Soil biological properties as influenced by long term manuring and fertilization under finger millet - maize cropping system in Alfisols

Introduction

Agriculture intensification with extensive use of chemical fertilizers has detrimental consequences on soils and the ecosystem, and poses problem on long-term sustainability. Overuse of fertilizers not only reduces fertilizer efficiency, but also causes environmental deterioration due to nutrient runoff and biodiversity loss (Zhang et al., 2012) [42] . Mineral fertilizer application lowers soil pH, which may affect nutrient availability by decreasing the microbial activities (Cai et al., 2015) [6] . Mineral fertilization was having negative impact on the bacterial and fungal community by reducing microbial diversity (Zhang et al., 2012) [42] . This is due to mineral fertilizers hasten the mineralization of soil organic carbon (SOC), their continued usage might deplete SOC which is the source of food for microorganisms (Ju et al., 2009). Mineral fertilizer can be replaced with manure as an alternative to mineral fertilizer application, which can improve soil biodiversity and quality (Jiang et al., 2018) [21] . The soil respiration and enzyme activity play major role in maintenance of productive soil. Soil microorganisms are the primary decomposers of organic matter, resulting in the formation of humus and thus the release of a various nutrients. One of the most important measures of soil quality is the microbial population. Soil microorganisms also help to improve the efficiency of chemical fertilizers by affecting the nutrient cycle mechanisms in the soil. The application of inorganic fertilizers, manures, irrigation, insect control techniques, and other factors affect soil biological parameters quickly. The simple, economical, and quicker way to determine soil biological and biochemical parameters related to organic matter decomposition and nutrient cycling, such as soil microbial population, microbial biomass C and N, and soil enzyme activity. The activity of dehydrogenase can be used as an excellent predictor of soil quality. As respiratory chain enzymes, dehydrogenases play an important role in the energy production of soil microbes. The addition of organic matter to the soil boosted the activity of soil dehydrogenase (Singh et al., 2007) [31] . Phosphatase plays an important function in phosphorus cycle, including phospholipid breakdown. The addition of organic matter to the soil boosted the activity of the phosphatase enzyme (Singh et al., 2007) [31] . Urease enzyme took part in the hydrolysis of urea to carbon dioxide and ammonia in soil, and it is a key part of the nitrogen cycle. The maximum urease enzyme activity was observed when NPK was combined with manure application (Bhatt et al., 2016) [4] . The activities of soil enzymes were positively associated with organic C and microbial population in the soil by Chhonkar and Tarafdar (1981) [11] . Nidaye and Dkhar (2000) reported soil enzyme activity has the potential to help in manage the ecosystems for long-term viability.

Abbasi and Khizar (2012) [1] reported that application of urea resulted in a drop in MBC, whereas the use of farmyard manure as a soil amendment resulted in an increase in MBC. Microbial diversity, microbial biomass, and enzyme activity are improved by the application of manure by increase in SOM (Lupwayi et al., 2019) [27] which involved in C, P, and N cycle. Improper nutrient management strategies, are one of the causes of increased soil degradation due to a decrease in SOC. These biological properties of soil have been extensively employed to investigate the effects of various soil and crop management strategies on soil health in a variety of habitats (Nannipieri et al., 1990; Aon et al., 2001; Masto et al., 2007; Trasar-Cepeda et al., 2008) [34,3,31,16] . Long term studies are useful for determining the effects of continued use of nutrient management strategies in crop production on soil productivity and quality of soil. Because soil biological and biochemical parameters are very sensitive to changes in land management methods, they are regarded valid indicators of soil quality (Gil-Sotres et al., 2005) [16] . As a result, keeping track of changes in diverse soil biological properties could lead to a better understanding of nutrient availability and soil quality. Therefore, the present investigation was undertaken to examine the effect of long term manuring and fertilization on soil biological properties under finger millet-maize cropping system in Alfisols.

