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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 1θ 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 79.2◦and 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 mWcm−2
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 60◦C 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).
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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 23◦C. 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 120◦C 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 23◦C 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
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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.
REFERENCES
Baveye, P. C. (2015). Grand challenges in the research on soil processes. Front.
Environ. Sci. 3:10. doi: 10.3389/fenvs.2015.00010
Baveye, P. C., Otten, W., Kravchenko, A., Balseiro Romero, M., Beckers, É.,
Chalhoub, M., et al. (2018). Emergent properties of microbial activity in
heterogeneous soil microenvironments: different research approaches are
slowly converging, yet major challenges remain. Front. Microbiol. 8:1364.
doi: 10.3389/fmicb.2017.01364
Baveye, P., Vandevivere, P., Hoyle, B. L., DeLeo, P. C., and de Lozada, D. S. (1998).
Environmental impact and mechanisms of the biological clogging of saturated
soils and aquifer materials. Crit. Rev. Environ. Sci. Technol. 28, 123–191.
doi: 10.1080/10643389891254197
Boddy, L. (1993). Saprotrophic cord-forming fungi: warfare
strategies and other ecological aspects. Mycol. Res. 97, 641–655.
doi: 10.1016/S0953-7562(09)80141-X
Boswell, G. P., and Davidson, F. A. (2012). Modelling hyphal networks. Fungal
Biol. Rev. 26, 30–38. doi: 10.1016/j.fbr.2012.02.002
Boswell, G. P., and Hopkins, S. (2008). Linking hyphal growth to colony
dynamics: spatially explicit models of mycelia. Fungal Ecol. 1, 143–154.
doi: 10.1016/j.funeco.2008.10.003
Cazelles, K., Otten, W., Baveye, P. C., and Falconer, R. E. (2013).
Soil fungal dynamics: parameterisation and sensitivity analysis
of modelled physiological processes, soil architecture and carbon
distribution. Ecol. Modell. 248, 165–173. doi: 10.1016/j.ecolmodel.2012.
08.008
Frontiers in Environmental Science | www.frontiersin.org 9July 2018 | Volume 6 | Article 68
Soufan et al. Fungal Growth in Soil-Like Micromodel
Choudhury, M. J. A., Trevelyan, P. M. J., and Boswell, G. P. (2018). A mathematical
model of nutrient influence on fungal competition. J. Theor. Biol. 438, 9–20.
doi: 10.1016/j.jtbi.2017.11.006
Cromack, K., and Caldwell, B. A. (1992). “The role of fungi in litter decomposition
and nutrient cycling,” in The Fungal Community — Its Organization and Role
in the Ecosystem, eds G. C. Carroll and D. T. Wicklow (New York, NY: Marcel
Dekker), 653–668.
Cruz, B. C., Furrer, J. M., Guo, Y. S., Dougherty, D., Hinestroza, H. F.,
Hernandez, J. S., et al. (2017). Pore-scale water dynamics during drying and the
impacts of structure and surface wettability. Water Resour. Res. 53, 5585–5600.
doi: 10.1002/2016WR019862
Deng, J., Orner, E. P., Chau, F., Anderson, E. M., Kadilak, A. L., Rubinstein, R.
L., et al. (2015). Synergistic effects of soil microstructure and bacterial EPS
on drying rate in emulated soil micromodels. Soil Biol. Biochem. 83, 116–124.
doi: 10.1016/j.soilbio.2014.12.006
Falconer, R. E., Battaia, G., Schmidt, S., Baveye, P., Chenu, C., and Otten,
W. (2015). Microscale heterogeneity explains experimental variability and
non-linearity in soil organic matter mineralisation. PLoS ONE 10:e0123774.
doi: 10.1371/journal.pone.0123774
Falconer, R. E., Houston, A. N., Otten, W., and Baveye, P. C. (2012).
Emergent behavior of soil fungal dynamics: influence of soil architecture
and water distribution. Soil Sci. 177, 111–119. doi: 10.1097/SS.0b013e31824
1133a
Fisher, M. C., Henk, D. A., Briggs, C. J., Brownstein, J. S., Madoff, L. C., McCraw, S.
L., et al. (2012). Emerging fungal threats to animal, plant and ecosystem health.
Nature 484:186. doi: 10.1038/nature10947.
Foster, R. C. (1988). Microenvironments of soil microorganisms. Biol. Fertil. Soils
6, 189–203.
