Geoderma 409 (2022) 115626

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Geoderma
journal homepage: www.elsevier.com/locate/geoderma

Rhizosphere influence on soil microbial biomass and enzyme activity in

banj oak, chir pine and banj oak regeneration forests in the
central Himalaya
Shailendra Kumar, Satish Chandra Garkoti *
School of Environmental Sciences, Jawaharlal Nehru University, New Delhi 110067, India

A R T I C L E I N F O A B S T R A C T

Handling Editor: Alberto Agnelli The present study aims to investigate the rhizosphere effect on soil organic carbon, nutrients and soil microbial
biomass in three forests viz, banj oak (Quercus leucotrichophora), chir pine (Pinus roxburghii) and banj oak
Keywords: regeneration forest (banj oak regeneration in chir pine) in the central Himalaya. Soil samples were collected
Temperate forest seasonally from three depths (0–10 cm, 10–20 cm, 20–30 cm) at two soil positions (i.e., rhizosphere and bulk
Rhizosphere effect
soil). The soil physicochemical and biological properties were compared between rhizosphere soil (RS) and bulk
Soil microbial biomass
soil (BS), among forest types using two-way ANOVA and comparison of means was assessed by Tukey’s HSD post-
Enzyme activity
Rhizosphere Soil Microbial Index (RSMI) hoc test. Correlation analysis was performed to observe the association among physical, chemical and biological
properties of the soil. Additionally, a Linear Mixed Effect Model (LMM) was applied to investigate the effect of
forest types, soil positions, seasons, depths and their interactions on soil biological properties. In the rainy
season, soil organic carbon (SOC) and total nitrogen (TN) in RS of banj oak were significantly higher (p < 0.05)
than the BS in the upper soil layer (0–10 cm). However, the difference was less prominent in the middle (10–20
cm) and lower soil layer (20–30 cm). Soil microbial biomass (Cmic, Nimc and Pmic) in RS of banj oak and chir
pine were significantly higher than the BS in upper and middle soil layers, while both RS and BS of banj oak
regeneration did not vary significantly. Forests differed significantly in the rhizosphere effect. Banj oak showed a
strong rhizosphere effect through the combined impact of favourable microclimatic conditions and high soil
microbial biomass. Chir pine grows in nutrient-depleted soil, showed a low rhizosphere effect compared to the
rhizosphere effect of banj oak. The SOC, Cmic and Pmic were generally higher in the RS of banj oak regeneration
forest than the RS of chir pine, reflecting that oak regeneration enhances microbial diversity through an
increased supply of root induced (root exudates) microbially mineralised nutrients. The rhizosphere effect
changed significantly with soil depths and was found higher in the upper soil layer (due to high soil nutrients and
microbial activity) and decreased with increasing soil depths. Across the forests, the rhizosphere effect was more
prominent in the rainy season than the winter and summer seasons, mainly driven by high soil moisture content.
Principal Component Analysis (PCA) was performed to obtain rhizosphere soil microbial index (RSMI). Our study
suggests that Cmic, Cmic: SOC and Pmic: AP were the most important parameters to evaluate rhizosphere soil
quality of this temperate forest of the central Himalaya.

1. Introduction cycling (Toberman et al., 2011). The rhizosphere soil is influenced by

The rhizosphere is one of the most complex and sensitive regions
influenced by plant roots and numerous soil microorganisms (De Feudis
et al., 2017; Proctor and He, 2021). It is a highly active part of the soil
ecosystem that influences the transfer of energy and nutrients from the
soil to the plant roots and vice versa (Hinsinger et al., 2009; Kuzyakov,
2002), and hence it plays a significant role in carbon and nutrient
rhizodeposition, plant nutrient uptake and microbial community har­
bouring in it (Massaccesi et al., 2015), which cause differentiation in
physical, chemical and biological properties of the rhizosphere soil (RS)
compared to the bulk soil (BS). Differentiation in the soil properties
between rhizosphere and bulk soil is called the “rhizosphere effect”. In
the rhizosphere soil, root exudates comprise low molecular-weight pri­
mary and secondary metabolites and high molecular-weight compounds

* Corresponding author.
E-mail address: sgarkoti@yahoo.com (S.C. Garkoti).

https://doi.org/10.1016/j.geoderma.2021.115626
Received 7 March 2021; Received in revised form 16 November 2021; Accepted 21 November 2021
Available online 3 December 2021
0016-7061/© 2021 Elsevier B.V. All rights reserved.
S. Kumar and S.C. Garkoti
such as mucilage (Phillips, 2002; Proctor and He, 2021). Secondary
metabolites establish an ecological relationship between plant roots and
soil microorganisms and play an essential role in improving soil mi­
crobial community structure and plant health (Proctor and He, 2021).
Plant root exudates constitute approximately 5–10% of the net photo­
synthetically fixed C (Ciccazzo et al., 2014; Farrar et al., 2003); how­
ever, the amount and quality of root exudates vary with the tree species
(Neumann and Römheld, 1999). Plants roots release a wide range of C
compounds, including root caps, border cells, dead root cells and root
exudates (Chen et al., 2017; Ciccazzo et al., 2014). Most of the root
exudates decompose to soil CO2 by the soil microorganisms, and only a
tiny fraction (2–5%) accumulate in soil organic carbon (SOC) pools
(Gunina and Kuzyakov, 2015).
The distribution pattern and composition of soil microbial commu­
nity in the RS primarily depends on the vegetation types, soil pH, soil C:
N ratio and environmental factors like temperature and soil moisture
(Chen et al., 2017; Devi et al., 2014; Li et al., 2018). The energy required
by the soil microbes in rhizosphere processes, including physical,
chemical, biochemical and biological processes, is supplied by the root
exudates (Ciccazzo et al., 2014; Wookey et al., 2009). The efflux and
nutrient uptake capacity of roots vary with tree species and depends
mainly on the root morphology and activity (Lovett et al., 2004). The
spatial distribution (allocation) of rhizodeposits vary with soil depth
because of the changing pedological, physicochemical and biological
properties (Hafner et al., 2014; Salome et al., 2010). In general, soil
organic matter concentrations, roots, soil microbes, and bioavailable
nutrients decrease with increasing soil depths. In comparison to surface
soil, the subsoil supports lower soil microbial biomass, resulting in
reduced mineralisation of root exudates (Proctor and He, 2021), which
affects the sorption of root exudates in the soil (Hafner et al., 2014;
Luster et al., 2009). Multiple studies have reported the rhizosphere ef­
fect of tree species (Li et al., 2021; Massaccesi et al., 2015) and shrubs
(Yuan et al., 2020). Pandey and Palni (2007) reported that the rhizo­
sphere: soil (R:S) ratio of bacteria, actinomycetes and fungi varied
significantly among forests of the central Himalaya, with no study
showing the influence of soil depths and season on the rhizosphere ef­
fect. The seasons significantly influence the rhizosphere soil (Li et al.,
2020), through the change in the microbial community structure,
nutrient content or both. Lower soil moisture content changes bacterial
community by altering membrane composition and favouring proteo­
lytic bacteria (Neumann and Römheld, 1999).
Interactions between plant-soil microbes at the interface of roots
helps to access the health of forest soils. Studies reported the importance
of Rhizosphere Soil Microbial Index (RSMI) values in accessing the soil
quality (Bastida et al., 2006; Sinha et al., 2009; Zhang et al., 2011). Some
previous studies have estimated RSMI values using principal component
analysis (PCA) to investigate the influence of various tree species on soil
health and suggested that species with high RSMI values could be
considered for afforestation in the degraded land ecosystems (Sinha
et al., 2009).
Due to the wide range of variations in topography and climatic
conditions, the central Himalaya exhibits a wide range of flora. The
region is mainly dominated at mid-latitude by two evergreen tree spe­
cies, banj oak (Quercus leucotrichophora) and chir pine (Pinus roxburghii).
Banj oak is a late-successional species found mainly in moisture and
nutrient-rich soils, while chir pine is an early successional species that
generally occupies moisture and nutrient-depleted soils (Singh and
Singh, 1987). Banj oak is found at an elevational range of 1500–2300 m
(Champion and Seth, 1968; Singh and Singh, 1987), and chir pine is
reported in a range of 900–1800 m (Chaturvedi and Singh, 1987; Singh
and Singh, 1987; Zobel and Singh, 1997), while banj oak regenerating
forests are reported at an elevation range of 1800–2200 m (Singh et al.,
2016; Verma and Garkoti, 2019).
In the central Himalayan, many studies were conducted indicating
vegetation structure and composition, fine root production, turnover
and litter decomposition. However, very few studies were performed
2
Geoderma 409 (2022) 115626
that revealed the rhizosphere impacts of different tree species on soil
nutrients, microbial biomass and enzyme activity. We hypothesized
that: (i) the physical, chemical, and biological soil properties in the RS
will significantly differ with the BS because of the carbon and nutrient
inputs to the rhizosphere soil from the plant roots and (ii) the rhizo­
sphere effect on soil properties would vary with the tree species, seasons
and soil depths.
To test the above hypotheses, the study was conducted with the
following objectives:
1. to assess physico-bio-chemical soil properties of the RS and BS.
2. to estimate the influence of forest types, seasonality and soil depths
on the RS soil properties.
3. to evaluate RSMI as an indicator of soil health.
2. Materials and methods
2.1. Study sites description
The study was carried out in the Almora district of the Kumaun re­
gion, Uttarakhand, in the central Himalaya (Fig. 1). Three representa­
tive forests, viz, banj oak (Quercus leucotrichophora), chir pine (Pinus
roxburghii) and banj oak regeneration (banj oak regeneration in chir
pine) were selected. Each forest was more than one hectare in size and
experienced similar climatic conditions. Detailed site characteristics are
given in Table 1.
The study area receives an average annual rainfall of 1100 mm
(range from 1000 to 1205 mm), and the average annual temperature is
17.7 ◦ C (mean annual temperature range from 16 to 19.2 ◦ C) (Mhalla
et al., 2019). The year can be categorised into three seasons, i.e., winter
(December-February), summer (April-June) and rainy (June-
September). In the central Himalayan region (CHR), banj oak (Quercus
leucotrichophora) and chir pine (Pinus roxburghii) are dominant tree
species between 1000 and 2000 m asl. Chir pine often forms pure stands,
while banj oak generally forms mixed stands with other associated tree
species such as Myrica nagi, Rhododendron arboreum, Lyonia ovalifolia.
Banj oak forests have a higher canopy cover that retains significantly
higher soil moisture than the chir pine forests with an open canopy
(Joshi and Garkoti, 2021).
The lithology of this region is a complex mixture of sedimentary and
low-grade metamorphic rocks. The parent rocks are mainly phyllites,
schist, and gneisses, containing mineral compositions like quartz, feld­
spar, mica, chlorite, and muscovite (Valdiya, 1980). Soil is brown in
colour and rich in soil organic matter (SOM) content. Soil texture is
predominantly sandy loam and slightly acidic.
2.2. Forest stands, tree species selections and soil sampling
In each selected forest, three representative trees of a similar
circumference at breast height (CBH) were randomly selected at about
8–10 m distance from each other and tagged. After removing the plant
litter, the soil samples were collected in triplicate from the RS and BS
during the rainy, winter and summer seasons from three soil depths
(0–10 cm, 10–20 cm, and 20–30 cm). Soil corer (diameter 5.6 cm) was
used to collect the soil samples. RS samples were extracted at 10–20 cm
away from the tagged tree, and the BS samples were collected at 1–2 m
away from the respective tree. Both RS and BS samples were brought to
the laboratory, and soil tightly adhering to the root (diameter < 2 mm)
was brushed on the paper bag and considered as rhizosphere soil. For BS
samples, a distance range of 1–2 m from the respective tree was kept to
minimise the influence of the roots (Li et al., 2020; Zhang et al., 2011). A
total of 54 cores were excavated, and each core was separated into three
soil depths. Thus, a total of 162 samples (3 forests × 3 seasons × 3
depths × 2 soil position, RS and BS) were collected for laboratory
analysis. Five replicates of soil bulk density (BD) were collected
following the same sampling procedure in each forest. Field collected
S. Kumar and S.C. Garkoti Geoderma 409 (2022) 115626

