganisms are well studied, but whether (and to what extent) soil structure impacts exposure and response of microorganisms to stressors remains poorly understood. In this thesis, it was hypothesised that environmental structures could influence the stressor exposure of cells (and hence stressor survival) within them, and that the extent of this protective effect would depend on the type and scale of environmental structure. To examine this overall hypothesis, the influence of environmental structures on microbial response (or survival) to stress were assessed: first in relation to soil aggregation, then within macroscale pores ranging from 0.5 – 2 mm in diameter and last within micrometre scale structures using microfluidic approaches. A method was developed to manufacture soil aggregates from natural soils with defined quantities of soil yeast in the aggregate exterior or interior. This was used to examine the impact of soil aggregation on microbial survival of a small panel of stressors (anoxia, lead nitrate, and heat stress). Results indicated that yeast cells inside aggregates were protected from acute heat stress relative to cells at the aggregate exterior, whereas effects of aqueous lead nitrate or anoxia were similar on cells at either location. The protective effect against heat stress was compromised after prolonged heat exposure but was accentuated within compacted versions of soil aggregates, providing evidence that soil compaction, a common consequence of agricultural activity, can influence microbial stress resilience. In further experiments, structured environments with millimetre-scale pores were developed by setting up vessels containing glass beads of different sizes. Yeast ii inocula and stressors were introduced to these to explore the relationship between the environmental pore size and stress survival. Here, it was demonstrated that survival of yeast in response to lead nitrate within these structures increased with decreasing average pore size. This trend was reproduced using additively manufactured (3D-printed) lattice structures, containing pores of similar size ranges to the less-uniform glass bead structures. Finally, microfluidic devices were used to determine whether structure at the microscale impacted microbial survival of stress. These devices contained either fabricated soil-like structures, or small microspheres to create simplified structures within otherwise homogeneous environments. At this scale, an impact of environmental structure was less clear. However, in the simplified microsphere environments, results suggested that cells within more confined spaces (I.e., more surrounded by protective structures) were less exposed to stressor (copper sulfate), which was introduced as a flowing solution within the microfluidic devices. Taken together, results from this thesis suggest that environmental structure can determine microbial (exposure to and) survival of stress, at scales of structure ranging from micrometres to millimetres. The new methodologies and results developed within this thesis provide a foundation upon which the relationship between microbial perturbation and environmental structure can be further explored.
... A recent study further demonstrated that bacterial motility in environments with various levels of confinement is jointly controlled by hydrodynamics and local steric interactions. 90 For fungal hyphae, it was observed that hyphae would frequently bend to adapt to the confinement of pillars in microfluidic chips (mimicking soil particles), 91 and the growth velocity decreased in confined channels. 92 Chemotaxis is the ability of motile cells to migrate toward or away from a chemical gradient in their local microenvironments. ...
... When fungal hyphae hit a wall, their Spitzenkörper shifts towards the wall and stays near the wall as hyphae grow 46 . Spitzenkörper seems to be responsible for the reorientation of hyphae after hitting an obstacle because it remains in the part of the hyphae that is close to the wall, in a phenomenon known as thigmotropism 47 as a way to maintain its original directionality after the obstacle is circumvented. In alternated turns channels, hyphae seem to maintain the original directionality regardless of the contact angle reducing the effect of the angles on fungal biomass. ...
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Microhabitat conditions determine the magnitude and speed of microbial processes but have been challenging to investigate. In this study we used microfluidic devices to determine the effect of the spatial distortion of a pore space on fungal and bacterial growth, interactions, and substrate degradation. The devices contained channels differing in bending angles and order. Sharper angles reduced fungal and bacterial biomass, especially when angles were repeated in the same direction. Substrate degradation was only decreased by sharper angles when fungi and bacteria were grown together. Investigation at the cellular scale suggests that this was caused by fungal habitat modification, since hyphae branched in sharp and repeated turns, blocking the dispersal of bacteria and the substrate. Our results demonstrate how the geometry of microstructures can influence microbial activity. This can be transferable to soil pore spaces, where spatial occlusion and microbial feedback on microstructures is thought to explain organic matter stabilization.
