Microbial dynamics as influenced by concentrate manure and inorganic fertilizer in alluvium soil of Varanasi , India

A field experiment was conducted during rabi season, 2009-2010 at Agricultural Research Farm, BHU, Varanasi, on alluvial soils to determine the effects of concentrate organic manure (wellgrow formulations) with levels of inorganic fertilizers. The results reveal that the higher soil microbial population was seen with the application of 100% NPK + 300 kg wellgrow soil ha -1 . Soil enzymes varied with the production systems. The urease, phosphatase and dehydrogenase activities were higher in wellgrow dose application with recommended dose of NPK. The soil enzymes and microbial population (bacteria, fungi and actinomycetes) were very responsive to organic manure application, but their levels and activities were not reflected in wheat crop under alluvial soils. Enzymatic activities were positively and significantly correlated with content of organic carbon.


INTRODUCTION
The role that microbial activity play in ecosystem processes is significant because approximately 80 to 90% of soil processes are mediated by microorganisms (Nannipieri and Badalucco, 2003).Soil microbial population are the driving force that regulate soil processes such as organic matter decomposition and nutrient cycling, it is imperative to have a better understanding of the factors that regulate its size, activity and structure (Masto et al., 2006).
Soils containing a high microbial diversity are characteristic of a healthy soil-plant relationship, whereas those with low microbial diversity are characterized as an unhealthy soil that often hardly responds to environ-mental changes (Tejada et al., 2011).Soil enzymatic activities can be used as an index of soil fertility and microbial functional diversity (Nannipieri et al., 2002;Maurya et al., 2011) in catalyzing several biochemical reactions which are necessary for the life processes of soil micro-organisms, organic wastes decomposition, organic matter formation and nutrients cycling (Tabatabai, 1994).
The microbial population dynamics is governed by interactions between plant type, climate and management practices.In addition, the soil microbial biomass in soil system responds more quickly to management practices than organic matter and is often used as an indi-cator of soil quality and health (Ge et al., 2010).The addition of organics and management practices greatly influence the microbial populations which is expected to cause changes in the soil enzymatic activities.
Incorporation of organic manures influenced soil enzymatic activity either because of the composition of the added materials themselves or because they increased micro-bial activity of the soil (DeForest et al., 2012).Improve-ment of the soil structure due to FYM application leads to a better environment for root development (Dejene and Lemlem, 2012) .FYM also improves soil water holding capacity ( Tadesse et al., 2013).The fact that the use of organic fertilizers maintains soil health has raised interests in organic farming ( Khan et al., 2010).The microbiological and biochemical conditions of a soil can serve as a marker of the soil status and is closely linked to its natural soil fertility.
Addition of the organic fraction stimulates the natural soil micro organisms and reac-tivates the biogeochemical cycles (Watts et al., 2010).Microbial population can also be increased through rhizosphere inoculation of bioagents.Soil enzymatic activity is responsible for farming stable organic molecules that contributes to the permanence of the soil ecosystem.Urease and phosphatase are two important enzymes involved in the N and P cycles, respectively (Badiane et al., 2001).Combined use of organic manures improved the microbial load of the soil rather than single organic manure application (Krishnakumar et al., 2005).The objective of this study was to evaluate the microbial population and enzymatic activities as influenced by manure and inorganic fertilizer in alluvium soil.

MATERIALS AND METHODS
Wellgrow is a plant product formulation in grain and in powder forms produced by an Indian Tobacco Company (ITC).In the case of wellgrow soil (certified organic input), organic manure (powder) from plant products with better nutritional value from non-timber forest product enhances efficiency of nitrogenous fertilizers and acts as a good nutritional media for the growth of bio-fertilizers and bio-pesticides to increase their performance (Table 1).Wellgrow grain is pelleted organic manure.It is also a certified organic input from plant products originating from non-timber forest product enhancing efficiency of nitrogenous fertilizers, improving soil fertility and crop vigor, especially targeted towards cereal crop (Meena et al., 2013)

Site description and field experiment
This study was conducted at the Agricultural Research Farm, Institute of Agricultural Sciences, BHU, Varanasi (25° 18  N latitude, 83° 03 E longitude and 128.93 m above MSL) The weekly mean maximum and minimum temperature during the experimentation ranged from 15.1 to 42.3°C and 7.1 to 29.7°C, respectively.Soil samples were collected from the experimental field and analyzed for phyco-chemical and biological properties.Some of the initial soil properties (0 to 15 cm) are present in Table 2.