Fricker, M. D., Heaton, L. L. M., Jones, N. S., and Boddy, L. (2017). The mycelium
as a network. Microbiol. Spect. 5. doi: 10.1128/microbiolspec.FUNK-0033-2017
Fries, N. (1973). Effects of volatile organic compounds on the growth
and development of fungi. Trans. Br. Mycol. Soc. 60, 1–21.
doi: 10.1016/S0007-1536(73)80055-5
Gow, N. A., and Gadd, G. M. (1995). The Growing Fungus. London: Chapman and
Hall.
Hanson, K. L., Nicolau, D. V. Jr., Filipponi, L., Wang, L., Lee, A. P., and
Nicolau, D. V. (2006). Fungi use efficient algorithms for the exploration
of microfluidic networks. Small 2, 1212–1220. doi: 10.1002/smll.2006
00105
Harris, K., Crabb, D., Young, I. M., Weaver, H., Gilligan, C. A., Otten,
W., et al. (2002). In situ visualisation of fungi in soil thin sections:
Problems with crystallisation of the fluorochrome FB 28 (CalcofluorM2R)
and improved staining by SCRI Renaissance 2200. Mycol. Res. 106, 293–297.
doi: 10.1017/S0953756202005749
Harris, K., Young, I. M., Gilligan, C. A., Otten, W., and Ritz, K. (2003).
Effect of bulk density on the spatial organisation of the fungus R.solani
in soil. Fems Microbiol. Ecol. 44, 45–56. doi: 10.1111/j.1574-6941.2003.
tb01089.x
Hawksworth, D. L. (2001). The magnitude of fungal diversity: the 1.5 million
species estimate revisited∗ ∗Paper presented at the Asian Mycological Congress
2000 (AMC 2000), incorporating the 2nd Asia-Pacific Mycological Congress
on Biodiversity and Biotechnology, and held at the University of Hong Kong
on 9-13 July 2000. Mycol. Res. 105, 1422–1432. doi: 10.1017/S09537562010
04725
Held, M., Edwards, C., and Nicolau, D. V. (2011). Probing the growth dynamics
of Neurospora crassa with microfluidic structures. Fungal Biol. 115, 493–505.
doi: 10.1016/j.funbio.2011.02.003
Held, M., Lee, A. P., Edwards, C., and Nicolau, D. V. (2010). Microfluidics
structures for probing the dynamic behaviour of filamentous
fungi. Microelectron. Eng. 87, 786–789. doi: 10.1016/j.mee.2009.
11.096
Hopkins, S., and Boswell, G. P. (2012). Mycelial response to spatiotemporal
nutrient heterogeneity: a velocity-jump mathematical model. Fungal Ecol. 5,
124–136. doi: 10.1016/j.funeco.2011.06.006
Jia, X., and Williams, R. A. (2001). A packing algorithm for particles of
arbitrary shapes. Powder Technol. 120, 175–186. doi: 10.1016/S0032-5910(01)
00268-6
Karadimitriou, N. K., and Hassanizadeh, S. M. (2012). A review of micromodels
and their use in two-phase flow studies. Vadose Zone Journal 11.
doi: 10.2136/vzj2011.0072.
Koebernick, N., Daly, K. R., Keyes, S. D., George, T. S., Brown, L. K., Raffan,
A., et al. (2017). High-resolution synchrotron imaging shows that root hairs
influence rhizosphere soil structure formation. New Phytol. 216, 124–135.
doi: 10.1111/nph.14705
Lilje, O., Lilje, E., Marano, A. V., and Gleason, F. H. (2013). Three dimensional
quantification of biological samples using micro-computer aided tomography
(microCT). J. Microbiol. Methods 92, 33–41. doi: 10.1016/j.mimet.2012.
10.006
Miller, R. M., and Jastrow, J. D. (2000). “Mycorrhizal fungi influence soil structure,”
in Arbuscular Mycorrhizas: Physiology and Function, eds Y. Kapulnik and D. D.
Douds (Dordrecht: Springer), 3–18.
Otten, W., and Gilligan, C. A. (1998). Effect of physical conditions on the spatial
and temporal dynamics of the soil-borne fungal pathogen Rhizoctonia solani.
New Phytol. 138, 629–637.
Otten, W., Bailey, D. J., and Gilligan, C. A. (2004a). Empirical evidence
of spatial thresholds to control invasion of fungal parasites and
saprotrophs. New Phytol. 163, 125–132. doi: 10.1111/j.1469-8137.2004.
