Phosphorus fertilization to the wheat-growing season only in a rice–wheat rotation in the Taihu Lake region of China

Field Crops Research 198 (2016) 32–39

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Phosphorus fertilization to the wheat-growing season only in a rice–wheat rotation in the Taihu Lake region of China Yu Wang a , Xu Zhao a , Lei Wang b , Pin-Heng Zhao c , Wen-Bin Zhu a,d , Shen-Qiang Wang a,∗ a

State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China Organic Food Development Center, Nanjing Institute of Environmental Sciences, Ministry of Environmental Protection, Nanjing 210042, China Changshu Institute of Agricultural Sciences, Changshu 215500, China d College of Resources and Environment, Anhui Agricultural University, Hefei, 230036, China b c

a r t i c l e

i n f o

Article history: Received 28 June 2016 Received in revised form 11 August 2016 Accepted 22 August 2016 Keywords: P fertilization Crop yield Soil P fraction Microbial community Paddy soil

a b s t r a c t Crop production in the Taihu Lake Region (TLR) of China has been greatly improved by increasing phosphorus (P) fertilizer input. However, overuse has led to accumulation of P and increased the risk of environmental pollution. In this study, we investigated the effects of four P fertilization regimes in paddy soils at two field stations (CS1 and CS2) during four years of a rice-wheat cropping system. The fertilization regimes were as follows: P fertilization only during the wheat-growing season (PW), P fertilization only during the rice-growing season (PR), P fertilization during both the rice- and wheat-growing seasons (PR + W, current farming practice), and no P fertilization during either season (Pzero, control). The PW treatment did not reduce crop yield in either CS1 or CS2 compared to the PR + W. Moreover, soil Olsen-P concentrations during each rice-wheat rotation were maintained, and a positive increase in phosphate utilization efficiency by 1.2–3.6% (p > 0.05) was observed over the four rice-wheat rotations. In contrast, the PR treatment reduced the straw and grain yield of wheat by 8.74–43.2 and 19.4–47.7%, respectively, as did the Pzero treatment by 7.21–60.0 and 19.6–84.1%, respectively (p < 0.05). During the rice season, the PW treatment showed no significant differences in P fractionation and microorganism communities compared to the PR and PR + W treatments. The results suggest that the PW fertilization regime is suitable for rice–wheat rotation systems in agro-ecosystems such as the Taihu Lake Region of China. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Phosphorous (P) is an essential plant nutrient, important for crop production; however, it is non-renewable and often in short supply. China is one of the world’s largest consumers and producers of P fertilizers (IFA, 2004). Available P rock deposits in China were previously estimated at 3.7 Pg P2 O5 (USGS, 2010; van Kauwenbergh, 2010). Nevertheless, approximately 80% of the deposits are of sedimentary origin and thus of low quality: 70% are classified as low grade, with less than 23% P2 O5 , and only 7% are classified as high-grade P rock resources, with more than 30% P2 O5 (Zhang et al., 2008). The production and consumption of P fertilizers in China have markedly increased during the past two decades (Zhang et al., 2008). Furthermore, excessive and frequent use of P fertilizer in conventional agriculture to attain high crop yields can reduce the efficiency of P use and increase the risk of environmental

∗ Corresponding author. E-mail address: [email protected] (S.-Q. Wang). http://dx.doi.org/10.1016/j.fcr.2016.08.025 0378-4290/© 2016 Elsevier B.V. All rights reserved.

pollution (Tilman et al., 2002; Ma et al., 2011). This phenomenon is particularly pronounced in the Taihu Lake Region of China (TLR) (Ma et al., 2011; Qiu, 2010; Zhang et al., 2011). The TLR primarily consists of paddy soils farmed under a rice–wheat crop rotation regime, which have been subjected to exceptionally high rates of chemical P fertilizer application (52.4 kg P ha−1 y−1 ) (Wang et al., 2015, 2012a,b). A previous survey indicated that overuse of chemical P fertilizer has led to P accumulation in TLR paddy soils (Wang et al., 2012a). However, despite these high concentrations of soil P, farmers continue to apply large amounts of P fertilizer, increasing P accumulation and the environmental risk. In fact, when the soil is flooded during the rice-growing season, the levels of available P in the soil increase significantly (Lu, 1998). Under such conditions, to efficiently reduce the environmental risk of P accumulation, omitting P application during the rice-growing season seems a good option. In a previous study, we investigated different P fertilizer regimes in a pot experiment. The results indicated that application of P fertilizer during the wheat season only could still supply enough available P sources for crop growth and sustain crop yield compared to the

