IMPACT OF N FERTILIZATION ON C BALANCE AND SOIL QUALITY IN MAIZE-DHAINCHA CROPPING SEQUENCE

Excess N fertilization to achieve high crop yield is a grand old practice in developing countries. However, inorganic nutrient sources considerably replenish soil organic C (SOC). In the present study, we applied six different levels of N keeping P and K constant for maize, grown under maize (Zea mays) – dhaincha (Sesbania aculeata) cropping sequence. We recorded high crop yield, profuse root biomass and SOC stock with increasing N fertilization. Moreover, water holding capacity, microbial biomass carbon and particulate organic carbon improved significantly with increasing levels of N. Conversely, bulk density, mineral associated organic carbon and pH decreased with increasing application of inorganic N. Furthermore, a significant positive correlation was recorded between root biomass and soil organic carbon. A study of the sensitivity index showed particulate organic carbon and microbial biomass carbon to be good indicators of nutrient management practices. Dhaincha cultivation accelerated C and N mineralization in soil, which is reflected in increased biomass and crop yield. Hence, we conclude that inorganic N fertilization rate (72 80 kg ha -1 ) in maizedhaincha cropping sequence successfully maintains the SOC balance and optimize N stock in soil.


Introduction
The atmospheric CO 2 concentration of the Earth is increasing rapidly.The 1997 Kyoto Protocol identified that the incorporation of atmospheric CO 2 into biomass and soil is highly effective in reducing CO 2 build up (Lal, 2004).The main pools of actively cycling C are atmospheric CO 2 , biota (mostly vegetation), soil organic matter (including detritus), and the ocean.Among these C pools, soil organic matter (SOM) plays a key role in global C cycling and is important in Banashree Sarma et al. 136 maintaining soil fertility that sustains agricultural productivity (Lou et al., 2011).It influences crop growth and yield, either directly by supplying nutrients or indirectly by modifying chemical, physical and biological conditions of soils (Hati et al., 2007).Improvement of soil biological properties affects growth of plants and soil microbial diversity and population, thereby creating a suitable environment for root growth (Lee et al., 2009).Hence, agricultural soils can constitute either a net significant source or sink of CO 2 .But decaying organic matters are biologically most useful.Therefore, a dilemma exists between the two states: whether to conserve SOM or use up for profit in terms of nutrient availability (Janzen, 2006).Sequestering more SOM by suppressing decomposition will cause energy deficiency for sustaining life of soil microbial population while mineralization will lead to the depletion of SOM and the release of CO 2 into the atmosphere.Therefore, effective management practice should be devised to maintain the SOC balance.
Crop response to N fertilization involves a change in biomass allocation.Therefore, this can significantly affect the input of C to the soil and subsequently SOC.There are many studies related to the rates and quantity of SOC levels due to N fertilization, nevertheless, the influence depends on management, soil type and climate (Alvarez, 2005).The incorporation of cover crops in the cropping sequence is another effective agricultural management practice that provides many services to agroecosystems, like improving soil nutrient retention and physical health (Lal, 2004).
Maize (Zea mays L.) is one of the popular cereal crops grown throughout the globe.Being an exhaustive crop, maize requires high quantity of N for its optimum growth.Moreover in India, the higher subsidy rates on nitrogenous fertilizers artificially reduced their cost compared to P and K fertilizers.This encourages the farmers to use nitrogenous fertilizers in abundance ignoring their long-term effects on soil quality.
Regarding these viewpoints, the present study was aimed at investigating the impacts of different levels of inorganic N fertilization on the changes in SOC and its fractions over a two-year period in a maize-dhaincha crop rotation.Moreover, we also analyzed the changes in soil physico-chemical properties and yield of maize.

Experimental design and site quality
The experiment was conducted at Tezpur located in north bank plain zone of Assam (26 o 14'N and 92 o 50'E), India having sandy loam type of soil.Maize (Zea mays L. cv.All rounder) was grown under irrigated conditions on experimental soil followed by dhaincha (Sesbania aculeata).The experiment was conducted in a randomized block design with three replicates for two consecutive years (2012 2014).We have applied various doses of N fertilizer in this study, keeping other management practices such as P and K rates, plant population, sowing methods, spacing and intercultural operations and harvesting time identical as per the standard practice recommended for this zone.The treatment combination used for the study was T 1 (Unfertilized); T 2 (N -100 kg ha -1 ); T 3 (N -80 kg ha -1 ); T 4 (N -72 kg ha -1 ); T 5 (N -60 kg ha -1 ); T 6 (N -40 kg ha -1 ).In all the treatments, the whole quantities of P and K along with half dose of N (urea) were applied as basal in furrows.The remaining quantity of N was applied in 2 split doses at 30 days after sowing (DAS) (knee high stage) and 70 DAS (silking stage) respectively.