01086.x
Otten, W., Gilligan, C. A., Watts, C. W., Dexter, A. R., and Hall, D. (1999).
Continuity of air-filled pores and invasion thresholds for a soilborne
fungal plant pathogen, R. solani. Soil Biol. Biochem. 31, 1803–1810.
doi: 10.1016/s0038-0717(99)00099-1
Otten, W., Hall, D., Harris, K., Ritz, K., Young, I. M., and Gilligan,
C. A. (2001). Soil physics, fungal epidemiology and the spread of
R.solani. New Phytol. 151, 459–468. doi: 10.1046/j.0028-646x.2001.
00190.x
Otten, W., Harris, K., Young, I. M., Ritz, K., and Gilligan, C. A. (2004b).
Preferential spread of the pathogenic fungus R. solani through structured
soil. Soil Biol. Biochem. 36, 203–210. doi: 10.1016/j.soilbio.2003.
09.006
Otten, W., Pajor, R., Schmidt, S., Baveye, P. C., Hague, R., and
Falconer, R. E. (2012). Combining X-ray CT and 3D printing
technology to produce microcosms with replicable, complex pore
geometries. Soil Biol. Biochem. 51, 53–55. doi: 10.1016/j.soilbio.2012.
04.008
Pajor, R., Falconer, R., Hapca, S., and Otten, W. (2010). Modelling and
quantifying the effect of heterogeneity in soil physical conditions on
fungal growth. Biogeosciences 7, 3731–3740. doi: 10.5194/bg-7-3731-
2010
Paulitz, T. C., and Schroeder, K. L. (2005). A new method for the
quantification of R.solani and R. oryzae from soil. Plant Dis. 89, 767–772.
doi: 10.1094/PD-89-0767
Riquelme, M., and Bartnicki-Garcia, S. (2004). Key differences between lateral
and apical branching in hyphae of Neurosora crassa.Fungal Genet. Biol. 41,
842–851. doi: 10.1016/j.fgb.2004.04.006
Riquelme, M., Reynaga-Peña, C. G., Gierz, G., and Bartnicki-García, S. (1998).
What determines growth direction in fungal hyphae? Fungal Genet. Biol. 24,
101–109. doi: 10.1006/fgbi.1998.1074
Roman, G. T., and Culbertson, C. T. (2006). Surface engineering
of poly(dimethylsiloxane) microfluidic devices using transition
metal sol–gel chemistry. Langmuir 22, 4445–4451. doi: 10.1021/la
053085w
Rubinstein, R. L., Kadilak, A. L., Cousens, V. C., Gage, D. J., and Shor,
L. M. (2015). Protist-facilitated particle transport using emulated soil
micromodels. Environ. Sci. Technol. 49, 1384–1391. doi: 10.1021/es50
3424z
Sposito, G. (2013). Green water and global food security. Vadose Zone J. 12.
doi: 10.2136/vzj2013.02.0041
Stamets, P. (2005). Mycelium Running: How Mushrooms Can Help Save the World.
Berkeley, CA: Ten Speed Press.
Stanley, C. E., Grossmann, G., Casadevall i Solvas, X., and deMello, A.
J. (2016). Soil-on-a-Chip: microfluidic platforms for environmental
organismal studies. Lab Chip 16, 228–241. doi: 10.1039/C5LC0
1285F.
Frontiers in Environmental Science | www.frontiersin.org 10 July 2018 | Volume 6 | Article 68
Soufan et al. Fungal Growth in Soil-Like Micromodel
Vidal-Diez de Ulzurrun, G., Baetens, J. M., Van den Bulcke, J., and De
Baets, B. (2017). Modelling three-dimensional fungal growth in response to
environmental stimuli. J. Theor. Biol. 414, 35–49. doi: 10.1016/j.jtbi.2016.11.020
Vidal-Diez de Ulzurrun, G., Baetens, J. M., Van den Bulcke, J., Lopez-
Molina, C., De Windt, I., and De Baets, B. (2015). Automated image-based
analysis of spatio-temporal fungal dynamics. Fungal Genet. Biol. 84, 12–25.
doi: 10.1016/j.fgb.2015.09.004
Watts, H. J., Véry, A. A., Perera, T. H., Davies, J. M., and Gow, N.
A. (1998). Thigmotropism and stretch-activated channels in the
pathogenic fungus Candida albicans.Microbiology 144 (Pt 3), 689–695.
doi: 10.1099/00221287-144-3-689
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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