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Soil Organic N Forms and N Supply as Affected by Fertilization Under Intensive Rice Cropping System1
Pedosphere 16(3): 345-353, 2006 ISSN 1002-0160/CN 32-1315/P @ 2006 Soil Science Society of China Published by Elsevier Limited and Science Press
Soil Organic N Forms and N Supply as Affected by Fertilization Under Intensive Rice Cropping System*' ZHANG Qi-Chun, WANG Guang-Huo*2 and XIE Weng-Xia College of .Environmental and Natural Resources Science, Zhejiang University, Hangzhou 31 0029 (China). [email protected]
E-mail:
(Received September 18, 2005; revised March 27, 2006)
ABSTRACT Changes of soil organic nitrogen forms and soil nitrogen supply under continuous rice cropping system were investigated in a long-term fertilization experiment in Jinhua, Zhejiang Province, China. The fertilizer treatments included combination of P-K, N-K, N-P, and N-P-K as well as the control. After six years of continuous double-rice cropping, total soil N and hydrolysable N contents remained stable in plots with N treatments, while the hydrolysable N contents were substantially reduced in those plots without N application. Compared t o the unbalanced fertilization treatments, P and K increased the percentage of hydrolysable ammonium N in the total soil N with the balanced application of N, and also maintained higher rice grain yields and nitrogen uptake. Grain yield was positively correlated with total N uptake (T = 0.875**), hydrolysable N ( T = 0.608**), hydrolysable ammonium N (T = 0.560**) and the hydrolysable unknown N (T = 0.417**). Total N uptake was positively correlated with hydrolysable N (T = 0.608**),hydrolysable ammonium N ( T = 0.440**) and hydrqlysable unknown N (r = 0.431**). Soil nutrient depletion and/or unbalanced fertilization to rice crop reduced N content in soil microbial biomass, and therefore increased C/N ratio, suggesting a negative effect on the total microbial biomass in the soil. Key Words:
continuous cropping, microbial biomass, organic N forms, rice
INTRODUCTION China is one of the main countries producing rice (Oryza sativa L.) in the world. The demand for rice has been kept rising owing t o continuous growth of population, resulting in more frequent applications of fertilizers in the rice production. Therefore, environmental pollution by nutrient leaching or runoff from rice fields has become a major concern (Zhang et al., 1988; Si et al., 2000). Acquiring detailed information about the soil fertility status is a prerequisite for accurately assessing the long-term impact of modern, intensive rice production techniques on paddy soils. Once applied into a paddy soil, fertilizer N may be absorbed by rice and eventually become part of the soil organic matter (SOM) or soil microbial biomass, or it remains in soluble N pools such as NHi-N. Smith et al. (1993) found that N derived from fertilizer was rapidly transformed into organic forms. As a basic component of all proteins and nucleic acids, nitrogen is essential for plant growth. Soil nitrogen is the main source of indigenous N supply t o the crop, contributing at least 50% to the total N uptake by rice (Shibahara and Inubushi, 1997). Organic nitrogen compounds account for well over 90% of the total nitrogen in most soils (Parson and Tinsley, 1975), and a considerable amount of organic N in soil is continually mineralized into NH$ forms through microbial decomposition of native organic matter, becoming available to plants. A better knowledge of the nature of soil nitrogen will allow us to better understand soil nitrogen cycle and to use nitrogen in soils more efficiently (Cassman et al., 1996). Because of the importance of nitrogen in soil biochemistry and soil fertility, a lot of researches regarding this nature have been conducted, including fractionation of soil N and assessment of N fractions for "lProject supported by the International Fertilizer Industry Association (IFI), France; the Potast & Phosphate Institute (PPI), USA and Canada; and the International Potassium Institute (IPI), Switzerland. *'Corresponding author. E-mail: ghwangQzju,edu.cn.
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plant nutrient availability. Chinese farmers had been using organic manure as fertilizer for crops for a very long time. But the traditional practice has been changed during the past 25 years because of the readily availability of chemical fertilizers, while manure is far less convenient to handle with for farmers. In Jinhua, Zhejiang Province, only few farmers still apply farmyard manure to their rice fields. Most rice fields have received very little manure for many years. It is not clear whether the change from organic manure to commercial fertilizers in rice fields affects the quality of soil organic matter and soil microbial activity, which controls the C and N cycling in the intensive rice production system (Li, 1993; Olk et al., 1996; Dawe et al., 2003). The soil microbial biomass is a labile pool of organic matter and comprises 1%-3% of total organic matter. The microbial biomass N is considered to act as both agent for biochemical changes in soil and repository of plant nutrients (Jenkinson, 1988; Akmal et al., 2004). Nitrogen mineralization is an essential function of the soil microbial system. The importance of soil microorganisms has stimulated the increase of the interests in measuring nutrients held in their biomass (Martikainen and Palojarvi, 1990; Das et aL, 1997). A long-term fertilization experiment in rice fields had been conducted since 1998 in Jinhua City, Zhejiang Province to investigate the effects of different fertilization on rice production and soil fertility over the time. Focused on the changes in organic N forms and N supply to the rice, the objectives of this study were to: 1) fractionate soil organic nitrogen and assess the dynamics of different soil N components, 2) evaluate the relationships between grain yield and N uptake by the rice and soil N fractions, and 3) quantify the effects of different fertilizer treatments on soil microbial biomass and indigenous N supply. MATERIALS AND METHODS
Experimental design A long-term field fertilization experiment was established in 1998 in a suburb of Jinhua City, Zhejiang Province. The soil at the study site belongs to Fluvisols. Initial soil samples were colleted from 0-15 cm in depth in 1998 and basic physico-chemical properties were determined (Table I). Except for the control, the experiment applied the soil plots with one of the four combined fertilizer treatments of P-K, N-P, N-K, and N-P-K as main plots and rice varieties as sub-plots. The latter was for a comparison of a conventional modern rice variety with a hybrid rice variety. The field trial was conducted in a split-plot design with three replications. The area of each subplot was 45 m2 and rice transplanting spacing was 20 cm x 20 cm. Urea was used as source of N at 150 kg N ha-’ for early rice and 180 kg N ha-’ for late rice. 50% of the N was applied as basal 1 day before transplanting, 25% a t pre-tillering and 25% at panicle initiation stage. Superphosphate (25 kg P ha-’) was applied as basal. Muriate of potash (100 kg K ha-’) was used as source of K with 50% applied as basal and 50% at panicle initiation stage. Soil samples (0-15 cm) of all plots were collected every year after the late rice harvest. Only soil samples from the hybrid rice plots were analyzed in this study. TABLE I Selected physico-chemical properties of the soil at Jinhua, Zhejiang Soil type
pHaf
Total N
Available-N
Olsen’s-P
Available-K
Sand
Paddy soil
4.8
g kg-l 2.7
123.4
mg kg-’ 16.5
54.6
278
Silt gkg-’ 562
Clay ~
160
a)The extraction ratio of soil Co H 2 0 is 1:l.
