Phosphorus availability and plants alter soil nitrogen retention and loss

Science of the Total Environment 671 (2019) 786–794

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Phosphorus availability and plants alter soil nitrogen retention and loss Kazi R. Mehnaz ⁎, Claudia Keitel, Feike A. Dijkstra Sydney Institute of Agriculture, School of Life and Environmental Sciences, The University of Sydney, 380 Werombi Rd, Brownlow Hill, NSW 2570, Australia

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Initially low but later high levels of P addition increased N2O emission rates. • Plants influenced N2O rates by affecting available resources for denitrification. • Increased net N mineralization favored cumulative N2O emission from planted pots. • Both P addition and plant presence reduced NO− 3 leaching and increased N retention.

a r t i c l e

i n f o

Article history: Received 10 December 2018 Received in revised form 26 March 2019 Accepted 26 March 2019 Available online 28 March 2019 Editor: Charlotte Poschenrieder Keywords: 15 N recovery Denitrification N mineralization N2O emission NO− 3 leaching P addition

a b s t r a c t Availability of phosphorus (P) can directly and/or indirectly affect nitrogen (N) retention and loss from soil by stimulating microbial and plant root activities. However, it is not clear how P availability and plant presence interact on nitrous oxide (N2O) emission and nitrate (NO− 3 ) leaching in soil. A mesocosm experiment was conducted to investigate the effect of P addition (0, 10 and 20 mg P kg−1) with and without plant presence 15 N recovery. Our results showed large variation (Phalaris aquatica, C3 grass) on N2O emission, NO− 3 leaching and in N2O emission with significant increases after leaching events. We observed that initially low but later (after 53 days of sowing) high levels of P addition increased N2O emission rates, possibly by stimulating nitrifiers and/or denitrifiers in soil. Plant presence decreased N2O emission at times when plants reduced water and − NO− 3 in the soil, but increased N2O emission at times when both water and NO3 in the soil were abundant, and where plants may have stimulated denitrification through supply of labile organic C. Furthermore, an increase in net N mineralization, possibly due to increased decomposition stimulated by root derived C, may also have contributed to the higher cumulative N2O emission with plant presence. P addition increased 15N recovery in soil, but reduced it in leachates, suggesting increased 15N fixation in microbial biomass. Our results showed that both P addition and plant presence stimulated N loss as N2O, but also increased N retention in the soilplant system and thus reduced N loss through leaching. © 2019 Published by Elsevier B.V.

⁎ Corresponding author. E-mail address: [email protected] (K.R. Mehnaz).

https://doi.org/10.1016/j.scitotenv.2019.03.422 0048-9697/© 2019 Published by Elsevier B.V.

K.R. Mehnaz et al. / Science of the Total Environment 671 (2019) 786–794

1. Introduction Nitrogen (N) is one of the most essential elements required by all living organisms to synthesize proteins, amino acids, nucleic acids and cofactors (Thomson et al., 2012). However, losses of N from soil through leaching, volatilization and denitrification not only affect primary productivity by reducing soil fertility but can also cause degradation of environmental quality (Cameron et al., 2013). Nitrous oxide (N2O) emission, an important pathway of N loss, can contribute to stratospheric ozone depletion (Ravishankara et al., 2009) and the greenhouse effect (Forster et al., 2007). Approximately 70% of global N2O emissions come from microbial nitrification and denitrification processes in the soil (Butterbach-Bahl et al., 2013). Leaching of nitrate (NO− 3 ) from soil to aquatic systems can affect human and animal health by contaminating drinking water, and cause death of fish and other aquatic organisms due to eutrophication (Brady and Weil, 2002). Plant growth and soil microbial processes are directly and indirectly responsible for N transformations and associated losses and retention, which in turn are affected by other nutrients in soil, such as phosphorus (P) (Martinson et al., 2013). Microbial nitrification and denitrification and associated N2O emissions are controlled by various factors in soil, such as soil ammonium − (NH+ 4 ) and NO3 concentrations, pH, temperature, and availability of oxygen, water and labile organic carbon (C) (Dalal et al., 2003; Firestone and Davidson, 1989; Signor and Cerri, 2013). Because of the high P requirement of microbes for building cellular and genetic components, as well as for metabolism and energy transfer (Sosa, 2017), availability of P in soil can also influence N2O production from microbially mediated processes. However, any change in the stoichiometric relation of P with other nutrients, such as N, can also affect losses and retention of N through different pathways by affecting microbial as well as plant root activities. For instance, N2O emission from P-limited forest soils significantly increased after both short-term and long-term N addition compared to the N-limited forest soils, possibly due to limited microbial growth and N immobilization associated with low availability of P (Hall and Matson, 1999). Similarly, P limitation of heterotrophic microorganisms in coastal environments showed the potential to increase N loss as N2O through denitrification (Sundareshwar et al., 2003). In contrast, other studies found an increase in N2O emission with increased availability of P, which was explained by alleviation of P limitation of the nitrifying and/or denitrifying microbial community (Mehnaz and Dijkstra, 2016; Mori et al., 2010), stimulation of N mineralization resulting in increased inorganic N substrates for nitrification and denitrification (Mori et al., 2010), enhanced organic matter decomposition providing soluble C to denitrifiers (Ullah et al., 2016), and/or an increase in heterotrophic microbial activities in general and hence increased oxygen consumption promoting anaerobic conditions suitable for denitrification (Mori et al., 2013). Plants can also affect N loss as N2O since their growth is dependent on availability of mineral N, which is the core substrate for nitrification and denitrification. Plant uptake of N can reduce N2O emission by lowering the mineral N concentration of soil and hence increase N retention. However, plant litter and root exudates are important sources of C and energy for heterotrophic denitrifiers (Henry et al., 2008). Any effect of P fertilization on plant growth can thus influence N loss and retention in soil. For instance, N retention increased in P-fertilized plots due to enhanced root and associated mycorrhizal fungal uptake of N (Blanes et al., 2012), while N2O emission was reduced due to increased N uptake by maize after P addition (Baral et al., 2014). P induced suppression of N2O emission due to root N uptake was also observed in other studies (Mori et al., 2014; Zhang et al., 2014). Conversely, P addition to a P-poor soil reduced total 15N recovery in plants, microbes and soil, due to increased gaseous loss of N (He and Dijkstra, 2015). A P induced plant growth can be responsible for increased gaseous loss of N as N2O through denitrification by providing labile C substrates from root exudation (Rheinbaben and Trolldenier, 1984).