Y. Wang et al. / Field Crops Research 198 (2016) 32–39

current farming practice (P fertilization during both the rice and wheat seasons; Wang et al., 2015, 2016). In this study, field experiments were conducted to determine whether the conclusions from our pot experiment could be put into practice on farms. To improve agricultural P management practices, we need to increase our understanding of the chemical and microbial processes involving P. Approximately 85–90% of the added inorganic P (Pi) becomes unavailable to plants during the year of application due to adsorption and precipitation with Fe, Al, and Ca in the soil (Lu 1998; Khan and Joergensen 2009; Li et al., 2013). The sequential P fractionation procedure can be used to separate various P pools and to record small changes in soil P (Hedley et al., 1982; Tiessen and Moir 1993). Phosphorous can also transform among pools by the conversion of organic P (Po) into inorganic P (via mineralization by microbial and root-released phosphatases) or by the conversion of residual P into NaOH-P (Malik et al., 2012; Wang et al., 2007). Dobermann et al. (2002) reported that application of P fertilizers to strongly weathered tropical soils substantially increased the absolute concentration and relative proportions of labile and moderately labile Pi as well as the HCl-P fractions. Paniagua et al. (1995) found that labile and moderately labile soil P pools were strongly dependent on Pi fertilization, and soils without P fertilization had the lowest levels of NaOH-Po and labile Pi. In addition, the application of P fertilizer can make the soil environment more favorable for microbial growth (Chu et al., 2007; Iovieno et al., 2009). Zheng et al. (2009) found that both bacterial and fungal populations were significantly larger under NPK fertilization compared with NK only and no fertilizer treatment. However, the effect of omitting P fertilization during the rice-growing season in a rice–wheat rotation system on chemical and microbial P processes remains unclear. Therefore, we performed a four-year field experiment in consecutive rice–wheat cropping seasons in two field stations in the TLR under four P fertilization regimes. Our objectives were as follows: (1) to investigate the effect of P fertilization during the wheat-growing season only in a rice–wheat rotation scheme and (2) determine the effect on chemical and microbial P processes under this fertilization regime.

2. Materials and methods 2.1. Field experiment The field experiment was conducted in paddy soils at Changshu National Agro-Ecosystem Observation and Research Station (CS1, 31◦ 32 N 120◦ 41 E) and at Changshu Institute of Agricultural Sciences (CS2, 31◦ 41 N 120◦ 40 E), located near northeastern Taihu lake. Physicochemical properties of the two soils are listed in Table 1. This region is a typical rice–wheat cropping area. The climate is subtropical monsoon with an average annual temperature of 15.4◦ C and annual rainfall of 1054 mm. The experiment was established at the start of the rice-growing season in June 2010 and continued for four consecutive integrated rice (Oryza sativa L.) and wheat (Triticum aestivum L.) rotations. Treatments were as follows: P fertilization only during the wheat-growing season (PW), P fertilization only during the rice-growing season (PR), P fertilization during both the rice- and wheat-growing seasons (PR + W, current farming practice), and no P fertilization (Pzero, control). The P fertilizer used was super phosphate applied at the soil surface. The P fertilizer application rates in each rice-wheat rotation were: 17.5 kg P ha−1 y−1 in the PR and PW treatments, respectively; 17.5 kg P ha−1 y−1 in the PR + W treatment, applied once in the rice season and once in the wheat season; and no P fertilizer in the Pzero treatment in either crop growing seasons. All treatments followed a randomized design and were replicated three times. Urea was applied consistently at a rate of 240 kg N ha−1 during each rice- and

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wheat-growing season in the two sites; 30% of the N was applied basally, and 40% was top-dressed at the tillering stage of both crops. The remaining 30% was top-dressed at the jointing or booting stages. Potassium fertilizers were applied basally in the form of potassium chloride at a rate of 50 kg K ha−1 . Local cultivation and field management practices were adopted (Table 2). During the rice-growing seasons, flooded water was generally maintained at a depth of 3–5 cm in the field, except for midseason aerations and final drainage. During the wheat-growing period, no irrigation was used, and rainwater was the only source of soil moisture. 2.2. Crop yield and soil analysis after each crop harvest Crops were manually harvested from the entire field at the soil surface, with no stubble remaining aboveground. The fresh weight of the grain was measured after mechanical threshing. Harvested plant samples from three 1 m2 subplots were evenly sampled and used for analysis of the grain/straw dry weight ratio and moisture and total P concentration of grain and straw. Per-unit-area yield of grain was then calculated by subtracting the moisture concentration from the fresh weight. Per-unit-area yield of straw was estimated based on the per-unit-area yield of grain and the grain/straw dry weight ratio. Soil samples (0–20 cm depth) from each treatment plot were collected after each crop harvest, air dried, ground, sieved (<2-mm sieve), and stored at room temperature for chemical analysis. Soil Olsen-P was estimated using sodium bicarbonate (NaHCO3 pH 8.5) (Olsen, 1954). 2.3. Physicochemical characteristics of the two original paddy soils and soils after the 2013 rice cropping season Total soil P was determined after digestion with sulfuric acid (H2 SO4 ) and perchloric acid (HClO4 ) and analyzed with an ultraviolet spectrometer (UV 2500 Japan) (Lu, 2000). Soil pH was measured in a 1:2.5 (w/v) soil/water solution using a pH meter (Lu, 2000). Soil organic C was determined using a Leco CN-2000 analyzer (LecoCorp., USA). Total soil N was measured using the Kjeldahl method (Lu, 2000). The soil CEC was measured using a modified NH4 acetate compulsory displacement method (Gaskin et al., 2008). Potassium in the soil was determined by flame emission after acid digestion (FP 640, China) (Lu, 2000). 2.4. Sequential chemical extraction of P from the soil after the 2013 rice cropping season P was extracted using the sequential extraction method described by Tiessen and Moir (1993). The soils used to determine P fractionation were sampled after the seventh season of crop cultivation (the rice-growing season in 2013). Samples of 0.5 g air-dry soil were used in the analysis. The P fractions were extracted in the following order: (1) resin-P, extracted with deionized water and anion exchange resin (Sinopharm Chemical Reagent Co., Ltd, pretreated with 10% NaCl, 5% HCl and 2–4% NaOH); (2) NaHCO3 -Pi and NaHCO3 -Po, extracted with 0.5 M NaHCO3 ; (3) NaOH-Pi and NaOHPo, extracted with 0.1 M NaOH; (4) HCl-Pi, extracted with 1 M HCl; (5) residual-P, remaining P in the soil after the above extractions was measured after digestion with H2 SO4 and H2 O2 at 360 ◦ C. At each extraction step, the samples were shaken for 16 h on a rotary shaker, and the soil and supernatant were then separated by centrifugation for 10 min at 10,000 × g at 0 ◦ C. The NaHCO3 and NaOH extracts were divided into two aliquots in order to measure the total P and inorganic P (molybdate-reactive P, Pi). The amount of organic P in each extract was calculated based on the difference between total P from digestion and inorganic P in the extracts. The sum of P extracted with anion-exchange resin and NaHCO3 is con-