Nitrogen analysis The soil samples were digested by the Kjeldahl method to extract the total organic soil N, which were
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SOIL N UNDER RICE CROPPING
then measured in the hydrolysate prepared by refluxing the soil sample with 6 mol L-l HC1 (Bremmer and Mulvaney, 1982). Total hydrolysable N was determined by steam distillation with 10 mol L-' NaOH after Kjeldahl digestion and acid hydrolyzation. Hydrolysable ammonium N (NH4-N) was measured by steam distillation with 3.5% MgO. Amino sugar N was calculated as the difference between the N content obtained from steam distillation of the hydrolysate with phosphate-borate buffer at pH 11.2 and the hydrolysable ammonium N content. Amino acid N (a-amino-N) was determined by steam distillation of an aliquot of the hydrolysate with phosphate-borate buffer and 5 mol L-' NaOH after treatment with 0.5 mol L-' NaOH at 100 "C to digest hexosamines and remove NH4-N. Ninhydrin powder was added to convert the amino-N to NH4-N. Acid insoluble nitrogen was estimated by subtracting hydrolysable N from total nitrogen. The amount of hydrolysable unknown N (HUN) was calculated as: HUN = total hydrolysable N - (NH4-N
+ amino acid N + amino sugar N)
(1)
Soil microbial biomass analysis After the late rice harvest in 2003, additional soil samples were collected from all treated plots. The moist field soil samples were slightly air-dried to pass through a 2-mm mesh sieve. The moisture of samples was adjusted to about 60% of soil water holding capacity, and then the samples were incubated for 7-10 d at 25 "C. Soil microbial biomass was measured using the fumigation extraction method (Brookes et al., 1985). Immediately after samples were collected, 15 g moist soil sample was extracted by shaking with 60 mL of 0.5 mol L-' KzS04 for 30 min. A parallel sample was fumigated for 24 h with ethanol-free chloroform and then extracted same as above. Microbial biomass C (MBC) was determined using a Shimadzu TOC-5000 analyzer and soil microbial biomass nitrogen (MBN) was determined by Kjeldahl digestion method (Brookes et al., 1985). MBC was estimated as MBC = EC/0.45 (Wu and Joergensen, 1990), where EC (extractable carbon) is the difference between carbon extracted from fumigated and un-fumigated samples, both expressed in the same measurement unit. MBN was estimated as MBN = EN/0.54 (Brookes et aL, 1985), where E N (extractable nitrogen) is the difference between N extracted from fumigated and un-fumigated samples.
Organic matter analysis The method developed by Olk et al. (1995) was employed to extract mobile humic acid (MHA) and calcium humate (CaHA). Briefly, air-dried soil was incubated for 24 h in 0.25 mol L-' NaOH at a ratio of so1ution:soil as 2.5:l. The solutions were centrifuged and the soluble MHA was decanted and acidified to pH 2. The soil was washed twice or more with water to maximize humic acid recovery. These precipitates were combined with the precipitate of the NaOH wash and considered as MHA. The soil sample was decalcified by washing with 0.1 mol L-' HC1 until the pH of the supernatant reached below 1.5, and then the soil sample was incubated for 24 h in 0.25 mol L-' NaOH. Like the MHA extraction, the solutions were centrifuged, and the solubilized CaHA was decanted and acidified to pH 2.
Statistical analysis Data were statistically analyzed by one-way ANOVA and significant differences were distinguished by the Tukey-HSD test at P < 0.05 and P < 0.01 levels. RESULTS AND DISCUSSION
Organic N forms in soil Table I1 presents the effect of different fertilization treatments on total soil organic N and its com-
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ponents in the paddy soil.. After 6 years of continuous double-rice cropping, the soil total N and the total hydrolysable N contents had maintained almost stable in the N-supply plots, while the contents of the total hydrolysable N and HUN were substantially reduced in plots without N treatment. Table I1 also showed that continuous balanced application of N, P, and K mineral fertilizers (N-P-K plot) maintained proper amount of hydrolysable ammonium compared t o the initial level or 25% higher than the control (CK). The greater amount of hydrolysable ammonium in N-P-K treatment is probably because of the retention of organic N in the form of crop residue even though most of the straw was removed with harvest. This might also be due to the increase of fixation of NH,f after application of mineral N fertilizer (Shen and Shi, 1990). TABLE I1 Contents of the total soil organic N and its components in the bulk soils after 6-year fertilization treatments under continuous double-rice cropping Year
1998 2003
Treatmenta)
Original soil CK P-K N-K N-P N-P-K
Hydrolysable N Total
Ammonium
Amino acid
Amino sugar
1450ab) 1078b 1089b 1354ab 1479a 1445a
376a 258c 253c 25% 270c 32313
523b 401c 568b 583b 622a 615a
59c 89ab 107ab lllab 123a 82ab
Non-hydrolysable N
Total soil N
434c 583b 703a 430c 460c 455c
1884a 1661b 1792ab 1784ab 1939a 1899a
Hydrolysable unknown N mg kg-' 492a 330b 161c 401ab 464a 425a ____
a)CK: the control; P-K: the combination of P and K fertilizers; N-K: the combination of N and K fertilizers; N-P: the combination of N and P fertilizers; and N-P-K: the combination of N, P, and K fertilizers. b)Means followed by the same letter(s) within each column are not significantly different at P = 0.05 level by Tukey-HSD.
Although free amino acids are only present in very small concentrations, amino acid polymers that can be hydrolyzed by acid are ubiquitous in soil and commonly make up 30% t o 45% of the nitrogen in surface soil. Amino acids are the building constituents of protein. Table I1 showed that the amount of hydrolysable amino acid N was markedly high in all soils with fertilizer treatments as compared to the control (CK) or the original soil. The percentages of various organic N forms to the total soil N are listed in Table 111. Compared with the original soil before the experiment in 1998, the percentages of hydrolysable ammonium N and HUN decreased for all treatments six years later. There was no significant change in the percentages of total non-hydrolysable N for different treatments. The percentage of hydrolysable amino acid N was low for the control, while it increased in other fertilizer treatments. TABLE I11 Percentages of different nitrogen forms to the total N in the soils after 6-year fertilization treatments under continuous double-rice cropping Year
Treatmenta)
Hydrolysable N
Non-hydrolizable N
Ammonium
Amino acid
Amino sugar
Hydrolysable unknown N
19.9 15.5 14.1 14.5 13.9 17.0
27.8 24.2 31.7 32.7 32.1 32.4
3.1 5.4 5.9 6.2 6.3 4.3
26.1 19.9 9.0 22.5 23.9 22.4
% 1998 2003
Original soil CK P-K N-K
N-P N-P-K
23.0 35.1 39.2 24.1 23.8 23.9
")CK: the control; P-K: the combination of P and K fertilizers; N-K: the Combination of N and K fertilizers; N-P: the combination of N and P fertilizers; and N-P-K: the combination of N, P, and K fertilizers.