Soil sampling for chemical and microbial analysis and preparation
Initial soil samples were collected in August 2009 prior to the start of the experiment.After harvesting of rice, soil samples were taken from the surface layer (0 to 15 cm) of nine treatments with three replications, second soil sampling at the time of flowering stage of wheat and third soil sampling was done after the harvest of wheat crop in May, 2010.The moist soil samples were sieved (2 mm) after removing plant material and roots.Half of the soil samples were airdried and stored at room temperature until chemical analysis.All chemical results are means of triplicate analyses and are expressed on an oven-dry basis.The rest of the sieved soil (2 mm) was immediately transferred to the laboratory for microbiological analysis.Soil samples were kept at 4°C in plastic bags for a few days to stabilize the microbiological activity disturbed during soil sampling and handling, and then analysed.

Microbiological analyses
Total bacteria, fungi and actinomycetes were estimated by the serial dilution and plating technique as described by Rolf and Bakken (1987).Total bacterial counts specific medium [1.0 g Dipotassium phosphate (K2HPO4), 0.2 g magnesium sulfate (MgSO4), 0.1 g calcium chloride (CaCl2), 0.1 g sodium chloride (NaCl), trace amount of ferric chloride (FeCl3), 0.5 g potassium nitrate (KNO3), 1.0 g aspargine, 0.1 g mannitol, 15 g agar-agar and 1000 mL distilled water], actinomycetes specific medium [1.0 g Dextrose, 0.10 g Di-potassium phosphate (K2HPO4), 0.10 g sodium nitrate, 0.10 g magnesium sulfate (MgSO4), 15 g agar and 1000 mL distilled water] and fungal specific medium [10 g glucose, 5.0 g peptone, 1.0 g di-potassium phosphate (K2HPO4), 0.5 g magnesium sulfate (MgSO4), 15 g agar and 1000 mL distilled water], streptomycin @ 30 mg mL -1 was added to melted medium after it has cooled to 50°C, then prepared and plated on the glass Petri dish.0.1 mL from 10 1 to 10 5 dilutions was poured on the surface of Petri dish with the help of a micropipette and was evenly spread with the help of a sterile spreader and incubated at 30±2°C.Colony count was carried out daily up to 10 days.
Alkaline phosphatase activity was assayed using 1 g of soil (wet equivalent), 4 mL of 0.1 M modified universal buffer (pH 11 for alkaline phosphatase) and 1 mL of 25 m M p-nitrophenyl phosphate (Tabatabai and Bremner, 1969).After incubation for 1 h at 37 ± 1°C, the enzyme reaction was stopped by adding 4 mL of 0.5 M NaOH and 1 mL of 0.5 M CaCl2 to prevent dispersion of humic substances.After centrifugation at 4000 rpm for 10 min, the absorbance was measured in the supernatant at 400 nm; enzyme activity was expressed as mg p-nitrophenol g -1 soil h -1 .

Statistical analysis
Data were assessed by Duncan's multiple range tests (Duncan, 1955) with a probability, P = 0.05.Least significant difference (LSD) between the mean values was evaluated by a one-way analysis of variance by using SPSS version 10.0.

Microbial population of bacteria, fungi and actinomycetes
Table 3 indicates that in the experiment, the microbial population was significantly increased in all the treatments.In the data of bacterial population before sowing of wheat crop, maximum bacterial population (58 × 10 5 g -1 soil) was observed with 100% NPK with + 200 kg wellgrow grain ha -1 , it may be due to the residual effect of rice crop at flowering stage of wheat, bacterial population was significantly influenced by wellgrow formulation with doses of inorganic fertilization and significant increase in population of bacteria over all other treatments (viz.control, 50, 75% NPK with wellgrow levels).Maximum population of bacteria was recorded with 100% NPK + 300 kg wellgrow grain/soil ha -1 . At harvest, lower bacterial population was observed in comparison with flowering stage of wheat, but at harvest, the maximum bacterial population was also observed in T 9 (68 × 10 5 g -1 soil) treatment (Table 3).This treatment significantly increased over all the treatments except T 8 (66 × 10 5 g -1 soil), at harvest decreasing bacterial population which was due to decrease in organic carbon.This finding is in accordance with the finding of Watts et al. (2010).This clearly revealed that organic material significantly increases the bacterial population.The soil microbial population in 100% NPK +300 kg wellgrow grain ha -1 , soil microbial biomass has been used as an index of soil fertility which depends on nutrient fluxes (Krishnakumar et al., 2005;Meena et al., 2013).
Fungi population increased with advancement of growth stages of crop with all treatments.Before sowing of wheat crop maximum fungal population was observed (50 × 10 4 g -1 soils).At flowering stage of wheat, highest population of fungi was registered (70 × 10 4 g -1 soil) with 100% NPK + 300 kg wellgrow soil ha -1 followed by 100% NPK + 300 kg wellgrow grain ha -1 (67 × 10 4 g -1 soil) (Table 3).Maximum fungal population was observed at flowering stage as compared to at harvest of wheat.Application of 100% NPK + 300 kg wellgrow soil ha -1 significantly increased in terms of percent over control which was 33%.At harvest, fungal population was maximum in T 9 (58 × 10 4 g -1 soil) which showed significant superiority over all the treatments and at par with T 8 (100% NPK + 300 kg wellgrow soil ha -1 ).Fungal population decreased at harvest due to lack of availability of nutrients and organic matter as compared to flowering stage of wheat.Fungi population increased with advancement growth stages of crop.It might be possible that increased total root biomass with the passage of time, might be instrumental to supporting higher fungi population.Similar results have been recently described in a study on wheat, in which total bacteria and fungi were evaluated (Vujanovic et al., 2012;Nedunchezhiyan et al., 2013).
registered (62 × 10 4 g -1 soil) with 100% NPK + 300 kg wellgrow grain/soil formulations.This is consistent with the finding of Bohme et al. ( 2005) who reported that microbial biomass was greater in soil after the application of farmyard manure.

