Integrated resource management improves soil glucosidase, urease, and phosphatase activities and soil fertility during rice cultivation in Indo-Gangetic plains

Abstract The sustainable cultivation of rice (Oryza sativa L.) without compromising yield is an emerging challenge. Field experiments were conducted at New Delhi, during 2007 and 2008 to investigate the effect of tillage, irrigation regimes, and integrated nutrient management practices on the soil enzymatic and microbial activities. The soil glucosidase (67.35%) and urease (106.75%) increased under conservation tillage compared with conventional tillage; largest increase was observed when a combination of 50% farm yard manure + 25% biofertilizer + 25% green manure (GM) was used in place of recommended dose of nitrogen (RDN) or when 25% RDN was replaced with biofertilizer or GM as nutrients in combination with conservation tillage and optimum water supply (three-irrigations) with a few exceptions. The present study has suggested that resource management practices significantly improved soil enzymatic and microbial activities under conservation tillage and optimal water supply which reduce the dependence on chemical fertilizers and make rice cultivation sustainable.


ABOUT THE AUTHORS
The activity of our research group at the Division of Microbiology, Indian Agricultural research institute (New Delhi), is mainly related to the improvement of rice-wheat cropping system in Indo-Gangetic plains by integrated resource management (IRM) practice, to minimize cost of production, reduce environmental damage in developing countries, and improving food security in the developing countries. The research reported in the paper pertains to observe the effect of resource management on soil enzyme activity and hence soil fertility during rice crop cultivation. High input of resources like chemical fertilizers, tillage, and water pollutes the environment and harms soil fauna. The present research demonstrates that integrated and optimum use of resources improves soil glucosidase, urease, and phosphatase activity and overall microbial activity of soil and thus fertility of soil by mixed use of organic and chemical fertilizers and optimum use of other high input resources.

PUBLIC INTEREST STATEMENT
Public concerns against input intensive agriculture include excessive use of chemical fertilizers, which reportedly leach into soil and water and pollute them. The present study demonstrates that integrated use of chemical and organic fertilizers, optimum irrigation, and minimum tillage can reduce the dependence on resources besides improving soil enzyme activity and fertility. It was observed in our study that important soil enzyme activities like glucosidase, urease, alkaline, and acid phosphatase which circulate carbon, nitrogen. and phosphorous. An increase in soil microbial activity was also observed. These observations confirmed increase in soil microbial activity and fertility. To check whether it affects overall metabolic activity, soil respiration and soil microbial biomass carbon were calculated. It was observed that overall metabolic quotient of soil decreased (i.e. increase in microbial activity) when organic fertilizers were used as compared to complete chemical fertilizer treatment suggesting the improvement of soil fertility.

Introduction
Rice is the staple diet of more than 65% of Indian population (Ghosh, 2009) which is grown on 44 million hectares of land (AIREA, 2012). The green revolution practices of the 1960s have been based on the application of relatively high rates of chemical fertilizers, tillage practices, and irrigation. However, the above-stated practices have proved to be insufficient in meeting the challenges of enhanced and sustainable productivity. Besides, the burgeoning problem of global warming also warrants the need to change conventional practices to reduce greenhouse gas emissions (You, Rosegrant, Fang, & Wood, 2005).
The excessive use of chemical fertilizers and energy-intensive processes, such as tillage and irrigation, has led to the distortion of soil physical and biological status in many agricultural ecosystems (Liu et al., 2005). On the other hand, resource-conservation technologies reduce dependence on resource-intensive processes (Erenstein, 2009) and make agriculture sustainable.
The soil enzymatic activities are considered a major index for soil microbial activity and soil organic carbon status (Bandick & Dick, 1999). The glucosidase and urease are among the prominent soil enzymes. The glucosidase reflects soil-management effects and has microbial significance because of its role in C-cycle (Bilen, Celik, & Altikat, 2010). Similarly, urease and phosphatase activity is responsible for N-and P-metabolism in the soil Rahmansyah, Antonius, & Sulistinah, 2009). There are only a few studies that have considered resource conservation (i.e. tillage, fertilizer, and irrigation) to determine improved soil and agricultural practices in Indo-Gangetic plains (IGP) for rice. Mahajan and Gupta (2009) reported the need for integrated nutrient management (INM) practices in South Asia to make agriculture sustainable. However, an integrated approach for tillage, water, and nutrient management for rice crop involving soil biological indices, i.e. soil glucosidase, urease, and phosphatase (for C, N, P circulation, respectively) has not been reported.
The sustainable cultivation of rice crop without compromising yield could help in reducing the overbearing pressure on resources, such as water and chemical fertilizers, without polluting the environment. The present study was planned to determine the optimum requirements for tillage, water, and nutrients in a sandy loam soil of IGP at Delhi, for rice crop, which can help in increasing the activity of soil glucosidase, urease, and phosphatase.