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High inputs of N through atmospheric deposition and/or fertilizer application compared to other nutrients can lead to ecosystem N saturation by exceeding its biotic demand which subsequently may result in increased leaching loss of N as NO− 3 (Aber et al., 1989; Dise and Wright, 1995). P limitation of plants and microbes can restrict their uptake and immobilization of N (Blanes et al., 2012) resulting in increased soil water NO− 3 concentration, while increased plant and/or microbial N utilization with P addition reduced NO− 3 leaching in grassland (Nielsen et al., 2009) and forest soils (Chen et al., 2017; Stevens et al., 1993; Wullaert et al., 2010). However, P addition can also stimulate mineralization of soil organic matter (Falkiner et al., 1993; White and Reddy, 2000) and nitrification (Hue and Adams, 1984), thereby increasing NO− 3 concentration with the potential for greater N leaching loss. On the other hand, enhanced denitrification with P addition (Mehnaz and Dijkstra, 2016) could potentially decrease leaching loss of N by keeping NO− 3 concentration low (Cameron et al., 2013). It is therefore not clear how P availability and plant presence interact on N loss through N2O emission and NO− 3 leaching, as well as on N recovery in soil plant systems. Here, we conducted a pot experiment to examine the effect of P addition (0, 10 and 20 mg P kg−1) and plant presence (Phalaris aquatica, C3 grass) on N2O emission and NO− 3 leaching from a P-fixing grassland soil. We added a 15N tracer (1 mg 15N kg−1) to the soil in all pots to assess P and plant presence effects on 15N recovery in soil, plant tissue and leachates. We hypothesized that increased addition of P will increase N2O emission due to stimulated nitrifying and denitrifying activities, induce greater uptake of N and 15N in plants and microbes, and thus less N and 15N would be recovered in leachates. We further hypothesized that, although plant presence could stimulate N2O emission by supplying C from root exudates, particularly at early stages of plant growth, N2O emission would be reduced at later stages of plant growth due to N uptake. 2. Materials and methods 2.1. Study area and soil used Soil was collected in April (mid-autumn) 2016 from a grassland at Westwood farm (latitude 33°59′46″S, longitude 150°39′16″E) near Camden in New South Wales, Australia. The mean annual precipitation of this area is 790 mm, and mean air temperature of July and January is 10.4 °C and 23 °C, respectively (Dijkstra et al., 2015). The dominating grass of the farm was Paspalum dilatatum Poir. (C4). The grassland was moderately grazed by cattle and not fertilized. The soil of this grassland was a sandy loam Red Kurosol (Australian Soil Classification, Isbell, 2002), or an Abruptic Acrisol (World Reference Base) with a pH of 5.58; 5.6% C; 0.4% N and 0.14% P. The soil also showed a P fixing capacity (Dijkstra et al., 2015) and relatively low extractable inorganic P concentrations (8.4 mg P kg−1 soil; extraction method described in Section 2.3). The extractable inorganic N content was 2.20 mg N kg−1 soil. Soil samples were collected at 0 to 20 cm depth from a single area of the grassland. After sieving through a 2 mm sieve, the soils were mixed well, and any visible roots were removed. 2.2. Experimental design Three kilogram of sieved soil was placed in each of the 24 polyvinyl chloride (PVC) pots (diameter 15 cm, height 20 cm) containing a 1 cm diameter plastic tube inserted at the bottom to facilitate leaching. The leaching tube inside the pot was covered by a layer of pebbles (500 g with 15–20 mm diameter) underneath the soil to prevent clogging of the pipe with soil particles. The pots were placed in a growth chamber at the Sydney Institute of Agriculture, in Camden, NSW. Temperature was set to 25 °C and 20 °C during day and night, respectively. Relative humidity was 70% during the day and 60% during the night. Twelve hours of daylight was generated using 1000 W metal halide lamps. A

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basal nutrient solution was added to all pots, containing the following nutrients: N (5.22 mg kg−1), K (113.69 mg kg−1), S (17.38 mg kg−1), Mg (12.66 mg kg−1), Ca (7.48 mg kg−1), Cu (0.04 mg kg−1), Mn (0.49 mg kg−1), Zn (0.27 mg kg−1), B (0.08 mg kg−1), and Fe (0.11 mg kg−1). We applied 15N labelled (99 atom%) potassium nitrate (KNO3; 1 mg N kg−1 soil) to all pots together with the basal nutrients. 0.4 g seeds of Phalaris aquatica, a widely used C3 grass for improved pasture in NSW, Australia, were sown in each of 24 pots, which resulted in approximately 50 seedlings per pot. 13 days after sowing (DAS), plants were removed from half of the pots whereas in the remaining half of the pots we maintained plants (plant presence treatment: without and with plants). This was due to enhance the probability of getting 12 identical pots with similar seedling growth, while plants were too small to cause any effect on the soil after removal. We added P in solution at three different levels (P addition treatment: 0, 10 and 20 mg P kg−1 soil) to all pots at three different times by equally splitting each amount. P was added as a mixture of monopotassium phosphate (KH2PO4) and dipotassium phosphate trihydrate (K2HPO4.3H2O) to get the final solution pH similar to soil pH, to avoid any P induced pH change in soil. The plant presence and P addition treatments were conducted in a full factorial design with four replicates for each treatment combination and randomly placed in the growth chamber. Soil moisture content of the pots was kept at 30% gravimetric (g/g) by watering the pots with DI water every second day to a specific target weight. 2.3. Sample collection, processing and analyses P was added at 19, 32 and 53 DAS. Gas samples were taken five times throughout the experiment at 20, 32, 34, 53 and 55 DAS. Leaching was induced twice at 33 and 54 DAS by adding a known amount of DI water to all pots and draining the water using a 50 kPa aquarium pump. We note that although soil moisture content of the pots was always maintained at 30%, it was slightly lower (27.8%) at 32 and 53 DAS, i.e., during the 2nd and 4th gas sampling day respectively, since the pots were watered less than the target weight on those days, allowing for addition of P solution just after gas sampling on the same day to bring soil moisture to 30%. In contrast, soil moisture contents were higher at 34 DAS (39.3% g/g) and 55 DAS (38.2% g/g for without plant pots and 34.2% g/g for planted pots), i.e., during the 3rd and 5th gas sampling day respectively, since the pots were watered with a high but constant amount of water to induce leaching in the previous day. On each day of gas sampling, pots were covered and temporarily sealed with similar PVC pots (diameter 15 cm, height 20 cm) equipped with sampling ports. First gas samples were taken immediately after coverage (time 0). The second and third gas samples were taken after 1 and 2 h, respectively. The gas samples were analysed immediately after collection for N2O and CO2 concentrations using an Agilent greenhouse gas chromatograph (7890 GC, Agilent Technologies Australia Pty Ltd., VIC, Australia) with dinitrogen (N2) as the carrier gas. N2O was measured on an Electron Capture Detector (ECD) at 300 °C. CO2 was converted to CH4 with a methanizer and then measured using a Flame Ionisation Detector (FID) at 250 °C. Columns were set at 60 °C. The gas concentrations of the standards were 0.42 and 4.2 ppm for N2O and 393 and 5060 ppm for CO2. The N2O and CO 2 fluxes were calculated as the slope of linear regressions from the measured gas concentrations over time (R2 N 0.90). Cumulative gas emissions were calculated by linear interpolation between sampling events. After each leaching event, the collected leachates were weighed, fil− tered and later analysed for pH, for extractable NH+ 4 and NO3 using a flow injection analyser (Lachat Instruments, Loveland, CO, USA), and for total dissolved N (TDN) using a TOC-VCPN analyser (Shimadzu Scientific Instruments, Sydney, NSW, Australia). To analyse the 15N in − leachate, the NH+ 4 and NO3 in the leachate samples were collected using acidified filter paper disks inside PTFE diffusion traps after