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Y. Wang et al. / Field Crops Research 198 (2016) 32–39

Table 1 Selected physicochemical characteristics of the two original paddy soils.

CS1 CS2

Soil typea

Total N (g kg−1 )

Total P (g kg−1 )

Total K (g kg−1 )

OM (g kg−1 )

Olsen-P (mg kg−1 )

CEC (cmol kg−1 )

pH

Hapli-Stagnic Anthrosols Fe-accumuli-Stagnic Anthrosols

2.45 1.93

0.69 0.56

16.8 16.0

46.6 35.9

7.05 10.2

19.2 17.5

7.48 5.56

OM, organic matter; CEC, cation exchange capacity; CS1 and CS2 indicate soils from Changshu National Agro-Ecosystem Observation and Research Station and Changshu Institute of Agricultural Sciences, respectively. a Soil type was classified according to Chinese Soil Taxonomy (CST) classification (Gong et al., 2003). Table 2 Dates of cultivation and management activities during four rice–wheat rotations from 2010 to 2014. Crop rotation

Activity

Date

Crop rotation

Activity

Date

2010 Rice

Basal fertilization Transplantation First top-dressing Mid-season aeration Second top-dressing Mid-season aeration Harvesting

June 10, 2010 June 15 June 22 July 10–15 July 20 August 8–16 October 31

2012 Rice

Basal fertilization Transplantation First top-dressing Mid-season aeration Second top-dressing Mid-season aeration Harvesting

June 13, 2012 June 20 June 27 July 16–25 July 29 August 16–23 November 2

2010/2011 Wheat

Basal fertilization Sowing First top-dressing Second top-dressing Harvesting

November 5, 2010 November 9 January 13, 2011 March 10 May 29

2012/2013 Wheat

Basal fertilization Sowing First top-dressing Second top-dressing Harvesting

November 13, 2012 November 16 January 8, 2013 March 12 May 28

2011 Rice

Basal fertilization Transplantation First top-dressing Mid-season aeration Second top-dressing Mid-season aeration Harvesting

June 16, 2011 June 19 June 26 July 16–25 July 21 August 12–19 November 3

2013 Rice

Basal fertilization Transplantation First top-dressing Mid-season aeration Second top-dressing Mid-season aeration Harvesting

June 11, 2013 June 15 June 21 July 15–24 July 26 August 15–24 November 6

2011/2012 Wheat

Basal fertilization Sowing First top-dressing Second top-dressing Harvesting

November 12, 2011 November 15 January 9, 2012 March 8 June 1

2013/2014 Wheat

Basal fertilization Sowing First top-dressing Second top-dressing Harvesting

November 9, 2013 November 12 January 12, 2014 March 16 May 29

Fertilizer application during the rice and wheat seasons: 240 kg N ha−1 (30% during basal fertilization, 40% during first top-dressing, and 30% during second top-dressing) and 50 kg K ha−1 for basal fertilization; during the rice season: P fertilizer (17.5 kg P ha−1 ) as basal fertilization for the PR and PR + W treatments; during the wheat season, P fertilizer (17.5 kg P ha−1 ) as basal fertilization for the PW and PR + W treatments.

