Light affects competition for inorganic and organic

Light affects competition for inorganic and organic nitrogen between maize and rhizosphere microorganisms ... tion for organic and inorganic nitrogen ...

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Plant Soil (2008) 304:59–72 DOI 10.1007/s11104-007-9519-7

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Light affects competition for inorganic and organic nitrogen between maize and rhizosphere microorganisms Xingliang Xu & Claus Florian Stange & Andreas Richter & Wolfgang Wanek & Yakov Kuzyakov

Received: 13 August 2007 / Accepted: 5 December 2007 / Published online: 23 December 2007 # Springer Science + Business Media B.V. 2007

Abstract Effects of light on the short term competition for organic and inorganic nitrogen between maize and rhizosphere microorganisms were investigated using a mixture of amino acid, ammonium and nitrate under controlled conditions. The amount and forms of N added in the three treatments was identical, but only one of the three N forms was labeled with 15N. Glycine was additionally labeled with 14C to prove its uptake by maize and incorporation into microbial biomass in an intact form. Maize out-competed microorganisms for 15 NO 3 during the whole experiment under low and high light intensity. Microbial uptake of 15N and 14C was not directly influenced by

the light intensity, but was indirectly related to the impact the light intensity had on the plant. More 15 NHþ 4 was recovered in microbial biomass than in plants in the initial 4 h under the two light intensities, although more 15N-glycine was incorporated into microbial biomass than in plants in the initial 4 h under low light intensity. Light had a significant effect on 15 NO 3 uptake by maize, but no significant effects 15 on the uptake of 15 NHþ N-glycine. High light 4 or intensity significantly increased plant uptake of 14 15 NO C. Based on 14C to 15N 3 and glycine recovery ratios of plants, intact glycine contributed at least 13% to glycine-derived nitrogen 4 h after

Responsible Editor: Angela Hodge. X. Xu (*) : Y. Kuzyakov Institute of Soil Science and Land Evaluation, University of Hohenheim, Emil-Wolff-Strasse 27, 70599 Stuttgart, Germany e-mail: [email protected] X. Xu Key Laboratory of Ecosystem Network Observation and Modelling, Institute of Geographic Sciences and Natural Resources Research, The Chinese Academy of Sciences, P.O. Box 9719, Beijing 100101, People’s Republic of China

A. Richter : W. Wanek Department of Chemical Ecology and Ecosystem Research, Vienna Ecology Centre University of Vienna, Althanstrasse 14, A-1090 Wien, Austria Y. Kuzyakov Department of Agroecosystem Research, University of Bayreuth, 95440 Bayreuth, Germany

C. F. Stange Helmholtz Centre for Environmental Research-UFZ, Department of Soil Physics, Theodor-Lieser-Str. 4, 06120 Halle/Saale, Germany

DO09519; No of Pages

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tracer additions, but it contributed only 0.5% to total nitrogen uptake. These findings suggest that light intensity alters the competitive relationship between maize roots and rhizosphere microorganisms and that C4 cereals such as maize are able to access small amounts of intact glycine. We conclude that roots were stronger competitor than microorganisms for inorganic N, but microorganisms out competed plants during a short period for organic N, which was mineralized into inorganic N within a few hours of application to the soil and was thereafter available for root uptake. Keywords Competition . Light . Inorganic nitrogen . Organic nitrogen . Soil microorganisms . Maize . 14C . 15N . Rhizosphere

Introduction Nutrients, light and water availability are the most important factors controlling primary production in terrestrial ecosystems (Grime 1979; Tilman 1988; Vitousek and Howarth 1991; Olff et al. 1993; Aerts and Chapin 2000). In order to test the resource competition theory which uses a minimum concentration (R*) of a limiting resource to predict the competitive outcome (Tilman 1982), attention has been paid to the competition for nutrients and light between plant species (De Montard et al. 1999; Schippers and Kropff 2001; Passarge et al. 2006, Miller et al. 2007). A growing number of studies have shown that a variety of terrestrial plants have the capacity to take up intact organic N in the form of low molecularweight substances (Melin and Nilsson 1953; Chapin et al. 1993; Kielland 1994; Schimel and Chapin 1996; Lipson and Monson 1998; Näsholm et al. 1998; Persson et al. 2003; Xu et al. 2004). It has been suggested that organic N serves as an important N source for plants in ecosystems with low inorganic N status (Chapin et al. 1993; Kielland 1994; Schimel and Chapin 1996; Henry and Jefferies 2003; Xu et al. 2006). Consequently, a large number of studies have focused on the comparison of inorganic and organic N acquisition by plants in a wide variety of terrestrial ecosystems. While some studies showed lower uptake rates of organic N than of inorganic N (Henry and Jefferies 2003), others demonstrated that many plants are able to take up organic N at similar rates or faster than inorganic N (Henry and Jefferies 2003; Cheng

