Variation of Soil Microbial Biomass and Enzyme Activities at Different Growth Stages of Rice (Oryza sativa)

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1 Rice Science, 2005, 12(4): Variation of Soil Microbial Biomass and Enzyme Activities at Different Growth Stages of Rice (Oryza sativa) ZENG Lu-sheng, LIAO Min, CHEN Cheng-li, HUANG Chang-yong (Department of Resources Science, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou , China) Abstract: A pot experiment was conducted under submerged conditions with hybrid rice Zhenong 7 to study the variation in the soil microbial biomass carbon (C mic), soil microbial biomass nitrogen (N mic), soil respiration rate, soil microbial metabolic quotient, soil enzyme activities, chlorophyll content, proline content and peroxidase activity (POD) in rice leaf at different growth stages. The soil C mic, N mic and soil respiration rate significantly increased at the early stage and then declined during rice growth, but ascended slightly at maturity. However, soil metabolic quotient declined at all the stages. Soil urease activity increased at first and then decreased, while acid phosphatase and dehydrogenase activities descended before ascended and then descended again. Soil urease activity and acid phosphatase activity showed a peak value at the tillering stage about 30 days after rice transplanting, but the peak value of dehydrogenase activity emerged at about 50 days after rice transplanting and the three soil enzymatic activities were significantly different at the different developmental stages. As rice growing, chlorophyll content in rice leaf descended at the early stage then ascended and a peak value appeared at about the 70th after rice transplanting, after that declined drastically, while POD activity increased gradually, but proline content declined gradually. There was a slight relation between rice physiological indices and soil biochemical indices, which indicated that soil biochemical characteristics were affected significantly by rice growth in the interaction system of the rice, soil and microorganisms. Key words: rice; soil microbial biomass; soil metabolic quotient; soil enzyme activity; chlorophyll; proline; peroxidase Soil microorganisms and soil enzymes not only play an active role in soil fertility as a result of their involvement in the cycle of nutrients like carbon and nitrogen, which are required for plant growth, but also are sensitive biological indicators for soil quality evaluation, which can sensitively reflect minute changes of the soil environment [1]. Soil microbial biomass carbon (C mic ) and soil microbial biomass nitrogen (N mic ) are the recycling and store pools of soil C and soil N, and also are the important source of soil available C and N, respectively [2]. Soil C mic comprises 1-4% of soil organic C [3], but due to its fast turnover (it is estimated to be 1-3 years), the microbial biomass pool plays a key role in controlling the nutrient cycling and energy flow in soil ecosystems [4]. Soil respiration rate and microbial metabolic quotient reflect soil microbial activities, and are obviously affected by the eco-environment changes [5]. Soil enzymatic activities are also the biological indices reflecting soil fertility. Soil urease showed close relations with urea hydrolyzation, and increases the Received: 22 August 2005; Accepted: 10 December 2005 Corresponding author: LIAO Min (liaomin@zju.edu.cn) [6, utilization rate of nitrogen fertilizer 7]. Acid phosphatase speeds up soil organic phosphorus decomposition and improves soil phosphorus validity, which is an important index to assess soil phosphorus bio-availability [8]. Dehydrogenase, produced by live organism, promoting soil organic matter mineralization, is also an important index to evaluate soil organic matter anaerobically decomposing [9]. Soil microorganism and soil enzymatic activities, influenced by their eco-environment, can sensitively reflected soil environment minute changes. The rice grows in the reciprocal and interaction ecosystem between soil-microorganism-rice and atmosphere, the rice development consequentially affect soil microorganism and soil enzymatic activities. However, similar studies mostly paid attention to soil microorganism and soil enzymatic activities at a certain growth stage, few to those at whole rice developmental stages. So the objectives of the present work were to investigate the changes of soil microbial biomass, metabolic quotient and soil enzymatic activities at whole rice growth stage; and to study the correlations between the changes of rice physiological