... The incompletely understood foraging behavior of fungal hyphae makes culturing multiple species at microscopic vicinity to each other risky, if cross-contaminations are to be avoided. It is also likely that we are still unaware of some challenges of culturing filamentous fungi in microfluidics, since this study direction is new (Soufan et al., 2018;Held et al., 2019). ...
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The pool of fungal secondary metabolites can be extended by activating silent gene clusters of cultured strains or by using sensitive biological assays that detect metabolites missed by analytical methods. Alternatively, or in parallel with the first approach, one can increase the diversity of existing culture collections to improve the access to new natural products. This review focuses on the latter approach of screening previously uncultured fungi for chemodiversity. Both strategies have been practiced since the early days of fungal biodiscovery, yet relatively little has been done to overcome the challenge of cultivability of as-yet-uncultivated fungi. Whereas earlier cultivability studies using media formulations and biological assays to scrutinize fungal growth and associated factors were actively conducted, the application of modern omics methods remains limited to test how to culture the fungal dark matter and recalcitrant groups of described fungi. This review discusses the development of techniques to increase the cultivability of filamentous fungi that include culture media formulations and the utilization of known chemical growth factors, in situ culturing and current synthetic biology approaches that build upon knowledge from sequenced genomes. We list more than 100 growth factors, i.e., molecules, biological or physical factors that have been demonstrated to induce spore germination as well as tens of inducers of mycelial growth. We review culturing conditions that can be successfully manipulated for growth of fungi and visit recent information from omics methods to discuss the metabolic basis of cultivability. Earlier work has demonstrated the power of co-culturing fungi with their host, other microorganisms or their exudates to increase their cultivability. Co-culturing of two or more organisms is also a strategy used today for increasing cultivability. However, fungi possess an increased risk for cross-contaminations between isolates in existing in situ or microfluidics culturing devices. Technological improvements for culturing fungi are discussed in the review. We emphasize that improving the cultivability of fungi remains a relevant strategy in drug discovery and underline the importance of ecological and taxonomic knowledge in culture-dependent drug discovery. Combining traditional and omics techniques such as single cell or metagenome sequencing opens up a new era in the study of growth factors of hundreds of thousands of fungal species with high drug discovery potential.
... These soil micromodels are designed to emulate the particle size distributions, aggregate sizes, and pore size distributions of a sandy loam soil and have three identical microstructured channels that provide built-in technical replicates for bacterial biofilm growth (Fig. S1) (20,21). Previously, these micromodels were used to measure the water retention properties of a soil bacterium's extracellular polymeric substance (21) and dynamic interactions of bacteria (21) and fungi (27) within structured microenvironments, while similar systems have been used to study transport of bacteria in flowing micromodel systems (28) and particle transport by soil protists in quiescent micromodel systems (29), in each case revealing phenomena that only occur within an appropriately scaled and suitably physiochemically complex soil pore microenvironment. The soil micromodels are fabricated from polymers that are electrically insulating (28). ...
