Availability of different nitrogen forms changes the microbial communities and enzyme activities in the rhizosphere of maize lines with different nitrogen use efficiency

Applied Soil Ecology 98 (2016) 30–38

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Availability of different nitrogen forms changes the microbial communities and enzyme activities in the rhizosphere of maize lines with different nitrogen use efficiency Laura Giagnonia,* , Roberta Pastorellib , Stefano Mocalib , Mariarita Arenellaa,b , Paolo Nannipieria , Giancarlo Renellaa a

Department of Agrifood Production and Environmental Sciences, University of Florence, Piazzale delle Cascine 28, 50144 Florence, Italy Consiglio per la Ricerca in Agricoltura e l’Analisi dell’Economia Agraria, Centro di Ricerca per l’Agrobiologia e la Pedologia (CRA-ABP), Piazza d’Azeglio 30, 50121 Florence, Italy b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 16 March 2015 Received in revised form 27 August 2015 Accepted 9 September 2015 Available online 27 October 2015

We studied how the Lo5 and T250 maize lines, characterized by high and low nitrogen use efficiency (NUE), respectively, modified the microbial biomass, enzymatic activities and microbial community structure in the rhizosphere after exposure to different N forms. The two maize lines were grown for 4 weeks in rhizoboxes allowing precise sampling of rhizosphere and bulk soil with no nutrient additions, and then exposed to with nitric-, ammonium- and urea-N. After N exposure, the plants were inserted back into their original rhizoboxes to allow the root exudates diffusion into the rhizosphere. After 24 h rhizosphere soil were sampled and analyzed. Microbial biomass and soil enzymatic activities were increased after the exposure to different N forms of both maize lines. The plant exposure to different N forms also induced changes in the rhizosphere bacterial and fungal communities composition. Plant responses to the availability of different N forms was a dominant factor regulating activity and composition of the rhizosphere microbial communities, likely due to changes in the rhizodepositions. Therefore different N forms used for fertilization of agriculturally relevant plants such as maize can result in different plant mediated effects on the microbial activity and community structure in the rhizosphere. ã 2015 Elsevier B.V. All rights reserved.

Keywords: Nitrogen uptake Maize rhizosphere DGGE Microbial activity

1. Introduction The rhizosphere, the thin soil layer profoundly modified by plant roots, hosts a larger and more active microbial communities as compared to the bulk soil, sustained by rhizodepositions which include both low molecular weight organic compounds (LMWOCs) such as carboxylic acids, sugars (Hawes et al., 2003) and more complex chemical molecules such as polyphenols (Tomasi et al., 2008), which account for a significant proportion of C fixed by photosynthesis (Uren 2007). Rhizodepositions create favorable conditions for root tip elongation and plant exploitation of soil resources, and they can vary depending on biotic and abiotic soil factors such as soil texture and soil moisture level (Neumann et al., 2009), presence of symbiotic or pathogenic microorganisms (Vivanco and Baluska 2012), presence of toxic compounds (Uren 2007) and nutrient availability (Nguyen, 2003).

* Corresponding author. E-mail address: laura.giagnoni@unifi.it (L. Giagnoni). http://dx.doi.org/10.1016/j.apsoil.2015.09.004 0929-1393/ ã 2015 Elsevier B.V. All rights reserved.

It is well-established that different plant species can select different microbial communities in the rhizosphere, also depending on the plant growth stage and season (Berg and Smalla 2009). For example, it has been reported that a-Proteobacteria can be more abundant in the rhizosphere than in bulk soil (McCaig et al., 1998) whereas actinomycetes and g-proteobacteria can be more abundant in bulk than rhizosphere soil (Heuer et al., 1997; Ulrich et al., 2008). Usually, studies on the activity and diversity of microbial community and biochemical activity in the rhizosphere have been carried out with plants under metabolically resting conditions, i.e. in the absence of specific stimulations, or using systems mimicking the root exudate release from model root surfaces (Baudoin et al., 2003; Renella et al., 2007). Despite it is nowadays possible to determine unculturable microorganisms in rhizosphere and bulk soil, changes in the activity, biomass and structure of the microbial communities in response to nutrient availability in the rhizosphere are still poorly understood. A reliable approach to analyze the active microbial population in complex and dynamic environment as the rhizosphere relies on the simultaneous characterization of both DNA and RNA profiles

