Bioresource Technology 98 (2007) 35853592

EVect of long-term application of manure and fertilizer on biological and biochemical activities in soil during crop development stages
Asit Mandal, Ashok K. Patra , Dhyan Singh, Anand Swarup, R. Ebhin Masto
Division of Soil Science and Agricultural Chemistry, Indian Agricultural Research Institute, New Delhi 110 012, India Received 6 September 2006; received in revised form 14 November 2006; accepted 15 November 2006 Available online 5 January 2007

Abstract A Weld experiment was conducted to investigate the eVect of six long-term (34-year) fertilizer and farmyard manure (FYM) treatments (Control, N, NP, NPK, NPK + S, NPK + FYM) and three physiological stages of wheat growth on the microbial biomass carbon (MBC), nitrogen (MBN) and dehydrogenase, mineralizable N and phosphatase activities in soil. It was found that a balanced application of NPK + FYM gave the highest values for the measured parameters and lowest at the control. Values were generally highest at tillering, followed by the Xowering and dough stages. A signiWcant positive interaction between fertilizer treatments and physiological stages of wheat growth was observed, being highest at maximum tillering due to application of NPK + FYM. Stepwise regressions have revealed that grain yield of wheat was signiWcantly associated with mineralizable N at tillering (R2 D 0.80), MBC at Xowering (R2 D 0.90) and alkaline phosphatase activity (R2 D 0.70) at dough stages of wheat growth. 2006 Elsevier Ltd. All rights reserved.
Keywords: Crop development stages; Farm yard manure (FYM); Long-term fertilizer use; Microbial biomass; Soil enzymes

1. Introduction In Indian agriculture, improving and maintaining soil fertility is of paramount importance to meet the demands of food grain production for an ever-increasing population in the country. Good soil fertility management ensures adequate nutrient availability to plants and increases yields. High aboveground biomass yield is obviously accompanied by an active root system, which releases an array of organic compounds into the rhizosphere (Bowen and Rovira, 1991). Plant roots release about 17% of the photosynthate captured, most of which is available to soil organisms (Nguyen, 2003). These compounds support the growth of the microbial community and result in increased population density (Bowen and Rovira, 1991; Kent and Triplett, 2002) and also aVect their level and diversity of functions (Read et al.,
*

Corresponding author. Tel.: +91 11 25841494; fax: +91 11 25841529. E-mail address: patraak@gmail.com (A.K. Patra).

2003; Patra et al., 2006). Microbial communities are important for the functioning of the ecosystem (Patra et al., 2005), both in relation to direct interactions with plants and with regard to nutrient and organic matter cycling. It has become increasingly recognized that interactions between the root and microorganisms are aVected by anthropogenic disturbances, such as agricultural practices, biological modiWcation and industrial pollution (Lynch, 2002). Among agricultural practices, ploughing, manuring and fertilization and crop rotation have beneWcial, harmful or neutral eVects on the trinity formed by plants, soil organisms (microbes and fauna) and soil (Bowen and Rovira, 1991; Hatch et al., in press). Since in a soil-plant system, the soils energy powerhouse is the rhizosphere, any alteration to the fertility management (e.g., balanced or imbalanced fertilization, use of organics, etc.) will have a strong impact (or feed back) at the soilplant interface, and subsequently on the agricultural productivity and sustainability of the ecosystem.