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and Bledsoe 2004; Thornton and Robinson 2005; Weigelt et al. 2005). It has also been shown that inorganic and organic N uptake by plants is influenced by temperature (Thornton and Robinson 2005) and plant species (Weigelt et al. 2005; Miller et al. 2007). However, plants compete for N not only with other plants, but also with microorganisms in the rhizosphere (Kaye and Hart 1997). Hence, competition for inorganic and organic N between plants and soil microorganisms has been explored under both field and controlled conditions. Although it has been suggested that soil microorganisms compete well with plants for organic and inorganic N inputs (Bardgett et al. 2003; Cheng and Bledsoe 2004), some plant species showed a better capacity to acquire inorganic N (Jaeger et al. 1999) and organic N (Lipson and Monson 1998; Lipson et al. 1999) in the face of microbial competition. As another important environmental factor, light has been suggested to mediate plant growth, e.g., Strengbom et al. (2004) have recently shown that light not nitrogen limits the growth of a grass species in boreal forests. Baligar et al. (2006) have reported that light intensity affects growth and nutrient uptake by legume cover crops. This implies that light affects the accumulation of organic matter and biomass via photosynthesis. Hence, light management has been considered as an effective approach in agroecosystems (Holt 1995). Light quantity and quality have an obvious role in plant biomass production however little is known about the effects on microbial competition for soil nutrients although inorganic N uptake by agricultural crops has been extensively studied (Mengel and Kirkby 1987; Haynes 1986; Loomis and Connor 1992). Due to the large quantities of inorganic N fertilizers that are applied to obtain high crop yields the importance of organic N may be insignificant for crops under conventional agriculture. Despite this, a growing body of evidence shows that some agricultural plant species are able to take up organic N in the form of amino acids under artificial conditions (Virtanen and Linkola 1946; Shobert and Komor 1987; Jones and Darrah 1994; Okamoto and Okada 2004), as well as under field conditions (Yamagata and Ae 1999; Matsumoto et al. 2000; Näsholm et al. 2000, 2001). Maize (Zea mays L.) is the third most important crop globally, after wheat and rice (FAO 2000). Many studies have been conducted to improve the under-

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standing of inorganic and organic N utilization by maize, e.g., it has been suggested that light regulates nitrate reductase in green leaves of maize (Beevers et al. 1965; Lillo 1994; Raghuram et al. 1999). Meanwhile, Meshram and Shende (1982) reported that Azotobacter inoculation enhances total N uptake by maize, while Schortemeyer et al. (1993) using hydroponics showed that mixed ammonium and nitrate nutrition is beneficial to maize growth. Moreover, it has been shown that maize can take up N from 15N labeled biomass of Paraserianthes facataria (Chintu and Zaharah 2003). Okamoto and Okada (2004) showed that maize roots have a lower capacity to take up protein N than sorghum and rice. The literature suggests that maize has the capacity to take up nitrate, ammonium and organic N which is corroborated by the evidence that transporters for nitrate, ammonium and amino acids are present in maize roots (Miller and Cramer 2004). However, up to date little is known about the effects of light on the competition for inorganic and organic N between maize and rhizosphere microorganisms especially in an environment with high N status. Therefore, the objectives of this study were to (1) determine if maize is able to access organic N in an inorganic N enriched environment, (2) compare organic and inorganic N uptake by maize, and (3) test if light affects the competition for organic and inorganic N between maize and soil microorganisms.

Materials and methods Soil Soil material was collected from the Ap horizon of a loamy Haplic Luvisol (long-term field experimental station Karlshof of Hohenheim University; 48°42′ 44.37″ N, 9°11′24.72″ E). The soil originated from loess, contained no CaCO3, and had the following characteristics: pH 6.0, organic C 1.2%, total N 0.13%, clay 23%, silt 73%, and sand 4.4%. The soil was air-dried and sieved through a 2-mm screen before the experiment. Experimental layout Pots (10 cm in height, 5 cm in diameter) were filled with 50 g air-dried soil. Single maize (Z. mays L.)

