Author's personal copy Journal of Plant Physiology 171 (2014) 164–172 Contents lists available at ScienceDirect Journal of Plant Physiology jo urnal homepage: www.elsevier.com/locate/jplph Physiology Ectomycorrhizal Pisolithus albus inoculation of Acacia spirorbis and Eucalyptus globulus grown in ultramafic topsoil enhances plant growth and mineral nutrition while limits metal uptake a, a a b Philippe Jourand ∗, Laure Hannibal , Clarisse Majorel , Stéphane Mengant , c d Marc Ducousso , Michel Lebrun a IRD, UR040 LSTM, TA A-82/J Campus International de Baillarguet, 34398 Montpellier Cedex 5, France b Université de Nouvelle-Calédonie, Laboratoire insulaire du vivant et de l’environnement, B.P. R4, 98851 Nouméa Cedex, New Caledonia c CIRAD, UR 82 LSTM, TA A-82/J Campus International de Baillarguet, 34398 Montpellier Cedex 5 France d Université Montpellier 2, UMR28 LSTM, TA A-82/J Campus International de Baillarguet, 34398 Montpellier Cedex 5, France a r t i c l e i n f o a b s t r a c t Article history: Ectomycorrhizal fungi (ECM) isolates of Pisolithus albus (Cooke and Massee) from nickel-rich ultramafic Received 13 September 2013 topsoils in New Caledonia were inoculated onto Acacia spirorbis Labill. (an endemic Fabaceae) and Euca- Received in revised form 25 October 2013 lyptus globulus Labill. (used as a Myrtaceae plant host model). The aim of the study was to analyze the Accepted 26 October 2013 growth of symbiotic ECM plants growing on the ultramafic substrate that is characterized by high and Available online 21 November 2013 toxic metal concentrations i.e. Co, Cr, Fe, Mn and Ni, deficient concentrations of plant essential nutrients such as N, P, K, and that presents an unbalanced Ca/Mg ratio (1/19). ECM inoculation was successful with Keywords: a plant level of root mycorrhization up to 6.7%. ECM symbiosis enhanced plant growth as indicated by Acacia spirorbis Ectomycorrhiza significant increases in shoot and root biomass. Presence of ECM enhanced uptake of major elements that are deficient in ultramafic substrates; in particular P, K and Ca. On the contrary, the ECM symbioses Eucalyptus globulus Pisolithus albus strongly reduced transfer to plants of element in excess in soils; in particular all metals. ECM-inoculated Ultramafic topsoil plants released metal complexing molecules as free thiols and oxalic acid mostly at lower concentrations than in controls. Data showed that ECM symbiosis helped plant growth by supplying uptake of deficient elements while acting as a protective barrier to toxic metals, in particular for plants growing on ultra- mafic substrate with extreme soil conditions. Isolation of indigenous and stress-adapted beneficial ECM fungi could serve as a potential tool for inoculation of ECM endemic plants for the successful restoration of ultramafic ecosystems degraded by mining activities. © 2013 Elsevier GmbH. All rights reserved. Introduction studies of the ecological traits of ultramafic soils have been reviewed to propose these soils as a model system in ecology and Ultramafic soils are produced by weathering and pedogene- conservation, mostly because of their high plant diversity (Harrison sis of ultramafic rocks that are characterized by high levels of Ni, and Rajakaruna, 2011). Cr, and sometimes Co, but contain low levels of essential nutri- In ultramafic ecosystems, most of the plants are known to be ents such as N, P, K and Ca (Rajkumar et al., 2009). The presence involved in mycorrhizal associations to face these extreme soil con- of heavy metals at high concentrations in these soils is toxic for ditions (Alexander et al., 2007; Schechter and Bruns, 2008; Branco many plants (Brooks, 1987; Harrison and Rajakaruna, 2011). Previ- and Ree, 2010). The endo- and ectomycorrhizal associations are ous studies have shown that ultramafic soils are characterized by a known to significantly enhance plant nutrition such as P assim- high biological diversity of plants able to adapt to this extreme envi- ilation, and to strongly reduce abiotic stresses on plants such as ronment (Proctor, 2003; Brady et al., 2005; Kazakou et al., 2008). metal toxicity (Finlay, 2008; Smith and Read, 2008). Previous stud- In addition, these soils contain a high diversity of microorganisms ies on ectomycorrhizal (ECM) fungal communities in ultramafic such as bacteria and fungi that use various mechanisms to cope soils showed a high diversity of fungal species developing ECM with the extreme soil conditions, in particular, adaptation to toxic symbioses with plants growing on these substrates (Moser et al., heavy metals (Rajkumar et al., 2009; Branco, 2010). Recently, major 2005; Urban et al., 2008). In addition, it was recently demonstrated that ultramafic soils do not limit and can even promote ectomycor- rhizal fungal diversity (Moser et al., 2009; Branco and Ree, 2010). Comparisons of ECM fungal diversity between ultramafic and