CSIRO PUBLISHING

Crop & Pasture Science, 2018, 69, 460–468

https://doi.org/10.1071/CP18016

Indigenous plant-growth-promoting rhizobacteria and chemical

fertilisers: impact on wheat (Triticum aestivum) productivity
and soil properties in North Western Himalayan region

Gaurav Sood A,C, Rajesh Kaushal B, Anjali Chauhan A, and Shaweta Gupta A
A
Department of Basic Sciences, College of Forestry, Dr. Y. S. Parmar University of Horticulture and Forestry,
Nauni, Solan, HP 173230, India.
B
Department of Soil Science and Water Management, Dr. Y. S. Parmar University of Horticulture and Forestry,
Nauni, Solan, HP 173230, India.
C
Corresponding author. Email: gauravsood5@gmail.com

Abstract. High levels of crop productivity cannot be sustained by chemical fertiliser application alone. In order to
mitigate this, a 2-year study was conducted to test the effects of combined application of indigenous plant-growth-
promoting rhizobacteria (PGPR) and chemical fertilisers on productivity of wheat and soil properties. Ten morphologically
distinct indigenous PGPR isolates from wheat roots and rhizosphere were evaluated at Solan, Himachal Pradesh, India,
during 2013–14. Three PGPR isolates (B2, SIR1 and BIS2) with maximum PGP traits were screened at different doses
of nitrogen (N) and phosphorus (P) (80%, 60% and 40% of recommended fertiliser dose, RFD) under net-house conditions.
Two isolates, B2 (Serratia sp.) and SIR1 (Bacillus subtilis), along with the optimum NP dose (i.e. 80% RFD) were selected
for field experimentation, which was performed over two consecutive years, 2014–16. Combined application of 80% RDF
of NP with PGPR (B2) significantly increased wheat yield by 9.4%, number of tillers per plant by 28.03%, grain number
per spike by 19.61%, 1000-grain weight by 10.5%, and biomass by 9.2% relative to the uninoculated control with 100%
RFD. Soil properties in the terms of available N, P and potassium, microbial biomass carbon, soil enzyme activities and
population of phosphate-solubilising bacteria in the wheat crop were significantly increased by the combined application
of bacterial inoculants with 80% RFD of NP in both years over the uninoculated control. Therefore, the results revealed
the potential of indigenous PGPR isolates to supplement ~20% of NP fertilisers without hampering the soil fertility and
productivity of wheat.

Additional keywords: grain yield, growth-promoting bacteria, nutrient uptake.

Received 2 August 2017, accepted 5 February 2018, published online 20 April 2018

Introduction scientists to explore renewable, low-cost, non-bulky inputs

Wheat (Triticum aestivum L.) is a member of Poaceae family
and is ranked among the top three cereal crops of the world.
Cereals constitute the staple food of Indians and provide ~61%
of the protein requirement (Sahi et al. 2012). In Himachal
Pradesh, wheat is grown under rainfed conditions over an area
of 352 000 ha with average production of ~414 000 Mt in the
rabi (winter) season (Kumar 2015).
Nitrogen (N) and phosphorus (P) are the primary constituents
of plant and animal life, playing active roles in many metabolic
processes and the phenology of crops and vegetables (Khan et al.
2013). Recommendations of chemical fertilisers for these crops
differ according to soil and agro-climatic conditions. Application
per ha of 90 kg N, 50 kg P, 30 kg potassium (K) and 10–15 t
farmyard manure (FYM) is recommended for obtaining higher
yields of wheat under mid-hill conditions in Himachal Pradesh
(Kumar et al. 2013).
The deterioration of soil health coupled with decreasing
nutrient-use efficiency is a matter of great concern, compelling
to bridge the gap of crop production, as well as improving the
quality of produce. Biofertilisers and plant-growth-promoting
rhizobacteria (PGPR) are well recognised as an important
component of integrated plant-nutrient management for
sustainable agriculture and they hold a great promise not only
to improve crop yield but also to sustain soil health (Pérez-
Montaño et al. 2014). PGPR consist of strains of genera such
as Pseudomonas, Azospirillum, Azotobacter, Bacillus, Serratia,
Rhizobium and Flavobacterium; they produce metabolites that
can promote growth and produce induced systemic resistance
against various phytopathogens. These PGPR are an important
component of the rhizosphere of many plants and provide
multiple benefits to these plants (Mehnaz and Lazarovits
2006). PGPR improve plant growth either directly by production
of plant growth regulators such as auxin and cytokinins and by
increasing plant uptake of some micro- and macro-nutrients in
the rhizosphere, or indirectly through induction of host defence
mechanisms (Glick 2012). Production of antifungal secondary

