Symbiosis

, Volume 60, Issue 3, pp 123–132

Hevea brasiliensis and Urtica dioica impact the in vitro mycorrhization of neighbouring Medicago truncatula seedlings

Authors

  • Tiffany Sosa-Rodriguez
    • Université Catholique de Louvain, Earth and Life Institute, Applied Microbiology, Mycology
  • Stéphane Declerck
    • Université Catholique de Louvain, Earth and Life Institute, Applied Microbiology, Mycology
  • Francoise Granet
    • Manufacture Française des Pneumatiques MICHELIN Place des Carmes-Déchaux
  • Sylvie Gaurel
    • Manufacture Française des Pneumatiques MICHELIN Place des Carmes-Déchaux
  • Els J. M. Van Damme
    • Department of Molecular BiotechnologyGhent University
    • Université Catholique de Louvain, Earth and Life Institute, Applied Microbiology, Mycology
Article

DOI: 10.1007/s13199-013-0248-9

Cite this article as:
Sosa-Rodriguez, T., Declerck, S., Granet, F. et al. Symbiosis (2013) 60: 123. doi:10.1007/s13199-013-0248-9

Abstract

Hevea brasiliensis is a mycotrophic tree for which root colonization by arbuscular mycorrhizal fungi (AMF) under in vitro or pot culture conditions can take several weeks. The reason for this slow colonization is still unknown, but the exudation of antifungal compounds such as hevein by the roots may be one of the causes. Here, the root colonization of Medicago truncatula, a highly mycotrophic plant, was assessed after 12 days of growth in the extraradical mycelium network of the AMF Rhizophagus irregularis in close vicinity of H. brasiliensis plantlets or Urtica dioica seedlings (also known to synthesize antifungal compounds of the hevein family). We hypothesized that a negative impact on the root colonization of a M. truncatula seedling developing close to H. brasiliensis and U. dioica may give indirect proofs for the exudation of inhibitory molecules. The percentages of total root colonization of M. truncatula were 30.1 % lower in the presence of H. brasiliensis than in the control plants, and 29.1 % lower in presence of U. dioica. The abundance of arbuscules in the roots of M. truncatula was also lower in plants grown in presence of H. brasiliensis plantlets than in the control plants. Similarly, the succinate dehydrogenase and the phosphatase activities measured in the extraradical mycelium of R. irregularis were significantly lower in the presence of both plants, compared with the controls. No root colonization was observed in H. brasiliensis and U. dioica within the time-frame of the experiments. The low root colonization of M. truncatula when grown in the presence of rubber or stinging nettle suggested the exudation of diffusible molecules which could also explain the delayed root colonization of H. brasiliensis and the absence of colonization of U. dioica.

Keywords

Arbuscular mycorrhizal fungiHevea brasiliensisUrtica dioicaMedicago truncatulaRhizophagus irregularisExtraradical mycelium network

1 Introduction

Arbuscular mycorrhizal fungi (AMF) are symbiotic soil microorganisms that colonize the roots of nearly 70 % of vascular plants (Brundrett 2009). In the roots, these fungi develop intercellular and intracellular hyphae (Bonfante and Genre 2010) that differentiate within cells into arbuscules, the typical structures involved in the bi-directional transport of carbohydrates from the plant to the fungus, and minerals (e.g. phosphorus or nitrogen) from the fungus to the plant. This active exchange is essential to the development of the obligate fungal symbiont and the improvement of plant growth. AMF also favor plant resistance to abiotic and biotic constraints and influence plant diversity and ecosystem productivity (van der Heijden et al. 1998; Smith and Read 2008).

Although symbiosis with AMF is reported in the vast majority of land plants, different families and species have been described as non-host (Brundrett 2002, 2009). Several factors have been suggested to explain the absence of colonization: (1) the plant is unable to produce essential signals for triggering the different steps leading to root colonization (Giovannetti and Sbrana 1998); (2) the plant cannot recognize fungal signals sent during the pre-symbiotic stage (Vierheilig and Bago 2005; Parniske 2008) (3) the plant has barriers against the mycorrhizal colonization that are intrinsic to the roots (i.e. related to physiological characteristics of the root cortex or epidermis; Fontenla et al. 1999); (4) the plant produces and exudates inhibitory or antifungal compounds (Vierheilig et al. 1996; Vierheilig and Bago 2005; Stinson et al. 2006) that impair the fungal growth.

A number of studies have reported on the impact of plant inhibitory signals and chemical compounds on different steps of the AMF development (from spore germination to root colonization and arbuscules formation) under in vitro, (Vierheilig and Bago 2005), greenhouse (Yun and Choi 2002; Stinson et al. 2006) and field (Barto et al. 2011) conditions. Interestingly, root exudates or extracts from non-host plants, such as the stinging nettle (Urtica dioica L.) have also been shown to influence the root colonization of neighbouring mycotrophic plants (Vierheilig et al. 1995, 1996, 2003; Fontenla et al. 1999).