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SOIL N UNDER RICE CROPPING
Changes of soil organic N The amount of hydrolysable ammonium showed a slight decrease in all treatments under continuous rice cropping, but not statistically significant (Table 11, Fig. la). HUN showed a general decrease in all treatments while decrease with years was obvious for P-K and CK treatments. The reduction in HUN followed the order of P-K > CK > N-P-K (Fig. lb). With continuous rice cropping, HUN decreased at a rate of about 24% per year for the 6-year experiment in the P-K treatment. The decrease was stabilized after the first four years for CK treatment. For the N-P-K treatment, it decreased in the first three years, increased after that, and stabilized at a high level. Table IV showed that the P-K plot had 500
-
200
-
100
-
u)
E 300
v
r
01
I
1998
1999
2000
2001
2002
2003
100
-
0' 1998
I
1999
2000
Year
2001
2002
2003
Year
15 + CK
+ P-K + N-P-K
1000
t
500 0' 1998
I
1999
2000
2001
2002
2003
Year The changes of soil organic nitrogen components from 1998 to 2003 under different fertilization treatments with continuous double-rice cropping. HUN: hydrolysable unknown N; CK: the control; P-K: the combination of P and K fertilizers; and N-P-K: the combination of N, P, and K fertilizers. TABLE IV Means of grain yields and total N uptake of hybrid rice from 1998 to 2003 under different fertilization treatmentsa) with continuous double-rice cropping a t Jinhua City, Zhejiang Province Year
CK 1998 1999 2000 2001 2002 2003
Total N uptake
Grain yield
4927ab) 3836b 4299ab 3624c 3659c 3660c
P-K 5460a 4119b 4198b 4254b 3799bc 4474b
N-K 5817a 4448c 6326a 5752ab 5117b 5565b
N-P
N-P-K
5789a 3563c 4163b 4535b 3813c 5597a
kg ha-l 5878a 57.30a 482913 50.27ab 5733a 48.27b 5911a 42.97~ 4754b 41.62~ 6369a 39.27~
CK
P-K
N-K
N-P
N-P-K
65.36a 49.66b 47.41~ 48.67bc 41.67d 50.49b
123.02a 90.22b 106.67ab 98.4613 85.46~ 92.09b
107.92a 73.91~ 74.62~ 90.75b 75.01~ 91.96b
116.8a 93.65b 110.81a 116.58a 86.3713 118.65a
&)CK:the control; P-K: the combination of P and K fertilizers; N-K: the combination of N and K fertilizers; N-P: the combination of N and P fertilizers; and N-P-K: the combination of N, P, and K fertilizers. b)Means followed by the same letter(s) within each column are not significantly different at P = 0.05 level by Tukey-HSD.
Q. C. ZHANG e t al.
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higher N uptake than the control, indicating that soil N pool depletion might occur faster under consecutive cropping when P, K and other nutrients are abundant. Overall, these data suggested that HUN might play an important role in the nitrogen supply of intensive rice cropping system. Total hydrolysable N contents were maintained constant in the N plots, but decreased slightly in CK and P-K treatments, indicating the sensitivity of N application with HUN (Fig. lc).
Correlation between organic N and rice yield In spite of yearly variation in grain yield and nitrogen uptake of hybrid rice, the balanced fertilizer treatment (N-P-K) always had higher grain yield and higher nitrogen uptake (Table IV) than the other treatments or the control. Total N uptake from mass balance was measured. It could concluded that the factors affected N leaching and runoff were the same in all the experiment plots due to their similar growth environment. All of the environmental factors could be offsetted each other so that, the factors might not affect the statistical results. Grain yield of rice was positively correlated with total N uptake in plant ( r = 0.875**), hydrolysable N ( r = 0.608**), hydrolysable ammonium N ( r = 0.560**) and HUN ( r = 0.417**) in soils (Table V). Total N uptake was positively correlated with hydrolysable N ( r = 0.608**), hydrolysable ammonium N ( r = 0.440**) and HUN ( r = 0.431**). These results suggested that organic forms of N in soil contributed greatly to the indigenous N supply in rice field. It should also be noted, however, that none of the different N fractions extracted explained more than 20% to 36% of rice N uptake. TABLE V Correlation coefficients ( r ) among different N forms and grain yield of hybrid rice after 6-year fertilization treatments with continuous double-rice cropping
Grain yield Total N uptake Ammonium Amino acid Amino sugar Hydrolysable unknown N Total hydrolysable N
Total N uptake
Ammonium
Amino acid
Amino sugar
Hydrolysable unknown N
Total hydrolysable N
Total soil N
0.875**
0.560** 0.440*
0.178 -0.175 -0.224
-2.53 -0.126 -0.786** 0.398*
0.417* 0.431* 0.413' -0.308 -0.269
0.608** 0.608** 0.420* 0.270 -0.056 0.808**
0.320 0.401* 0.040 0.300 -0.029 0.151 0.295
*,**Significant at P = 0.05 and P = 0.01 levels, respectively (n = 25).
Soil organic matter in paddy soil Hutnic acids extracted by NaOH can be separated into Ca-bound (i.e. CaHA) and non-bound (i.e. MHA) fractions in the characterization of soil organic matter properties (Shinkarev et al., 1987). Although both are labile, MHA is considerably enriched in N and hydrolysable amino acids compared with CaHA in all soils (Olk et al., 1995 and 1996). Both MHA and CaHA contribute to N mineralization and immobilization that govern the N supplying capacity of paddy soils (Olk et al., 1996). The contents of MHA and CaHA in the soils with different fertilizer treatments are shown in Table VI. No significant difference was found in the contents of MHA or CaHA among the different treatments, and the amounts of MHA and CaHA did not decline with rice cropping for six years possibly because continuous rice cropping promoted the conversion of lablile N materials into stable and humified organic substance, which was similar t o the findings by Janzen (1987). It is not well known at present if the MHA and CaHA in acidic paddy soil have similar properties as in calcareous soil reported by Olk et al. (1995). Therefore, this investigation focused on the effect of fertilizer and continuous rice cropping on soil organic N and inherent soil fertility. The fact of increase in certain N forms coupled with the decrease in others indicated conversion from one form t o the other. The exact mechanism of the conversion of various N forms could only be evidenced through 15N tracer technology. Detailed characterization of
351
SOIL N UNDER RICE CROPPING
these two humic acid fractions need be conducted in the further study. TABLE VI Contents of humic substances in the bulk soils of different fertilization treatmentsa) with continuous double-rice cropping Year
Mobile humic acid
Calcium humate
CK
P-K
N-K
N-P
N-P-K
6.86 7.36 8.42 6.22
6.47 6.08 10.14 6.07
6.87 5.81 10.09 6.74
6.34 7.00 10.48 6.75
6.60 7.67 9.93 6.45
CK
P-K
N-K
N-P
N-P-K
2.38 1.62 1.95 2.20
2.73 2.26 2.48 2.40
2.65 2.34 2.16 2.09
2.73 1.91 2.32 2.51
2.12 2.09 2.19 2.53
mg g-l
1999 2000 2001 2002
a)CK: the control; P-K: the combination of P and K fertilizers; N-K: the combination of N and K fertilizers; N-P: the combination of N and P fertilizers; and N-P-K: the combination of N, P, and K fertilizers.
