Studies on nutrient uptake of rice and characteristics of soil microorganisms in a long-term fertilization experiments for irrigated rice

Studies on nutrient uptake of rice and characteristics of soil microorganisms in a long-term fertilization experiments for irrigated rice first-author †E-mail:ghwang@zjuem.zju.edu.cn †E-mail:ghwang@zjuem.zju.edu.cn ghwang@zjuem.zju.edu.cn Department of Natural Resources, Zhejiang University, Hangzhou 310029, China Department of Natural Resources, Zhejiang University, Hangzhou 310029, China The ecosystem characteristics of soil microorganism and the nutrient uptake of irrigated rice were investigated in a split-block experiment with different fertilization treatments, including control (no fertilizer application), PK, NK, NP, NPK fertilization, in the main block, and conventional rice and hybrid rice comparison, in the sub block. Average data of five treatments in five years indicated that the indigenous N supply (INS) capacity ranged from 32.72 to 93.21 kg/ha; that indigenous P supply (IPS) capacity ranged from 7.42 to 32.25 kg/ha; and that indigenous K supply (IKS) capacity ranged from 16.24 to 140.51 kg/ha, which showed that soil available nutrient pool depletion might occur very fast and that P, K deficiency has become a constraint to increasing yields of consecutive crops grown without fertilizer application. It was found that soil nutrient deficiency and unbalanced fertilization to rice crop had negative effect on the diversity of the microbial community and total microbial biomass in the soil. The long-term fertilizer experiment (LTFE) also showed that balanced application of N, P and K promoted microbial biomass growth and improvement of community composition. Unbalanced fertilization reduced microbial N and increased C/N ratio of the microbial biomass. Compared with inbred rice, hybrid rice behavior is characterized by physiological advantage in nutrient uptake and lower internal K use efficiency. Rice Nutrient uptake Fertilization Soil microorganism Diversity INTRODUCTION 1 China is one of the main countries producing rice ( Oryza sativa L.) in the world. The demand for rice continues to increase owing to continued growth of population. It is predicted that a 50% to 60% increase in rice production will be required to meet demand from population growth by 2025. Rice yield increases are likely to occur through fine-tuning of crop management. Nitrogen supply commonly limits grain yield in irrigated rice systems. The demand of the rice plant for other macronutrients mainly depends on the N supply (Dobermann et al., 1998 1998 ). On the other hand, the average plant recovery of fertilizer N is only about 30% (Dobermann, 2000 2000 ), although much knowledge has been gained about the nitrogen cycle in lowland rice environments. Environmental pollution by nutrient leaching or runoff from rice fields has become another concern (Ahmad et al., 1996 1996 ; Zhang et al., 1988 1988 ). Application of other macronutrients such as potassium has lagged behind, leading to imbalanced plant nutrition and negative potassium input-output balances in many parts of Asia (Dobermann et al., 1998 1998 ) Due to negative nutrient balances, significant depletion of soil nutrients such as K or P seems to occur in irrigated rice areas of tropical Asia (Dobermann et al., 1996 1996 ). However, most current knowledge about nutrient balances and soil fertility status in irrigated rice systems is based on data from a few long-term experiments. There are considerable uncertainties about crop N, P and K requirements because the internal efficiencies vary greatly depending on variety, nutrient supply, crop management and climatic conditions. Several investigators suggested that genetic variation in internal efficiency of P and K may exist in rice (Fageria et al., 1988 1988 ), but systematic studies are few. Soil microorganisms are an important part of the soil. Recognition of the importance of soil microorganisms has led to increased interest in measuring the nutrients held in their biomass (Jenkinson and Powlson, 1976 1976 ). Soil microbial biomass is considered to act both as the agent of biochemical changes in soil and as a repository of plant nutrients such as nitrogen (N) and phosphorus (P) in agricultural ecosystems (Jenkinson and Ladd, 1981 1981 ). In paddy fields, in which N fertility had been sustained over a long period of time, about 60%–70% of N absorbed by rice plants was