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seedlings of similar size were planted into them two days after germination. The plants were grown in a growth chamber for 21 days, set to a 14 h daytime period, light intensity 800 µmol m−2 s−1, with daytime and nighttime temperature of 27±1°C and 22±1°C. Soil moisture was maintained at 30% of soil dry weight by watering to weight daily. All treatments were supplied with 10 ml 0.2 mmol l-1 NH4NO3 solution per pot two weeks after planting, equal to 56 μg N g-1 soil. Three weeks after planting treatments were imposed, a mixture of glycine, NHþ 4 ,    þ and NO3 1 : 1 : 1 N  glycine N  NH4 N  NO 3 was injected into the soil, equal to 19 μg N g−1 soil. In each of the three treatments only one of the three N forms was labeled with 15N (98.2% 15N enrichment 15 for 15 NO NHþ 3 , 98.4% enrichment for 4 , and 95.0% enrichment for glycine). Uniformly labeled 14C glycine was applied at 0.90 kBq per pot (equivalent to 0.90 kBq per plant) to allow determination of intact organic N uptake over the experimental period as 15 N-labeled amino acids cannot represent actual intact amino acid uptake over long-term periods. Plants which were not injected with a N tracer were supplied with equivalent amounts of water and were taken as controls. After fertilizer injection plants were immediately divided into two groups: one group was maintained under the same light conditions (high light intensity), the other group was subject to shading by reducing light levels to 10% of the incoming light (low light intensity). Therefore, a two factor design was construed: the first factor was N form with three þ treatments: 15N in NO 3 , NH4 or glycine; and the second factor was light intensity with two levels, 800 and 80 µmol m −2 s −1, during daytime. Three replicates were collected from each treatment and at each sampling time. Sampling and analyses Three individual pots from each treatment were randomly destructively sampled at 4, 24 and 48 h after tracer additions. The roots were carefully removed from the soil and quickly rinsed with water, then soaked in 0.5 mmol l−1 CaCl2 solution for 30 mins, and again rinsed with distilled water. The roots and leaves were microwaved for 90 s, 600 W, and dried at 60°C for 48 h. Dried samples were weighed and ground to a fine powder using a ball mill (MM2, Fa Retsch).

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N incorporation into microbial biomass was determined by chloroform fumigation (Brookes et al. 1985)–15 g fresh soil was fumigated with chloroform for 24 h and then immediately extracted with 60 ml 0.5 M K2SO4. An additional soil sample (15 g) was extracted without fumigation. This extract was used for determination of ammonium and nitrate concentration. Total dissolved N in extracts was converted to nitrate by a modified alkaline persulfate digestion method (Sollins et al. 1999). Nitrate concentrations and isotope ratios in extracts and persulfate digests were analysed by SpinMas system (Stange et al. 2007). In the SpinMas system nitrate in the aqueous samples (up to 10 ml) was reduced to NO with V(III) Cl3 solution (Merck, Darmstadt, Germany) in acidic medium (HCl; Merck, Darmstadt, Germany)  3þ NO þ 4Hþ ! NO þ 3V4þ þ 2H2 O . The 3 þ 3V NO gas produced was transferred in a permanent He carrier stream (10 ml min−1, Linde, Germany) from the vial via an open split to the inlet capillary of the quadrupole mass spectrometer (GAM 400, InProcess Instruments GmbH, Bremen, Germany). Water and CO2 were removed by a cryo trap (−100°C). For nitric oxide the ion currents [I] at m/ z 30 and 31 were measured allowing the calculation of the 15N abundance of the NO 3 . A correction was made for the presence of naturally abundant 17O (0.037 at.%). Ammonium concentrations and isotope ratios were also quantified by SpinMas based on the chemical conversion of NHþ 4 to N2 i.e. oxidation by NaOBr solution in alkaline medium (Stange et al. 2007). Gross N mineralization and nitrification rates were measured by 15N pool dilution technique. Dissolved organic C (DOC) in extracts was measured by a Dimatoc automatic analyzer (Dimatec Essen, Germany). For 14C analyses, plant samples (50 mg) were combusted in an oxidizer (Model 307, Canberra Packard Ltd., USA). 14CO2 was trapped in CarboSorb E (Perkin Elmer Inc., USA) and scintillation cocktail (Permafluor E+, Perkin Elmer Inc., USA). 14 C activities in these samples were then measured by liquid scintillation counting (Rackbeta, 1419 LKB, Wallac). Dry ground plant samples were weighed (2 mg) into tin cups for elemental N and 15N analysis, which was performed by continuous-flow gas isotope ratio mass spectrometry (CF-IRMS). The CF-IRMS system consisted of an elemental analyser (EA 1110, CE Instruments, Milan, Italy), a ConFlo II interface

(Finnigan MAT, Bremen, Germany) and a gas isotope ratio mass spectrometer (DeltaPLUS, Finnigan MAT). Calculation and statistics 15