+ converting the NO− 3 into NH4 by adding Devarda's alloy, and magnesium oxide to convert NH+ 4 to ammonia (Stark and Hart, 1996). The filter disks were dried and analysed on a mass spectrometer for 15N enrichment. Pots were harvested at 58 DAS. Shoots were clipped at the soil surface, dried (60 °C) and weighed to determine above ground biomass. Roots were separated from the soil, washed, dried (60 °C) and weighed. After grinding, shoot and root samples were burned into ash in a muffle furnace at 500 °C. The ashed samples were dissolved in 6 N HCl and analysed for P calorimetrically using ammonium paramolybdate/vanadate reagent (Jackson, 1958) at 400 nm on a UV-VIS Spectrophotometer (UVmini-1240, Shimadzu Scientific Instruments, Sydney, NSW, Australia). − Soils were analysed for extractable P, NH+ 4 and NO 3 , microbial biomass C, N and P, 15 N recovery (see analysis and calculation below) and pH. Soil samples were exposed to chloroform for 24 h for microbial P (fumigation-extraction method; Brookes et al., 1982) and 48 h for microbial C and N analysis (fumigation-extraction method; Bruulsema and Duxbury, 1996). For microbial biomass P, fumigated and non-fumigated samples were extracted with 0.03 N NH4F - 0.025 N HCl and analysed for P colorimetrically using (NH4) 6Mo 7O 24.4H 2 O and SnCl 2 reagent (Olsen and Sommers, 1982) on a spectrophotometer at 660 nm (UV-VIS spectrophotometer, Shimadzu, Kyoto, Japan). Microbial biomass P was calculated from the difference of P concentration of fumigated and non-fumigated samples and divided by an extraction efficiency of 0.40 (Brookes et al., 1985). For microbial biomass C and N, fumigated and nonfumigated samples were extracted with 0.05 M potassium sulphate (K2SO4) and analysed for total C and N using a TOC-VCPN analyser (Shimadzu Scientific Instruments, Sydney, NSW, Australia). Microbial biomass C and N were calculated from the difference of their concentrations in fumigated and non-fumigated samples divided by an extraction efficiency of 0.45 (Beck et al., 1997) and 0.54 (Brookes et al., 1985), respectively. Non-fumigated samples were − analysed for extractable NH+ 4 and NO 3 using a flow injection analyser. The pH of soils (1:5 soil: deionised water) and leachates were measured using a pH meter (Mettler Toledo SevenMulti™). The approximate net amount of N mineralized in each pot was calculated similar to the method by Dijkstra et al. (2009):

Net N mineralization
¼ Nafter þ Nleachate þ Ngas þ Nplant −ðNinitial þ Nadded Þ

ð1Þ

where, Nafter, Ninitial and Nadded represent inorganic N of soil observed after harvesting, present initially before the experiment started, and added at the beginning of the experiment in mg pot−1, respectively. Nleachate, Ngas and Nplant are the losses of inorganic N from soil through leaching, N2O emission and plant uptake in mg pot−1, respectively. This calculation does not account for losses of inorganic N as N2, or any other gaseous forms of N, except N2O. However, these unaccounted losses of inorganic N were most likely relatively small compared to the net N mineralization. 2.4. Isotope analysis and calculation Stable isotope and elemental analysis of dried (60 °C) and ground (Retsch Micromill, Retsch, Haan, Germany) plant and soil material were performed on a Delta V Advantage isotope ratio mass spectrometer (IRMS, Thermo Fisher Scientific, Bremen, Germany). Soil samples were analysed for total N and 15N, whereas dried plant samples were analysed for total C, total N and 15N using the following internal standards with known elemental (IVA Soil Standards 1–5 and Algal Standard) and known isotopic composition, or calibrated against primary isotope standards from the IAEA (against AIR): Proline (+4.19‰), Lglutamate (−5.5‰), IAEA-305 (Vial A: +39.8‰; Vial B: +375.3‰).