Materials and Methods Experimental site and treatment details

The In a randomised block design with individual plot sizes of 16 m × 9 m, the experiment was carried out having eleven treatments which are replicated four times. However, for the present study only three replications and one more additional treatment of fallow land were considered i.e. 50% NPK (T 1 ), 100% NPK (T 2 ), 150% NPK (T 3 ), 100% NPK + Hand weeding (T 4 ), 100% NPK + Lime (T 5 ), 100% NP (T 6 ), 100% N (T 7 ), 100% NPK + FYM (T 8 ), 100% NPK (-S) (T 9 ), 100% NPK + FYM + Lime (T 10 ), Control (T 11 ), Uncultivated land (T 12 ). The treatment details with NPK doses and fertilizer sources are given in Table 2. The recommended dose of NPK used in finger millet was 100: 22: 42 kg ha -1 and maize 100: 33: 82 kg ha -1 . All eleven treatments employed chemical weed management, with the exception of 100% NPK + HW. About 15 t ha -1 of well decomposed farmyard manure (FYM) on dry weight basis was mixed into the soil 10-15 days just before the transplanting of finger millet crop to treatments T 8 and T 10 .

Lime has been applied based on lime requirement following the method given by Shoemaker et.al. (1961) at 15 days before sowing of finger millet crop to the treatments T 5 and T 10 . However, if the soil pH exceeds 6.00 then lime was applied @ 500 kg ha -1 . After 25 to 30 days of sowing / transplanting of crops, 50 percent of the recommended nitrogen dose, 100 percent of the recommended P and K doses were applied as a basal, and the remaining 50 percent nitrogen dose was administered in two equal splits as a top dress. Diammonium phosphate (DAP) is applied as a source of P and N along with urea and muriate of potash (MOP) as a source of K in sulphur free treatment [T 9 ]. The single superphosphate was not used to avoid S for treatment T 9 . In rest of the treatments (except T 9 ), urea, single super phosphate (SSP) and MOP are used as sources of NPK, respectively. Neither any chemical fertilizer nor any organic manure is used in absolute control treatment (T 11 ). 00 -00 -00 00 -00 -00 ----------T12: Fallow land 00 -00 -00 00 -00 -00 ----------

Soil sampling, processing and analysis

After the maize crop was harvested, composite soil samples were collected from each plot using a soil tube auger, taken at two depths of 0-15 and 15-30 cm. Soil samples for the treatment fallow land were collected in three places near experimental plots that had not been cultivated. The collected soil samples were air-dried, ground to pass through 2 mm sieve and were subjected for analysis of biological properties of the soil. Soil microbial biomass carbon (SMBC) and nitrogen (SMBN) was estimated by fumigation-extraction method as proposed by Carter (1991) in which Ninhydrin-reactive N, as a measure of microbial biomass carbon released during soil fumigation by using ninhydrin reagent. The dehydrogenase activity in the soil was determined by the reduction of 2,3,5-triphenyltetrazolium chloride to 1,3,5-triphenylformazan (TPF) given by Casida et al. (1964) [9] . The acid phosphatase enzyme activities were determined as per the method given by Eivazi and Tabatabai, 1977 [14] using p-nitrophenyl phosphate tetrahydrate solution of pH 6.5. The urease activity was determined by following the method described by Watts and Crisp (1954) by using trishydroxymethylaminometane (THAM) buffer.

Statistical analysis

For the interpretation of results, the data obtained in the laboratory studies will be subjected to statistical analysis using the technique of analysis of variance for randomised block design as outlined by Gomez and Gomez (1984). The level of significance used in "F" and "t" test was P = 0.05.

Critical difference (CD) values were calculated for the P = 0.05 whenever "F" test was found significant.

