Sheoran et al.

BMC Plant Biology (2025) 25:117 BMC Plant Biology

https://doi.org/10.1186/s12870-025-06128-2

RESEARCH Open Access

Organic manure and fertilizer N management

strategies improve soil health at different
growth stages of pearl millet under pearl
millet-wheat sequence
Sunita Sheoran1†, Dhram Prakash1†, Dev Raj1†, Parmod Kumar Yadav1, Rameshwar Singh1, Rajeev Kumar Gupta2,3*,
Saud Alamri4, Manzer H. Siddiqui4 and Shahbaz Khan5

Abstract
Background Incorporating organic manure improves soil properties and crop productivity. A long-term study
started in October 1967 examined the effects of farmyard manure and nitrogen fertilization on the soil at key growth
stages of pearl millet in a pearl millet-wheat cropping system over its 51st cycle.
Results Applying 15 Mg of farmyard manure (FYM) per hectare in both growing seasons significantly boosted soil
organic carbon (SOC), dissolved organic carbon (DOC), and key nutrients compared to one-season application.
SOC levels reached 9.1–11.0% with FYM15, outperforming FYM10 and FYM5. The effectiveness order was
FYM15 > FYM10 > FYM5 > FYM0. Chemical nitrogen fertilization also enhanced soil properties, with FYM15 improving
DOC, available nitrogen, and enzyme activities during various growth stages.
Conclusions Principal component analysis (PCA) effectively distinguished soil variables and treatments, with
β-glucosidase and arylsulfatase activity (ARA) showing the highest loading (0.294) in the first principal component
(PC1), followed by dissolved organic carbon (DOC) at 0.292 and dehydrogenase activity (DHA) at 0.291. A strong
polynomial relationship was noted between soil organic carbon (SOC) and various soil properties, with R² values
between 0.93 and 0.99. The FYM15 x N120 treatment improved soil health in North-West India’s sandy loam soils during
both seasons.
Keywords Long-term, Manure, Growth stages, Soil fertility, Enzymes

2
†
Sunita Sheoran, Dhram Prakash and Dev Raj contributed equally to Department of Agronomy, School of Agriculture, Lovely Professional
this work. University, Jalandhar, Punjab 144001, India
3
Department of Soil Science, Punjab Agricultural University, Ludhiana,
*Correspondence: Punjab 141004, India
Rajeev Kumar Gupta 4
Department of Botany and Microbiology, College of Science, King Saud
[email protected] University, Riyadh 11451, Saudi Arabia
1
Department of Soil Science, Chaudhary Charan Singh Haryana 5
Colorado Water Center, Colorado State University, Fort Collins, CO, USA
Agricultural University, Hisar, Haryana 125004, India

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Sheoran et al. BMC Plant Biology (2025) 25:117 Page 2 of 16

Introduction fertilization’s effects on soil health, there is a lack of

Pearl millet (Pennisetum glaucum)-wheat (Triticum aes- research on the combined impact of manures and fer-
tivium) is a dominant cropping sequence after rice-wheat tilizers on soil properties and enzyme activities [1, 18].
in India, especially in Haryana’s semi-arid regions, cover- We hypothesized that a combined approach of organic
ing about 2.26 million ha [1]. While India has achieved manures and fertilizers would enhance soil fertility and
food production self-sufficiency, soil quality is declin- enzymatic activities over time in a pearl millet-wheat
ing due to nutrient mining from intensive farming. To system. This investigation aims to assess the long-term
address this, revitalizing organic manure alongside fer- effects of levels of FYM during different modes and
tilizers is essential for maintaining soil fertility and crop chemical N application on soil properties (soil reaction,
productivity [2–4]. EC, SOC, DOC, available N, P and S) and enzyme activ-
Long-term experiments have shown that combining ity (DHA, β-glucosidase, Urease, APA and ARA) at vari-
organic manures with fertilizers enhances soil nutrient ous growth stages of pearl millet in Haryana, India.
availability and microbial activity [5–7]. For instance,
integrating NPK with FYM has resulted in the highest Materials and methods
soil organic carbon content and enzyme activity in vari- Experimental site
ous cropping systems [8]. Soil enzymes play a critical role The present study was conducted at Research Farm,
in nutrient transformation and organic residue decompo- Department of Soil Science, CCSHAU, Hisar (India), in
sition, biochemical functions of soils and preserving soil an ongoing long-term field experiment established in
structure, and are sensitive indicators of SOM turnover October 1967. The experimental site is situated at a lati-
[9]. The extent of enzyme activity is influenced by soil tude of 29o16’N and longitude of 75o75’E in the North-
properties, microbial communities, and environmental Western region of India. The study area is characterized
factors [10]. by a semi-arid climate with a mean annual precipitation
Seasonal changes in moisture, temperature and sub- of 443 mm and a mean annual temperature of 24.8oC.
strate availability affect microbial biomass and enzyme Approximately 90% of rainfall is obtained from the
activity, particularly during crop growth stages [11]. Southwest monsoon during May to October. The low-
Nutrient demand and enzymatic activities fluctuate. est mean temperature was recorded in January, while
More intense microbial activity is often observed at the the mean maximum temperature was recorded in May.
vigorous crop growth stage, generally the flowering stage, The soil type was sandy loam, classified explicitly as
and then gradually declines up to crop harvest [12]. Stud- Typic Ustochrept. The chemical properties of surface soil
ies indicate peaks in enzyme activity, such as dehydroge- (0–15 cm) at the start of the experiment (1967) were as
nase and alkaline phosphatase, during specific stages in follows: soil pH of 8.20, SOC of 0.47%, CaCO3 of 1.10%,
long-term cropping systems, highlighting the importance available N, P and K were 200.00, 26.00, and 498.00 kg
of managing nutrients to meet crop demands effectively ha− 1, respectively [19].
[13, 14]. Additionally, organic manure application has
enhanced soil health indicators over time. In a long-term Experimental design
experiment (> 100 years) of semi-arid sub-tropical condi- The experiment focuses on the compensation of fertil-
tions in alfisols, organic manure application beneficially izer N through FYM under pearl millet-wheat cropping
favoured SOC accrual, NPK availability, DHA, urease, system. The experiment was carried out in a split-plot
APA, and ARA over INM and control at various growth design with 10 m x 5 m subplots and three replications.
stages of maize [15]. The treatments in the main plots involved three methods
The key challenge in developing a sustainable agricul- of applying FYM: applying it to the Rabi crop (winter sea-
ture system is maintaining long-term soil organic mat- son), to the Kharif crop (summer season), and both crops
ter (SOM), which enhances soil microbial activity and (Rabi and Kharif seasons). Each crop received three levels
biochemical processes. While inorganic fertilizers boost of FYM (15, 30, and 45 Mg ha− 1 yr− 1 until 2007-08, and 5,
soil fertility and alter its properties, combining manure 10, and 15 Mg ha− 1 yr− 1 from 2008 to 09 onwards, based
and fertilizers can help prevent soil organic carbon on dry weight). One FYM control plot was also assigned
(SOC) declines and close the yield gap [16]. Thus, inte- to each main plot. FYM was added to the plough layer
grating nutrient sources offers a logical approach to sus- (15 cm) before sowing the crops. The FYM utilized in the
taining soil fertility and crop productivity [17]. Proper experiment was analyzed annually, and the average nutri-
management of nutrient practices and microbiological ent composition of FYM applied during 2017-18 was as
activities can further enhance productivity. Long-term follows: C (38.10%), N (0.85%), P (0.97%), K (1.87%), S
use of farmyard manure (FYM) and nitrogen fertilizers is (0.36%), Na (0.68%) and Ca (1.25%). The main plot was
expected to improve microbial health and soil fertility in subdivided into two subplots, with one receiving no fer-
semiarid conditions. Despite various studies examining tilizer N (0 kg N ha− 1) and another receiving 120 kg N
Sheoran et al. BMC Plant Biology (2025) 25:117 Page 3 of 16

