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Research Article
Mutualism Within a Simulated Microgravity Environment - Piriformospora
indica Promotes the Growth of Medicago truncatula
Martin W. Hayes1, Gary W. Stutte1,2, Michelle McKeon-Bennett1, and Patrick G. Murray1
1Shannon Applied Biotechnology Centre, Department of Applied Science, Limerick Institute of
Technology, Limerick, Ireland; 2Space Life Science Lab, Kennedy Space Center, FL
ABSTRACT
The endophytic fungus, Piriformospora
indica, developed a subepidermal infection
within Medicago truncatula at 1 g and at
simulated microgravity over a period of 15 days,
resulting in intracellular colonization of mature
host tissue. At 1 g, P. indica inoculation
affected the growth and morphology of M.
truncatula, predominantly roots. Inoculated M.
truncatula had a significantly greater number of
roots (102%), total root length (88%), and dry
root weight (25%) than non-inoculated plants.
Effects on shoot morphology of P. indica
inoculated M. truncatula included longer (31%)
and heavier (30%) shoots, along with increased
leaf surface area (98%). P. indica retained the
ability to promote the growth of M. truncatula
under simulated microgravity conditions upon
two dimensional clinostatic rotation, signi-
ficantly increasing root number by 51% and root
length by 48%. These physiological and
morphological changes may mitigate biotic and
abiotic stresses that would otherwise limit crop
productivity.
INTRODUCTION
Leguminous plants (beans, peas, alfalfa,
vetch, etc.) are important to humans and natural
and agricultural ecosystems, as they account for
27% of primary crop production and are
cultivated on 12% to 15% of the Earth’s arable
soil (Graham and Vance, 2003). M. truncatula
(Barrel medic) is extensively used as a model
species of the legume family due to its relatively
small genome size, ease of growth, and ability
to induce transformations (Trinh et al., 1998).
Legumes develop a symbiotic relationship with
rhizobia bacteria, which enables the fixation of
atmospheric nitrogen, replenishing depleted
soils for future cultivations, and achieving
sustainability under environmental conditions
that would otherwise limit crop productivity
(Graham and Vance, 2003).
Plant/microbe systems will play a major role
in long-term bioregenerative life support
systems on board spacecraft and long duration
space missions by providing fresh food and
recycling air, water, and waste products (Ferl et
al., 2002). Establishing successful plant/microbe
mutualisms would improve and increase plant
growth, stress tolerance under microgravity, and
increase flight crew independence by reducing
re-supply costs during long-term missions. P.
indica, a root endophytic fungus isolated from
the soils of the Indian Thar Desert, Rajasthan
(Varma et al., 1999; Verma et al., 1998), has
been shown to induce host growth promotion,
increase seed yield, and increase resistance to
abiotic and biotic stresses (Bagde et al., 2011;
Deshmukh et al., 2006; Peskan-Berghofer et al.,
2004; Varma et al., 1999; Waller et al., 2005),
Key words: Fungus; Legume; Endophyte;
Infection; Microgravity; Growth Promotion
Correspondence to: Martin W. Hayes
Shannon Applied Biotechnology Centre
Department of Applied Science
Limerick Institute of Technology
Limerick, Ireland
Telephone: +35361293503
E-mail: martin.hayes01@gmail.com
Gravitational and Space Research Volume 2 (2) Dec 2014 221
Hayes et al. – Mutualism Within a Simulated Microgravity Environment
thus displaying its potential as a plant growth-
promoting and stress-alleviating bioinoculant
(Sarma et al., 2011). P. indica displays a lack of
host-specificity by establishing symbiotic
relationships with a wide range of plant hosts,
including monocots and dicots (Deshmukh et
al., 2006; Peskan-Berghofer et al., 2004; Varma
et al., 1999). This seemingly non-specific host
recognition directly contrasts with the highly
specific symbiosis between Sinorhizobium
meliloti and specific members of Leguminosae
(Jones et al., 2007). The non-specific root
colonization strategies of P. indica have been
described for barley (Deshmukh et al., 2006)
and Arabidopsis (Peskan-Berghofer et al.,
2004). Both species display similar infection
patterns, resulting in intracellular colonization
of the host’s mature cortical cells (Deshmukh et
al., 2006; Peskan-Berghofer et al., 2004; Schäfer
and Kogel, 2009). This intracellular root
colonization is associated with host cell death
(Deshmukh et al., 2006; Jacobs et al., 2011),
which is intriguing as detrimental effects do not
arise with infection.
