Plant species identities and fertilization influence on arbuscular mycorrhizal fungal colonisation and soil bacterial activities

Plant species identities and fertilization influence on arbuscular mycorrhizal fungal colonisation and soil bacterial activities

G Model APSOIL 2299 No. of Pages 8 Applied Soil Ecology xxx (2015) xxx–xxx Contents lists available at ScienceDirect Applied Soil Ecology journal h...

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G Model APSOIL 2299 No. of Pages 8

Applied Soil Ecology xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Applied Soil Ecology journal homepage: www.elsevier.com/locate/apsoil

Plant species identities and fertilization influence on arbuscular mycorrhizal fungal colonisation and soil bacterial activities N. Legaya,* , F. Grasseinb , M.N. Binetc , C. Arnoldia , E. Personenib , S. Perigona , F. Polyd, T. Pommierd , J. Puissanta , J.C. Clémenta , S. Lavorela , B. Mouhamadoua a

Laboratoire d’Ecologie Alpine, CNRS UMR 5553, Université Joseph Fourier, BP 53, 38041 Grenoble Cedex 09, France Université de Caen Basse-Normandie, UMR950—INRA, Ecophysiologie Végétale Agronomie et Nutrition N, C, S, Esplanade de la Paix, 14032 CAEN Cedex, France c UMR Agroécologie INRA 1347/AgroSup/Université de Bourgogne, Pôle Interactions Plantes Microorganismes ERL CNRS 6300, 17 rue Sully, BP 86510, 21065 Dijon Cedex, France d Ecologie Microbienne, Université Lyon1, Université de Lyon, UMR CNRS 5557, USC INRA 1364, Villeurbanne Cedex, France b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 1 June 2015 Received in revised form 11 September 2015 Accepted 10 October 2015 Available online xxx

Plant species influence soil microbial communities, mainly through their functional traits. However, mechanisms underlying these effects are not well understood, and in particular how plant/ microorganism interactions are affected by plant identities and/or environmental conditions. Here, we performed a greenhouse experiment to assess the effects of three plant species on arbuscular mycorrhizal fungal (AMF) colonization, bacterial potential nitrification (PNA) and denitrification activities (PDA) through their functional traits related to nitrogen acquisition and turnover. Three species with contrasting functional traits and strategies (from exploitative to conservative), Dactylis glomerata (L.), Bromus erectus (Hudson) and Festuca paniculata (Schinz and Tellung), were cultivated in monocultures on soil grassland with or without N fertilization. Fertilization impacted some plant traits related to nutrient cycling (leaf and root N concentration, root C:N) but did not affect directly microbial parameters. The highest PDA and PNA were observed in D. glomerata and F. paniculata monocultures, respectively. The highest AMF colonization was obtained for F. paniculata, while B. erectus exhibited both the lowest AMF colonization and bacterial activities. Bacterial activities were influenced by specific above-ground plant traits across fertilization treatments: above-ground biomass for PDA, shoot:root ratio and leaf C:N ratio for PNA. Mycorrhizal colonization was influenced by below-ground traits either root dry matter content or root C:N. Hence, AMF colonization and bacterial activities were impacted differently by species-specific plant biomass allocation, root traits and nutrient requirement. We suggest that such effects may be linked to distinct root exudation patterns and plant abilities for nutrient acquisition and/or nutrient competition. ã 2015 Elsevier B.V. All rights reserved.

Keywords: Leaf traits Root traits Denitrification enzyme activity Nitrification enzyme activity Mycorrhizal colonization Nutrient availability

1. Introduction Soil microbial communities by their activities and diversity are involved in a wide range of ecosystem processes, such as carbon and nitrogen cycling (Kowalchuk and Stephen, 2001; van der Heijden et al., 2008), and influence plant growth through nutrient availability (van der Heijden et al., 2008). Identifying the drivers of the diversity and activity of soil microbial communities is therefore crucial to understand ecosystem functioning and to anticipate ecosystem responses to global changes (Allison and Martiny,

* Corresponding author. E-mail address: [email protected] (N. Legay).

2008). At the individual plant level, previous studies have reported the importance of species identity on both fungal (Kardol et al., 2007; Rooney and Clipson, 2009) and bacterial communities (Patra et al., 2006). For example, Orwin et al. (2010) demonstrated that leaf traits and litter quality of fast growing species favoured bacteria over fungi in the rhizosphere. At the community and ecosystem levels, the diversity and structure of plant communities can affect soil microbial community size and composition as well as their enzymatic activities (Grayston et al., 1998; Hedlund et al., 2003; Harrison and Bardgett, 2010; De Deyn et al., 2011; Le Roux et al., 2013). The activity of soil microbial communities partly depends on resource availability, notably N and C (Attard et al., 2011; Falcão Salles et al., 2012; Cantarel et al., 2015). Therefore, plant influence on soil microbial communities can be mediated

http://dx.doi.org/10.1016/j.apsoil.2015.10.006 0929-1393/ ã 2015 Elsevier B.V. All rights reserved.

