Biol Fertil Soils DOI 10.1007/s00374-013-0810-x ORIGINAL PAPER Effects of two grass species on the composition of soil fungal communities B. Mouhamadou & J. Puissant & E. Personeni & M. Desclos-Theveniau & E. M. Kastl & M. Schloter & L. Zinger & J. Roy & R. A. Geremia & S. Lavorel Received: 17 January 2013 /Revised: 5 April 2013 /Accepted: 10 April 2013 # Springer-Verlag Berlin Heidelberg 2013 Abstract Many studies have shown effects of plants spe- in fungal communities coincide with differing strategies cies on fungal communities, but these are often confounded of plant root growth, with D. glomerata having greater with soil effects. Thus, the specific role of plant species in root mass, length, and area than F. paniculata. Our study, by structuring rhizospheric and soil fungal communities is dissociating soil effects from plant effects, demonstrated poorly described. Our study used microcosms in which that plant species exert a key control on soil fungi. We plants were grown under artificial conditions to bridge this suggest that such effects may be linked to inter-specific gap. Two perennial grasses dominating subalpine grass- differences in root traits and their consequences on lands, Festuca paniculata and Dactylis glomerata,were nitrogen uptake. grown at two levels of fertilization on standard soil. Fungal communities were determined by 454 pyrosequencing of the Keywords Plant species . Fungal community structure . internal transcribed spacer 1 region. Among the fungal Nitrogen fertilization . Plant growth strategy communities characterized by the primers used, original communities were associated to each plant species and also diverged between rhizosphere and bulk soils within each Introduction plant species, though there were no significant fertilization effects. Differences regarded global composition of the Soil fungi are involved in various biological processes and fungal communities and abundant molecular operational control transformation of organic matter and nutrient avail- taxonomic units (MOTUs). Both plant species and loca- ability (Falkowski et al. 2008;Singhetal.2010). The tion effects were reflected more in the abundance than network of fungal hyphae is essential for the conservation in the composition of MOTUs. The observed differences of the soil structure (Rillig and Mummey 2006). Mycorrhi- zal fungi colonize plant roots and enhance plant productivity by facilitating plant nutrient uptake (Wardle et al. 2004; Electronic supplementary material The online version of this article van der Heijden et al. 2008), as well as conferring plant (doi:10.1007/s00374-013-0810-x) contains supplementary material, which is available to authorized users. resistance or tolerance to biotic and abiotic stresses : : : : (root pathogens, heavy metals, drought) (Clark and Zeto B. Mouhamadou J. Puissant L. Zinger J. Roy 2000; Turnau and Haselwandter 2002). Pathogenic fungi R. A. Geremia (*) : S. Lavorel Laboratoire d’Ecologie Alpine, UMR 5553 UJF/CNRS, Université may nevertheless have negative effects on ecosystem Joseph Fourier, BP 53, 38041 Grenoble cedex 9, France functioning by reducing plant productivity (van der Heijden e-mail: [email protected] et al. 2008). Because of this importance of fungi for ecosystem pro- E. Personeni : M. Desclos-Theveniau INRA, UMR950 Ecophysiologie Végétale Agronomie et nutrition cesses, a number of studies have sought to identify and N, C, S, 14032 Caen cedex, France quantify the key factors that regulate the structure and : diversity of soil fungal communities. Among these, it has E. M. Kastl M. Schloter been shown that plant species identity influences the com- Research Unit for Environmental Genomics, Helmholtz Zentrum München, Ingolstädter Landstr. 1, position and the abundance of soil fungal communities. For 85758 Oberschleissheim, Germany example, studies conducted by Costa et al. (2006) using Biol Fertil Soils molecular fingerprints have shown the effect of plant spe- structure in bulk soil and rhizosphere under different cies on fungal rhizosphere communities of strawberry and N-fertilization regimes. oilseed. Similarly, Arfi et al. (2012)demonstratedina mangrove ecosystem that the distribution of fungal commu- nities was influenced by plant host specificity. More gener- Methods ally, numerous studies have indicated the effects of plant species on soil fungal communities (Grayston et al. 1998; Experimental setup Bardgett et al. 1999; Innes et al. 2004). In general, the ability of plant species to influence soil fungal communities may be Living plant individuals were collected in summer 2009 related to their ecophysiological traits (Wardle et al. 1998) from a semi-natural grassland site in the upper Romanche or to their response to stress (Bouasria et al. 2012). Plants valley (Central French Alps, 45.041 N 6.341E, 1,650– may influence soil fungi through species-specific interac- 2,000 m a.s.l.), where D. glomerata and F. paniculata are tions (van der Heijden et al. 2008) and through their pro- among the dominants. Their roots were thoroughly washed duction