Mutualism Within a Simulated Microgravity Environment - Piriformospora indica Promotes the Growth of Medicago truncatula

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 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 30°C 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. 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 haemocytometer measured the spore concentration to be 3.3 × 10⁵/mL. The spore solution was diluted with 3.3 mL of the control inoculum to generate a final working stock of 1.0 ×10⁵ spores per mL as the fungal inoculant.

Sterile M. truncatula seeds were soaked in 15 μL of the fungal inoculum (1.0 × 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.

All seeds were aseptically transferred to 10 mL of MS media, within individual sterile Petri dishes 15 mm × 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 experimentation: relative humidity 50%, temperature 22°, 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.

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 × 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

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).

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 100°C 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).

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 −80°C 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).

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).

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 intracellulary, 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 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

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 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

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.

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 (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 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 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 lipochitioligosaccharides (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.

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 × 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.

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 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

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 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 nonpathogenic 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