N calculations were based on atom percent excess (APE), calculated as the difference between 15N treated and control plants. 15N recovery in plants (15NRplant), as percentage of input, was calculated as the amount of 15N taken up by plants divided by total 15 N added per pot. 15N recovery in inorganic pools, as percentage of input, was calculated as the amount of 15 N recovered in the nitrate/ammonium pool divided by total 15N added per pot. Because 15N uptake is located in the roots, 15N uptake rates were calculated by dividing total plant 15N at time t by root dry weight in grams. In situ uptake rates of the three  N  forms were calculated as follows: Root NHþ 4 NO3 þ uptake was calculated dividing root 15 NH4 15 NO 3  15  uptake by at % N in the NHþ 4 NO3 pool after 4 h in  the NHþ 4 NO3 treatment, whereas root glycine uptake was estimated dividing root 15N-glycine uptake by 95.0 at % 15N and multiplying with ratio of 14CR to 15NR of plants. 15N in microbial biomass (15NRMB) was calculated as the difference in 15N mass between fumigated and non-fumigated soil samples, divided by total 15N added per pot (Zogg et al. 2000). A KEN factor (0.54) has been used to correct for the incomplete extraction of microbial N in a large number of studies, but the correction factor often varies dependent on the analysis method (Joergensen 1996). In this study the correction factor didn’t alter the relationship between plants and soil microorganisms as well as microbial uptake of the three N species (Fig. 6), only changing the value of the microbial 15N uptake. Furthermore, in short-term 15 N uptake experiments soluble 15N and insoluble 15N are in disequilibrium and the KEN factor would therefore lead to overestimation of the microbial 15 N sink. We therefore did not apply the correction factor to correct microbial 15N uptake. 14C in plants (14CRplant) was calculated as the amount of 14C taken up by plants divided by total 14C added per pot. 14C in microbial biomass (14CRMB) was calculated as the difference in 14C between fumigated and nonfumigated soil samples, divided by total 14C added per pot. 14C recovery in dissolved organic matter (DOM, 14CRDOM) was calculated as the 14C amount recovered in extracts of unfumigated soil samples

divided by total added 14C. Gross mineralization and nitrification rates were calculated based on the 15N pool dilution approach using the equations developed by Kirkham and Bartholomew (1954). Repeated measures analysis of variance was used to test for the effects of 15N form added, light, time and their interactions on 15N uptake by plants and by soil microorganisms. Two-way analysis of variance was used to test for the effects of light, time and light x time on 14C uptake by plants. All results were considered significant at the P<0.05 level. Statistical calculations were run using a SPSS 11.5 statistical software package. SE of means are presented in the figures as a variability parameter.

Results Plant biomass Light intensity significantly affected total plant biomass 48 hours after imposing the light treatment (P<0.01, Fig. 1). Root to shoot ratios were not significantly influenced by light treatment and were on average about 0.43 (Fig. 1). N turnover in the soil Due to rapid N turnover in the soil, the variation between the replicates was high in both ammonium 250 Shoots

Low light High light

Biomass (mg per pot)

200

**

150 100 50 0 42

4

48

24

48

50 100

Roots

150 4

Time (h)

Fig. 1 Shoot and root biomass of maize seedlings under low and high light intensity. A mixture of three N forms was amended to each pot, where only one N form was labeled with 15 N at a time. Asterisks indicate significant differences of plant biomass under the two light conditions. Values are means (±SE) of three replicates

Gross mineralization (µg N g-1 soil d.w. d-1)

63 7

Gross nitrification (µg N-NO3- g-1 soil d.w. d-1)

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10

a 6

Low light High light

5 4 3 2 1 0

b

**

8

6

** 4

2

0 24

48

Time (h)

Fig. 2 Gross mineralization a and nitrification b rates under low and high light intensity. Asterisks indicate significant differences of gross mineralization/nitrification rates under the two light conditions. Values are means (±SE) of three replicates at each time

concentration (4.7±0.3 μg N g−1 soil d.w.) and 15N abundance of NHþ 4 . Therefore gross N mineralization rate using the pool dilution approach in the 15 NHþ 4 labeled treatments yielded high standard errors. No statistical difference was observed between high and low light treatments and gross mineralization rates averaged 2.1±4.3 μg N g−1 soil d.w. day−1 (Fig. 2a). 15 N recovery in the NHþ 4 fraction decreased to <12% of tracer input after 4 h (Fig. 3). During the chase period, 15N recovery in the ammonium pool in the 15 NO 3 treatment was low while there was no significant difference for 15N recovered in the ammonium pool in the 15N-glycine and -ammonium treatments 24 h after tracer additions (Fig. 3 e,f). Fast turnover of the NO 3 pool allowed an accurate calculation of gross nitrification rates in the soil using 15 N pool dilution techniques. It was shown that high light significantly enhanced gross nitrification rates. During the first 24 h period after tracer injection,

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a

15

Glycine N labeled 15 Ammonium N labeled 15 Nitrate N labeled

9

6

3

0 15

b 12

9

6

recovered in the ammonium pool (as % of added input)

12

15

15N

15N

recovery by soil microorganisms (as % of added input)

15

3

0 80

12

9

6

3

0 15

f 12

9

6

3

0 80

g recovered in the nitrate pool (as % of added input)

c

40

20

0 80

d 60

40

15N

recovery by plants (as % of added input)