Understanding the impacts of reduced-
gravity environments on symbiotic plant/
microbe associations is critical before inclusion
in a functioning regenerative life support
system. As plant growth and metabolism are
altered in microgravity, leading to physiological
changes which may affect host defenses, plants
become more susceptible to infection by
pathogens and organisms that normally do not
cause disease (Bishop et al., 1997; Tripathy et
al., 1996). The effect of the unique space
environment on cell-to-cell communications
and biochemical interaction between
organisms – like those involved in symbiotic
associations – can result in an alternative
ecological balance that redefines the
relationship between host and commensal, or
between host and opportunistic pathogen
(Stutte and Roberts, 2012). The ability of P.
indica to override plant defense
mechanisms during infection (Jacobs et al.,
2011) is thus of concern during microgravity
simulation, as there is evidence that the
virulence of plant pathogens increases under
such conditions (Ryba-White et al., 2001). A
previous study by Bishop et al. (1997) showed
that a non-pathogenic fungal
species (Neophodium) can become
pathogenic during spaceflight; however, a
more recent study carried out on the
well-defined mutualistic symbiosis between
M. truncatula and root nodule forming
bacteria S. meliloti during spaceflight
suggested that both organisms cultivated
in microgravity still have the potential to
establish a symbiotic interaction (Stutte and
Roberts, 2012).
A better understanding of the limitations of
these mutualistic associations and their adaption
to this unique environment is required, since
such investigations may also uncover insights
into how microbial populations may be
managed in terrestrial settings to enhance crop
production. Clinorotation is used on the ground
for investigations as an analog for microgravity
on biological systems (Albrecht-Buehler, 1992).
This vertical rotation through the gravity vector
overwhelms the organisms with an orientational
stress, similar to that perceived in
microgravity (Albrecht-Buehler, 1992;
Dedolph and Dipert, (1971).
The aims of the present investigation were:
1) to test the hypothesis that M. truncatula
growth would increase through a mutualistic
association with P. indica, 2) to establish the
timeline for infection and colonization strategy
within the M. truncatula roots, and 3) to test
whether this mutualistic association develops
under simulated microgravity conditions.
MATERIALS AND METHODS
M. truncatula Seed Scarification and
Sterilization
Seeds of M. truncatula Gaertn cv. Jemelong
A17 (gift from Carroll Vance, USDA-ARS, St.
Paul, MN) were extracted from seed pods
(typically 6-8 seeds/pod) and seed coats were
gently, mechanically scarified on fine-grade
(150 grit) sandpaper until there were visible
signs of seed coat abrasion. Seeds were then
sterilized in a 33% bleach solution for three
minutes and rinsed with several washes of
sterile H2O (Garcia et al., 2006).
P. indica Axenic Cultivation and Inoculum
Preparation
P. indica was obtained from the DSMZ
(German Collection of Microorganisms and Cell
Cultures, Braunschweig, Germany) and initially
grown from spores from freezer stocks on 60
mm Petri dishes containing MS media
(Murashige and Skoog, 1962) within a 30oC
incubator for 6 days. P. indica was then
harvested by adding 8 mL of sterile, deionized
(DI) H2O to Petri dishes, and gently agitating
and carefully removing hyphae and spores from
the agar surface with a sterile plate spreader.