U. dioica has been classified as a non-host plant, since colonization of its roots by AMF has never been observed (Renker et al. 2005; Fontenla et al. 1999; Vierheilig et al. 1996; Helgason et al. 2007; Bainard et al. 2011). U. dioica plants produce and accumulate a monomeric 8 kDa protein called Urtica dioica agglutinin (UDA) in their roots and rhizomes (Peumans et al. 1984; Broekaert et al. 1989), which was shown to inhibit the mycelial growth of several saprophytic and pathogenic fungi such as Botrytis cinerea, Septoria nodorum, Trichoderma hamatum and Phycomyces blakesleeanus (Van Parijs et al. 1991). The antifungal properties of UDA have been attributed to its capacity to bind chitin oligosaccharides, which are main components of the fungal wall (Broekaert et al. 1989). UDA has also been reported to inhibit the growth of hyphae arising from germinated spores of the AMF Fulleniformis mosseae (formerly Glomus mosseae) under in vitro culture conditions as well as the root colonization of Glycine max (Vierheilig et al. 1996) and Pisum sativum (Fontenla et al. 1999).

Interestingly, the amino acid sequence of UDA presents high homology with hevein, a smaller protein that is a major component of the latex of natural rubber, Hevea brasiliensis Müll. Arg. (Van Parijs et al. 1991; Gidrol et al. 1994). Antimicrobial properties of latex have been observed in vitro (Kanokwiroon et al. 2008) and the capacity of purified hevein to inhibit the mycelial growth of B. cinerea, S. nodorum, T. hamatum and P. blakesleeanus was reported by Van Parijs et al. (1991).

H. brasiliensis is a mycotrophic plant (Ikram et al. 1992, 1993). However, its colonization by AMF under greenhouse conditions may take several weeks (Schwob et al. 2000). Root colonization of young plantlets under in vitro culture conditions growth in a dense extraradical mycelium network also took a long time, as the plantlets were colonized only after a period of at least 7 weeks (Sosa-Rodriguez et al. 2013). The reasons for this delay are still unknown, but the exudation of hevein (or another antifungal compound) from roots could be a realistic explanation. As reported for U. dioica (Vierheilig et al. 1996; Fontenla et al. 1999), studying the colonization of a highly mycotrophic plant developing in close vicinity of H. brasiliensis may thus indirectly support this hypothesis.

Here, we used the mycelium donor plant (MDP) in vitro culture system developed by Voets et al. (2009) to investigate the effects of H. brasiliensis or U. dioica on the root colonization of Medicago truncatula by Rhizophagus irregularis MUCL 41833 and the metabolic activity in the extraradical mycelium of the AMF.

2 Materials and methods

Urtica dioica seedlings

Seeds of stinging nettle, U. dioica L., (Herbiseed, Berkshire, UK) were surface-sterilized by immersion in ethanol (70 %) for 5 min followed by rinsing in deionized sterile water for 10 min and immersion in sodium hypochlorite (8 % active chloride) for 15 min. The seeds were finally rinsed three times in deionized sterile water and germinated in 107x94x65 mm sterile containers (Duchefa Biochemie B.V., Haarlem, the Netherlands) filled with 100 ml of modified Strullu-Romand (MSR) medium (Declerck et al. 1998) lacking sucrose and vitamins, and solidified with 3 g l−1 Phytagel™ (Sigma-Aldrich, St. Louis, USA). Thirty seeds were plated in each container. The containers were placed in a growth chamber under controlled conditions (22 °C/18 °C day/night, 70 % relative humidity, 16 hd−1 photoperiod and a photosynthetic photon flux of 225 μmol m−2 s−1). Seeds germinated within 8–10 days and seedlings were ready to use after 20 days, when the length of the shoots was approximately 20 mm.

Hevea brasiliensis plantlets

Twelve-week-old plantlets of H. brasiliensis Müll. Arg. genotype PB 260 (Prang Besar, Malaysia) were produced by somatic embryogenesis (MFP MICHELIN & CIRAD, Clermont-Ferrand and Montpellier, France). Before transfer to the MDP in vitro culture systems (see below), the plantlets were grown under mixotrophic culture conditions inside glass tubes (25 mm diameter × 200 mm height) filled with 30 ml of an agar-gelled culture medium adapted to the embryo germination and growth of H. brasiliensis. This medium contained 234 mM of sucrose, but no growth regulators (Lardet et al. 2007). The lid of the tubes had a porous filter (3 M™ Micropore™Tape) that allowed the gas exchange, maintaining the sterility in the tubes. The plantlets were placed in a growth chamber set at 27 °C, with a 12 hd−1 photoperiod, and a photosynthetic photon flux of 60 μmol m−2 s−1. At the time of the experiment, the plantlets had 1–3 leaves, 4–6 cm stem height, 4–6 cm taproot length and 5–10 lateral roots of 1–3 cm length.