Experimental location
Field experiments were conducted at the research farm of Indian Agricultural Research Institute, New Delhi, (Latitude: 28°38′ N, Longitude: 77°12′ E, Altitude: 216 msl) during the summer (kharif) season of 2007 and 2008. New Delhi has semi-arid and sub-tropical climate with hot and dry summers and cold winters. It falls under the agro-climate zone called "Trans-Gangetic plains," where summer months (May and June) are the hottest, with the maximum temperature ranging between 41 and 48°C, whereas January is the coldest, with the minimum temperature ranging between 3 and 7°C.
The temperature rises gradually through the months of February and March and it reaches the maximum during June, and falls slightly with the advent of southwest monsoon rain. The mean precipitation for Delhi is 650 mm, which is mostly received during July to September and occasionally during winter.

Experimental design
Two experiments were laid out in a split-plot design with three replicates each. Two tillage treatments (i.e. conservation and conventional), were assigned to main plots and three irrigation treatments [i.e. sub-optimum (two-irrigation regime), optimum (three-irrigation regime), and supra-optimum (five-irrigation regime)] were assigned to sub-plots (each at a gap of 20 days). The sub-plots were further divided into sub-sub-plots for different nutrient treatments as shown in Table 1.
During the period of 2003-2007 tillage, water and nutrient management had been practiced in the field on different crops (i.e. all crops were subjected to the same practices) ( Table 2). The rice cultivar (cv.) "Pusa Sugandh 3" seeds were sown during the first week of June 2007 and 2008 at a spacing of 20 × 15 cm. A uniform dose of 33 kg ha −1 P was made available through single superphosphate and 37 kg ha −1 of K made available through muriate of potash. Both single superphosphate and muriate of potash were applied to the plots before sowing. The chemical N fertilizer (urea) was applied in three split doses, i.e. half as basal dose and the other two doses in two equal parts as top dressing at tillering and at panicle initiation stages of rice. The six INM treatments are given in Table 2. The control plot refers to no input of N, whereas blank plot refers to uncultivated soil.
The well decomposed farm yard manure (FYM) containing 0.5% N (i.e. 0.05 kg N kg −1 of FYM) on a dry-weight basis calculated by Kjeldahl's method was incorporated in the soil as green manure (GM) before sowing, as a substitute for chemical fertilizers. Similarly, Sesbania aculeate was added as a GM at 10.5 t ha −1 before seeding of rice in the same experiment. One fourth of urea was replaced by biofertilizer (Azospirillium brasilense) CDJA received from Dr J. Dobereiner, Brazil. The A. brasilense strain fixes 25-30% [of recommended dose of nitrogen (RDN)] N ha −1 . The coating of seeds was done by dipping the seeds in an aqueous suspension culture of the biofertilizer. The inoculum density was 10 8 cells ml −1 (Zorita & Canigia, 2008

Soil sampling and physical and chemical characteristics of soil
The soil type in IARI fields (New Delhi) is Typic Haplustept (Saha et al., 2010). The moist soil was sieved (2 mm mesh size), homogenized, and stored at 4°C. The gravimetric moisture content was determined immediately. Microbial analysis of the soil samples was done using fresh soil samples, and the results were expressed on dry weight basis. Some important physical and chemical characteristics of soil are given in Table 3.