sidered labile P, and that extracted with NaOH as moderately labile P. P extracted with HCl and residual P after digestion are considered stable P (Hedley et al., 1982; Tiessen and Moir 1993). The P concentration in the supernatant was determined with an ultraviolet spectrometer (UV 2500, Japan; 700 nm) using the ascorbic acid molybdenum blue method (Murphy and Riley, 1962). 2.5. Microbial analysis of soil P after the 2013 rice cropping season Fresh surface soil samples (0–20 cm depth) were collected after the 2013 rice-growing season. Acid phosphatase activity (ACP) and alkaline phosphatase activity (ALP) were measured using pnitrophenyl phosphate (Sigma-Aldrich Co., America) as substrate (Alef and Trazar-Cepeda, 1995). Soil phospholipid fatty acid (PLFA) composition was analyzed to investigate the effects of different P treatments on microbial community composition. As described in a previous study (Wang et al., 2016), PLFAs were extracted from freeze-dried soil samples with a single-phase mixture of chloroform-methanol-citrate buffer (1:2:0.8, v/v/v; 0.15 M, pH 4.0) (Frostegard et al., 1993). The resulting fatty acid methylesters were then separated and identified on a gas chromatograph (Agilent 7890 N, Wilmington, DE) fitted with a MIDI Sherlock microbial identification system (Version 4.5, MIDI, Newark, NJ). Separation was accomplished with an Agilent 19091B-102E Ultra 25% phenyl methyl siloxane column (25.0 m × 200 ␮m × 0.33 ␮m). The oven temperature was held at 190 ◦ C, increased to 285 ◦ C with

a gradient of 10 ◦ C min−1 , and then increased to 310 ◦ C with a gradient of 60 ◦ C min−1 and held for 2 min. The temperatures of the injector and detector were 250 ◦ C and 300 ◦ C, respectively. Ultra high purity hydrogen was used as the FID makeup gas at a flow rate of 30 mL min−1 . Individual fatty acids were quantified as the percentages of total fatty acids recovered from the samples. The abundances of individual PLFAs were expressed as mg g−1 soil. Concentrations of individual PLFAs were calculated based on the 19:0 internal standard concentrations and were designated in terms of the total number of carbon atoms: number of double bonds, followed by the position (u) of the double bond from the methyl end of the molecule. The prefixes “a”, “i”, and “cy” indicate anteiso- and iso-branched-chain fatty acids, and cyclopropane groups, respectively. The presence of the main microbial taxa, including gram-positive (G+ ) bacteria, gram-negative (G− ) bacteria, actinomycetes, fungi, and arbuscular mycorrhizal fungal (AMF), was determined based on the presence of specific individual PLFA biomarkers as follows: the sum of i14:0, i15:0, a15:0, i16:0, i17:0, and a17:0 was calculated and used as an indicator of G+ bacteria; G− bacteria were identified based on PLFAs 16:1w9c, 16:1w7c, 17:1w8c, 18:1w5c, 18:1w7c, cy17:0, and cy19:0; 10Me16:0, 10Me17:0, and 10Me18:0 were used as indicators of actinomycetes; fungi were identified by the PLFAs 18:2w6c and 18:3w6c; and the PLFA 16:1w5c was used as a marker for AMF (Olsson, 1999; Zelles, 1999). The sum of the PLFAs was based on the abovementioned microbial fatty acids only, rather than all of the PLFAs detected.

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Table 3 Sequentially-extracted P fractions in two paddy soils subjected to four phosphate treatments in the seventh crop season (2013/Rice) in field stations CS1 and CS2. PW, P fertilization only during the wheat growing season; PR, P fertilization only during the rice season; PR + W, P fertilization during both the rice and wheat seasons; Pzero, no P fertilization during either season. Soil type

Treatment

Labile Pa

Moderately labile Pb

Stable Pc

Resin-P mg kg−1

NaHCO3 -Pi

NaHCO3 -Po

NaOH-Pi

NaOH-Po

HCl-P

Residual-P

CS1

Pzero PW PR PR + W

4.68 ± 1.41d 4.57 ± 0.73 4.11 ± 0.89 6.67 ± 3.32

2.63 ± 0.87 9.54 ± 3.27 7.13 ± 2.35 11.7 ± 2.08

9.57 ± 2.34 20.2 ± 2.22 10.8 ± 1.76 17.1 ± 6.35

15.1 ± 4.57 10.5 ± 0.34 10.4 ± 2.21 12.3 ± 1.43

24.8 ± 4.44 20.3 ± 8.19 25.6 ± 7.60 44.5 ± 14.6

111.2 ± 22.9 124.5 ± 27.3 91.4 ± 25.8 120.3 ± 5.48

374.0 ± 125.5 335.5 ± 72.5 274.1 ± 55.9 341.0 ± 34.8

CS2

Pzero PW PR PR + W

7.56 ± 1.51 11.4 ± 2.12 12.9 ± 4.26 11.7 ± 4.98

14.6 ± 1.34 16.9 ± 3.76 16.2 ± 3.07 21.1 ± 3.34

39.3 ± 7.61 51.2 ± 5.88 52.8 ± 2.06 57.8 ± 14.8

13.9 ± 0.78 16.5 ± 3.50 14.3 ± 3.52 15.0 ± 3.73

34.1 ± 3.85 52.5 ± 23.6 33.5 ± 1.80 25.6 ± 6.41

114.0 ± 9.2 122.5 ± 17.7 117.8 ± 4.34 112.8 ± 18.8

235.6 ± 8.92 251.5 ± 5.32 244.1 ± 5.33 244.5 ± 17.0

0.001 0.276 0.549

0.000 0.016 0.937

0.000 0.117 0.163

0.028 0.638 0.217

0.189 0.736 0.040

0.000 0.551 0.449

0.524 0.380 0.429

Significance level (p) Soil type Treatment Soil type × Treatment a b c d

Labile P is the sum of Resin-P, NaHCO3 -Pi and NaHCO3 -Po. Moderately labile P is the sum of NaOH-Pi and NaOH-Po. Stable P is the sum of HCl-P and Residual-P. Data represent means of triplicates ± standard deviation.