Medicago truncatula seedlings

Seeds of barrel medic, M. truncatula L., cv. Jemalong strain A17 (SARDI, Adelaide, Australia), were surface-sterilized by immersion in sodium hypochlorite (8 % active chloride) for 15 min followed by rinsing three times in deionized sterile water. Seeds were germinated in Petri plates (90 mm diameter) filled with 30 ml of MSR medium. Fifteen seeds were plated per Petri plate and further incubated at 18–22 °C in the dark. Seeds germinated within 1–2 days and were ready to use after 4 days when the length of the shoots was approximately 20 mm.

Arbuscular mycorrhizal fungus

Rhizophagus irregularis (Błaszk, Wubet, Renker & Buscot) Walker and Schuessler (2010) [as ‘irregulare’] MUCL 41833 (Synonymy: Glomus irregulare) was purchased from the Glomeromycota in vitro collection (GINCO www.mycorrhiza.be/ginco-bel). This AMF was initially associated and sub-cultured using the Ri T-DNA transformed carrot (Daucus carota L.) roots clone DC1 (for details, see Cranenbrouck et al. 2005). Afterwards, the strain was mass-produced in association with M. truncatula in the Half-Closed Arbuscular Mycorrhizal–Plant (HAM–P) in vitro culture system (Voets et al. 2005). Several thousands of spores were produced in the hyphal compartment (HC) of this system within a period of 10 weeks.

2.1 Experimental set-up

Experiment 1: in vitro mycorrhization of Medicago truncatula in presence of Urtica dioica

Mycelium Donor Plant (MDP) in vitro culture systems were established associating a M. truncatula plantlet with spores of R. irregularis MUCL 41833 to produce an extraradical mycelium (ERM) network (for details, see Voets et al. 2009). Briefly, each MDP in vitro culture system consisted of a bi-compartmented Petri plate of 90 mm diameter allowing the physical separation of a root compartment (RC) from a hyphal compartment (HC; Fig. 1). Both compartments were filled with 20 ml of MSR medium without sucrose and vitamins. The M. truncatula seedling was transferred to the RC and inoculated with a plug of gel of 0.4 cm3 containing approximately 100 spores of R. irregularis. The MDP in vitro culture systems were placed in a growth chamber under controlled conditions (22 °C/18 °C day/night temperature, 70 % relative humidity, 16 hd−1 photoperiod and a photosynthetic photon flux of 225 μmol m−2 s−1). Starting from week 3, 10 ml MSR medium lacking sucrose and vitamins was added weekly to the RC (Voets et al. 2009).
https://static-content.springer.com/image/art%3A10.1007%2Fs13199-013-0248-9/MediaObjects/13199_2013_248_Fig1_HTML.gif
Fig. 1

Mycelium Donor Plant (MDP) in vitro culture system adapted from Voets et al. (2009) to study the impact of Urtica dioica on the mycorrhization of Medicago truncatula by Rhizophagus irregularis MUCL 41833. a Schematic representation of the system showing the disposition of the U. dioica plants, the root compartment (RC) with the opening (Op) of aprox. 2 mm diam. made to allow the development of the plant shoot outside the Petri plate, the partition wall (separation between the compartments), and the hyphal compartment (HC). bUrtica dioica treatment (UD) system showing the disposition of the 3M™ Micropore™ Tape filters (f) on the system lid, the M. truncatula donor plant, and the M. truncatula test plant

After a period of 8 weeks, hyphae crossed the partition wall between the two compartments and spread on the HC. To assure the homogeneous growth of the ERM into the HC, the medium of this compartment was almost entirely removed, but a band of medium (approximately 2 mm width) parallel to the partition wall was kept to maintain the mycelium connection between the RC and the HC. Twenty-five ml fresh MSR medium without sucrose and vitamins was then added to the HC. Two weeks later, the systems in which the mycelium had covered the complete surface of the HC were selected for the experiment.