derived from native soil N rather than fertilizer N. A few studies revealed that microbial biomass was an important source of mineralizable soil organic N in paddy soils (Jenkinson and Ladd, 1981 1981 ; Marumoto, 1984 1984 ). The composition of the microbial community, in particular the proportion of bacteria and fungi, may also influence C and N turnover of soil, although the microbial characteristics of paddy soils have been little investigated. This research focused attention on crop nutrient requirements, assessment of soil nutrient supplying capacity, and characteristics of soil microorganisms in a long-term fertilization experiment based on nutrient balance concepts. Furthermore, the relationships between the microbial biomass and chemical properties of the soil, such as available N, P and K contents were investigated. The investigation aimed at analyzing the effects of fertility on soil microbial biomass and soil nutrient supplying capacity, and at considering implications for the long-term management of paddy soil fertility. MATERIALS AND METHODS 1 Description of the long-term fertilization experiment 2 The experimental field located near the suburb of Jinhua City, Zhejiang Province. Soil at the study site is alluvial soil. The experiment was conducted on late rice (LR) of 1998 to early rice (ER) of 2003. Before the experiment, initial soil samples were collected in the spring of 1998 to determine general soil properties at 0.0 to 0.15 m depth (Table 1 1 ). The experiment was arranged as a split-block with three replicates in all treatments, including control (CK, no fertilizer application), PK (only P and K were applied), NK (only N and K were applied), NP (only N and P were applied) and NPK (N, P, and K were applied) fertilization, in the main block, and inbred rice and hybrid rice comparison in the sub block. ER and LR of inbred rice were Jia293 and Xiushui11, respectively. ER and LR of hybrid rice were Weiyou402 and Xieyou46, respectively. Plot size was 45 m 2 . Nitrogen was applied as urea at before transplanting (75 kg N/ha), early tillering (37.5 kg N/ha) and panicle initiation (37.5 kg N/ha). Phosphorus (25 kg P/ha) as single super phosphate was applied before transplanting. Potassium was applied as KCl before transplanting (50 kg K/ha) and at panicle initiation (50 kg K/ha). pH (H 2 O:soil=1:1) 4.8 Total N (%) 0.27 Olsen’s-P (mg/kg) 16.5 Available-K (mg/kg) 54.6 Sand (g/kg) 278 Silt (g/kg) 562 Clay (g/kg) 160 Plant sampling procedures followed a standard procedure in all treatments (Witt et al., 1999 1999 ). A 12-tiller plant sample was collected at physiological maturity to determine nutrient concentrations in plant tissue. Grain yields were obtained from a central 5-m 2 . Nitrogen concentrations in grain and straw were measured by micro-kjeldahl digestion, distillation, and titration (Bremmer and Mulvaney, 1982 1982 ). Tissue P was measured by the molybdenum-blue colorimetric method and tissue-K by atomic adsorption spectrophotometry after wet digestion (Walinga et al., 1995 1995 ). Soil microbial biomass determination 2 After the late rice of 2002 was harvested, samples were collected from all treated soils. The moist field soils were first dried partly to pass through a 2 mm mesh sieve, then adjusted to about 60% of full water holding capacity, and conditioned for 7–10 d at 25 °C. Soil microbial biomass was measured using the fumigation extraction method (Brookes et al., 1985 1985 ). Fifteen grams moist soil were extracted immediately after sampling by shaking for 30 min with 60 ml of 0.5 mol/L K 2 SO 4 and 15 g were fumigated for 24 h with ethanol-free chloroform and then extracted as described above. Microbial biomass C (MBC) was determined using a Shimadzu TOC-5000 analyzer and soil microbial biomass nitrogen (MBN) was determined by kjeldahl digestion (Brookes et al., 1985 1985 ). MBC was estimated as MBC = EC /0.45 (Wu and Joergensen, 1990 1990 ), where EC (extractable carbon) is the difference between carbon extracted from fumigated and un-fumigated sample, MBN was estimated as MBN = EN /0.54 (Brookes et al., 1985 1985 ), where EN (extractable nitrogen) is the difference between N extracted from fumigated and un-fumigated samples. Soil microbial communities determination 2 Patterns of potential C source utilization by microbial communities of paddy soil with different fertilization treatments can be readily generated from direct incubation of environmental samples