Table 4 The sum of selected phospholipid fatty acid (PLFA) levels in two paddy soils under four different phosphate fertilization treatments in the seventh cropping season (2013/Rice). Significance level (p)

G− bacteria

G+ bacteria

Fungi

AMF

Actinomycetes

Soil type Treatment Soil type × Treatment

0.002 0.120 0.431

0.521 0.135 0.761

0.364 0.308 0.928

0.000 0.052 0.602

0.060 0.213 0.534

Gram-positive (G+ ) bacteria (i14:0 + i15:0 + a15:0 + i16:0 + i17:0 + a17:0); Gram-negative (G− ) bacteria (16:1w7c + 16:1w9c + 17:1w8c + 18:1w5c + 18:1w7c + cy17:0 + cy19:0); fungi (18:2w6c + 18:3w6c); actinomycetes (10Me16:0 + 10Me17:0 + 10Me18:0); arbuscular mycorrhizal fungi (AMF) (16:1w5c).

2.6. Statistical analysis General soil properties and other measurements were tested by one-way analysis of variance (ANOVA). To compare the mean values of treatments, the Duncan multiple range test was used at a significance level of p < 0.05. Phosphorous fractions and the sum of selected PLFA levels were analyzed by two-factorial ANOVA, with soil types, treatments and their interaction as factors. Principal component analysis (PCA) and canonical correspondence analysis (CCA) were used to ordinate plots according to PLFA profiles. All statistical analyses were conducted with the SPSS 16.0 CORRELATE and REGRESSION and FACTOR procedures. The phosphate utilization efficiency (PUE) was calculated by ((Ptreatment − Pzero)/P-input) × 100, where P-treatment is P uptake from the soil treated with fertilizer P (fertilizer P + soil P), and Pzero is P uptake from the control (soil-P). Phosphorus uptake refers to the amount of P removed from the field of crop grain portions (Chien et al., 2012).

3. Results 3.1. Crop yield and total P concentrations The PW and PR + W treatments produced significantly higher grain and straw yields than the Pzero treatment during the wheatgrowing seasons (p < 0.05); however, no notable changes were observed during the rice-growing seasons (Figs. 1 , S1). This was observed at both field stations (CS1 and CS2) over the four-year period. Furthermore, the PW treatment produced no significant difference in crop yields compared to the PR + W treatment (Figs. 1, S1). In field station CS1, which had lower soil Olsen-P concentrations compared to CS2, wheat yield was more sensitive to the different fertilization regimes. During the four-year study period,

the PR treatment compared with the PR + W treatment reduced straw and grain yields of wheat by 8.74–43.2 and 19.4–47.7%, respectively. In comparison, the Pzero treatment reduced wheat straw and grain yields by 7.21–60.0 and 19.6–84.1%, respectively (p < 0.05; Fig. 1). 3.2. Crop total P concentrations and phosphate utilization efficiency In stations CS1 and CS2, the total P concentration was higher in the grains than in the straw by 1.79–5.23-fold in rice and 7.63–20.9fold in wheat (Fig. S2). Compared to the PR + W treatment, the PW and PR treatments consistently showed an increase in PUE (p > 0.05). For example, values of PUE under the PW treatment increased by about 1.15 and 3.61% in the CS1 and CS2 field stations, respectively (Table S1). 3.3. Soil Olsen-P concentration and soil P fractionation Compared to the PR + W treatment, the PW treatment reduced or maintained soil Olsen-P concentrations in the CS1 and CS2 stations over the whole four-year period (Fig. 2). Table 3 illustrates the differences in soil P fractions for the four P fertilizer treatments in CS1 and CS2 after the 2013 rice harvest. The labile P fractions (resin-P, NaHCO3 -Pi, and NaHCO3 -Po) added up to 3.11–6.54% and 13.4–18.5% of total P concentration in CS1 and CS2, respectively, for the four fertilization regimes, while the moderately labile P (NaOHPi and NaOH-Po) fraction ranged from 5.86–10.5% and 8.30–13.2%. The stable P fraction (HCl-extractable P and residual P) ranged from 85.1–87.7% and 52.4–54.4% of total P, respectively. The PW, PR, and PR + W treatments increased labile P levels significantly (p < 0.05). Total P levels in the soil were higher in CS1 than in CS2, which is consistent with the initial total P levels measured four years

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Y. Wang et al. / Field Crops Research 198 (2016) 32–39

-1

Yields in CS1 (kg ha )

20000 2010R

2010/2011W

2011/2012W

2012R

Straw

2012/2013W

2013R

2013/2014W

16000 12000 8000 4000

A A A A

A A A A

A A A A

A A A A B B B

A A A A a a a a a ab bc

0 20000

2010R

c

2010/2011W

a ab b ab

2011R

C C B A a ab b b

a a a a

A

2011/2012W

2012R

BC C

a a a a B

b b b

A bc c a ab

a

-1

Yields in CS2 (kg ha )