L. Giagnoni et al. / Applied Soil Ecology 98 (2016) 30–38

and the comparison of 16S rRNA fingerprint profiles (Griffiths et al., 2000). This approach allows to achieve information on total microbial community structure (DNA fingerprinting) and on active microbial population (RNA-fingerprinting) (Griffiths et al., 2000; Saleh-Lakha et al., 2005). Nitrogen is the main nutrient limiting plant growth and crop yield (Raun and Gohnson 1999), due to both the inherent plant N use efficiency (NUE), N losses by leaching, run off and volatilization, and to microbial N immobilization in the rhizosphere. While progress on the understanding of the plants mechanisms responsible for NUE have been made using model (Xu et al., 2012) and agriculturally relevant plants (Zamboni et al., 2014), limited information on the changes of microbial community composition, microbial and biochemical activity in the rhizosphere upon increase of N availability are available. For example it is known that several plant species can influence the N availability in soil by releasing microbial nitrification inhibitors in the root exudates (Subbarao et al., 2012). Recently, Zamboni et al. (2014) studied the genetic responses of maize lines Lo5 and T250 characterized by high and low NUE, respectively, and reported that exposure to NO3 –N induced a larger genetic response in the high NUE Lo5 (6.2  103 transcripts) than in the low NUE T250 (2.0  103 transcripts) maize line, with only 368 transcripts shared by these two maize lines. These maize lines have been shown to deplete inorganic N in the rhizosphere with different trends, and presented differences in the microbial communities and enzymatic activities in the rhizosphere (Pathan et al., 2015). Because such genome wide responses were not only limited to genes related to N absorption and organication, but also involved genes responsible for the synthesis of sugars, proteins, secondary metabolites and cofactors, it was reasonable to hypothesize that the N exposure for plant roots resulted in differences in the root exudate profiles of these two plants. Therefore, we hypothesized that different N forms exposure for maize plant should result in different plant responses which could in turn cause short term responses of activity, biomass and structure of microbial communities in the rhizosphere. Moreover, because the plant genetic response was greater in the higher NUE Lo5 than in the low NUE T250 maize line (Zamboni et al., 2014) we also hypothesized that larger microbial stimulation should occur in the rhizosphere of plants with higher NUE and that could also depend on the N form used to induce the plants. To test our hypothesis, we used two maize lines with different NUE, grown in rhizoboxes. In our work we induced the plants with NO3 –N, NH4+–N and urea, and evaluated the changes in the bacterial and fungal communities composition and enzyme activities of rhizosphere, following the plant responses to the availability of different N forms. Our methodological approach aimed at understating how the plant responses to the various N forms, could influence the enzymatic activities and microbial communities in the rhizosphere. 2. Materials and methods 2.1. Soil, plants, rhizobox set up and soil sampling The sandy clay loam soil, classified as a Eutric Cambisol (FAO), was sampled from the Ap horizon (0–25 cm) from an experimental farm under conventional maize crop regime located at Cesa (Tuscany, Central Italy). The soil had the following properties: 32.1% sand, 42.2% silt, 25.7% clay, 10.8 g kg 1 total organic C (TOC), 1.12 g kg 1 total N and 6.45 g kg 1 total P. The soil was sieved at field moisture (<2 mm), after removing visible plant material and immediately used for filling the two soil compartments of the rhizoboxes before the plantlet insertion in the plant compartment. The plants used for the experiments were the maize (Zea mays L.) inbred lines Lo5 and T250 with high and low NUE, respectively