Soil microbial biomass The soil microbial biomass acts as both source and sink of the plant nutrient, and regulates the function of soil system. Marumoto et al. (1982) found that the quantities of nutrients mobilized in two German upland soils were closely related to the amounts available in freshly killed biomass; and they showed a scheme for the transformation of dead microbial biomass C and N in arable soil during 4 weeks. Soil microbial biomass nitrogen contents and C/N ratio in different treatments are shown in Fig. 2. Microbial biomass N varied from 23.7 to 73.3 mg N kg-' soil and accounted for 2.5% to 3.7% of the total nitrogen in the soil. The N-P-K treatment had much higher soil microbial biomass N than other treatments (N-P, P-K and N-K) (Fig. 2a). The C/N ratios of the microbial biomass in N-P-K and N-K treatments (7.6 and 8.7, respectively) were much lower than in other treatments (12-15.8) (Fig. 2b). The C/N ratio of microbial biomass in different treatments followed the order: N-P > P-K > CK > N-K > N-P-K. For the late rice in the year of 2003, N-P plots exhibited serious K deficiency, while N-K plot did not show obvious P deficiency symptoms, suggesting that K might be more important limited factor than P in these soils. This could also be partially confirmed by the fact that N-K plots always had higher yields and higher N uptake than N-P plots (Table IV). The C/N ratio of the soil microbial biomass in the N-P treatment was also much higher than that in the N-K treatment. It seemed that nutrient deficiency and/or nutrient unbalance supply not only reduced the soil microbial biomass, but also increased the C/N ratio of soil microbial biomass. Sufficient supply of K seemingly facilitated the uptake of N in microbial biomass and rice, and therefore enhanced the grain yield. Since total biomass N and C/N ratio of microbial biomass revealed the quantity, activity and composition of the biomass
z
1
loo
a
b
il L L
P-K
N-P
CK
N-P-K Treatment
N-K
11
P-K
N-P
CK N-P-K Treatment
N-K
Fig. 2 Soil microbial biomass N (a) and C/N ratio of microbial biomass (b) in different fertilization treatments with continuous double-rice cropping. CK: the control; P-K: the combination of P and K fertilizers; N-K: the combination of N and K fertilizers; N-P: the combination of N and P fertilizers; and N-P-K: the combination of N, P, and K fertilizers.
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Q. C. ZHANG et al.
of the soil, they might serve as indexes of soil nutrient status. The effects of N, P, K treatments on N uptake and C/N ratio in microbial biomass need to be studied thoroughly in the future. CONCLUSIONS The forms of organic N varied with the different fertilization. Long-term balanced application of N, P and K promoted the amount of total hydrolysable organic N and hydrolysable ammonium, whilst unbalanced fertilization to rice resulted in a decline in organic N pools that led to decrease of N uptake by crop and soil microbial biomass. Although the exact chemical nature of HUN was unknown, this fraction apparently played a significant role in the nitrogen uptake of rice. Total hydrolysable N and HUN might be two useful and key indexes of N supply to rice in paddy soil. During the experiment, rice straw was removed from the field after crop harvest, and only short rice stubble (2-5 cm in soil depth) remained in the field. Our results indicated that with proper application of mineral N fertilizer without presence of organic manure, soil total N and hydrolysable N contents could still be well maintained. However, sustainability of the hydrolysable ammonium N and HUN was not guaranteed. Balanced application of N, P and K promoted microbial biomass, whereas unbalanced fertilization reduced microbial N and increased the C/N ratio of the microbial biomass. The reduction of N uptake may be due to insufficient supply of K instead of P. Fertilizer effects on specific groups of microorganisms and microbial activities in paddy rice systems remain to be studied in the future. ACKNOWLEDGEMENTS The constructive comments and suggestions by Dr. R. Buresh and Dr. C. Witt in the International Rice Research Institute, Philippines, and Dr. A. Dobermann in the University of Nebraska, USA, are gratefully acknowledged. We are also grateful to Mr. Sun Qing-Zu and Mr. Fu Rong-Xing in the Jinhua Agricultural Bureau, Zhejiang for their excellent management of the field experiment. REFERENCES Akmal, M., Khan, K. S. and Xu, J. M. 2004. Dynamics of microbial biomass in a rainfed soil under wheat cultivation. Pedosphere. 14(1): 53-62. Bremmer, J. M. and Mulvaney, C. S. 1982. Nitrogen. I n Page, A. L., Miller, R. H. and Keeney, D. R. (eds.) Methods of Soil Analysis. Part 2. Chemical and Microbiological Properties. ASA and SSSA, Madison, USA. pp. 595-624. Brookes, P. C., Andrea, L., Pruden, G. and Jenkinson, D. S. 1985. Chloroform fumigation and release of soil nitrogen: A rapid direct extraction method t o measure microbial biomass nitrogen in soil. Soil Boil. Biochem. 17(6): 837-842. Cassman, K. G., Dobermann, A., Sta. Cruz, P. C., Gines, G. C., Samson, M. I., Descalsota, J. P., Alcantara, J. M., Dizon, M. A. and Olk, D. C. 1996. Soil organic matter and the indigenous nitrogen supply of intensive irrigated rice systems in the tropics. Plant and Soil. 182: 267-278. Das, A. K., Boral, L., Tripathi, R. S. and Pandey, H. N. 1997. Nitrogen mineralization and microbial biomass-N in a subtropical humid forest of Meghalaya, India. Soil Biol. Biochem. 29(9): 1609-1 612. Dawe, D., Dobermann, A., Ladha, J . K., Yadav, R. L., Bao, L., Gupta, R. K., Lal, P., Panaullah, G., Sariam, O., Singh, Y . , Swarup, A. and Zhen, Q. X. 2003. Do organic amendments improve yield trends and profitability in intensive rice systems? Field Crops Res. 83(2): 191-213. Janzen, H. H. 1987. Soil organic matter characteristics after long-term cropping to various spring wheat rotations. Canadian Journal of Soil Science. 67: 845-856. Jenkinson, D. S. 1988. Determination of microbial biomass carbon and nitrogen in soil. In Wilson, J. R. (ed.) Advances in Nitrogen Cycling in Agricultural Ecosystems. Commonwealth Agricultural Bureau International, Wallingford. pp. 368-385. Li, S. Y. 1993. Yield stability and fertilizer efficiency of long-term triple cereal cropping in paddy fields of China. Biol. Fertil. Soils. 16: 151-153. Martikainen, P. J. and Palojarvi, A. 1990. Evaluation of the fumigation-extraction method for the determination of microbial C and N in a range of forest soils. Soil Biol. Biochem. 22: 797-802. Marumoto, T., Anderson, J. P. E. and Domsch, K. H. 1982. Mineralization of nutrients from soil microbial biomass. Soil Biol. Biochem. 14: 469-475.