Fig. 1. Geographical location of study sites in central Himalaya representing banj oak, chir pine and banj oak regeneration forest.

polyethylene bags and analysed separately in three replicates.

Table 1
Geographical location of study sites in the central Himalaya, Uttarakhand.
2.3. Analysis of soil physico-bio-chemical properties
Sites Location Altitude Aspect Slope Forest types
(m) (⁰)
Soil texture was estimated by the hydrometer method (Bouyoucos,
Kapkot 29◦ 32′ 1795 North 5–10 Q. leucotrichophora 1962). The soil bulk density (g cm− 3) was estimated using a known core
49′′ N
volume, and soil moisture content (%) was measured after oven drying
79◦ 45′
50′′ E
fresh soil at 105 ◦ C till constant weight. The soil pH was measured in a
Dabri 29◦ 32′ 1714 East 20–25 P. roxburghii 1:2.5 (w/v) soil to solution ratio (Jackson, 1958). The soil organic car­
78′′ N bon (SOC) was estimated using the chromic acid wet oxidation method
79◦ 45′ (Walkley and Black, 1934). Soil total nitrogen (TN) content (%) was
48′′ E
calculated using the micro-Kjeldahl digestion and distillation method
That 29◦ 31′ 1810 North- 5–10 Q. leucotrichophora and
67′′ N West P. roxburghii (Jackson, 1958), and Bray and Kurtz (1945) were followed for the
79◦ 44′ estimation of available phosphorus (AP).
72′′ E Soil microbial biomass carbon (Cmic), nitrogen (Nmic) and phos­
phorus (Pmic) were estimated by the chloroform fumigation-extraction
(FE) method (Brookes et al., 1985; Wu et al., 1990). Ethanol-free chlo­
fresh soil samples (from the RS and BS) were sieved through a 2-mm
roform was used for the fumigation process. Cmic and Nmic soil samples
mesh and divided into two parts, samples in the first part were air-
were extracted with the 0.5 M K2SO4 solutions.
dried and used for soil physical and chemical analysis and the second
Cmic was calculated using the formula as below:
part were stored at 4 ⁰C (for a period not exceeding one week) for bio­
logical analysis (microbial biomass and enzyme). To minimise the EC(f ) − EC(nf )
Cmic =
moisture loss and other interferences, soil samples were sealed in double KEC

3
S. Kumar and S.C. Garkoti Geoderma 409 (2022) 115626

Table 2
Physicochemical characteristics of rhizosphere (RS) and bulk soil (BS) in rainy, winter and summer seasons (mean ± se, n = 3).
Attributes Seasons

Soil Soil Depth Rainy Winter Summer

Parameters Positions (cm)
Banj- Chir- Banj-Oak Banj- Chir- Banj-Oak Banj- Chir- Banj-Oak
Oak Pine Regeneration Oak Pine Regeneration Oak Pine Regeneration