Journal compilation
CSIRO 2018 www.publish.csiro.au/journals/cp

Biofertilisers and inorganic nutrients
metabolites such as 2,4-diacetylphloroglucinol, pyrrolnitrin,
hydrogen cyanide (HCN), siderophore and lytic enzymes
(protease) is a prominent feature of many PGPR (van Loon
et al. 1998).
Although PGPR are of great interest in sustainable crop
production and sustenance of soil health, the extent of their
benefits depends on their number and efficiency, which is
governed by many soil and environmental factors (Saber et al.
2012). It has also been observed that PGPR isolated from native
rhizosphere are more effective than other strains in growth
enhancement and crop protection because of better adaptability
of these local bacterial strains (Sood et al. 2018). Therefore,
considering the cropping area and productivity of wheat in the
North Western Himalayan region, particularly under rainfed
conditions, the present study was undertaken. We isolated
indigenous strains of PGPR with various PGP traits, and then
studied the effects of inoculation of selected PGPR with
different levels of N and P fertilisers on growth and yield of
wheat under controlled pot and field conditions.
Materials and methods
The experiment was carried out in the laboratory at the
Department of Basic Sciences, Dr. Y. S. Parmar University of
Horticulture and Forestry, and in a field of a local farmer at
Dharja, in Nauni, Solan, Himachal Pradesh, during 2013–16.
The study area was at an altitude of 1150 m amsl. Wheat variety
HPW-42 was used as test crop. The investigations were divided
into three parts: (i) isolation, screening and characterisation
of PGPR; (ii) pot experiment; and (iii) field studies.
Isolation, screening and characterisation of PGPR
In 2013, indigenous PGPR showing various PGP traits were
isolated from the rhizosphere and roots of wheat from multiple
sites in the four agro-climatic zones of Himachal Pradesh:
zone I (low hills, subtropical), zone II (mid hills, subhumid),
zone III (high hills, wet temperate), and zone IV (high hills, dry
temperate) (Table 1).
Standard serial-dilution and pour-plating techniques were
used for isolation of endophytic and rhizospheric microbes on
nutrient agar (NA), Pikovskaya’s medium (PVK) and Jensen’s
medium. On purification, only ten of the most predominant and
morphologically distinct isolates (Table 2) able to form clear
halo zone on PVK medium and growth on Jensen’s medium
were screened for other PGP traits, viz. phosphate solubilisation,
HCN production, growth on N-free medium, auxin production
and siderophore production, by adopting standard procedures.
Phosphate solubilisation activity was estimated by using PVK
medium (Pikovskaya 1948). Production of indole-3-acetic acid
(IAA) was determined by the method of Gordon and Palleg
(1957). The ability of isolates to produce siderophore and
HCN was assessed by methods of Schwyn and Neilands
(1987) and Bakker and Schippers (1987), respectively.
The biocontrol potential of the bacterial isolates against test
fungal pathogens (Fusarium graminearum, Claviceps purpurea
and Alternaria triticina) was ascertained by the agar streak plate
method on potato dextrose agar medium, and growth inhibition
(%) was calculated as described by Vincent (1947).
Crop & Pasture Science 461
On the basis of 16S rRNA sequencing, isolates B2 and
SIR1 (the two best performing isolates in the pot experiment;
see below) were identified as Serratia spp. (accession
number KT427416) and Bacillus subtilis (accession number
KX379531), respectively.
Pot experiment
Of the 10 morphological and biochemical distinct isolates, three
(B2, SIR1 and BIS2) possessing maximum PGP activities were
selected for a pot experiment in the 2014. Potting mixture
was prepared by mixing sand, soil and well-rotted FYM in
a ratio of 1 : 2 : 1. The mixture’s properties were: pH 7.21;
electrical conductivity 0.41 dS m–1; rhizospheric bacterial
population 70.00  106 cfu g–1 soil; high organic carbon (1.03%);
available N, P and K contents 272.21, 20.30 and 309.18 kg ha–1,
respectively. Wheat seeds (variety HPW-42) was obtained from
the local market of University of Horticulture and Forestry,
Nauni, Solan, and surface sterilised (0.1% HgCl2 for 2 min
and rinsed five times with sterilised water). Pure cultures of
B2, SIR1 and BIS2 were grown in NA for the experiments.
Single colonies from each strain were transferred into 100-mL
flasks containing nutrient broth and grown aerobically in the
flasks overnight on a rotating shaker (300 rpm) at 308C. Bacteria
grown in nutrient broth were then diluted with sterilised,
distilled water to a final concentration of 108 cfu mL–1. Wheat
seeds were placed in a bacterial suspension of 108 cfu mL–1 for
30 min before sowing.
Seeds were then sown in pots and allowed to grow for 45 days
under net-house conditions. The following 10 treatments were
arranged in a completely randomised block design with three
replications: T1, control, recommended fertiliser dose (RFD) of
NPK (i.e. 100%); T2, B2 + 80% RFD (of N and P); T3, B2 + 60%
RFD; T4, B2 + 40% RFD; T5, SIR1 + 80% RFD; T6, SIR1 + 60%
RFD; T7, SIR1 + 40% RFD; T8, BIS2 + 80% RFD; T9, BIS2 +
60% RFD; T10, BIS2 + 40% RFD. Recommended doses and
fertilisers used were as described below for the field studies.
Observations on plant growth parameters such as germination
percentage, shoot and root length (cm) and biomass (g), and
number of leaves per plant were recorded by following standard
methods. Of the three isolates, the two best performing (B2 and
SIR1) with optimum fertiliser dose (i.e. 80% RFD of N and P)
were selected for further evaluation under field conditions.
Field studies
Field trials were conducted during 2014–16, using the same
treatments in both years. The experimental soil was sandy
loam with nearly neutral pH (7.13), electrical conductivity
0.35 dS m–1, rhizospheric bacterial population 123.50  105
cfu g–1 soil, high organic carbon content (1.23%), and available
N, P and K contents 295.37, 21.14 and 321.34 kg ha–1,
respectively. PGPR inocula were applied by dipping seeds for
30 min into a liquid culture of B2 and SIR1 isolates (cell
density ~108 cfu mL–1). Individual plot size was 2 m2 (2.0 m
by 1.0 m) and seeds were planted with 25-cm row-to-row
spacing, so that there were five rows per plot. An area 1 m by
1 m of each plot was selected for collection of data.
Three treatments were laid out in randomised block design
with seven replicates: T1, control, RFD of NPK (i.e. 100%);
T2, B2 + 80% RFD (of N and P); T3, SIR1 + 80% RFD.
462 Crop & Pasture Science G. Sood et al.