ha− 1 through urea. Application of fertilizer N was divided was measured by determining the amount of p-nitro-
into two splits: half at the sowing time and the remain- phenol produced when the soil was incubated with
ing half after 25–30 days after sowing. Throughout the p-nitrophenyl-β-D-glucoside (PNG) (pH 6.0) buffer and
trial, only urea has been used as a chemical fertilizer. toluene [27]. The urease activity was measured using a
Pearl millet (variety HHB 223) was sowed on 5 July 2017, potassium chloride-phenylmercuric acetate (KCl-PMA)
right after the field was prepared following the measures solution [28]. The activity of alkaline phosphatase (APA)
recommended by the institution. The hybrid (HHB 223) was assessed using substrate p-nitrophenyl phosphate
used in this experiment was released for cultivation by (PNP) [29]. Aryl sulfatase activity was measured by
the Central Variety Release Committee, Ministry of Agri- determining the p-nitrophenol emitted when the soil was
culture Government of India (GOI), and notified vide incubated with buffered potassium p-nitrophenol sulfate
S.O. 211 (E), dated 29th January 2010. Chaudhary Charan (pH 5.8), acetate buffer, and toluene [30].
Singh Haryana Agricultural University, Hisar Haryana
(India), is the sole proprietor for this hybrid (HHB 223). Statistical analysis
The crop was irrigated using canal water, and an irriga- The statistical analysis was conducted using the software
tion of approximately 7.5 cm was applied as and when STATISTICA 6.0 by Stat Soft, Inc. (2001). A box plot and
required based on visual inspection of the field. The crops principal component analysis (PCA) were used to evalu-
were harvested around 2–5 cm above ground level, and ate the variation accounted for by the principal compo-
roots and stubble were incorporated in situ in the field. nents (PCs) using Origin (Pro) Version 2024. The two
most prominent principal components, representing the
Soil sampling most significant variation, were visually illustrated in a
Two sets of soil samples were collected from each plot in two-dimensional graph.
triplicate from surface layer (0–15 cm) at the following
growth stages of pearl millet: S1 (at sowing), S2 (tiller- Results
ing), S3 (heading), S4 (maturity), and S5 (after harvest). Soil reaction
One batch/set of soil samples was dried in the shade by Soil pH ranged from 7.49 to 7.77, 7.38 to 7.71, 7.24 to
exposure to air, pulverized, filtered through a 2 mm sieve, 7.68, 7.42 to 7.60, and 7.38 to 7.73 at S1, S2, S3, S4, and
and stored in a plastic bag under normal environmen- S5 of pearl millet growing period, respectively (Table 1).
tal conditions for physicochemical analysis. The second Modes of FYM application showed a significant impact
fresh, moist soil sample batch/set was placed in a freezer on soil pH regardless of the growth stages of pearl millet.
at 4◦C temperature to analyze soil enzyme activity. Among modes of FYM application, the significantly low-
est soil pH was recorded with FYM application during
Soil fertility and enzyme activities analysis both seasons at all the stages, and the order was obtained:
Soil pH was measured using a glass electrode and poten- rabi > kharif > both seasons. Levels of FYM application
tiometric method (1:2 soil-water suspension). Soil exhibited a significant influence on soil pH. The highest
electrical conductivity (EC) was determined using the reduction of soil pH was recorded with the addition of 15
conductometric method in a 1:2 soil-to-water superna- Mg FYM ha− 1 compared to its lower application rates at
tant [20]. The wet oxidation method was employed to all studied stages of pearl millet (Table 1). Mode x level of
estimate the SOC content [21]. To determine the dis- FYM significantly interacted with soil reaction during the
solved organic carbon concentration (DOC), 50 ml growing period of pearl millet (Supplementary Table 1).
distilled water was added to 10 g soil and agitated in a Soil pH ranged from 7.41 to 7.77, 7.30 to 7.71, and 7.10 to
horizontal shaker for one hour, followed by centrifuga- 7.68 at stages 1, 2 and 3, respectively. Regardless of stages,
tion for 30 min at 8000 rpm. The DOC was analyzed in the lowest soil pH among different treatment combina-
filtrate using the dichromate acid oxidation method [22]. tions was observed with both seasons x FYM15. Further,
The available N was determined using the Kjeldahl-distil- the FYM x fertilizer N level also interacted significantly
lation method [23]. Available P was extracted with 0.5 M with soil pH, ranging between 7.47 and 7.78 (Supplemen-
sodium bicarbonate solution (pH 8.5) and the P content tary Fig. S1). All three factors showed decreased soil pH
in the extract was then measured at 660 nm wavelength at S5 over S1 (Table 1). A box plot showed 50% soil pH
of spectrophotometer [24]. The availability of S was values ranging between 7.76 and 7.48 at S1 as opposed to
assessed using turbidimetric analysis at 420 nm wave- 7.66 and 7.41 at S5. However, the lowest pH was found at
length [25]. S3, with 50% values ranging from 7.63 to 7.30 (Fig. 1A).
Dehydrogenase activity (DHA) was assessed by esti-
mating the rate at which tri-phenyl formazan (TPF) Soil electrical conductivity
was produced from 2,3,5-triphenyl tetrazolium chloride Soil EC ranged from 0.29 to 0.46, 0.31 to 0.55, 0.33 to
(TTC) [26]. The activity of the β-glucosidase enzyme 0.58, 0.27 to 0.43, and 0.26 to 0.39dS m− 1 at S1, S2, S3,
Sheoran et al. BMC Plant Biology (2025) 25:117 Page 4 of 16

S4 and S5, respectively (Table 1). Modes, levels of FYM,

Table 1 Long-term impact of mode, level of FYM, and fertilizer N application on soil reaction, electrical conductivity (EC), and organic carbon at various growth stages of pearl millet

1.23
1.25
1.43
0.04
0.52
1.42
1.55
1.72
0.05
1.25
1.35
0.02
S5
and chemical N application showed a significant impact
on soil EC. In both seasons, the FYM application showed

1.30
1.32
1.46
0.05
0.55
1.49
1.63
1.78
0.06
1.31
1.41
0.03
S4 the highest value of EC (0.34 to 0.49dS m− 1) and signifi-
cantly increased EC compared to the rabi season. Over
Soil organic carbon (%)

S1, soil EC decreased by 28.2, 8.8, and 17.1% with FYM

1.37
1.40
1.53
0.06
0.59
1.58
1.70
1.87
0.07
1.38
1.49
0.05
S3

application during rabi, kharif, and both seasons. The

FYM application rates significantly impacted soil EC at
1.32
1.34
1.49
0.06
0.57
1.52
1.65
1.80
0.06
1.33
1.43
0.05
all stages, and treatment FYM15 showed the highest soil
S2