Please cite this article in press as: N. Legay, et al., Plant species identities and fertilization influence on arbuscular mycorrhizal fungal colonisation and soil bacterial activities, Appl. Soil Ecol. (2015), http://dx.doi.org/10.1016/j.apsoil.2015.10.006

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through their functional traits related to soil nutrients cycling (Mouhamadou et al., 2013; Legay et al., 2014a), and more specifically through: (i) the amount and quality of litter and root exudates as a provider of nutrient for soil microbial communities (Wardle et al., 1998; Cesco et al., 2012) and (ii) their ability to compete for nutrient uptake and thus depleting the nutrient available for microbial communities (Cantarel et al., 2015). These plant effects have been related to plant traits such as plant growth rate and size (Wardle et al., 1998), specific leaf area and leaf dry matter content (Cornelissen et al., 1999; Kazakou et al., 2006), and below-ground traits including root turnover, root morphology, exudation (Innes et al., 2004). Plant functional traits are an intrinsic property of species reflecting their environmental niche (Grime et al., 1997), determining their response to environmental changes (Lavorel and Garnier, 2002; Amatangelo et al., 2014), and also varying within a given species in response to environmental variations (Albert et al., 2010; Kichenin et al., 2013). For example, a management practice such as nitrogen fertilization influences plant leaf traits such as vegetative height, leaf dry matter content and nitrogen concentration (Lavorel et al., 2011; Gardarin et al., 2014), and thereby ecosystem functioning (Wardle et al., 1998; Pakeman, 2011; Lienin and Kleyer, 2012). Consequently, the effects of fertilization on soil microorganisms may also be indirectly mediated through plant trait changes (De Deyn et al., 2011). Hence, plant influences on soil microbiota may result directly from intrinsic functional traits i.e. the morphological, phenological and physiological traits and/or indirectly through modified traits by environmental conditions. The underlying mechanisms are not well understood, in particular uncertainties remain about which plant traits, (specific to plant species or mediated by environment), are involved in the response of soil microorganisms to plant species. The purpose of our study was: (i) to investigate the effects of three plant species on soil bacterial activities and mycorrhizal status, (ii) to determine how plant/microorganism interactions are affected by plant identities through their functional traits, and (iii) how fertilization could impact these interactions. We focused on AMF colonization and N-cycling bacteria representing key soil microbial groups regulating nitrogen cycling in terrestrial ecosystems (Veresoglou et al., 2011). Three dominant subalpine grass species from the Central French Alps with contrasting functional traits were cultivated under similar soil nutrient availabilities with and without nitrogen addition in greenhouse conditions. Festuca paniculata is a conservative species with traits indicative of slow nutrient turnover while Dactylis glomerata is a more exploitative species with traits indicative of a faster nutrient turnover and Bromus erectus has intermediate characteristics (Grassein et al., 2010). Above- and below-ground traits, bacterial enzymatic activities (nitrification and denitrification) and mycorrhizal colonization (frequency and intensity) were measured for each plant species under low and high nutrient availability. We hypothesized that (i) AMF colonization and bacterial activities are impacted differently by the three plant species with contrasting nutrient turnover and that (ii) fertilization impacts bacterial activities and AMF colonization directly through change in nutrient availability and indirectly through change of plant traits (root biomass and nutrient status). 2. Materials and methods 2.1. Soil and plant species Soil and three co-occurring grasses, D. glomerata (L.), B. erectus (Hudson) and F. paniculata (Schinz and Tellung), differing in their relative growth rates and in their resource use strategy (Grassein et al., 2010), were sampled in the upper Romanche valley of the central French Alps (45.041 N 6.341 E, 1650–2000 m a.s.l.). During