Table 1. Rhizospheric and endophytic viable bacterial population associated with wheat in different
agro-climatic zones of Himachal Pradesh

Site Rhizospheric soil bacterial population Endophytic bacterial population

(104 cfu g–1 soil) (102 cfu g–1 root)
Nutrient agar Pikovskaya’s Jensen’s Nutrient Pikovskaya’s Jensen’s
medium medium agar medium medium
Agro-climatic zone I
Dehra 220.30 91.60 74.60 72.60 56.60 45.00
Fatehpur 208.30 89.30 65.30 73.60 59.30 39.30
Rakkar 225.00 80.00 68.60 61.30 53.00 40.00
Mehatpur 205.60 95.00 61.00 62.60 47.00 38.00
Nadaun 197.00 68.30 56.30 55.60 38.00 41.00
Neri 209.30 96.30 67.60 77.30 42.30 50.60
Ghumarwin 211.00 98.60 62.00 102.00 57.60 43.00
Jhalta 180.30 79.30 72.00 61.00 45.00 44.60
Parwanoo 192.30 80.00 67.00 60.30 38.60 37.00
Nalagarh 199.60 92.40 60.60 67.60 42.30 38.30
Sarahan 184.00 88.00 55.00 65.00 38.00 50.30
Ponta sahib 169.00 90.30 54.30 58.00 37.30 37.00
Agro-climatic zone II
Sihunta 163.30 85.00 60.60 61.00 41.60 51.60
Banikhet 146.00 87.00 63.60 59.30 38.60 42.60
Palampur 205.00 91.60 72.00 100.30 48.30 47.60
Mataur 195.30 90.60 74.00 96.00 35.00 54.30
Sarkaghat 181.30 85.60 58.30 72.30 36.60 44.30
Sundernagar 199.00 86.30 62.30 69.60 33.60 39.60
Arki 193.60 80.30 56.30 71.60 41.60 38.30
Kandaghat 188.00 85.00 54.00 52.60 37.00 41.30
Rajgarh 197.30 91.60 58.00 59.30 50.60 37.00
Nahan 184.60 88.00 55.00 57.30 41.00 47.00
Agro-climatic zone III
Bharmour 138.00 75.00 53.30 50.60 39.60 32.60
Bhadra 117.30 73.30 50.30 49.60 36.30 37.30
Bajaura 167.30 84.00 55.30 56.60 43.00 46.30
Patlikhul 158.60 83.60 57.30 63.30 39.30 43.00
Theog 126.60 75.00 49.00 53.60 32.60 40.60
Rohru 119.00 69.30 48.60 54.30 31.30 30.0
Agro-climatic zone IV
Kaza 91.00 62.60 44.00 45.00 19.60 28.60
Kukumseri 58.60 60.30 43.60 50.30 22.30 24.30
Pangi 52.30 45.30 46.60 48.30 20.60 27.30
Kalpa 55.60 50.60 33.00 47.00 18.30 33.60

Recommended doses of N, P2O5 and K2O were 90, 50 and 30 kg
ha–1, respectively. Urea (46% N), single superphosphate (16%
P2O5) and muriate of potash (60% K2O) were used as source of
chemical fertilisers. All other intercultural practices including
application of FYM (15 t ha–1) and insect-pest and disease
management were followed as per standard recommendations.
Observations were recorded of number of tillers per plant,
grain number per spike, 1000-grain weight, grain yield and
straw yield. Total N, P and K contents of plants were
determined as per method suggested by Jackson (1973).
Nutrient uptake (kg ha–1) was calculated by multiplying the
total NPK concentration of the whole plant by total dry matter
content. Available N, P and K contents of soil were determined
following standard procedures (Tandon 2009). Estimation of
soil enzymes (phosphatase, dehydrogenase and phytase) was
carried out by the methods of Tabatabai and Bremner (1969),
Casida (1977) and Boyce et al. (2004), respectively. Microbial
biomass carbon was determined by the soil fumigation
extraction method detailed by Vance et al. (1987).
Statistical analyses
Data obtained from field studies were pooled for different years
in order to obtain more precise estimates and to meet the
more general and broader objective of common interpretation.
Study of the treatment  year interaction was carried out as
per Bartlett’s test and the interaction was found to be non-
significant (see Supplementary material table available at
journal’s website). Statistical analyses of data were done by
using SPSS version 16 (SPSS Inc., Chicago, IL, USA) and
Microsoft Excel (Microsoft, Redmond, WA, USA) at P = 0.05
level of significance.
Biofertilisers and inorganic nutrients Crop & Pasture Science 463