Moisture RS 0–10 25.16 ± 18.70 ± 23.42 ± 20.78 ± 18.36 ± 20.86 ± 0.13aA 23.58 ± 15.13 ± 17.34 ± 1.81aB
(%) 1.47aA 0.57aB 0.75aAB 0.67aA 0.61aA 1.00aA 0.43aB
10–20 17.98 ± 14.01 ± 16.48 ± 0.10aA 14.77 ± 16.18 ± 15.96 ± 1.90aA 15.68 ± 8.06 ± 11.23 ±
0.33aA 0.81aA 0.86aA 2.38aA 0.95aA 1.13aB 1.72aAB
20–30 14.65 ± 13.00 ± 15.99 ± 0.32aA 12.88 ± 12.99 ± 12.13 ± 0.24aA 13.47 ± 8.19 ± 9.71 ± 0.59aA
1.67aA 0.74aA 0.39aA 0.15aA 0.69aA 1.57aA
Mean 19.26 15.23 ± 18.89 ± 0.58 16.14 ± 15.84 ± 16.31 ± 0.84 17.57 ± 10.46 ± 12.76 ± 0.78
± 1.03 0.58 0.79 0.52 1.02 0.78
BS 0–10 22.47 ± 15.41 ± 18.52 ± 19.32 ± 15.11 ± 14.46 ± 0.18bA 18.61 ± 21.11 ± 13.93 ± 1.35bB
1.46aA 1.14aB 0.32bAB 0.61aA 0.40aA 1.60aAB 1.67aA
10–20 20.43 ± 15.17 ± 12.68 ± 17.59 ± 14.52 ± 12.70 ± 0.45aA 11.72 ± 13.43 ± 10.71 ± 0.93aA
0.11aA 0.79aAB 1.20bAC 0.74aA 2.02aA 1.83aA 1.46aA
20–30 18.59 ± 18.12 ± 14.79 ± 0.25aA 15.68 ± 12.36 ± 11.47 ± 0.20aA 9.50 ± 7.86 ± 7.03 ± 0.47aA
2.15aA 1.23aA 2.22aA 0.13aA 1.15aA 1.46aA
Mean 20.49 16.23 ± 15.33 ± 0.67 17.53 ± 13.99 ± 12.87 ± 0.29 13.27 ± 14.13 ± 10.55 ± 0.66
± 0.37 0.31 0.35 0.28 0.91 1.28
pH RS 0–10 5.19 ± 6.08 ± 5.79 ± 0.03aA 5.33 ± 6.10 ± 6.21 ± 0.07aA 5.52 ± 6.06 ± 5.54 ± 0.06aB
0.03aB 0.06aA 0.03aC 0.01aB 0.06aB 0.10aA
10–20 5.16 ± 6.06 ± 5.79 ± 0.05aAB 5.23 ± 6.16 ± 6.35 ± 0.03aA 5.14 ± 5.92 ± 5.54 ± 0.12aA
0.00aC 0.01aA 0.03aC 0.02aB 0.07aB 0.07aA
20–30 5.09 ± 5.83 ± 5.74 ± 0.02aA 5.20 ± 6.11 ± 6.25 ± 0.04aA 5.24 ± 6.17 ± 5.50 ± 0.03aB
0.28aA 0.01aA 0.06aC 0.00aB 0.05aB 0.13aA
Mean 5.14 ± 5.99 ± 5.77 ± 0.02 5.25 ± 6.12 ± 6.27 ± 0.03 5.30 ± 6.05 ± 5.52 ± 0.01
0.14 0.07 0.04 0.02 0.11 0.07
BS 0–10 5.25 ± 6.07 ± 6.01 ± 0.01aAB 5.53 ± 6.14 ± 6.09 ± 0.01aA 5.57 ± 6.12 ± 5.85 ± 0.07bA
0.01aC 0.03aA 0.15aB 0.03aA 0.31aA 0.09aA
10–20 5.17 ± 5.95 ± 5.93 ± 0.03aAB 5.83 ± 6.18 ± 6.00 ± 0.05bA 5.66 ± 5.99 ± 5.80 ± o.04bA
0.01aC 0.01aA 0.03aB 0.01aA 0.28A 0.03aA
20–30 5.19 ± 5.96 ± 5.88 ± 0.02aAB 5.27 ± 6.21 ± 6.24 ± 0.03aA 5.66 ± 6.12 ± 6.03 ± 0.06bA
0.03aC 0.10aA 0.03aA 0.05aA 0.23aA 0.16aA
Mean 5.21 ± 5.99 ± 5.94 ± 0.07 5.54 ± 6.17 ± 6.11 ± 0.07 5.63 ± 6.07 ± 5.89 ± 0.07
0.01 0.03 0.18 0.02 0.00 0.07
SOC (%) RS 0–10 3.04 ± 2.21 ± 2.18 ± 0.28aBC 2.17 ± 1.92 ± 1.95 ± 0.03aA 2.99 ± 1.77 ± 1.78 ± 0.02aBC
0.25aA 0.06aB 0.02aA 0.04aA 0.01aA 0.02aB
10–20 1.66 ± 1.25 ± 1.59 ± 0.27aA 1.66 ± 1.19 ± 1.20 ± 0.02aBC 1.21 ± 0.95 ± 1.53 ± 0.06aBC
0.02aA 0.01aA 0.13aA 0.06aB 0.03aA 0.06aB
20–30 1.09 ± 0.84 ± 0.70 ± 0.07aA 0.77 ± 0.79 ± 0.67 ± 0.02aA 0.96 ± 0.47 ± 0.66 ± 0.01aBC
0.02aA 0.04aA 0.04aA 0.01aA 0.06aA 0.01bB
Mean 1.93 ± 1.43 ± 1.49 ± 0.43 1.53 ± 1.3 ± 1.27 ± 0.37 1.72 ± 1.06 ± 1.32 ± 0.20
0.58 0.41 0.41 0.33 0.64 0.38
BS 0–10 1.29 ± 2.17 ± 1.75 ± 0.02aB 1.49 ± 1.70 ± 1.72 ± 0.07aA 1.26 ± 1.40 ± 1.70 ± 0.09aA
0.02bC 0.05aA 0.07bA 0.05aA 0.02bA 0.14aA
10–20 1.10 ± 0.92 ± 0.72 ± 0.08bAC 0.57 ± 0.70 ± 0.62 ± 0.06bA 0.57 ± 0.80 ± 0.44 ± 0.02bB
0.02bA 0.08aA 0.06bA 0.09bA 0.03bAB 0.04aA
20–30 0.90 ± 0.65 ± 0.65 ± 0.04aBC 0.31 ± 0.61 ± 0.46 ± 0.03aA 0.51 ± 0.80 ± 0.53 ± 0.03aA
0.02bA 0.02aB 0.05bA 0.04aA 0.03bA 0.06aA
Mean 1.64 ± 1.24 ± 1.04 ± 0.36 0.79 ± 1.0 ± 0.93 ± 0.40 0.78 ± 1.0 ± 0.89 ± 0.22
0.11 0.47 0.36 0.35 0.224 0.20
TN (%) RS 0–10 0.37 ± 0.24 ± 0.29 ± 0.02aA 0.36 ± 0.25 ± 0.23 ± 0.00aA 0.32 ± 0.19 ± 0.18 ± 0.04aAB
0.01aA 0.00aA 0.01aA 0.00aA 0.03aA 0.06aB
10–20 0.20 ± 0.16 ± 0.15 ± 0.00aA 0.17 ± 0.14 ± 0.14 ± 0.01aA 0.17 ± 0.10 ± 0.15 ± 0.03aA
0.01aA 0.01aA 0.00aA 0.02aA 0.02aA 0.03aA
20–30 0.16 ± 0.15 ± 0.30 ± 0.15aA 0.16 ± 0.22 ± 0.15 ± 0.00aAB 0.13 ± 0.09 ± 0.10 ± 0.03aA
0.01aA 0.00aA 0.00aB 0.09aA 0.03aA 0.04aA
Mean 0.26 ± 0.18 ± 0.25 ± 0.05 0.19 ± 0.18 ± 0.17 ± 0.04 0.20 ± 0.12 ± 0.15 ± 0.03
0.06 0.03 0.03 0.02 0.06 0.03
BS 0–10 0.25 ± 0.23 ± 0.18 ± 0.00aB 0.22 ± 0.21 ± 0.18 ± 0.00aB 0.13 ± 0.16 ± 0.13 ± 0.04aA
0.00bA 0.00aA 0.02bA 0.00aA 0.01bA 0.05aA
10–20 0.20 ± 0.17 ± 0.14 ± 0.00aC 0.21 ± 0.16 ± 0.11 ± 0.01aC 0.10 ± 0.09 ± 0.11 ± 0.02aA
0.00aA 0.00aB 0.00aA 0.00aB 0.01aA 0.03aA
20–30 0.21 ± 0.16 ± 0.11 ± 0.00aC 0.09 ± 0.11 ± 0.13 ± 0.00aA 0.10 ± 0.06 ± 0.06 ± 0.02aA
0.01aA 0.01aB 0.00aA 0.00aA 0.01aA 0.02aA
Mean 0.22 ± 0.18 ± 0.14 ± 0.02 0.17 ± 0.16 ± 0.14 ± 0.04 0.11 ± 0.10 ± 0.10 ± 0.01
0.01 0.02 0.04 0.03 0.01 0.03
AP (%) RS 0–10 0.07 ± 0.04 ± 0.03 ± 0.00aBC 0.05 ± 0.02 ± 0.02 ± 0.00aBC 0.04 ± 0.05 ± 0.06 ± 0.01aA
0.01aA 0.00aB 0.01aA 0.01aB 0.01aA 0.03aA
10–20 0.05 ± 0.03 ± 0.03 ± 0.00aA 0.04 ± 0.01 ± 0.01 ± 0.01aBC 0.02 ± 0.01 ± 0.01 ± 0.00aA
0.00aA 0.01aA 0.01aA 0.00aB 0.01aA 0.00aA
20–30 0.04 ± 0.03 ± 0.04 ± 0.01aA 0.03 ± 0.01 ± 0.01 ± 0.01aA 0.01 ± 0.03 ± 0.02 ± 0.01aA
0.00aA 0.01aA 0.00aA 0.00aA 0.00aA 0.01aA
(continued on next page)

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S. Kumar and S.C. Garkoti Geoderma 409 (2022) 115626

Table 2 (continued )
Attributes Seasons

Soil Soil Depth Rainy Winter Summer

Parameters Positions (cm)
Banj- Chir- Banj-Oak Banj- Chir- Banj-Oak Banj- Chir- Banj-Oak
Oak Pine Regeneration Oak Pine Regeneration Oak Pine Regeneration