the autumn 2010, three mother tussocks for each species were collected in grassland which was lightly grazed. Plants and especially roots were carefully washed to remove soil particles prior to vegetative multiplication in a nutritive solution and perlite as described in (Grassein et al., 2015). After one month, each replicate of plant species was clipped at 6-cm for the above-ground part and 4-cm for the below-ground part prior to the plantation in order to standardize plant size and to reduce the carryover of fungi from the field soil. 2.2. Growing conditions Clipped tillers were transplanted in an air-dried and sieved (5.6mm mesh) grassland soil collected in autumn 2010 at a depth of 5– 20 cm, in an area of 2 m in diameter in the same grassland (45.041 N 6.341 E, 1650–2000 m a.s.l.). Perlite (25 g per pot) was added to limit soil compaction since all gravels and stones were removed. Initial physical and chemical soil properties were as follows: clay, 30%; silt 46%; sand 24%; total carbon content: 44.4 g kg 1; total nitrogen content, 4.14 g kg 1; total phosphorus content, 1.79 g kg 1; pH (H2O), 5.5–6. Plants were cultivated in mesocosms constituted by cylindrical PVC boxes (7 cm of diameter and 16 cm of height; 617-cm3). In each mesocosm, two individuals per plant species were grown in 500 g of the perlite/soil mix. Mesocosms were placed in a glasshouse with air temperature kept at 20/16  2  C (day/night), additional light from artificial lighting (400-W high-pressure sodium lamps, Philips Son-T-pia Agro) provided 450-mmol m 2 s 1 photosynthetically active radiation (PAR) at plant height with a 16/8 h photoperiod cycle. Soils were watered daily to keep humidity at 20 g water 100 g 1 dry soil, and two treatments were used: no fertilized or fertilized with 50 kg N ha 1 (14 mg N kg 1 of dry soil) in the form of a urea-based slow release N fertilizer (Osmocote1) applied at the beginning of the experiment. This type of fertilization was chosen to simulate organic manure fertilization applied in mountain grassland. A total of 18 pots (3 plant species  2 treatments  3 replicates) were set up and moved twice a week to avoid any positional effect. 2.3. Harvest and plant trait measurements After three months, rhizospheric soil, aerial and root parts were harvested separately. We gently washed the roots with water on a 0.5-mm sieve to avoid any loss of fine roots. The root biomass was split into three aliquots, one aliquot was dried at 60  C, the two others were kept in an alcoholic solution (ethanol 10%, acetic acid 5% v: v) until (i) arbuscular mycorrhizal determination (see below) and (ii) root trait analyses. Plant functional traits for leaf and root were measured following standardised protocols (Cornelissen et al., 2003). For leaves, we measured fresh leaf area (LICOR Li-3100), fresh biomass and dry biomass after 72 h at 60  C, in order to calculate specific leaf area (SLA), leaf dry matter content (LDMC) and above-ground biomass (ABM). For roots, we measured root length (WINRHIZO software (Regent Instruments Inc., Canada)), fresh biomass and dry biomass after 72 h at 60  C, in order to calculate specific root length area (SRL), root dry matter content (RDMC) and root biomass (RBM). Above- and below-ground biomasses were used to compute the shoot root ratio (SRR). Leaf and root dried masses were finely ground (5-mm diameter) for analysis of N, C using an isotope ratio mass spectrometer (IRMS, Isoprime, GV Instrument) for leaf N and C content (LNC and leaf C:N ratio) and root N and C content (RNC, root C:N ratio) determination. 2.4. Soil analysis At the harvest, the root density was so high after three months of growth that all the soils in the PVC box were considered as under

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the influence of the rhizosphere. Fresh soils were weighed and sieved through a 5.6-mm mesh, stored at 4  C until the measurement of soil properties (within 48 h). From three air dried and ground soil subsamples we measured total N and C contents using an elemental analyser (FlashEA 1112, Thermo Electron Corporation). Soil nutrient contents (ammonium (NH4+-N), nitrate (NO3 -N) and total dissolved nitrogen (TDN)) were measured from K2SO4 (0.5 M) extracts of fresh soil subsamples (Jones and Willett, 2006) using a FS-IV colorimetric chain (OI-Analytical Corp., TX, USA). 2.5. Microbial enzymatic activities Potential nitrification activity (PNA) was estimated following the protocol of Dassonville et al. (2011). Briefly, 3 g fresh soil from each pot was incubated under aerobic conditions (180 rpm, 28  C, 10 h) in 30 mL of a (NH4)2SO4 solution (2 mg N L 1). Rates of NO2 and NO3 production were measured after 2, 4, 6, 8 and 10 h by ionic chromatography (DX120; Dionex, Salt Lake City, UT, USA). PNA was calculated from the slope of the linear regression curve of nitrate plus nitrite production versus time. Potential denitrification activity (PDA) was determined according to Attard et al. (2011). Briefly, c. 10 g dw soil was placed at 28  C under anaerobic conditions using HeC2H2 (90:10) mixture inhibiting N2O-reductase activity. Each flask was supplemented with c. 3-mL KNO3 (50-mg N NO3 g 1 dw), glucose (0.5-mg C g 1 dw) and sodium glutamate (0.5-mg C g 1 dw), completed with distilled water to reach the water-holding capacity. N2O was measured at 2, 3, 4, 5 and 6 h using a gas chromatograph (microGC RS3000; SRA instruments, Marcy l’Etoile, France). PDA was calculated from the slope of the linear regression curve of N2O production versus time.