IAA, Indole-3-acetic acid; SPE, siderophore production efficiency; HCN, hydrogen cyanide. Values in parentheses are arc sine-transformed. Within a column, means followed by the same letter are
not significantly different (critical difference (CD) at P = 0.05). For HCN production: +, light brown; ++, dark brown; –, no activity. For ammonium production: +, light brown colour; ++, dark brown colour;

37.33(37.65)de
32.67(34.84)fg

26.67(31.07)hi
25.00(29.98)ij
28.67(32.33)h
38.67(38.43)d

62.64(52.31)b
Results

67.08(54.97)a
55.50(48.14)c

33.00(34.97)f
Alternaria
triticina

3.50
Isolation and screening of PGPR isolates
The microbial count in rhizosphere varied from 52.3  104 to
225.0  104 cfu g–1 soil on NA medium. A similar trend was
observed on Jensen’s medium (33.0  104 to 74.6  104 cfu g–1
% Antagonism against:

soil) and PVK medium (45.3  104 to 98.6  104 cfu g–1 soil) by

38.00(38.03)efg

38.67(38.43)def
39.33(38.82)de

32.33(34.63)hi
40.33(39.41)d
57.58(49.35)b

33.33(35.25)h
66.71(54.75)a
42.87(40.88)c
23.00(28.63)j
Claviceps

using the replica plate method. The population of endo-

purpurea

rhizobacteria varied from 45.0  102 to 102.0  102 cfu g–1

2.31
root on NA medium and similarly on Jensen’s medium
(24.3  102 to 54.3  102 cfu g–1 root) and PVK medium
(18.3  102 to 59.3  102 cfu g–1 root). Total microbial counts
were predominantly in the rhizosphere compared with the
38.33(38.23)cde

30.33(33.40)ghi

36.67(37.24)def
30.67(33.61)gh

30.67(33.61)gh
39.00(38.63)cd
40.67(39.60)bc
graminearum

endophytic population. Maximum bacterial counts in both

43.11(47.66)b

31.00(33.82)g
54.67(41.02)a
Fusarium

rhizospheric soil and the endo-rhizosphere of wheat were

2.50
found in agro-climatic zone I, except for endo-rhizospheric
count on the Jensen’s medium. Minimum counts were found
Table 2. Screening of bacterial isolates for various plant-growth-promoting activities

in agro-climatic zone IV (Table 1).

Ten morphologically distinct PGPR isolates, eight from the
rhizosphere (B2, MAS1, UNS1, HAR3, CHS1, BIS2, KIS3 and
production
Ammonia

+++
+++

+++

+++

LSR1) and two from wheat roots (SIR1 and SHR1), having
++
++

++
+
–

diverse PGP activities were selected from 127 isolates from all
four agro-climatic zones (Table 2). Isolates B2, UNS1, HAR3
and BIS2 were from zone I; SIR1 and MAS1 from zone II;
+++, orange brown colour; –, no activity

production

CHS1 and SHR1 from zone III; and LSR1 and KIS3 from
HCN

zone-IV. The PGPR isolates showed significant variation in

++

++
+

+
–

–
–

phosphate solubilisation, ranging from 91.67 to 205.33 mg mL–1

in liquid PVK medium. Production of IAA and siderophore
ranged from 19.33 to 31.70 mg mL–1 and from 29.0% to
65.0%, respectively. Only five isolates (B2, SIR1, UNS1,
41.00(39.79)efg

32.67(34.84)hi
42.00(40.38)ef
57.00(49.01)b

33.00(35.04)h

46.67(43.07)d
65.00(53.71)a

51.33(45.75)c

43.33(41.15)e
29.00(32.57)j
SPE (%)

CHS1 and BIS2) were HCN producers. All isolates except SHR1
and LSR1 produced ammonia. All isolates showed antagonism
2.50

against F. graminearum, A. triticina and C. purpurea under

in vitro conditions. Furthermore, isolate B2 showed maximum
antagonism against all three test fungi. Three bacterial isolates
(B2, SIR1 and BIS2) showed maximum multifarious PGP traits
25.33bcde
(mg mL–1)

25.67bcd

19.33fgh
26.33bc

(Table 2), and hence they were selected for pot experimentation.
20.33fg

20.33fg
20.33fg
IAA

27.33b
31.70a

21.33f
2.68

Pot experiment
Maximum seed germination (91.67%) and leaf number (6.17
leaves plant–1) were recorded in T2, and minimum seed
Growth on
Jensen’s
medium

germination (56.67%) and leaf number (3.67 leaves plant–1) in

+
+
+
+
+
+
+
+
+
+

T10 (Table 3). Maximum shoot length (45.90 cm) and shoot
biomass (2.5 g plant–1) were also recorded in T2, and minimum
shoot length (25.67 cm) and shoot biomass (1.09 g plant–1) in
solubilisation in liquid