Mean 0.05 ± 0.03 ± 0.03 ± 0.00 0.04 ± 0.01 ± 0.01 ± 0.00 0.02 ± 0.03 ± 0.03 ± 0.01
0.01 0.00 0.01 0.00 0.01 0.01
BS 0–10 0.05 ± 0.04 ± 0.03 ± 0.00aA 0.05 ± 0.02 ± 0.03 ± 0.01aA 0.02 ± 0.03 ± 0.04 ± 0.00bA
0.00aA 0.00aA 0.00aA 0.00aA 0.01aA 0.02aA
10–20 0.03 ± 0.03 ± 0.03 ± 0.01aA 0.03 ± 0.01 ± 0.02 ± 0.01aA 0.02 ± 0.01 ± 0.01 ± 0.00aA
0.01aA 0.00aA 0.00aA 0.00aA 0.01aA 0.00aA
20–30 0.05 ± 0.03 ± 0.01 ± 0.01aA 0.04 ± 0.02 ± 0.01 ± 0.01aA 0.02 ± 0.02 ± 0.03 ± 0.00aA
0.01aA 0.01aA 0.01aA 0.00aA 0.00bA 0.00aA
Mean 0.03 ± 0.03 ± 0.02 ± 0.00 0.04 ± 0.01 ± 0.02 ± 0.01 0.02 ± 0.02 ± 0.02 ± 0.01
0.01 0.00 0.01 0.00 0.00 0.01
C: N RS 0–10 8.24 ± 9.11 ± 11.68 ± 1.27aA 8.31 ± 10.16 ± 8.46 ± 0.09aA 9.41 ± 9.12 ± 10.47 ± 0.94aA
0.71aA 0.38aA 0.16aA 0.44aA 0.86aA 0.10aA
10–20 8.15 ± 7.65 ± 8.25 ± 1.72aA 9.59 ± 8.64 ± 8.85 ± 0.50aA 7.36 ± 9.56 ± 11.51 ± 3.5aA
0.42aA 0.31aA 0.48aA 1.48aA 0.49aAC 1.28aAB
20–30 6.86 ± 5.63 ± 3.20 ± 0.89aA 4.84 ± 4.60 ± 6.50 ± 0.32aA 9.69 ± 7.27 ± 6.53 ± 0.49aBC
0.21aA 0.14aA 0.36aA 1.2aA 0.51aA 2.11aAB
Mean 7.25 ± 7.46 ± 7.71 ± 1.46 7.58 ± 7.80 ± 7.94 ± 0.73 8.82 ± 8.65 ± 9.50 ± 1.52
0.45 1.01 1.42 1.66 0.73 0.70
BS 0–10 5.25 ± 9.43 ± 9.86 ± 0.31aAB 6.78 ± 7.92 ± 9.78 ± 0.53aA 9.55 ± 8.72 ± 14.16 ± 1.70aA
0.15aC 0.38aA 0.19aAC 0.37aAB 0.79aAB 0.19aB
10–20 5.46 ± 5.55 ± 5.33 ± 0.60aA 2.81 ± 4.30 ± 5.59 ± 0.29aA 5.64 ± 9.14 ± 7.33 ± 0.61aA
0.14aA 0.50aA 0.32bAC 0.44aAB 0.63aA 0.40aA
20–30 4.41 ± 4.17 ± 5.62 ± 0.28aA 3.42 ± 5.70 ± 5.15 ± 0.58aA 5.99 ± 15.10 ± 8.99 ± 1.15aAB
0.18aA 0.22aA 0.59aB 0.12aA 1.6aB 2.6aA
Mean 5.04 ± 6.38 ± 6.94 ± 1.47 4.33 ± 5.98 ± 6.84 ± 1.47 7.06 ± 11 ± 10.16 ± 2.06
0.32 1.57 1.24 1.05 1.25 2.06
Cmic:SOC RS 0–10 1.08 ± 0.73 ± 0.79 ± 0.07aA 0.78 ± 0.43 ± 0.65 ± 0.03aA 0.82 ± 0.70 ± 0.86 ± 0.09aA
0.11bA 0.04aA 0.01bA 0.05aA 0.06aA 0.16aA
10–20 1.28 ± 1.21 ± 1.14 ± 0.27bA 0.83 ± 0.60 ± 0.98 ± 0.01aA 1.58 ± 0.80 ± 0.81 ± 0.25aA
0.08aA 0.14aA 0.12aA 0.03aA 0.34aA 0.18aA
20–30 1.44 ± 1.19 ± 1.65 ± 0.21aA 1.24 ± 0.53 ± 0.33 ± 0.19aC 1.42 ± 1.64 ± 0.51 ± 0.15aA
0.06aA 0.08aA 0.10aA 0.01aB 0.63aA 0.49aA
Mean 1.27 ± 1.04 ± 1.19 ± 0.25 0.95 ± 0.52 ± 0.65 ± 0.19 1.27 ± 1.05 ± 0.73 ± 0.11
0.10 0.16 0.15 0.05 0.23 0.30
BS 0–10 1.49 ± 0.51 ± 0.95 ± 0.05aAB 1.73 ± 0.44 ± 0.52 ± 0.03aA 1.27 ± 0.75 ± 0.79 ± 0.16aA
0.07aA 0.03aB 0.07aA 0.02aA 0.25aA 0.11aA
10–20 1.49 ± 0.68 ± 2.04 ± 0.19aA 1.19 ± 0.78 ± 1.43 ± 0.23aA 1.86 ± 1.05 ± 2.03 ± 0.73aA
0.09aA 0.12bB 0.20aA 0.11aA 0.27aA 0.23aA
20–30 1.13 ± 1.55 ± 1.60 ± 0.22aA 2.17 ± 0.74 ± 0.24 ± 0.14aBC 3.12 ± 0.88 ± 1.83 ± 0.28aA
0.07aA 0.17aA 0.40bA 0.08aB 0.85aA 0.12aA
Mean 1.37 ± 0.91 ± 1.53 ± 0.32 1.69 ± 0.65 ± 0.73 ± 0.36 2.08 ± 0.90 ± 1.55 ± 0.38
0.12 0.11 0.43 0.11 0.55 0.09
Nmic:TN RS 0–10 0.87 ± 0.84 ± 1.23 ± 0.35aA 0.57 ± 0.48 ± 0.42 ± 0.07aA 0.87 ± 0.71 ± 0.79 ± 0.13aA
0.09aA 0.16aA 0.07aA 0.09aA 0.13aA 0.06aA
10–20 1.24 ± 0.99 ± 1.05 ± 0.08aA 0.63 ± 0.48 ± 0.53 ± 0.06aA 0.98 ± 0.89 ± 1.04 ± 0.27aA
0.25aA 0.23aA 0.10aA 0.07aA 0.16aA 0.22aA
20–30 1.20 ± 0.73 ± 0.58 ± 0.22aA 0.59 ± 0.25 ± 0.13 ± 0.07aA 0.83 ± 1.94 ± 0.78 ± 0.08aA
0.10aA 0.14aA 0.06aA 0.07aA 0.27aA 0.40aA
Mean 1.11 ± 0.85 ± 0.95 ± 0.19 0.59 ± 0.40 ± 0.36 ± 0.12 0.68 ± 1.18 ± 0.87 ± 0.09
0.12 0.08 0.02 0.08 0.21 0.38
BS 0–10 0.53 ± 0.67 ± 1.30 ± 0.27aA 0.27 ± 0.32 ± 0.61 ± 0.16aA 0.98 ± 0.59 ± 1.06 ± 0.29aA
0.07aA 0.06aA 0.05aA 0.03aA 0.10aA 0.08aA
10–20 0.61 ± 0.85 ± 1.15 ± 0.23aA 0.27 ± 0.38 ± 0.64 ± 0.17aA 0.93 ± 0.53 ± 1.08 ± 0.31aA
0.21bA 0.12aA 0.10aA 0.04aA 0.21aA 0.02aA
20–30 0.26 ± 0.51 ± 0.81 ± 0.27aA 0.30 ± 0.39 ± 0.24 ± 0.14aA 1.35 ± 0.83 ± 1.31 ± 0.40aA
0.04aA 0.17aA 0.07aA 0.10aA 0.50aA 0.37aA
Mean 0.47 ± 0.68 ± 1.08 ± 0.15 0.28 ± 0.35 ± 0.50 ± 0.13 1.09 ± 0.65 ± 1.15 ± 0.08
0.10 0.10 0.01 0.02 0.13 0.09
Pmic:AP RS 0–10 1.62 ± 1.21 ± 1.65 ± 0.06aA 1.89 ± 0.67 ± 2.81 ± 0.29aA 1.09 ± 0.49 ± 0.59 ± 0.28a A
0.14aA 0.18aA 0.12aA 0.19aB 0.22aA 0.32aA
10–20 1.96 ± 0.86 ± 0.94 ± 0.04aBC 1.39 ± 0.46 ± 2.11 ± 0.09aA 0.57 ± 0.79 ± 1.15 ± 0.15aA
0.18aA 0.15aB 0.15aB 0.09aB 0.14aA 0.08aA
20–30 2.02 ± 0.21 ± 0.31 ± 0.04aBC 1.35 ± 0.56 ± 0.24 ± 0.14aAB 0.38 ± 0.73 ± 0.85 ± 0.38aA
0.22aA 0.05aB 0.50aA 0.04aA 0.08aA 0.57aA
Mean 1.87 ± 0.76 ± 0.97 ± 0.39 1.54 ± 0.56 ± 1.72 ± 0.77 0.68 ± 0.67 ± 0.86 ± 0.16
0.12 0.29 0.18 0.06 0.21 0.09
BS 0–10 0.78 ± 1.22 ± 1.38 ± 0.14aA 0.54 ± 0.69 ± 1.63 ± 0.72bA 1.55 ± 1.22 ± 0.13 ± 0.01aB
0.10bA 0.34aA 0.05bB 0.53aB 0.48aA 0.52aA
10–20 1.38 ± 0.24 ± 1.21 ± 0.24aA 0.96 ± 0.29 ± 1.23 ± 0.22aA 1.00 ± 1.02 ± 0.70 ± 0.01aA
0.18aA 0.06aB 0.04aA 0.02aB 0.24aA 0.19aA
20–30 0.22 ± 0.04aA 0.92 ± 0.53aA 0.20 ± 0.03aA
(continued on next page)

5
S. Kumar and S.C. Garkoti Geoderma 409 (2022) 115626

Table 2 (continued )
Attributes Seasons

Soil Soil Depth Rainy Winter Summer

Parameters Positions (cm)
Banj- Chir- Banj-Oak Banj- Chir- Banj-Oak Banj- Chir- Banj-Oak
Oak Pine Regeneration Oak Pine Regeneration Oak Pine Regeneration

0.36 ± 0.20 ± 0.63 ± 0.39 ± 0.60 ± 0.54 ±

0.04bA 0.02aA 0.17aA 0.06aA 0.06aA 0.06aA
Mean 0.84 ± 0.56 ± 0.94 ± 0.36 0.71 ± 0.46 ± 1.26 ± 0.21 1.05 ± 0.92 ± 0.34 ± 0.18
0.30 0.33 0.13 0.12 0.28 0.20

Capital letters (ABC): significant differences (p < 0.05) among the three forests within the season at the same soil position of same depths; small letters (ab): significant
differences (p < 0.05) between soil positions (RS vs BS) at same soil depths. SOC = soil organic carbon, TN = total nitrogen, AP = available phosphorus, Cmic =
microbial biomass carbon, Nmic = microbial biomass nitrogen, Pmic = microbial biomass phosphorus.