0960-8524/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2006.11.027

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In the context of soil fertility management, long-term fertilizer experiments (LTFEs) are valuable assets for determining yield trends, changes in nutrient dynamics and balances, predicting soil carrying capacity, assessing soil quality and system sustainability. Trends in soil fertility changes in many of the short-term (Marcote et al., 2001; Blaise et al., 2005) or LTFEs have been reported from samples obtained at the beginning or at the end of the cropping sequence (Dalal and Mayer, 1986; Swarup et al., 1998; Albiach et al., 2000; Marinari et al., 2000; Kautz et al., 2004; Manna et al., 2005; Hati et al., 2006; Masto et al., 2006). In contrast, information on the biological processes, such as soil enzymatic activities, which mediate nutrients cycling and inXuence their acquisition during active crop growth stages, is limited (Barnard et al., 2006). Since microbial processes are dynamic, patterns of temporal Xuctuation during crop growth are of great importance in relation to the nutrient supplying capacity of the ecosystem and the crop demands. The present research, therefore, was undertaken to assess the impact of long-term (34 years) application of mineral fertilizers and farm yard manure and physiological stages of wheat growth on soil biological and biochemical activities, and their relationships with crop yields in a semi-arid, tropical agro-ecosystem. 2. Methods 2.1. Experimental site and treatments This experiment was conducted in the rabi season (cooler winter-spring period, November 2004 to April 2005) under a wheat crop of the long-term fertilizer experiment (LTFE) at the Indian Agricultural Research Institute (IARI), New Delhi, located at 2837 2839 N latitude and 779 7711 E longitude (about 250 m above mean sea level). The experimental area was characterized as semi-arid sub-tropical region having hot summers and cold winters with a mean annual maximum and minimum air temperature of 40.5 and 6.5 C, respectively. The mean annual precipitation was about 670 mm, most of which was conWned to a three-month period from July to September (monsoon). The total precipitation during the course of this study was 189.9 mm and average temperatures ranged from 9.9 to 23.3 C. The on-going LTFE at IARI, New Delhi was set up in 1971 with intensive cropping sequence of pearl millet wheat-cowpea (fodder). Later in 1982, pearl millet was replaced by maize, and in 2002 cowpea was dropped. Hence, the cropping sequence since 2002 was maize wheat. The soil was an Inceptisol (Typic Haplustept) with the following initial properties: sand 69%, silt 16%, clay 14%, pH (1:2.5) 8.3, electrical conductivity (EC, 1:2.5) 0.45 dS m1, CEC 10.6 cmol (p+) kg1, soil organic carbon (WalkleyBlack) 4.4 g kg1, NaHCO3 extractable P 16 kg ha1, NH4OAc-extractable K 155 kg ha1. There were original 10 treatments, out of which only six contrasting treatments were selected for this study; control no manure or fertilizer, 100% N, 100% NP, 100% NPK, 100% NPK + S, and 100% NPK + FYM. Based on the soil test values, the optimum fertilizer dose (100% NPK) was

120 kg N, 26 kg P and 33 kg K per hectare for maize and wheat. Farm yard manure (FYM) with a mean nutrient content of 350 g kg1 C, 5 g kg1 total N, 2.5 g kg1 total P, 15 g kg1 total K and 30 mg kg1 Zn was applied at a rate of 15 Mg ha1 once in a cropping cycle, i.e., each year before the sowing of maize. The treatments were laid out in a randomised block design (RBD), having a plot size of 21 8 m. There were three replications for this study. The fertilizer sources of N, P and K were urea/diammonium phosphate (DAP), DAP/single superphosphate and potassium chloride (muriate of potash), respectively. Sulphur in the 100% NPK + S treatment was applied through single super phosphate (containing 12% S). The soil samples were studied under the wheat crop in the cropping sequence of maize (cv. Ganga Safed-2) wheat (cv. HD-2329). Wheat was sown on 25th November 2004 with a row spacing of 22 cm using a seed drill. For fertilization, 50% of the N and all of the P and K fertilizers were applied as a basal dose and the remaining half of the N was applied after one month from sowing. A total of six irrigations were given during the crop growth. Other agronomic practices were adopted as necessary. 2.2. Soil sampling and analysis Composite surface (015 cm) soil samples (i.e., 10 random core samples from each plot were thoroughly mixed together) were collected from each plot (i.e., replicate) at three physiological stages of the wheat crop, viz. tillering, Xowering and dough, on 12 January, 18 February and 14 March 2005, respectively. The composite samples were placed in plastic bags and transported to the laboratory, where Weld moist soil was sieved (2 mm mesh size), homogenized and stored at 4 C. The gravimetric moisture content was determined immediately. Total organic carbon (TOC) in soil was determined by wet digestion method (Snyder and Trofymow, 1984), and total N by Kjeldahl digestiondistillation method (Bremner and Mulvaney, 1982). The microbial biomass C was estimated by chloroform fumigation extraction method as described by Jenkinson and Ladd (1981) using a Kc value of 0.25 (Bremner and Kesssel, 1990). The microbial biomass N was determined by the fumigation extraction following the modiWed procedure of Brookes et al. (1985), with a Kc value of 0.45 (Bremner and Kesssel, 1990). Dehydrogenase activity in soil was determined by monitoring the rate of production of tri-phenyl formazon (TPF) from tri-phenyl tetrazolium chloride (TTC), using the method of Klein et al. (1971). Mineralizable N was estimated by the method as given by Subbiah and Asija (1956). Acid and alkali phosphatase enzyme activities were determined using p-nitro phenol (PNP) method of Tabatabai and Bremner (1969). 2.3. Statistical analysis A two-factor analysis of variance (ANOVA) was performed to determine the eVects of fertilizer treatments,