In half of the selected MDP in vitro culture systems, four U. dioica seedlings (UD treatment) were plated on the surface of the HC (Fig. 1a). The shoot of these plants remained inside the HC. In the other half, no U. dioica seedlings were plated in the ERM of the HC (control treatment—CT-UD). The lids of the systems were replaced by new ones with two holes of 0.6 mm diameter covered by a sterilized (121 °C for 15 min.) double-layer white strip of 20 × 20 mm (3 M™ Micropore™ Tape) allowing gas exchanges and thus photosynthesis of U. dioica, but avoiding microbial contamination (Fig. 1b). One week later, a 4-day-old M. truncatula seedling was introduced in the HC of each MDP in vitro culture system with the roots in contact with the ERM and the shoot extending outside the system (Fig. 1). The M. truncatula seedlings were grown for 4, 8 and 12 days in the presence (UD) or the absence (CT-UD) of U. dioica. The systems were placed in the growth chamber under the same growth conditions as described above. Four replicates (i.e. seedlings of M. truncatula) per treatment were collected at each time.

Experiment 2: in vitro mycorrhization of Medicago truncatula in presence of Hevea brasiliensis

The MDP in vitro culture system mentioned above was slightly modified following the methodology described by Sosa-Rodriguez et al. (2013). Briefly, the bi-compartmented system was set up by placing a small Petri plate (55 mm diameter × 8 mm height—the RC) into a large Petri plate (145 mm diameter × 20 mm height—the HC; Fig. 2a). The lid of the system was also modified with the addition of a screw cap tube (30 mm diameter × 35 mm length; Fig. 2b) to facilitate the addition of medium. Starting from week 3, 10 ml MSR medium lacking sucrose and vitamins was added weekly to the RC (Sosa-Rodriguez et al. 2013). The systems were gamma sterilized (25 KGy) before utilization. M. truncatula was used as donor plant and inoculated with spores of the AMF as described above. The systems were incubated during 10 weeks to obtain a profuse ERM network in the HC.
https://static-content.springer.com/image/art%3A10.1007%2Fs13199-013-0248-9/MediaObjects/13199_2013_248_Fig2_HTML.gif
Fig. 2

Mycelium Donor Plant (MDP) in vitro culture system adapted from Voets et al. (2009) to study the impact of Hevea brasiliensis Müll. Arg. on the mycorrhization of Medicago truncatula by Rhizophagus irregularis MUCL 41833. a Schematic representation of the system showing the disposition of the plants, the root compartment (RC), the openings (Op) of aprox. 2 mm diam. made to allow the development of the plant shoots outside the Petri plate, the partition wall (separation between the compartments), and hyphal compartment (HC), with the extraradical mycelium network (ERM) extending from the RC. bHevea brasiliensis treatment (HB) system showing the position of the screw cap tube (P) on the system lid, the M. truncatula donor plant, and the H. brasiliensis test plant

Only the systems in which the mycelium had covered 70 % of the HC surface (approximately 75 cm2) were used. In half of these MDP in vitro culture systems, a twelve-week-old H. brasiliensis plantlet (HB treatment) was placed in the HC with the roots in contact with the ERM network and the shoot extending outside the systems (Fig. 2), while in the other half, the HC remained free (control treatment—CT-HB). All the systems were then placed in a growth chamber under controlled conditions (26 °C/23 °C day/night temperature, 70 % relative humidity, 12 hd−1 photoperiod and a photosynthetic photon flux of 60 μmol m−2 s−1) adapted to the acclimatization of H. brasiliensis plantlets produced in vitro.

One week later, a 4-day-old M. truncatula seedling was introduced in the HC of each MDP in vitro culture system (next to the H. brasiliensis plantlet in the HB treatment), with the roots in contact with the ERM and the shoot extending outside the system (Fig. 2). The M. truncatula plantlets were grown for 12 days in the presence (HB) or the absence (CT-HB) of H. brasiliensis. During this period, the systems were placed in a growth chamber under controlled conditions (22 °C/18 °C day/night temperature, 70 % relative humidity, 16 hd−1 photoperiod and a photosynthetic photon flux of 225 μmol m−2 s−1). Four replicates (i.e. seedlings of M. truncatula) per treatment were collected after 12 days of contact with the ERM.

Harvest and data collection

To study the impact of U. dioica (Exp. 1) and H. brasiliensis (Exp. 2) on the root colonization of M. truncatula, the entire root systems of the M. truncatula seedlings were collected and intraradical colonization estimated after 4, 8 and 12 days of contact with the ERM for U. dioica, and after 12 days for H. brasiliensis. The root systems of U. dioica and H. brasiliensis were also harvested. In parallel, culture medium of the HC containing the mycelium of R. irregularis was sampled in each system to evaluate the proportion of metabolically active hyphae by measuring the activity of the succinate dehydrogenase enzyme (SDH; Saito 1995; Vierheilig et al. 2005), and the phosphate metabolism by determining the activities of the alkaline phosphatase (ALP) and acid phosphatase (ACP) enzymes (van Aarle et al. 2002; Zocco et al. 2011). These analyses were done at day 4, 8 and 12 in Exp. 1 and day 12 in Exp. 2.