where EC (f) = extracted carbon from fumigated soil sample, EC (nf) =
extracted carbon from non-fumigated soil, KEC = 0.45 (Vance et al.,
1987).
2.3.1. Microbial biomass nitrogen (Nmic)
The 20 ml extractant of 0.5 N K2SO4 was digested and distilled in the
micro-Kjeldahl system to determine the ammonia gas.
Nmic was calculated using the formula as below:
Nmic =
EN(f ) − EN(nf )
KN
where EN(f) = extracted nitrogen from fumigated soil, EN (nf) =
extracted nitrogen from non-fumigated soil, KN = 0.54 (Brookes et al.,
1985).
2.3.2. Microbial biomass phosphorus (Pmic)
Pmic was measured as described by Wu et al. (2000). In 20 g of both
fumigated and non-fumigated soil samples, 80 ml of extracting solution
(NH4F-HCl) was added. Prepared soil solutions were centrifuged at
3,000 rpm for 10 min, then shaken until the complete mixing of soil with
the suspension and filtered using Whatman no. 42 filter paper. The ex­
tracts were analysed for inorganic P using the ammonium-molybdate
blue method (Olsen and Sommer, 1982). Pmic was calculated using
the formula as below:
EP(f ) − EP(nf )
Pmic =
KP
where EP(f) = extracted phosphorus in fumigated soil, EP (nf) =
extracted phosphorus from non-fumigated soil, KP = fraction of biomass
phosphorus extracted after fumigation (0.40) (Brookes et al., 1982).
The soil microbial quotients including Cmic: SOC, Nmic: TN and
Pmic: AP were calculated (Fan et al., 2010) for estimating the microbial
efficiency of soil C, N and AP.
2.3.3. Dehydrogenase enzyme activity (DHA)
The soil dehydrogenase enzyme activity was determined by the
following Klein et al. (1971). One gram of fresh soil sample was placed in
screw-capped tubes with 0.2 ml 3% TTC (triphenyl tetrazolium chloride)
and 0.5 ml 1% glucose solution and incubated for 24 h at 28 ◦ C. After the
incubation, 10 ml methanol was added and incubated for 6 h at 28 ◦ C.
Triphenyl tetrazolium formazan (TPF) formation was measured at 485
nm wavelength in a spectrophotometer (Shimadzu UV-1800).
2.4. Rhizosphere soil microbial index (RSMI)
soil properties, PCA was performed. PCs retaining high eigenvalues (≥1)
mainly represents variations in the forest types. Under each PC, soil
properties having higher loading values represent that property contri­
bution in PC composition. Only the highly weighted soil properties were
retained from each PC for the indexing (Table 5). Highly weighted
variables were described as having absolute values within 10% of the
highest factor loading (Andrews et al., 2002). When more than one soil
property retained higher loading under a single PC, then correlation
(using Pearson correlation) was checked. Among highly correlated soil
variables, the variable with the highest factor loading was considered for
the indexing (Andrews et al., 2002). To transform the microbial property
values into the score (S) following equation was used.
[ ]
S = a/ 1 + (x/x0 )b (1)
where S is the score of the proposed microbial property after trans­
formation; a is the maximum score (here a = 1), x is the value of the
microbial property, and x0 is the mean value of each microbial property,
b represents the value of the slope of the equation. PCA has two kinds of
properties, positive ‘+’ (more is better) and negative ‘-’ (less is better).
To obtain the sigmoid curve tending to 1 for all microbial properties,
− 2.5 and 2.5 were used as a b value for ‘more is better’ and ‘less is
better’, respectively (Sinha et al., 2009).
PCA based RSMI equation is as follow
∑n
RSMI = WiSi (2)
i=1
W is the weighting factor of the microbial property obtained from PCA,
and S is the respective score. The equation was finally normalised to get
a maximum RSMI value of 1.
2.5. Statistical analysis
A two-way analysis of variance (ANOVA) was performed between
soil positions (RS and BS), seasons, soil depths and forest types (3 types),
and a Tukey HSD post-hoc test was used to compare the means. Linear
mixed-effect model (LMM) was performed using package “lme4” to
observe the differences in the soil microbial biomass between forest
types, soil positions, seasons and soil depths. (Table 4). To obtain chi-
square (χ2) and p-value, the likelihood-ratio test was performed. Pear­
son correlation was used to check out the association among physico-
bio-chemical properties of the soils. PCA was performed using the
package “Factomini”, and “Factoextra” in R. Entire statistical analysis
was performed in R studio statistical software version 3.6.3 (R core team
2020).

Principally, the method was described by Bastida et al. (2006) and
Sinha et al. (2009), and an integrated procedure was developed by Sinha
et al. (2009) to calculate the RSMI. The indexing is generally performed
in three steps: (1) Selection of appropriate soil properties (physico-bio-
chemical), (2) transformation and weighting of properties; and (3)
Combining the score (S) into an index. For the selection of appropriate
3. Results
3.1. Physicochemical characteristics of rhizosphere soil and bulk soil of
different forest types
The RS of banj oak has a significantly (p < 0.05) higher SOC than the

6
S. Kumar and S.C. Garkoti Geoderma 409 (2022) 115626

BS, while RS of chir pine and banj oak regeneration generally showed

Soil properties represents BD = bulk density, SOC = soil organic carbon, TN = total nitrogen, AP = available phosphorus, DHA = dehydrogenase enzyme activity, Cmic = microbial biomass carbon, Nmic = microbial
Nmic:TN
higher SOC than the BS across the seasons. Among the forests, SOC was
significantly higher in banj oak RS than the RS of chir pine and banj oak

0.07
regeneration in the upper soil layer (0–10 cm) of the rainy and summer
season (Table 2). However, no difference of SOC was found between RS
Cmic:SOC

of chir pine and RS of banj oak regeneration (Table 2). The total nitrogen

0.38**
− 0.14
(TN) content in RS of banj oak, chir pine and banj oak regeneration was
higher than BS in the upper soil layer. Across the seasons, available
phosphorus (AP) was almost similar in both RS and BS in all three for­

0.51***
ests. AP was significantly higher in RS of banj oak compared to RS of chir

− 0.21
DHA

0.16
pine and banj oak regeneration in the upper soil layer of the rainy and
winter season. In the upper soil layer, the RS of banj oak and chir pine
generally has lower soil pH than the BS. Unlike the upper soil layer, no

0.76***

0.75***
trend was observed in the middle (10–20 cm) and lower soil layers

− 0.06
0.12
Pmic

(20–30 cm). RS of banj oak regeneration showed lower pH in rainy and

summer, mainly in upper soil layers, while the opposite was observed in
the winter season. In rainy and winter, RS of chir pine and banj oak
0.68***
0.78***

0.50***
0.38** regeneration significantly had higher soil pH than the RS of banj oak in
− 0.05
Nmic

upper and middle soil layers. SOC: TN ratio was similar in both RS and
BS soil. Across the season, RS of banj oak regeneration significantly had
0.86***
0.71***
0.79***

higher soil moisture content than BS in the upper soil layer. Both RS and
0.39**
0.34*
Cmic

0.15

BS of banj oak and chir pine showed almost similar soil moisture content
in all seasons. The sand content in the RS (73–79.5%) of banj oak, chir
pine and banj oak regeneration forests was lower than the BS (77–80%),
0.36**
0.27*

0.29*
0.22

0.10

− 0.42

0.17

while silt and clay contents were higher in the RS (15–21% and 9–12%)
C: N

compared to the BS (11–17% and 7–12%). The RS of banj oak and banj
oak regeneration forests showed a lower bulk density (0.87 ± 0.10–0.96
Pearson correlation coefficients between soil physical, chemical and biological properties of rhizosphere (RS) and bulk soil (BS).

± 0.04 g cm− 3) than the BS (0.98 ± 0.02–1.01 ± 0.02 g cm− 3) (sup­

0.57***
0.55***
0.56***
0.58***
− 0.06

0.01
0.07
0.05

plementary Table 1).

AP

3.2. Soil biological properties

0.61***

0.60***
0.60***
0.57***
0.69***
− 0.10

− 0.33
− 0.25
0.26

Across the seasons, the RS of banj oak had significantly (p < 0.05)
TN

higher soil microbial biomass (Cmic, Nmic and Pmic) and dehydroge­
nase enzyme activity (DHA) compared to the BS in upper and middle soil
0.73***
0.48***
0.52***
0.68***
0.73***
0.61***
0.81***

0.42**

layers (Fig. 2 a-l). In the lower soil layer, soil microbial biomass was
− 0.53
0.03
SOC

almost similar in both RS and BS soil. The RS of chir pine had signifi­
biomass nitrogen, Pmic = microbial biomass phosphorus. p-value 0.001(***), 0.01(**), 0.05(*).

cantly higher Cmic and Pmic in the upper and middle soil layers during
− 0.14
− 0.25
− 0.38

− 0.47
− 0.32
− 0.30
− 0.43
− 0.19
− 0.13

the rainy season (Fig. 2 a, g). Banj oak regeneration forest showed
0.05

0.00
pH

almost similar values of soil microbial biomass for both the RS and BS in
all seasons. Across the seasons, the RS of banj oak had significantly
Porosity

higher soil microbial biomass compared to RS of chir pine in upper and

0.08
0.19
0.10
0.09
0.13
0.07
0.13
0.22
0.18
− 0.16
0.03
0.13

middle soil layers. Soil microbial biomass did not vary between RS of
chir pine and RS of banj oak regeneration forests. The RS of banj oak had
significantly higher microbial biomass compared to the RS of banj oak
− 0.82
− 0.05
− 0.33
− 0.16
− 0.07
− 0.23
− 0.10
− 0.15
− 0.15
− 0.29