2011R

Grain

2012/2013W

2013R

2013/2014W

16000 A AA A A

A A A A

12000

A A A A

A A A A

8000 4000

A

A

A

A

AB

B B

a a a a

a a a a a

ab b b

a a a

a

a a

a

a

Pze ro PR P PR W +W Pze ro PR P PR W +W Pze ro PR P W PR +W Pze ro PR PRPW +W Pze ro PR P PR W +W Pze ro PR P PR W +W Pze ro PR P PR W +W Pze ro PR P PR W +W

0

A

A

a a a a

a a a a a a a a

A A A

B B AB

A

Fig. 1. Variation in grain and straw yields with time after application of four different phosphate fertilization treatments in field stations CS1 and CS2 over a four-year period. For all figures, PW indicates P fertilization only during the wheat-growing seasons, PR indicates P fertilization only during the rice-growing seasons, PR + W indicates P fertilization during both the rice- and wheat-growing seasons, and Pzero indicates no P fertilization during either season. CS1 and CS2 represent soils from Changshu National Agro-Ecosystem Observation and Research Station and Changshu Institute of Agricultural Sciences, respectively. Means followed by the same lowercase and capital letters compare effects of different phosphate treatments on straw and grain yield, respectively. Different letters indicate significant differences (p < 0.05, n = 3). Error bars indicate ± standard deviation of the mean (n = 3).

PR

PW

PR+W

20 CS1 15 10 5 25 20

CS2

15 10

1000 Fungi AMF Actionmycetes + G bacteria G bacteria

-1

Pzero

The sum of selected PLFAs (µg g )

Soil Olsen-P concentration varitation -1 with time (mg kg )

25

800

600

400

200

0

5

Pzero PR R W 1 R W 2 R W 3R W 3 1 4 1 1 2 1 10 20 /201 20 /201 20 /201 20 /201 2 0 3 1 1 1 1 1 20 20 20 20

Fig. 2. Variation in soil Olsen-P concentrations in field stations CS1 and CS2 after harvesting in each crop season over four rice–wheat crop rotations under four different phosphate fertilization regimes. PW, P fertilization only during the wheatgrowing season; PR, P fertilization only during the rice-growing season; PR + W, P fertilization during both the rice- and wheat-growing seasons; Pzero, no P fertilization during either season. Error bars indicate ± standard deviation of the mean (n = 3).

PW PR+W

CS1

Pzero PR

PW PR+W

CS2

Fig. 3. The sum of selected phospholipid fatty acid (PLFA) levels in two paddy soils under four different phosphate fertilization treatments in the seventh cropping season (2013/Rice). Symbols: Gram-positive (G+ ) bacteria (i14:0 + i15:0 + a15:0 + i16:0 + i17:0 + a17:0); Gram-negative (G− ) bacteria fungi (16:1w7c + 16:1w9c + 17:1w8c + 18:1w5c + 18:1w7c + cy17:0 + cy19:0); (18:2w6c + 18:3w6c); actinomycetes (10Me16:0 + 10Me17:0 + 10Me18:0); arbuscular mycorrhizal fungi (AMF) (16:1w5c). Error bars indicate ± standard deviation of the mean (n = 3).

3.4. Microbial community composition ago. However, labile P and moderately labile P concentrations were lower in CS1 than in CS2, which is also similar to the Olsen-P concentrations. Two-way ANOVA revealed a significant effect of the two soils on resin-P, NaHCO3 -Pi, NaHCO3 -Po, NaOH-Pi and HClP (p < 0.05); however, no significant effect of P treatment or the interactions between soil types and treatments was observed.

We used 19 PLFAs as biomarkers to examine the sizes of specific microbial populations in soils under different P treatments in the rice season (Fig. 3). P fertilization (the PW, PR and PR + W treatments) increased the total relative abundance of selected PLFAs significantly in both CS1 and CS2 (p < 0.05), which included PFLAs

Y. Wang et al. / Field Crops Research 198 (2016) 32–39

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labile P, moderately labile P (MLP), acid phosphatase enzyme (ACP) activity, and alkaline phosphatase enzyme (ALP) activity in soils. In addition, crop properties including crop yield and total crop P concentration in response to different treatments also influenced the microbial composition of the soils at both field stations (Fig. 4b). In CS1, the microbial community was related to crop yield, including straw yield, grain yield, and Olsen-P concentration, whereas in CS2, the microbial composition was related to total crop P, labile P, moderately labile P, phosphatase enzyme including acid and alkaline phosphatase enzyme activity, soil pH, total C, and total N.