Results and Discussion

Dehydrogenase activity

The dehydrogenase activity among different fertilizer treatments at different depths varied significantly due to long term application of inorganic fertilizers and organic manures in finger millet-maize cropping sequence over 33 years of cultivation is presented in Table 3. Among different treatments uncultivated land recorded significantly higher dehydrogenase activity (80.43 and 39.04 μg TPF g-1 24 h -1 at 0-15 and 15-30 cm soil depth respectively) followed by 100% NPK + FYM + Lime (72.71 and 34.55 μg TPF g-1 24

h -1 at 0-15 and 15-30 cm soil depth respectively) and 100% NPK + FYM (67.81 and 31.96 μg TPF g-1 24 h -1 at 0-15 and 15-30 cm soil depth respectively) compared to control and only inorganic fertilizer treated plots. This would imply that the activity of dehydrogenase had a direct relationship with the organic matter content of the soil. The significantly lower dehydrogenase activity was recorded in treatments with imbalanced application of fertilizer 100% N (32.96 and 14.37 μg TPF g-1 24 h -1 at 0-15 and 15-30 cm soil depth respectively), followed by 100% NP (43.10 and 21.64 μg TPF g-1 24 h -1 at 0-15 and 15-30 cm soil depth respectively) and control (40.39 and 18.69 μg TPF g-1 24 h -1 at 0-15 and 15-30 cm soil depth respectively). Application of lime 100% NPK + Lime (59.50 and 27.24 μg TPF g-1 24 h -1 at 0-15 and 15-30 cm soil depth respectively) and 100% NPK + FYM + Lime (72.71 and 34.55 μg TPF g-1 24 h -1 at 0-15 and 15-30 cm soil depth respectively) increases the dehydrogenase activity compared to without lime treatment 100% NPK (56.49 and 24.06 μg TPF g-1 24 h -1 at 0-15 and 15-30 cm soil depth respectively) and 100% NPK + FYM (67.81 and 31.96 μg TPF g-1 24 h -1 at 0-15 and 15-30 cm soil depth respectively). Dehydrogenase activity is higher in no-till soil due to presence of higher organic matter, less disturbance to microbes than ploughed soil. FYM alone or in combination with inorganic fertilizers resulted in a considerable increase in dehydrogenase activity due to that the addition of an organic source, microorganisms utilize the native pool of organic carbon as a source of carbon, which acts as substrate for dehydrogenase (Rai and Yadav, 2011 and Nath et al., 2012) [35] . The decrease in enzymatic activities in sub surface layer of soil could be attributed to the lower organic matter content with increase in soil depth (Martens et al., 1992) [29] . The application of fertilizers affects the microbial activity in turn reduces the enzyme activity (Kandeler et al., 1999) [23] . When nitrogenous fertilizers are applied to soil alone, dehydrogenase activity, which is a measure of soil microbial activity, is greatly influenced by the presence of nitrate, which serves as an alternative electron acceptor, resulting in low activities (Casida et al., 1964) [9] . This could be reason for the dehydrogenase activity in the soils of the alone N treated treatment was the lower. Application of lime, in combination with a balanced NPK, promoted the activity of microorganisms to use the local pool of organic carbon as a source of carbon, which functions as a substrate for dehydrogenase, and also liming increases pH, which is advantageous to microbiological growth.