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Olk, D. C., Cassman, K. G., Randall, E. W., Kinchesh, P., Sanger, L. J. and Anderson, J. M. 1996. Changes in chemical properties of organic matter with intensified rice cropping in tropical lowland soil. European Journal of Soil Science. 47: 293-303. Olk, D. C., Cassman, K. G. and Fan, T. W. M. 1995. Characterization of two humic acid fractions from a calcareous vermiculitic soil: Implications for the humification process. Geodema. 6 5 : 195-208. Parson, J . W. and Tinsley, J. 1975. Nitrogenous substances. In Gieseking, J. E. (ed.) Soil Components. Volume 1. Springer Verlag, New York. pp. 263-304. Shen, Q. R. and Shi, R. H. 1990. Study on the distribution and bioavailability of forms of organic N. J. Soil Sci. (in Chinese). 21(2): 54-57. Shibahara, F. and Inubushi, K. 1997. Effects of organic matter application on microbial biomass and available nutrients in various types of paddy soils. Soil Sci. Plant Nutr. 43(1): 191-203. Shinkarev, A. A., Kostyukevich, I. I., Hinkst, G. and Fischer, T. 1987. Kinetics of the dissolving of humic acids free and bound to calcium in the A1 horizon of Chernozems and Sod-Podzolic soils. Soviet Soil Science. 19: 78-80. Si, Y. B., Wang, S. Q. and Chen, M. H. 2000. Leaching of nitrogen and phosphorous in field and nutrient accumulation in water. Soils (in Chinese). 4: 188-191. Smith, C. J., Freney, J. R. and Mosier, A. R. 1993. Effect of acetylene provided by wax-coated calcium carbide on transformation of urea nitrogen applied to an irrigated wheat crop. Biol. Fertil. Soils. 16: 86-92. Wu, J . and Joergensen, R. G. 1990. Measurement of soil microbial biomass C-An automatic procedure. Soil Biol. Biochern. 22: 1167-1 169. Zhang, S. L., Zhu, Z., Xu, Y., Chen, R. and Li, A. 1988. On the optimal rate of application of nitrogen fertilization for rice and wheat in Tai-lake region. Soils (in Chinese). 20: 5-9.
" N " "
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Soil Organic N Forms and N Supply as Affected by Fertilization Under Intensive Rice Cropping System*' ZHANG Qi-Chun, WANG Guang-Huo*2 and XIE Weng-Xia College of .Environmental and Natural Resources Science, Zhejiang University, Hangzhou 31 0029 (China). [email protected]
E-mail:
(Received September 18, 2005; revised March 27, 2006)
ABSTRACT Changes of soil organic nitrogen forms and soil nitrogen supply under continuous rice cropping system were investigated in a long-term fertilization experiment in Jinhua, Zhejiang Province, China. The fertilizer treatments included combination of P-K, N-K, N-P, and N-P-K as well as the control. After six years of continuous double-rice cropping, total soil N and hydrolysable N contents remained stable in plots with N treatments, while the hydrolysable N contents were substantially reduced in those plots without N application. Compared t o the unbalanced fertilization treatments, P and K increased the percentage of hydrolysable ammonium N in the total soil N with the balanced application of N, and also maintained higher rice grain yields and nitrogen uptake. Grain yield was positively correlated with total N uptake (T = 0.875**), hydrolysable N ( T = 0.608**), hydrolysable ammonium N (T = 0.560**) and the hydrolysable unknown N (T = 0.417**). Total N uptake was positively correlated with hydrolysable N (T = 0.608**),hydrolysable ammonium N ( T = 0.440**) and hydrqlysable unknown N (r = 0.431**). Soil nutrient depletion and/or unbalanced fertilization to rice crop reduced N content in soil microbial biomass, and therefore increased C/N ratio, suggesting a negative effect on the total microbial biomass in the soil. Key Words:
continuous cropping, microbial biomass, organic N forms, rice
INTRODUCTION China is one of the main countries producing rice (Oryza sativa L.) in the world. The demand for rice has been kept rising owing t o continuous growth of population, resulting in more frequent applications of fertilizers in the rice production. Therefore, environmental pollution by nutrient leaching or runoff from rice fields has become a major concern (Zhang et al., 1988; Si et al., 2000). Acquiring detailed information about the soil fertility status is a prerequisite for accurately assessing the long-term impact of modern, intensive rice production techniques on paddy soils. Once applied into a paddy soil, fertilizer N may be absorbed by rice and eventually become part of the soil organic matter (SOM) or soil microbial biomass, or it remains in soluble N pools such as NHi-N. Smith et al. (1993) found that N derived from fertilizer was rapidly transformed into organic forms. As a basic component of all proteins and nucleic acids, nitrogen is essential for plant growth. Soil nitrogen is the main source of indigenous N supply t o the crop, contributing at least 50% to the total N uptake by rice (Shibahara and Inubushi, 1997). Organic nitrogen compounds account for well over 90% of the total nitrogen in most soils (Parson and Tinsley, 1975), and a considerable amount of organic N in soil is continually mineralized into NH$ forms through microbial decomposition of native organic matter, becoming available to plants. A better knowledge of the nature of soil nitrogen will allow us to better understand soil nitrogen cycle and to use nitrogen in soils more efficiently (Cassman et al., 1996). Because of the importance of nitrogen in soil biochemistry and soil fertility, a lot of researches regarding this nature have been conducted, including fractionation of soil N and assessment of N fractions for "lProject supported by the International Fertilizer Industry Association (IFI), France; the Potast & Phosphate Institute (PPI), USA and Canada; and the International Potassium Institute (IPI), Switzerland. *'Corresponding author. E-mail: ghwangQzju,edu.cn.
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plant nutrient availability. Chinese farmers had been using organic manure as fertilizer for crops for a very long time. But the traditional practice has been changed during the past 25 years because of the readily availability of chemical fertilizers, while manure is far less convenient to handle with for farmers. In Jinhua, Zhejiang Province, only few farmers still apply farmyard manure to their rice fields. Most rice fields have received very little manure for many years. It is not clear whether the change from organic manure to commercial fertilizers in rice fields affects the quality of soil organic matter and soil microbial activity, which controls the C and N cycling in the intensive rice production system (Li, 1993; Olk et al., 1996; Dawe et al., 2003). The soil microbial biomass is a labile pool of organic matter and comprises 1%-3% of total organic matter. The microbial biomass N is considered to act as both agent for biochemical changes in soil and repository of plant nutrients (Jenkinson, 1988; Akmal et al., 2004). Nitrogen mineralization is an essential function of the soil microbial system. The importance of soil microorganisms has stimulated the increase of the interests in measuring nutrients held in their biomass (Martikainen and Palojarvi, 1990; Das et aL, 1997). A long-term fertilization experiment in rice fields had been conducted since 1998 in Jinhua City, Zhejiang Province to investigate the effects of different fertilization on rice production and soil fertility over the time. Focused on the changes in organic N forms and N supply to the rice, the objectives of this study were to: 1) fractionate soil organic nitrogen and assess the dynamics of different soil N components, 2) evaluate the relationships between grain yield and N uptake by the rice and soil N fractions, and 3) quantify the effects of different fertilizer treatments on soil microbial biomass and indigenous N supply. MATERIALS AND METHODS
Experimental design A long-term field fertilization experiment was established in 1998 in a suburb of Jinhua City, Zhejiang Province. The soil at the study site belongs to Fluvisols. Initial soil samples were colleted from 0-15 cm in depth in 1998 and basic physico-chemical properties were determined (Table I). Except for the control, the experiment applied the soil plots with one of the four combined fertilizer treatments of P-K, N-P, N-K, and N-P-K as main plots and rice varieties as sub-plots. The latter was for a comparison of a conventional modern rice variety with a hybrid rice variety. The field trial was conducted in a split-plot design with three replications. The area of each subplot was 45 m2 and rice transplanting spacing was 20 cm x 20 cm. Urea was used as source of N at 150 kg N ha-’ for early rice and 180 kg N ha-’ for late rice. 50% of the N was applied as basal 1 day before transplanting, 25% a t pre-tillering and 25% at panicle initiation stage. Superphosphate (25 kg P ha-’) was applied as basal. Muriate of potash (100 kg K ha-’) was used as source of K with 50% applied as basal and 50% at panicle initiation stage. Soil samples (0-15 cm) of all plots were collected every year after the late rice harvest. Only soil samples from the hybrid rice plots were analyzed in this study. TABLE I Selected physico-chemical properties of the soil at Jinhua, Zhejiang Soil type
pHaf
Total N
Available-N
Olsen’s-P
Available-K
Sand
Paddy soil
4.8
g kg-l 2.7
123.4
mg kg-’ 16.5
54.6
278
Silt gkg-’ 562
Clay ~
160
a)The extraction ratio of soil Co H 2 0 is 1:l.