60

15N

e

20

0

4

24

48

Time (h)

gross nitrification rate was 6.7±0.4 μg g−1 dry soil day−1 under low light intensity and 8.8±0.7 μg g−1 dry soil day−1 under high light. During the second 24 h, gross nitrification rates decreased strongly. They

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40

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0 80

h 60

40

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0

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Time (h)

were 1.5±0.3 μg g−1 dry soil day−1 under low light and 3.6±1.2 μg g−1 dry soil day−1 under high light (Fig. 2). Fast turnover of glycine and ammonium and high nitrification rates led to high concentrations (9.6±

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R

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Fig. 3 Proportion of 15N recovered by maize seedlings and soil microorganisms as well as inorganic N pools under different light intensities. a Soil microorganisms under low light intensity; b soil microorganisms under high light intensity; c plants under low light intensity; d plants under high light intensity; e soil ammonium pool under low light intensity, f soil ammonium pool under high light intensity, g soil nitrate pool under low light intensity, h soil nitrate pool under high light intensity. A mixture of three N forms was amended to each pot, where only one N form was labeled with 15N at a time. Values are means (±SE) of three replicates. Note different Y scales for the different pools

acquisition by microorganisms was similar for NHþ 4 and NO 3 (Fig. 3a). Microorganisms took up more 15 N from NHþ 4 than from either of the other N forms under high light intensity (except 48 h after tracer injection). 15NRMB for 15 NHþ 4 decreased substantially from 10.5±0.5% at 4 h to 0.2±0.02% at 48 h. Uptake of 15N-glycine by microorganisms showed a similar 15 15 trend as for 15 NHþ N-glycine declining 4 , NRMB of from 3.2±0.02% at 4 h to 1.1±0.05% at 48 h. In contrast, 15NRMB for 15 NO 3 slightly increased from 4 h to 24 h after tracer addition and decreased thereafter (Fig. 3b). These results show that the maximal uptake of 15N by soil microorganisms peaked within 4 h for amino acids and NHþ 4 and within 24 h for NO . 3 Under low light intensity, maize seedlings acquired three to four times more 15N from NO 3 than from 15 either 15 NHþ or N-glycine during the first 4 and 4 24 h. However, plants almost acquired an equivalent fraction of 15N from the three N forms at 48 h after tracer addition (Fig. 3c). 15 NHþ 4 uptake by plants substantially increased from 1.3±0.2% at 4 h to 40.7 ±1.8% at 48 h. Under low light, 15N-glycine uptake by plants showed very similar values as 15N uptake from NHþ 4 (Fig. 3c). Under high light, plants showed a substantial increase in 15NRplant with time for all three N forms (Fig. 3d). Maize seedlings took up 15 much more 15 NO N-glycine or 15 NHþ 3 than either 4 during the whole experimental period whereas 15Nglycine and 15 NHþ 4 uptake were similar under high light conditions.

0.6 μg N g−1 soil d.w.) and 15N recoveries of NO 3 in 15 N-glycine and 15 NHþ treatments, almost equal to 4  15 the 15N recoveries in NO of the NO treatment 3 3 (>50%; Fig. 3). 15N recovery in the nitrate pool during the initial 4 h was not affected by N form and light level. With time less 15 NO 3 was recovered (Fig. 3 g,h) due to rapid uptake for NO 3 by plant roots. 15

N recovery and N uptake on a plant basis

Repeated measures analysis of variance indicated significant effects of 15N form, light and time as well as their interactions on 15NRplant of maize seedlings (except N form × light) and 15NRMB of soil microorganisms (P<0.05, Table 1). Light therefore had a significant effect on 15N uptake by maize seedlings and soil microorganisms (Fig. 3). Under low light intensity, microorganisms immobilized more 15N 15  from glycine than from NHþ N 4 or NO3 , and

Table 1 Results of repeatedmeasures ANOVA for the effects of 15N form added, light, time and their interactions on 15N uptake by plants and by soil microorganisms

Components

Sources of variation

F values

P values

Plants

15

72.52 4.37 185.74 2.94 8.66 6.51 3.55 285.36 135.05 352.08 578.31 147.15 207.77 159.48

0.00 0.04 0.00 0.07 0.00 0.00 0.02 0.00 0.00 0.00 0.00 0.00 0.00 0.00

Microbial biomass

N form Light Time 15 N form x light 15 N form x time Light x time 15 N form x light x time 15 N form Light Time 15 N form × light 15 N form × time Light × time 15 N form × light × time

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Table 2 Results of repeatedmeasures ANOVA for the effects of light, time, and light × time on 14C uptake by plants