22 Gravitational and Space Research Volume 2 (2) Dec 2014
Hayes et al. – Mutualism Within a Simulated Microgravity Environment
The solution was transferred to a 50 mL sterile
centrifuge tube for use as an inoculant. A similar
process was carried out on non-inoculated MS
plates for use as treatment for non-inoculated
control plants. A spore count via haemo-
cytometer measured the spore concentration to
be 3.3 x 105/mL. The spore solution was diluted
with 3.3 mL of the control inoculum to generate
a final working stock of 1.0 x105 spores per mL
as the fungal inoculant.
M. truncatula Seed Inoculation
Sterile M. truncatula seeds were soaked in
15 μL of the fungal inoculum (1.0 x 105/mL or
1500 spores), or 15 μL of control treatment
solution for five hours within the laminar flow
hood for inoculated and non-inoculated plants,
respectively.
Growth Conditions
All seeds were aseptically transferred to 10
mL of MS media, within individual sterile Petri
dishes 15 mm x 60 mm (i.e., one seed per dish).
The Petri dishes were sealed with Parafilm®
and punctured with pin holes to allow additional
gaseous exchange. The plates were then set up
on a 2-D clinostat (described in next section)
and transferred to a controlled environment
chamber (CEC) (EGC model no. M48,
Environmental Growth Chamber, Chagrin Falls,
Ohio) for 15 days. The CEC was set to the
following parameters throughout experiment-
ation: relative humidity 50%, temperature 22oC,
CO2 ambient, 16 hr light/8 hr dark photoperiod
at 200 µmol m2 s-1 photosynthetically active
radiation (PAR), under GE F96T8/SPX41/HO
fluorescent lamps.
Two-Dimensional (2-D) Clinostat Setup
Each individual Petri dish (containing one
seed) was attached to a 15 cm (diameter) plastic
disk. Eight Petri dishes in total (four control
and four P. indica inoculated seeds) fit onto
each disk and their formation lined the outer
region of the disks (Figure 1). Four orientational
treatments were prepared to complete the 2-D
clinostat setup–vertical static (VS), vertical
rotating (VR), horizontal static (HS), and
horizontal rotating (HR) (Figure 1). Both of the
rotating treatments were attached to an 8 rpm
motor spinning at a centrifugal force of 5.3 x 10-
3g throughout the entire experiment, whereas
both static treatments remained fixed. The VR
treatment simulates the microgravity stress due
to the constant reorientation of cellular gravity
perception as the disk spins (Perbal and Driss-
Ecole, 2003). The other three treatments (VS,
HS, HR) were set up as controls to account for
the effect of centrifugal force/rotation and
orientation on the interaction separately.
Figure 1. 2-D clinostat set up within the ECG, M. truncatula seeds are placed in the center of each Petri
dish on MS media. Setup from a-d are horizontal static (HS), horizontal rotating (HR), vertical static
(VS), and vertical rotating (VR), respectively. The VR (d) setup is an analog to simulate microgravity
stress.
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Hayes et al. – Mutualism Within a Simulated Microgravity Environment
Biometric Image Analysis
All plant length measurements were
determined from digital images using Image J
(Abràmoff et al., 2004) for image analysis.
Scales were set using digital calibration
(Abràmoff et al., 2004).
P. indica Staining and Host Colonization
Analysis Procedure
Over a period of days (day 4, 11, and 13)
post-inoculation, individual control and treated
samples were removed from CEC and their root
systems were analyzed for P. indica infection. A
simple ink-vinegar staining process was carried
out (Pitet et al., 2009) on the root samples,
followed by analysis under a light microscope
(Nikon Labophot). Briefly, the protocol required
clearing the sample tissue of phenolics and
unwanted pigments by heating in 10% KOH at
100oC and rinsing with water. The tissue was
then fixed with 5% acetic acid, followed by
additional rinsing with water. Each sample was
then stained for 2 hours at room temperature in a
5% acetic acid/ 5% Schaeffer black ink solution.