K.R. Mehnaz et al. / Science of the Total Environment 671 (2019) 786–794

The soil 15N recovery (15 Nrec,soil) was calculated using the following equation: 15

Nrec;soil ¼ Nsoil 



15


Npost;soil −15 Npre;soil =

15

Nlabel −15 Npre;soil




ð2Þ

where Nsoil is the total N content of soil; 15Npost,soil and 15Npre,soil are the 15 N atom% measured in the soil after and before labelling respectively; and 15Nlabel is the 15N atom% of the applied label. The 15N recovery in shoot, root and leachate were calculated as in the soil using Eq. (2). Total 15N recovery was calculated by summing the plant, soil and leachate 15N recovery. 2.5. Statistical analysis We used a two-way ANOVA to test for main effects of P addition (0, 10 and 20 mg P kg−1), plant presence (with and without plants) and their interactive effects on N2O and CO2 emission rates, microbial biomass C, N and P, soil and leachate inorganic N, extractable P, pH and 15 N recovery, net N mineralization, and total 15N recovery in soilplant-leachate. For plant parameters we used a one-way ANOVA to test for main effects of P addition only. Linear regression was used to test for the relationship of N2O emission rate with leachate and soil extractable NO− 3 , and the relationship of cumulative N2O emission with net N mineralization and soil pH. When main or interactive effects of P were significant at P ≤ 0.05, pairwise post hoc tests were used to

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compare the means of each treatment combination. P values between 0.05 and 0.1 were considered marginally significant. When necessary, data were log-transformed to improve normal distribution and to reduce heteroscedasticity (examined with the Brown-Forsythe test for equal variance). All statistical analyses were conducted in JMP (v. 10.0.0; SAS Institute, Cary, NC, USA). 3. Results P addition and plant presence had no effect on N2O emission rates at 20 DAS after 1st P addition (Fig. 1a). N2O emission rates decreased with plant presence (P = 0.003) at 32 DAS but increased with both P addition (P = 0.03) and plant presence (P = 0.0001) at 34 DAS, before and after 2nd P addition and 1st leaching event, respectively (Fig. 1b, c). However, P induced increases in N2O emission rates at 34 DAS were only significant for the low level of P addition. During the last two days of gas sampling, at 53 DAS and 55 DAS, before and after 3rd P addition and 2nd leaching event, respectively, N2O emission rates increased with high level of P addition (P = 0.03 and 0.04 respectively) but decreased with plant presence (P = 0.03 and b0.0001, respectively, Fig. 1d, e). CO2 emission rates significantly increased with plant presence at all sampling dates (Fig. 2a–e). Both low and high level of P addition increased CO2 emission rates at 32 and 34 DAS (P = 0.007 and 0.0003 respectively) with largest increases in the presence of plants (significant P × plant interactive effect, P = 0.03 and 0.009 respectively) and 12 N2O (ug N pot-1 hr-1)

20 DAS 10 8

a

0P 10 P 20 P

6 4 2 0

N2O (µg N pot-1 hr-1)

2.5

2.0

Without plants

34 DAS

b N2O (ug N pot-1 hr-1)

32 DAS

Plant: 0.003

1.5

1.0

0.5

Without plants

With plants 53 DAS

P: 0.03 Plant: 0.0001

30 20 10

P: 0.03 Plant: 0.03

Without plants

d N2O (ug N pot-1 hr-1)

N2O (µg N pot-1 hr-1)

1.0

c

0

0.0 1.2

40

With plants

0.8

0.6

0.4

55 DASWith plants

e

40 P: 0.04 Plant: <0.0001

30 20 10

0.2

0

0.0

Without plants

With plants

Without plants

With plants

Fig. 1. Mean N2O emission rates from soil with 0, 10 and 20 mg kg−1 P with and without plant presence at 20, 32, 34, 53 and 55 days after sowing (DAS) (a–e). Error bars represent standard error (n = 4). P values for ANOVA are reported when significant (P b 0.05).

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20 DAS 0P 10 P 20 P

CO2 (mg C pot-1 hr-1)

32 DAS 8

6

P: 0.007 Plant: <0.0001 P * Plant: 0.03

b

b

Plant: <0.0001

Without plants 34

DAS

P: 0.0003 Plant: <0.0001 P * Plant: 0.009

a ab

a

With plants

a

c

a

b

4

2

c

c

c

c

c

c

0 Without plants

CO2 (mg C pot-1 hr-1)

53 DAS

With plants

d

Without plants

55 DAS

With plants

e

8

6

P: 0.07 Plant: <0.0001

Plant: <0.0001

4

2

0

Without plants

With plants

Without plants

With plants

Fig. 2. Mean CO2 emission rates from soil with 0, 10 and 20 mg kg−1 P with and without plant presence at 20, 32, 34, 53 and 55 days after sowing (DAS) (a–e). Error bars represent standard error (n = 4). P values for ANOVA are reported when significant (P b 0.05) and marginally significant (P b 0.1). Bars with different letters are significantly different, pairwise post hoc test.

marginally increased CO2 emission rates at 53 DAS (P = 0.07) (Fig. 2b– d). Cumulative N2O emission increased with low level of P addition (P = 0.04) and plant presence (P = 0.0008) (Fig. 3a). On the other hand, cumulative CO2 emission increased with both low and high level of P addition (P = 0.0006) as well as with plant presence (P b 0.0001) (Fig. 3b). Leachates collected at both 33 and 54 DAS showed reduced (P b 0.0001) TDN and NO− 3 concentrations in the presence of plants, although NH+ 4 concentration increased (P = 0.02) with plant presence at 33 DAS (Table 1). The N2O emission at 55 DAS was positively related 2 to the NO− 3 concentration in leachate (R = 0.58, P b 0.0001) collected at 54 DAS. Surprisingly, P addition did not show any significant effect on P concentration in leachates collected on both days, whereas plant presence increased P concentration (P = 0.02) in leachates collected at 33 DAS (Table 1). High level of P addition and plant presence increased leachate pH at 54 DAS (P = 0.03 and 0.02 respectively). Total 15 N recovery in leachate was strongly reduced with both low and high level of P addition as well as with plant presence (P b 0.0001). However, P-induced reduction of 15N was largest in leachates of pots without plants (significant P × plant interactive effect, P = 0.0006) (Table 1). P addition increased shoot and root biomass (P = 0.05 and 0.02 respectively) and their C content (P = 0.05 and 0.02 respectively) with the largest increase with the highest amount of P addition (Table 2).