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physiological stages and their interactions on biological and biochemical properties. Whereas, one-way ANOVA was used for analysis of total organic C, N and grain yield of wheat. Least signiWcant diVerence (LSD at P D 0.05) was used to determine whether means diVered signiWcantly. For statistical analysis of data, Microsoft Excel (Microsoft Corporation, USA) and SPSS window version 10.0 (SPSS Inc., Chicago, USA) packages were used. Unless otherwise stated, the level of signiWcance referred to in the results is P < 0.05. 3. Results and discussion 3.1. Total organic C (TOC), N (TN) and C to N ratio

Total organic carbon (g kg-1)

13 12 11 10 9 8 7 6 5 4

e cd b bc d

Co

ntr

ol

NP

K NP

S YM K+ +F NP PK N

Treatment
1400

Total N (mg kg -1)

1200 1000 800 600 400 200 0

c c b a b b

Total organic C (TOC) and total N (TN) were determined at the maximum tillering stage only, presuming that TOC and TN were not likely to be changed signiWcantly during crop growth. The data on TOC and TN at tillering stage showed a signiWcant (p < 0.01) eVect of treatments (Fig. 1). The TOC was lowest (6.1 g kg1) in control and highest (12.9 g kg1) in 100% NPK + FYM treatment. For TOC, the treatment eVect can be arranged in the following order (based on statistical signiWcance): 100% NPK + FYM > 100% NPK + S D 100% NPK D 100% NP D 100% N > control. The data on total N ranged from 420 mg N kg1 in control to 1.07 g kg1 in 100% NPK + FYM. There is no signiWcant diVerence in total N concentration between NPK + S and NPK + FYM treatments, and between N, NP and NPK treatments after 34 years. The values of C/N ratios were not signiWcantly diVerent. The highest SOC and TN in 100% NPK + FYM supported results obtained in an earlier study at the same site (Masto et al., 2006) and other LTFE centers in India (Manna et al., 2005). In the present experiment, the aboveground biomass was harvested and no residues incorporated in the soil, except FYM in 100% NPK + FYM treatment. The increase in soil organic matter with application of inorganic fertilizer nutrients was because of greater input of root biomass due to increased crop productivity. In the treatment with manure, an additional ca 5250 kg C ha1 yr1 was added which resulted in signiWcantly higher organic matter compared to the rest of the treatments. In an earlier study, Manjaiah and Singh (2001) estimated an annual input of organic carbon from the cropping sequence to the tune of 8190 and 2780 kg ha1 in 100% NPK + FYM and 100% NPK treatments, respectively. An understanding of the fate of this C and associated N inputs and their contribution to the C and N sequestration/budget on a time scale would be an important area of future research. The values of C/N ratio of the soils ranged from 11.1 to 14.7, but interestingly the treatment eVect was found not signiWcant (Fig. 1c). The C/N ratio in soil was determined by the state of organic matter, and microbial populations and activities in soil. Similar ratio in all the treatments in this study indicates that the proportion of increases or decreases in C and N was similar in all the treatments.