Nitrogen analysis The soil samples were digested by the Kjeldahl method to extract the total organic soil N, which were
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SOIL N UNDER RICE CROPPING
then measured in the hydrolysate prepared by refluxing the soil sample with 6 mol L-l HC1 (Bremmer and Mulvaney, 1982). Total hydrolysable N was determined by steam distillation with 10 mol L-' NaOH after Kjeldahl digestion and acid hydrolyzation. Hydrolysable ammonium N (NH4-N) was measured by steam distillation with 3.5% MgO. Amino sugar N was calculated as the difference between the N content obtained from steam distillation of the hydrolysate with phosphate-borate buffer at pH 11.2 and the hydrolysable ammonium N content. Amino acid N (a-amino-N) was determined by steam distillation of an aliquot of the hydrolysate with phosphate-borate buffer and 5 mol L-' NaOH after treatment with 0.5 mol L-' NaOH at 100 "C to digest hexosamines and remove NH4-N. Ninhydrin powder was added to convert the amino-N to NH4-N. Acid insoluble nitrogen was estimated by subtracting hydrolysable N from total nitrogen. The amount of hydrolysable unknown N (HUN) was calculated as: HUN = total hydrolysable N - (NH4-N
+ amino acid N + amino sugar N)
(1)
Soil microbial biomass analysis After the late rice harvest in 2003, additional soil samples were collected from all treated plots. The moist field soil samples were slightly air-dried to pass through a 2-mm mesh sieve. The moisture of samples was adjusted to about 60% of soil water holding capacity, and then the samples were incubated for 7-10 d at 25 "C. Soil microbial biomass was measured using the fumigation extraction method (Brookes et al., 1985). Immediately after samples were collected, 15 g moist soil sample was extracted by shaking with 60 mL of 0.5 mol L-' KzS04 for 30 min. A parallel sample was fumigated for 24 h with ethanol-free chloroform and then extracted same as above. Microbial biomass C (MBC) was determined using a Shimadzu TOC-5000 analyzer and soil microbial biomass nitrogen (MBN) was determined by Kjeldahl digestion method (Brookes et al., 1985). MBC was estimated as MBC = EC/0.45 (Wu and Joergensen, 1990), where EC (extractable carbon) is the difference between carbon extracted from fumigated and un-fumigated samples, both expressed in the same measurement unit. MBN was estimated as MBN = EN/0.54 (Brookes et aL, 1985), where E N (extractable nitrogen) is the difference between N extracted from fumigated and un-fumigated samples.
Organic matter analysis The method developed by Olk et al. (1995) was employed to extract mobile humic acid (MHA) and calcium humate (CaHA). Briefly, air-dried soil was incubated for 24 h in 0.25 mol L-' NaOH at a ratio of so1ution:soil as 2.5:l. The solutions were centrifuged and the soluble MHA was decanted and acidified to pH 2. The soil was washed twice or more with water to maximize humic acid recovery. These precipitates were combined with the precipitate of the NaOH wash and considered as MHA. The soil sample was decalcified by washing with 0.1 mol L-' HC1 until the pH of the supernatant reached below 1.5, and then the soil sample was incubated for 24 h in 0.25 mol L-' NaOH. Like the MHA extraction, the solutions were centrifuged, and the solubilized CaHA was decanted and acidified to pH 2.
Statistical analysis Data were statistically analyzed by one-way ANOVA and significant differences were distinguished by the Tukey-HSD test at P < 0.05 and P < 0.01 levels. RESULTS AND DISCUSSION
Organic N forms in soil Table I1 presents the effect of different fertilization treatments on total soil organic N and its com-
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ponents in the paddy soil.. After 6 years of continuous double-rice cropping, the soil total N and the total hydrolysable N contents had maintained almost stable in the N-supply plots, while the contents of the total hydrolysable N and HUN were substantially reduced in plots without N treatment. Table I1 also showed that continuous balanced application of N, P, and K mineral fertilizers (N-P-K plot) maintained proper amount of hydrolysable ammonium compared t o the initial level or 25% higher than the control (CK). The greater amount of hydrolysable ammonium in N-P-K treatment is probably because of the retention of organic N in the form of crop residue even though most of the straw was removed with harvest. This might also be due to the increase of fixation of NH,f after application of mineral N fertilizer (Shen and Shi, 1990). TABLE I1 Contents of the total soil organic N and its components in the bulk soils after 6-year fertilization treatments under continuous double-rice cropping Year
1998 2003
Treatmenta)
Original soil CK P-K N-K N-P N-P-K
Hydrolysable N Total
Ammonium
Amino acid
Amino sugar
1450ab) 1078b 1089b 1354ab 1479a 1445a
376a 258c 253c 25% 270c 32313
523b 401c 568b 583b 622a 615a
59c 89ab 107ab lllab 123a 82ab
Non-hydrolysable N
Total soil N
434c 583b 703a 430c 460c 455c
1884a 1661b 1792ab 1784ab 1939a 1899a
Hydrolysable unknown N mg kg-' 492a 330b 161c 401ab 464a 425a ____
a)CK: the control; P-K: the combination of P and K fertilizers; N-K: the combination of N and K fertilizers; N-P: the combination of N and P fertilizers; and N-P-K: the combination of N, P, and K fertilizers. b)Means followed by the same letter(s) within each column are not significantly different at P = 0.05 level by Tukey-HSD.
Although free amino acids are only present in very small concentrations, amino acid polymers that can be hydrolyzed by acid are ubiquitous in soil and commonly make up 30% t o 45% of the nitrogen in surface soil. Amino acids are the building constituents of protein. Table I1 showed that the amount of hydrolysable amino acid N was markedly high in all soils with fertilizer treatments as compared to the control (CK) or the original soil. The percentages of various organic N forms to the total soil N are listed in Table 111. Compared with the original soil before the experiment in 1998, the percentages of hydrolysable ammonium N and HUN decreased for all treatments six years later. There was no significant change in the percentages of total non-hydrolysable N for different treatments. The percentage of hydrolysable amino acid N was low for the control, while it increased in other fertilizer treatments. TABLE I11 Percentages of different nitrogen forms to the total N in the soils after 6-year fertilization treatments under continuous double-rice cropping Year
Treatmenta)
Hydrolysable N
Non-hydrolizable N
Ammonium
Amino acid
Amino sugar
Hydrolysable unknown N
19.9 15.5 14.1 14.5 13.9 17.0
27.8 24.2 31.7 32.7 32.1 32.4
3.1 5.4 5.9 6.2 6.3 4.3
26.1 19.9 9.0 22.5 23.9 22.4
% 1998 2003
Original soil CK P-K N-K
N-P N-P-K
23.0 35.1 39.2 24.1 23.8 23.9
")CK: the control; P-K: the combination of P and K fertilizers; N-K: the Combination of N and K fertilizers; N-P: the combination of N and P fertilizers; and N-P-K: the combination of N, P, and K fertilizers.