As expected, both shoot and root P increased with increasing level of P addition (P = 0.0003 and 0.02 respectively). Total N and 15N recovery in shoot and root biomass were not affected by P addition. Soil extractable P increased with increasing P addition (P b 0.0001) and decreased with plant presence (P b 0.0001) (Table 3). Both low and high level of P addition increased soil extractable NH+ 4 (P = 0.01) and marginally increased soil extractable NO− 3 (P = 0.06) analysed after harvesting. Plant presence decreased both extractable NH+ 4 and NO− 3 from soil (P = 0.01 and b0.0001 respectively) suggesting increased plant inorganic N uptake. As with the leachates, the N2O emission at 55 2 DAS was positively related to NO− 3 concentration in soil (R = 0.58, P b 0.0001) analysed just after harvesting at 58 DAS. This suggests that the source of N2O emission was likely from denitrification. The 15N recovery in soil strongly increased with both low and high level of P addition (P b 0.0001) but decreased in the presence of plants (P b 0.0001). Higher addition of P and plant presence increased soil pH (P = 0.05 and b0.0001 respectively), although the P (high level only) induced pH increase was only significant in the presence of plants (significant P × plant interactive effect, P = 0.02) (Table 3). However, soil pH showed a positive relationship (R2 = 0.22, P = 0.02) with cumulative N2O emission. Net N mineralization increased with plant presence (P = 0.004, Table 3) and also showed positive relationship (R2 = 0.4, P = 0.0008) with

b0.0001 b0.0001 0.0006 ns b0.0001 ns ns b0.0001 ns ns ns ns ns ns ns 0.03 0.02 ns ns b0.0001 ns ns b0.0001 ns

5.84 ± 0.44 a 2.72 ± 0.53 b 3.27 ± 0.13 b 0.66 ± 0.10 c 0.15 ± 0.02 c 0.29 ± 0.12 c

(%) )

74.99 ± 11.0 75.44 ± 11.0 60.08 ± 7.34 1.06 ± 0.13 1.61 ± 0.31 1.61 ± 0.64

(mg N L )

54.25 ± 5.50 53.58 ± 4.79 46.68 ± 4.09 0.09 ± 0.04 0.07 ± 0.02 0.08 ± 0.02

(mg N L )

0.14 ± 0.03 0.16 ± 0.03 0.16 ± 0.03 0.14 ± 0.08 0.08 ± 0.02 0.11 ± 0.03

(mg N L )

88.12 ± 5.50 75.67 ± 2.83 73.15 ± 5.62 85.60 ± 6.33 89.24 ± 3.15 80.01 ± 5.46

(mg L

2.58 ± 0.05 2.64 ± 0.01 2.74 ± 0.07 2.67 ± 0.02 2.81 ± 0.10 2.87 ± 0.07 )

57.59 ± 6.76 53.93 ± 5.73 50.50 ± 6.85 24.57 ± 2.02 12.08 ± 3.20 17.52 ± 5.65

(mg N L )

36.48 ± 5.92 36.85 ± 5.88 30.23 ± 3.84 22.13 ± 1.80 10.61 ± 3.24 13.25 ± 5.01

(mg N L ) (mg N L

0.13 ± 0.03 0.13 ± 0.05 0.10 ± 0.04 0.22 ± 0.05 0.18 ± 0.03 0.22 ± 0.04

Total 15N recovery

−1

TDN

−1

NO− 3

−1

NH+ 4

−1

)

ns 0.02 ns ns 0.02 ns ns ns 0.07 ANOVA P-values P Plant P × plant

Higher addition of P increased plant root and shoot biomass as well as their C and P contents (Table 2) suggesting P limitation of plant growth. However, the P induced plant growth did not stimulate plant N uptake (Table 2) and thus did not reduce N2O emission by limiting N availability for nitrifiers and/or denitrifiers, unlike observed in other studies (Baral et al., 2014; Chen et al., 2017). Instead, a stimulation of N2O emission rates was observed with low level of P addition during the early stage of plant growth and with high level of P addition at later stages of the experiment (Fig. 1). Cumulative N2O emission also increased with low level of P addition (Fig. 3a). It has been suggested that high availability of P can increase N2O emission by stimulating the whole N cycle, including N mineralization and thus providing N substrates for nitrifiers and denitrifiers (Mori et al., 2010). Although ex− tractable NH+ 4 and NO3 was higher in P added soils after harvesting, net N mineralization was not significantly increased with P addition (Table 3). However, an alleviation of P limitation of nitrifying and/or denitrifying bacteria due to increased availability of P could have led to increased N2O emission (Mori et al., 2010). Mehnaz and Dijkstra

(mg L

4.1. Effects of P addition and plant presence on N2O emission and NO− 3 leaching

98.76 ± 4.68 94.98 ± 12.0 93.16 ± 7.53 101.97 ± 5.44 122.31 ± 4.86 117.65 ± 13.7

4. Discussion

2.61 ± 0.05 2.64 ± 0.03 2.71 ± 0.02 2.69 ± 0.05 2.58 ± 0.06 2.56 ± 0.06

cumulative N2O emission (Fig. 4). P addition did not affect microbial biomass C, N and P, whereas plant presence increased microbial biomass C and N (P = 0.007 and b0.0001 respectively) but decreased microbial biomass P (P = 0.01) (Table 4). Total 15N recovered in soil, plant and leachate was increased with P addition (P = 0.007, particularly with low level of P addition), and marginally increased with plant presence (P = 0.07) (Fig. 5).

Plant presence

Fig. 3. Mean cumulative N2O (a) and CO2 (b) emissions from soil with 0, 10 and 20 mg kg−1 P with and without plant presence. Error bars represent standard error (n = 4). P values for ANOVA are reported when significant (P b 0.05).

Without Without Without With With With

with plant

soil)

without plant

0P 10 P 20 P 0P 10 P 20 P

0

P addition (mg kg

1

P

2

pH

3

−1

P: 0.0006 Plant: <0.0001

−1

4

−1

Cumulative CO2 (g C pot-1)

b 5

−1

0

−1

2

54 DAS

4

TDN

6

NO− 3

8

NH+ 4

P: 0.04 Plant: 0.0008

P

a

33 DAS

10

0P 10 P 20 P

791

pH

12

Treatments

Cumulative N2O (mg N pot-1)

14

Table 1 15 − Mean ± standard error of leachate pH, P, NH+ N recovery collected at 33 and 54 days after sowing (DAS) in response to 0, 10 and 20 mg kg−1 P addition with and without plant presence. P b 0.05, significant; P 4 , NO3 , total dissolved N (TDN) and total b 0.1 marginally significant; ns, not significant. Different letters in each column indicate significant differences among treatment combinations (P b 0.05, pairwise post hoc test).