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Mass spectrometry imaging (MSI) is becoming an increasingly popular analytical technique to investigate microbial systems. However, differences in the ionization efficiencies of distinct MSI methods lead to biases in terms of what types and classes of molecules can be detected. Here, we sought to increase the molecular coverage of microbial colonies by employing metal-assisted laser desorption/ionization (MetA-LDI) MSI, and we compared our results to more commonly utilized matrix-assisted laser desorption/ionization MALDI MSI. We found substantial (∼67%) overlap in the molecules detected in our analysis of Bacillus subtilis colony biofilms using both methods, but each ionization technique did lead to the identification of a unique subset of molecular species. MetA-LDI MSI tended to identify more small molecules and neutral lipids, whereas MALDI MSI more readily detected other lipids and surfactin species. Putative annotations were made using METASPACE, Metlin, and the BsubCyc database. These annotations were then confirmed from analyses of replicate bacterial colonies using liquid extraction surface analysis tandem mass spectrometry. Additionally, we analyzed B. subtilis biofilms in a polymer-based emulated soil micromodel using MetA-LDI MSI to better understand bacterial processes and metabolism in a native, soil-like environment. We were able to detect different molecular signatures within the micropore regions of the micromodel. We also show that MetA-LDI MSI can be used to analyze microbial biofilms from electrically insulating material. Overall, this study expands the molecular universe of microbial metabolism that can be visualized by MSI. IMPORTANCE Matrix-assisted laser desorption/ionization mass spectrometry imaging is becoming an important technique to investigate molecular processes within microbial colonies and microbiomes under different environmental conditions. However, this method is limited in terms of the types and classes of molecules that can be detected. In this study, we utilized metal-assisted laser desorption/ionization mass spectrometry imaging, which expanded the range of molecules that could be imaged from microbial samples. One advantage of this technique is that the addition of a metal helps facilitate ionization from electrically nonconductive substrates, which allows for the investigation of biofilms grown in polymer-based devices, like soil-emulating micromodels.
... In the case of fungal dispersal rates, hyphal expansion and branching angles have been observed on 2D surfaces and incorporated into fungal models (Stacey, Truscott, & Gilligan, 2001). In theory these types of parameterization can be expanded to 3D; however, it has been shown that branching angles in a 3D pore structure can significantly deviate from those reported in literature, suggesting that expansion of fungal colonies has more complex feedback mechanisms with the environment than is currently considered in models (Soufan et al., 2018). ...
Article
Macroscopic models of soil organic matter (SOM) turnover have faced difficulties in reproducing SOM dynamics or in predicting the spatial distribution of carbon stocks. These models are based on a largely inadequate linear response of soil microorganisms to bulk concentrations of nutrients and it is clear that a new approach to SOM modeling is required. Introducing explicit microbial activity and OM reactivity in macroscopic models represents a challenge because of the fine spatial scales at which the processes occur. To get a better grasp on interactions that take place at the micro‐scale, a new generation of SOM models have been developed at the spatial scale of the soil micro‐environments where microorganisms evolve. They are well adapted to challenge traditional hypotheses about the influence of soil architecture on soil microbial activity. Soil architecture provides the scene for a dynamic spatial accessibility of resources to microbes and the emergence of interactions between the actors of SOM decomposition. In this context, we review microscale models of microbial activity that have been designed for soils and soil analogs. To understand how these models account for spatial accessibility, we have looked in detail at how soil micro‐environments are described in the different approaches and how microbial colonies are spatialized in these micro‐environments. We present the advantages and disadvantages of the developed strategies and we discuss their limits.
... Thus, given the broad colonization by fungi after 42 days of incubation (Fig. 5), we suggest that fungi play an important role in the transfer of plant OM into the surrounding mineral soil, besides possible diffusion of plant-derived moieties. While fungi rely on existing pores of sufficient size (>10 μm, ideally > 100 μm) to explore soil resources (Soufan et al., 2018;Erktan et al., 2020), their ability to bridge air filled pores facilitates the expansion of the hyphal network (Griffin, 1985) and, thus, OM translocation into the mineral soil (Frey et al., 2003;Poll et al., 2006;Kaiser et al., 2015;Gorka et al., 2019). Fungi exude carbon-rich substances, including polysaccharides, lipids, and proteins (Cairney, 1992;Jastrow et al., 2007). ...