− 0.15

regeneration in upper soil depths (in all seasons); however, the values
0.25
0.01
BD

did not vary in middle and lower soil layers. DHA was significantly
higher in the RS of banj oak than the RS of chir pine in the upper soil
0.46***

0.43**

layer across the seasons (Fig. 2 j-l). In RS, microbial biomass and DHA
− 0.21

− 0.01

− 0.54
− 0.07
− 0.05
Clay

0.15
0.22

0.21
0.04

0.03
0.20

0.15

decreases significantly between upper and lower soil depths in banj oak
and banj oak regeneration forests (Fig. 3 a-d). Chir pine showed signif­
0.80***

0.47***

icant variations in DHA between upper and lower soil depths. Season­
0.39**
0.28*
− 0.22
0.16

0.23
0.10

0.07
0.24
− 0.01
0.20
− 0.44
− 0.01
− 0.04

ally, Nmic and Pmic in the RS of banj oak changed significantly (Fig. 3 b-
Silt

c). The RS of banj oak and chir pine significantly has higher soil mi­
crobial biomass in the rainy season than in the winter, particularly in the
0.51***

upper soil layer (Fig. 3 a-c). The outcome of the linear mixed effect
− 0.95
− 0.95

− 0.16
− 0.26
− 0.49
− 0.23
− 0.07
− 0.43
− 0.05
− 0.23

− 0.18
Sand

0.22

0.01

0.04
0.05

model (LMM) revealed that Cmic, Nmic and Pmic were significantly (p
< 0.05) influenced by forest types, soil positions, seasons, soil depths
and their interactions (Table 4). Our results revealed that in the RS of
Moisture

0.69***
0.78***
0.56***

0.64***
0.58***
0.60***
0.72***

0.39**

banj oak and banj oak regeneration forests, microbial biomass and DHA
− 0.10

− 0.17

− 0.33

− 0.01

− 0.26
− 0.22
0.09
0.10

0.13

decreased more rapidly with the soil depths than the BS (Fig. 3 a-d).
DHA was found to be higher across the forests, seasons, and soil depths
in the RS (2.90 to 5.37 µg TPF g− 1 soil hr− 1) than the BS (2.78 to 3.68 µg
Parameters

Cmic:SOC

TPF g− 1 soil hr− 1). Across the seasons and soil depths, the RS of the banj
Nmic:TN
Pmic:AP
Porosity
Table 3

oak forest showed maximum DHA activity (2.40 to 8.51 µg TPF g− 1 soil
Nmic
Cmic

Pmic
Sand

DHA
Clay

C: N
SOC
Silt

TN
BD

pH

AP

hr− 1) followed by the banj oak regeneration (1.04 to 7.67 µg TPF g− 1 soil

7
S. Kumar and S.C. Garkoti Geoderma 409 (2022) 115626

Table 4
Summary of LMM, showing relationship between soil biological properties and different attributes (soil positions, depths, seasons). Forest was considered as a random
effect. p value obtained by likelihood-ratio test. Table contains chi-square value (χ2), from the likelihood-ratio test, degree of freedom (df), and the associated p value.
Soil Parameters Forest types Position Depth Season Position × Depth Position × Season Depth × Season
(df = 2) (df = 1) (df = 2) (df = 2) (df = 3) (df = 3) (df = 6)

Cmic χ2 = 93.0*** χ2 = 21.2*** χ2 = 74.1*** χ2 = 76.3*** χ2 = 30.7*** χ2 = 63.1*** χ2 = 80.9***

Nmic χ2 = 22.9*** χ2 = 27.1*** χ2 = 68.1*** χ2 = 98.6*** χ2 = 28.7*** χ2 = 38.0*** χ2 = 82.8***
Pmic χ2 = 71.1*** χ2 = 17.0*** χ2 = 45.5*** χ2 = 58.4*** χ2 = 21.9*** χ2 = 65.2*** χ2 = 57.6***
Cmic/SOC χ2 = 28.4*** χ2 = 11.5*** χ2 = 36.1*** χ2 = 13.9** χ2 = 14.9** χ2 = 13.5** χ2 = 37.0***
Nmic/TN χ2 = 3.8 ns χ2 = 2.0 ns χ2 = 0.54*** χ2 = 35.8*** χ2 = 2.3 ns χ2 = 4.0 ns χ2 = 10.3*
Pmic/AP χ2 = 18.4*** χ2 = 18.8*** χ2 = 19.3*** χ2 = 19.0*** χ2 = 11.8** χ2 = 11.8** χ2 = 22.8***

Soil properties represents Cmic = microbial biomass carbon, Nmic = microbial biomass nitrogen Pmic = microbial biomass phosphorus, SOC = soil organic carbon, TN
= total nitrogen, AP = available phosphorus. p-value 0.001(***), 0.01(**), 0.05(*).

hr− 1) and the chir pine forest (1.10 to 5.17 µg TPF g− 1 soil hr− 1)
(Fig. 3d).
Across the forests, soil positions, seasons and soil depths, the Cmic:
SOC, Nmic: TN and Pmic: AP ranged from 0.52 to 1.65, 0.28 to 1.18 and
0.34 to 1.72, respectively (Table 2). In banj oak forest, the soil microbial
quotient (Cmic: SOC and Pmic: AP) varied significantly (p < 0.05) be­
tween the RS and the BS in upper soil layer of rainy and winter seasons
(Table 2). Nmic: TN ratio was almost similar in both the RS and the BS in
each forest. Cmic: SOC and Pmic: AP mean values were generally higher
in RS of banj oak compared to the RS of chir pine and banj oak regen­
eration. In the RS of banj oak, Cmic: SOC, Nmic: TN and Pmic: AP values
were higher in the rainy season than in the winter and summer, while no
seasonal trend was found in the RS of chir pine and banj oak regener­
ation. In brief, LMM showed that Cmic: SOC, Pmic: AP ratios were
affected by forests, soil positions, seasons and soil depths, while Nmic:
TN was affected by the soil depths (Table 4).
3.3. Rhizosphere soil microbial index (RSMI)
The RS soil properties having significant (p < 0.05) differences
among forest types were selected for PCA analysis (Table 6). PCA results
showed that the first three PCs eigenvalues were more than one and the
cumulative variation of PCs were 76.9%, which indicated almost all the
RS soil properties contributions (Table 7). In PC-1, soil properties with
higher factor loadings were Cmic, Nmic and SOC (Table 7). Cmic has the
highest loading and is significantly correlated with Nmic (r = 0.92), and
SOC (r = 0.78); therefore, Cmic was retained for RSMI (Table 5). Like­
wise, Cmic: SOC and Pmic: AP have the highest loading under PC-2 and
PC-3, respectively and retained for indexing. Weights of selected pa­
rameters were calculated with the help of the percent variance obtained
from the PCs. Final normalised PCA based RSMI equations are:
⎡ ⎤ ⎡ ⎤ ⎡ ⎤
⎢
⎢ 1 ⎥
⎥
⎢
⎢ 1 ⎥
⎥
⎢
⎢ 1
RSMI =0.67⎢ ( )
⎣
⎥ +0.17⎢ ( )
⎦ ⎣
⎥ +0.11⎢
⎦ ⎣
( )
X1 X2 − X3
1+ 130.4 − 2.5 1+ 1.0 2.5 1+
1.07
x1, x2 and x3 represent Cmic, Cmic: SOC and Pmic: AP, respectively, and
coefficients are weighting factors obtained from PCA results. The pa­
rameters in Eq. (3) are observed as the most critical indicators to express
the soil quality of forests. Different parameters for the scoring curves are
presented in Table 8. RSMI values of rhizosphere soil varied from 0.11 to
0.67 and found maximum for banj oak forest (0.67) followed by chir
pine (0.45) and banj oak regeneration (0.44) forests. Variation in RSMI
values shows that the RS of each tree species acts differently and mainly
depends on the soil nutrient status of the species.
4. Discussion
4.1. Rhizosphere influence on physicochemical soil properties.
Our study showed that both forest types and soil positions (RS and
BS) significantly influence the soil properties, mainly in the upper soil
layer (0–10 cm) (Tables 2 and 4). The quality and quantity of plant litter,
roots (from above-ground) and root exudates (from belowground) play
an essential role in driving the changes in rhizosphere SOC and nutrients
(Garkoti and Singh 1999, Kumar et al., 2021, Dhyani et al., 2019).
Consistent with the finding of Dhyani et al. (2019), banj oak showed
significantly (p < 0.05) higher rhizosphere effect (i.e., RS was signifi­
cantly different from the BS) relative to the chir pine and banj oak
regeneration forests (Fig. 2 a-l). Lower pH values in the RS of banj oak
than the BS reflect root exudates acidifying nature (Hinsinger et al.,
2003). In addition, the release of CO2 through the mineralisation pro­
cess, root respiration and acids produce by saprophytic fungi contributes
in the acidification of RS (Casarin et al., 2004; Dhyani et al., 2019). Banj
oak forest had low pH in RS compared to RS of chir pine and banj oak
regeneration, which may be due to the release of more CO2 (because of
higher fine root density) in banj oak (Verma et al., 2021). Low pH value
in the rainy season compared to winter and summer was consistent with
other studies in the region (Kumar et al., 2021; Verma and Garkoti,
2019), indicating the influence of precipitation in decomposition pro­
cesses and release of humic substances that may contribute in the
acidification of soil. Dimri et al. (1997) reported that high humus con­
tent in soil is responsible for declining soil pH.
Higher rhizosphere effect in banj oak was displayed by low soil pH,
bulk density and high soil moisture, TN, AP, Cmic, Pmic and DHA in
root-associated soil than the BS (Fig. 2 a-l and Table 2). Similar to the
present findings, Dhyani et al. (2019) also reported that the banj oak
forest has a higher rhizosphere effect than the chir pine due to nutrient-
enriched soil conditions. The soil moisture content is an essential factor
⎥
⎥ regulating the composition of the soil microbial community, which be­
⎥
⎦ comes more prominent at the root-soil interface (Cui et al., 2019; Zhu
et al., 2014). The higher soil moisture in the RS than the BS may be due
− 2.5
to higher clay and soil organic matter content, enhancing the water-
(3)
retaining capacity than the BS (supplementary Table 1). In both RS
and BS, high soil moisture content in banj oak and banj oak regeneration
forests than the chir pine confirm the influence of the high canopy cover
in banj oak and banj regeneration in retaining the soil moisture (Joshi
and Garkoti, 2021; Verma and Garkoti, 2019).
The root exudates play a key role in mediating soil nutrient abun­
dance in terrestrial ecosystems by chelating nature and stimulating
microorganisms (Lynch, 1990; Marschner, 2011). The rhizosphere
priming effect on soil organic matter (SOM) has been widely reported for
different ecosystems (Cheng and Kuzyakov, 2005; Colin-Belgrand et al.,