Soil sampling
Soil samples were collected at harvest of the crops.Soil samples from each treatment consisted of three composite sub samples that were taken with a probe (6.0-cmdiameter core) from the upper 0-15 cm depth.Field-moist soil samples were divided into two sub samples.One sub sample was air dried at room temperature and sieved through a 0.5-mm sieve and used for chemical analysis.The other sub sample was oven-dried at 105°C for 24 hours, sieved through 2-mm sieve and analysed for the physical parameters.

Physico-chemical analysis
Soil physical properties such as bulk density (BD) and water holding capacity (WHC) were analysed using standard methods by following Page et al. (1982).Soil pH was estimated in 1:2 soil-water suspension using a digital pH meter (HI96107).SOC was determined by oxidation with potassium dichromate and by titrating the excess dichromate with ferrous ammonium sulphate (Baruah and Borthakur, 1997).Particulate Organic Carbon (POC) was determined with modifications of the method described by Cambardella and Elliott (1992).Ten grams of dry soil was dispersed in 30 ml of sodium hexametaphosphate (5 g L -1 ) with shaking on a reciprocating shaker (90 r min -1 ) for 18 hours.The soil suspension was poured over a 53-mm screen.Water in the slurry was evaporated in a forced air oven at 45°C and the dried sample was ground with a mortar and pestle and analyzed for mineral associated organic C (AOC).POC content was calculated by subtracting AOC from SOC (Divito et al., 2011).Soil microbial biomass carbon (MBC) was determined using CHCl 3 fumigation-extraction method (Vance et al., 1987).Analysis of available N, P and K of soil was done according to the method given in Baruah and Borthakur (1997).Avaiable N was determined using alkaline potassium permanganate method in Kelplus (Model-Elite Ex VA) using NaOH, H 2 SO 4 and methyl red as indicator.Available P was estimated by using Bray's reagent.Available K was estimated using flame photometer.For this, 2 g of soil is extracted with neutral 1N ammonium acetate followed by analysis of K in the solution in a flame photometer.

Plant parameters
Both above-ground and below-ground plant biomasses were recorded at regular interval.Root biomass was obtained by excavating root and then by drying the samples in an oven at 105°C for 12 hours.After drying, samples were kept in a desiccator and dry weights were taken.Maize was harvested at its physiological maturity and dhaincha was harvested 10 weeks after sowing and yield was recorded at harvest.

Data handling and statistical analysis
The Sensitivity Index (SI) was computed using the following formula according to Yang et al. (2012): All data presented are the mean of two years.Statistical analysis was performed with SPSS for Windows 16.0.20.All data obtained were subjected to one-way analysis of variance (ANOVA) using the randomized block design.Relationships among the variables were studied using Pearson's correlation coefficients.LSD and Duncan's Multiple Range Test (DMRT) at the 5% level of probability were used to test the significance of differences between treatment means.