Article
The interface between decaying plant residues and soil minerals represents an essential soil microenvironment at which soil organic matter forms. The high amount of microbial products and residues within this hot spot of microbial activity fosters the formation of mineral-associated organic matter. Besides classical quantitative analyses, our understanding of processes controlling soil organic matter formation greatly benefits from microscopic observations and measurements, which provide spatially resolved information at a meaningful scale for microbial processes and for the association between organic and mineral particles. We studied carbon and nitrogen transfer from fresh-plant residues to the mineral soil, through a litter decomposition experiment in an artificial soil mixture. Needles of Norway spruce (Picea abies L.) were placed in microbatch containers filled with an artificial soil mixture free of soil organic matter. Containers were buried in fresh organic layer material from a Norway spruce stand and incubated for 14 and 42 days. We applied nanoscale secondary ion mass spectroscopy (NanoSIMS) to investigate the spatial distribution of mineral and organic compounds at the needle vicinity and into the mineral soil (0–550 μm from the needle). After 14 days, we depicted the formation of mineral-associated organic matter in the surrounding of the decaying needles. After 42 days, we observed substantial colonization of the needles and the detritusphere by saprotrophic fungi. The fungal hyphae extended into the mineral matrix of the artificial soil acting as vectors for the transfer of litter-derived carbon and nitrogen into the bulk soil. This resulted in an increase of the area covered by organic matter in the detritusphere, with up to 10% of the total investigated area classified as organic matter closely associated with mineral surfaces. Our results provide evidence that the carbon and nitrogen derived from litter decomposition transformed by microorganisms is transferred as mineral-associated organic matter, heterogeneously distributed from the litter source, and still detected 550 μm away from the latter. The close association of newly formed soil organic matter and fine sized minerals suggests that the formation of mineral-associated OM and likely also microaggregates is directly driven by microbial activity in the vicinity of hot spots for plant carbon input (e.g. the detritusphere).
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Microbes govern most soil functions, but investigation of these processes at the scale of their cells has been difficult to accomplish. Here we incubate microfabricated, transparent ‘soil chips’ with soil, or bury them directly in the field. Both soil microbes and minerals enter the chips, which enables us to investigate diverse community interdependences, such as inter-kingdom and food-web interactions, and feedbacks between microbes and the pore space microstructures. The presence of hyphae (‘fungal highways’) strongly and frequently increases the dispersal range and abundance of water-dwelling organisms such as bacteria and protists across air pockets. Physical forces such as water movements, but also organisms and especially fungi form new microhabitats by altering the pore space architecture and distribution of soil minerals in the chip. We show that soil chips hold a large potential for studying in-situ microbial interactions and soil functions, and to interconnect field microbial ecology with laboratory experiments.
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Over the last 60 years, soil microbiologists have accumulated a wealth of experimental data showing that the bulk, macroscopic parameters (e.g., granulometry, pH, soil organic matter, and biomass contents) commonly used to characterize soils provide insufficient information to describe quantitatively the activity of soil microorganisms and some of its outcomes, like the emission of greenhouse gasses. Clearly, new, more appropriate macroscopic parameters are needed, which reflect better the spatial heterogeneity of soils at the microscale (i.e., the pore scale) that is commensurate with the habitat of many microorganisms. For a long time, spectroscopic and microscopic tools were lacking to quantify processes at that scale, but major technological advances over the last 15 years have made suitable equipment available to researchers. In this context, the objective of the present article is to review progress achieved to date in the significant research program that has ensued. This program can be rationalized as a sequence of steps, namely the quantification and modeling of the physical-, (bio)chemical-, and microbiological properties of soils, the integration of these different perspectives into a unified theory, its upscaling to the macroscopic scale, and, eventually, the development of new approaches to measure macroscopic soil characteristics. At this stage, significant progress has been achieved on the physical front, and to a lesser extent on the (bio)chemical one as well, both in terms of experiments and modeling. With regard to the microbial aspects, although a lot of work has been devoted to the modeling of bacterial and fungal activity in soils at the pore scale, the appropriateness of model assumptions cannot be readily assessed because of the scarcity of relevant experimental data. For significant progress to be made, it is crucial to make sure that research on the microbial components of soil systems does not keep lagging behind the work on the physical and (bio)chemical characteristics. Concerning the subsequent steps in the program, very little integration of the various disciplinary perspectives has occurred so far, and, as a result, researchers have not yet been able to tackle the scaling up to the macroscopic level. Many challenges, some of them daunting, remain on the path ahead. Fortunately, a number of these challenges may be resolved by brand new measuring equipment that will become commercially available in the very near future. Introduction
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Fungi have a well-established role in nutrient cycling and are widely used as agents in biological control and in the remediation of polluted landscapes. Competition for resources between different fungal communities is common in these contexts and its outcome impacts on the success of such biotechnological applications. In this investigation a mathematical model is constructed to represent competition between two fungal colonies that have access to different resources. It is shown that the model equations display a multitude of travelling wave solutions and that the outcome of competition between two fungal biomasses can be controlled through the simple manipulation of the nutrient resources available to each. The model equations are also numerically integrated to illustrate the range of outcomes arising from fungal competition and these results are placed in context of established experimental observations.