Estimation of soil respiration, dehydrogenase, and soil microbial biomass carbon
Soil samples were collected from a depth of 0-15 cm immediately after the harvest of rice crop. Six sub-samples per treatment were composited. The field-moist samples were sieved to 2 mm and analyzed for: respiration, dehydrogenase activity (DHA), and soil microbial biomass carbon (SMBC) content in the soil. The SMBC was estimated following chloroform fumigation extraction method of Vance, Brookes, and Jenkinson (1987). The soil respiration (SR) was measured according to Stotzky (1965). The soil dehydrogenase enzyme (DH) activity was determined via the method used by Casida, Klein, and Santoro (1964). The metabolic quotient (qCO 2 ; i.e. the respiration to biomass ratio) was calculated from basal respiration (CO 2 -C h −1 ) per unit SMBC (C-mineralized) (Tischer, 2005).

Number of grains per panicle
From the 10 selected panicles of rice plants, cleaned, filled, and unfilled grains were separated. The filled grains were counted with the help of a seed counter. The mean value of filled grains per panicle was computed.

Statistical analysis
All the data recorded were analyzed using the standard procedure of statistical analysis for a splitplot design (Gomez & Gomez, 1984). Analysis of variance (ANOVA) was used to determine the effect of each treatment. A multiple-mean comparison was performed using CD (critical difference) (0.05 probability level) values. The data were analyzed via MSTAT statistical package.

Soil glucosidase activity
The soil glucosidase activity increased significantly (i.e. 67%) in zero-tillage plots compared to the conventional tillage plots (Table 4). Among INM treatments, the highest rate of soil glucosidase activity was recorded in the treatment where N was made available through a sole organic source of N (i.e. 32.85 μg NPG (p-nitrophenyl-β-D-glucoside) per g −1 soil), followed by the treatment where 25% RDN was replaced with FYM (i.e. 32.11 μg NPG per g −1 soil). The increase in soil glucosidase activity has been significantly (p < 0.05) greater compared to the control plots. For a given nutrient-management practice, the three different water regimes significantly affected the soil glucosidase activity. Soil receiving sole organic source for the three-irrigation regime (optimum) recorded 16 and 22% higher soil glucosidase activity as compared to the two (sub-optimum) and five-irrigation regime (supra-optimum), respectively (Table 5). The use of urea as a sole N-source caused a 17% reduction in soil glucosidase activity under two-irrigation regime compared to the five-irrigation regime; whereas, soil glucosidase activity under five-irrigation regime was statistically similar to that under three-irrigation regime. The use of sole organic source of N caused an increase in about 39% in soil glucosidase activity compared to the control. Other plots receiving RDN in various forms also had higher soil glucosidase activity compared to control plots.

Soil urease activity
A significantly greater soil urease activity (107%) was detected in zero-tillage plots compared to the conventional-tillage plots (Table 4). Among the INM treatments, the highest rate of soil urease activity was recorded for treatments where the N requirement was met through a sole organic source of N (i.e. 189.481 μg N g −1 soil h −1 ), followed by the treatment where 25% RDN was replaced with biofertilizer (i.e. 174.74 μg N g −1 soil h −1 ), which was further followed by the treatment where 25% RDN was replaced with sewage sludge (i.e.152.79 μg N g −1 soil h −1 ). These values showed significantly higher (p < 0.05) soil urease activity for treatments where RDN was replaced with biofertilizer and where sole organic source was used as compared to the control plots. For a given nutrient-management practice, the three different water regimes significantly affected the soil urease activity. Soil receiving sole organic source (FYM, Biofertilizer, and GM, i.e. T 6 ) for the three-irrigation regime recorded 14 and 31% higher soil urease activity compared to two-irrigation regime and five-irrigation regime, respectively ( Table 6). The highest urease activity in plots with urea as N-source was observed for the five-irrigation regime (i.e. 207.87 μg N g −1 soil h −1 ), which was 32% higher than that for three-irrigation plots and 41% higher than that for two-irrigation plots. The use of sole organic source of N caused a 100% increase in soil urease activity compared to the control. Other plots having RDN in various forms also had higher soil urease activity compared to control plots.