Basic soil characteristics of experimental site
Table 1 shows the basic soil characteristics.The experimental soil was slightly acidic with SOC content of 1.41% with medium nutrient status (N, P and K).The soil was sandy loam with 44.4% water holding capacity and 1.1 Mg m -3 bulk density.Impact of inorganic N on SOC content of cropped soil Soil organic C content increased with increasing N levels (Figure 1).The highest SOC was recorded under T 2 (1.47%) followed by T 3 (1.45%) (P=0.03) in the upper soil zones at the end of maize cultivation.This increase in SOC compared to basic soil (Table 1) can be attributed to higher biomass production and return of residue from maize plants due to N fertilization (Lee et al., 2009).The results of a positive correlation between root biomass of maize and SOC in our experiment support this finding (r=0.958).N fertilization also stimulates soil microbial activity, thereby changing the dynamics of SOC in soil (Lou et al., 2011).However, dhaincha cultivation showed a negative impact on SOC concentration.SOC decreased in T 2 , T 3 and T 5 after dhaincha cultivation while the rest of the treatments maintained the value at par with maize cultivation.Decreased SOC during cover crop cultivation can be credited to a high nutrient extraction rate by the crop without any external input of fertilizer.In our experiment, the increased substrate availability from maize cultivation leads to higher microbial activity which lowers the SOC content during dhaincha cultivation i.e. increased C mineralization.Similar results of reduction in SOC pool by cover crops grown for a short time was also reported by other researchers (Lal, 2004).The results from our experiment suggested that this cropping sequence (maize-dhaincha) maintains the C dynamics by avoiding C saturation in the soil.Thus, the soil can act as a sink of CO 2 for a relatively longer period of time.The positive influence of N fertilization on SOC sequestration was also reported by other researchers (Guzman et al., 2006;Lee et al., 2009).
Different lower case letters indicate significant differences between treatments at the 5% level of significance according to DMRT.
The response of MBC to N fertilization was inconsistent throughout the experiment.Such inconsistency in MBC under different levels of N fertilizer has also been reported earlier (Lou et al., 2011).However, the positive impact of N fertilization on MBC was prominent at the end of maize harvest.The highest significant MBC was obtained in T 2 (P=0.00).Compared to maize, dhaincha cultivation recorded lower soil MBC content in all the plots.This is due to higher root biomass (Table 3) and availability of soil N (Figure 3) for microbial utilization that provided a good substrate for microbial growth and activity.This contradicts the findings of Jia et al. (2010) while working with Larix gmelinii and Fraxinusm andshurica plantations.The reduction of MBC content can be attributed to the changes of soil nutrient properties (N, P, K and SOC) and pH (Yu et al., 2013).

Physical properties
Soil bulk density decreased with increasing levels of N fertilizers (Figure 2a).This decrease in BD was more pronounced under maize cultivation compared to dhaincha.Nitrogen of 100 kg ha -1 (T 2 ) recorded the highest decline in BD followed by 80 and 72 kg ha -1 (T 3 and T 4 ), respectively.BD showed a negative correlation with root biomass (r for maize=0.912;dhaincha=0.942) and SOC (r for maize=0.983;dhaincha=0.674).The reduction of BD was due to greater biomass production (Table 3) and return of residues to the soil under N fertilization.This is in agreement with the findings of Hati et al. (2007).Another reason for the reduction of BD could be due to application of more inorganic N fertilizer which increases SOC and subsequently enhances microbial and faunal activity resulting in improvement of soil porosity (Haynes and Naidu, 1998).This in turn also improved the WHC.
Improved WHC was observed in T 2 with higher dose of N fertilizer up to 100 kg ha -1 (Figure 2b).This rise in WHC was more prominent during maize cultivation than that of cover crop, dhaincha.More considerable increase of WHC in maize than in dhaincha can be ascribed to surface application of fertilizers which resulted in more root biomass in maize than in dhaincha.However, at the end of dhaincha cultivation, T 2 recorded the highest value of WHC while the least increase was observed in control (T 1 ).WHC showed a positive correlation with SOC (r for maize=0.929;dhaincha=0.688).A significant increase of WHC due to application of farmyard manure and inorganic fertilizers was reported by Rasool et al. (2008) in maize and wheat crops.
Different lower case letters indicate significant differences between treatments at the 5% level of significance according to DMRT.