Soil enzymatic activities
Soil enzyme activity is an indirect indication of the activities of microbes which is directly correlated with soil microbial dynamics.Enzyme activity in the soil environment is considered to be a major contributor of overall soil microbial activity (Burns et al., 2013).
In the present investigation, significantly increased enzyme activity of urease, dehydrogenase and alkaline phosphatase was noticed due to application of wellgrow soil, wellgrow grain and nutrient levels (Table 4).Due to the effects of external disturbance on their activity, enzymes can serve as sensitive indicators of soil quality (Dick et al., 1994;Nedunchezhiyan et al., 2013;Meena et al., 2013).

Urease activity
Urease is an important enzyme responsible for the hydrolysis of urea fertilizer applied to the soil, NH 3 and CO 2 with the concomitant rise in soil pH (Byrnes and Amberger, 1989).Before sowing of wheat crop, maximum urease activates registered 100% NPK + 200 kg wellgrow grain ha -1 (320 μg UH g -1 soil hr -1 ), this treatment was followed by 100% NPK + 300 kg wellgrow soil ha -1 and 100% NPK + 300 kg wellgrow grain ha -1 (306 and 316 μg UH g -1 soil hr -1 , respectively).At flowering stage of wheat, highest urease activities was registered with 100% NPK + 300 kg wellgrow soil (327 μg UH g -1 soil hr - 1 ) followed by 100% NPK + 300 kg wellgrow grain ha -1 (324 μg UH g -1 soil hr -1 ).This could be attributed to their higher N content and faster decomposition and release  of NH 4 -N (Saha et al., 2008;Meena et al., 2013) Soil enzymes regulate the transformation process of elements required for plant growth in soil (Burns, 1982).Crop growth stages also influenced the urease activity.Under field conditions, urease activity was highest at flowering stage but under greenhouse conditions, the activity was more pronounced at tillering stages (Watts et al., 2010).
At harvest, lower urease activity in comparison with at flowering stage of wheat, maximum urease activity was registered (264 μg UH g -1 soil hr -1 ) with treatment 100% NPK + 300 kg wellgrow soil ha -1 and at par with 100% NPK + 300 kg wellgrow grain soil ha -1 (261 μg UH g -1 soil hr -1 ), the lower activity of urease at harvest of crop could be related to lower microbial biomass and decreasing content of soil organic carbon.The correlation between urease and organic carbon at flowering and at harvest stages (r = 0.511 and r = 0.901, respectively) were positively significant (Table 5).Maestre et al. (2011) reported a decrease in the urease activity with addition of inorganic N whereas crop residues and organic manure additions increased it.Enzyme activities of soils are usually correlated with their organic carbon and available N contents (Ndubuisi-Nnaji et al., 2011).Higher levels oforganic carbon stimulate microbial activity, and therefore enzyme synthesis.