Acid phosphatase activity
The soil acid-phosphatase activity was significantly higher (115%) in zero-tillage plots, compared to the conventional-tillage plots (Table 4). Among INM treatments, the highest rate of soil acid-phosphatase activity was recorded in treatments where the N requirement was met through a sole organic source of N (i.e. 61.55 μg PNPP (p-nitrophenyl phosphate) g −1 soil h −1 ), followed by the treatment where 25% RDN has been replaced with FYM (i.e. 55.14 μg PNPP g −1 soil h −1 ), which is further followed by the treatment where 25% RDN was replaced with biofertilizer (i.e. 50.33 μg PNPP g −1 soil h −1 ). These treatments showed significantly (p < 0.05) higher soil acid-phosphatase activity compared to the control. For any given nutrient-management practice, the three different irrigation regimes significantly affected the soil acid-phosphatase activity. Soil receiving N from a sole organic source in three-irrigation regime recorded 26 and 32% higher soil acid phosphatase activity compared to twoand five-irrigation regimes, respectively (Table 7). The use of urea as a sole N-source decreased soil acid-phosphatase activity under two-and five-irrigation treatments by 45 and 50%, respectively, compared to three-irrigation treatment.

Alkaline phosphatase activity
For rice crop, a significantly greater soil alkaline phosphatase activity (105%) was noted in zero-tillage plots compared to conventional-tillage plots (Table 4). Among the INM treatments, the highest rate of soil alkaline-phosphatase activity was recorded for the treatment where 25% N requirement was met through FYM of N (i.e. 135.54 μg PNPP g −1 soil h −1 soil), followed by the treatment where urea was used (i.e.134.36 μg PNPP g −1 soil h −1 soil), which was further followed by the treatment where whole organic source of N was used as RDN (i.e.134.03 μg PNPP g −1 soil h −1 soil). Significantly (p < 0.05) higher soil alkaline-phosphatase activity in treatments with organic amendments was detected than in the control plots. For any given nutrient-management practice, the three different water regimes significantly increased the soil alkaline-phosphatase activity. Soil receiving RDN as urea in the three-irrigation treatment recorded 8 and 24% higher soil alkaline-phosphatase activity compared to two-and five-irrigation treatments, respectively (Table 8). The use of sole organic source for N with three irrigation regimes gave statistically similar result as that from the plots where urea was used as the source of RDN. The use of RDN as urea caused a 9% increase compared to the control. Other plots receiving RDN in various forms also had higher soil alkaline-phosphatase activity compared to control plots.

Effect of resource management practices on soil microbial activities (soil respiration, dehydrogenase, and soil microbial biomass carbon) and grain number of rice
The resource management practices (RMPs) such as conservation tillage, water, and nutrient management, had a significant positive effect on soil microbial activities such as SR, SMBC, and DHA. All parameters of soil microbial activities registered a significant increase, i.e. SR (36.46%), SMBC (95.38%), and DHA (74.34%), in plots where sole organic source was used for N, along with conservation tillage and three irrigations compared to the plots where RDN as urea was used as a source of N (Table 9). Similarly, conventional-tillage plots showed a reduction in SR, SMBC, and DHA in relation to conservation-tillage plots across all three irrigation regimes and nutrient-management practices. In general, Notes: RDN denotes recommended dose of nitrogen and CD denotes critical difference. Values are mean of the data (n = 6) and are statistically significant at p < 0.05. Data analyzed by one way ANOVA at p < 0.05. Data followed by the same letter within a row are not significantly different at p ≤ 0.05 level as determined by Duncan's multiple range test. Different alphabets indicate significant difference between two values.
among the irrigation practices, the three-irrigation treatment showed higher enzymatic activities than did two-and five-irrigation treatment, in both the tillage systems. Among the nutrient treatments, the sole organic source treatment in conjunction with conservation tillage and three-irrigation regime showed significantly higher microbial activities compared with other INM treatments.
The increase in grain number was observed following the use of conservation-tillage practices (Table 10). The grain number increased significantly when 25% biofertilizer had been replaced with RDN or when sole organic source of N was used with conservation tillage.