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fr/mychintec/Mycocalc-prg/download.html). The intensity of mycorrhizal colonization (%M) gives an estimation of the amount of colonized root cortex in the whole root system. The mycorrhizal frequency (F%) gives the percentage of root fragments colonized by AMF community. 2.7. Data analysis A principal component analysis (PCA) was conducted to explore the relationships between plant above- and below-ground traits, and their variability within species. The effects of species and fertilization on plant, microbial and soil properties were tested using two-way ANOVAs, followed by Tukey’s HSD post hoc tests. PCA and ANOVAs were performed in JMP 7.0 (SAS Institute, Cary, NC). We applied a generalized linear regression with a forward selection analyses (R library MASS using the stepAIC procedure) with plant and soil variables as explanatory variables, and soil microbial activities as response variables in order to identify the best plant traits and soil parameters explaining microbial activities. The quality of the model was determined based on the bayesian information criterion (BIC) to avoid overestimation of the amount of variance explained. This analysis was performed in R 2.14.0 (R Core team 2012) using the library ‘MASS’ (Oksanen et al., 2012). When necessary, data were transformed to verify the assumptions of normality and homoscedasticity required for analyses. 3. Results 3.1. Covariation of plant functional traits, microbial parameters and soil properties according to the plant species identity with or without fertilization

2.6. Mycorrhizal colonization After digestion in KOH solution, root colonization by arbuscular mycorrhizal fungi was determined by staining roots with trypan blue in lactophenol (Phillips and Hayman, 1970). For each root system, AMF colonization was estimated by optical microscopy from sixty root fragments of approximately 1 cm. Mycorrhizal development was evaluated according to the method by Trouvelot et al. (1986) using the MYCOCALC program (http://www.dijon.inra.

All plant traits differed between species (Fig 1), with different positions in the multivariate space defined by leaf and root traits revealing contrasted nutrient use strategies. The first axis of the PCA was defined by plant C and N concentrations, with RNC and LNC on one side and root and leaf C:N ratio on the opposite side. The second axis was defined by plant traits related to plant nutrient use strategies and discriminated the most exploitative species, D. glomerata from the most conservative species of this

Fig. 1. Principal components analysis (PCA) of plant functional traits of Bromus erectus (BE), Dactylis glomerata (DG), and Festuca paniculata (FP) under fertilized (black symbol) and unfertilized (white symbol) conditions. Abbreviations: aboveground biomass (ABM), root biomass (RM), specific leaf area (SLA), leaf nitrogen concentration (LNC), leaf dry matter content (LDMC), leaf C:N ratio (LC:N), specific root length (SRL), root nitrogen concentration (RNC), root dry matter content (RDMC) and root C:N ratio (RC:N).

Please cite this article in press as: N. Legay, et al., Plant species identities and fertilization influence on arbuscular mycorrhizal fungal colonisation and soil bacterial activities, Appl. Soil Ecol. (2015), http://dx.doi.org/10.1016/j.apsoil.2015.10.006

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Table 1 Plant traits, microbial parameters and soil properties for each species and N treatments after 90 days of growth in greenhouse. Values correspond to the mean of the three pots per species and treatments (n = 3  SE, p < 0.05) and those that are not significantly different between species have the same letter (Tukey post-hoc test). F. paniculata

a) Plant traits Above-ground biomass (g pot 1) Root biomass (g pot 1) Shoot:root ratio Specific leaf area (mm2 mg 1) Specific root length (m g 1) Leaf N Content (%) Leaf C:N Leaf dry matter content (mg g 1) Root N content (%) Root C:N Root dry matter content (mg g 1) b) Microbial parameters PDA (mg N N2O g 1 dw h 1) PNA (mg N (NO2 + NO3 ) g Mycorrhizal frequency (%) Mycorrhizal intensity (%)

1

dw h

1

)

c) Soil properties Nitrate concentration (mg N NO3 g 1 dw) Ammonium concentration (mg N NH4+ g 1 dw) Soil water content (%) Total dissolved N (mg N g 1 dw)

B. erectus

D. glomerata

N

N+

N

N+

N

N+

2.14  0.29A 0.63 + 0.13C 3.53  0.30A 8.23  0.05C 119.15  12.01A 1.69  0.20C 27.28  2.84A 354.12  9.94A 0.69  0.06D 67.52  6.24A 0.21  0.01A