Lastly the ink/vinegar stain was decanted and
the root tissue was de-stained by several washes
with an acidified glycerol solution. The roots
were then viewed under a light microscope and
their images recorded with a handheld camera
(Nikon Coolpix S3100).
Chlorophyll Pigment Extraction and Analysis
Total chlorophyll (a, b) and carotenoid
concentrations per dry weight of sample were
measured from dimethyl sulfoxide (DMSO)
extracts (Lichtenthaler and Wellburn, 1983;
Wellburn, 1994). Harvested shoot samples were
frozen at -80oC and freeze dried. Samples
pigments were then extracted in 4 mL DMSO
and its UV-visible absorbance read from a
Biotek Gen 5 plate reader over a spectra range
of 300-700 nm and at individual spectra –
665, 649, and 480 nm. Chlorophyll a and
b concentrations were then determined from
three replicate mean absorbance values
using the Wellburn equation described
for DMSO extractions (Wellburn, 1994).
Statistical Analysis
The experiment was carried out with 4
repeats (4 un-inoculated and 4 P. indica
inoculated) for each orientation, and was
replicated four times. All of the collected data
was combined together, giving a total of 16 un-
inoculated and 16 P. indica inoculated samples
for each clinostatic setup described previously.
Initially, to identify outliers, all data obtained
was subjected to an interquartile outlier test
using Excel (Microsoft Office Excel 2010),
whereby a number of outliers were identified
and removed from the data set. The remaining
(post outlier removal) sample size (n) is
displayed on respected growth characteristic
figures. Therefore, based on the validity of the
underlying assumptions (normality and
homoscedasticity (both assessed using Minitab,
Version 17)), the appropriate tool for statistical
analysis was chosen (i.e., parametric, non-
parametric tests) to measure the effect of P.
indica inoculation on M. truncatula growth,
clinorotation on M. truncatula growth, and
interactions between P. indica inoculation and
clinorotation. Data, which was found to meet the
requirements for analysis of variance
(ANOVA), was subjected to a one-way
ANOVA with a critical difference of P <0.05,
whereas non-normal and data of unequal
variance was subjected to the Kruskal-Wallis
test, also with a critical difference of P <0.05
using Minitab (Version 17).
RESULTS AND DISCUSSION
M. truncatula Root Colonization Strategy by
P. indica Under 1 g and Simulated
Microgravity Stress
To date, P. indica has not been reported to
display distinct host-specificity, as it will
colonize the root systems of many plant species,
including monocots and dicots (Peskan-
Berghofer et al., 2004; Varma et al., 1999;
Waller et al., 2005), both inter- and intra-
cellulary, with asymptomatic results. The
infection strategy by P. indica during host
colonization has been described from its
association with barley and Arabidopsis
(Deshmukh et al., 2006; Jacobs et al., 2011;
Peskan-Berghofer et al., 2004; Schäfer and
Kogel, 2009). In both species, P. indica initially
colonizes root rhizodermal cells, followed by
cortical tissue and intracellular colonization of
cortex cell layers.
We report, to the best of our knowledge, the
previously unrecorded infection strategy of P.
indica within M. truncatula. P. indica and M.
truncatula develop a mutualistic relationship,
through fungal colonization of root tissue, which
results in the intracellular establishment of P.
indica within the maturation zone of the plant
24 Gravitational and Space Research Volume 2 (2) Dec 2014
Hayes et al. – Mutualism Within a Simulated Microgravity Environment
root. P. indica’s propagation is not exclusive to
host cells–it also grows externally on agar and
root surface. This infection strategy is consistent
with the infection strategies reported for barley
(Deshmukh et al., 2006) and Arabidopsis
(Peskan-Berghofer et al., 2004). The infection
process is initiated by an extracellular
establishment of P. indica along the topography
of the outer epidermal layer of the root by days
after inoculation (DAI) 4 (Figure 2A). By DAI
11, subepidermal fungal colonization is
observed within the cortical tissue (Figure 2B),
along with intracellular sporulation (Figure 2C).