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In this paper, we provide direct evidence of the importance of root hairs on pore structure development at the root–soil interface during the early stage of crop establishment. This was achieved by use of high‐resolution (c. 5 μm) synchrotron radiation computed tomography (SRCT) to visualise both the structure of root hairs and the soil pore structure in plant–soil microcosms. Two contrasting genotypes of barley (Hordeum vulgare), with and without root hairs, were grown for 8 d in microcosms packed with sandy loam soil at 1.2 g cm⁻³ dry bulk density. Root hairs were visualised within air‐filled pore spaces, but not in the fine‐textured soil regions. We found that the genotype with root hairs significantly altered the porosity and connectivity of the detectable pore space (> 5 μm) in the rhizosphere, as compared with the no‐hair mutants. Both genotypes showed decreasing pore space between 0.8 and 0.1 mm from the root surface. Interestingly the root‐hair‐bearing genotype had a significantly greater soil pore volume‐fraction at the root–soil interface. Effects of pore structure on diffusion and permeability were estimated to be functionally insignificant under saturated conditions when simulated using image‐based modelling.
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Plants and microbes secrete mucilage into soil during dry conditions, which can alter soil structure and increase contact angle. Structured soils exhibit a broad pore size distribution with many small and many large pores, and strong capillary forces in narrow pores can retain moisture in soil aggregates. Meanwhile, contact angle determines the water repellency of soils, which can result in suppressed evaporation rates. Although they are often studied independently, both structure and contact angle influence water movement, distribution, and retention in soils. Here drying experiments were conducted using soil micromodels patterned to emulate different aggregation states of a sandy loam soil. Micromodels were treated to exhibit contact angles representative of those in bulk soil (8.4° ± 1.9°) and the rhizosphere (65° ± 9.2°). Drying was simulated using a lattice Boltzmann single-component, multiphase model. In our experiments, micromodels with higher contact angle surfaces took 4 times longer to completely dry versus micromodels with lower contact angle surfaces. Microstructure influenced drying rate as a function of saturation and controlled the spatial distribution of moisture within micromodels. Lattice Boltzmann simulations accurately predicted pore-scale moisture retention patterns within micromodels with different structures and contact angles.
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The Mycelium as a Network, Page 1 of 2 Abstract The characteristic growth pattern of fungal mycelia as an interconnected network has a major impact on how cellular events operating on a micron scale affect colony behavior at an ecological scale. Network structure is intimately linked to flows of resources across the network that in turn modify the network architecture itself. This complex interplay shapes the incredibly plastic behavior of fungi and allows them to cope with patchy, ephemeral resources, competition, damage, and predation in a manner completely different from multicellular plants or animals. Here, we try to link network structure with impact on resource movement at different scales of organization to understand the benefits and challenges of organisms that grow as connected networks. This inevitably involves an interdisciplinary approach whereby mathematical modeling helps to provide a bridge between information gleaned by traditional cell and molecular techniques or biophysical approaches at a hyphal level, with observations of colony dynamics and behavior at an ecological level.