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(Balconi et al., 1997; Zamboni et al., 2014). Maize seeds were germinated in a closed chamber at 25  C, and the seedlings were inserted into the plant compartment of the rhizoboxes, enclosed by 0.22 mm mesh nylon tissue, avoiding the soil passing into in the plant compartment. Then plants were grown in climatic chambers with 16:8 light/dark period, 200 mE m 2 s 1 light intensity and of 22/25  C for the dark and light periods, and a relative humidity of 70%. Twelve rhizoboxes were prepared for each of the two maize lines. Plants were manually watered with distilled H2O to avoid nutrient additions and the Lo5 and T250 maize lines were grown for 21 and 28 d, respectively, the growth periods allowing the full colonization of the plant compartment by plant roots without nutrient starvation. The N content of the soil solution was monitored by Rhizon1 probes into the soil in contact with the plant compartment; this measurement allowed to evaluate the maize plants NUE and to prevent plant starvation. At the end of the growth period (t0), three out of twelve rhizoboxes for each plants were destructively sampled for immediate analysis of chemical properties, microbial biomass, enzyme activities, and microbial community structure of the rhizosphere and bulk soil. The used rhizoboxes allowed precise sampling of the 0–2 mm soil layer adhering to the plant compartment, considered as the rhizosphere, whereas the soil at distance greater than 22 mm was considered as bulk soil. For testing the responses to plant metabolic effects with different N forms, the plant compartment of each maize line were taken out from the rhizoboxes and immersed into sterile glass beakers containing sterile solutions of 0.1 M NH4SO4, 0.1 M KNO3 or 0.1 M urea, and 200 mE m 2 s 1 light intensity, for 4 and 8 h for the LO5 and T250 maize lines, respectively, prepared in three replicates from each N form. After the N exposure, the plant compartments were thoroughly washed with sterile deionized H2O and inserted back into their original rhizoboxes, by ensuring the full contact with soil. Under the adopted experimental conditions the urea hydrolysis could be considered as negligible, and the plants were mainly absorbing intact urea. The rhizoboxes with the N induced plants were incubated for 24 h in climatic chambers under the same light, radiation, temperature and humidity conditions described above. After 24 h both rhizosphere was sampled as previously described. Since preliminary experiments showed that microbial biomass, enzymatic activities and microbial community structure of the bulk soil were not influenced after the N forms exposure, only the bulk soil of plants exposed to H2O (t0) and sampled after 24 h after their reinsertion into the rhizoboxes were showed in the present paper. 2.2. Analysis of soil microbial biomass and enzyme activities of the rhizosphere and bulk soil The NH4+–N and NO3 –N concentrations of the rhizosphere solution were analyzed by ion selective electrodes (Crison) on soil solution extracted using the Rhizon1 probes. Soil microbial biomass was determined by the ATP content according to Ciardi and Nannipieri (1990). Arylesterase activity was determined as described by Zornoza et al. (2009). Acid and alkaline phosphomonoesterase activities were assayed according to Tabatabai and Bremner (1969), and phosphodiesterase activity as reported by Browman and Tabatabai (1978). b-glucosidase activity was assayed according to Tabatabai (1982). All hydrolase activities were determined at 37  C for 1 h, followed by centrifugation at 6000 g at 4  C, and quantification of the p-nitrophenol (p-NP) released by the enzyme activities determined at by spectrophotometry at 400 nm wavelength (Lambda 2, PerkinElmer). Urease activity was determined according to Nannipieri et al. (1974); the released NH4+–N was extracted with 1 M KCl and quantified at 660 nm after reaction with the Nessler reagent. The