Root colonization

After sampling, the root length of the M. truncatula seedlings was measured with a ruler. Roots of M. truncatula and U. dioica were then cleared in 10 % KOH at 80 °C for 20 min, rinsed with distilled water and stained with an ink-vinegar solution (5 % v:v vinegar with 5 % v:v Parker® blue ink; Vierheilig et al. 1998) at 80 °C for 5 min. For the H. brasiliensis plantlets, roots were cleared in 10 % KOH at 80 °C for 4 h, rinsed with distilled water, bleached with 3 % H2O2 at 80 °C for 3 min, and stained with the ink-vinegar solution at 80 °C for 5 min.

Roots were examined for AMF colonization under a compound bright-field light microscope (Olympus BH2-RFCA, Olympus Optical GmbH, Hamburg, Germany) at x125 magnification. The percentage of total root colonization and percentages of arbuscules and intraradical spores/vesicles were estimated using the magnified intersects method (McGonigle et al. 1990). For each plant, a minimum of 150 root intersections were analyzed.

Enzyme histochemical staining of the AMF mycelium

The mycelium of R. irregularis in the HC of each system was extracted following medium solubilization with a citrate buffer (Doner and Bécard 1991). The hyphae were thereafter rinsed to remove the buffer and then stained for detection of succinate dehydrogenase (SDH), alkaline phosphatase (ALP) and acid phosphatase (ACP) enzymatic activities.

For the SDH activity, the samples were incubated overnight in the staining solution (0.25 M sodium succinate, 0.05 M Tris–HCl buffer (pH 7.4), 0.5 mM MgCl2 and 1 mg.ml−1 Nitrotetrazolium blue chloride; Saito 1995) at room temperature in the dark (Zocco et al. 2011). For the phosphatase activities, a solution of 0.1 M Tris–HCl buffer (pH 8.5) for ALP or 0.1 M sodium acetate buffer (pH 5.5) for ACP was mixed with 1.1 mg.ml−1of alpha-Naphthyl phosphate Na salt (Sigma-Aldrich, Steinheim, Germany) and 1.0 mg.ml−1 fast blue RR salt (Sigma-Aldrich, Steinheim, Germany). The samples were incubated for 90 min at room temperature in the dark (Zocco et al. 2011).

After the enzymatic staining, samples for SDH, ALP or ACP activities were washed with deionized water for 15 min, counterstained with acid fuchsin (0.1 % w/v acid fuchsin/lactic acid) for 15 min, and finally transferred to lactoglycerol (1:2:1 v:v:v lactic acid:glycerol:water) for de-staining and storage (Zocco et al. 2011). For the microscopic observations, the samples were mounted on microscope slides with lactoglycerol. Formazan (for SDH) or Fast blue RR granular precipitation (for ALP and ACP) was assessed with a compound bright-field light microscope (Olympus BH2-RFCA, Olympus Optical GmbH, Hamburg, Germany) at x500 magnification. For each sample, at least 150 hyphal intersections were observed following the magnified intersects method (McGonigle et al. 1990). Intersections of hyphae were classified as active or non-active according to the presence or absence of granular precipitates, and the percentage of SDH, ACP and ALP active hyphae was determined.

Statistical analyses

Data analysis was performed with the statistical software Statistica® for Windows (StatSoft 2001). For the percentage of SDH, ACP and ALP active hyphae, the percentage of total colonization, and the percentages of arbuscules and intraradical spores/vesicles, the values were arcsine √(x/100) transformed before statistical analysis. ANOVA analyses were conducted after the verification of the normal distribution, homogeneity of the variances and residuals’ independency of the data.

3 Results

Impact of Urtica dioica or Hevea brasiliensis on the root colonization of Medicago truncatula

The presence of U. dioica or H. brasiliensis negatively impacted the root colonization of M. truncatula (Tables 1 and 2). In presence of U. dioica, the percentages of total colonization and arbuscules were significantly reduced in the M. truncatula seedlings as compared to the controls (CT-UD). The percentage of intraradical spores/vesicles colonization was also lower, but the difference was not significant. In presence of H. brasiliensis both, the percentages of arbuscules and intraradical spores/vesicles colonization were significantly reduced as compared to the controls (CT-HB). The total root colonization was also lower in presence of H. brasiliensis, but the difference was not significant. At the end of the experiments (day 12), the total root colonization was reduced by 30.1 % in presence of H. brasiliensis and by 29.1 % in presence of U. dioica as compared with the CT-HB or CT-UD treatments, respectively. In addition, M. truncatula roots contained 46.3 % and 19.3 % less arbuscules in presence of H. brasiliensis and U. dioica respectively, as compared to the plantlets in the CT-HB or CT-UD treatments (Tables 1 and 2). Independently of the treatment (UD and CT-UD), the first intraradical hyphae were present after 4 days of contact with the mycelium network, and the first arbuscules and intraradical spores/vesicles appeared after 8 days (Table 1). The total colonization and percentages of arbuscules and intraradical spores/vesicles significantly increased with time in presence or absence of U. dioica (Table 1).
Table 1