EC, significantly higher (9.5 to 21.9%) than FYM10 at

various growth stages. Fertilizer N application increased
1.26
1.21
1.40
0.06
0.54
1.40
1.53
1.69
0.07
1.24
1.33
0.06
S1

considerably soil EC (except S5) by 5.4 to 9.1% at differ-

ent stages. The highest soil EC (0.52dS m− 1) was found
0.28
0.31
0.34
0.03
0.26
0.27
0.32
0.39
0.04
0.30
0.32
when 15 Mg FYM ha− 1 was applied during both seasons,
S5

NS
followed by the same dose of FYM given in rabi (0.48dS
m− 1) and kharif (0.40dS m− 1) season (Supplementary Fig.
0.32
0.34
0.37
0.02
0.27
0.31
0.36
0.43
0.03
0.33
0.36
0.02
S4

S2). A box plot showed that 50% values of soil EC ranged

between 0.44 and 0.30 dS m− 1 at S1 as opposed to 0.35 to
0.45
0.47
0.49
0.03
0.33
0.44
0.52
0.58
0.03
0.46
0.49
0.02
Soil EC (dS m− 1)

0.26 dS m− 1 at S5 (Fig. 1B).

S3

Soil organic carbon

(the subscript figures in treatment indicate the dose of FYM in t ha− 1 and the dose of fertilizer nitrogen in kg ha− 1)

0.42
0.44
0.46
0.03
0.31
0.41
0.49
0.55
0.03
0.43
0.46
0.02
S2

During the 51st cycles of pearl millet under the pearl mil-
let-wheat sequence, SOC ranged between 0.54 and 1.60,
0.39
0.34
0.41
0.02
0.29
0.36
0.42
0.46
0.02
0.37
0.39
0.01
S1

0.57 to 1.80, 0.59 to 1.87, 0.55 to 1.78 and 0.52 to 1.72%

at S1, S2, S3, S4 and S5, respectively (Table 1). Modes of
7.56
7.54
7.48
0.06
7.73
7.53
7.47
7.38
0.07
7.54
7.51

FYM, levels of FYM, and fertilizer N application signifi-

S5

NS

cantly influenced SOC content in these sandy loam soils.

Irrespective of the stages of pearl millet, both seasons of
7.60
7.58
7.52
0.05
7.55
7.59
7.51
7.42
0.05
7.58
7.55
S4

NS

FYM application showed the highest SOC and obtained

Pearl millet growth stage

order, both seasons > rabi season > kharif season. Applica-
tion of 15 Mg FYM ha− 1 showed significantly the high-
7.51
7.47
7.33
0.04
7.68
7.48
7.35
7.24
0.05
7.46
7.42
0.04
S3

est SOC content (S: 10.5, S2: 9.1, S3: 10.0, S4: 9.2, and S5:
11.0%) over 10 t FYM ha− 1 application. Long-term data
Soil pH (1: 2)

7.56
7.55
7.44
0.04
7.71
7.53
7.45
7.38
0.04
7.53
7.50
0.03
S2

indicated that modes x levels of FYM significantly influ-

enced SOC content at stages 1, 4, and 5 of the pearl millet
7.71
7.60
7.53
0.04
7.77
7.62
7.57
7.49
0.05
7.62
7.60

growing period (Supplementary Table 1). Soil OC ranged

S1

NS

from 0.54 to 1.86, 0.55 to 1.92, and 0.52 to 1.91% at S1,

S4 and S5, respectively. Both seasons x FYM15 exhibited
Rabi and Kharif, both seasons

significantly higher SOC content at these growth stages

than single season x FYM15. Soil OC slightly increased
Season/level of input

with the growing period up to S3 and decreased there-

after; however, there was a slight increase in SOC at S5
over S1. A box plot indicated that 50% values of SOC
ranged from 1.61 to 0.95% at S1 (Fig. 1C).
FYM10
FYM15
Kharif

FYM0
FYM5
Rabi

N120
N0

Soil-dissolved organic carbon

Dissolved OC in soil ranged from 50.5 to 243.1, 66.1 to
Level of fertilizer N (kg ha− 1)

282.5, 69.0 to 298.5, 57.5 to 277.3, and 45.6 to 268.6 mg

Mode of FYM application

kg− 1 at different growth stages of pearl millet (Supple-

Level of FYM (t ha− 1)

mentary Table 2). The application of FYM in both sea-

sons significantly increased DOC content compared to
CD (p = 0.05)

CD (p = 0.05)

CD (p = 0.05)
Treatment

the single-season application, regardless of the stages.

Application of 15 Mg FYM ha− 1significantly accrued
DOC content (S1: 33.4, S2: 25.0, S3: 21.4, S4: 44.0 and S5:
Sheoran et al. BMC Plant Biology (2025) 25:117 Page 5 of 16

Fig. 1 Box plot of soil pH (A), EC (B), SOC (C), DOC (D), available N (E), P (F), S (G), dehyrdogenase activity (H), β-Glucosidase (I), urease (J), alkaline phos-
phatase activity (K) and aryl sulfatase activity (L) at different growth stages of pearl millet in soils under pearl millet-wheat cropping system

50.3%), over application of 10 Mg FYM ha− 1. Similar to Available nutrients in the soil
SOC, adding chemical N significantly increased the DOC Available nitrogen
content by 14.4, 11.4, 10.9, 10.8, and 12.8% at different Among different treatments, soil available N ranged
growing stages of pearl millet. Interaction of mode x level between171 to 259,179 to 278, 196 to 296, 169to 264,
of FYM varied from 57.7 to 235.1, 72.7 to 276.5, 74.6 to and 164to 261 kgha− 1atS1, S2, S3, S4 and S5, respec-
294.1, 64.1 to 265.2, and 56.7 to 256.1 mg kg− 1 at stages tively (Table 2). Modes of FYM application exhibited a
1–5, respectively (Supplementary Table 2). Regardless of significant impact on available N. In both seasons, FYM
growth stages, the application of 15 Mg FYM ha− 1 during application enhanced the N availability of soil by 4.5 to
both seasons was significantly superior among all other 10.0% over the rabi season at different growth stages.
treatment combinations. Interaction of both seasons x The application of 15 Mg FYM ha− 1 accounted for the
N120 was significantly the best combination for increas- highest available N, but it could not significantly sustain
ing DOC in soil (Supplementary Table 2). At stages 2 and N availability over the application of 10 Mg FYM ha− 1.
3, DOC ranged from 66.1 to 244.6 and 69.0 to 269.3 mg Chemical N fertilization (N120) proved best to increase
kg− 1, and FYM15 x N120 was significantly superior for soil N availability (10.2 to 11.5%) in soil at different growing
DOC content (Supplementary Table 2). The abundance stages of pearl millet in these semi-arid soils of Haryana.
of DOC with nutrient management practices was lowest Treatment both seasons x FYM15 was found consider-
at S1 and highest at S3; a box plot exhibited that 50% of ably superior to enhance available N (316 kg ha− 1) in soil
DOC ranged between 239.9 and 110.7% at S3 (Fig. 1D). as compared to the same dose of FYM applied in either
single season (Supplementary Fig. S3). FYM x fertilizer
N level significantly interacted with available N of soil,
Sheoran et al. BMC Plant Biology (2025) 25:117 Page 6 of 16

ranging from 150 to 266, 156 to 290, 174 to 306, and 146

51.0
54.1
65.1

25.4
46.1
67.4
88.1

51.5
62.0
3.7

4.3

3.3
S5
to 270 kg ha− 1 at stages 1 to 4, respectively (Supplemen-
Table 2 Long-term impact of mode, level of FYM, and fertilizer N application on available nutrients (NPS, kg ha− 1) at various growth stages of pearl millet (the subscript figures in

tary Fig. S4). The addition of 15 Mg FYM and 120 kg fer-

54.1
57.1
69.1

26.9
49.2
73.0
91.3

54.9
65.3
tilizer N ha− 1 showed the highest available N in the soil.