T10. Likewise, maximum root length (7.0 cm) was recorded in

medium (mg mL–1)

T2, and minimum (3.73 cm) in T10. Maximum root biomass

Phosphate

129.00gh
172.67cd

(0.35 g plant–1) was recorded in T2, statistically on par with

137.33fg

146.33ef
91.67ij
193.67b
205.33a

154.00e
174.67c

95.67i
11.50

T5 (0.33 g plant–1) and T1 (0.31 g/plant–1), and again the

minimum (0.16 g plant–1) was recorded in T10.
None of the tested isolates influenced soil pH, electrical
conductivity or organic carbon significantly relative to the
uninoculated control (data not shown), whereas in some cases
SHR1 (endophytic)

there was a significant increase (or decrease) in available nutrient

SIR1 (endophytic)

contents of soil due to inoculation with bacterial isolates

CD (P = 0.05)

(Table 4). For example, relative to the control, significantly

higher content of available N (289.74 kg ha–1) was recorded
MAS1
Isolate

HAR3
UNS1

CHS1

LSR1

in T2, and significantly lower content in T6, T7 and T9, with

KIS3
BIS2

the lowest (253.67 kg ha–1) in T10. Maximum available

B2
464 Crop & Pasture Science G. Sood et al.

Table 3. Effect of liquid bacterial inoculum (B2, SIR1, BIS2) and fertiliser (N, nitrogen; P, phosphorus; K, potassium) on seed germination and
growth parameters of wheat under net-house conditions
Values in parentheses are angular-transformed. Within columns, means followed by same letter are not significantly different (critical difference (CD) at P = 0.05)

Treatment (inoculum + Seed germination Shoot length Root length No. of leaves Root biomass Shoot biomass
%recommended (%) (cm) per plant (g plant–1)
fertiliser dose)
T1, control (100% NPK) 88.33(70.70)abc 40.97bcd 6.25bc 5.83abc 0.31abc 2.17bc
T2, B2 + 80% NP 91.67(76.18)a 45.90a 7.00a 6.17a 0.35a 2.50a
T3, B2 + 60% NP 73.33(58.98)e 36.18e 5.28de 5.33bcde 0.21de 1.82d
T4, B2 + 40% NP 66.67(54.76)efgh 30.00gh 4.33fgh 4.67efg 0.19defg 1.27f
T5, SIR1 + 80% NP 89.33(70.92)ab 45.37ab 6.37ab 6.00ab 0.33ab 2.21b
T6, SIR1 + 60% NP 72.67(58.56)ef 35.80ef 5.55cd 5.50abcd 0.20def 2.21b
T7, SIR1 + 40% NP 63.33(52.75)fghi 29.23ghi 3.97hi 4.33fgh 0.18defgh 1.20fgh
T8, BIS2 + 80% NP 86.00(68.21)abcd 42.35abc 4.93def 5.83abc 0.23cd 1.76de
T9, BIS2 + 60% NP 70.00(56.97)efg 32.87efg 4.88defg 5.00def 0.19defg 1.21fg
T10, BIS2 + 40% NP 56.67(48.82)ij 25.67hij 3.73hij 3.67hi 0.16defghi 1.09fghi
CD (P = 0.05) 9.67 4.63 0.71 0.73 0.09 0.20