K.R. Mehnaz et al. / Science of the Total Environment 671 (2019) 786–794

792

K.R. Mehnaz et al. / Science of the Total Environment 671 (2019) 786–794

Table 2 Mean ± standard error of shoot and root biomass, C, N, P and 15N recovery in response to 0, 10 and 20 mg kg−1 P addition in soil. P b 0.05, significant; P b 0.1 marginally significant; ns, not significant. Different letters in each column indicate significant differences among treatment combinations (P ≤ 0.05, pairwise post hoc test). Treatments

Shoot biomass

Shoot C

Shoot N

Shoot P

Shoot 15N recovery

Root biomass

Root C

Root N

Root P

Root 15N recovery

P addition (mg kg−1 soil)

(g pot−1)

(g pot−1)

(mg pot−1)

(mg pot−1)

(%)

(g pot−1)

(g pot−1)

(mg pot−1)

(mg pot−1)

(%)

0P 10 P 20 P

4.13 ± 0.15 b 4.58 ± 0.15 ab 4.75 ± 0.18 a

1.71 ± 0.06 b 1.91 ± 0.06 ab 1.98 ± 0.07 a

76.88 ± 4.37 78.18 ± 5.07 81.66 ± 4.84

6.71 ± 0.28 c 8.93 ± 0.26 b 10.27 ± 0.52 a

35.51 ± 1.06 33.64 ± 1.48 33.29 ± 0.82

1.65 ± 1.65 b 1.93 ± 1.93 ab 2.22 ± 2.22 a

0.75 ± 0.01 b 0.87 ± 0.04 ab 1.00 ± 0.07 a

18.20 ± 0.66 19.89 ± 1.34 22.30 ± 2.12

2.36 ± 0.03 b 3.07 ± 0.26 a 3.43 ± 0.28 a

8.03 ± 0.46 8.28 ± 0.34 8.66 ± 0.49

ANOVA P-values P

0.05

0.05

ns

0.0003

ns

0.02

0.02

ns

0.02

ns

(2016) also demonstrated that potential denitrification as well as N2O emission from denitrification were limited by P in a high P-fixing grassland soil. Moreover, the stimulated microbial respiration with P addition, supported by increased cumulative CO2 emission (Fig. 3b) after P addition, could also have increased oxygen (O2) consumption thereby creating anaerobic conditions to facilitate denitrifying activities and associated N2O emission (Mori et al., 2013). Temporal changes in N2O emission were observed in the presence of plants depending on the direct or indirect effects of plants on N2O producing microorganisms. Denitrification and associated N2O emission can increase with progressing plant age in presence of sufficient NO− 3 in soil (Rheinbaben and Trolldenier, 1984). However, in this experiment plant presence showed reduced N2O emission rates at 32 and 53 DAS (Fig. 1b, d), just before the 1st and 2nd leaching events, respectively. A reduction in N2O emission from planted pots at both these days could be due to a rapid water uptake of the plant roots from the soil with low moisture content (27.8%), which subsequently reduced denitrification and associated N2O emission. In contrast, plant presence significantly increased N2O emission rates at 34 DAS (Fig. 1c), the day after the 1st leaching event, when soil moisture content was high (39.3%). A stimulation of denitrifying activity may have occurred in high soil moisture condition due to a possible increase in soluble organic C substrates from root exudation, as denitrifiers are heterotrophic and re− quire C. Although plant NO− 3 uptake may have reduced the soil NO3 content, which is supported by the decreased NO− concentration mea3 sured in the planted pot leachates at 33 DAS (Table 1), the high N2O emissions from the planted pots at 34 DAS suggest that the denitrifiers were not limited by NO− 3 in the soil, but rather by soluble organic C. At 55 DAS with high soil moisture conditions (38.2% for pots without plants and 34.2% for planted pots), just a day after the 2nd leaching event, N2O emission rates were found to be low in the planted pots and also significantly lower than in the pots without plants (Fig. 1e). This large reduction in N2O emission from the planted pots suggests that denitrifiers were no longer limited by C, but rather by NO− 3 in the

soil. Increased plant growth may have reduced denitrification through more rapid uptake of NO− 3 from the soil. This is supported by the large reduction in NO− 3 concentration in leachates collected at 54 DAS (Table 1) and also in soils (Table 3) extracted at 58 DAS, just after harvesting. Furthermore, both leachate NO− 3 concentration at 54 DAS and soil NO− 3 concentration after harvesting showed positive relationships with N2O emission rates at 55 DAS, suggesting a reduction in N2O emission due to low availability of soil NO− 3 in planted pots. Although N2O emission rates showed large temporal fluctuations, cumulative N2O emission increased in presence of plants (Fig. 3a), suggesting that plants may have stimulated denitrifiers by providing them with labile C through root exudates (Henry et al., 2008). The cumulative N2O emission was based on a limited number of flux measurements, and we therefore interpret these results with caution. However, an increased input of plant root derived C can stimulate N mineralization (Qian et al., 1997; Zak et al., 1993), resulting in increased N availability for denitrifiers. Indeed, the net N mineralization during the experiment was found to be higher with plant presence (Table 3). Greater availability of C in planted pots supplied by the growing roots may have stimulated the biomass and activity of the microbial population, which can be supported by the increased soil microbial biomass C (Table 4) and CO2 emission (Figs. 2a–e, 3b) in presence of plants. However, the higher CO2 emissions from planted pot soils could also be due to root respiration in addition to soil heterotrophic activity. An increase in microbial biomass and activity may have subsequently increased microbial N turnover and/or N release from native organic matter in soil (Grayston et al., 1996; Zak et al., 1993) resulting in increased net N mineralization. Cumulative N2O emission showed a positive relationship with net N mineralization (Fig. 4), further suggesting that the increase in cumulative N2O emission in planted pots could be due to an increase in N availability that resulted from stimulated N mineralization in presence of plants. We note that we may have underestimated net N mineralization by not accounting for gaseous N loss other than N2O. However, we assume that gaseous N loss was relatively small compared to net N

Table 3 15 − Mean ± standard error of soil pH, extractable (extr.) P, NH+ N recovery and net N mineralization measured after harvesting in response to 0, 10 and 20 mg kg−1 P addition 4 and NO3 , soil with and without plant presence. P b 0.05, significant; P b 0.1 marginally significant; ns, not significant. Different letters in each column indicate significant differences among treatment combinations (P b 0.05, pairwise post hoc test). Treatments P addition (mg kg−1 soil)