3.2

3.8

2.9
S4
Available N was highest at S3 of the pearl millet growing
period, and a box plot exhibited that 50% values of avail-

106.7
64.5
69.6
81.3

35.0
60.3
85.2

65.9
77.6
Available S (kg ha− 1)

4.5

5.2

3.3
S3

able N ranged from 297 to 236 kg ha− 1 at S3 (Fig. 1E).

54.1
57.2
68.2

23.4
49.5
71.6
94.8

55.1
64.6
Available phosphorus
3.4

3.9

2.8
S2

Available Pin soil ranged from 23.7 to 101.2, 26.9 to

111.0, 31.4 to 129.3, 24.7 to 106.7, and 21.4 to 102.3 kg
50.5
47.8
60.4

21.9
42.0
63.7
84.1

48.5
57.4
3.3

3.9

3.2
S1

ha− 1at pearl millet growing S1, S2, S3, S4 and S5, respec-
tively (Table 2). All three factors exhibited significant
102.3
63.6
64.8
91.9

21.4
77.5
92.6

70.8
76.1
effects on P availability in soils. Irrespective of the grow-
3.9

4.4

2.6
S5

ing stages, the application of FYM during both seasons

significantly increased available P by 35.2 to 47.7% over
106.7
65.5
68.4
94.2

24.7
77.8
95.0

72.8
79.3
4.6

5.3

3.0
S4

the rabi season at various growth stages of pearl millet

(Table 2). However, the FYM application in kharif was
115.8

114.6
129.3

slightly better than the rabi season application, except

78.4
83.2

31.4
94.6

88.6
96.4
4.9

5.6

2.8
S3
Available P (kg ha− 1)

for S1. During the 51st cycle of pearl millet under pearl
millet-wheat cropping, FYM applications remarkably
101.3
111.0
71.8
74.4
97.1

26.9
85.3

78.1
84.2

influenced P availability in soil and followed the order,

3.9

4.5

2.3
S2

FYM15 > FYM10 > FYM5 > FYM0 (Table 2). Relative to
applying 10 Mg FYM ha− 1, 15 Mg FYM ha− 1 application
101.2
66.2
63.8
90.1

23.7
76.8
91.9

70.7
76.1

significantly enhanced available P by 10.1, 9.6, 12.8, 12.3,

3.2

3.7

2.2
S1

and 10.5% at S1, S2, S3, S4, and S5, respectively. Further,
chemical N application increased available P by 7.6, 7.8,
226
227
239

164
245
254
261

218
243
S5

11

13

10

8.8, 8.9 and 7.5% at pearl millet growing S1, S2, S3, S4,
treatment indicate the dose of FYM in t ha− 1 and the dose of fertilizer nitrogen in kg ha− 1)

and S5, respectively. Interaction of mode x level of FYM

229
231
243

169
248
256
264

222
246
S4

10
Pearl millet growth stage

for soil available P varied from 23.7 to 127.2, 26.9 to

9

135.4, 31.4 to 169.0, 24.7 to 134.6, and 21.4 to 130.9 kg

Available N (kg ha− 1)

254
261
279

196
279
288
296

252
278

ha− 1 at stage 1–5, respectively (Supplementary Table 1).

S3

Treatment in both seasons x FYM15 was significantly

238
243
259

179
260
270
278

233
260

superior to other combinations for increasing soil P avail-

S2

ability throughout the growing period of pearl millet.

A box plot also indicated that available P was lowest at
229
226
240

171
243
253
259

220
243
S1

10
9

S1 and highest at S3, whereas 50% ranged from 114.8 to

55.21 kg ha− 1 at S3 (Fig. 1F).
Rabi and Kharif, both seasons

Available sulphur
Season/level of input

Available S in soil varied between21.9 to 84.1, 23.4 to

94.8, 35.0 to 106.7, 26.9 to 91.3, and 25.4 to 88.1 kg ha− 1
at pearl millet growing S1, S2, S3, S4 and S5, respectively
(Table 2). Regardless of stages, applying FYM during both
FYM10
FYM15
Kharif

FYM0
FYM5

seasons significantly enhanced available S content in soil

Rabi

N120
N0

(19.6 to 27.7%) compared to rabi season and obtained

the order, both seasons > kharif season > rabi season.
Level of fertilizer N (kg ha− 1)

Over the application of 10 Mg FYM ha− 1, its higher rate

Mode of FYM application

(15MgFYM ha− 1) application significantly increased

Level of FYM (t ha− 1)

available S by 32.0, 32.4, 25.2, 25.1 and 30.7% at S1, S2,

S3, S4, and S5, respectively. Among various stages of
CD (p = 0.05)

CD (p = 0.05)

CD (p = 0.05)
Treatment

pearl millet, chemical N fertilized plots showed a sig-

nificant increase (17.2 to 20.4%) in soil available S than
unfertilized (N0) plots. Interaction of mode x level of
Sheoran et al. BMC Plant Biology (2025) 25:117 Page 7 of 16

FYM significantly impacted available S, and it ranged

16.8
18.2
21.0

14.2
18.4
19.9
22.2

17.5
19.9
2.2

2.5

2.1
Urease activity (µg NH4+-N g− 1 h− 1)

S5
between 21.9 and 97.3, 23.4 to 108.6, 35.0 to 123.3, 26.9 to

Table 3 Long-term impact of mode, level of FYM and fertilizer N application on soil enzymes activity at different growth stages of pearl millet (The subscript figures in treatment
107.7, and 25.4 to 103.2 kg from stages 1–5, respectively

44.1
48.9
55.5

21.6
49.9
59.1
67.4

43.7
55.3
2.7

3.2

2.8
S4
(Supplementary Table 1). Irrespective of growth stages,
both seasons x FYM15 significantly performed better

57.6
64.5
71.7

23.5
71.8
78.1
84.9

58.6
70.6
than the rest of the treatment combinations in increasing

3.5

4.1

2.9
S3
soil S availability (Supplementary Table 1). The periodic
abundance of available S was similar to that observed for

71.4
74.9
83.7

40.6
82.1
88.7
95.2

70.0
83.4
3.4

3.9

3.1
S2
available P, and a box plot revealed that 50% of values of
available S ranged from 94.6to42.9 at S3 (Fig. 1G).

18.4
17.6
20.8

15.5
18.2
20.2
21.8

17.6
20.2
1.8

2.1

1.8
S1
Soil enzyme activities

221.0
235.5
263.8

118.2
248.5
280.9
312.9

230.9
249.3
Dehydrogenase activity

19.0

22.0

NS
S5
β-Glucosidase activity (µg PNP g− 1 h− 1)
The impact of different modes, levels of FYM, and fer-
tilizer N application on dehydrogenase activity (DHA)

306.5
320.0
339.7

146.7
346.8
384.6
410.2

311.3
332.8
11.8

13.7
in soil ranged from 16.1 to 24.1, 48.3 to 108.9, 80.1 to

9.7
S4
154.8, 52.5 to 126.8 and 40.8 to 83.3 µg TPF g− 1 24 h− 1
at pearl millet growing S1, S2, S3, S4 and S5, respectively

122.8
132.0
177.0

142.4
168.3
200.6

137.0
150.8
14.4
64.3

16.6

13.2
S3
(Table 3). Various modes of FYM application significantly
improved DHA activity in the soil and followed the order:

154.4
160.5
189.0

166.7
200.3
224.3

160.0
175.9
both seasons > kharif season > rabi season (except S1).