Figure 2C shows P. indica to have intracellulary
colonized the more mature regions of the plant
root tissue, completely occupying its host’s cells
with chlamydospores. By DAI 13, heavy fungal
colonization/sporulation was observed within
the maturation zone of M. truncatula roots
(Figure 2D).
Figure 2. Infection strategy of P. indica within M. truncatula at 1 g. The infection was monitored with the
use of an ink-vinegar stain, which adheres to chitin within P. indica appearing as blue. Root sections are
marked as follows: epidermis (e), cortex (c), and vascular bundle (vb). (A) P. indica grows along the M.
truncatula root topography during an initial extracellular establishment by DAI 4. (B) By DAI 11,
subepidermal P. indica root colonization is witnessed within the inner cortical cells. (C) DAI 11, P. indica
colonizes the mature cortex root tissue intracellularly, completely occupying its host cells with
chlamydospores. (D) At DAI 13, heavy fungal colonization is seen within the maturation zone of the M.
truncatula root. Scale bars on all images represent 50 μm in length.
These findings are consistent with that
reported from P. indica’s association with
barley and Arabidopsis (Deshmukh et al., 2006;
Jacobs et al., 2011), whereby P. indica
sporulation increased within mature root tissue
and was associated with the occurrence of host
cell death. This suggests that P. indica either
interferes with programmed cell death when
forming a mutualism within its host, or actively
senses the process and invades targeted dead
cells. This recently reported colonization
strategy of the endophyte is intriguing, as it
achieves this high volume of root cell death
associated infection without causing detrimental
effects to its host (Deshmukh et al., 2006;
Jacobs et al., 2011). The same strategy is
witnessed within M. truncatula, accompanied by
an increase in M. truncatula growth (Figure 4).
Upon investigating whether or not P. indica
was capable of infecting M. truncatula under
Gravitational and Space Research Volume 2 (2) Dec 2014 25
Hayes et al. – Mutualism Within a Simulated Microgravity Environment
simulated microgravity, it was found that P.
indica retained the ability to form an
endosymbiotic association (Figure 3), with
intracellular sporulation being observed within
mature host tissue by DAI 12.
Figure 3. Infection strategy of P. indica within M. truncatula at simulated microgravity. Root sections are
marked as follows: epidermis (e), cortex (c), and vascular bundle (vb). (A) By DAI 12, subepidermal P.
indica root colonization is witnessed within the inner cortical cells of M. trunatula. (B) Inner cortical cells
of M. truncatula are intracellularly colonized by P. indica chlamydospores by DAI 12. Scale bars on all
images represent 50 μm in length.
Very little literature exists relating to the
microbial colonization of plants within
microgravity environments. An experiment in
the 1970s had a similar clinostatic setup,
investigated the effects of gravity compensation
on crown gall formation, and found that under
simulated microgravity conditions the infection
occurred (Kleinschuster et al., 1975). Along
with infection, the crown gall developed tumors
that were said to be larger on gravity-
compensated samples than those grown at 1 g
(Kleinschuster et al., 1975). Bishop et al. (1997)
previously reported the presence of a strain of
Neotyphodium (a known endophyte) within
space-grown wheat. The fungal strain had
colonized the wheat samples; however, in
contrast to our observations, its colonization
strategy could not be reported. P. indica’s
colonization strategy involves an override of
host defenses (Jacobs et al., 2011) and is
associated with host cell death (Deshmukh et al.,
2006), and is thus a concern during microgravity
simulation (Ryba-White et al., 2001).
Interestingly, P. indica is closely associated
with an endosymbiotic bacteria (Rhizobium
radiobacter), which is known to also cause
crown gall disease within hosts (Sharma et al.,
2008). This is a further concern under
microgravity stress, as crown gall tumors are
reported to enlarge under gravity compensation
(Kleinschuster et al., 1975). In this study,
however, colonized M. truncatula appeared
healthy post-clinorotation.