Acid phosphatase activity

Perusal of the data on acid phosphatase activity (Table 3) revealed that among different treatments significantly higher acid phosphatase activity was recorded in uncultivated land (121.14 and 49.90 µg of PNP g -1 of soil h -1 at 0-15 and 15-30 cm soil depth respectively) followed at par with 100% NPK + FYM (110.23 and 47.80 µg of PNP g -1 of soil h -1 at 0-15 and 15-30 cm soil depth respectively). The significantly lower acid phosphatase activity was recorded in control (80.93 and 32.60 µg of PNP g -1 of soil h -1 at 0-15 and 15-30 cm soil depth respectively) followed by 100% N (89.72 and 34.91 µg of PNP g -1 of soil h -1 at 0-15 and 15-30 cm soil depth respectively) and 100% NP (90.13 and 36.94 µg of PNP g -1 of soil h -1 at 0-15 and 15-30 cm soil depth respectively). Application of lime decreased the acid phosphatase activity non significantly both in 100% NPK + Lime (90.24 and 38.38 µg of PNP g -1 of soil h -1 at 0-15 and 15-30 cm soil depth respectively) and 100% NPK+FYM + Lime (99.85 and 45.17 µg of PNP g -1 of soil h -1 at 0-15 and 15-30 cm soil depth respectively) when compared to respective treatments without lime 100% NPK (94.37 and 42.86 µg of PNP g -1 of soil h -1 at 0-15 and 15-30 cm soil depth respectively) and 100% NPK + FYM (110.23 and 47.80 µg of PNP g -1 of soil h -1 at 0-15 and 15-30 cm soil depth respectively). The activities of acid phosphatase enzyme was more in uncultivated land when compared to cultivated land due to higher organic matter availability as a result of less disturbance to the microbial activity in turn increases the phosphatase activity. Similar results were reported by Dick and Tabatabai (1984) [13] . The increased acid phosphatase activity in combined application fertilizers with organic manure treatment than others is due to increased availability of substrates like organic phosphorous, organic carbon, and microbial biomass as a result of continuous application FYM over the years (Singh et al., 2007) [31] . Mishra et al.

(2008) [32] , while analysing the results of a long-term study in Alfisols, also reported low activities of the phosphatases enzymes in soil due to imbalanced use of chemical fertilizers which affect microbial growth. The application of lime reduced the acid phosphatase activity in soils of both soil depth which was associated with increase in bacteria but decreases the number of fungi. Similar results are reported by Halstead et al., 1963. Casida (1959) [19,8] reported that fungi produce enzyme capable of dephosphorylating organic phosphorous compounds in soil.

Urease activity

The urease activity among different fertilizer treatments at different depths varied significantly due to long term application of inorganic fertilizers and organic manures in finger millet-maize cropping sequence over 33 years of cultivation is presented in Table 3 and Fig. 3. Among different treatments uncultivated land recorded significantly higher urease activity (42.32 and 21.09 µg of NH 4 + -N g -1 of soil h -1 at 0-15 and 15-30 cm soil depth respectively) followed by 100% NPK + FYM + Lime (37.58 and 19.10 µg of NH 4 + -N g -1 of soil h -1 at 0-15 and 15-30 cm soil depth respectively) and 100% NPK + FYM (34.46 and 15.81 µg of NH 4 + -N g -1 of soil h -1 at 0-15 and 15-30 cm soil depth respectively) compared to control and all inorganic fertilizer alone treated plots. The significantly lower urease activity was recorded in 100% N (26.95 and 10.91 µg of NH 4 + -N g -1 of soil h -1 at 0-15 and 15-30 cm soil depth respectively) followed by control (27.66 and 11.09 µg of NH 4 + -N g -1 of soil h -1 at 0-15 and 15-30 cm soil depth respectively). Application of lime increased the urease activity non significantly both in 100% NPK + Lime (31.40 and 15.31 µg of NH 4 + -N g -1 of soil h -1 at 0-15 and 15-30 cm soil depth respectively) and 100% NPK + FYM + Lime (37.58 and 19.10 µg of NH 4 + -N g -1 of soil h -1 at 0-15 and 15-30 cm soil depth respectively) when compared to corresponding treatments without lime 100% NPK (29.96 and 13.93 µg of NH 4 + -N g -1 of soil h -1 at 0-15 and 15-30 cm soil depth respectively) and 100% NPK + FYM (34.46 and 15.81 µg of NH 4 + -N g -1 of soil h -1 at 0-15 and 15-30 cm soil depth respectively). Significantly higher urease activity in uncultivated compare to cultivated land was due to higher organic matter availability and less disturbance in uncultivated land, which promotes microbial activity, which in turn raises urease activity. The urease enzyme activity was significantly and positively correlated with organic C and microbial population in the soil. The highest urease activity in manure treated soil over control and decrease with long-term addition of minerals (Dick et al., 1988) [12] . Significantly lower urease activity observed in imbalanced fertilizer application due to the lack of sufficient amount of organic substrate required for microbes to carry out urease activity in fertilizer treated soil (Islam et al., 2019) [20] . Application of lime along with fertilizers increases the urease activity by promoting microbial activities in soil.