in BIOLOG micro-plates (Zak, 1994 1994 ). The BIOLOG Gram-negative (GN) micro-plates used in this study rely on redox dye tetrazolium violet to detect respiration of sole carbon sources. The 96-well GN micro-plate consisted of 95 substrate-containing wells and a control well without a carbon source. Fifteen grams of soil sample (prepared as described above) were diluted to a 10 −4 solution, and inoculated with a multipipettor into the plates kept at room temperature (25 °C). Color formation in micro-plate wells (absorbance at 405 nm) was analyzed using a Biotek EL 320 micro-plate reader with automated stracker-loader cassette. Overall color development in BIOLOG plates was expressed as average well color development (AWCD). AWCD was derived from the mean difference among gray scale values of the 95 response wells (containing sole carbon source), R, and the gray scale value of the control well without a carbon source), C: [∑( C − R )]/95. We used two forms of ordination-principal component analysis (PCA), and canonical variate analysis (CVA), to visualize the relationships among samples by Genstat 5.3 (NAG Ltd., Oxford, UK) (Yao et al., 2000 2000 ). RESULTS 1 Yields and indigenous nutrient supply 2 Nutrient uptake of rice influences yields directly. To get more knowledge about nutrient uptake in rice, we studied yields for 5 consecutive years of planted rice with different fertilization treatments. There were marked differences only between applied-N treatments and no applied-N treatments during the first season (1998LR), and no significant difference between other treatments could be detected (at the 0.05 P -level). The grain yield was 4043 kg/ha in the PK treatment, compared to 3647 kg/ha in the CK treatment, which indicated that fertilizer P and K began to show efficiency from the fifth season (2000LR). Compared with NPK, yields of NP and NK began to decrease significantly from the third season (1999LR) and the eighth season (2002ER), respectively. The higher grain yield in the NPK treatment compared with NP seemed to result from differences in K caused by fertilizer application (Table 2 2 ). In a column, grain yield means followed by the same letters (a, b or c) are not significantly different by DMRT (Duncan’s multiple range test) at 5% level. E means early rice, L means late rice Grain yield 2 98L 2 99E 2 99L 2 00E 2 00L 2 01E 2 01L 2 02E 2 02L 2 03E CK 5373 b 3230 b 3349 b 4266 b 3647 c 3670 d 3313 c 2790 c 3759 c 3204 c PK 5696 b 3569 b 3599 b 4535 b 4013 b 4091 c 4075 b 3163 c 3991 bc 3273 c NK 6736 a 4887 a 4407 a 6152 a 6046 a 5740 a 5578 a 4477 b 5391 a 4502 b NP 6495 a 4758 a 2828 c 5938 a 4272 b 4602 b 4392 b 4310 b 4201 b 4115 b NPK 6654 a 5107 a 4328 a 6074 a 5564 a 5107 a 5538 a 4922 a 5090 a 5168 a Soil indigenous nutrients supply was estimated in nutrient omission plots (PK, NK, NP) (Jacssen et al., 1990 1990 ). Average data of five treatments in five years indicated that the indigenous N supply (INS) capacity ranged from 32.72 to 93.21 kg/ha, indigenous P supply (IPS) capacity ranged from 7.42 to 32.25 kg/ha, indigenous K supply (IKS) capacity ranged from 16.24 to 140.51 kg/ha. Table 3 3 shows that INS, IPS and IKS had decreasing trend with advancing season of planting rice. The PK plot providing higher N compared to no fertilizer plot indicated that soil N pool depletion might occur faster in crops grown consecutively when P and K nutrients were in abundance. Similarly, the NK plot depleted more P and the NP plot depleted more K compared the no fertilizer plot. Tables 2 2 and 3 3 indicate that P and K deficiency had become a constraint to increasing yield of consecutively planted rice. 2 Season 4 N uptake 4 P uptake 4 K uptake No fertilizer SD Applied PK SD No fertilizer SD Applied NK SD No fertilizer SD Applied NP SD 1998L 59.8 9.76 71.4 18.3 18.1 2.7 27.8 3.5 82.7 21.7 101.2 11.6 1999E 32.1 5.93 36.6 4.0 9.3 1.4 15.5 1.5 74.3 9.5 100.6 19.4 1999L 47.6 7.46 46.5 6.8 9.6 1.5 15.8 2.4 59.5 23.9 36.5 14.7 2000E 34.1 7.6 36.7 5.2 10.1 1.6 15.3 2.5 80.7 8.6 84.8 9.6 2000L 32.3 5.1 33.9 2.2 8.8 2.3 14.2 1.3 45.4 11.9 39.2 7.3 2001E 35.9 4.2 39.5 3.6 8.4 0.8 8.9 0.8 52.9 4.7 56.0 11.3 2001L 39.8 7.3 44.7 8.5 11.2 3.0 18.5 3.8 50.8 17.9 54.8 42.5 2002E 44.2 5.6 44.5 6.0 9.7 1.5 14.3 2.8 54.8 7.5 69.9 16.8 2002L 43.6 3.1 42.5 4.3 12.2 1.2 17.4 3.6 49.1 6.3 47.0 36.6 2003E 33.0 6.5 33.3 3.6 10.9 2.7 13.8 3.2 61.1 12.0 65.8 8.1 Total 402.4 62.6 429.6 62.5 108.3 18.7 161.5 