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protease activity was determined by the hydrolysis of Na-caseinate (Ladd and Butler, 1972), and quantified by spectrophotometric determination of the released tyrosine after reaction with the Folin reagent by a calibration curve. 2.3. Analysis of the microbial community structure The soil microbial community was analyzed by the polymerase chain reaction-denaturing gradient gel electrophoresis (PCRDGGE) technique. Rhizosphere and bulk soil samples of all replicates were stored at -80  C immediately after sampling. From each soil sample total RNA and DNA were co-extracted by using the RNA PowerSoil1 Total Isolation Kit (MoBio, Solano Beach, CA, USA) from 2 g of soil, according to the manufacturer instructions. RNA was eluted first and successively from the same column DNA was eluted using the PowerSoil1 DNA elution Accessory Kit (MoBio, Solano Beach, CA, USA). To check for the integrity of total RNA, an aliquot of the RNA sample was run on a agarose gel (1% w/vol) stained with ethidium bromide (EtBr). The RNAs extracted were subjected to DNase digestion to remove the potential DNA contamination and the reverse transcription (RT) was carried out on RNA samples/DNA free to generate cDNA, as previously described (Pastorelli et al., 2009). The extracted DNA was visualized on an agarose gel (1% w/vol) and DNA yields were estimated by comparison to bacteriophage l DNA dilutions (200, 100, 50 ng) using the Chemidoc Apparatus (Bio-Rad). From DNA extracted, bacterial 16S rRNA and fungal 18S rRNA gene sequences were amplified by PCR using the specific primers, GC986f–UNI1401r (Felske and Akkermans, 1998) and EF390– GCFR1 (Vainio and Hantula, 2000), respectively. Since the effect on bacterial composition after nitrogen exposure was more evident than the fungal composition changes, the bacterial 16S rRNA transcripts were amplified from cDNA generated from the rhizosphere before and after nitrogen treatments. Amplicons were checked by agarose gel (1% w/vol) electrophoresis and yields were estimated by comparing amplified DNA to Low DNA mass ladder (Invitrogen) using the Chemidoc Apparatus (Bio-Rad). Both 16S and 18S amplicon pools were separated by DGGE, loading 500 ng of amplicons onto a polyacrilamide gel (acrylamide/bis 37,5:1; Euroclone), with a linear denaturing gradients (from 55% to 65% and from 35% to 58% for 16S and 18S rRNA gene fragments, respectively) obtained with an 100% denaturing solution containing 40% formamide (Euroclone) and 7 M Urea (Euroclone). The polyacrilamide gel concentration and denaturating gradient were the optimal for the separation of 16S and 18S rRNA gene fragments with different molecular weight and C–G content. Electrophoresis was run in 1X TAE buffer at constant voltage (75 V) and temperature (60  C) for 17 h using the INGENY phorU-2 System (Ingeny International BV). At the end, gels were stained with SYBR1GOLD (Molecular Probes) diluted 1:1000 in 1X TAE and the gel images digitalized using the Chemidoc Apparatus (BioRad).

PAST software and used for further statistical analysis. One-way analysis of similarity (ANOSIM) and permutational multivariate analysis of variance (PERMANOVA) were used to examine statistical significance between DGGE profiles in order to determine difference in the microbial community structure due to exposure by different N forms. ANOSIM and PERMANOVA analysis were performed in PAST with the Bray–Curtis distance measure and 9999 permutation tests. Principal component analysis (PCA) were performed by PAST software to represent the distance between each sample in a two-dimensional space. We analyzed the DGGE results of t0 (before N exposure) for both plants by PCA to check the microbial communities changes in relation to different plants. Then we analyzed the DGGE results after N exposure for both plants, separately. 3. Results 3.1. Inorganic N availability and enzyme activities The Lo5 maize line showed significantly faster the uptake of NH4+–N and NO3 –N than the T250 maize, particularly in the first 15 d of growth (Fig. 1). Before the plant exposure to the various N forms, the bulk soil significantly showed lower ATP content than the rhizosphere of both maize lines (Fig. 2). Plants exposure by different N forms did not significantly change the ATP content as compared to control plants (Fig. 2). Before exposure to the various N forms, the rhizosphere of the Lo5 maize line showed significantly higher acid and alkaline phosphomonoesterase, b-glucosidase, protease and urease activities than the corresponding bulk soil, whereas the rhizosphere soil of the T250 maize line showed significantly higher alkaline phosphomonoesterase and arylsulfatase activities than the corresponding bulk soil (Fig. 2). Exposure to NH4+–N a significant increase of alkaline and acid phosphomonoesterase, phosphodiesterase and arylsulfatase activities in the rhizosphere of both maize line, as compared to control plants (Fig 2). Exposure to NH4+–N significantly increased the b-glucosidase activity in the rhizosphere of the Lo5 maize line, whereas significantly increased the arylesterase and protease activities in the rhizosphere of the T250 maize line (Fig. 2). Plant exposure to NO3 –N resulted in variable effects on the enzyme activities in the rhizosphere of the two maize lines. Exposure to NO3 –N significantly increased phosphodiesterase, b-glucosidase, arylsulfatase, arylesterase activities in Lo5 rhizosphere whereas only alkaline phosphomonesterase and protease

2.4. Data analysis Data of soil chemical properties, microbial biomass and enzyme activities were analyzed by one-way ANOVA followed by the Tukey test for assessing the significance of differences between mean values (p < 0.05), using the PAST3 software (Hammer 2009; http:// folk.uio.no/ohammer/past). DGGE images gels were normalized and analyzed using Gel Compare II software v 4.6 (Applied Maths). Band-matching data with band intensities were standardized by calculating relative intensity of each band (ratio of intensity of each band versus the total band intensity), the obtained matrices were imported into

Fig. 1. Ammonium and nitrate concentrations in the rhizosphere solution of the Lo5 and T250 maize lines during the plant growth. Symbols * indicate significant differences between mean values (n = 3) of Lo5 and T 250 maize lines rhizosphere.