Dehydrogenase activity
Dehydrogenase is an enzyme that occurs in all intact viable microbial cells.These soil enzymes function as a measurement of the metabolic state of soil microorganisms by relating it to the presence of viable microorganisms and their oxidative capacity.Therefore, dehydrogenase can be used as a measure of microbial respiration and a reliable index of microbial activity in soil (Tejada et al., 2011).Data on dehydrogenase activities before sowing of crop maximum observed with 100% NPK + 300 kg wellgrow grain ha -1 (188 μg TPF g -1 soil day -1 ) followed by T 8 and T 7 may be due to higher organic matter content and relatively higher organic carbon (Wlodarczyk et al., 2002).Similar trend was also observed in flowering stage of wheat and maximum dehydrogenase activities was registered (313 μg TPF g -1 soil day -1 ) with 100% NPK + 300 kg wellgrow grain ha -1 at flowering stage of wheat.Crop growth stage also greatly impacted dehydrogenase activity.Dehydrogenase activity measured during flowering stage was almost double that measured before sowing of the crop (Table 4).The higher dehydrogenase activity after addition of concentrate manure could be due to increased microbial activity, which is known to stimulate the dehydrogenase activity (Watts et al., 2010).At harvest of wheat, lower urease activity was seen in comparison with at flowering stage of crop, highest dehydrogenase activity was registered as 252 μg TPF g -1 soil day -1 with treatment 100% NPK + 300 kg wellgrow soil ha -1 and it is at par with T 9 , T 7 and T 6 (225, 225 and 215 μg TPF g -1 soil day -1 respectively), the increase in activity during the flowering stage compared well with harvest stage, suggesting that greater microbial biomass occurred with a change in growth stage.These results suggest that changes in the size of microbial populations and respiratory activity occurred in response to the increase in available substrate.In addition, an increase in available substrate corresponds to more readily available C and N pools, which were most likely disproportionally enhanced as a result of manure addition.
This was confirmed by the significant positive correlation between dehydrogenase and organic carbon at flowering and harvest (r = 0.464, 0.873, respectively) (Table 5).In the present study, lowest dehydrogenase activity measured after harvest can be attributed to oxidation status of the soil as water was drained at maturity.

Alkaline phosphatase activity
Alkaline phosphatase is an enzyme of great agronomic value because it hydrolyses compounds of organic phosphorus and transforms them into different forms of inorganic phosphorus that are assimilated by plants (Maestre et al., 2011).Data on alkaline phosphatase tend to be lowest in the control treatment before sowing, flowering and at harvest of crop (Table 4), and highest (86 μg p-NP g -1 soil h -1 ) with the application of 100% NPK + 200 kg wellgrow grain ha -1 before sowing of crop.At flowering stage, maximum alkaline phosphatase activates (83 μg p-NP g -1 soil h -1 ) 100% NPK + 300 kg wellgrow grain ha -1 . Sriramachandrasekharan and Ravichandran (2011) reported that the addition of organic substances to the soil served as a carbon source that enhanced microbial biomass and phosphatase activity, showing that these enzymes are of microbiological origin and crop growth stage also significantly influenced soil enzyme activities (Bohem et al., 2005).
This hypothesis is also supported by the alkaline phosphatase activity and organic carbon highly significant and positively correlated at flowering and harvest stages (r = 0.635 and r = 0.919, respectively), at the 0.01 level (2-tailed).The importance of organic carbon in nutrient cycling was evident, the enzyme activity quantified in the present study showed positive correlation with organic carbon.This indicates that organic material significantly increases the enzymatic activity in soil (Table 5).Several studies have observed inverse relationships between inorganic P availability and phosphatase activity although this depends on initial bio-available P (DeForest et al., 2012).

Conclusions
Integrated use of organic manure (wellgrow) and inorganic fertilizers improved the enzymatic activities as well as microbial population of bacterial, fungal and actinomycetes with the best application at 100% NPK + 300 kg wellgrow grain/soil ha -1 .Decomposition of organic matter and recycling of carbon have substantial effect on the activity of enzyme evolved in mineralization of nutrients.Soil enzymes significantly contribute to soil health.The activities of urease, dehydrogenase and phosphatase were significantly influenced by the crop growth stages.Enzymatic activity increased up to flowering stage of wheat and declined thereafter.Hence judicious application of 100% NPK + 300 kg wellgrow grain/soil ha -1 emerged as the best treatment for both flowering and harvest stage for enzymatic activity as well as microbial population.Manure together with reduced dose of nutrient levels not only improved crop growth but also significantly buildup nutrient in the soil; it also maintained a balanced enzymatic activity with a lesser pollution potential then high dose of nutritional levels.

Table 1 .
Characteristics of wellgrow soil and grain.

Table 3 .
Soil microbial population (bacteria, fungi and actinomycetes) at different growth stages (cfu g -1 of soil) of wheat as influenced by concentrate manure and inorganic fertilizers.

Table 4 .
Soil enzymes activities [urease (UA) dehydrogenase (DHA) and alkaline phosphatase (APA)] at different growth stages of wheat in the study soils as influenced by concentrate manure and inorganic fertilizers.

Table 5 .
Correlations of organic carbon with enzyme activities in wheat at flowering and harvest of crop as influenced by concentrate organic manure and inorganic fertilization.