25.4 611.3 124 655.8 177.9 Internal N, P and K use efficiencies 2 Hybrid rice has a physiological advantage in nutrient uptake in contrast to the inbred rice. In late rice, average total N uptake of hybrid rice increased by 7.9%, total P uptake increased by 20.7%, and total K uptake increased by 23.9%, compared with inbred rice (Table 4 4 ). The results indicated that genetic variation in the internal efficiency of N, P and K might exist in different rice cultivars. Treatments with N had physiology advantage in N uptake compared to without N treatments, and also improved P and K uptake, especially P uptake. In a column, UK means followed by the different letters (a, b) are significantly different by DMRT at 5% level. UK means nutrient uptake 2 2 Nutrient uptake 2 Variety 5 Treatment CK PK NK NP NPK 6 Early rice 2 NUK * * Inbred 51.02a 50.17 96.90 94.44 95.93 Hybrid 45.91b 52.06 88.71 86.97 91.61 2 PUK Inbred 12.46 11.90 18.71 17.60 21.42 Hybrid 11.57 13.94 16.79 18.70 21.41 2 KUK Inbred 64.42 78.85 125.12 77.11 129.20 Hybrid 64.28 84.72 122.38 73.77 124.75 6 Late rice 2 NUK Inbred 43.69b 47.75 101.56 87.14 95.28b Hybrid 50.11a 52.86 105.11 88.05 108.96a 2 PUK Inbred 11.78 15.02 19.17 17.50a 19.36 Hybrid 13.32 15.53 20.60 30.57b 22.87 2 KUK Inbred 46.03b 63.32b 105.51 46.46b 97.84b Hybrid 69.95a 85.32a 113.08 65.04a 125.41a Definitions of nutrient use efficiency vary greatly (Gourley et al., 1993 1993 ). We restrict our discussion to aggregate physiological efficiency, or internal nutrient use efficiency (PE), defined as the amount of grain yield produced per kilogram of nutrient present in above ground plant biomass. Table 5 5 shows physiological nutrient use efficiency of inbred and hybrid rice under different fertilizer treatments in the LTFE. PE estimation covers a wide period range of fertilization treatments and rice cultivars. The PE in the five years period ranged from 42 to 94 kg grain/kg N, from 128 to 457 kg grain/kg P, and from 66 to 109 kg grain/kg K, respectively. The PEK of inbred rice was significantly greater than that of hybrid rice. The PE of late rice being generally greater than that of early rice may be related to physiological differences between cultivars and the season of soil nutrient supply. In a column, PE means followed by the different letters (a, b) are significantly different by DMRT at 5% level 2 2 Internal nutrient use efficiency 2 Variety 5 Treatment CK PK NK NP NPK 6 Early rice 2 PEN Inbred 70.13 73.10 55.83 50.54b 56.76 Hybrid 72.09 73.23 56.21 54.10a 56.20 2 PEP Inbred 296.05 324.83a 295.71 278.27 266.15 Hybrid 296.47 280.99b 300.37 256.57 250.93 2 PEK Inbred 56.37a 46.55 43.36a 65.11 42.85 Hybrid 52.01b 45.16 40.34b 66.66 41.58 6 Late rice 2 PEN Inbred 78.48b 85.42 55.11 48.67b 54.96 Hybrid 85.01a 87.16 54.98 51.91a 52.13 2 PEP Inbred 300.13b 270.56b 295.41 246.27 273.86 Hybrid 325.61a 306.20a 292.93 249.86 261.43 2 PEK Inbred 76.63a 64.63a 52.88a 101.05a 53.97a Hybrid 64.69b 54.57b 50.78b 91.13b 45.36b Results of microbial biomass analysis 2 The levels of soil microbial biomass carbon and nitrogen in the different treatments are shown in Fig. 1 1 . Microbial biomass C and N varied from 480 to 760 mg C/kg soil and 23.7 to 73.3 mg N/kg soil. DMRT showed that the microbial carbon and nitrogen for different treatment were significantly different at 5% level. Soil microbial C was maximum in the NP or PK treatments, with CK treatment being second. The soil microbial biomass N showed a marked increase in the NPK treatment as compared to values in the unbalanced nutrient treatments (NP, PK and NK). CK treatment could also support a higher level of soil microbial biomass N. The amount of biomass carbon in hybrid rice was significantly higher than that in inbred rice. In contrast, the values of biomass N did not show appreciable difference among rice cultivars. The differences between the changes in MBN and MBC values affected the C to N ratio (range 7.6–18.4). The C to N ratio in NK and NPK treatment (7.6–8.99) were markedly lower than that in other treatments (Table 6 6 ). After four crops, the NP plot showed potassium deficiency; the PK plot showed nitrogen deficiency at the very beginning while the NK plot did not show phosphorus deficiency after 4 years of cropping. From these results, it was assumed that the C to N ratio was lower when N, P and K was sufficient; but that when nitrogen and potassium stressed the microbial population, the C to N ratio increased, especially, for hybrid rice, which required more K (Table 6 6 ). This may indicate that soil indigenous nutrient supply and nutrient balance can influence the soil microbial biomass C/N ratio, which may be used as an index of soil fertility. 