P (29.12 kg ha–1) was recorded in T2, followed by T5, T3, T6 and
T8, and minimum available P (17.63 kg ha–1) was recorded in
T10, with low values also recorded in T9 and T7. These groups of
treatments were significantly higher and lower, respectively,
than the control. Among the inoculated treatments, maximum
available K (377.63 kg ha–1) in soil was recorded in T2 and
minimum (307.00 kg ha–1) in T10; however, all were significantly
higher than the control.
The joint application of bacterial inoculant with chemical
fertilisers significantly influenced soil enzymes and microbial
biomass carbon relative to the uninoculated control (Table 4).
Maximum dehydrogenase activity (11.50 mg TPF g–1 h–1) was
recorded in T2 followed by T5 (both significantly higher than
the control) and T8 (statistically on par with the control),
whereas minimum dehydrogenase activity (6.94 mg TPF g–1
h–1) was recorded in T7. Maximum phosphatase activity
(425.00 mmol L–1 g–1 h–1) was recorded in T2, which was
statistically on par with T5. The minimum (334.33 mmol L–1
g–1 h–1) was recorded in T10; the other treatments were not
significantly different from the control. Maximum phytase
activity (5419.00 mmol L–1 g–1 h–1) was found in T2 and
minimum (4009.00 mmol L–1 g–1 h–1) in T10. Maximum microbial
biomass carbon (102.00 mg 100 g–1 soil) was recorded in T2,
which was statistically on par with T5, whereas the minimum
(47.67 mg 100 g–1 soil) was recorded with T10.
Total rhizospheric and endophytic bacterial counts varied
from 58.67 to 81.33  106 cfu g–1 soil and from 30.33 to
50.67  101 cfu g–1 root, respectively, on NA medium
(Table 4). The maximum microbial count was recorded in the
rhizosphere of plants whose seeds received T2, whereas the
minimum count was recorded in T10. A similar trend was
observed in the case of endophytic bacterial population.
Field studies
Maximum number of tillers per plant (5.35) was observed in T2
(i.e. joint application of 80% RFD of NP and seed inoculation
with B2 isolate; Table 5), resulting in a 28.03% increase in
tillering over the uninoculated control. Similarly, T2 proved
most effective with 19.61% and 10.5% increases in grain
number per spike and 1000-grain weight, respectively, over
the uninoculated control (i.e. with 100% RFD of NPK) during
both years. T3 (i.e. joint application of 80% RFD of NP and
seed inoculation with SIR1 isolate) also resulted in significantly
higher number of tillers per plant and 1000-grain weight than
the control.
Maximum values of grain yield (3.51 t ha–1) and straw yield
(10.56 t ha–1) were observed in T2, with 9.4% and 9.2% increases
over the uninoculated control. T3 also had a significant positive
effect on grain and straw yields.
Uptake of N, P and K increased significantly with joint
application of PGPR along with an economic dose of fertilisers
during both years (Table 5). T2 resulted in maximum N uptake
(247.83 kg ha–1), which was statistically on par with T3.
Minimum N uptake (220.56 kg ha–1) was recorded in the control.
Similarly maximum P (26.28 kg ha–1) and K (235.38 kg ha–1)
uptakes were recorded in T2, followed by T3 (not significantly
different from T2), with minimum P (21.41 kg ha–1) and K
(213.56 kg ha–1) uptakes recorded in the uninoculated control.
The isolate treatments did not significantly influence soil
pH, electrical conductivity or organic carbon compared with
the uninoculated control under field conditions, even after
two years (data not shown). Available N, P and K contents
were increased with the joint application of PGPR and optimum
N and P fertilisers in both years (statistically significant in all
cases except for available N in T3; Table 6). Maximum values
of available N (331.88 kg ha–1) and P (32.99 kg ha–1) were
recorded in T2 and were statistically on par with values in T3.
The uninoculated control had minimum values of available N
(304.47 kg ha–1) and P (27.41 kg ha–1). Maximum available K
(347.4 kg ha–1) was recorded in T2, significantly greater than
T3, and again the minimum (322.0 kg ha–1) was recorded in the
uninoculated control. Contents of available N, P and K at the
termination of experiment were increased over the initial status
(from Methods: 295.37, 21.14 and 321.34 kg ha–1, respectively).
Maximum significant increases in available N, P and K content
(12.36%, 56.05% and 8.11%) were recorded in T2.
Combined application of PGPR with chemical fertilisers
significantly influenced soil enzymes and microbial biomass
carbon in all cases compared with the uninoculated control
Table 4. Effect of liquid bacterial inoculums (B2, SIR1, BIS2) and fertiliser (N, nitrogen; P, phosphorus; K, potassium) on soil and microbial properties (at termination of experiment)
under net-house conditions
MBC, Microbial biomass carbon; RBP, rhizospheric bacterial population; EBP, endophytic bacterial population. Within columns, means followed by same letter are not significantly different (critical
difference (CD) at P = 0.05)

Treatment (inoculum + Available soil nutrients (kg ha–1) Soil enzymes Microbial properties
%recommended N P K Dehydrogenase Phosphatase Phytase MBC RBP EBP
fertiliser dose) (mg TPF g–1 h–1) (mmol L–1 g–1 h–1) (mg 100 g–1 soil) (106 cfu g–1 soil) (101 cfu g–1 root)
T1, control (100% NPK) 276.14bc 20.16f 302.10j 8.50cdef 378.00cdefg 4532.00f 68.30cdef 72.33cde 43.67def
T2, B2 + 80% NP 289.74a 29.12a 377.63a 11.50a 425.00a 5419.00a 102.00a 81.33a 50.67a
T3, B2 + 60% NP 268.88cde 24.64c 357.48e 9.20c 389.00c 4976.00c 73.00c 72.33cde 44.00de
T4, B2 + 40% NP 267.18cdef 19.77fg 331.00g 7.30hi 381.00cd 4234.33gh 60.00fg 68.00 g 35.67h
Biofertilisers and inorganic nutrients

T5, SIR1 + 80% NP 278.64b 26.88b 373.80b 10.40b 413.00ab 5290.00ab 95.70ab 77.00b 50.00ab
T6, SIR1 + 60% NP 263.18efg 22.40d 364.55c 8.63cde 380.00cdef 4925.00cd 71.30cd 74.00bcd 45.00d
T7, SIR1 + 40% NP 258.83fghi 17.99h 312.33h 6.94hij 374.70cdefgh 4090.33hi 53.30ghi 64.67h 30.67i
T8, BIS2 + 80% NP 275.88bcd 21.28e 361.80d 9.00cd 380.70cde 4871.33cde 70.70cde 75.33bc 49.67abc
T9, BIS2 + 60% NP 262.42efgh 17.92hi 356.23ef 8.30defg 366.70defghi 4415.70fg 58.30gh 72.00def 43.33defg
T10, BIS2 + 40% NP 253.67hij 17.63hij 307.00i 7.43h 334.33j 4009.00hij 47.70ij 58.67i 30.33j
CD (P = 0.05) 9.00 0.95 1.84 0.83 18.91 267.83 9.70 3.12 3.53