Discussion
The tillage (i.e. no-tillage) and nutrient management have brought about increase in soil microbial and enzymatic activities viz. soil glucosidase, urease, acid and alkaline phosphatase (Tables 1, 2, 4 and 5). The soil enzymatic activities have shown positive synergistic effect of the RMPs studied i.e. conservation tillage with three-irrigations (at the gap of 20-days). The organic nutrient forms affect the rhizospheric microbial properties in different tillage and water regimes (Singh et al., 2009).
Our study has shown an improvement in glucosidase enzyme activity with tillage, water, and nutrient resource management, which subsequently resulted in higher available C in the soil and improved the microbial population in soil under rice cultivation. The role of glucosidase enzyme during carbon (C) cycle has been suggested by Deng and Tabatabai (1996). The increased soil glucosidase activity in conservation tillage system might be attributable to increased SR and soil organic matter (SOM) while in the conventional tillage system, soil disturbance results in the rapid rate of plant residue decomposition which exacerbates the mineralization of C and N in the soil which further led to loss of organic matter and enzyme activity of soil. This view is supported by the idea forwarded by Balosa, Kanashiro, Filho, Andrade, and Dick (2004) and Mohanty, Painuli, Misra, and Ghosh (2007). Thus, it appears that glucosidase enzyme activity of C-cycle increases with the use of resource management and organic nutrients which subsequently results in high available C in the soil and improves the microbial population in the soil. Similar results have been reported by Zhang et al. (2010).
Tillage practices have been shown to affect soil nutrient content, enzymatic activity, and microbial activity under the Indian sandy clay loam soil (Mathers & Nash, 2009). The agroecosystem functioning and global element cycling has been affected by microbial communities of rhizosphere of plants. Thus, the rhizosphere of plants contributes significantly to biogeochemical cycles (circulation of elements like C, N, P, S etc.) in biosphere. It has been reported that no-tillage (i.e. conservation tillage) practices increases availability of soil enzymes (like acid phosphatase, amylase, cellulase, urease, etc.) in rhizosphere and hence nutrient cycling (Davis, Parton, Dohleman, & Smith, 2010). The urease activity was higher in the conservation tillage fields across all the nutrient treatments as compared to the conventional tillage fields (Table 6). Individual effects of resource optimization in improving soil urease activity and sustained growth of plant have been reported by previous workers (Davis et al., 2010;Mathers & Nash, 2009). Our results emphasized that conservation tillage and three-irrigations enhanced the soil urease activity, synergistically. The soil urease activity increased with reduced tillage, optimal (three-) irrigations and mixed use of chemical and organic nutrients as shown in Tables 4 and 6. This might be attributable to improvement in intracellular and extracellular soil urease activity (Qin, Hu, Wang, Li, & He, 2010).
The present day agriculture is dependent on chemical (i.e. N, P, K) fertilizers and energy intensive processes (Bosede, 2010). It has been established that chemical fertilizers cause many problems like soil alkalinity, acidity, yield stagnation, etc., and are the major source of environmental pollution. So, nowadays, emphasis of research has been shifted toward exploring the alternative organic options for inorganic N, P, K fertilizers (Mahajan & Gupta, 2009). Our work highlighted that INM practices could reduce dependence on chemical fertilizers, irrigation, and tillage. Further, we observed that integrated use of conservation tillage, three-irrigation, and sole organic source resulted in a 20% increase in acid phosphatase activity compared to urea treatment while the increase was 60.89% compared to the control when urea was used as RDN. Our results showed that acid-phosphatase 107.00 ± 4.39e 85.67 ± 3.59c Notes: T 1 -T 7 have their usual meanings as given in activity was positively related to the integrated use of conservation tillage, three-irrigations, and various combinations