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SOIL N UNDER RICE CROPPING
Changes of soil organic N The amount of hydrolysable ammonium showed a slight decrease in all treatments under continuous rice cropping, but not statistically significant (Table 11, Fig. la). HUN showed a general decrease in all treatments while decrease with years was obvious for P-K and CK treatments. The reduction in HUN followed the order of P-K > CK > N-P-K (Fig. lb). With continuous rice cropping, HUN decreased at a rate of about 24% per year for the 6-year experiment in the P-K treatment. The decrease was stabilized after the first four years for CK treatment. For the N-P-K treatment, it decreased in the first three years, increased after that, and stabilized at a high level. Table IV showed that the P-K plot had 500
-
200
-
100
-
u)
E 300
v
r
01
I
1998
1999
2000
2001
2002
2003
100
-
0' 1998
I
1999
2000
Year
2001
2002
2003
Year
15 + CK
+ P-K + N-P-K
1000
t
500 0' 1998
I
1999
2000
2001
2002
2003
Year The changes of soil organic nitrogen components from 1998 to 2003 under different fertilization treatments with continuous double-rice cropping. HUN: hydrolysable unknown N; CK: the control; P-K: the combination of P and K fertilizers; and N-P-K: the combination of N, P, and K fertilizers. TABLE IV Means of grain yields and total N uptake of hybrid rice from 1998 to 2003 under different fertilization treatmentsa) with continuous double-rice cropping a t Jinhua City, Zhejiang Province Year
CK 1998 1999 2000 2001 2002 2003
Total N uptake
Grain yield
4927ab) 3836b 4299ab 3624c 3659c 3660c
P-K 5460a 4119b 4198b 4254b 3799bc 4474b
N-K 5817a 4448c 6326a 5752ab 5117b 5565b
N-P
N-P-K
5789a 3563c 4163b 4535b 3813c 5597a
kg ha-l 5878a 57.30a 482913 50.27ab 5733a 48.27b 5911a 42.97~ 4754b 41.62~ 6369a 39.27~
CK
P-K
N-K
N-P
N-P-K
65.36a 49.66b 47.41~ 48.67bc 41.67d 50.49b
123.02a 90.22b 106.67ab 98.4613 85.46~ 92.09b
107.92a 73.91~ 74.62~ 90.75b 75.01~ 91.96b
116.8a 93.65b 110.81a 116.58a 86.3713 118.65a
&)CK:the control; P-K: the combination of P and K fertilizers; N-K: the combination of N and K fertilizers; N-P: the combination of N and P fertilizers; and N-P-K: the combination of N, P, and K fertilizers. b)Means followed by the same letter(s) within each column are not significantly different at P = 0.05 level by Tukey-HSD.
Q. C. ZHANG e t al.
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higher N uptake than the control, indicating that soil N pool depletion might occur faster under consecutive cropping when P, K and other nutrients are abundant. Overall, these data suggested that HUN might play an important role in the nitrogen supply of intensive rice cropping system. Total hydrolysable N contents were maintained constant in the N plots, but decreased slightly in CK and P-K treatments, indicating the sensitivity of N application with HUN (Fig. lc).
Correlation between organic N and rice yield In spite of yearly variation in grain yield and nitrogen uptake of hybrid rice, the balanced fertilizer treatment (N-P-K) always had higher grain yield and higher nitrogen uptake (Table IV) than the other treatments or the control. Total N uptake from mass balance was measured. It could concluded that the factors affected N leaching and runoff were the same in all the experiment plots due to their similar growth environment. All of the environmental factors could be offsetted each other so that, the factors might not affect the statistical results. Grain yield of rice was positively correlated with total N uptake in plant ( r = 0.875**), hydrolysable N ( r = 0.608**), hydrolysable ammonium N ( r = 0.560**) and HUN ( r = 0.417**) in soils (Table V). Total N uptake was positively correlated with hydrolysable N ( r = 0.608**), hydrolysable ammonium N ( r = 0.440**) and HUN ( r = 0.431**). These results suggested that organic forms of N in soil contributed greatly to the indigenous N supply in rice field. It should also be noted, however, that none of the different N fractions extracted explained more than 20% to 36% of rice N uptake. TABLE V Correlation coefficients ( r ) among different N forms and grain yield of hybrid rice after 6-year fertilization treatments with continuous double-rice cropping
Grain yield Total N uptake Ammonium Amino acid Amino sugar Hydrolysable unknown N Total hydrolysable N
Total N uptake
Ammonium
Amino acid
Amino sugar
Hydrolysable unknown N
Total hydrolysable N
Total soil N
0.875**
0.560** 0.440*
0.178 -0.175 -0.224
-2.53 -0.126 -0.786** 0.398*
0.417* 0.431* 0.413' -0.308 -0.269
0.608** 0.608** 0.420* 0.270 -0.056 0.808**
0.320 0.401* 0.040 0.300 -0.029 0.151 0.295
*,**Significant at P = 0.05 and P = 0.01 levels, respectively (n = 25).
Soil organic matter in paddy soil Hutnic acids extracted by NaOH can be separated into Ca-bound (i.e. CaHA) and non-bound (i.e. MHA) fractions in the characterization of soil organic matter properties (Shinkarev et al., 1987). Although both are labile, MHA is considerably enriched in N and hydrolysable amino acids compared with CaHA in all soils (Olk et al., 1995 and 1996). Both MHA and CaHA contribute to N mineralization and immobilization that govern the N supplying capacity of paddy soils (Olk et al., 1996). The contents of MHA and CaHA in the soils with different fertilizer treatments are shown in Table VI. No significant difference was found in the contents of MHA or CaHA among the different treatments, and the amounts of MHA and CaHA did not decline with rice cropping for six years possibly because continuous rice cropping promoted the conversion of lablile N materials into stable and humified organic substance, which was similar t o the findings by Janzen (1987). It is not well known at present if the MHA and CaHA in acidic paddy soil have similar properties as in calcareous soil reported by Olk et al. (1995). Therefore, this investigation focused on the effect of fertilizer and continuous rice cropping on soil organic N and inherent soil fertility. The fact of increase in certain N forms coupled with the decrease in others indicated conversion from one form t o the other. The exact mechanism of the conversion of various N forms could only be evidenced through 15N tracer technology. Detailed characterization of
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SOIL N UNDER RICE CROPPING
these two humic acid fractions need be conducted in the further study. TABLE VI Contents of humic substances in the bulk soils of different fertilization treatmentsa) with continuous double-rice cropping Year
Mobile humic acid
Calcium humate
CK
P-K
N-K
N-P
N-P-K
6.86 7.36 8.42 6.22
6.47 6.08 10.14 6.07
6.87 5.81 10.09 6.74
6.34 7.00 10.48 6.75
6.60 7.67 9.93 6.45
CK
P-K
N-K
N-P
N-P-K
2.38 1.62 1.95 2.20
2.73 2.26 2.48 2.40
2.65 2.34 2.16 2.09
2.73 1.91 2.32 2.51
2.12 2.09 2.19 2.53
mg g-l
1999 2000 2001 2002
a)CK: the control; P-K: the combination of P and K fertilizers; N-K: the combination of N and K fertilizers; N-P: the combination of N and P fertilizers; and N-P-K: the combination of N, P, and K fertilizers.