of litter and root exudates (Bais et al. 2006; Millard under tap water, and the plants were clipped at 6 cm above- and Singh 2010), which vary quantitatively and qualitative- ground and 4 cm belowground. The plants were then grown ly between plant species (Gransee and Wittenmayer 2000), for 2 months in hydroponic conditions (i.e., perlite with 1/4 resulting in a unique and singular environment for soil strength Hoagland solution renewed every week). Root microorganisms (Bertin et al. 2003; Berg and Smalla washing, root clipping, and growth in hydroponic condi- 2009). The array of root-derived substances is diverse and tions were intended to reduce the carryover of fungi from includes signal molecules such as flavonoids that directly the original soil. [or indirectly] influence fungi and bacteria (Akiyama and Plants for the quantification of fungal communities were Hayashi 2006; Standing and Killham 2007; Steinkellner et grown in root boxes made of polyvinyl chloride. They al. 2007; Cesco et al. 2012). consisted of a soil compartment (29×17.5×1 cm) The degree of plant-derived effects on fungal communi- (Personeni et al. 2007). The soil compartment was filled ties, however, is strongly linked to soil organic matter con- with 1 kg of sieved (5 mm) soil, adjusted to 40 % of the tent (Zinger et al. 2011), to management (fertilization or maximal water holding capacity. We used a soil with loamy tillage), and to local climate conditions (Lekberg et al. sand texture, collected from a grassland at the Helmoltz 2007; Millard and Singh 2010; Lekberg et al. 2011; Schnoor Zentrum experimental station in Scheyern (Germany, et al. 2011). For example, Bezemer et al. (2006) demonstrat- 479 m a.s.l., 48°30′N, 11°28′E). Soil was taken in a depth ed that effects of plant species on microbial communities of 10–20 cm and is characterized by a pH value of 6.1, a significantly differed when they were grown in different soil total N content of 0.15 %, a total C content of 1.47 % (C/N types. Hence, it is very difficult to dissociate specific plant ratio 10.1), a nitrate content of 5.4 mg kg−1, and an ammo- effects from soil effects and to conclude that plant effects are nium content of 6.5 mg kg−1. The soil texture has been modulated by soil properties. characterized as 54 % sand, 32 % silt, and 14 % clay. In addition to plant species, N fertilization has also been Two plants of the same species were placed in the soil postulated as a factor that influences microbial communities compartment of each root box. Plants were grown for 4 weeks by promoting the relative abundance of bacteria over fungi, under artificial lighting (400-W high-pressure sodium lamps, as well as affecting the diversity within bacterial and fungal Philips Son-T-pia Agro) providing 450 μmol m−2 s−1 photo- functional groups (Bardgett et al. 1999; Allison et al. 2007). synthetically active radiation at plant height with a 16/8-h Nitrogen fertilization could directly modify soil microorgan- photoperiod cycle. Air temperature was kept at 20/16±2 °C isms by affecting mycorrhizal fungi and decomposers (day/night). Throughout the experiment (4 weeks), soil mois- through the repression of enzyme activity (Fog 1988; ture was kept constant by watering the boxes every 2 days. Donnison et al. 2000; Frey et al. 2004). Moreover, because Boxes were randomized each day to avoid any positional fertility alters plant traits and thereby ecosystem functioning effect. Root boxes were supplied three times with the 1/4 (Wardle et al. 1998; Lavorel et al. 2011; Pakeman 2011), Hoagland solution without N. Half of the boxes received fertilization effects on soil microorganism communities may overall 18.92 mg of NH4NO3. In total, 12 root boxes (2 plant also be mediated by plant trait effects on soil microorgan- species×2 fertilization levels×3 replicates) were used. Plants isms (De Deyn et al. 2011). were harvested after 4 weeks for measurements of root In this study, we used microcosms for plant growth morphology. and a barcoding approach based on 454 sequencing of The mycorrhizal status of the root of each plant sample the internal transcribed spacer (ITS) 1 region, to inves- was examined by microscopic observation after staining tigate the influence of two perennial grasses, Festuca with Trypan Blue (Phillips and Hayman 1970; Trouvelot paniculata and Dactylis glomerata, on fungal community et al. 1986). For the analysis of fungal community structure, Biol Fertil Soils soil sticking to roots was considered as rhizosphere soil. It positions correspond to the different tags. The PCR reac- was collected after strongly shaking the root systems. Bulk tions were carried out in 25 μl reaction mixtures containing soil was sampled in the soil zones where no root was 10 ng of fungal DNA, 4 mM of both primers, 200 mM of detected. Rhizosphere and bulk soils for fungal analysis each dNTP, 1 U of Ampli Taq Gold DNA polymerase, and were stored immediately after sampling at −80 °C; bulk 2.5 μl of PCR buffer (Applied Biosystems, Courtaboeuf, soils for the chemical analysis were kept at 4 °C.