non- ∗ Corresponding author. Tel.: +33 4 67 59 39 55; fax: +33 4 67 59 38 02. E-mail address: [email protected] (P. Jourand). ultramafic soils showed differences within the fungal population 0176-1617/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.jplph.2013.10.011 Author's personal copy P. Jourand et al. / Journal of Plant Physiology 171 (2014) 164–172 165 structure (Brearley, 2006), sometimes with the presence of unique Mycelial culture and inoculum species (Moser et al., 2005). The study of physiological behaviours such as metal tolerance within the same fungal species present on Stock cultures from each sporocarp of P. albus isolates were both ultramafic and non-ultramafic soils have suggested adaptive obtained by aseptically transferring a piece of the pileus trama evolution, raising questions about the adaptation and evolution of to solid modified Melin-Norkrans Medium (MNM) (Marx, 1969) 1 1 fungal species on these soils (Gonc¸ alves et al., 2007, 2009; Jourand containing: KH2PO4 (0.5 g L− ), (NH4)2HPO4 (0.25 g L− ), CaCl2 1 1 1 et al., 2010a,b). The abundance of ECM found on plant roots living (0.05 g L− ), NaCl (0.025 g L− ), MgSO4 7H2O (0.15 g L− ), thi- 1 · 1 on ultramafic soils has also raised questions about their specific amine hydrochloride (100 ␮g L− ), FeCl3 6H2O (0.03 g L− ), glucose 1 1 · 1 roles in plant nutrition and adaptation to such extreme soil condi- (10 g L− ), malt extract (3 g L− ) and agar (14 g L− ). The pH was tions (Kazakou et al., 2008), in particular ECM symbioses which are adjusted to 5.6 with 1 M HCl and the medium autoclaved for 20 min known to limit metal accessibility and uptake by the plant (Colpaert at 120 ◦C. All fungal strains were maintained as sub-cultures at et al., 2011). 24 ◦C on the same medium. Vegetative inocula were prepared as Here, we present the results of a set of experiments carried out described in Brundrett et al. (1996). Briefly: large scale inocula on the ECM fungi Pisolithus albus (Cooke and Massee) collected from were produced by incubating each fungal mycelium from cultures ultramafic soils in New Caledonia, a tropical archipelago located on Petri dishes inoculated into a sterile solid substrate (peat- in the South Pacific Ocean (Fig. 1). In New Caledonia, the ultra- vermiculite mixture, 1:10, w:w), saturated with MNM liquid media mafic soils cover one-third of the main island because of natural in large plastic bags (1.5 L). The incubation was carried out at 30 ◦C geological evolution (Fig. 1). As a result of the presence of such in a dark room for 8 weeks. ultramafic outcrops, specific biological endemic ecosystems have developed (Jaffré, 1992) making the main island a biodiversity Plant growth and treatments hot spot (Myers et al., 2000). In addition, in the neo-Caledonian ultramafic ecosystem, it has been reported that the ectotrophic Eucalyptus globulus Labill. subsp. bicostata (Maiden et al.) J.B. mycorrhizal symbioses are dominant and might play an important Kirkp. seed lot N◦16731 was obtained from the Australian Tree Seed role in plant adaptation to the extreme soil conditions (Prin et al., Centre (Kingston, ACT, Australia). Acacia spirorbis subsp. spirorbis 2012). In this study, P. albus ultramafic ecotype isolates were inocu- Labill. seed lot N◦036/11 was obtained from the New Caledonian lated onto (i) the endemic neo-Caledonian Fabaceae, Acacia spirorbis Agronomic Institut (IAC, Païta, New Caledonia). A set of experi- Labill. and (ii) the Myrtaceae, Eucalyptus globulus Labill., which used ments was carried out in a glasshouse as described in Brundrett as a model plant to study the ECM symbiosis between Pisolithus et al. (1996). Briefly, seeds were pre-treated for 2 min in 70% ethanol and its host-plant at physiological and molecular levels (Duplessis supplemented with 0.1% (v/v) Tween 20, then surface sterilized for et al., 2005; Bellion et al., 2006; Jourand et al., 2010b; Majorel 5 min in H2O2 (30%) and washed 3 times in sterile water. Germina- et al., 2012). The aim of the study was to assess the importance tion occurred after 1 week at 28 ◦C in the dark. Seedlings were then of the ECM symbiosis on plant growth parameters, uptake of major placed for a week in a growth chamber with an 18/6 h day/night 2 1 mineral elements, control of heavy metal uptake and exudation of cycle at a light intensity of 200 ␮mol m− s− photosynthetic active molecules involved in metal binding, when cultivated in ultramafic radiation and at temperature 25/18 ◦C. Two-week-old seedlings substrate. The final objective was to evaluate the potential of ECM were transplanted into plastic containers of 1 litre capacity and symbioses in plant adaptation to ultramafic soils containing high filled with (i) neutral growth substrate composed of vermiculite concentrations of heavy metals, which is a prerequisite for their use or (ii) ultramafic substrate from site 4 (Mont Dore).