2.19  0.23A 0.73 + 0.07BC 3.01  0.05A 9.91  0.5C 102.56  2.46ABC 1.99  0.08BC 22.62  0.95A 340.15  13.76ABC 0.72  0.03CD 63.53  2.23A 0.21  0.01A

1.12  0.1B 1.09 + 0.03B 1.03  0.07C 11.86  1.56B 121.82  30.86AB 2.62  0.20B 17.92  1.18AB 347.40  11.23AB 1.05  0.22B 40.69  4.95B 0.16  0.02B

0.79  0.09B 0.70 + 0.15B 1.20  0.16BC 13.41  1.16B 112.34  26.88ABC 3.47  0.17A 13.59  0.72B 314.96  8.75CD 1.68  0.05A 24.53  1.41C 0.17  0.01B

2.41  0.14A 1.78 + 0.05A 1.35  0.05B 17.89  0.78A 67.25  2.89C 1.66  0.05C 27.30  0.80A 293.85  3.00D 0.84  0.05BC 51.70  2.95B 0.16  0.00B

2.58  0.26A 1.93 + 0.12A 1.33  0.08B 16.37  0.24A 72.14  2.95BC 1.85  0.33C 26.29  5.64A 318.77  5.00BCD 0.89  0.05BC 49.67  3.18B 0.15  0.01B

0.37  0.04BC 0.29  0.08AB 92.38  5.59A 40.85  7.99A

0.33  0.02CD 0.60  0.27A 88.43  6.69A 32.69  13.17AB

0.31  0.02CD 0.13  0.05BC 60.84  5.84AB 9.68  1.58B

0.28  0.01D 0.20  0.08ABC 57.01  17.32B 12.53  8.14B

0.40  0.02AB 0.09  0.03C 73.36  12.31AB 13.52  7.67B

0.45  0.02A 0.12  0.03BC 73.20  2.24AB 14.70  4.23B

39.49  5.93AB 25.86  2.39B 26.21  3.79BC 76.07  7.09BC

41.28  6.93AB 35.65  7.30AB 33.13  0.88AB 88.85  13.53AB

19.30  3.41B 26.65  0.97AB 29.26  5.74ABC 71.39  9.70BC

68.64  17.08A 39.98  6.92A 32.57  1.04ABC 112.90  19.45A

0.08  0.05C 25.48  1.19B 36.49  3.92A 46.11  2.11C

3.74  3.24C 25.89  2.56B 23.58  2.48C 48.27  5.97C

were higher than under F. paniculata and B. erectus (Table 1). At the opposite, nitrification activities were higher for F. paniculata than for D. glomerata, and B. erectus. AMF colonization significantly differed between plant species (Table 2). For instance, highest and lowest mycorrhizal frequencies in the roots of F. paniculata and of B. erectus were observed respectively (Table 1). The colonization in roots of D. glomerata being intermediate and no differences with the two other species were found. Mycorrhizal intensity was higher in the roots of F. paniculata than in B. erectus and D. glomerata roots.

study, F. paniculata. For example, D. glomerata was characterized by the highest values of ABM, RM and SLA, but the lowest values of LDMC and SRL; B. erectus had the highest values of LNC and RNC, but the lowest values of ABM and C:N ratios; lastly F. paniculata had the highest values of SRR, LDMC, RCN and RDMC but the lowest values of SLA (Table 1). Among plant traits investigated, only three traits, LNC, RNC and root C:N ratio were influenced by the fertilization treatment for the three plants species (Table 2). No microbial parameters were affected directly by fertilization (Table 2). In details, denitrification activities under D. glomerata

Table 2 Effect of species, fertilization and their interactions on plant traits, microbial parameters and soil properties after 90 days of growth in greenhouse with or without fertilization. Values are results from ANOVAs (F) and significance (***p < 0.001, **p < 0.01, *p < 0.05, ns: not significant). The (+) after the asterisk indicates a positive response to fertilization. Species

a) Plant traits Above-ground biomass (g pot 1) Root biomass (g pot 1) Shoot:root ratio Specific Leaf Area (mm2 mg 1) Specific Root Length (m g 1) Leaf N Content (%) Leaf C:N Leaf dry matter content (mg g 1) Root N content (%) Root C:N Root dry matter content (mg g 1) b) Microbial parameters PDA (mg N N2O g 1 dw h 1) PNA (mg N (NO2 + NO3 ) g Mycorrhizal frequency (%) Mycorrhizal intensity (%)

1

dw h

1

)

c) Soil properties Nitrate concentration (mg N NO3 g 1 dw) Ammonium concentration (mg N NH4+ g 1 dw) Soil water content (%) Total dissolved N (mg N g 1 dw)

Sp.  Ferti.