Changes in soil chemical properties
In all the treatments, soil pH decreased with increasing doses of N fertilizer (Table 4).Treatment T 2 recorded the lowest pH (6.09) while T 1 and T 6 maintained the highest pH (6.14) at the end of two years (P=0.00).Such decrease was significant in the fertilized plots compared to control plots.This soil acidification was found to be continued even after the harvest of dhaincha (Table 4).The stimulating effect of urea in the process of nitrification under subtropical conditions leads to soil acidification (Darilek et al., 2009).This acidifying effect of N fertilizer has already been demonstrated by other researchers (Hati et al., 2007;Guzman et al., 2006;Miglierina et al., 2000).Interestingly, the correlation data suggested an inverse relation between pH and SOC (r for maize=0.938;dhaincha=0.725).
More available soil N was found with the application of higher N fertilizer while in control available N decreased over time (Figure 3).The highest accumulation of available N was observed in T 2 and T 3 (Figure 3) (P=0.00).Our results are in agreement with the findings of other researchers (Jagadamma et al., 2008;Miglierina et al., 2000).Similar to SOC, available N decreased during dhaincha cultivation.The sluggish N uptake by maize plants during maturity resulted in accumulation of N in the upper soil layer (Peng et al., 2010).On the other hand in dhaincha, increased uptake of available N with no external application of fertilizer resulted in reduction of available soil N concentration.Nitrogen availability showed a positive correlation with SOC (r=0.974) and an inverse correlation with soil pH (r=0.971) during maize cultivation, however, this relation was weaker for dhaincha.Availability of P and K increased in all the treatments at the end of two years (Figure 3).The highest available P was found in T 3 while T 1 recorded the lowest.A decreasing level of available P was noticed after the harvest of maize, i.e. during dhaincha cultivation in all the treatments except in T 1 .However, we observed a significant increase in available soil K content in treatment T 4 followed by T 2 .After the harvest of maize, an increase in K content was recorded in all the treatments except in T 5 .Lower uptake of K by maize relative to other crops resulted in accumulation of K. Another reason of surface accumulation of K was probably due to removal of K from lower depths and decomposition of crop residue at the soil surface (Guzman et al., 2006).The observed variation in availability of both P and K under different levels of N fertilizer might be due to its effect on microbial activity and population leading to reduced availability of these nutrients.Positive responses of available P to basal application of N and P fertilizers have also been reported by Miglierina et al. (2000).High levels of N fertilization disturbed the N: P: K balance of soil.Edmeades (2003) reported that imbalanced fertilization of P and K confines the supply of these nutrients that hampers the uptake for crop growth and finally yield of maize.

Plant parameters and yield
Crop yield was the highest in treatment T 2 followed by T 3 (Table 5) and T 1 recorded the lowest yield (P=0.003).Improvement in soil physical health (Figure 2a and 2b) and nutrient availability status (Figure 3) might be the cause of increased yield under higher levels of N fertilization.A highly significant positive correlation of yield was observed with SOC (r=0.889) and available N (r=0.933).However, soil pH showed a significant negative correlation with yield (r=0.986).Irrespective of treatments, an increase in root biomass was observed throughout the crop growing season (Table 3).Nitrogen fertilization had a substantial role in increasing the root biomass.This can be explained by the direct role of N fertilizer in plant growth and development and indirectly by improving the soil health.The strong positive correlation between below-ground biomass (root biomass) and available N (r for maize=0.905;dhaincha=0.641) also supports this.However, in the long run this positive influence of higher N fertilizer on improvement of maize yield and biomass production will not continue.The increased N fertilization might result in higher availability of residual N in soil (Figure 3) and this subsequently will increase the risk of nitrogen loss by leaching and nitrification leading to soil acidity (Malhi and Lemke, 2007).

Sensitivity index of carbon fractions
Fertilizer treatments and crop have a significant impact on the SI of various fractions of SOC (Figure 4).During dhaincha cultivation, lower sensitivity of SOC fractions was observed compared to maize.This is due to application of fertilizer during maize cultivation only.The fractions were more sensitive to the high fertilizer doses.The SI of POC was the highest followed by MBC.This showed that these labile fractions are more sensitive to management practices.Hence, monitoring of these fractions serves as a better way to understand the SOC dynamics in relation to management practices such as fertilizer treatments.Yan et al. (2007) also reported POC and MBC to be more sensitive indicators of SOC changes in the tested soil induced by nutrient management.

Conclusion
Results of the present experiment showed that SOC balance is maintained to a certain degree in maize-dhaincha cropping sequence.Increased nitrogenous fertilization of 100 kg ha -1 resulted in higher yield with improved soil C stock as well as physical health, but in the long term, this fertilization rate will stimulate soil

Figure 1 .
Figure 1.Impact of various levels of N fertilization on SOC content.

Figure 2 .
Figure 2. Impact of various levels of N fertilization on (a) bulk density, (b) water holding capacity.

Figure 3 .
Figure 3. Impact of various levels of N fertilization on nutrient availability (N, P and K) under different crops.

Figure 4 .
Figure 4. Impact of various levels of N fertilization on sensitivity indices of various C fractions.

Table 1 .
General characteristics of basic soil.

Table 2 .
Changes in fractions of organic carbon under different levels of N fertilization.

Table 3 .
Changes in root biomass (g plant -1 ) under different levels of N fertilization during the maize growing season.

Table 4 .
Changes in pH under different N levels.

Table 5 .
Maize yield under different N levels.