Plant presence

0P 10 P 20 P 0P 10 P 20 P

Without Without Without With With With

ANOVA P-values P Plant P × plant

Extr. P

Extr. NH+ 4

Extr. NO− 3

15

(mg pot−1)

(mg N pot−1)

(mg N pot−1)

(%)

(mg pot−1)

5.00 ± 0.07 cd 4.92 ± 0.00 d 4.95 ± 0.03 d 5.13 ± 0.02 bc 5.18 ± 0.03 b 5.39 ± 0.08 a

23.04 ± 1.42 39.53 ± 1.57 45.66 ± 1.46 16.98 ± 1.07 27.93 ± 1.67 38.17 ± 1.75

1.71 ± 0.18 3.76 ± 0.29 3.33 ± 0.89 0.74 ± 0.56 2.27 ± 0.39 2.02 ± 0.65

52.93 ± 0.43 77.48 ± 12.0 72.41 ± 13.0 1.64 ± 0.26 1.90 ± 0.14 2.22 ± 0.25

59.79 ± 1.42 69.75 ± 2.19 67.24 ± 0.29 24.42 ± 2.66 35.37 ± 1.11 30.91 ± 0.46

48.57 ± 4.32 74.91 ± 14.0 67.22 ± 15.0 83.02 ± 4.47 91.46 ± 7.06 95.00 ± 9.42

0.05 b0.0001 0.02

b0.0001 b0.0001 ns

0.01 0.01 ns

0.06 b0.0001 ns

b0.0001 b0.0001 ns

ns 0.004 ns

Soil pH

N recovery

Net N mineralization

K.R. Mehnaz et al. / Science of the Total Environment 671 (2019) 786–794

100

20

R2 = 0.40 P = 0.0008

0 P, without plants 10 P, without plants

16

Total 15N recovery (%)

Cumulative N2O (mg N pot-1)

18

20 P, without plants 0 P, with plants

14

10 P, with plants

12

20 P, with plants

10 8 6 4

80

793

Leachate Soil Root Shoot

P: 0.007 Plant: 0.07

60

40

20

2 0 20

40

60

80

100

120

0

140

0P

Net N mineralization (mg N pot-1) Fig. 4. Relationship of cumulative N2O emission with net N mineralization in response to 0, 10 and 20 mg kg−1 P with and without plant presence.

mineralization (i.e., Fig. 3a and Table 3), while net N mineralization may have particularly been underestimated in planted pots if gaseous N loss was higher in planted pots (as observed for N2O). The increase in N2O emission rates with higher addition of P (at later stages of the experiment) as well as increased cumulative N2O emission with plant presence could also be partly attributed to an increase in soil pH. High level of P addition in planted pots and plant presence increased soil pH, observed at the end of the experiment (Table 3). Leachate collected at 54 DAS also showed an increase in pH with both high P addition and plant presence (Table 1). It is unclear why P addition increased soil pH and thus leachate pH, since P was added in solution with the same pH as in the soil, but the plant induced pH increase 3− could be due to uptake of NO− 3 and PO4 by the plants and thereby releasing equivalent amounts of OH– or HCO–3 into the soil to counterbalance the excess of negative charge moving into the plant cells (Hinsinger et al., 2003; Nye, 1981). Since a reduction in soil acidification can cause favourable conditions for microbial growth and activity (Chen et al., 2017) as well as for denitrification (Knowles, 1982), high level of P and plant induced pH increase may have stimulated the biomass and activity of nitrifying and/or denitrifying communities and thus N2O emission from planted pots. Although the ratio of N2O to N2 frequently decreases with increasing pH (Wrage et al., 2001), an overall increase in denitrification rate (Knowles, 1982) may have caused a net increase in N2O production in response to elevated soil pH. P addition did not show an increase in microbial biomass C after harvesting. However, increased cumulative CO2 emission with both low and high level of P addition could suggest higher microbial activity with the addition of this nutrient. An increase in microbial biomass C (Table 4) and cumulative CO2 emission (Fig. 3b) with plant presence, observed at the end of the

10 P

20 P

Without plants

0P

10 P

20 P

With plants

Fig. 5. Mean ± standard error (error bars) of total 15N recovery in leachate, soil, root and shoot in response to 0, 10 and 20 mg kg−1 P with and without plant presence. P values for ANOVA are reported when significant (P b 0.05) and marginally significant (P b 0.1).

experiment, could suggest a stimulation of the growth and activity of microbial communities, possibly including nitrifiers and denitrifiers, due to a decrease in soil acidification. Indeed, soil pH showed a positive relationship with cumulative N2O emission providing further support for this hypothesis. 4.2. Effects of P addition and plant presence on 15N recovery Both low and high levels of P addition significantly decreased 15N recovery in leachates (collected at 33 and 54 DAS, Table 1), suggesting a P induced reduction in N loss through leaching. However, addition of this nutrient had no effect on 15N recovery in plants (Table 2), but increased 15 N recovery in soils (Table 3). Since it is unlikely that P addition would 15 enhance abiotic NO− N recovery in soils can be 3 retention, the higher attributed to increased microbial N immobilization, as was observed in different forest soils (Hall and Matson, 1999; Vesterdal and RaulundRasmussen, 2002). An increase in NO− 3 -N utilization by denitrifiers for respiration as well as possible NO− 3 immobilization by the heterotrophic microflora in general may have reduced 15N loss through leachates in Padded pots. As expected, soil 15N recovery significantly decreased in planted pots (Table 3) due to plant 15N uptake (Table 2). Plant uptake of N along with a higher N consumption by the increased microbial population, supported by increased soil microbial biomass C and N with plant presence (Table 4), and a greater N loss as N2O were possibly responsible for the significantly reduced 15N recovery in planted pot leachates (Table 1). Although low level of P addition significantly increased cumulative N2O emission, a higher total 15N recovery (15N recovered in soil, plants and leachates, Fig. 5) in low P-added pots could

Table 4 Mean ± standard error of microbial biomass C, N and P observed after harvesting in response to 0, 10 and 20 mg kg−1 P addition with and without plant presence. P b 0.05, significant; ns, not significant. Treatments