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Fig. 2. Enzyme activities and ATP content of the rhizosphere and bulk soil of the Lo5 and T250 maize lines before and after the plant exposure to NO3 –N, NH4+–N and urea. Different letters indicate significant differences between mean concentration values (n = 3) for each enzyme activity and for each plant.

activities significantly increased in T250 rhizosphere, as compared to the respective control plants (Fig. 2). Plant exposure to urea also resulted in variable effects on the enzyme activities in the rhizosphere of the two maize lines. Exposure to urea significantly increased the alkaline phosphomonesterase, phosphodiesterase, b-glucosidase, arylsulfatase, arylesterase and urease activities in Lo5 rhizosphere, whereas the acid phosphomonesterase, phosphodiesterase, arylsulfatase, arylesterase and protease activities significantly increased in T250 rhizosphere, as compared to the respective control plants (Fig. 2).

3.2. Microbial community structure and activity The DGGE genetic fingerprinting of both bacterial and fungal communities and the analysis of the active microbial communities by cDNA before and after the plant exposure by different N forms showed complex banding patterns for the rhizosphere and bulk soil of both maize lines (Supplemental Fig. A1a, A1b, A2a, A2b, A3). The ANOSIM and PERMANOVA analyses showed that the rhizosphere and bulk soil of the Lo5 and T250 lines not exposed to N forms had significantly different bacterial (Lo5 P < 0.005, T250

Table 1 Analysis of similarity (ANOSIM) and permutational multivariate analysis of variance (PERMANOVA) global test evidencing differences in bacterial and fungal communities derived from DGGE band profiles for the two inbread maize lines.

16S 16S 18S 18S 16S 16S 18S 18S 16S 16S

rRNA Bacteria—T250 rhizo vs bulk rRNA Bacteria—LO5 rhizo vs bulk rRNA Fungi—T250 rhizo vs bulk rRNA Fungi—LO5 rhizo vs bulk rRNA Bacteria—T250 rhizo t0 vs rhizo N treatment rRNA Bacteria—LO5 rhizo t0 vs rhizo N treatment rRNA Fungi—T250 rhizo t0 vs rhizo N treatment rRNA Fungi—LO5 rhizo t0 vs rhizo N treatment rRNA-RNA trascripts Bacteria—LO5 rhizo t0 vs rhizo N treatments rRNA-RNA trascripts Bacteria—T250 rhizo t0 vs rhizo N treatments

ANOSIM R

Significance

PERMANOVA F

Significance

0.7942 1.00

P < 0.05 P < 0.005

7.4 20.7

P < 0.05 P < 0.005

1 0.835 0.932 0.967 0.877 0.911 0.639

P < 0.005 P < 0.0001 P < 0.0001 P < 0.005 P < 0.0001 P < 0.0005 P < 0.0005

9.8 8.5 17.2 9.6 6.3 5.45 3.81

P < 0.005 P < 0.0001 P < 0.0001 P < 0.005 P < 0.0001 P < 0.0005 P < 0.0005

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P < 0.05) and fungal communities (P < 0.005) (Table 1), and the PCA confirmed that the rhizosphere bacterial and fungal communities of the Lo5 and T250 clustered separately from those of the respective bulk soils (Fig. 3, Table 1). One-way ANOSIM and PERMANOVA analysis showed that the bacterial and fungal communities of both maize lines were significantly influenced by N treatments (Table 1). Concerning the effects of the N forms on rhizosphere bacterial communities of Lo5 maize line exposed to NO3 –N and urea clustered separately from those of plants exposed to NH4+–N, whereas in the rhizosphere of the T250 maize line after the NO3 – N and urea exposure, the soil bacterial communities clustered separately from those of rhizosphere of T250 maize line exposed to NH4+–N and control plants (Fig. 4). The exposure to different N forms showed that the rhizosphere fungal communities of Lo5 maize line exposed to the NO3 –N and urea, clustered separately from the rhizosphere fungal communities of those of plants exposed to NH4+–N and of control plants, whereas the rhizosphere fungal communities of T250 maize line exposed to all N forms clustered together but separately from the control (Fig. 4). The effect of N treatment on fungal community structure of both maize lines was not as evident as for bacterial community composition, even if a less marked but still significant separation