8
S. Kumar and S.C. Garkoti Geoderma 409 (2022) 115626

2003; Kuzyakov, 2002). The results of this study provide evidence

Soil properties represents BD = bulk density, SOC = soil organic carbon, TN = total nitrogen, AP = available phosphorus, DHA = dehydrogenase enzyme, Cmic = microbial biomass carbon, Nmic = microbial biomass
Nmic:TN
supporting the activation of microbes due to the rhizosphere priming
effects. We found strong positive correlations between SOC and DHA (r

0.05
= 0.85, p < 0.001), between Cmic and DHA (r = 0.81, p < 0.001) and
between SOC and Cmic (r = 0.78, p < 0.001) (Table 5). These results
Cmic:SOC

support the stimulations of soil microbes by the labile C released by the

0.49**
roots in the soil, which lead to SOM decomposition (Cheng and Kuzya­

− 0.13
kov, 2005; Zhu et al., 2014). Compared to the chir pine, the rhizosphere
effect is more pronounced in the banj oak and banj oak regeneration,
indicating the higher abundance of il microbes in RS of the banj oak and

0.52**
− 0.13
DHA

0.11
banj oak regeneration. Additionally, in banj oak and banj oak regener­
ation forests, fine root biomass production was higher than the chir pine
(Usman et al., 1998, Usman, 2002), which may result in higher amounts

0.75***

0.76***
of production of organic carbon and nutrients in the rhizosphere of banj
Pmic

0.04
0.13
oak and banj oak regeneration compared to chir pine. The Higher silt
and clay content in the RS increases the surface area of the soil and
0.71***
0.77***

hence increases nutrients retaining capacity of soil (Brady et al., 2008).

Nmic

0.41*
0.14

0.35 RS of banj oak contain 1.3 times and 1.2 times more SOC than the SOC of
chir pine RS and banj oak regeneration forest, respectively. Similarly,
TN in RS of banj oak is 1.3 times and 1.1 times higher than the TN of chir
0.92***
0.75***
0.81***

pine RS banj oak regeneration forest, respectively. The high rhizosphere

0.40*
Cmic

0.22
0.22

effect of banj oak and banj oak regeneration forest compared to chir pine
is probably the result of the high SOM, soil porosity and higher microbial
population, which was favoured by the presence of high SOM and high
0.42*
0.22
0.23
0.13

− 0.43
0.22
0.24
C: N

soil porosity (low bulk density). In a similar kind of study, it was found
that microbial counts were higher in banj oak rhizosphere than the chir
pine indicating that favourable microclimatic conditions in banj oak
0.68***
0.72***
0.64***
0.64***
− 0.04

favour the proliferation of microbes (Dhyani et al., 2019).

0.19
0.25
0.14
AP

To understand the regeneration of banj oak in chir pine forest, we

tried to collect below-ground information. Literature on these species
(banj oak and chir pine) reported that the high nutrient conservation
0.64***

0.70***
0.65***

0.65***
0.57**
− 0.13

− 0.07
− 0.23
0.26

ability of chir pine creates difficulties for the other species to invade in
Pearson correlation coefficients between soil physical, chemical and biological properties of rhizosphere soil (RS).
TN

the pure stand of chir pine (Garkoti, 2015; Singh et al., 1984). The RS of
banj oak regeneration forest exhibits lower soil bulk density (high
porosity) and higher soil moisture, SOC, TN and Cmic, Pmic than the RS
0.74***

0.78***
0.76***
0.65***
0.85***
0.55**
0.50**

0.44*
− 0.37
− 0.05

of chir pine forests (Fig. 2 and Table 2). These findings are similar to (Li
SOC

et al., 2021), who reported that in the rhizosphere, soil moisture, AP,
TN, SOC of conifers were significantly increased after mixing with
broadleaf tree species. We found lower RS pH in the banj oak regener­
− 0.13
− 0.12
− 0.34
− 0.06
− 0.38
− 0.36
− 0.30
− 0.42
− 0.25
− 0.21
0.01
pH

ation forest than the RS of chir pine forests in the rainy and summer
nitrogen, Pmic = microbial biomass phosphorus. p-value 0.001(***), 0.01(**), 0.05(*).

season, which may be due to the high secretion of acidic root exudates
by the regenerating banj oak tree species.
Porosity

0.02
0.08
− 0.07
0.01
0.29
0.08
0.07
0.16
0.09
− 0.01
0.15
0.08

Further regeneration of banj oak enhanced the fine root biomass

production, consequently increasing the amount of root exudates in RS.
Because fine root biomass production aids in the accumulation of soil
nutrients in the RS through the rapid turnover of fine roots (Brzostek
− 0.72

− 0.30

− 0.58
− 0.15
− 0.11
− 0.07
− 0.28

− 0.14
− 0.10
0.04

0.03
0.04

0.15
BD

et al., 2013; Garkoti, 2011; Joshi and Garkoti, 2021; Verma et al., 2021).
The higher soil nutrient content in RS is beneficial for the proliferation of
soil fungi (Yang et al., 2019), which decompose a wide variety of
− 0.12

− 0.08

− 0.28
− 0.14
− 0.22
Clay

0.05
0.09
0.22
0.07
0.09
0.20
0.04
0.09

0.03

complex and recalcitrant organic matter (Jirout et al., 2011). Thus, the
regeneration of banj oak in chir pine improved the RS physicochemical
properties by increasing the soil nutrients and microbial biomass by
0.90***

decomposing complex organic matter by the diverse group of microbes.

− 0.18
0.06
0.37
0.27
0.11
0.05
0.27
− 0.06
0.03
− 0.17
0.01
− 0.47
− 0.20
− 0.23
Silt

4.2. Rhizosphere influence on soil microbial biomass

− 0.98
− 0.97

− 0.06
− 0.25
− 0.25
− 0.09
− 0.07
− 0.24

− 0.06

− 0.02
0.39*
Sand

0.16

0.02

0.13

0.18
0.23

In comparison to the BS, RS is more sensitive to changing environ­

mental factors, soil nutrients, quality and quantity of root exudates due
to living biomass (Berg and Smalla, 2009; Liu et al., 2012; Zhang et al.,
Moisture

0.88***
0.80***

0.78***
0.72***
0.72***
0.85***

2011). The obtained results support our hypothesis that rhizosphere

0.59**

0.52**
− 0.01

− 0.21

− 0.20

− 0.16
− 0.13
0.01
0.00

0.12

0.27

effects would induce the soil nutrients and microbial biomass. We found
a strong positive correlation between soil moisture and Cmic (r = 0.64,
p < 0.001) (Table 3) which indicates that high soil moisture content in
Parameter

Cmic:SOC
Nmic:TN

RS supports the growth of the microbial community (Cui et al., 2019).

Pmic:AP
Porosity
Table 5

Nmic
Cmic

Pmic

Among the three forest types, RS of the banj oak significantly influenced
Sand

DHA
Clay

C: N
SOC
Silt

TN
BD

pH

AP

soil nutrients and microbial biomass more than the chir pine (Fig. 2 a-l).