80.4
6.0

7.0

5.4
S2
Both seasons’ FYM application significantly improved
DHA by 14.4 to 37.1% over the rabi season FYM appli-

110.6

105.0
134.1
81.0
74.5

35.7
80.0

81.6
95.8
cation. Treatment FYM15 enhanced the DHA by 12.4,

6.8

7.8

6.0
S1
11.1, 16.1, and 12.4% at S2, S3, S4, and S5 over treatment
Dehydrogenase (DHA,µg TPF g− 1 24 h− 1)

FYM10. Regardless of growing stages, chemical N appli-

56.9
63.2
78.0

40.8
65.9
74.1
83.3

61.9
70.2
6.3

7.3

5.0
S5

cation was found essential and significantly helped to

improve DHA at different stages by 10.6 to 14.7%. There-
107.6

109.2
126.8
80.3
92.9

52.5
85.8

87.4
99.8
7.8

9.0

6.1
fore, all three factors individually played crucial roles in
S4

enhancing and maintaining DHA during the pearl millet

Pearl millet growth stage

111.9
121.8
137.2

120.4
139.3
154.8

115.2
132.1
growing period (Table 3). Modes x level of FYM signifi-
80.1
indicate the dose of FYM in t ha− 1 and the dose of fertilizer nitrogen in kg ha− 1)

8.1

9.4

6.5
S3

cantly interacted with DHA activity at stages 4 and 5, and

DHA ranged from 80.1 to 176.6, 52.5 to 157.6, and 40.8
108.9

to 99.9 µg TPF g− 1 24 h− 1, respectively (Supplementary

77.9
83.6
91.3

48.3
82.9
96.9

78.9
89.5
8.1

9.3

6.3
S2

Table 3). The impact of 15MgFYM application during

both seasons significantly improved DHA at these stages
20.1
19.5
23.0

16.1
20.9
22.3
24.1

19.8
21.9
2.4

2.8

1.8
S1

compared to other combinations (Supplementary Table

3). The trend of DHA was similar to that recorded for
Rabi and Kharif, both seasons

SOC and available nutrients, and a box plot showed that

50% of DHA values ranged between 144.4 and 94.3 µg
Season/level of input

TPF g− 1 24 h− 1 at S3(Fig. 1H).

β-glucosidase activity
The β-glucosidase activity of soil varied from 35.7 to
FYM10
FYM15
Kharif

FYM0
FYM5

134.1, 80.4to 224.3, 64.3 to 200.6, 146.7 to 410.2, and

Rabi

N120
N0

118.2 to 312.9 µg PNP g− 1 h− 1at pearl millet growing S1,

S2, S3, S4 and S5, respectively (Table 3). Irrespective of
Level of fertilizer N (kg ha− 1)

the stages, a significant effect of modes, levels of FYM,

Mode of FYM application

and chemical N (except S5) application was recorded

Level of FYM (t ha− 1)

on β-glucosidase activity under the pearl millet growing

period. Regardless of stages, both seasons FYM applica-
CD (p = 0.05)

CD (p = 0.05)

CD (p = 0.05)
Treatment

tion showed higherβ-glucosidase activity (S1: 36.5; S2:

22.4; S3: 44.1; S4: 10.8 and S5: 19.4%) over rabi season
and seasons obtained order, both seasons > kharif > rabi.
Sheoran et al. BMC Plant Biology (2025) 25:117 Page 8 of 16

Application of 15 Mg FYM ha− 1 was found to be sig- between 40.6 and 107.1, 23.5 to 93.6, and 21.6 to 75.9 µg
nificantly better than other lower doses of FYM and NH4+-N g− 1 h− 1, respectively (Supplementary Table 3).
increased β-glucosidase activity by 27.7, 12.0, 19.2, 6.7, Further, the level of FYM x fertilizer N also consider-
and 11.4% at stages 1 to 5, respectively, over 10 Mg FYM ably interacted with urease activity, and it varied between
ha− 1. Treatment N120significantly increasedβ-glucosidase 21.2 and 92.8 µg NH4+-N g− 1 h− 1 and 20.1 to 74.5 µg
activity by 17.4, 9.9, 10.1, 6.9, and 8.0% at stages 1–5, NH4+-N g− 1 h− 1 at stages 3 and 4, respectively (Supple-
respectively, over treatment N0. The interaction effect of mentary Fig. S5). Treatment FYM15 x N120 was best for
modes x FYM on β-glucosidase activity ranged from 35.7 increasing urease activity at stages 3 and 4 compared to
to 167.5, 80.4 to 253.3, and 64.3 to 246.0(µg PNP g− 1 h− 1) the rest of the treatment combinations. Among the stages
at S1, S2, and S3, respectively (Supplementary Table 3). of pearl millet growth, urease activity followed the order
The impact of both seasons x FYM15 on β-glucosidase S2 > S3 > S4 > S5 > S1, and a box plot showed that 50% val-
activity was significantly higher than their other combi- ues of urease activity ranged from 93.8 to 58.3 µg NH4+-
nations. Relative abundance showed an irregular trend, N g− 1 h− 1 at S2 (Fig. 1J).
and a box plot exhibited thatβ-glucosidase activity was
highest at S4, and a box plot showed that 50% values of Alkaline phosphatase activity
β-glucosidase activity ranged from 400.9 to 236.6 at S4 Among different treatments, APA ranged between354to
(Fig. 1I). 563, 472 to 911, 530 to 1005, 449 to 820, and 396 to
691 µg PNP g− 1 h− 1at pearl millet growing S1, S2, S3, S4,
Urease activity and S5, respectively (Table 4). Among different stages,
Urease activity of soil fluctuated between 15.5 and APA increased by 16.4, 5.7, 6.0, 4.6, and 8.1% with both
21.8, 40.6 to 95.2, 23.5to 84.9, 21.6 to 67.4, and 14.2 to seasons FYM application over rabi season and obtained
22.2 µg NH4+-N g− 1 h− 1 at pearl millet growing S1, S2, order, both seasons > kharif > rabi (except S1). Regard-
S3, S4 and S5, respectively (Table 3). Relative to either less of the stages, each level of FYM showed a favourable
single season, both seasons’ FYM applications signifi- impact on APA, and 15 Mg FYM ha− 1 application was
cantly improved urease activity and application of FYM superior to enhancing APA than its lower doses. Chemi-
in both seasons increased urease activity by 13.0, 17.2, cal N application also significantly increased APA at dif-
24.5, 25.9, and 25.0% at stages 1 to 5, respectively, over ferent stages of pearl millet (Table 4). Among interactive
the rabi season. Overtreatment, FYM10, and treatment effects, mode x level of FYM played a beneficial role on
FYM15 significantly increased urease activity by 7.9, 7.3, APA and varied between 354 and 653, 472 to 949, 530
8.7, 14.0, and 11.6% at stages 1–5, respectively. Similar to 1048, 449 to 851, and 396 to 719 µg PNP g− 1 h− 1 at
to available N, plots treated with chemical N showed a stages1 to 5, respectively (Supplementary Table 3). Fur-
significant increase in urease activity by 14.8, 19.1, 20.5, ther, FYM x fertilizer N also significantly interacted at
26.5, and 13.7% at stages 1–5, respectively, compared stages 1 and 5, and it ranged from 311 to 576 and 368 to
to control plots (N0). The interaction of mode x level of 704 at these stages, respectively (Supplementary Fig. S6).
FYM was significant at stages 2, 3, and 4, and it ranged Treatment FYM15 x N120 was significantly superior for