Co

ol nt r

NP

K NP

M S FY K+ NP K+ NP

Treatment
18 16 14 12 10 8 6 4 2 0

NS (P = 0.61)

C to N ratio

ol nt r Co

NP

K NP

S YM K+ +F NP PK N

Treatment

Fig. 1. (a) Total organic C, (b) total N and (c) C/N ratio in soils at tillering (48 d after sowing) of wheat growth under the long-term fertiliser experiment. Bars indicate standard errors (n D 3). Means not sharing a letter in common diVer signiWcantly (P D 0.05) from each other. NS D not signiWcant.

3.2. Soil microbial biomass C and N Microbial biomass carbon (MBC) in soil samples showed a signiWcant eVect of diVerent fertilizer treatments and stages of wheat growth and their interactions (Table 1). Tillering stage of wheat crop had the maximum impact in enhancing MBC (414 mg kg1) in the soil followed by the Xowering (396 mg kg1) and dough (297 mg kg1) stages. Mean values of the treatments for MBC ranged from 261 mg kg1 in the control to 517 mg kg1 in 100% NPK + FYM. Addition of S with 100% NPK also signiWcantly increased MBC and was the next highest after 100% NPK + FYM. Like MBC, the microbial biomass nitrogen (MBN) in soil was also signiWcantly (P < 0.05) aVected by diVerent fertilizer treatments and stages of wheat growth (Table 2), but interaction eVect was not signiWcant.

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Table 1 Microbial biomass carbon (mg C kg1) in soil as aVected by long-term fertilizer application and physiological stages of wheat crop Treatments Stage of wheat growth Tillering Control 100% N 100% NP 100% NPK 100% NPK + S 100% NPK + FYM Mean 288 338 400 424 458 576 414 Flowering 260 252 392 422 504 544 396 Dough 236 242 254 282 336 432 297 Mean 261 277 349 376 433 517

LSD (P D 0.05): Treatment (T) D 37.10; Stage (S) D 26.24; T S D 64.27.

Table 2 Microbial biomass nitrogen (mg N kg1) in soil as aVected by long-term fertilizer application and physiological stages of wheat crop Treatments Stage of wheat growth Tillering Control 100% N 100% NP 100% NPK 100% NPK + S 100% NPK + FYM Mean 47.7 62.2 68.4 74.4 74.7 85.0 68.7 Flowering 51.8 56.0 60.1 66.4 70.5 78.8 64.0 Dough 53.9 53.9 56.0 60.1 66.4 78.8 61.5 Mean 51.2 57.4 61.5 67.0 70.5 80.9

LSD (P D 0.05): Treatment (T) D 7.94; Stage (S) D 5.61; T S D not signiWcant.