Fertilization

F

p

F

p

F

p

26.2 70.83 55.33 65.36 5.72 24.34 7.79 10.54 24.28 36.33 19.81

>0.001 >0.001 >0.001 >0.001 0.021 >0.001 0.005 0.003 >0.001 >0.001 >0.001

0.042 0.28 0.11 0.13 0.31 7.36 1.99 0.89 6.31 5.69 0.13

ns ns ns ns ns 0.027(+) ns ns 0.041(+) 0.049(+) ns

0.65 3.39 0.91 2.06 0.27 1.45 0.26 4.92 1.99 1.89 0.09

ns ns ns ns ns ns ns 0.029 ns ns ns

17.48 7.30 4.52 5.40

>0.001 0.009 0.033 0.023

0.06 1.88 0.098 0.038

ns ns ns ns

2.44 0.08 0.02 0.24

ns ns ns ns

42.66 1.50 0.08 8.44

>0.001 ns ns 0.004

8.78 3.8 0.12 3.95

0.020(+) ns ns ns

3.42 0.08 6.19 1.44

ns ns 0.015 ns

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Fig. 2. Relationship between potential denitrification activity and above-ground plant biomass. Bromus erectus (circle), Dactylis glomerata (square) and Festuca paniculata (triangle) in fertilized (dark symbols) or unfertilized (grey symbols) condition. Table 3 Percentages of variance of bacterial activities and AM fungal colonization explained by plant traits and soil properties. All species and treatments were used in the models. Response variable

Retained effect variables

% Variation explained

T-value

p-value

Potential denitrification activity

Above-ground biomass Soil nitrate

68.5

2.54 2.49

* *

Potential nitrification activity

Shoot:root ratio Leaf C:N ratio

66.82

5.44 3.33

*** **

Mycorrhizal frequency

Root C:N

38.32

3.31

**

Mycorrhizal intensity

Root dry matter content Total dissolved nitrogen Soil nitrate Soil ammonium

74.54

3.90 4.57 3.25 2.62

** *** ** *

For soil properties, nitrate concentrations remaining in the soil at the end of the growing period increased with fertilization (Table 2). We also observed that under D. glomerata, soil nitrate concentration was very low despite N fertilization, confirming that it has a more exploitative strategy (high nutrient acquisition abilities) than the other two species. Plant species also modified total dissolved N, with higher values in the soils cultivated with F. paniculata and B. erectus (Table 1). 3.2. Responses of bacterial activities and AM fungal colonization to plant functional traits

regression analysis. For instance, PDA was positively correlated to above-ground biomass (Fig. 2 and Table 3) and negatively correlated to soil nitrate (Fig. 3 and Table 3). PNA was positively related to shoot:root ratio (Fig. 4) and leaf C:N ratio with 66.8% of variation (Table 3). Finally, root C:N ratio explained 38.3% of variation in mycorrhizal frequency and 74.5% of mycorrhizal intensities were explained by three soil properties (soil nitrate, ammonium and total dissolved N contents) and RDMC (Table 3). These relationships were positive across all the three plant species (Fig. 5 and Fig. 6). Among plant traits that affect microbial activities and fungal colonization, only root C:N ratio was modified by fertilization.

The interactions between plant species identities, fertilization and microbial properties were studied using a multiple linear

Fig. 3. Relationship between soil nitrate concentration and potential denitrification activity. Bromus erectus (circle), Dactylis glomerata (square) and Festuca paniculata (triangle) in fertilized (dark symbols) or unfertilized (grey symbols) condition.

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Fig. 4. Relationship between shoot:root ratio and potential nitrification activity (R2 = 0.47); Bromus erectus (circle), Dactylis glomerata (square) and Festuca paniculata (triangle) in fertilized (dark symbols) or unfertilized (grey symbols) condition.

Fig. 5. Bromus erectus (circle), Dactylis glomerata (square) and Festuca paniculata (triangle) in fertilized (dark symbols) or unfertilized (grey symbols) condition.

4. Discussion We found that N fertilization had low but significant effects on root C:N ratio and on the N concentration in roots and leaves of the three studied species (Table 2). The fact that leaves and roots N concentrations increased in plants cultivated with fertilization confirmed that growing tillers used exogenous nitrogen during the three months of growth. Comparable results have been reported in the studies conducted by Ryser and Lambers (1995) or Legay et al. (2014b). Moreover, fertilization did not have any effects on bacterial activities and AMF colonization indicating that

fertilization in our system was not the main driver for soil microbial communities as demonstrated by previous studies (Bardgett et al., 1999; Mouhamadou et al., 2013). Consequently and as previously reported (Bais et al., 2006; van der Heijden et al., 2008) plant species identity significantly affected soil bacterial activities and AMF colonization. The different bacterial responses could be explained by distinct pathways. Root exudates of individual plant species can be quantitatively and qualitatively different (Gransee and Wittenmayer, 2000; Michalet et al., 2013), resulting in very distinct environments for soil microorganisms (Berg and Smalla, 2009). The array of root-derived

Fig. 6. Relationship between root C:N ratio and mycorrhizal frequency. Bromus erectus (circle), Dactylis glomerata (square) and Festuca paniculata (triangle) in fertilized (dark symbols) or unfertilized (grey symbols) condition.