2 284 Rice Science, Vol. 12, No. 4, 2005 indices and the changes of soil microorganisms and soil enzymatic activities. MATERIALS AND METHODS Soils and pretreatment Paddy soilⅠwas marine sediment silty loam [Loamy mixed active thermic typic endoaqualfs (US taxonomy)], taken from Huajiachi experimental field, Zhejiang University, Hangzhou, China. Paddy soilⅡ was yellowish red earth [Loamy mixed active thermic typic humaquepts (US taxonomy)], taken from Deqing county, Zhejiang Province, China. Two types of soil were collected from surface layer (0-20 cm), air-dried, ground, sieved through a 3.2-mm mesh, to detemine the basic physico-chemical properties (Table 1). The experiment was carried out with three replications, in plastic pots (80 cm 2 20 cm large) filled with air-dried soils (equivalent of 5.0 kg oven-dried soil), which prior to the experiment was mixed with 0.4 g urea / kg soil and 0.2 g K 2 HPO 4 / kg soil. The pots were irrigated for 15 d prior to transplanting seedlings. Rice cultivation Hybrid rice Zhenong 7 was grown in a greenhouse of Zhejiang University, Hangzhou (30º16' N, 120º12' E), China. Selected good quality seeds, soaked for two days in water, disinfected for three hours with 40% formalin, then, set in a incubation box with constant moisture at 25 for one week, and then sowed the pre-germinated seeds (with sprout of cm long) into seedbed. About 25 d later, when Table 1. Basic physicochemical properties of the soils studied. Characteristics Paddy soil Ⅰ Paddy soil Ⅱ ph (H 2O) Total organic carbon(g/kg) Available P (mg/kg) Total N (g/kg) Cation exchange capacity (cmol/kg) Fe 2O 3 (g/kg) Base saturation (%) Granule composition (g/kg) mm mm <0.002 mm Paddy soilⅠ: derived from marine sediment silty loam; Paddy soil Ⅱ: derived from yellowish red earth. rice seedlings grew to 10 cm long, the seedlings were transplanted into pre-treated pots, with six seedlings per pot. During the whole growth period, a water level of 2 cm above the soil surface was maintained. Apply 0.5 g urea and 0.5 g K 2 HPO 4 in each pot at 15, 30 and 50 days after transplanting. Sample collection Soil samples of the surface layer (0-10 cm) were collected using a device like a injector on the 0, 15, 30, 50 and 90 days after transplanting, and then put into sterilized plastic bags, homogenized with a glass stick, and brought back to the laboratory immediately to analyse soil C mic, N mic, respiration rate and soil enzymatic activities or stored at 4 until analysis within ten days. Meanwhile, the third rice leaves (from the top) were sampled with a pair of stainless scissors on the 15, 30, 50, 70 and 90 days after transplanting to determine chlorophyll content, proline content and peroxidase activities. Analysis methods Soil basic physico-chemical properties were determined according to the conventional methods [10] ; The fumigation-extraction method was used to determine C mic and N [11, 12] mic ; Soil basal respiration rate was estimated by using the 0.1 mol/l NaOH trap method [13] ; Urease activity was measured using urea as the substrate, as described by Gianfreda et al [14] ; Acid phosphatase activity was assayed by Hoffman s method [15] and soil dehydrogenase activity by the reduction of triphenyltetrazolium chloride (TTC) to triphenylformazan (TPF) [16]. All results were expressed on an oven-dry soil basis (105, 24 h) and were the mean of three replications. Chlorophyll (Chl) was extracted with acetone and ethanol solution (V/V = 1 : 1) and the absorbance of the extract was estimated at 645 nm and 663 nm. Proline content and guaiacol peroxidase was assayed according to Zhang [17]. Statistical analysis Differences between mean values were evaluated by a one-way analysis of variance (ANOVA), followed by the Student-Newman-Keuls test of significant differences. Comparison of multiple means was performed using the Duncan s multiple range test at P=0.05. The relationship between soil