tion. However, it is well established that rhizodeposits stimulate biological activity (which can be concluded from this study), and beneWts plants by enhancing nutrient availability (Kuzyakov, 2002). With regard to MBC to MBN ratios, there were no treatment or interaction eVects (data not shown). However, their ratio was signiWcantly aVected by the stages of wheat growth. The ratio was highest at the tillering stage (6.25) and lowest in the dough stage (4.8). Since there is a strong relationship between MBC and MBN (Table 7), the reasons discussed for the variable amounts of MBC recorded in this study are perhaps applicable to MBN as well. The signiWcant decrease in the ratio of MBC to MBN at the dough stage could be possibly due to changes in community structure and size which might be the result of changing inputs of metabolizable N from plant residues (Gomez et al., 2006) and soil conditions arising out of plant growth inXuences (Patra et al., 2006). High MBC to MBN at tillering and Xowering compared to the dough stage might be due to temporal changes in the proportion of bacteria relative to fungi in soils; bacteria have a lower C:N ratio compared with fungi (Anderson and Domsch, 1980; Bardgett et al., 1999), or due to greater decrease in MBC compared to MBN, indicating C limiting in later stages. Further studies would be required in this agro-ecosystem under LTFE conditions to determine temporal changes in microbial communities in soil and the functional signiWcance of these changes during plant growth stages. 3.3. Dehydrogenase activity Data on dehydrogenase activity in soil samples showed a signiWcant (P < 0.05) eVect of diVerent fertilizer treatments and stages of wheat growth and their interactions (Table 3). EVect of interaction was at higher amplitude with NPK + FYM, NPK + S, NPK than the mean values of the treatments, at tillering followed by dough and Xowering stages. As with MBC and MBN, the maximum tillering stage of wheat coincided with the highest (average of six treatments) dehydrogenase activity (20.0 mg TPF kg1 h1) in the soil. However, unlike MBC and MBN, dehydrogenase activity was signiWcantly higher in the dough (16.3 mg TPF kg1 h1) than in the Xowering (14.0 mg TPF kg1 h1) stage
Table 3 Dehydrogenase ( g TPF g1 h1) activity in soil as aVected by long-term fertilizer application and physiological stages of wheat crop Treatments Stage of wheat growth Tillering Control 100% N 100% NP 100% NPK 100% NPK + S 100% NPK + FYM Mean 15.3 19.3 20.0 21.4 21.6 22.2 20.0 Flowering 9.4 11.5 12.6 13.7 18.1 19.0 14.0 Dough 14.7 16.2 16.0 16.4 16.7 18.0 16.3 Mean 13.1 15.6 16.2 17.2 18.8 19.7

The range of MBC was consistent with several other workers (Kautz et al., 2004; Manna et al., 2005). The observations on the eVect of physiological stages clearly indicated that microbial biomass C or N was subject to the time of sampling; highest values being associated with the tillering stage and the lowest at the dough stage. Microbial biomass was a small but very dynamic component of soil organic matter Xuctuating with weather, crop, input and season (Garcia and Rice, 1994) by as much as 40% in native ecosystems (Garcia and Rice, 1994) and agricultural systems (Buchanon and King, 1992). In this study, soil moisture was not signiWcantly diVerent among treatments, nor there was any signiWcant correlations with the microbial biomass (data not presented). Temperature was important, but in spite of lower temperatures at tillering (14 January 2004, average about 13 C), maximum microbial biomass and enzyme activities (Table 1) were recorded. Thus, it could be suggested that the interaction eVect of treatments and rhizosphere conditions induced by plant growth stages played a major role in the enhancement of microbial biomass to a maximum around tillering. On an average, shoots export to belowground about half of the C Wxed by photosynthesis (Nguyen, 2003). Thus, the extra plant growth achieved by long-term balanced fertilization (NPK + S or NPK + FYM) would stimulate more below ground Xux of C Wxed by photosynthesis. The release of organic C from roots into the soil might be regarded as a lost pool of reduced C that no longer contributed to dry matter produc-

LSD (P D 0.05): Treatment (T) D 1.44; Stage (S) D 1.02; T S D 2.50.