2 Treatments 2 C/N ratio Inbred rice Hybrid rice NPK 8.20c 7.60c NK 8.99bc 8.73c CK 9.96bc 11.95b NP 11.06b 15.83a PK 18.44a 12.12b Results of microbial communities assessment using the BIOLOG GN plate 2 The relationship between AWCD and incubation time are shown in Fig. 2 2 . In the first 32 h of incubation, AWCD changed very little but afterwards increased continuously. This suggested that C sources were not used in the first 30 h of incubation. At the same time, AWCD of NPK treatment was the highest in all treatments. AWCD of CK treatment increased gradually and was higher than that of NP, PK and NK treatments at the late stage of incubation. Soil available nutrient pool depletion might occur very fast under consecutive crops grown without fertilization and result in the decrease in grain yield (Table 2 2 ). It was shown here that deficiency of the necessary nutrients for plants also adversely influences microbial communities. To further analyze the microbial communities, BIOLOG data after 158 h were subjected to canonical variate analysis (CVA) (Fig. 3 3 ). In inbred rice, PK and NK treatments had similar values for CV1, which explained 55.69% of the variance in the data, and all treatments had different coordinate values for CV2, which explained 31.07% of the variance in the data (Fig. 3a 3a ). In hybrid rice, CV1 explained 52.20% of the variance in the data, and CV2 explained 30.45% of variance in the data (Fig. 3b 3b ). These results suggested that the structure of microbial community changed under different fertilization treatments. Treatment with balanced nutrients (NPK) had significantly increased soil biological activity, and promoted the enlargement of the microbial number utilizing different carbon resources. DISCUSSION 1 In this long-term continuous cropping experiment, there is enormous year to year and season to season variation in grain yield and nutrient uptake in treatment plots. Average rice yields of NPK treatment were about 5.4 t/ha in our study, but they must be raised to 8.0 t/ha by 2020 to meet the demand for rice in Asia (Hossain and Fischer, 1995 1995 ). Nitrogen is the most limiting nutrient in irrigated rice systems (De Datta et al., 1988 1988 ), but P and K deficiency will also be a constraint to increasing yield for consecutive planting of rice. The goal of responsive nutrient management is congruence of nutrient supply and demand. Congruent nutrient management requires prediction of the effective indigenous nutrient supply and crop nutrient uptake requirements to determine fertilizer application according to timing and space. Tolerance of genotypes to slight differences in soil fertility or nonhomogeneous fertilizer application may be an important adaptive trait to account for within field variation in soil fertility or rice growth (Dobermann, 1994 1994 ). Whether there is significant variation among rice genotypes, e.g. hybrid and inbred, in their plasticity to smooth out small scale spatial variation in nutrient supply needs further investigation in the field. The soil microbial biomass is a labile pool organic matter and comprises 1%–3% of total soil organic matter (Jenkinson and Ladd, 1981 1981 ). The soil microbial biomass acts as a source and sink of plant nutrient and regulates the functioning of the soil system. Microbial population in the soil-floodwater-rice system is perhaps one of the most complex biological systems in agriculture. Marumoto et al.( 1982 1982 ) found that the quantities of nutrients of mobilized in two German upland soils were closely related to 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. We concluded that fertilization influenced microbial biomass and community diversity. 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On the optimal rate of application of nitrogen fertilization for rice and wheat in Tai-lake region 10.1631/jzus.2005.B0147 Project supported by the International Fertilizer Industry Association (IFIA), the Potash and Phosphate Institute, Canada (PPIC), and International Potash Institute (IPI) 2004-05-09 2004-06-20 2005-01-20 2022-01-05 Copyright © 2005, Journal of Zhejiang University Science 2005 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 02 6B