3 ZENG Lu-sheng, et al. Variation of Soil Microbial Biomass and Enzyme Activities at Different Growth Stages in Rice 285 microorganisms, enzymes and rice physiological indices were tested by linear correlation analysis using SPSS program package (SPSS 11.5). RESULTS Changes of soil C mic, N mic and metabolic quotient at different rice growth stages Compared with the transplanting day, the C mic increased by 9.91% and 18.27% in paddy soilⅠand paddy soilⅡat the 15 days after transplanting (DAT), respectively, then decreased by 49.38% and 38.25% on 50 DAT, and then increased slowly as the rice developed. At rice maturity, the C mic increased to 83.50% and 77.45% of those on the transplanting day in paddy soilⅠand paddy soilⅡ, respectively. Soil N mic had the similar changes with the soil C mic. That was, soil N mic increased at the early two weeks (the rice was just at seedling stage), and then decreased with rice development. On the 50 DAT, the soil N mic were just only 46.43% and 55.46% of those on the transplanting day in paddy soilⅠand paddy soil Ⅱ, respectively. However, on the 90 DAT (the rice was just at maturity), the soil N mic increased by 22.38% and 8.19% in paddy soilⅠand paddy soilⅡ, respectively, compared with that on 50 DAT. Soil respiration rate was closely correlated to the soil microbial biomass and its activity. At the early stage after rice transplanting, soil respiration rate increased slowly and then decreased onwards (Table 2). On the 50 DAT, soil respiration rate were just 28.60% and 26.44% in paddy soilⅠand paddy soil Ⅱ respectively compared with the rice transplanting day. But at rice maturity, soil respiration rate increased slightly. However, unlike soil C mic and N mic, soil respiration rate was higher in paddy soilⅠthan in paddy soilⅡat a certain rice growth stage. Soil C mic, N mic and soil respiration rate were significantly different according to LSD test at different rice growth stages (Table 2). After rice transplanting, along with rice development, soil metabolic quotient declined slowly, and the soil metabolic quotient was not significantly different in both paddy soils at different rice growth stages. Changes of soil enzymatic activities at different rice growth stages Soil urease activity ascended gradually with rice growth at early stage and reached the highest value on the 30 DAT (Table 3). Compared with the rice transplanting day, soil urease activity increased by 47.88% and 21.61% in paddy soilⅠand paddy soilⅡ respectively. However, along with rice continuous growth, soil urease activity declined gradually. At maturity, soil urease activities were only 95.9% and 64.81% of those on the rice transplanting day in paddy soilⅠand paddy soil Ⅱ respectively. During the whole growing process, the soil urease activity was higher in paddy soil Ⅱ than in paddy soilⅠ. After transplanting, soil acid phosphatase activity decreased gradually at early two weeks, and then with the similar change tend to that of the urease, that is, along with rice development, soil acid phosphatase activity went up gradually and reached the highest value in 30 DAT or so. The acid phosphatase activity increased by 18.36% and 86.43% in paddy soilⅠand paddy soil Ⅱ respectively. However, along with rice continuous Table 2. Soil microbial biomass and soil metabolic quotient at different rice growth stages. Microbial activity Paddy Days after transplanting soil type 0 d 15 d 30 d 50 d 90 d Mean Standard deviation CV (%) C mic (g/kg) Ⅰ b a c 37.8 e d Ⅱ c a b e d N mic (g/kg) Ⅰ b a c 7.64 e 9.35 d Ⅱ b a c e d Respiration rate(co 2-C g / kg h -1 ) Ⅰ b a 9.48 c 3.20 e 6.19 d Ⅱ 9.38 a 9.46 a 8.32 b 2.48 d 3.30 c Soil metabolic quotient (h -1 ) Ⅰ a a a c b Ⅱ a a a a a Within a row, data followed by the same letters indicate no significant difference at 0.05 level according to LSD test. The same as in the tables below.