between fungal communities could be observed (Fig. 4b and d; Table 1). Since the effect of N treatments on bacterial communities composition was more evident than on fungal composition in both plant lines, cDNAs generated by reverse transcription of RNA extracted from Lo5 and T250 rhizosphere, were amplified and DGGE was performed to detect changes in active bacterial community composition (Fig. A3). The ANOSIM and PERMANOVA analyses showed that exposure to the different N forms induced changes in the active bacterial communities in the rhizosphere of both maize lines (Table 1). In particular, for the Lo5 maize line active bacterial communities of plants exposed to urea and NO3 –N clustered separately from those exposed to NH4+–N and control plants, whereas for the T250 maize line active bacterial communities of plants exposed to urea clustered separately from those exposed to NH4+–N and NO3 –N and from control plants (Fig. 5). 4. Discussion The Lo5 maize line confirmed to have a higher NUE than the T250 as previously reported by Pathan et al. (2015), particularly in the early growth period (Fig. 1). Zamboni et al. (2014) showed that the Lo5 maize line responded faster to NO3 –N availability and

Fig. 3. Principal Component analysis (PCA) of bacterial (a) and fungal (b) community of Lo5 maize line and bacterial (c) and fungal (d) community of T250 maize line before N treatment.

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Fig. 4. Principal Component analysis (PCA) of bacterial (a) and fungal (b) community of Lo5 maize line and bacterial (c) and fungal (d) community of T250 maize line after N treatment.

Fig. 5. Principal Component analysis (PCA) of active bacterial community of Lo5 maize line (a) and of T250 maize line (b) after N treatment.

with different metabolic and physiological mechanisms as compared to the T250 maize line. Increase of N availability in the soil can induce genome wide responses in the plant (Krouk et al., 2010; Bouguyon et al., 2012), with up- or down-regulation that in maize can involve up to 10% of the plant genome (Liu et al., 2008). Since different N fertilizers are used in agriculture and we have shown that maize lines with higher NUE show faster uptake of both nitrate and ammonium (Fig. 1), further studies are required

to better understand the genomic responses of important crop plants to different N forms. Soil hydrolase activities and ATP content were generally higher in the rhizosphere than the bulk soil for both the maize lines not previously exposed to N, particularly for the high NUE Lo5 maize line (Fig. 2). This result was related to the release of root exudates, in line with previous results (Baudoin et al., 2003; Renella et al., 2007). However, different patterns were observed in the