Components

Sources of variation

F values

P values

Plants

Light Time Light × time Light Time Light × time Light Time Light × time

52.99 30.65 2.10 0.15 3.88 0.92 1.15 27.84 1.88

0.00 0.00 0.13 0.70 0.03 0.41 0.29 0.00 0.17

Microbial biomass

Dissolved organic carbon

1.0

a

.6

.4

.2

0.0 6

b 5 4 3 2 1 0 18

NR ratio of plants and microbial biomass C recovered in DOC (as % of added input)

Under low light, 14CR to 15NR ratios of microbial biomass were in the range of 0.34 and 0.53, with the lowest value at 48 h and the highest value at 24 h (Fig. 5a). The values between 24 and 48 hours were significantly different (P<0.05, Fig. 5a). By comparison, 14CR to 15NR ratios of microbial biomass increased with time from 0.60±0.13 at 4 h to 3.40± 0.14 at 48 h under high light (Fig. 5a). Under low light, 14CR to 15NR ratios of plants were 0.18±0.02 in the initial 4 h after tracer injection, which was nearly nine times higher than the values at 24 and 48 h. 14CR to 15NR ratios were similar for plants under high light and low light, except 24 h after tracer injection (Fig. 5a).

16

14

CR to

15

14 C uptake by plants (as % of added input)

Repeated measures analysis of variance indicated significant effects of light and time on 14CRplant (P < 0.05, Table 2). Time significantly affected 14 CRMB and 14CRDOM while light and interactions of light x time had no significant effects on them (Table 2). In contrast, high light significantly enhanced 14C uptake by maize seedlings (Fig. 4a). 14 CRplant increased from 0.48±0.06% at 4 h to 0.91± 0.06% at 48 h under high light intensity. Uptake of glycine (measured as 14C) under low light intensity reached a maximum after 24 h and was 30–50% less than under high light (Fig. 4a). 14CRDOM was much higher than 14CRMB at all times and in both light treatments. 14CRMB increased while 14CRDOM declined slightly with time (Fig. 4b,c).

C uptake by microorganisms (as % of added input)

C recovery in maize seedlings, microbial biomass and DOM

14

14

14

Low light High light

.8

c

14 12 10 8 6 4 2 0 4

24

48

Time (h)

Fig. 4 Percent of added 14C input recovered in a maize seedlings, b soil microorganisms and c dissolved organic carbon under low and high light intensity. Values are means (±SE) of nine replicates. Note different Y scales for plants, microorganisms and DOM

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4

14

C to 15N recovery ratio

a Low light High light

3

**

2

** 1

0 .25

14

C to 15N recovery ratio

b .20

.15

.10

.05

** 0.00 4

24

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Time (h)

Fig. 5 14CR to 15NR ratio of a microbial biomass and b maize plants. Values are means (±SE) of three replicates. Note different Y scales for microorganisms and plants. Asterisks indicate significant differences of 14CR to 15NR ratio of microbial biomass and maize plants between the two light conditions

Discussion Competition for inorganic and organic N between soil microorganisms and plants Numerous studies have shown that plant roots and rhizosphere microorganisms compete intensely for available N in the rhizosphere and that microorganisms are stronger competitors in the short-term (Jackson et al. 1989; Schimel et al. 1989; Zak et al. 1990; Schimel and Chapin 1996; Kaye and Hart 1997; Hodge et al. 1999; Owen and Jones 2001; Bardgett et al. 2003; Cheng and Bledsoe 2004). However, a few studies also showed contradictory results e.g. that some plant species were better able to acquire inorganic (Jaeger et al. 1999) or organic N (Lipson and Monson 1998) in the face of microbial competition. A possible explanation for this is that the competition for N between plants and soil micro-

organisms is strongly dependent on available N supplies in the soil, based on the hypothesis that only N in surplus of microbial growth is available for plants. In the current study we added inorganic N to the soil in the form of NH4NO3 at a rate of 56 μg N g−1 dry soil, resulting in high available N supplies for plant roots and rhizosphere microorganisms before tracer additions. This is confirmed by high mean NO 3 concentration (9.6±0.6 μg N g−1 soil d.w.) during the trace period. Hence, it was not surprising that maize out-competed soil microorganisms for 15N from NO 3 both under low and high light intensities (Fig. 3c,d). Higher 15NRplant and greater 15N acquisition by plants from 15 NO 3 treatment can result from preferential NO uptake by maize or because the NO 3 3 ion is more mobile in soil solutions (Jackson et al. 1989; Schimel et al. 1989; Owen and Jones 2001) therefore often serving as the most convenient N source for crop plants (Raven 2003). What’s more, there exists a high affinity nitrate transport system in maize roots (Aslam et al. 1992; Glass and Siddiqi 1995) that confers a high capacity for root NO 3 uptake. Although a large amount of inorganic N was amended one week before tracer additions, plant uptake of 15N-glycine during the initial 4 h (0.03 and 0.06 mg N g−1 root h−1 under low and high light, respectively, Fig. 6) was comparable to uptake rates published for other grass species (ca 0.03 to 1.35 mg N g−1 root h−1, Raab et al. 1999; ca 0.01 to 0.12 mg N g−1 root h−1, Xu et al. 2004). This clearly indicates that maize is able to access organic N even in an inorganic N enriched environment. 15N-glycine uptake was very similar to 15 NHþ 4 uptake by maize seedlings under the two light conditions. This might be explained by rapid decomposition of glycine by microorganisms to inorganic N (Kuzyakov and Demin 1998; Jones 1999; Jones and Kielland 2002; Nordin et al. 2004) and thereafter uptake of 15N from  glycine in the form of NHþ 4 or NO3 4 h following tracer addition. In this study we didn’t determine the rate of glycine turnover in the soil. However, nearly 60% of added 15N from glycine appeared in the nitrate pool in the initial 4 h following tracer addition (Fig. 3 g,h). This clearly implies that rapid decomposition of glycine occurred in soils as observed by other studies (Kuzyakov and Demin 1998; Jones 15 1999), providing available 15 NHþ NO 3 for plant 4 or uptake. As a result, the contribution of organic N to plants might be strongly overestimated by using