Table 4 Long-term impact of mode, level of FYM, and fertilizer N application on soil enzymes (alkaline phosphatase activity, APA and
aryl sulfatase activity, ARA) activity at different growth stages of pearl millet (the subscript figures in treatment indicate the dose of
FYM in t ha− 1 and the dose of fertilizer nitrogen in kg ha− 1)
Treatment Season/level of input Alkaline phosphatase activity (µg Aryl sulfatase activity (µg PNP g− 1 h− 1)
PNP g− 1 h− 1)
Pearl millet growth stage
S1 S2 S3 S4 S5 S1 S2 S3 S4 S5
Mode of FYM application Rabi 464 746 823 694 558 7.80 22.16 20.67 17.06 14.23
Kharif 456 761 847 702 575 7.28 23.30 21.78 18.66 14.97
Rabi and Kharif, both seasons 540 789 872 726 603 9.93 25.29 23.27 20.16 16.96
CD (p = 0.05) 7 9 5 7 7 1.40 1.46 1.29 1.52 1.11
Level of FYM (t ha− 1) FYM0 354 472 530 449 396 5.32 16.33 14.69 10.67 9.17
FYM5 497 817 905 768 591 7.69 23.96 22.07 18.61 14.47
FYM10 533 862 949 792 637 9.08 25.62 24.31 21.02 17.11
FYM15 563 911 1005 820 691 11.26 28.43 26.57 24.22 20.79
CD (p = 0.05) 9 10 6 8 8 1.62 1.68 1.48 1.75 1.28
Level of fertilizer N (kg ha− 1) N0 469 753 833 699 562 7.92 22.82 21.30 18.02 14.64
N120 505 778 862 715 595 8.76 24.35 22.52 19.23 16.13
CD (p = 0.05) 6 7 7 5 5 0.51 0.58 0.84 0.91 0.84
Sheoran et al. BMC Plant Biology (2025) 25:117 Page 9 of 16

APA in soil than other combinations involving the use of of salts and ionic species in the soil solution. The miner-
FYM-coupled fertilizer N under pearl millet (Supplemen- alization of FYM during the early stages of crop growth
tary Fig. S6). APA increased up toS3 and declined with might increase salt concentration in soil solution and re-
advancement of the growth stage. A box plot showed adsorption of ions on the soil surface, in addition to crop
that 50% values varied from 973 to 698 µg PNP g− 1 h− 1 at uptake and decreased soil EC during later crop growth
growth S3 of pearl millet (Fig. 1K). stages [34].

Aryl sulfatase activity Soil organic carbon

Among different treatments, ARA ranged between 5.32 Applying FYM for 51 years significantly increased OC
and 11.26, 16.33 to 28.43, 14.69 to 26.57, 10.67 to 24.22, content in the soil compared to the initial value due to
and 9.17 to 20.79 µg PNP g− 1 h− 1 at pearl millet grow- the direct addition of organic matter in the soil. Irrespec-
ing S1, S2, S3, S4 and S5, respectively (Table 4). Similar tive of the mode of application, OC content was accrued
to soil available S, modes of FYM application significantly with an increasing application rate of FYM due to higher
influenced the ARA and obtained the order, both sea- quantity addition. Further, adding fertilizer N signifi-
sons > kharif > rabi (except S1). Among different growth cantly increased OC content over control due to higher
stages, ARA increased by 27.3, 14.1, 12.6, 18.2, and 19.2% crop productivity with N addition, leading to higher SOC
with both seasons FYM application over rabi season. accumulation [35]. The higher build-up of OC with the
The addition of 15 Mg FYM ha− 1 showed an increase of integrated application of FYM and fertilizer N as com-
ARA (9.3 to 24.0%) compared to 10 Mg FYM ha− 1 appli- pared to FYM alone may be due to more generation of
cation at different stages. Chemical N application also root biomass, rhizodeposition, left-over residues, and
significantly increased ARA from 5.7 to 10.6% at vari- ultimately, incorporation of more organic matter in soil
ous stages of pearl millet. It was lowest at S1 and sharply [36]. Irrespective of modes and levels of FYM, OC con-
increased at S2; however, thereafter, a regular decrease tent in soil was increased from sowing to heading due
was obtained up to S5, and a box plot showed that 50% to the addition of carbon substances through FYM and,
values varied from 27.1 to 19.5 µg PNP g− 1 h− 1 at growth thereafter, decreased up to harvesting due to the greater
S2 of pearl millet (Fig. 1L). utilization of carbonaceous compounds by microbes and
more losses of organic matter as CO2 [12]. This study also
Discussion observed a polynomial relationship between SOC and
Soil reaction and electrical conductivity soil properties, with R2 = 0.93 to 0.99 (Fig. 2).
Long-term nutrient management practices decreased soil
pH under the pearl millet-wheat sequence compared to Dissolved organic carbon
the antecedent value due to the release of organic acids During the 51st year of continuous fertilization and crop-
during the decomposition of organic matter. The decrease ping under the pearl millet-wheat system, DOC content
in soil pH with the addition of FYM might also be due was significantly increased with FYM application because
to the oxidation of organic matter that liberates CO2 in of abundant food availability that stimulated microbial
the soil [31]. The addition of N fertilizer also decreased growth, accelerated organic matter decomposition and
soil pH compared to control plots due to oxidation of N increased the DOC content in soil [37, 38]. The lowest
fertilizer, i.e. transformation of urea to ammonium ion DOC content was observed in control plots due to low
releasing H+ in soil solution [32]. The decrement in soil inherent fertility. The application of fertilizer N improved
pH with fertilizer N might also be attributed to the rapid DOC content in the soil compared to the control, which
release of nutrients, which plants could take up. The might be attributed to increased organic matter content
increased uptake led to less accumulation of exchange- due to increased crop growth and more rhizo-deposi-
able bases in soil that otherwise could neutralize soil tions. The higher DOC content in integrated plots com-
acidity [33]. Irrespective of the mode of application, the pared to the sole application of nutrient sources might
decrease in soil pH was more significant with the increas- also be due to the priming effect of fertilizer N to FYM
ing application rate of FYM, which might be attributed [4, 39]. An increase in DOC content during initial stages
to a higher quantity of addition of FYM, thereby releas- may be due to increased mineralization, and a decrease
ing more organic acids. Irrespective of modes and levels at later stages might be due to oxidation loss of carbon as
of FYM, soil pH was lowered during the growth stages of CO2 along with utilization by the crop. Another reason
the crop compared to sowing time due to acids released for higher content at the initial stages may be the favour-
by plants’ roots in addition to SOM decomposition. Irre- able temperature and moisture conditions for microbial
spective of mode and levels of FYM and fertilizer N, a proliferation [40]. In this study, a polynomial relation-
decrease in soil EC during crop growth was attributed ship between SOC and DOC content of the soil was also
to a change in equilibrium between the concentration observed, with R2 = 0.98 (Fig. 2).
Sheoran et al. BMC Plant Biology (2025) 25:117 Page 10 of 16