4 286 Rice Science, Vol. 12, No. 4, 2005 growth, soil phosphatase activity declined gradually. At maturity, soil phosphatase activity were only 13.43% and 59.71% in paddy soilⅠand paddy soil Ⅱ respectively compared with the rice transplanting day. Throughout the rice growth period, paddy soil Ⅱ had higher soil phosphatase activity and smaller coefficient of variation than paddy soilⅠ(table 3). After rice transplanting, soil dehydrogenase activities also decreased gradually at early two weeks, then increased. However, differred from soil urease and acid phosphatase activities, the highest value appeared at the booting stage, with values of and µg/g d (TPF, 37 ) in paddy soilⅠand paddy soil Ⅱ respectively. From then on, the soil dehydrogenase activities continuously declined with rice development. Similar to acid phosphatase activity, through the whole growing process, there were higher soil dehydrogenase activity and smaller coefficient of variation in paddy soil Ⅱ than in paddy soilⅠ. According to LSD test, soil urease activity, acid phosphatase activity and dehydrogenase activity were significantly different at rice different growth stages in both soils (Table 3). Changes of rice physiological indices at different growth stages Table 4 implied that after transplanting, from the 15 to the 50 DAT, rice chlorophyll content declined slowly in paddy soil Ⅰ and increased first then decreased in paddy soil Ⅱ. From then on, the chlorophyll content increased in both soils and peaked on the 70 DAT. Whereas, on the 90 DAT, the chlorophyll content decreased rapidly. Rice proline content changed a little in paddy soilⅠuntil the 50 DAT, and then declined rapidly and reached the lowest value on the 70 DAT, then trended to be stable. However, in paddy soilⅡ, the rice proline content decreased slowly and reached the lowest value on the 70 DAT, then enhanced a little on the 90 DAT. After transplanting, the rice peroxidase activities increased slowly from 15 to 50 DAT and 70 to 90 DAT, but increased rapidly from the 50 to the 70 DAT in paddy soilⅠ. However, in paddy soilⅡ, the rice peroxidase activities remained relatively stable till the 70 DAT and then increased evidently. In general, the coefficients of variation of rice chlorophyll content, proline content and peroxidase activities were comparatively higher in paddy soilⅠthan in paddy soilⅡ. Correlations analysis indicated that there were no significant correlations between the changes of rice physiological indices and the changes of soil biochemical indices at different rice growth stages in tested paddy soils. DISCUSSION Soil microbial biomass, which can be reflected by C mic or N mic, is an important index to assess ability of soil microorganism taking part in the recycling of Table 3. Soil enzymatic activities at different rice growth stages. Soil enzymatic activity Paddy soil Days after transplanting Standard Mean type 0 d 15 d 30 d 50 d 90 d deviation CV(%) Urease (NH 3-N mg/g d) Ⅰ 0.22 c 0.23 b 0.32 a 0.22 c 0.21 d Ⅱ 0.48 c 0.55 b 0.59 a 0.37 d 0.31 e Acid phosphatase (mg/g d) Ⅰ 0.87 c 0.57 b 1.03 a 0.49 d 0.12 e Ⅱ 1.40 b 0.91 d 2.61 a 1.03 c 0.84 c Dehydrogenase (TPF µg/g d) Ⅰ b d c a e Ⅱ c d b a e Table 4. Changes of physiological indices at different rice growth stages. Physiological index Paddy soil Days after transplanting Standard Mean type 15 d 30 d 50 d 70 d 90 d deviation CV (%) Chlorophyll (mg/g) Ⅰ 4.80 b 4.58 c 3.25 d 5.19 a 1.05 e Ⅱ 4.05 b 4.68 a 3.80 c 4.65 a 1.13 d Proline (µg/g) Ⅰ a c b d d Ⅱ a b c e d Peroxidase (OD 470/g min) Ⅰ e d c b a Ⅱ d d c b a

5 ZENG Lu-sheng, et al. Variation of Soil Microbial Biomass and Enzyme Activities at Different Growth Stages in Rice 287 carbon and nitrogen and their substance in soils. Table 1 and Table 2 indicated that the soil microbial biomass were affected by soil properties. The soil C mic in paddy soil Ⅱ, accounting for mg/kg was almost eight times higher than that in paddy soilⅠ, averaging mg/kg, which indicated that the soil organic matter and clay content were likely to affect the soil C mic. The microbial metabolic quotient has been used as a bioindicator of environmental stress on microbial communities, disturbance and ecosystem development [18]. When soil environment comes in for stress or disturbed