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of wheat, though the interaction eVect of NPK + FYM and NPK + S with these stages were at par (Table 3). Mean values of the treatments for dehydrogenase activity ranged from 13.1 mg TPF kg1 h1 in control to 19.7 mg TPF kg1 h1 in 100% NPK + FYM. Addition of S with 100% NPK was as eVective as 100% NPK + FYM. Biological and biochemical properties of the soil have often been proposed as early and sensitive indicators of soil ecological stress or other environmental changes (Dick, 1994). Since dehydrogenase activity is only present in viable cells, it is thought to reXect the total range of oxidative activity of soil microXora and consequently may be considered to be a good indicator of microbial activity (Nannipieri et al., 1990). Generally the enzyme activities in the soil are closely related to the organic matter content (Beyer et al., 1993). The application of balanced amounts of nutrients and manures improved the organic matter and MBC status of soils, which corresponded with higher enzyme activity. It has been reported that the increase in dehydrogenase activity and microbial biomass were proportional to the addition of number and amount of nutrients (Manjaiah and Singh, 2001; Masto et al., 2006). In the present study, a similar trend was also observed. According to Pancholy and Rice (1973), dehydrogenase activity is inXuenced more by the quality than by the quantity of organic matter incorporated into soil. Thus, the stronger eVects of FYM or sulphur on dehydrogenase activity might be due to the more easily decomposable components of crop residues on the metabolism of soil microorganisms. The observation that dehydrogenase activity is less inXuenced by mineral nitrogen fertilization is consistent with the studies of Kautz et al. (2004) and Marinari et al. (2000). Concerning temporal variability and interaction eVects between fertility treatments and growth stages, dehydrogenase activity was highest at tillering (sampled in January), similar to MBC. However, a signiWcant reduction in dehydrogenase activity at the Xowering stage is an interesting observation, reasons for which could not be obtained in the present study and requires further investigation. 3.4. Mineralizable N Highest mineralizable N (133.9 mg kg1) was observed due to interactive eVect of 100% NPK + FYM treatment and maximum tillering stage (Table 4). Like MBC or MBN, mineralizable N in soil was highest (105.8 mg kg1) at tillering followed by Xowering (91.8 mg kg1) and dough (79.5 mg kg1) stage. Mean values of the treatments for mineralizable N ranged from 78.5 mg kg1 in control to 105.8 mg kg1 in 100% NPK + FYM. Addition of S with 100% NPK has exhibited as the next best value for mineralizable N. The data have revealed that 100% NP and 100% N were at par, and so were 100% NPK and 100% NPK + S. It may be mentioned that the method used for measuring mineralizable N in this study was chemical based, to represent that N which can be mineralized during the crop growth period. This is one of the most accepted methods,

Table 4 Mineralizable N (mg kg1) in soil as aVected by long-term fertilizer application and physiological stages of wheat crop Treatments Stage of wheat growth Tillering Control 100% N 100% NP 100% NPK 100% NPK + S 100% NPK + FYM Mean 74.2 90.5 104.5 112.0 119.9 133.9 105.8 Flowering 85.9 86.8 89.1 95.2 96.1 97.5 91.8 Dough 75.3 76.1 77.0 78.9 84.0 85.9 79.5 Mean 78.5 84.5 90.2 95.3 100.0 105.8

LSD (P D 0.05): Treatment (T) D 6.69; Stage (S) D 4.73; T S D 11.58.

particularly in India; which represents strong correlations with crop yields in many previous soil test crop response studies. Mineralisation/immobilization of organic N is mediated by several enzymatic processes, such as amino acid dehydrogenases, amino acid oxidase, amidohydrolases and amidinohydrolases (Nannipieri et al., 1990). The signiWcant correlation with MBC, MBN and enzymic activities (Table 7) in our study is indicative of the fact that the method represents that fraction which is mediated by enzymic activities. Though except dehydrogenase activity none has been studied here, it can be presumed that activity of several enzymes might have been elevated and enhanced mineralizable N due to enhanced interactive eVect of NPK + FYM treatment and rhizospheric feed back at tillering. 3.5. Phosphatase activity The data showed no signiWcant interaction eVect between treatments and stages of crop growth on acid phosphatase activity (Table 5). The Xowering stage of wheat crop had the highest value (30.2 mg PNP kg1 h1), followed by the tillering (19.6 mg PNP kg1 h1) and dough (14.9 mg PNP kg1 h1) stages. Mean values for the treatments for acid phosphatase activity ranged from 19.0 mg PNP kg1 h1 in control to 24.4 mg PNP kg1 h1 in 100% NPK + FYM. With regard to alkaline phosphatase activities in soil (Table 6), interactive eVect of 100% NPK + FYM treatment
Table 5 Acid phosphatase activity ( g PNP g1 h1) in soil as aVected by longterm fertilizer application and physiological stages of wheat crop Treatments Stage of wheat growth Tillering Control 100% N 100% NP 100% NPK 100% NPK + S 100% NPK + FYM Mean 16.1 19.3 17.1 21.3 21.3 22.4 19.6 Flowering 29.8 28.9 29.3 30.0 31.4 31.8 30.2 Dough 11.1 14.3 12.7 13.6 18.9 18.8 14.9 Mean 19.0 20.8 19.7 21.6 23.9 24.4

LSD (P D 0.05): Treatment (T) D 1.69; Stage (S) D 1.19; T S D not signiWcant.