Microbial biomass carbon

The data on microbial biomass carbon (MBC) among different fertilizer treatments and at different depths varied significantly due to long term application of inorganic fertilizers and organic manures in finger millet-maize cropping sequence over 33 years of cultivation and is presented in Table 4. Among different treatments microbial biomass carbon was significantly higher in treatment uncultivated land (324.76 and 231.85 mg kg -1 at 0-15 and 15-30 cm soil depth respectively) followed 100% NPK + FYM + Lime (286.53 and 215.94 mg kg -1 at 0-15 and 15-30 cm soil depth respectively) and 100% NPK + FYM (262.16 and 201.53 mg kg -1 at 0-15 and 15-30 cm soil depth respectively). The significantly lower content of microbial biomass carbon was recorded in control (195.92 and 140.72 mg kg -1 at 0-15 and 15-30 cm soil depth respectively) and found at par 100% N (205.18 and 151.57 mg kg -1 at 0-15 and 15-30 cm soil depth respectively). The significant decline in MBC in cultivated soils than uncultivated soil is due to agricultural use. The disturbance caused by cultivation changes the size, composition, and activity of the microbial community (Angers et al., 1993 [2] , Fauci and Dick 1994 [15] and Granatstein et al., 1987 [17] . Uncultivated soil, on the other hand, has less organic matter degradation, which increases organic carbon and act as source of food for microorganisms in tern promotes microbial activity. The higher MBC value due to the combined application of FYM and NPK fertilizer is attributable to a higher turnover of root biomass produced under the FYM + NPK treatment (Moharana et al., 2012) [33] . These findings are also consistent with those of Grego et al. (1998) [18] . Higher stress caused by insufficient or no nutrient utilization in control and unbalanced fertilization limited the crop output and hence carbon substrate (root exudates), resulting in a loss in biomass carbon. The MBC of the surface layer was much greater than that of subsurface soil. The downward migration of soluble OM from top soil, is critical for maintaining subsoil microbial activity which in turn sustains microbial biomass carbon. These findings are also consistent with (Chantigny, 2003) [10] . [28] , who found that combining inorganic and organic fertilizers had a substantial impact in increasing MBN (Kumari et al., 2011) [24] . Imbalanced fertilization limited the crop production and hence nitrogen substrate (root exudates), resulting in a loss in biomass nitrogen.

Microbial biomass nitrogen:

Conclusion

The present study demonstrates that in comparison to using NPK fertilizers alone, using an optimal dose of NPK (100%) combined with FYM in a finger millet-maize cropping system improved soil microbial status at both depths. This treatment increased the biomass of microorganisms and the activity of several enzymes in the soil, both of which are significant in nutrient cycling. All enzyme activities (dehydrogenase, urease activity and acid phosphatase) and microbial biomass carbon and nitrogen were recorded more in the uncultivated land compared cultivated land followed by treatment receiving 100% NPK + FYM + Lime and 100% NPK + FYM, however the continuous cropping without application of manure and imbalanced fertilizer application recorded lowest biological activities. The soil health gets deteriorated due to continuous cultivation without application of fertilizers. With the depth of the soil, all of the soil's biological properties decreased. It may thus be inferred that the balanced application of organic manure and chemical fertilizers, as well as effective weed management, is critical for maintaining finger millet-maize system production and soil biological health in Alfisols.

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