Soil microbial biomass The soil microbial biomass acts as both source and sink of the plant nutrient, and regulates the function of soil system. Marumoto et al. (1982) found that the quantities of nutrients mobilized in two German upland soils were closely related to the amounts available in freshly killed biomass; and they showed a scheme for the transformation of dead microbial biomass C and N in arable soil during 4 weeks. Soil microbial biomass nitrogen contents and C/N ratio in different treatments are shown in Fig. 2. Microbial biomass N varied from 23.7 to 73.3 mg N kg-' soil and accounted for 2.5% to 3.7% of the total nitrogen in the soil. The N-P-K treatment had much higher soil microbial biomass N than other treatments (N-P, P-K and N-K) (Fig. 2a). The C/N ratios of the microbial biomass in N-P-K and N-K treatments (7.6 and 8.7, respectively) were much lower than in other treatments (12-15.8) (Fig. 2b). The C/N ratio of microbial biomass in different treatments followed the order: N-P > P-K > CK > N-K > N-P-K. For the late rice in the year of 2003, N-P plots exhibited serious K deficiency, while N-K plot did not show obvious P deficiency symptoms, suggesting that K might be more important limited factor than P in these soils. This could also be partially confirmed by the fact that N-K plots always had higher yields and higher N uptake than N-P plots (Table IV). The C/N ratio of the soil microbial biomass in the N-P treatment was also much higher than that in the N-K treatment. It seemed that nutrient deficiency and/or nutrient unbalance supply not only reduced the soil microbial biomass, but also increased the C/N ratio of soil microbial biomass. Sufficient supply of K seemingly facilitated the uptake of N in microbial biomass and rice, and therefore enhanced the grain yield. Since total biomass N and C/N ratio of microbial biomass revealed the quantity, activity and composition of the biomass
z
1
loo
a
b
il L L
P-K
N-P
CK
N-P-K Treatment
N-K
11
P-K
N-P
CK N-P-K Treatment
N-K
Fig. 2 Soil microbial biomass N (a) and C/N ratio of microbial biomass (b) in different fertilization treatments with continuous double-rice cropping. CK: the control; P-K: the combination of P and K fertilizers; N-K: the combination of N and K fertilizers; N-P: the combination of N and P fertilizers; and N-P-K: the combination of N, P, and K fertilizers.
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of the soil, they might serve as indexes of soil nutrient status. The effects of N, P, K treatments on N uptake and C/N ratio in microbial biomass need to be studied thoroughly in the future. CONCLUSIONS The forms of organic N varied with the different fertilization. Long-term balanced application of N, P and K promoted the amount of total hydrolysable organic N and hydrolysable ammonium, whilst unbalanced fertilization to rice resulted in a decline in organic N pools that led to decrease of N uptake by crop and soil microbial biomass. Although the exact chemical nature of HUN was unknown, this fraction apparently played a significant role in the nitrogen uptake of rice. Total hydrolysable N and HUN might be two useful and key indexes of N supply to rice in paddy soil. During the experiment, rice straw was removed from the field after crop harvest, and only short rice stubble (2-5 cm in soil depth) remained in the field. Our results indicated that with proper application of mineral N fertilizer without presence of organic manure, soil total N and hydrolysable N contents could still be well maintained. However, sustainability of the hydrolysable ammonium N and HUN was not guaranteed. Balanced application of N, P and K promoted microbial biomass, whereas unbalanced fertilization reduced microbial N and increased the C/N ratio of the microbial biomass. The reduction of N uptake may be due to insufficient supply of K instead of P. Fertilizer effects on specific groups of microorganisms and microbial activities in paddy rice systems remain to be studied in the future. ACKNOWLEDGEMENTS The constructive comments and suggestions by Dr. R. Buresh and Dr. C. Witt in the International Rice Research Institute, Philippines, and Dr. A. Dobermann in the University of Nebraska, USA, are gratefully acknowledged. We are also grateful to Mr. Sun Qing-Zu and Mr. Fu Rong-Xing in the Jinhua Agricultural Bureau, Zhejiang for their excellent management of the field experiment. REFERENCES Akmal, M., Khan, K. S. and Xu, J. M. 2004. Dynamics of microbial biomass in a rainfed soil under wheat cultivation. Pedosphere. 14(1): 53-62. Bremmer, J. M. and Mulvaney, C. S. 1982. Nitrogen. I n Page, A. L., Miller, R. H. and Keeney, D. R. (eds.) Methods of Soil Analysis. Part 2. Chemical and Microbiological Properties. ASA and SSSA, Madison, USA. pp. 595-624. Brookes, P. C., Andrea, L., Pruden, G. and Jenkinson, D. S. 1985. Chloroform fumigation and release of soil nitrogen: A rapid direct extraction method t o measure microbial biomass nitrogen in soil. Soil Boil. Biochem. 17(6): 837-842. Cassman, K. G., Dobermann, A., Sta. Cruz, P. C., Gines, G. C., Samson, M. I., Descalsota, J. P., Alcantara, J. M., Dizon, M. A. and Olk, D. C. 1996. Soil organic matter and the indigenous nitrogen supply of intensive irrigated rice systems in the tropics. Plant and Soil. 182: 267-278. Das, A. K., Boral, L., Tripathi, R. S. and Pandey, H. N. 1997. Nitrogen mineralization and microbial biomass-N in a subtropical humid forest of Meghalaya, India. Soil Biol. Biochem. 29(9): 1609-1 612. Dawe, D., Dobermann, A., Ladha, J . K., Yadav, R. L., Bao, L., Gupta, R. K., Lal, P., Panaullah, G., Sariam, O., Singh, Y . , Swarup, A. and Zhen, Q. X. 2003. Do organic amendments improve yield trends and profitability in intensive rice systems? Field Crops Res. 83(2): 191-213. Janzen, H. H. 1987. Soil organic matter characteristics after long-term cropping to various spring wheat rotations. Canadian Journal of Soil Science. 67: 845-856. Jenkinson, D. S. 1988. Determination of microbial biomass carbon and nitrogen in soil. In Wilson, J. R. (ed.) Advances in Nitrogen Cycling in Agricultural Ecosystems. Commonwealth Agricultural Bureau International, Wallingford. pp. 368-385. Li, S. Y. 1993. Yield stability and fertilizer efficiency of long-term triple cereal cropping in paddy fields of China. Biol. Fertil. Soils. 16: 151-153. Martikainen, P. J. and Palojarvi, A. 1990. Evaluation of the fumigation-extraction method for the determination of microbial C and N in a range of forest soils. Soil Biol. Biochem. 22: 797-802. Marumoto, T., Anderson, J. P. E. and Domsch, K. H. 1982. Mineralization of nutrients from soil microbial biomass. Soil Biol. Biochem. 14: 469-475.
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