Table 5. Effect of PGPR (B2, SIR1) and chemical fertilisers (N, nitrogen; P, phosphorus; K, potassium) on wheat growth and yield parameters under field conditions
Values are averages of 2 years of data. Within columns, means followed by same letter are not significantly different (critical difference (CD) at P = 0.05)

Treatment (inoculum + No. of tillers Grain no. 1000-grain Yield (t ha–1) Nutrient uptake (kg ha–1)
%recommended fertiliser dose) per plant per spike weight (g) Grain Straw N P K
T1, control (100% NPK) 3.85c 37.42bc 48.1c 3.18c 9.59c 220.56c 21.41c 213.60c
T2, B2 + 80% NP 5.35a 46.57a 53.74a 3.51a 10.56a 247.84a 26.30a 235.40a
T3, SIR1 + 80% NP 4.78ab 41.85ab 51.58ab 3.36ab 10.17b 235.40ab 23.95ab 227.60ab
CD (P = 0.05) 0.67 4.96 2.23 0.15 0.30 13.20 2.40 12.71

Table 6. Effect of PGPR (B2, SIR1) and chemical fertilisers (N, nitrogen; P, phosphorus; K, potassium) on soil and microbial properties associated with wheat rhizosphere (under
field conditions)
MBC, Microbial biomass carbon; RBP, rhizospheric bacterial population; EBP, endophytic bacterial population. Values are averages of 2 years of data. Within columns, means followed by same letter are
not significantly different (critical difference (CD) at P = 0.05)

Treatment (inoculum + Available soil nutrient status (kg ha–1) Soil enzymes Microbial properties
%recommended N P K Dehydrogenase Phosphatase Phytase MBC RBP EBP
fertiliser dose) (mg TPF g–1 h–1) (mmol L–1 g–1 h–1) (mg 100 g–1 soil) (105 cfu g–1 soil) (101 cfu g–1 roots)
T1, control (100% NPK) 307.47bc 27.41c 322.0c 8.69c 468.00c 5529.71c 103.79c 134.00c 77.71bc
T2, B2 + 80% NP 331.88a 32.99a 347.4a 11.39a 526.64a 6744.36a 123.36a 183.86a 86.43a
Crop & Pasture Science