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A. Mandal et al. / Bioresource Technology 98 (2007) 35853592 Table 7 Correlation (r values) matrix for MBC, MBN and soil enzyme activities during wheat growth Variables Dough 137.4 148.5 175.0 194.6 217.3 230.2 183.8 Mean 146.8 159.9 180.9 198.2 213.4 226.4 MBC MBN D-ase MN Acid p-ase Alk p-ase MBC 1.00 MBN 0.72 1.00

Table 6 Alkaline phosphatase activity ( g PNP g1 h1) in soil as aVected by longterm fertilizer application and physiological stages of wheat crop Treatments Stage of wheat growth Tillering Control 100% N 100% NP 100% NPK 100% NPK + S 100% NPK + FYM Mean 184.0 197.4 198.4 215.5 225.0 243.7 210.7 Flowering 118.9 133.7 168.9 184.5 197.8 205.3 168.2

D-ase 0.58 0.57 1.00

MN 0.75 0.57 0.59 1.00

Acid p-ase 0.48 NS NS 0.32 1.00

Alk p-ase 0.66 0.63 0.77 0.58 NS 1.00

LSD (P D 0.05): Treatment (T) D 11.57; Stage (S) D 8.18; T S D 20.04.

MBC, microbial biomass C; MBN, microbial biomass N; D-ase, dehydrogenase activity; MN, mineralizable N; Acid p-ase, acid phosphatase; Alk p-ase, alkaline phosphatase. , Marked correlations are signiWcant at P D 0.05 and 0.01, respectively (n D 54, data pooled from all three stages). NS, not signiWcant at P D 0.05.

and maximum tillering stage showed highest value (243.7 g PNP g1 h1), whereas control and Xowering stage showed the lowest (118.9 g PNP g1 h1). Interaction eVect was found to be at higher order with NPK + FYM > NPK + S > NPK than the mean values of the treatments at tillering followed by dough and Xowering stages. Unlike acid phosphatase activity, alkaline phosphatase was more active at the tillering (210.7 mg PNP kg1 h1) followed by dough (183.8 mg PNP kg1 h1) and Xowering stages (168.2 mg PNP kg1 h1). Examination of the data indicates that alkaline phosphatase activities at diVerent stages followed the same pattern as dehydrogenase activity in the soil samples. Although the mean data for alkaline phosphatase activity in soils under diVerent treatments gave the following order (based on statistical signiWcance): 100% NPK + FYM > 100% NPK + S > 100% NPK > 100% NP > 100% N > control, but there was aberration on this trend when the eVect of interactions between treatments and stages was examined. For example, NPK + FYM D NPK + S at Xowering stage, and 100% N D 100% NP at tillering. Activity of phosphatases is important in studying the P cycle because this can provide a route for P mineralization and plant uptake. However, similarity in their activities was not persistent, and sometimes even contrasting: for example, at the Xowering stage of wheat (Table 5). The acid phosphatase activity was much lower than alkaline phosphatase activity, irrespective of the treatments, which may be due to the alkaline reaction of the soil. Earlier studies also proved that phosphatase activity was strongly inXuenced by soil pH (Eivazi and Tabatabai, 1977; Dick, 1994). The signiWcantly greater activities of alkaline phosphatase activity in the FYM treated soils may be due to enhanced microbial activity and perhaps diversity of phosphate solubilizing bacteria due to manure input over the years. The phosphatase activity was also closely related to the microbial biomass C content (Table 7), thus consistent with the observations of Parham et al. (2002). 3.6. Wheat yield With respect to wheat grain yield, there was no signiWcant diVerence between control and 100% N treatment