Fig. 2 Relationship between soil organic carbon (SOC) and dissolved OC (DOC), available N, P, S, dehydrogenase activity (DHA), β-Glucosidase (Beta glu),
urease, alkaline phosphatase activity (APA) and aryl sulfatase activity (Aryl)of soil

Nutrient availability in soil [41]. The lower N content during later stages might also
Continuous rotation of pearl millet-wheat for 51 years be due to reduced N-mineralization because of decreased
could not sustain the initial level of N in control plots, labile organic matter, microbial activity, and toxic sub-
which might be attributed to N leaching and volatiliza- stance accumulation during later stages [44, 45]. A poly-
tion losses in addition to plant utilization [8]. Applying nomial relationship between urease activity and available
FYM improved available N content in the soil because N content of soil was also observed (R2 = 0.97; Fig. 3).
of the direct addition of organic matter and reduced N The lowest available P content in the control plot may
losses that build up the available N in the soil. Moreover, be attributed to the fixation of inherited P in soil in addi-
it might also be attributed to the stimulation of microbial tion to crop removal. A significant build-up of available
activity, which mineralizes organic matter and releases N P with FYM application may be due to mineralization
in soil solution [5]. The probable reason for the increased and solubilization processes of P compounds in soil [7].
availability of N with the INM compared to FYM alone The organic amino compounds added through FYM
may be the high C: N ratio of FYM, which caused immo- also retard the fixation of P in soil and cause desorp-
bilization with the sole application of FYM [41]. Add- tion of P from mineral compounds [46]. The high C: N
ing N fertilizer and FYM narrowed the C: N ratio and ratio of FYM promotes fungal feeding populations, so
increased mineralization [42]. Another reason might be the availability of P could be improved due to pictorial-
that excess C supply may lead to variations in the propor- fungal associations. Adding FYM over long period also
tions of fungi and bacteria that alter the N cycling in soil decreased the activity of polyvalent cations such as Ca,
[43]. The plots receiving FYM in both seasons recorded Fe, and Al due to chelation, which ultimately reduces
the highest available N content due to higher quantity P-fixation [47]. The increase in available P content dur-
addition and urease activity. The available N content in ing early growth stages might be due to decomposition
soil was increased from sowing to heading due to rapid of SOM and solubilization of phosphates. The decline in
hydrolysis of urea and increased mineralization of FYM P content after attaining the peak might be due to plants’
[8, 33, 44]. The lower available N content from heading utilization, and also the reduction in easily degradable
to maturity may be due to plants’ uptake and losses of N organic phosphates [44]. The P availability in soil is also
through biotic and abiotic processes after mineralization governed OC content of soil and alkaline phosphatase
Sheoran et al. BMC Plant Biology (2025) 25:117 Page 11 of 16

Fig. 3 Relationship between urease activity and available N of soil

Fig. 4 Relationship between alkaline phosphatase activity and available P of soil

activity (APA) [48]. A polynomial relationship between FYM increased available S content in the soil. The lower
SOC and available P; APA and available P content of the available S content during the early stages might be due
soil had R2 = 0.97 (Figs. 2 and 4). to higher leaching losses of soluble sulfate because of
The incorporation of FYM for 51 cycles of pearl mil- increased moisture availability [8]. The increase in avail-
let-wheat crop rotation significantly increased microbial able S content at later stages could be due to favourable
biomass and enzyme activities that enhanced the avail- temperature that increased microbial activity and min-
ability of S in soil [4]. Similar to N and P, irrespective eralization. A polynomial relationship between SOC and
of modes of application, the higher application rate of available S (Fig. 2; R2 = 0.95); ARA and available S content
Sheoran et al. BMC Plant Biology (2025) 25:117 Page 12 of 16

(Fig. 5; R2 = 0.96) of the soil was observed in the present Incorporating FYM increased cellulose and hemi-
study. cellulose degrading β-glucosidases enzyme activity by
increasing substrate availability and stimulating the
Soil enzymes microbial population [51]. The higher β-glucosidase
FYM application significantly increased DHA com- activity with the combined application of FYM and fer-
pared to control or fertilizer N application because tilizer N was possibly due to increased nutrient availabil-
FYM supply carbon and energy sources for microbes ity and intensified root biomass [8]. Temporal variations
that improved autochthonous and exogenous micro- in enzyme activity might be attributed to alteration in
bial diversity ultimately the higher dehydrogenase activ- macro and microclimatic conditions, nutrient availabil-
ity [18, 44]. Dehydrogenase activity increased with the ity, and substrate inputs [52, 53]. The gradual decline in
increasing application rate of FYM due to higher sub- β-glucosidase activity might be due to decreasing C sub-
strate addition for microbes. The lowest dehydrogenase strates with crop progress [54]. The irregularly decreasing
activity in control pots might be due to insufficient sub- trend during pearl millet might also be due to elevated
strate for microbial growth. Comparatively, higher activ- soil moisture that exceeded optimal conditions for
ity with integrated application of FYM and fertilizer N microbes. The higher β-glucosidase activity at harvest-
might be due to increased decomposition of organic mat- ing stage might be due to an increased decomposition
ter because of lowered C: N ratio and more availability of residues with time and, ultimately, accumulation of
of nutrients for micro-organisms [39]. The deficiency of SOM [55, 56]. A polynomial relationship between SOC
available P in soil resulted in decreased enzyme activity and β-glucosidase activity of soil was observed, having
at the initial stage [49]. The highest activity at heading R2 = 0.99 (Fig. 2).
stage might be attributed to increased root exudates lead- The incorporation of FYM increased substrate avail-
ing to more microbial activity, which gradually decreases ability that increased the microbial population and
when the crop matures [12]. Moreover, applied FYM urease. The addition of fertilizer N also increased the
gets mineralized rapidly providing sufficient nutrition enzyme activity due to the addition of substrate, i.e.
for the proliferation of microbes in at early stages of crop urea. The increased enzyme activity with increased FYM
growth, and scarcity of easily decomposable substrate at application rate may be attributed to enhanced micro-
the end of crop season gradually decreased the enzyme organisms’ populations because of more availability of
activity [50]. The lowest dehydrogenase activity after har- carbon-releasing extra-cellular enzymes [57]. However,
vest could be due to less moisture availability at maturity the higher application rate of FYM may also decrease the
[44]. A good correlation between SOC and DHA content enzyme activity due to inhibitory effect of the end prod-
of the soil was observed, having R2 = 0.96 (Fig. 2). ucts [58]. The highest urease activity with FYM applica-
tion in both seasons was due to higher substrate addition