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a Glycine 15N labeled Ammonium 15N labeled Nitrate 15 N labeled

3

2

1

15

N uptake (mg

15

-1

N g root d.w.)

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N uptake (mg

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-1

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0 4

0 4

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Time (h)

Fig. 6 Root 15N uptake by maize seedlings under a low light intensity and b high light intensity. A mixture of three N forms was amended to each pot, where only one N form was labeled with 15N at a time. Values are means (±SE) of three replicates

only 15N-labelled amino acids without 14C or 13C tracer because of rapid mineralization of added amino acids. The rapid mineralization of glycine and the rapid nitrification of ammonium (Fig. 3 g,h) hamper a similar interpretation of the measurements because 4 h after application of tracers all pools were highly enriched with 15N. This implies that the competition for nitrate in this soil is more important than for glycine and ammonium and therefore the maize roots benefited from fast mineralization and nitrification compared to microorganisms. Effects of light on the competition for inorganic and organic N between maize and rhizosphere microorganisms Light had a significant effect on 15 NO 3 uptake by maize seedlings (P<0.01, Table 1 and Fig. 6). This is

corroborated by other studies showing that light is involved in the regulation of nitrate reductase gene expression in maize (Raghuram et al. 1999) and in the uptake of NO 3 by plant roots (Miller and Cramer 2004). Light had no significant effect on 15N uptake by maize from NHþ 4 or glycine pools (Fig. 6), whereas it significantly affected 14C uptake by maize seedlings (Table 2 and Fig. 4). A possible explanation is that 15N uptake derived from rapid decomposition of glycine conceals the uptake of intact glycine because plant roots compete directly for glycine in the first several hours following tracer additions and thereafter, after glycine depletion, plant roots acquire 15N released from rhizosphere microorganisms as NHþ 4 (McFarland et al. 2002). Moreover, in this study plant roots might take up more 15N from glycine as NO 3 derived from the rapid decomposition of glycine and subsequent nitrification (Fig. 3 g,h). Compared to the effect of light on inorganic and organic N uptake by plants, light cannot directly affect rhizosphere microorganisms. However, there is a difference in the uptake of 15N  from NHþ 4 , NO3 and glycine by rhizosphere microorganisms between low and high light (Table 1). We here ascribe the difference to two major reasons. Firstly, under low light plants have lower transpiration and uptake rates and therefore rhizosphere microorganisms have more chance to take up N than under high light condition, e.g. more glycine was incorporated into microbial biomass under low light whereas this was reversed under high light. Besides, glycine is often considered as a relatively poor substrate for soil microorganisms (Lipson et al. 1999), though it contains two “C” atoms per molecule which can trigger microbial uptake of glycine (and 15N) as a carbon source under low light. Secondly, under high light intensities plants release more C into the soil (Kuzyakov and Cheng 2001). Root exudates contain many amino acids and carbohydrates and are more appropriate for microorganisms than glycine (Yano et al. 1998; Lipson et al. 1999) which may facilitate the microbial acquisition of nutrients from soil (Jones et al. 2004). This might have been the case, since more 15 N from NHþ 4 was recovered in microbial biomass under high light, except 48 h after tracer addition (Fig. 3). The decrease of 15N from NHþ 4 in microbial biomass might result from rapid microbial oxidation

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contribution of intact glycine molecules to glycinederived 15N acquisition by maize seedlings, based on 14 CR to 15NR ratios of plants. Although 14C uptake by maize indicates that light had a significant positive effect on organic N uptake, it had no significant effect on the contribution of intact glycine molecules to glycine-derived 15N acquisition by maize (Fig. 4b). Based on 14CR to 15NR ratios of plants, intact glycine