conditions, soil microbes need more energy to maintain survival, and result in the metabolic quotient s augment [19]. The results above indicated that there were complex relations between the growth of rice aerial part and the soil microbe and biochemical characteristics of the rice roots. This might be attributed to that on the 15 DAT, rice was just at the seedling-tillering-rooting stage and growing slowly, which resulted in weak absorbing ability to soil nutrients and benefited to soil microbe development [20]. From 30 to 50 DAT, the rice was just at the tillering-filling stage, the rice aerial part grew rapidly and roots were just at expanding stage, thus, with a strong ability of rice absorbing soil nutrients such as N, P, S, etc and competiton with soil microorganism [21]. Microorganism assimilation and microbial biomass and soil respiration rate were reduced. On the 90 DAT, rice was just at the maturity stage, the rice roots grew slowly or even stagnated, which weakened the competition with soil microorganism and resulted in the increase of microbial biomass and soil respiration rate. The complex relations between the rice growth and soil microorganism need further study. Microbial biomass level is influenced by many ecological factors, such as plant community composition, soil organic matter level, soil contamination, soil moisture and temperature [22]. Soil C mic and N mic taking on positive correlations with the soil organic matter content under natural conditions, abundant carbon resource can transform more soil microbial biomass. However, soil respiration rate and metabolic quotient manifest the soil microbe activity, the values were relative not only to soil microbial biomass but also to soil texture, mineral composing, soil ph and environmental stress such as soil contamination. Consequently, more soil microbial biomass didn t mean better microbial activities. In the tested soils, paddy soil Ⅱ with more organic matter resulted in more microbial biomass. Meanwhile, the granule composition in paddy soil Ⅱ was mainly of silt and clay, with strong water holding capacity and weak aerating, and adverse energy metabolic activities in soil microorganisms, as a result, the soil had higher microbial biomass, lower respiration rate and metabolic quotient. Throughout the rice growth period, the soil urease activity was higher in paddy soilⅡthan in paddy soilⅠ, which might be attributed to the higher total nitrogen content and abundant organic matter in paddy soil Ⅱ. Therefore, it could be concluded that more nitrogen resource for soil microorganism resulted in higher soil urease activity [23]. The results in this study suggested that in the early two weeks after rice transplanting, except increase of the urease activity, both soil acid phosphatase and dehydrogenase activities decreased. From the tillering to filling stages, the rice was just at the most flourished stage and the soil enzymatic activities were at strongest stage, the rice roots excreted more organic acid and carbohydrate, which stimulated the correlative soil enzymatic activities. Both soil urease and acid phosphatase were extracellular enzymes with the similar changes, but soil dehydrogenase only occurred within living cells, come from the life activities of soil microorganism and rice growth, thus, with the different change trends from the former two enzymes. The results above also showed that the soil enzymatic activities were affected not only by rice growth but also by the soil properties. Soil enzymes were soil active protein, on the one hand, coming from soil microbial exudation. Consequently, in the tested soils, more soil microbial biomass and microbe exudation resulted in higher soil enzymatic activities in paddy soil Ⅱ than in paddy soilⅠ. On the other hand, soil enzymes coming from rice roots exudation, dominated by rice life activities at different growth stages [24]. When rice life activity was at strong stages, its roots exudation and soil enzymatic activities were increased, but, to a certaint extent, soil microbe activities were constrained. When rice was at the maturity stage, its roots decrepituded and its exudation reduced. As a result, soil enzymatic