(data not shown). The treatments can be arranged (based on statistical signiWcance) in the following order: 100% NPK + FYM (4940 kg ha1) D 100% NPK + S (4687 kg ha1) D 100% NPK (4336 kg ha1) D 100% NP (3939 kg ha1) > 100% N (3181 kg ha1) D control (2593 kg ha1). Wheat yield in NPK + FYM was almost double than that in control and 55% more than that in the N treatment. The data on crop yields from this experiment over the years have been reported earlier (Singh et al., 1998), indicating the beneWts of organic manure on crop performance. 3.7. Correlations Correlation studies have shown the existence of a signiWcant relationship (P D 0.01) between the soil enzyme activities, except with acid phosphatase (Table 7). Stepwise regression equations were developed to Wnd out the critical biological and biochemical factors at a particular physiological stage, which signiWcantly associated with grain yield of wheat. It was observed that mineralizable N at tillering (R2 D 0.80), MBC at Xowering (R2 D 0.90) and alkaline phosphatase activity (R2 D 0.70) at dough stages (Table 8) were signiWcant at the respective stages to predict the grain yield of wheat crop. Crop growth and demand for N at the maximum tillering stage is high. This was evident from the study on plant uptake of N during wheat growth, which was highest (31.6 g kg1, oven dry weight basis) at maximum tillering
Table 8 Stepwise regression of grain yield of wheat with MBC, MBN, dehydrogenase, MN, acid and alkaline phosphatase activities (n D 18) at a particular physiological stage Physiological stage Predictor variable Constant 107.8 1273.4 184.4 CoeYcient 38.3 6.8 20.5 R2 P (model)

Maximum tillering MN Flowering MBC Dough Alk p-ase

0.80 0.000 0.90 0.000 0.70 0.000

The values are Wtted in the equation: y D a + bx1 + cx2. y D grain yield; x1 and x2 are the independent variables; a, b, c and d are coeYcients. MN, mineralizable N; MBC, microbial biomass C; Alk p-ase, alkaline phosphatase.

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stage than at Xowering (20.6 g kg1) and dough stages (17.6 g kg1) (Mandal, 2005). High MBC to MBN ratios at this stage, however, indicates the chances of more N immobilization by microbes than its availability by mineralization and that warrants for external supply or better management of N. The signiWcance of MBC at Xowering stage is not clear; perhaps indicates its inXuence to all the microbial processes that may be responsible for balanced nutrient supply at this stage. The phosphatase activity in soil at dough stage is important, perhaps to meet the high demands of P at the dough stage. In cereals, during seed and grain development period, content of phytate (storage form of P, synthesized from myoinositol, phytic acid and phosphate groups) sharply rises (Ogawa et al., 1979). Since phytates are involved in regulation of starch synthesis during grain Wlling, an association with alkaline phosphatase in such soil cannot be ruled out. 4. Conclusions This study has shown that the microbial biomass and soil enzyme activities were controlled by the long-term manure and fertilizer treatments and physiological stages of wheat crop (rhizosphere feed back) and their interactions. Microbial biomass and microbial activities were highest in NPK + FYM treatment at maximum tillering, followed by Xowering and dough stages. In general, the amplitude of interaction eVect was at higher order in NPK + FYM, NPK + S and NPK than the mean values of the treatments at tillering, followed by Xowering and dough stages. Among the biological and biochemical parameters, mineralizable N at maximum tillering, MBC at Xowering and alkaline phosphatase activity at dough stages were signiWcantly associated with the grain yields of crop. Thus, while studying the impact of long-term fertility management on soil biological and biochemical parameters, the interaction with crop physiological stages and their importance to nutrient cycling, crop yields and sustainability needs to be addressed. Acknowledgements The authors are thankful to Dr. S. Bhadraray and Dr. T.J. Purakayastha for their helpful discussion during the experiment. Also thanks are due to Dr. S.C. Kaushik and Dr. V. Verma for technical advice. Asit Mandal is highly grateful to IARI (ICAR), New Delhi, for awarding him a Junior Research Fellowship. References
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