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stimulation of the enzyme activities in the Lo5 and T250 maizes lines rhizosphere after exposure to the different N forms. The high NUE Lo5 maize line induced significant increases of the rhizosphere enzyme activities after NO3 –N and urea exposure, versus the T250 maize lines (Table 2), whereas the T250 maize line showed an significant increase in enzyme activities after NH4+–N exposure in comparison to Lo5 maize line (Table 2). Responsiveness of the enzyme activities in the rhizosphere after exposure of the Lo5 and T250 maize lines to the different N forms ranked in the following order: phoshodiesterase, arylsulfatase > alkaline phosphomonoesterase, arylesterase > b-glucosidase, protease > urease (Table 2). The large response of the phosphodiesterase after plant exposure and low responses phosphomonoesterase activities after plant exposure could reflect the relative proportions P monoesters and diesters in the rhizosphere, with P diesters as predominant P forms, and the high availability of labile C (Turner and Haygarth, 2005; Renella et al., 2007). In fact, the rhizosphere is a soil portion with high microbial activity sustained by the release of root exudates and it has been reported that the proportion of diesters of microbial origin in soils increased upon increasing of microbial activity and nutrient availability (Wang et al., 2014). The lack of response or inhibition of the acid phosphomonoesterase activity after plant exposure could be related to the relatively low activity of fungi in the rhizosphere of the two studied maize lines, since fungi are major producers of acid phosphomonoesterase activity in soil (Quiquampoix and Mousain, 2005). Similarly, because of the arylsulfatase activity in soil is mainly of microbial origin (Gianfreda and Bollag, 1996), the arylsulfatase activity stimulation in the rhizosphere after plant exposure to the various N forms could be related to active microbial communities and to relatively high availability labile C. Strong responses of arylsulfatase to the release of low molecular weight organic compounds has been reported (Renella et al., 2007) and stronger responses of arylsulfatase as compared to other hydrolytic enzyme activities in organically amended soils were reported by Perucci et al. (1984). Moreover, arylsulfatase catalyze the hydrolysis of different complex sulfate esters and also act as sulfotransferase for various arylamines and phenols producing sulfurilated aromatics (George and Fitzgerald, 1981), therefore we hypothesize that the responsiveness of arylsulfatase activity upon plant exposure to various N forms could be connected to the formation of sulfate esters in the rhizosphere from phenolic substances released by the plant roots as intermediates of S storage. This hypothesis should be proven by the study of the root exudate profile of the two maize lines. The significant increase of the b-glucosidase in the rhizosphere of the Lo5 maize line after all N treatments could be due to the

Table 2 Responses of hydrolase activity in the rhizosphere of the Lo5 and T250 maize lines after plant exposure to NH4+–N, NO3 –N and urea. Symbols , + and = indicate significant reduction (P < 0.05), significant increase (P < 0.05) and same levels, respectively, as compared to rhizosphere of plants not exposed to N. Nitrogen forms Enzyme activity

NH4+–N

NO3 –N

Urea

Increases

Lo5 T250 Lo5 T250 Lo5 T250 Acid phosphomonoesterase Alkaline phosphomonoesterase Phosphodiesterase b-glucosidase Arylsulfatase Arylesterase Protease Urease Increases

+

+ +

=

= +

= +

+ =

2 4

+ + + = = = 4

+ = + + + = 6

+ + + + = = 4

= = = = + = 2

+ + + + = + 6

+ = + + + = 5

5 3 5 4 3 1

stimulation of microbial b-glucosidase synthesis upon the glucosides release in the rhizosphere in response to N assimilation in plants, as reported by Lugtenberg and Bloemberg (2004). The significant increase of the protease activity in the T250 maize line rhizosphere and of the urease activity in the Lo5 maize line rhizosphere could indicate differences in the N cycling mechanisms in the rhizosphere of the two maize lines. The majority of the N pool in soil is of peptidic or protein origin (Nannipieri and Paul, 2009) and there are increasing evidences that plant NUE is deeply dependent to the microbial activity in rhizosphere, particularly due to the proteolytic communities (Mooshammer et al., 2014). The N phytoavailability in soil also depends on the hydrolysis of other organic N forms such as urea and chitin catalyzed by the urease and chitinases (Metcalfe et al., 2002). Increased N mineralizing activities in response to the root exudates release has been reported (Renella et al., 2007), but in spite of their importance in determining N availability to plants, studies on the link between the diversity of protease encoding genes and protease activities in the rhizosphere are still scarce. Soil properties and management influence the abundance and distribution of proteolytic microorganisms (Fuka et al., 2009). Taken together the changes of the protease and urease activities in the rhizosphere of the Lo5 and T250 maize lines indicated that their exposure to N rapidly induced changes in the potentially available N in their rhizosphere. This may be due to differences in urea uptake by plants by transport systems having high or low affinity for urea (Kojima et al., 2006), which also operate urea absorption in maize exposed to different urea concentrations (Zanin et al., 2015). Therefore, the responses of protease and urease activities after the plant exposure to different N forms could be due to differences in root exudate profiles, because when plants absorb charged N forms (e.g. NH4+, NO3 ) generally release organic or inorganic ions with the same charge to balance the internal cell charge whereas urea has no charge and may result in the release of different molecules, mainly due to differences in the expression of urea utilization pathways within the plant (Mérigout et al., 2008). There is general consensus on the important role of hydrolase activities in the rhizosphere for plant nutrition and crop production, because they can release inorganic N, P and S which can be taken up by plants (Nannipieri et al., 2012). Higher enzyme activities induced by plant responses to N absorption could have a cooperative effect on plant acquisition of other nutrients such as P, S thus keeping an optimal C:N:P:S absorption ratio required for the correct metabolic activity, avoiding eventual nutrient imbalance after N acquisition. The microbial community analysis demonstrated that plant responses to N availability induced rapid (24 h) and significant changes in the microbial communities, with bacterial being more responsive than fungi (Fig. 4). These results confirm that the plant root activity is an important factor in shaping the rhizosphere microbial communities (Berg and Smalla, 2009). Changes in the microbial community composition after release of root exudates has been obtained using model systems mimicking the rhizosphere (Baudoin et al., 2003; Landi et al., 2006), whereas to our knowledge information on the microbial communities responses in the rhizosphere of plants induced by N forms is still poor. Plants release a wide diverse array of low molecular weight secondary metabolites and in the rhizosphere (Bais et al., 2004), but more studies on the effects of different N forms on the genomic responses as well as the characterization of the root exudate profiles of the these two maize lines are needed to understand the relation between root exudate profiles and changes in the bacterial and fungal communities in the rhizosphere. Furthermore, the RNA analysis showed that exposure of maize plants to different N forms induced responses of different bacterial communities, likely due to differences in root exudate profiles, and