9
S. Kumar and S.C. Garkoti
Fig. 2. Two-way ANOVA with Tukey HSD post hoc test (p < 0.05, n = 3) illustrates variation of soil microbial biomass carbon (Cmic), nitrogen (Nmic), phosphorus
(Pmic) and dehydrogenase enzyme activity (DHA) among three forests (banj oak, chir pine and banj oak regeneration) within the season. Values shown are mean of
three replicates with standard error (±SE). Capital letters (ABC): significant differences (p < 0.05) among the three forests at the same soil position (rhizosphere soil
(RS) and bulk soil (BS)) of the same depth; small letters (abc): significant differences (p < 0.05) between soil positions (RS versus BS) at same soil depths.
This may be primarily due to the higher quantity of SOC,
nutrient-enriched root exudates, and higher fine root biomass produc­
tion and turnover rates in RS of the banj oak, compared to chir pine RS
(Usman, 2002; Verma et al., 2021). The soil C: N ratio had a great in­
fluence on the diversity and microbial community composition (Cui
et al., 2019; Shen et al., 2013) and a higher C: N ratio indicates a broader
range of microbial population (Bell et al., 2014). The higher C: N ratio in
the RS over BS in banj oak (Table 2) indicates a higher rate of microbial
immobilisation for nitrogen. The Pearson correlation shows a significant
correlation between C: N ratio and Nmic (r = 0.27, p < 0.05). Among the
three forests, the RS of the oak regeneration forest showed a higher C: N
ratio than the banj oak and chir pine forests, indicating that the banj oak
regeneration forests support a wide range of microbial populations
compared to pure stands banj oak and chir pine forests. Li et al. (2021)
reported that conifer mixed broadleaf forests showed increase in the
abundance and diversity of rhizosphere soil fungi. A microbial quotient
is a useful tool for assessing the microbial activity and soil health of the
ecosystems (Bell et al., 2014; Diaz-Ravina et al., 1995). Our results
showed that microbial quotients (Cmic: SOC, Nmic: TN) was lower in the
RS (0.85–1.16%) than the BS (0.95–1.66%) in banj oak and banj oak
regeneration forests. In contrast, chir pine RS had a higher microbial
quotient value than the BS. A lower value in the RS indicates higher
microbial mineralisation of root exudates and plant litter than the BS,
while in chir pine rhizosphere, less secretion of root exudates may be the
possible reason for the higher microbial quotient. Colin-Belgrand et al.
(2003) found that microbes do not get adequate energy to decompose
SOM due to the fewer secretions of root exudates.
4.3. Rhizosphere influence on soil dehydrogenase enzyme activity
Dehydrogenase enzyme represents the overall soil microbial activity
(Gu et al., 2009; Salazar et al., 2011) because of intracellular origin
(Bing-Cheng and Dong-Xia, 2012; Zhao et al., 2010), and plays an
10
Geoderma 409 (2022) 115626
essential role in the oxidation of soil organic matter (Zhang et al., 2010).
In the present study, banj oak RS has 1.6 times higher dehydrogenase
enzyme activity than the BS, while it was 2 times higher in RS of banj
oak regeneration and 1.1 times in RS of chir pine forest. Higher soil
microbial biomass (Fig. 2) in RS indicated higher microbial counts than
in the BS (Dhyani et al., 2019). The higher rhizosphere effect in banj oak
regeneration forest may be due to nutrient-rich broad-leaved, and the
fast turnover rate of the fine root of the banj oak supports a biologically
diverse soil microbial community. The banj oak and banj oak regener­
ation RS has higher SOC, moisture, and low bulk density, promoting
microbial biomass and enzyme activity than the RS of chir pine con­
taining lower SOC, Cmic and Nmic. Pearson correlation matrix also
showed a strong positive correlation between SOC and DHA (Table 3).
Present results agree with the findings of Dhyani et al. (2019) and Kumar
et al. (2013), who found that the species with high SOC also have higher
soil dehydrogenase enzyme activity.
4.4. Influence of seasons and soil depths on rhizosphere effect
In the RS, microorganisms play a vital role in the decomposition of
root exudated organic compounds, SOM and plant litter (Progar et al.,
2000) and reflect nutrient and health condition of the soils. Confirming
our second hypothesis, we found that soil depths and seasons affect the
rhizosphere processes in all studied forests. Across the forests, the RS
showed higher SOC, soil nutrient (TN, AP), and microbial biomass in the
rainy seasons compared to the summer and winter (Fig. 3 and Table 2),
indicating that seasonal variation is one of the major influencing factors
on physicochemical and biological properties of RS particularly in the
upper soil layer (0–10 cm). Our results also showed that RS microbial
biomass and DHA decreased with the soil depths (Fig. 3 a-d). However,
there was no difference between the RS and BS at lower soil depths
(20–30 cm), which may be due to decreased amount of root exudates in
the deeper soil across the forests. Increasing soil bulk density (low
S. Kumar and S.C. Garkoti Geoderma 409 (2022) 115626

Fig. 3. Change in soil microbial biomass carbon (Cmic), nitrogen (Nmic), phosphorus (Pmic) and DHA in three forests with seasons (rainy, winter, summer) at three
soil depths (10 cm, 20 cm, 30 cm). Values are the mean of three replicates (±SE). Upper-case letters (ABC): significant differences (p < 0.05) among three seasons at
same depth; lower-cases letters (abc): significant differences (p < 0.05) among three depths within the same season. Red colour (solid line): rhizosphere soil, green
colour (dotted line): bulk soil. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Table 6 Table 7
Statistical analysis of the rhizosphere soil (RS) properties. Results of principal component analysis (PCA) of rhizosphere soil quality
Soil parameters DF F value P SE (n ¼ 3)
parameters.
Principal Components PC-1 PC-2 PC-3
Moisture 2 6.89 0.001 0.49
pH 2 45.8 0.000 0.04 Eigenvalue 2.26 1.17 1.08
SOC 2 4.76 0.01 0.07 Variance (%) 0.514 0.137 0.117
TN 2 3.19 0.04 0.00 C. Variation (%) 0.514 0.652 0.769
AP 2 6.38 0.002 0.00 Eigenvectors
Cmic 2 23.89 0.000 6.92 Moisture − 0.382 0.197 − 0.061
Nmic 2 11.04 0.000 0.76 pH 0.160 0.492 0.175
Pmic 2 21.71 0.00 0.32 SOC ¡0.382 0.285 − 0.129
DHA 2 0.23 0.79 0.08 TN − 0.303 0.105 − 0.286
Cmic/SOC 2 3.51 0.04 0.28 AP − 0.303 − 0.209 − 0.350
Nmic/TN 2 0.18 0.83 0.05 Cmic − 0.401 − 0.190 0.081
Pmic/AP 2 8.27 0.000 0.06 Nmic ¡0.387 − 0.172 − 0.001
Pmic − 0.376 0.021 0.304
Cmic:SOC 0.008 − 0.669 0.381
porosity) with increasing soil depths may reduce the mobilisation of Pmic:AP − 0.212 0.260 0.706
solutes for root uptake (Proctor and He, 2021), which may further lower Normalised RSMI = 0.514Cmic + 0.514Nmic + 0.514SOC + 0.137Cmic: SOC +
the microbial biomass and enzyme activity. 0.117Pmic: AP / 0.768 = 0.67Cmic + 0.17 Cmic: soc + 0.11Pmic: AP.
Final RSMI ¼ 0.67Cmic þ 0.17 Cmic: SOC þ 0.11 Pmic: AP.
4.5. Rhizosphere soil microbial index (RSMI) Boldface eigenvalues correspond to the PCs examined for the index.
Boldface factor loading are considered highly weighted.
Assessment of soil quality is an emerging topic for researchers Bold-italic factor loadings correspond to the parameters included in the index.
(Bünemann et al., 2018; Gil-Sotres et al., 2005; Zhang et al., 2011). Due
to the intricate nature of the soil, it is not easy to identify soil quality

11
S. Kumar and S.C. Garkoti
Table 8
Mean values, and R2 coefficients of scoring curves parameters.
Parameters Cmic Cmic: SOC Pmic: AP
Curve type More is better More is better More is better
Slope − 2.50 − 2.50 − 2.50
Mean (X0) 130.39 1.00 1.07
R2 0.98 0.96 0.75
F. significance 0.000 0.000 0.001
indicators, yet we have selected some statistically significant parameters
which may indicate soil health. Our findings indicated that the soil
health of studied forests was majorly governed by parameters such as
Cmic, Cmic: SOC, Pmic: AP (Eq. (3)). Soil microbial biomass is an active
fraction of soil organic matter that plays a key role in the decomposition
and transformation of soil organic matter and acts as both a ‘source’ and
‘sink’ of the soil nutrients. Therefore, soil microbial biomass is consid­
ered an essential indicator of soil health. The tree species with an RSMI
value greater than 0.5 can be recommended for afforestation in the
degraded forest (Sinha et al., 2009). In our study, RSMI value was
maximum for banj oak forest (0.68) followed by chir pine (0.45) and
minimum for banj oak regeneration forest (0.44). Therefore, banj oak
with a high RSMI value improves soil health and promotes understory
vegetation and could be recommended for the afforestation in the
degraded forests of the central Himalaya.
5. Conclusion
In conclusion, we found that the RS of banj oak significantly in­
fluences SOC, nutrients, microbial biomass and dehydrogenase enzyme
activity in the upper soil layer (0–10 cm), while less effect was observed
in middle and lower soil layers. Forests with high soil nutrient levels (e.
g., banj oak) has a significantly more substantial rhizosphere influence
than a forest with nutrient-poor soil (e.g., chir pine). The soil nutrients
and microbial biomass reveal that the rhizosphere effect of tree species
significantly varied across the soil depths and found maximum in the
rainy season. In the present study, soil nutrient seems to be a significant
factor influencing the rhizosphere effect. Cmic, Cmic: SOC and Pmic: AP
could be most significant for assessing rhizosphere soil quality in
temperate forests of the central Himalaya.
The banj oak regeneration in chir pine forest improved RS quality
through increasing soil moisture content, SOC, soil microbial biomass
and dehydrogenase enzyme activity compared to monoculture stand of
chir pine forests. Based on a high RSMI value (0.65), banj oak may be
considered for afforestation in the degraded forests of the region.
Further research is needed at the taxonomic level to investigate the
microbial shift during the regeneration of the natural forest ecosystems.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
The authors are highly thankful to University Grants Commission
(UGC) and DST-PURSE, India, for providing financial support (Ref. No.
420/(CSIR-UGC NET, DEC. 2016).
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