4. Discussion 4.1. Feasibility of P fertilization during the wheat-growing season only in a rice–wheat rotation system

Fig. 4. a, Principal components analysis (PCA) of phospholipid fatty acid (PLFA) data under four different phosphate treatments in the seventh cropping season in field stations CS1 and CS2 (2013/Rice); b, Canonical correspondence analysis (CCA) relating PLFA biomarkers to crop yield (G-Yield indicates grain yield, S-Yield indicates straw yield); crop total P (G-TP indicates grain total P, S-TP indicates straw total P), and soil properties including TC (total organic C), TN (total N), TK (total K), Olsen-P, Labile P, MLP (moderately labile P), pH, and ACP (acid phosphatase enzyme) and ALP (alkaline phosphatase enzyme) concentrations in soils in field stations CS1 and CS2 under four phosphate treatments.

from G+ bacteria, G− bacteria, actinomycetes, and fungi. The total microbial concentration was higher in CS1 soil than in CS2 soil (Fig. 3). Two-way ANOVA revealed a significant effect of the two soil types on G− bacteria and AMF (p < 0.05); however, there was no significant effect of the four P treatments or the interactions between soil types and P treatments (Table 4) (p > 0.05). Principal components analysis (PCA) of the selected biomarker PLFAs revealed changes in microbial composition in soils with different P treatments (Fig. 4a). The first two principal components (PC1 and PC2) accounted for 84.5% of the variation in the data, which separated the variables in terms of soil P treatments. Clustering of the component weights of both PC1 and PC2 revealed strong covariation in CS1 and CS2. Correspondingly, the relative abundance of the individual PLFAs exhibited regular patterns of change. Canonical correspondence analysis (CCA) revealed that the microbial community was mostly influenced by different environmental variables such as soil properties, including pH, total C (TC), total N (TN), and total K (TK) concentrations, as well as Olsen-P,

Different management options to improve P-use efficiency while reducing P losses have been proposed, with the overall goal of obtaining long-term sustainable crop production (Djodjic et al., 2005). In this study, despite a 50% reduction in P application rate, the PW treatment produced similar yields of rice and wheat over the four-year period compared to the PR + W treatment (Figs. 1 and S1). Moreover, the PW treatment maintained Olsen-P concentrations in both CS1 and CS2 soils (Fig. 2), consistently resulting in slight increases (1.2–3.6%) in PUE compared to the PR + W treatment, although no statistical differences were found due to the variability among replicates (Table S1). These results suggest that P fertilization applied during the wheat-growing season only met the P requirements under the current rice–wheat rotation system. When applied during the wheat season, soil available P in the following rice season increased, probably due to the reduction in Fe3+ to Fe2+ under anaerobic conditions during the rice season, which increased the dissolution and availability of P salts. Iron P (Fe-P) and aluminum P (Al-P) in acidic soils can also dissolve with increasing pH. In addition, chelation of organic acid with Ca, Al, and Fe, the ion exchange effect of organic anion and Fe-P and Al-P, the decreasing positive charge of the soil surface, desorption of adsorbed P in acidic soil after flooding, and the effect of P diffusion in the soil can also contribute to the increased availability of P in the soil (Lu 1998; Wang et al., 2015; Hinsinger, 2001). In contrast to the PW treatment, the PR treatment reduced the wheat crop yield over the four-year experiment (p < 0.05; Fig. 1). These results provide strong support of our hypothesis that omitting P fertilization of flooded rice in rice–wheat paddy fields is a suitable management strategy in TLR, sustaining crop yields and improving P use efficiency while reducing the environmental risk posed by excessive soil P accumulation. The results of the field conditions therefore confirm the results of our previous pot trials (Wang et al., 2015, 2016) and, for the first time, provide evidence of the positive effects of omitting P fertilization of flooded rice in rice–wheat rotation in the long-term. Management of P fertilization must aim to optimize the efficiency of P input to secure yield production while maintaining soil P levels within acceptable limits by avoiding depletion or accumulation and, at the same time, limiting the off-field transport of P to the surface and/or groundwater (Djodjic et al., 2005; MacDonald et al., 2011). Such management practices should not only be beneficial for the local environment, but they should also be economically sustainable for the farmers. The traditional scheme is neither sustainable nor efficient and leads to larger P losses compared to PW treatment. The common use of high concentrations of NPK fertilizer by farmers in TLR regardless of soil P situation and crop season therefore needs to be addressed (NP2 OK2 O ratio: 151515 or 161616). Our pot trials (Wang et al., 2015, 2016) and subsequent field experiments demonstrate that PW and current farming