Effect of P. indica Inoculation on M.
truncatula Growth and Morphology at 1 g
The mechanism by which P. indica elicits
its beneficial effect to its host is not well
defined. Studies have linked various diffusible
factors, such as extracellular phytohormone
production by P. indica with the increase in
plant growth (Sirrenberg et al., 2007).
Vadassery et al. (2008) found that P. indica
produces low levels of auxin and high levels of
cytokinin, and colonized Arabidopsis plants
contained higher levels of cytokinin than un-
colonized.
This study showed a positive effect of P.
indica root colonization on the growth and
morphology of M. truncatula. M. truncatula
plants treated with P. indica appeared visibly
healthy, while also displaying an increase in
shoot and root length. Growth promotion in the
aerial organs consisted of a 31% increase in
total stem length (control 83 mm vs. treated 109
mm), 30% increase in shoot dry weight (control
15 mg vs. treated 20 mg), and 98% increase in
total leaf surface area (control 126 mm2 vs.
treated 250 mm2) (Figure 4 D-F). The
subterranean organs displayed the highest level
of growth promotion, which consisted of a
102% increase in root number (control 23 vs.
treated 47), 88% increase in total root length
26 Gravitational and Space Research Volume 2 (2) Dec 2014
Hayes et al. – Mutualism Within a Simulated Microgravity Environment
(control 322 mm vs. treated 606 mm), and a
25% increase in dry root weight (control 7 mg
vs. treated 9 mg) (Figure 4 A-C). This increased
root growth promotion may enhance stress
tolerance, such as within drought related areas
where water is held deeper within the soil, as
well as produce a greater yield of legume crop
for forage. Similar growth effects have been
reported from P. indica’s association with
micropropagated Feronia limonia (L.) Swingle
(Vyas et al., 2008). Also, the treatment with P.
indica culture filtrate alone has been reported to
induce and stimulate the same growth
promoting response (Bagde et al., 2011).
Figure 4. The effect of P. indica inoculation on M. truncatula growth characteristics after 15 days
inoculation at 1 g (HS). All displayed data was found to be significant (P < 0.05), respective P values are
included on each graph. Sample size is displayed as n.
The morphological differences witnessed in
this investigation could be a result of hormone
alterations, such as auxin (Davies, 2004).
Exogenous auxin produced by rhizobacteria
tends to promote root growth and branching
(Shahab et al., 2009), and is consistent with
Sirrenberg et al., (2007) findings that auxin
plays a role in P. indica’s growth-promoting
effects. In contrast, Sirrenberg et al., (2007)
reported that auxin levels were not up-regulated
Gravitational and Space Research Volume 2 (2) Dec 2014 27
Hayes et al. – Mutualism Within a Simulated Microgravity Environment
within treated Arabidopsis plants – while
cytokinin levels were – and concluded that
cytokinins were required for P. indica-induced
cell division and growth promotion (Vadassery
et al., 2008). Another rhizospheric diffusible
factor capable of eliciting such a growth-
promoting response could be lipochitio-
ligosaccharides (LCO) (Maillet et al., 2011).
Rhizobacteria and mycorrhizal fungi, which
produce LCOs during host infection, have been
shown to induce LCO-dependent lateral root
formation (Maillet et al., 2011; Olah et al.,
2005), similar to that induced by P. indica. LCO
exudates alone are also capable of promoting the
same induced lateral root formation response
(Olah et al., 2005), similar to P. indica fungal
exudate (Bagde et al., 2011). Interestingly
enough, P. indica colonization is associated
with an up-regulation of calcium levels within
host tissue (Vadassery et al., 2009), which
happens to be essential for nodulation within M.
truncatula and linked to the LCO signal
transduction pathway (Olah et al., 2005).
However, to our knowledge, no investigation
into the role of LCOs as a diffusible factor
responsible for P. indica-induced growth
promotion has been carried out.