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that more marked effects were observed in the active bacterial communities of the high NUE Lo5 than the low NUE T250 maize line rhizosphere (Fig. 5). Analysis of microbial community composition by next generation sequencing is needed to identify the microbial populations activated by the maize response to different N forms and for understanding their relation with the plant NUE. Globally, the microbial community composition and the bacterial active communities profile analysis indicated that microbial selection in the rhizosphere occurs when the plant responds to nutrient acquisition: plants with higher NUE may have a stronger ‘rhizosphere selective’ effects than plants with low NUE. Moreover, the plant response to different N forms trigger the activity of different microbial populations. Results of the active bacterial communities in the rhizosphere paralleled those of the enzyme activities, with stronger effects observed after exposure of the high NUE Lo5 maize to NO3 –N and urea (cfr Table 2, Fig. 5). However, the used approach did not permit to establish a direct link between changes in the microbial community structure and enzyme activities in the rhizosphere. Studies based on the analysis on the diversity and expression of enzyme encoding genes is needed to understand the origin of enzymes synthesized by the rhizosphere microbial communities during the maize response to different N forms and relation with the maize NUE. In conclusion, the obtained results confirmed our working hypotheses that maize lines with low and high NUE hosted different microbial communities and some differences in the enzymatic activities in the rhizosphere. Moreover, our experimental approach allowed to demonstrate that maize exposure to different N forms induced rapid increased enzymatic activities and significantly changed in the microbial communities in the rhizosphere, and that stimulated distinct bacterial communities, depending on the N form presumably in response to increase and chemical changes in root exudation. Overall, our results showed that plant-mediated effects on microbial communities and enzymatic activities in the rhizosphere may be important for defining the plant NUE. A better understanding of the effects in the rate of release and chemical composition of root exudates after plant exposure to different N forms and on the knowledge of the microbial diversity in the rhizosphere of agriculturally relevant plants using nucleic acid sequencing may improve the fertilizer use and minimize the impact of agriculture in the environment and possibly improve the plant growth and crop yields. Acknowledgement This research was supported by the Ministry for Education and Research project ‘PRIN 2009MWY5F9’. The Department of Agrifood Production and Environmental Sciences, Florence, thanks the Ente Cassa di Risparmio di Firenze for the financial support for the acquisition of new instruments. We also thank two anonymous referees for their constructive comments on the present manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. apsoil.2015.09.004. References Bais, H.P., Park, S.W., Weir, T.L., Callaway, R.M., Vivanco, J.M., 2004. How plants communicate using the underground information superhighway. Trends Plant Sci. 9, 26–32. Balconi, C., Brosio, D., Motto, M., 1997. Analysis of nitrogen partitioning in maize. Maize Gen. Coop. Newsl. 71, 10–11.

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