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substances is widely diversified, including chemicals inhibitors that could influence bacterial nitrification (Subbarao et al., 2009) and denitrification (Bardon et al., 2014) activities. Moreover, we observed strong positive correlation between PDA and aboveground biomass. This pattern suggests a trophic mechanism, where high above and below-ground biomasses promote large exudation acting as carbon and energy sources for denitrifying bacteria (Pausch et al., 2013). Interestingly, the fact that D. glomerata had a greater root and shoot development likely led to greater root exsudation (Baptist et al., 2015), resulting in a larger occurrence of denitrifiers in its rhizosphere soil. Together, these high root biomasses coupled with high PDA activities whatever N fertilization also explained the very lower soil nitrate concentrations under D. glomerata in comparison to those observed in the soils under the other two species. The low PDA obtained in the rhizosphere of F. paniculata, which produced a low root biomass, is consistent with the trophic hypothesis. In contrast, B. erectus showed the lowest PDA. This species had trait values intermediate between D. glomerata and F. paniculata, with a root biomass similar to F. paniculata and a shoot biomass similar to D. glomerata. We assume that the exceptionally high root C:N observed in B. erectus compared to the other two species may result in low exudation rates (Valé et al., 2005). Potential nitrifying activity (PNA) was positively correlated with shoot:root ratio. This link between PNA and plant species identities through their functional traits may reflect nutrient competition between plant species and nitrifiers. This competition is in favour of exploitative species (D. glomerata) and to the detriment of conservative species (F. paniculata). Nevertheless, B. erectus exhibited a pattern similar to D. glomerata presumably due to its high capability to take up and concentrate N in its roots (Grassein et al., 2015) despite its low root biomass. Moreover, although the distribution of microbial resources appears to be linked to effects of plant identity and some ‘hard’ functional traits such as inorganic N forms and quantities uptakes, quantity and types of compounds exuded, the literature also suggests alternative mechanisms, in particular different types of interactions between bacteria and AMF (Veresoglou et al., 2012). AMF colonization also showed differences between plant species with lower degree of AMF colonization in D. glomerata and B. erectus compared to F. paniculata. We found that frequency and intensity of AMF colonization were positively linked to root C: N and RDMC, which are root functional traits known to favour mycorrhizal colonization (Urcelay et al., 2009). The highest mycorrhizal intensity was found for the more conservative species F. paniculata which presents reduced ability to prospect soil (low root biomass) (Gross et al., 2010). F. paniculata interacted with mycorrhizal fungi which can be assumed to lead to a better efficiency in nitrogen uptake. In contrast the exploitative strategy of D. glomerata resulting in greater root development did not seem to require an important AM contribution to N acquisition. In the intermediate species, B. erectus, the pattern was different and did not reflect the relationship between root development and mycorrhizal colonization. This outcome, which associates low mycorrhizal colonization to low root development could probably result from the weak mycorrhizal dependency of this species (van der Heijden et al., 2008). The surveys of interactions between fertilization, plant species through their functional traits and soil microbial parameters revealed distinct mechanisms affecting bacterial and fungal properties. While bacterial denitrification and nitrification activities were sensitive to above-ground plant traits in particular ABM, shoot:root ratio and leaf C:N ratio, fungal frequency and intensity were rather influenced by below-ground plant traits, RDMC and root C:N, respectively. As for the influences of root traits on AMF, they reflected either species identity or were mediated by N