Microbial C

Microbial N

Microbial P

P addition (mg kg−1 soil)

Plant presence

(mg pot−1)

(mg pot−1)

(mg pot−1)

0P 10 P 20 P 0P 10 P 20 P

Without Without Without With With With

695.13 ± 17.67 826.24 ± 91.59 706.38 ± 58.27 854.87 ± 50.66 973.81 ± 68.32 871.41 ± 71.75

33.09 ± 8.80 20.59 ± 7.67 30.37 ± 6.71 70.88 ± 4.53 79.10 ± 5.58 71.94 ± 6.04

52.55 ± 2.46 64.40 ± 2.51 65.83 ± 16.0 39.17 ± 1.36 47.81 ± 9.99 50.35 ± 3.63

ns 0.007 ns

ns b0.0001 ns

ns 0.01 ns

ANOVA P-values P Plant P × plant

794

K.R. Mehnaz et al. / Science of the Total Environment 671 (2019) 786–794

suggest a possible reduction in gaseous loss of N as N2 but an increase in the N2O/N2 ratio, also hypothesized by Mehnaz et al. (2018). Unfortunately, N2 emission was not measured in this experiment. Further studies should be conducted to understand any effect of P on the N2O/N2 ratio in denitrification. 5. Conclusion Our results showed that initially low and later (after 53 days of sowing) high level of P addition increased N2O emission rates possibly by stimulating nitrifiers and/or denitrifiers and also heterotrophic microbial activities in general to provide anoxic conditions for denitrification. Plant presence showed temporal fluctuations in N2O emission as limiting factors for denitrification – such as water, soluble organic C and NO− 3 content of soil – changed over time due to plant root activities. However, an increase in net N mineralization due to increased heterotrophic activities was found to be responsible for higher cumulative N2O emission from planted pots. Although both P addition and plant presence stimulated N loss as N2O, they increased N retention in the soil-plant system and thus reduced N loss through leaching. Acknowledgement We thank Hero Tahaei and Janani Vimalathithen for their assistance in soil chemical analyses. This research was financially supported by the Australian Research Council (FT100100779). References Aber, J.D., Nadelhoffer, K.J., Steudler, P., Melillo, J.M., 1989. Nitrogen saturation in northern forest ecosystems. BioSci 39, 378–386. Baral, B.R., Kuyper, T.W., Van Groenigen, J.W., 2014. Liebig's law of the minimum applied to a greenhouse gas: alleviation of P-limitation reduces soil N2O emission. Plant Soil 374, 539–548. Beck, T., Joergensen, R., Kandeler, E., Makeschin, F., Nuss, E., Oberholzer, H., Scheu, S., 1997. An inter-laboratory comparison of ten different ways of measuring soil microbial biomass C. Soil Biol. Biochem. 29, 1023–1032. Blanes, M.C., Emmett, B.A., Viñegla, B., Carreira, J.A., 2012. Alleviation of P limitation makes tree roots competitive for N against microbes in a N-saturated conifer forest: a test through P fertilization and 15N labelling. Soil Biol. Biochem. 48, 51–59. Brady, N.C., Weil, R.R., 2002. The Nature and Properties of Soil. 13th edn. Pearson Education, Singapore. Brookes, P.C., Powlson, D.S., Jenkinson, D.S., 1982. Measurement of microbial biomass phosphorus in soil. Soil Biol. Biochem. 14, 319–329. Brookes, P.C., Landman, A., Pruden, G., Jenkinson, D., 1985. Chloroform fumigation and the release of soil nitrogen: a rapid direct extraction method to measure microbial biomass nitrogen in soil. Soil Biol. Biochem. 17, 837–842. Bruulsema, T., Duxbury, J., 1996. Simultaneous measurement of soil microbial nitrogen, carbon, and carbon isotope ratio. Soil Sci. Soc. Am. J. 60, 1787–1791. Butterbach-Bahl, K., Baggs, E.M., Dannenmann, M., Kiese, R., Zechmeister-Boltenstern, S., 2013. Nitrous oxide emissions from soils: how well do we understand the processes and their controls? Philos. Trans. R. Soc. B 368, 20130122. Cameron, K.C., Di, H.J., Moir, J.L., 2013. Nitrogen losses from the soil/plant system: a review. Ann. Appl. Biol. 162, 145–173. Chen, H., Zhang, W., Gurmesa, G.A., Zhu, X., Li, D., Mo, J., 2017. Phosphorus addition affects soil nitrogen dynamics in a nitrogen-saturated and two nitrogen-limited forests. Eur. J. Soil Sci. 68, 472–479. Dalal, R.C., Wang, W., Robertson, G.P., Parton, W.J., 2003. Nitrous oxide emission from Australian agricultural lands and mitigation options: a review. Soil Res. 41, 165–195. Dijkstra, F.A., Bader, N.E., Johnson, D.W., Cheng, W., 2009. Does accelerated soil organic matter decomposition in the presence of plants increase plant N availability? Soil Biol. Biochem. 41, 1080–1087. Dijkstra, F.A., He, M., Johansen, M.P., Harrison, J.J., Keitel, C., 2015. Plant and microbial uptake of nitrogen and phosphorus affected by drought using 15N and 32P tracers. Soil Biol. Biochem. 82, 135–142. Dise, N.B., Wright, R.F., 1995. Nitrogen leaching from European forests in relation to nitrogen deposition. For. Ecol. Manag. 71, 153–161. Falkiner, R.A., Khanna, P.K., Raison, R.J., 1993. Effect of superphosphate addition on N mineralization in some Australian forest soils. Soil Res. 31, 285–296. Firestone, M.K., Davidson, E.A., 1989. Microbiological basis of NO and N2O production and consumption in soil. In: Andreae, M.O., Schimel, D.S. (Eds.), Exchange of Trace Gases Between Terrestrial Ecosystems and the Atmosphere. John Wiley & Sons, New York, pp. 7–21. Forster P, Ramaswamy V, Artaxo P, et al. (2007) Changes in atmospheric constituents and in radiative forcing. In: Solomon S, Qin D, Manning M, Chen Z, Marquis M, Averyt KB, Tignor M, Miller HL (eds) Climate Change 2007: The Physical Science Basis. Contribution of Working Group I to the Fourth Assessment Report of the Intergovernmental

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