The current investigation suggests that M.
truncatula’s association with P. indica may also
result in alterations to host physiology, as
increased levels of host photosynthetic tissue
were found with P. indica inoculation. Treated
plants showed a 25% increase in total
chlorophyll a and b levels, in comparison to
non-treated control samples; however, this
finding was not found to be significant (data not
shown). Previous studies on P. indica’s effect
on Arabidopsis under drought-stress also
showed higher chlorophyll content and
increased photosynthetic efficiency with P.
indica inoculation (Sherameti et al., 2008).
Similar to arbuscular mycorrhizal fungi (AMF)
symbiosis (Ceccarelli et al., 2010; Yano-Melo et
al., 1999), such increases in leaf perimeter
length, photosynthetic potential, and chlorophyll
may result in increased carbon assimilation in P.
indica-colonized plants, leading to faster
development and higher biomass production.
Effect of Clinorotation on the Growth of M.
truncatula
The effect of clinorotation on M. truncatula
was investigated to determine if the rate
of rotation and or the orientation of the 2-D
set up had a significant effect on plant
development, and ultimately if analog
microgravity conditions had an effect on the
growth of M. truncatula. The HS (1 g)
treatment was used as a control whereby,
HR (1 g), VS (1 g), and VR
(microgravity) were all used as comparisons.
Analysis of M. truncatula plant growth on all
four 2-D clinostatic setups yielded significant
variations among HS and VS
treatments (orientation change) at 1 g, and
also between HS and VR treatments (from 1 g
to microgravity) (Figure 5 A-F, HS vs. VS).
Decreases in M. truncatula growth on the VS
in comparison to the HS treatment (orientation
change) may have been associated with a
lower availability of nutrients to root zones,
i.e., plants growing on the horizontal axis
would fully penetrate into media, whereas
those on the vertical axis may grow along
the media. Nevertheless, the VR axis which
simulates microgravity was found to be the
strongest inhibitor of M. truncatula growth
in relation to root growth, with
significant differences recorded for root count
(-42%, HS 36 vs. VR 25), root length (-53%,
HS 458 mm vs. VR 299 mm), and dry root
weight (-70%, HS 8 mg vs. VR 5 mg) (Figure 5
A-C, HS vs. VR). M. truncatula total stem
length (-17%, HS 97 mm vs. VR 83 mm),
shoot dry weight (-39%, HS 18 mg vs. VR 13
mg), and total leaf surface area (-46%, HS
180 mm2 vs. VR 124 mm2) were also found
to be strongly inhibited under analog
microgravity conditions (Figure 5 D-F, HS vs.
VR). The rate of rotation (5.3 x 10-3g) was
found to not have a significant effect on
M. truncatula development on the horizontal
axis (1 g), as HS samples did not alter
significantly to HR (Figure 5 A-F, HS vs. HR
A-F). Taking all observations into account
suggests that the orientational change
from horizontal to vertical elicited an
inhibitory effect on M. truncatula
development, while rotation displayed a
larger and significant inhibitory effect on
the vertical axis only, showing a M.
truncatula growth inhibitory effect under
analog microgravity conditions.
Effect of P. indica’s Association with M.
truncatula on the Morphological and
Physiological Development of M. truncatula
Under 2-D Clinorotation
Multiple studies have shown P.
indica’s ability to mitigate biotic and abiotic
stresses that would otherwise inhibit plant
growth, with P. indica inoculation inducing
salt tolerance, drought tolerance, pathogen
28 Gravitational and Space Research Volume 2 (2) Dec 2014
Hayes et al. – Mutualism Within a Simulated Microgravity Environment
resistance, transfer shock tolerance, and
resistance to low temperatures (Murphy et al.,
2014; Murugan, 2011; Sherameti et al., 2008;
Varma et al., 1999; Waller et al., 2005; Zarea
et al., 2012). To our knowledge, this is the first
instance whereby P. indica’s association with
a host plant grown under simulated
microgravitational stress has been reported.