Percentages of root colonization of Medicago truncatula seedlings after 4, 8 and 12 days of contact with the extraradical mycelium of Rhizophagus irregularis MUCL 41833, in presence (UD) or absence (CT-UD) of Urtica dioica

U. dioica

Time (days)

Total root colonization (%)

Arbuscules (%)

Intraradical spores/vesicles (%)

Present (UD)

4

4.2 ± 0.5

0

0

8

11.4 ± 1.3

3.1 ± 0.6

2.1 ± 0.7

12

20.7 ± 0.6

19.6 ± 1.4

11.6 ± 0.2

Absent (CT-UD)

4

6.5 ± 0.9

0

0

8

18.4 ± 1.3

5.7 ± 0.7

3.1 ± 0.5

12

29.2 ± 1.2

24.3 ± 1.4

14.2 ± 2.2

Significance of the treatment (P value obtained by analysis of variance: two-way ANOVA, P ≤ 0.05)

UD

<0.001

<0.05

NS

Time

<0.001

<0.001

<0.001

UD × Time

NS

NS

NS

NS not significant. Data represent means +/− SE of four replicates

Table 2

Percentages of root colonization of Medicago truncatula seedlings after 12 days of contact with the extraradical mycelium of Rhizophagus irregularis MUCL 41833, in presence (HB) or absence (CT-HB) of Hevea brasiliensis

H. brasiliensis

Total root colonization (%)

Arbuscules (%)

Intraradical spores/vesicles (%)

Present (HB)

15.3a ± 1.2

5.1a ± 0.8

2.0a ± 0.3

Absent (CT-HB)

21.9a ± 4.1

9.5b ± 1.4

3.3b ± 0.3

Data represent means +/− SE of four replicates. Significant differences are identified with a different letter for each parameter (one-way ANOVA; P ≤ 0.05)

Neither hyphopodium at the root surface nor root colonization were noticed in H. brasiliensis and U. dioica. Nonetheless, for H. brasiliensis, some hyphae were observed growing along the roots. The total root lengths of M. truncatula seedlings were 62.8 ± 9.8 cm and 38.4 ± 5.6 cm in presence of U. dioica (UD) and H. brasiliensis (HB), respectively and were not significantly different when the seedlings were grown alone (CT-UD or CT-HB treatments; One Way ANOVA, P ≤ 0.05).

Impact of Urtica dioica or Hevea brasiliensis on the enzymatic activities of Rhizophagus irregularis

In presence of U. dioica seedlings (UD treatment) and H. brasiliensis plantlets (HB treatment), the succinate dehydrogenase (SDH) enzymatic activity of the extraradical mycelium growing into the HC (Tables 3 and 4) was significantly lower as compared to the controls. A reduction of 23.6 % in the proportion of SDH active hyphae in presence of U. dioica, and of 15.6 % in presence of H. brasiliensis as compared with the control treatments (CT-UD or CT-HB) after 12 days of contact with the ERM network were observed. Observations of M. truncatula enzymatic activities at 4 and 8 days in the U. dioica experiment (Table 3), showed that the proportion of SDH-active hyphae decreased significantly with time in both UD and control (CT-UD) treatments.
Table 3

Percentages of succinate dehydrogenase (SDH), acid phosphatase (ACP) and alkaline phosphatase (ALP) active hyphae of Rhizophagus irregularis MUCL 41833 after 4, 8 and 12 days of in vitro growth in presence (UD) or absence (CT-UD) of Urtica dioica

U. dioica

Time (days)

SDH activity (%)

ACP activity (%)

ALP activity (%)

Present

4

60.3 ± 2.8

29.5 ± 4.9

66.0 ± 1.6

8

58.5 ± 3.1

28.5 ± 3.5

65.5 ± 3.5

12

44.5 ± 5.0

30.2 ± 7.6

59.1 ± 0.9

Absent (CT-UD)

4

71.3 ± 4.3

34.5 ± 2.7

73.2 ± 2.0

8

64.9 ± 6.2

30.1 ± 2.0

71.4 ± 2.7

12

58.3 ± 2.1

29.2 ± 3.4

68.1 ± 2.8

Significance of the treatment (P value obtained by the analysis of variance; two-way ANOVA; P ≤ 0.05)