Fig. 5 Relationship between aryl sulfatase activity and available S of soil

Sheoran et al. BMC Plant Biology (2025) 25:117 Page 13 of 16

and C availability [59]. Variations at growth stages indi-

cated that temperature and moisture conditions influ-
enced enzyme activities by affecting microbial growth
and substrate availability. The enhanced urease activity
at tillering stage might be attributed to higher N con-
tent due to faster decomposition of SOM and release of
ammonium [60]. The lower enzyme activity at harvesting
stage was due to low temperature, lower microbial bio-
mass, and decreasing SOC content [44]. In this study, a
polynomial relationship between SOC and urease activity
of soil having R2 = 0.96 was also observed (Fig. 2).
A significant improvement of APA in manure treated
soil could be related to enhanced microbial activity and
diversity of phosphate-solubilizing bacteria [61]. The
addition of FYM provides sources of C, N, and P for Fig. 6 Plot of principal component analysis (PCA) on the soil properties
soil microorganisms which might stimulate the alkaline including soil pH, electrical conductivity (EC), soil organic carbon (SOC),
dissolved OC (DOC), available N, P, S, dehyrdogenase activity (DHA),
phosphatase activity [58]. Alkaline phosphatase activity β-Glucosidase (Beta glu), urease, alkaline phosphatase activity (APA) and
was higher with conjunctive use of FYM and fertilizer N aryl sulfatase activity (Aryl)in soils (pooled data of five growth stages of
because of faster decomposition of FYM in presence of pearl millet) under pearl millet-wheat cropping system in North-western,
fertilizer N that resulted in more release of organically India. PC = principal component
bound P. The balanced fertilization also promotes higher
microbial population and more secretion of enzymes processes within soils and their broader environmental
[39, 44]. The higher phosphatase activity from sowing implications.
to heading might be attributed to sufficient temperature
and moisture availability in the rainy summer. The rain- Principal component analysis (PCA)
fall during summer season often leads to nutrient loss The principal component analysis (PCA) separated the
and low available P content, which enhances phosphatase soil variables and treatments in orthogonal space (Fig. 6).
activity and fulfils the increasing P demand for plant and The PC1 and PC2 explained 98.36% variability in the data
microbe growth. In this study, a polynomial relationship set (95.7% and 2.66% of total variability, respectively). The
between SOC and APA activity of soil was also observed, PC1 and PC2 had eigen values of 11.48 and 0.32, respec-
with R2 = 0.99 (Fig. 2). tively. Each soil variable had a corresponding factor
Like other enzymes, arylsulfatase activity is also asso- loading or eigenvector weight value and contribution to
ciated with the application rate of organic manures, variability (Supplementary Table S4). The β-glucosidase
and increased activity with FYM application is due to a and ARA both had the highest loading value (0.294), fol-
favourable environment for C-degrading microorgan- lowed by DOC (0.292) and DHA (0.291) in PC1 and also
isms and enhanced substrate [51]. With FYM appli- correlated with other high loading indicators. In the sec-
cation, elevated activity indicates the differences in ond PC (PC2), available N was the most highly loaded
microbial community and higher fungal biomass. The indicator (loading value = 0.462) followed by EC (-0.444)
higher enzyme activity with combined use of FYM and and available S (-0.431) in PC2. The percentage of total
fertilizer N than FYM alone could be due to enhanced variance estimated based on the weight of each PC, the
organic matter input and C, N, P, and S availability [8, 62]. weighted factor for two distinct minimum data sets
Variations at different stages indicated that enzyme activ- (MDS) followed PC1 (0.97) > PC2 (0.03). High levels of
ity is influenced by temperature and moisture conditions β-glucosidase activity indicated a robust microbial popu-
[40]. Significantly higher enzyme activity was noticed in lation capable of breaking down organic materials, essen-
the wet season than in dry season. The higher activity tial for maintaining soil health and productivity. DHA
at tillering might also be ascribed to an increase in root play a key role in the respiration processes of microorgan-
exudates that leads to more intense microbial growth, isms by transferring hydrogen from organic substrates to
which gradually declines with the advancement of crop electron acceptors. High DHA is often associated with
growth [12]. This study also reported a polynomial rela- increased microbial metabolic rates, indicating a thriving
tionship between SOC and ARA activity of soil, with microbial community contributing to decomposition and
R2 = 0.98 (Fig. 2). The polynomial relationships between nutrient cycling of organic materials. Elevated levels sug-
SOC and enzyme activity are essential for understand- gest enhanced soil structure and fertility, while low levels
ing soil dynamics, fertility, and ecosystem health [63]. may indicate poor soil conditions [64]. DOC represents
They highlight the interconnectedness of biological primary energy source for soil microbes, influencing their
Sheoran et al. BMC Plant Biology (2025) 25:117 Page 14 of 16

Author contributions
growth and activities. Thus, high levels of DOC indicate S S and D P: Writing-original draft, Investigation, Data curation. S S, D
healthy soil ecosystems and reflect active organic mat- R, R K G, MHS and SK: Writing-original draft, Supervision, Methodology,
ter turnover and nutrient cycling processes [65]. In PCA, Conceptualization D P; P K Y and R S: Writing-review & editing, Visualization,
Methodology, Data curation.S A, SK and M HS: Writi ng-review & editing,
PC1 often captures the most variance among the data set, Software. SA, SK: Critically revised and funding acquisition. All authors have
highlighting these variables’ importance in distinguishing read and agreed to the published version of the manuscript. SS, DP & DR
between different soil conditions or treatments [64]. The contributed equally.

high loadings on PC1 suggest that β-glucosidase, DHA Funding

and DOC are interrelated factors that collectively influ- The authors appreciate the Researchers Supporting Project number
ence the biological processes within the soil ecosystem. (RSP2025R347), King Saud University, Riyadh, Saudi Arabia.

The β-glucosidase and DHA activities alongside DOC Data availability

and their interactions facilitate essential processes such Data is provided within the manuscript or supplementary information files.
as organic matter decomposition, nutrient cycling, and
overall ecosystem functioning, making them critical indi- Declarations
cators for assessing soil health.
Ethics approval and consent to participate
This article does not contain any studies with human or animal subjects. The
Conclusions current experimental research and field study including the collection of plant
Long-term application of FYM and chemical N signifi- material, is complying with relevant institutional, national, and international
guidelines and legislation and used for research and development.
cantly influenced the soil properties during the 51st cycle
of the pearl millet-wheat system. Application of 15 Mg Consent for publication
FYM ha-1 showed the highest available N, P, and S con- Not applicable.

tent, DHA, urease, β-Glucosidase, APA, and ARA activ- Clinical trial number
ity significantly at different growth stages over FYM10. NA.
Chemical N application significantly accrued soil OC
Competing interests
and increased nutrient flush to the equilibrium soil solu- The authors declare no competing interests.
tion. A synergistic favour and significant enrichment
in SOC, available nutrients, and enzyme activity were Received: 19 October 2024 / Accepted: 20 January 2025
also recorded with treatment in both seasons x FYM15.
Interaction of FYM15 x N120 was also significantly supe-
rior for maintaining the highest level of DOC, available
N, APA, and urease at different growth stages. Under References
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