Book
This book is about the growth and differentiation processes underlying the growth and differentia­ of filamentous fungi. The impetus for this work tion of fungi and that it provides the reader with stems from our perception that the coverage of adequate source references for further information. this highly diverse and important group of organ­ It is estimated conservatively that there are more isms has been neglected in recent years, despite than 1. 5 million species of fungi - more than five many significant advances in our understanding of times the number of vascular plants and second the underlying mechanisms of growth. This situ­ only in diversity to the insects. The extreme ation contrasts with the treatment of Saccharomyces diversity of form in the fungi has always been a cerevisiae, for example, which because of its ideal source of inspiration for mycologists. This book is properties for genetic analyses, has established concerned mainly with those systems that have itself as the model eukaryote for the analysis of the been well characterized from the biochemical, cell cycle, and basic studies of biochemical and physiological or genetic points of view. Although genetic regulation. This book does not deal with it has not been possible to illustrate the breadth of the detailed growth phYSiology of S.
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Most fungi grow by developing complex networks that enable the translocation of nutrients over large distances. Spatially explicit mathematical models are able to capture both the complexity of the fungal network and the biomass evolution, as such providing a powerful alternative to classical modelling paradigms. Unfortunately, most of these models restrict growth to two dimensions or confine it to a lattice, thereby resulting in unrealistic representations of fungal networks. In addition, interactions between fungi and their environment are often neglected.
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Soil is the habitat of countless organisms and encompasses an enormous variety of dynamic environmental conditions. While it is evident that a thorough understanding of how organisms interact with the soil environment may have substantial ecological and economical impact, current laboratory-based methods depend on reductionist approaches that are incapable of simulating natural diversity. The application of Lab-on-a-Chip or microfluidic technologies to organismal studies is an emerging field, where the unique benefits afforded by system miniaturisation offer new opportunities for the experimentalist. Indeed, precise spatiotemporal control over the microenvironments of soil organisms in combination with high-resolution imaging has the potential to provide an unprecedented view of biological events at the single-organism or single-cell level, which in turn opens up new avenues for environmental and organismal studies. Herein we review some of the most recent and interesting developments in microfluidic technologies for the study of soil organisms and their interactions with the environment. We discuss how so-called "Soil-on-a-Chip" technology has already contributed significantly to the study of bacteria, nematodes, fungi and plants, as well as inter-organismal interactions, by advancing experimental access and environmental control. Most crucially, we highlight where distinct advantages over traditional approaches exist and where novel biological insights will ensue.
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Due to their ability to grow in complex environments, fungi play an important role in most ecosystems and have for that reason been the subject of numerous studies. Some of the main obstacles to the study of fungal growth are the heterogeneity of growth environments and the limited scope of laboratory experiments. Given the increasing availability of image capturing techniques, a new approach lies in image analysis. Most previous image analysis studies involve manual labelling of the fungal network, tracking of individual hyphae, or invasive techniques that do not allow for tracking the evolution of the entire fungal network. In response, this work presents a highly versatile tool combining image analysis and graph theory to monitor fungal growth through time and space for different fungal species and image resolutions. In addition, a new experimental set-up is presented that allows for a functional description of fungal growth dynamics and a quantitative mutual comparison of different growth behaviours. The presented method is completely automated and facilitates the extraction of the most studied fungal growth features such as the total length of the mycelium, the area of the mycelium and the fractal dimension. The compactness of the fungal network can also be monitored over time by computing measures such as the number of tips, the node degree and the number of nodes. Finally, the average growth angle and the internodal length can be extracted to study the morphology of the fungi. In summary, the introduced method offers an updated and broader alternative to classical and narrowly focused approaches, thus opening new avenues of investigation in the field of mycology.