80

a

Low light High light

60

40

Ratios of 15N uptake by plants to 15N uptake by microorganisms

of NHþ 4 i.e. nitrification and subsequent uptake by plants (Figs. 2, 3). In this study very high 15NR from 15 N-glycine or 15 NHþ 4 in the nitrate pool also indicates the occurrence of rapid N transformation processes in this experiment. In contrast, 15 N recovered by microbial biomass from NO 3 decreased with time under high light (Fig. 3). This also can be ascribed to a stimulation of 15N uptake by maize from NO 3 pool by high light, thus reducing the microbial sink for 15N from NO 3. A number of studies have shown that exudates released by roots in the form of a wide variety of organic compounds are most directly linked to photosynthesis (Meharg 1994; Uren 2001; Kuzyakov and Cheng 2001, 2004). These exudates are rapidly utilized by microorganisms in the rhizosphere and regulate rhizomicrobial activity (Anderson and Domsch 1985; Smith and Paul 1990; Kuzyakov and Cheng 2001). In the current study high light enhanced nitrification rates and thereby resulted in high NO 3 concentrations in soil solution. This indicates that high light intensity might indirectly stimulate the activity of nitrobacteria by increasing the amount of root exudates. In order to provide a better insight into the competition for inorganic and organic N between plants and soil microorganisms, we here calculated the ratios of 15N uptake by plants to that by rhizosphere microorganisms (Fig. 7). These results clearly indicate that light strongly mediates the competition for inorganic and organic N between plants and rhizosphere microorganisms. Plant roots out-competed rhizosphere microorganisms for 15N from NO 3 under both light conditions over the whole experimental period (Fig. 7c). Although rhizosphere microorganisms out-competed plants for ammonium and glycine under low light during the initial 4 h, high light facilitated effective competition for glycine by plants (Fig. 7a). By comparison, high light enhanced the competition for NHþ 4 by rhizosphere microorganisms in the initial 24 h whereas it strongly stimulated the competition by plants during the later 24 h (Fig. 7b).

69

20

0 180 Time (h)

b 150 120 90 60 30 0 80

c 60

40

20

0 4

24

48

Time (h)

Contribution of organic and inorganic N to N acquisition by plants In this study, application of dual-labeled (14C, 15N) glycine permitted a rough estimation of the minimal

Fig. 7 Ratios of 15N uptake by plants to that by soil microorganisms 4, 24 and 48 h after injecting 15N labeled glycine a, ammonium b and nitrate c. A mixture of three N forms was amended to each pot, where only one N form was labeled with 15N at a time. Values are means (±SE) of three replicates

70

molecules contributed at least 13% to the total N acquisition derived from glycine 4 h after tracer addition (Fig. 4b). This value was slightly lower compared to estimates for field grown wheat by Näsholm et al. (19–23%; 2001), using 13C and 15N labeled glycine. One day after tracer addition the contribution of intact glycine to glycine-derived 15N uptake declined sharply (Fig. 4b). This is a result of 14 C release by respiration but also of continued 15N uptake from inorganic N derived from rapid decomposition of glycine. Similar processes may have hindered the detection of intact dual-labeled glycine uptake in tundra plants 24 h after tracer addition (Schimel and Chapin 1996). Consequently, we should be cautious to draw conclusions when estimating organic N uptake by plants using 13C or 14C labeled amino acids over longer time scales. The most critical points to accurately estimate organic N uptake are the fraction of C isotopes taken up by plants that is subsequently lost via plant respiration and the fast turnover and breakdown of amino acids in soils (Jones 1999; Jones and Kielland 2002). Uptake rates of glycine, ammonium and nitrate were estimated to be about 0.04±0.02, 2.62±2.33 and 4.28 ± 1.20 mg g−1 root d.w. under high light conditions over 4 h. Under low light conditions they were estimated to be 0.02±0.01, 1.13±0.52 and 2.55± 0.52 mg g−1 root d.w., respectively. Based on these uptake rates, the contribution of nitrate to the total N uptake was estimated to be 69% under low light and 62% under high light whereas it was 31% under low light and 38% under high light for ammonium. Although at least 13% of glycine-derived 15N acquisition was due to uptake of intact glycine molecules, the contribution of organic N in the form of intact glycine to total N uptake was very small. It only contributed about 0.6% under low light and 0.5% under high light to total N uptake by maize. This calculation does not consider respiratory losses of the 14C label taken up by plants, but implies that crop uptake of organic N is only of minor importance in agricultural soils amended with a large amount of inorganic fertilizer. Acknowledgements We kindly thank DAAD K. C. Wong Fellowships for awarding Dr. Xu a fellowship to support this study in University of Hohenheim (Germany). The German Research Foundation supported the study. We also thank the two anonymous reviewers for their helpful comments and Rebecca Hood for language improvement.

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