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fertilization. To our knowledge, this is one of the few studies describing the mechanisms concerning plant species effects on both bacterial denitrification/nitrification activities and AMF colonization through their functional traits. 5. Conclusions Our study clearly demonstrated that, in monocultures under greenhouse conditions, plant species identity were the key determinants for bacterial activities and AMF colonization, and that these effects could be linked to species functional traits. The distinct and specific mechanisms involved in these microbial responses could reflect distinct root exudate patterns, plant abilities to prospect soil for nutrient acquisition and/or nutrient competition. Future studies are required to demonstrate such effects across greater arrays of plant species and to confirm their relevance to field conditions. Acknowledgements This study was conducted as part of ERA-Net BiodivERsA project VITAL, ANR-08-BDVA-008. We thank Servane Lemauviel-Lavenant, Anne-Françoise Ameline & Josette Bonnefoy for help and assistance in greenhouse and lab measurements. We also thank Nadine Guillaumaud for her help for the measurements of microbial activities on the AME platform (UMR5557-USC1364). We are really grateful to Miss Viviane Barbreau for her critical reading of the manuscript and her correction of the language and address our special thanks to Lila, Ilian and Nael for their help. References Albert, C.H., Thuiller, W., Yoccoz, N.G., Soudant, A., Boucher, F., Saccone, P., Lavorel, S., 2010. Intraspecific functional variability: extent, structure and sources of variation. J. Ecol. 98 (3), 604–613. Allison, S.D., Martiny, J.B.H., 2008. Resistance, resilience, and redundancy in microbial communities. Proc. Natl. Acad. Sci. U. S. A. 105, 11512–11519. Amatangelo, K.L., Johnson, S.E., Rogers, D.A., Waller, D.M., 2014. Trait–environment relationships remain strong despite 50 years of trait compositional change in temperate forests. Ecology 95 (7), 1780–1791. Attard, E., Recous, S., Chabbi, A., De Berranger, C., Guillaumaud, N., Labreuche, J., Philippot, L., Schmid, B., Le Roux, X., 2011. Soil environmental conditions rather than denitrifier abundance and diversity drive potential denitrification after changes in land uses. Glob. Change Biol. 17 (5), 1975–1989. Bais, H.P., Weir, T.L., Perry, L.G., Gilroy, S., Vivanco, J.M., 2006. The role of root exudates in rhizosphere interations with plants and other organisms. Annu. Rev. Plant Biol. 57, 233–266. Baptist, F., Aranjuelo, I., Legay, N., Lopez-Sanjil, L., Molero, G., Rovira, P., Nogués, S., 2015. Rhizodeposition of organic carbon by plants with contrasting traits for resource acquisition: responses to different fertility regimes. Plant Soil 394 (1), 391–406. Bardon, C., Piola, F., Bellvert, F., Haichar, F.e.Z., Comte, G., Meiffren, G., Pommier, T., Puijalon, S., Tsafack, N., Poly, F., 2014. Evidence for biological denitrification inhibition (BDI) by plant secondary metabolites. New Phytol. 204 (3), 620–630. Bardgett, R.D., Mawdsley, J.L., Edwards, S., Hobbs, P.J., Rodwell, J.S., Davies, W.J., 1999. Plant species and nitrogen effects on soil biological properties of temperate upland grasslands. Funct. Ecol. 13 (5), 650–660. Berg, G., Smalla, K., 2009. Plant species and soil type cooperatively shape the structure and function of microbial communities in the rhizosphere. FEMS Microbiol. Ecol. 68 (1), 1–13. Cantarel, A.A.M., Pommier, T., Desclos-Theveniau, M., Diquélou, S., Dumont, M., Grassein, F., Kastl, E.-M., Grigulis, K., Laîné, P., Lavorel, S., Lemauviel-Lavenant, S., Personeni, E., Schloter, M., Poly, F., 2015. Using plant traits to explain plantmicrobe relationships involved in nitrogen acquisition. Ecology 96 (3), 788–799. Cesco, S., Mimmo, T., Tonon, G., Tomasi, N., Pinton, R., Terzano, R., Neumann, G., Weisskopf, L., Renella, G., Landi, L., Nannipieri, P., 2012. Plant-borne flavonoids released into the rhizosphere: impact on soil bio-activities related to plant nutrition. A review. Biol. Fert. Soils 48 (2), 123–149. Cornelissen, J.H.C., Lavorel, S., Garnier, E., Diaz, S., Buchmann, N., Gurvich, D.E., Reich, P.B., ter Steege, H., Morgan, H.D., van der Heijden, M.G.A., Pausas, J.G., Poorter, H., 2003. A handbook of protocols for standardised and easy measurement of plant functional traits worldwide. Aust. J. Bot. 51 (4), 335–380. Cornelissen, J.H.C., Perez-Harguindeguy, N., Diaz, S., Grime, J.P., Marzano, B., Cabido, M., Vendramini, F., Cerabolini, B., 1999. Leaf structure and defence control litter decomposition rate across species and life forms in regional floras on two continents. New Phytol. 143 (1), 191–200.

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Please cite this article in press as: N. Legay, et al., Plant species identities and fertilization influence on arbuscular mycorrhizal fungal colonisation and soil bacterial activities, Appl. Soil Ecol. (2015), http://dx.doi.org/10.1016/j.apsoil.2015.10.006