of organic and inorganic fertilizers (Table 8). The soil acid-phosphatase activity was higher for the conservation-tillage treatment compared to the conventional-tillage treatment across all the nutrient treatments and irrigation regimes. Roldan, Garcia, and Alguacil (2005) have reported that no-tilled soils had increased values of acid phosphatase activity as compared to the tilled soils. The increased acid phosphatase activity might be attributable to increased organic matter which further led to phosphorus (P) stress. The P-stress stimulated plant roots to secrete acid phosphatase for their hydrolyzation (Balemi & Negisho, 2012).
Soil alkaline-phosphatase activity was significantly higher in case of conservation-tillage treatment for the three-irrigation regime across various nutrient sources (T 1 -T 6 ) as compared to the conventional-tillage treatment and other two irrigation regimes (two-and five-irrigation regime) (Table 8). These results implied that conservation tillage, optimum (three-) irrigations, and nutrient-management practices improve soil alkaline phosphatase activity. The observed increase in alkaline-phosphatase activity might be due to increased contents of SOM, total N, P, K, ammonia ion (NH + 3 ) and available P, K which further led to increased activity of phosphatase (alkaline) enzyme . This suggests a relationship between conservation tillage, optimum (i.e. three-) irrigations, and organic fertilizers with alkaline phosphatase activity. Similar, results in cereals have been reported by Mikanová, Javůrek, Šimon, Friedlová, and Vach (2009).
The major soil microbiological parameters such as SR and soil microbial biomass showed an increase, along with an increase in the organic content of the fertilizer mixture (Table 9). Our study showed that the low qCO 2 values during the INM practices correspond with an increased microbial activity which is directly related to soil fertility. Thus, the decreased qCO 2 with increase in the organic content of the fertilizer (Table 9) suggested an increase in the microbial efficiency of soil as both parameters (i.e. qCO 2 and microbial efficiency) has been found to be inversely related (Hu & Cao, 2007). The increase in microbial content and decrease in qCO 2 indicated a shift toward stability in the Leucaena leucocephala and Arachis hypogaea system (Balota & Chaves, 2011). In our study, the SMBC, SR, and DHA increased and qCO 2 values decreased with an increase in organic content in the fertilizer mixture, which indicated an increase in the fertility of soil and thus increase in yield of rice crop (Table 10). The variations on qCO 2 values for various nutrient treatments in our experiment could be due to differences in the accessibility of C substrates to the micro-organisms, changes in the microbial metabolic rates, and changes in the microbial community composition and their physiological standards (Islam & Weil, 2000).
The water has been a precious resource for agriculture. But excessive use of water has created a condition of shortage. The excessive water can harm soil microbes and produces anaerobic conditions like hypoxia (a condition of oxygen deficiency). The reproductive growth of plant is negatively affected by hypoxic conditions. The hypoxic conditions also reduces yield of rice plant (Prasad & Nagarajan, 2004). Therefore, optimization is essential for higher enzymatic activities which may further increase soil microbial activities and yield of plant (Singh, Dahiya, & Jaiwal, 2006). We have also observed that three irrigations enhance soil enzymatic activities (Table 5) compared to the two-and five-irrigation treatments.
Our results indicate INM practices, especially, when whole organic source treatment replaces recommended dose of nutrients or when 25% RDN is replaced by biofertilizer or when 25% RDN is replaced by FYM along with conservation/reduced tillage and water management maintains high soil glucosidase, urease, and phosphatase which are essential for C, N, and P circulation in soil (Tables 4-9) during rice cultivation. Further, microbial activity of soil and yield of rice plants (Table 10) have also been increased with the use of RMPs. Similar results in cereals have been reported by Mikanová et al. (2009).