Figure 5. The effect of clinorotation on the development of M. truncatula. HR, VS, and VR (microgravity)
growth measurements are all compared to HS (1 g). P values displayed above each column describe the
level of significant difference between the respective columns and the HS column. P values < 0.05 were
accepted as significant, whereas non-significance is depicted as NS. Sample size is represented as n.
Inoculation with P. indica had a significant
effect on growth and morphology of M.
truncatula under analog microgravity
conditions. The most influential root count
promotion was recorded on the HS (1 g)
treatment (102%), while the simulated
microgravity (VR)-treated associates resulted in
a 51% promotion (Figure 6 A). The same was
found for total root length, with HS (1 g)
displaying an 88% increase in growth, and
Gravitational and Space Research Volume 2 (2) Dec 2014 29
Hayes et al. – Mutualism Within a Simulated Microgravity Environment
simulated microgravity (VR) samples displaying
a lesser, but still substantial 48% increase in
growth (Figure 6 B), all of which shows that
analog microgravity conditions have a ‘nulling’
effect on P. indica induced M. truncatula root
growth promotion. In addition, P. indica
inoculated plants grown on the simulated
microgravity setup showed similar growth
stimulatory effects as that seen on the 1g plane,
such as increased root weight, shoot weight, and
total leaf surface area. These findings, however,
were found to not be significant (data not
shown).
Bishop et al. (1997) reported that a non-
pathogenic strain of Neotyphodium (endophyte)
became pathogenic under spaceflight, leading to
detrimental effects on wheat growth. Here we
report that although mitigated, P. indica still
retains the ability to induce the root growth
promotion of M. truncatula under analog
microgravity conditions (Figure 6 A-B). This is
an interesting observation as it suggests that
gravity’s influence upon M. truncatula and or P.
indica may play a role within the mutualistic
association.
Figure 6. Root growth stimulatory effect of P. indica on the growth of M. truncatula at 1 g and simulated
microgravity. P values shown represent the level of significant difference between non-inoculated and P.
indica inoculated M. truncatula on HS (1 g) and VR (microgravity) growth conditions. Sample size is
represented as n.
CONCLUSIONS
These findings demonstrate that the legume
M. truncatula is colonized by the endophytic
fungus P. indica, and that this infection results
in the promotion of root and shoot growth of the
host. The interaction represents an ideal
combination to study the effect of P. indica on
leguminous plants. The infection strategy is
similar to that reported for barley and
Arabidopsis, suggesting a common mode of
infection between monocots and dicots.
Colonization by P. indica was also observed
under analog microgravity conditions, and the
infection was similar to that of 1 g. P. indica
infection also resulted in significant increase in
growth of M. truncatula under analog
microgravity conditions, albeit these were less
than observed for 1 g. With regard to P.
indica’s effect on host morphology, our
observations suggest that P. indica LCOs (a
previously un-associated diffusible factor) may
be contributing to root morphological
alterations.
These results demonstrate that establishing
plant/microbe symbiosis has potential to
enhance the growth of plants under analog
microgravity conditions, under mission-relative
environmental conditions. Additional research
is needed to understand how the analog
microgravity environment mitigates the effect of
P. indica on M. truncatula growth.
ACKNOWLEDGEMENTS
We thank Dr. Carroll Vance (USDA-ARS,
St. Paul, MN) for providing the seeds of M.
truncatula Gaertn cv. Jemelong A17. We also
thank Dr. Michael Roberts (CSS-Dynamac) and
the sustainable systems research group (Space
Life Science Lab, Kennedy Space Center, FL),
for their support and training during the
Limerick Institute of Technology’s life science
research programme of Martin Hayes. We
30 Gravitational and Space Research Volume 2 (2) Dec 2014
Hayes et al. – Mutualism Within a Simulated Microgravity Environment
acknowledge the help of Thomas O’Brien
(Enterprise Research Centre, University of
Limerick, Ireland) for carrying out the statistical
analysis on the recorded growth data. The
authors also acknowledge Marie Curie IIF
programme for support of Professor Gary Stutte.
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