T3, SIR1 + 80% NP 318.46ab 32.82ab 336.12b 10.30b 484.29b 6358.64b 116.29ab 157.14b 80.36b
CD (P = 0.05) 13.43 1.67 7.03 0.35 8.16 190.97 8.71 7.79 3.43
465
466 Crop & Pasture Science
(Table 6). Maximum dehydrogenase activity (11.39 mg TPF g–1
h–1) was recorded in T2, followed by T3, with the minimum
(8.69 mg TPF g–1 h–1) recorded in the uninoculated control.
Likewise, maximum phosphatase activity (526.64 mmol L–1
g–1 h–1) was recorded in T2, followed by T3, with the
minimum (468.00 mmol L–1 g–1 h–1) recorded in the
uninoculated control. Maximum phytase activity (6744.36 mmol
L–1 g–1 h–1) was similarly shown in T2, followed by T3, and the
minimum (5529.71 mmol L–1 g–1 h–1) was recorded in the
uninoculated control. Maximum microbial biomass carbon
(123.36 mg 100 g–1 soil) was recorded in T2, which was on
par with T3, with the minimum (103.79 mg 100 g–1 soil) again
recorded in the uninoculated control.
Total bacterial and endophytic bacterial populations were
increased by combined application of PGPR and optimised
dose of fertilisers in both years compared with the uninoculated
control (significant in all cases except endophytic population
in T3; Table 6). Total bacterial and endophytic bacterial count
varied from 134.00 to 183.86  105 cfu g–1 soil and from 77.71
to 86.43  101 cfu g–1 root on NA medium. The maximum
microbial count in the rhizosphere was recorded for plants
whose seeds received T2 and the minimum count was in the
uninoculated control. A similar pattern was observed for
endophytic bacterial population.
Discussion
Isolation and screening of PGPR isolates
Great variation in the microbial population in both rhizosphere
and roots (endorhizosphere) of wheat was observed. Dominance
of total microbial counts occurred in the rhizosphere compared
with the endophytic population in all three growth mediums (NA,
Jensen’s and PVK medium). The variation in populations of
both rhizosphere soil bacteria and endophytes may be attributed
to location, age of plant, time of sampling, physico-chemical
properties of soil and environmental conditions (Donn et al.
2015). The results are in accord with those of Chiarini et al.
(1998), who also reported that plant development and soil
characteristics have a marked influence on the rhizosphere and
endophytic microflora of maize.
Phosphate solubilisation by bacterial isolates was in the
range 91.67–205.33 mg mL–1, IAA 19.33–31.70 mg mL–1 and
siderophore production 29.00–65.00%. PGP and biocontrol
activities of PGPR have well been established by Gupta et al.
(2017) and Mia et al. (2010). They found that the majority
of rhizospheric bacteria solubilised tri-calcium phosphate, and
produced siderophore, HCN and IAA. Many microorganisms
capable of solubilising insoluble phosphates have been isolated
from the root region or rhizosphere of cereal crops, and their
population in general is greater in rhizosphere (20–40%)
than in non-rhizosphere (10–15%) soil (Kapoor et al. 1989).
Inhibitory effects of many PGPR against F. graminearum and
A. triticina have also been reported by Nourozian et al. (2006)
and Siddiqui (2007).
Pot experiment
Joint application of bacterial inoculants with chemical fertilisers
significantly influenced growth parameters of wheat. Better
shoot and root length and biomass might be due to increases
G. Sood et al.
in the availability of the soil nutrients, particularly N,
P (P solubilisation) and iron (siderophore production), to the
plant by direct mechanisms of application of PGPR inoculum in
the rhizosphere. This affects plant metabolism (increasing the
uptake of water and minerals), enhancing root development
and increasing enzymatic activity of the plant by suppressing
plant pathogens (Abbasi et al. 2011; Baris et al. 2014; Moustaine
et al. 2016).
The increase in available N and P contents in soil may be due
to mineralisation of organic matter and fixation of atmospheric
N2 by the asymbiotic bacterial isolates. These results are in
agreement with those of Ahmad et al. (2008).
Significant increases in soil enzyme activities and microbial
biomass carbon of the treatment T2 over the uninoculated
control are very important because they are direct link of the
soil microbial community to metabolic requirements and available
nutrients in soil. Higher enzyme activities in soil indicate the
potential of soil to effect the biochemical transformations
necessary for the maintenance of soil fertility (Kamlesh et al.
1991; Rao et al. 1990), which supports our results.
The total bacterial population at the termination of experiment
was greater than the initial bacterial population. Application
of efficient inoculum of PGPR with high viable counts
(108–1010 cfu g–1) coupled with adequate amounts of N and
other nutrients might have contributed to the increase in total
microbial population. These results agree with those of
Thakuria et al. (2004), who isolated a large number of
microorganisms capable of solubilising insoluble phosphates
from the root region or rhizosphere of cereal crops and found
that the population of P-solubilisers is greater in rhizosphere
(20–40%) than in non-rhizosphere (10–15%) soil as compared to
total population.
Field studies
The combined application of 80% RFD of N and P along with
seed inoculation with B2 isolate (i.e. T2) proved most effective,
with increases of 28.03% in tillering, 19.61% in grain number
per spike, 10.5% in 1000-grain weight, 9.4% in grain yield
and 9.2% in straw yield. This increase might be due to the
action of PGPR possessing multiple PGP traits, which helped
in colonising the root hair and cortical cells and enhanced the
root surface area, meaning greater acquisition of nutrients as well
as plant hormones by the plant. These results are in agreement
with the findings of many other workers (Adjanohoun et al. 2011;
Jarak et al. 2012; Turan et al. 2012; Afzal et al. 2014).
The increased uptake of N and P may have been due to
increases in different fractions of mineral N and P in soil or to
increases in plant growth-stimulating mechanisms as result
of asymbiotic N2 fixation and P solubilisation. These processes
can positively influence plant growth and crop yields (Asghar
et al. 2002; Schoebitz et al. 2013; Kumar et al. 2014).
Increases in available N and P contents may be attributed to
higher microbial activity of integrated nutrient management
treatments, which favoured the conversion of atmospheric N2
by asymbiotic N2 fixation, and mineralisation of organically
bound N to inorganic forms. Higher available P in PGPR
treatments may be attributed to phosphate-solubilising activity
of microorganisms, which might have brought some P from
Biofertilisers and inorganic nutrients
unavailable to available form by the secretion of organic acids
and phosphatase enzymes. Our results conform with the findings
of many other workers (Antoun et al. 1998; Riggs et al. 2001;
Ogut and Er 2016).
High soil enzyme activity and microbial biomass carbon
content were observed especially in the treatment receiving
seed inoculation with B2 isolate along with 80% RFD of
N and P. Our studies are in accordance with Kaur and Reddy
(2015), who also demonstrated that inoculation of phosphate-
solubilising bacteria, together with rock phosphate fertilisation,
increased the activities of soil enzymes such as dehydrogenase,
acid phosphatase, alkaline phosphatase and phytase compared
with di-ammonium phosphate treatment in a wheat crop.
Microbial biomass and microbial activity are often closely
related because biomass plays an important role in the
transformation of organic elements (C, N and sulfur).
The total bacterial population at the termination of experiment
was greater than the initial population. The increase in microbial
counts may be attributed to artificial inoculation of efficient
microbes and their proliferation in the rhizosphere under the
influence of biotic and abiotic factors on the dynamic structure
of endophytic and rhizospheric microbes (Hallmann et al. 1997).
Conclusion
PGPR (especially B2) increased not only the availability and
uptake of N, P and K and the soil-enzyme status but also the
microbial properties of the soil, thereby resulting in higher
growth and yield of wheat. The productivity of wheat with
80% RFD of chemical fertilisers plus PGPR was found to be
significantly higher than sole application of 100% RFD. The
study therefore indicated the potential of isolated PGPR strain
B2 in partial replacement of N and P (~20%) applied through
chemical fertilisers, besides higher productivity of crops.
Conflicts of interest
The authors declare that there are no conflicts of interest.
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