6 288 Rice Science, Vol. 12, No. 4, 2005 activities and the restriction to soil microbe activities were weakened. Therefore, soil enzymatic activities and soil microbial biomass were performed differently. Under nutrient nonsterile conditions, the changes of rice physiological indices at different growth stages were dominated by the heredity characteristic. Consequently, there were no significant correlations between the changes of rice physiological indices at different growth stages and the changes of soil biochemical indices. ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China ( and ) and China National Basic Research Program (2002CB410804). We wish to thank the College of Agriculture and Biotechnology, Zhejiang University, for the generous supply of rice seeds. REFERENCES 1 Huang C Y. Soil Science. Beijing: Chinese Agricultural Press, (in Chinese) 2 Insam H, Mitchell C C, Dormaar J F. Relationship of soil microbial biomass and activity with fertilization practice and crop yield of three Ultisols. Soil Biol Biochem, 1991, 23: Anderson T H, Domsch K H. Ratios of microbial biomass carbon to total organic carbon in arable soils. Soil Biol Biochem, 1989, 21: He Z L, Yao H Y, Chen G C. Relationship of crop yield to microbial biomass in highly-weathered soils of China. In: Ando T ed. Plant Nutrition for Sustainable Food Production and Environment. Tokyo, Japan: Kluwer Academic Publishers, Alvarez R, Diaz R A, Barbero N, Santanatoglia O J, Blotta L. Soil organic carbon, microbial biomass and CO 2 -C production from three tillage systems. Soil & Till Res, 1995, 33: Cookson P, Lepiece A G. Urease enzyme activities in soils of the Batinah region of the Sultanate of Oman. J Arid Environ, 1996, 32: Klose S, Tabatabai M A. Urease activity of microbial biomass in soils. Soil Biol Biochem, 1999, 31: Pascual J A, Moreno J L, Hernandez T. Persistence of immobilized and total urease and phosphatase activities in a soil amended with organic wastes. Bioresource Technol, 2002, 82: Brzezinaska M, Stepniewska Z, Stepniewski W. Soil oxygen status and dehydrogenase activity. Soil Biol Biochem, 1998, 30: Anderson J M, Ingram J S I. Tropical Soil Biology and Fertility: A Handbook of Methods. 2nd ed. Wallingford, UK: CAB International, Vance E D, Brookes P C, Jenkinson D S. An extraction method for measuring soil microbial biomass C. Soil Biol Biochem, 1987, 19: Brookes P C, Landman A, Pruden G. Chloroform fumigation and the release of soil nitrogen: A rapid extraction method to measure microbial biomass nitrogen in soil. Soil Biol Biochem, 1985, 17(6): Coleman D C, Anderson R V, Cole C V, Elliott E T, Woods Y, Campion M K. Trophic interactions in soils as they affect energy and nutrient dynamics. IV. Flows of metabolic and biomass carbon. Microbial Ecol, 1978, 4: Gianfreda L, Sannino F, Ortega N, Nannipieri P. Activity of free and immobilized urease in soil: effects of pesticides. Soil Biol Biochem, 1994, 26: Alef K, Nannipieri P. Methods in Applied Soil Microbiology and Biochemistry. London: Academic Press, Min H, Ye Y F, Chen Z Y, Wu W X, Du Y F. Effects of butachlor on microbial populations and enzymatic activities in paddy soil. J Environ Sci & Health, 2001, 36: Zhang X Z. Research Methods on Crop Physiology. Beijing: Agricultural Press, (in Chinese) 18 Anderson T H, Domsch K H. The metabolic quotient for CO 2 (qco 2 ) as a specific activity parameter to assess the effects of environmental conditions, such as ph, on the microbial biomass of forest soils. Soil Biol Biochem, 1993, 25: Wardle D A, Ghani A. A critique of the microbial metabolic quotient (qco 2 ) as a bioindicator of disturbance and ecosystem development. Soil Biol Biochem, 1995, 27: Liao M, Xie X M, Wu L H. Effects of dry-cultivated and plastic film-mulched rice planting on microorganism ecological quality in a paddy field soil. Chinese J Rice Sci, 2002, 16(3): (in Chinese with English abstract) 21 Zheng P Y. Introduction of Crop Physiology. Beijing: Beijing Agricultural University Press, (in Chinese) 22 Wardle D A. A comparative assessment of factors which influence microbial biomass carbon and nitrogen levels in soil. Biol Rev Cambridge Philos Soc, 1992, 67: Lose S, Tabatabai M A. Urease activity of microbial biomass in soils. Soil Biol Biochem, 1999, 31: Guan S Y. Soil Enzyme and Its Research Methods. Beijing: Agricultural Press, (in Chinese)