UD

<0.05

NS

<0.05

Time

<0.05

NS

NS

UD × Time

NS

NS

NS

NS not significant. Data represent means +/− SE of four replicates

Table 4

Percentages of succinate dehydrogenase (SDH), acid phosphatase (ACP) and alkaline phosphatase (ALP) active hyphae of Rhizophagus irregularis MUCL 41833 after 12 days of growth in presence (HB) or absence (CT-HB) of Hevea brasiliensis

H. brasiliensis

SDH activity (%)

ACP activity (%)

ALP activity (%)

Present (HB)

67.4a ± 1.7

36.4a ± 2.7

64.4a ± 1.7

Absent (CT-HB)

74.9b ± 2.0

37.0a ± 2.0

56.4a ± 2.0

Data represent means +/− SE of four replicates. Significant differences are identified with a different letter for each parameter (one-way ANOVA; P ≤ 0.05)

Phosphatase activity of the mycelium was only partially affected. Mycelium growing next to the stinging nettle roots had an ALP activity that was significantly lower in comparison with the control (CT-UD; Table 3), but no significant difference was found between the UD and CT-UD treatments for the proportion of ACP active hyphae. Neither the alkaline (ALP) nor the acid (ACP) phosphatase enzymatic activities were significantly influenced by the presence of H. brasiliensis (Table 4).

4 Discussion

The establishment of a functional symbiosis between an AMF and the roots of a host plant involves a sequence of events leading to the morphological and physiological integration of the two symbionts (Bonfante and Genre 2008). In plants, colonization may be impaired by a number of factors among which the production of inhibitory molecules (Parniske 2008). Here, we observed that the root colonization of M. truncatula seedlings grown in the vicinity of H. brasiliensis or U. dioica was lower than in the control plants, as well as the metabolic activity of the extraradical mycelium network of the AMF. Similarly, no root colonization was observed in H. brasiliensis and U. dioica within the time-frame of the experiments (i.e. 12 days). These results suggested that molecules with potential antifungal properties (e.g. UDA and hevein), may be exuded, and thus could limit the AMF colonization of M. truncatula roots. The presence of such molecules may further explain the absence or the delay in the colonization previously reported for U. dioica and H. brasiliensis roots, respectively.

The exudation of inhibitory molecules by H. brasiliensis was indirectly detected by growing highly mycotrophic plants in close vicinity of its roots, as was previously reported for U. dioica by Vierheilig et al. (1996) and Fontenla et al. (1999). These authors tested the effect of stinging nettle on the root colonization of Glycine max and Pisum sativum grown in compartmented pots, and observed reduced root colonization of both plants in presence of the stinging nettle.

M. truncatula is a highly mycotrophic plant which colonization in the MDP in vitro culture system starts within 2–3 days and reaches a plateau after 12–15 days (Voets et al. 2009). In our experiment, the M. truncatula seedlings grown in the presence as well as in the absence of U. dioica or H. brasiliensis were colonized by R. irregularis. Typical intraradical structures (arbuscules, intraradical spores/vesicles and hyphae) were observed. However, the total root colonization of M. truncatula was significantly lower in the presence of U. dioica than in the control plants, and a significant lower proportion of arbuscules was observed in the presence U. dioica and H. brasiliensis, suggesting an indirect effect on the intraradical phase of the symbiosis. Other studies conducted with sugar maple seedlings (Barto et al. 2011) and tomato (Roberts and Anderson 2001) grown in the vicinity of Alliaria petiolata, another non-host plant, reported a significant reduction of the root colonization and the arbuscule formation. Such inhibitory effects were also associated to the exudation of antifungal compounds produced by A. petiolata.

In our experiments, the low percentages of root colonization and the reduced formation of arbuscules may be associated with the reduction in the enzymatic activity observed in the ERM in presence of both stinging nettle and rubber tree plantlets. Arbuscules are specialized structures involved in nutrient exchange between AMF and plant cells (Parniske 2008). Their formation is considered as a worthy indicator of an active mycorrhizal symbiosis (Brundrett 2004; Bonfante and Genre 2008). The lower percentages of arbuscular colonization in M. truncatula roots observed in the presence of H. brasiliensis and U. dioica could be a consequence of the impairment of the metabolic activity of the ERM network. Our results suggested that the exudates produced by these plants may indirectly impact the nutrient exchange within the roots of the receiver M. truncatula plant via a reduction of viability and phosphorus metabolism in the ERM network. The lower values of SDH activity observed in the presence of U. dioica and H. brasiliensis indicated a reduced proportion of viable hyphae in the HC. Furthermore, the reduced activity of ALP in the hyphae of the AMF indicated that the tested plants significantly affected the metabolism of P in the fungal hyphae.