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practice of P fertilization results in similar yields in rice-wheat rotation cropping paddy fields in the TLR. Adjusting the structure of the fertilizers by reducing or eliminating compounds of NPK fertilizers in the rice season might therefore be an efficient way to achieve this PW regime on a large scale. Moreover, this would be beneficial both to the environment and the economy. Estimations suggest that 0.246 million tons of P2 O5 could be retained in 1.02 million square hectares of paddy soil in four years. Based on the current price of superphosphate (12% P2 O5 , 600 RMB t−1 ), 1.2 billion RMB could be saved in the TLR (Wang et al., 2012a, 2015). In China, the challenge of P management is to balance resource limitations and pollution problems at the field/regional scale. This regime could also be expanded to large-scale application in other upland ricecrop rotated paddy regions in China with excessive P input and high soil P levels. 4.2. Response of chemical and microbial processes to the PW regime during the rice season Advancing our understanding of the agricultural P cycles require us to determine how much total P, inorganic P, or organic P is present in the soil system and which P species or molecules are present at which concentrations (Kruse et al., 2015). Biological processes regulate the movement and distribution of labile forms of P (Stewart and Tiessen, 1987). Our study revealed that despite no P fertilization during the rice season, the PW treatment supplied sufficient available P similar to the PR and PR + W treatments (Table 4). Moreover, the PW treatment had no significant effect on microbial processes compared to the PR and PR + W treatments as determined by profiling of microbial PLFAs (Fig. 3). This was possibly due to the fact that bacteria and fungi are able to liberate orthophosphate from inorganic substrates by releasing organic anions, protons, phosphatases, and cation-chelating compounds (Wakelin et al., 2004; Taurian et al., 2010; Richardson et al., 2011). P fertilization increased levels of the most labile Pi fraction (resin P and NaHCO3 -Pi) compared to the Pzero treatment. In addition, P fertilizer significantly increased the total relative abundance of selected PLFAs in both CS1 and CS2 (p < 0.05), including bacteria, actinomycetes and fungi. The PW treatment increased the values of resin P and NaHCO3 -Pi in CS1 and CS2, respectively, as well as the PR + W treatment. These results suggest that soil P availability is the limiting factor in P unfertilized soils, and that an adequate supply of P fertilizer such as in the PW treatment is essential for maintaining crop productivity in paddy soils in the TLR. Similarly, Zheng et al. (2002) found that the absolute concentration of resin-P increased by 2.6 mg P kg−1 with continuous application of P over 8 years. Malik et al. (2012) reported that the size of inorganic P pools increased significantly after addition of P sources to the soil. Moreover, labile and moderately labile P levels were found to increase after continuous-term P fertilizer application (Negassa and Leinweber, 2009). Additionally, chemical fertilizer application increased microbial biomass and activity without significantly changing bacterial community structure (Chu et al., 2007; Islam et al., 2011) and the population sizes of soil organic P-mineralizing bacteria and inorganic P-solubilizing bacteria increased when mineral fertilizer was applied (Hu et al., 2009). In this study, two-way ANOVA revealed no significant effect of the four P treatments or interaction between soil types and P treatments; however, a significant effect of the two soil types on P fractions and microorganisms was observed (p < 0.05; Tables 3 and 4). The tested soils were collected after the ricegrowing season, and thus the PW treatment involved no P fertilizer during the rice season, unlike the PR and PR + W treatments, which did employ P fertilizer. However, P fractions and microorganisms under the PW treatment showed no significant difference compared to the PR and PR + W treatments. To meet the P requirements

of crops and reduce soil accumulation of unavailable P in rice-wheat rotated paddy soils, these results suggest that P application during the wheat season may be more effective than during the rice season. The microbial concentration was higher in CS1 soil than in CS2 soil due to their different response to soil properties, soil P fractionation, soil phosphatase enzyme activities, and crop responses. The microbial composition of CS2 was more influenced by total crop P, soil labile P and moderately labile P, phosphatase enzymes, soil pH, total C, and total N than that of CS1 (Figs. 3 and 4). In our previous pot study, we found similar results, with soil P pools and microorganisms from different sites showing different responses to crop and soil properties (Wang et al., 2016). DeForest et al. (2012) experimentally increased available P and/or pH in several acidic eastern deciduous forests underlain by glaciated and unglaciated soils, and similarly, found that all treatments had minimal influence on the microbial biomass, although available pools of P were strongly correlated with microbial composition. However, in this study, we failed to find a relationship between microorganisms and P availability because of the complex environment in the field (Table S2) and low-resolution of the PLFA. In our future research, we therefore aim to focus on gaining a more robust understanding of P biogeochemistry and the mechanisms controlling P availability.

5. Conclusions In the intensive rice-wheat rotated paddy soil of the Taihu Lake Region (TLR), farmers excessive use of P fertilizer has led to a dramatic spike in P accumulation. Management options aimed at improving P utilization efficiency while reducing P losses are therefore urgently needed. Our four-year field study of two paddy soils with varying P supply capacities clearly highlights the feasibility of omitting P fertilizer application during the rice season in ricebased cropping paddy fields in the TLR, reducing the environmental impacts related to soil P accumulation without compromising rice yield. Moreover, in the rice season, a P regime without P fertilization not only ensures an adequate supply of labile P and moderately labile P in the soil, but also maintains microbial diversity for P transformation compared to treatments with P fertilization in the rice growing season. To save P resources and balance P needed for optimum crop growth while minimizing P accumulation, our results suggest that omitting P fertilizer during the rice season may be a promising strategy, complementing the more conventional approaches of fertilization, cultivation, breeding and other agricultural practice.

Acknowledgments We especially thank the anonymous reviewers and Dr. Marie Louise Bornø for their constructive comments and language revision, which have greatly improved the manuscript. This work was supported by grants from the Chinese National Basic Research Program (No. 2015CB150403), the Strategic Priority Research Program of the Chinese Academy of Sciences (No. XDB15020402), the Jiangsu Agriculture Science and Technology Innovation Fund (CX(15)1004), and the National Natural Science Foundation of China (No. 41671304, 41571294).

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.fcr.2016.08.025.

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