During the 12 days of growth into the MDP in vitro culture systems, neither U. dioica nor H. brasiliensis were colonized, supporting previous observations (Sosa-Rodriguez et al. 2013; Vierheilig et al. 1996; Fontenla et al. 1999). The MDP in vitro culture system was developed for the fast and homogenous colonization of seedlings or micropropagated plantlets (Voets et al. 2009). This system was successfully applied to barrel medic seedlings (Voets et al. 2009), but also to potato (Gallou et al. 2010), banana (Koffi et al. 2012) and rubber plantlets (Sosa-Rodriguez et al. 2013). While the three first plant species reached heavy root colonization values within the in vitro period of sub-cultivation (~3–4 weeks), H. brasiliensis root colonization markedly exceeded this duration (~7–8 weeks).

Thus, the observed absence of roots colonization in U. dioica by the active ERM network of R. irregularis was a confirmation of its status of non-host plant. For H. brasiliensis, the results suggested the existence defense mechanisms via the exudation of inhibitory molecules that may impair early root colonization by the AMF. However, this inhibition seems to be transient, as H. brasiliensis could be colonized after 7 weeks of contact with an ERM network under similar experimental conditions (Sosa-Rodriguez et al. 2013).

The exudation of inhibitory compounds was probably linked to a defense strategy against fungal pathogens (hevein was indeed reported to reduce the growth of several fungi; Van Parijs et al. 1991), but could also a defense response elicited in the young H. brasiliensis trees by the AMF itself (Kloppholz et al. 2011). The transient non-host plant behavior displayed by the H. brasiliensis plantlets towards AMF would thus be a collateral effect of a defense strategy developed by the tree to facilitate its establishment. Kloppholz et al. (2011) found that if AMF and plants have a long history of mutual association, during an early phase of the symbiosis the plant can react to the colonization by the AMF, inducing transient defense and stress responses. It was therefore suggested that AMF, to establish long-term biotrophic relationships with their hosts, must either actively suppress or avoid these plant defense responses.

Schwob et al. (2000) already observed a transitory enhancement of some enzymes involved in the lignin metabolism in roots of H. brasiliensis seedlings inoculated with F. mosseae. They hypothesized that the AMF elicited a temporary defense on the natural rubber roots. Whatever the mechanism involved, our hypothesis that H. brasiliensis roots produce and exude molecules with antifungal properties is therefore plausible. The antifungal capacity of hevein, a protein similar to UDA present in the latex of H. brasiliensis has already been established (Van Parijs et al. 1991; Martin 1991; Kanokwiroon et al. 2008).

However, clarification of the involvement of hevein would need to be performed as its exudation by H. brasiliensis roots was never documented. The detection of UDA, hevein (or other antifungal compounds) exuded during the growth of the M. truncatula seedlings in the presence of U. dioica or H. brasiliensis could also be quantified. The dynamic of root colonization could be paralleled with the detection of specific compounds in the roots during the time course of the experiment. In this case, specific protein detection techniques as Western blot could be used (using the suitable antibody; Laukkanen et al. 2003). It is also worth to notice that the compounds exuded by H. brasiliensis could be either less active against R. irregularis than UDA, or exuded in lesser amounts than by U. dioica, as the ERM was able to grown close to H. brasiliensis roots during the first 12 days of contact (this behavior was never observed with U. dioica), and the P metabolism (i.e. the ACP and ALP enzymatic activities) of the fungus was not significantly reduced in the presence of the rubber plantlets.

As a conclusion, we demonstrated that the highly-mycotrophic plant M. truncatula presented lower root colonization in the presence of H. brasiliensis or U. dioica than control plants grown alone. This result, along with the lower enzymatic activity noticed in the ERM network of the AMF, suggested the exudation of diffusible molecules from the roots of H. brasiliensis as well as U. dioica having antifungal activity, and thus leading to a lower colonization of M. truncatula. The exudation of these antifungal molecules by the roots of H. brasiliensis and U. dioica, could also explain the delay or the absence of root colonization in these plants, respectively. However, the physiological mechanisms and ecological reasons that would explain the transient or non-host behavior of these plants need to be further explored.

Acknowledgments

We are grateful to Dr. Marc-Philippe Carron of the UPR Systèmes de Pérennes, CIRAD (France) for his technical advices. Sosa-Rodriguez T. acknowledges the support of the “coopération au développement” PhD. Scholarship of the Université catholique de Louvain during this work.

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© Springer Science+Business Media Dordrecht 2013